G Model
PHAREP 113 1–8 Pharmacological Reports xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep 1 2 3 4 5 6
Original research article
Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms Q1 Mariusz
Sikora, Adrian Drapala, Marcin Ufnal *
Department of Experimental and Clinical Physiology, Medical University of Warsaw, Warsaw, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 May 2013 Received in revised form 7 April 2014 Accepted 8 April 2014 Available online xxx
Background: Increasing evidence suggests that disturbances in H2S homeostasis may participate in the development of hypertension. In this study we compared hemodynamic responses to intracerebroventricular (ICV) infusions of sodium hydrosulfide (NaHS), a H2S donor, between normotensive rats (WKY), spontaneously hypertensive rats (SHR) and angiotensin II – induced hypertensive rats (WKY-Ang II). Methods: We tested the effects of NaHS on mean arterial blood pressure (MABP) and heart rate (HR) in 12–14-week-old, male rats. MABP and HR were continuously recorded at baseline and during ICV infusion of either vehicle (Krebs–Henseleit buffer) or NaHS. Results: ICV infusions of the vehicle did not affect MABP and HR. WKY rats infused with 30 nmol/h of NaHS showed a mild decrease in MABP and HR. ICV infusion of 100 nmol/h produced a biphasic response i.e. mild hypotension and bradycardia followed by an increase in MABP and HR, whereas, the infusion of 300 nmol/h of the H2S donor caused a monophasic increases in MABP and HR. In contrast, SHR rats as well as WKY-Ang II rats showed a decrease in MABP and HR during ICV infusions of NaHS. Conclusions: The results provide further evidence for the involvement of H2S in the neurogenic regulation of the circulatory system and suggest that alterations in H2S signaling in the brain could be associated with hypertension. ß 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Keywords: Angiotensin II Blood pressure Hydrogen sulphide Hypertension Brain
7 8
Introduction
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
The toxic effects of exposure to high concentrations of hydrogen sulfide (H2S) have been known for many centuries. However, only over the last two decades have the physiological functions of H2S as gaseous mediator been extensively investigated. This was triggered by Abe and Kimura describing the enzymatic production of H2S in the brain and showing its ability to influence long-term potentiation [1]. A growing body of data suggests that H2S is an important biological mediator involved in various physiological processes [2,3]. Recently, several H2S releasing compounds were developed; and their pharmacotherapeutic potential in cardiovascular, gastrointestinal and immunological diseases are being tested [4]. In the circulatory system H2S has been found to affect vascular tone [5,6] and cardiac functions [7–10]. Several studies have shown that peripherally administered H2S donors produce hypotensive effect [11,12]. It seems, however, that the effects of
* Corresponding author. E-mail addresses: [email protected], [email protected] (M. Ufnal).
H2S in the circulatory system depend on the dose as well as animal species [9,13]. In this context, H2S has been found to dilate mesenteric [14] and hepatic [15] arteries in rats as well as human corpus cavernosum [16] but contract rat and duck aorta [13]. Furthermore, it has been reported that H2S contracts rat and mouse aorta and human arteries at 10–100 mM concentrations while relaxes these vessels at 100–1000 mM concentrations [17–19]. Similarly, H2S may exert both positive as well as negative chronotropic effect on the heart [9,10]. It has been postulated that decreased synthesis of endogenous H2S plays a role in the development of hypertension. Yang and collaborators found that genetic deletion of cystathionine gamma-lyase (CSE), an enzyme which generates H2S, produces hypertension in mice [20]. Furthermore, it has been found that peripheral administration of H2S donors and precursors decreases blood pressure in several experimental models of hypertension such as hypertension induced by chronic inhibition of nitric oxide synthase, two-kidney-one-clip and in spontaneously hypertensive rats (SHR) [21–23]. Accumulating evidence highlights the crucial role of cardiovascular centers in the brain regulation of the circulatory system [24]. An increasing number of mediators, including
http://dx.doi.org/10.1016/j.pharep.2014.04.004 1734-1140/ß 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Please cite this article in press as: Sikora M, et al. Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.004
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
G Model
PHAREP 113 1–8 M. Sikora et al. / Pharmacological Reports xxx (2014) xxx–xxx
2
47 48 49 50 51 52 53 54 55 56 57
gaseous transmitters are being added to the group of mediators involved in the neurogenic control of arterial blood pressure [25]. In our previous studies we found that intracerebroventricular (ICV) infusions of NaHS, a H2S donor exerts significant hemodynamic effect in normotensive WKY rats [9], which imply that H2S plays a role in the brain control of arterial blood pressure. In this study we compared hemodynamic responses to ICV infusions of NaHS between normotensive rats (WKY), spontaneously hypertensive rats (SHR) and WKY rats with angiotensin II (Ang II) – induced hypertension.
58
Materials and methods
59 60 61 62 63
The study was carried out in accordance with domestic and European Union guidelines of animal welfare. The experimental design, animal care and procedures were approved by the Second Local Animal Research Ethics Committee at the Medical University of Warsaw.
64
Animals
65 66 67
We performed the experiments on 12–14 weeks old, conscious male Wistar Kyoto (WKY) and Spontaneous Hypertensive (SHR) rats.
68
Drugs
69 70 71 72 73 74 75 76 77 78
The following drugs were used: Hexamethonium (Hexamethonium bromide, Sigma–Aldrich, Switzerland), Angiotensin II (Sigma–Aldrich, USA), Krebs–Henseleit bicarbonate-buffer (Sigma–Aldrich, USA), sodium hydrosulfide (NaHS; Sigma–Aldrich, Germany). NaHS was used as a H2S donor and was prepared 10 min. before the onset of ICV infusion. Due to limited buffering capacity of the buffer, in order to maintain pH 7.45–7.5 the maximum 10 mM NaHS was prepared. Administration of increasing doses of NaHS was achieved by infusing increasing volumes of NaHS solution at a rate of 3–30 ml/h.
79
Surgical preparation
80 81 82 83 84 85 86 87 88
All rats were implanted with a stainless steel cannula into the lateral cerebral ventricle and week later an arterial catheter was inserted through the femoral artery into the abdominal aorta as previously described [9]. All surgical procedures were performed under general anesthesia with ketamine 100 mg/kg of bw IP (Bioketan, Vetoquinol Biowet, Poland) and xylazine 10 mg/kg of bw IP (Xylapan, Vetoquinol Biowet, Poland). After surgery the animals were given benzathine penicillin 30 000 IU IM (Debecylina, Polfa Tarchomin, Poland).
89
Hemodynamic measurements
90 91 92 93 94 95 96 97 98 99 100 101 102
Measurements were performed 2 days after the second surgery. Mean arterial blood pressure (MABP) was recorded on-line through the arterial catheter connected to the blood pressure recording system (Biopac MP100 unit; Biopac Systems, Goleta, CA). All measurements were done in animals freely moving in their living cages after stabilization of hemodynamic parameters (45 15 min). MABP and HR were recorded at baseline (10 min) and during ICV infusions (45 min). Heart rate (HR) was calculated from the consecutive systolic peaks on the blood pressure tracing by the AcqKnowledge v3.7 Biopac software. ICV infusions were performed through a stainless steel infusion tube which was inserted into the previously implanted cannula.
Experimental series
103
WKY and SHR rats were randomly assigned to experimental series. In control series normotensive WKY rats (WKY-Control; n = 6) and hypertensive SHR rats (SHR-Control; n = 6) were infused with the Krebs–Henseleit buffer at a rate of 30 ml/h. ICV infusions of 10 mM NaHS was administered to WKY and SHR rats either at a dose of 30 nmol/h (WKY-30H2 S series, n = 6 and SHR-30H2 S series, n = 6), 100 nmol/h (WKY-100H2 S series, n = 6 and SHR-100H2 S series, n = 6) or 300 nmol/h (WKY-300H2 S series, n = 6 and SHR-300H2 S series, n = 6). Additionally, to prove that hemodynamic effects of ICV infused NaHS are mediated by the brain mechanisms and not by peripheral action of NaHS, we did the two series of experiments. In the first experiment WKY and SHR rats were concomitantly infused ICV with 300 nmol/h of NaHS and IV with hexamethonium, a ganglionic blocker (10 mg bolus followed by continuous infusion at the dose of 0.5 mg/min/rat, WKY-300H2 SþHex series, n = 6 and SHR-300H2 SþHex series, n = 6, respectively). In the second experiment WKY and SHR rats were infused IV with 300 nmol/h of NaHS. Finally, to compare the pattern of hemodynamic response to NaHS in two different animal models of hypertension, we checked the response to ICV infusion of either: 300 nmol/h of NaHS or the Krebs–Henseleit buffer at a rate of 30 ml/h in SHR rats (SHR-300H2 S series, n = 6 and SHR-Control series, n = 6) and in WKY rats with hypertension induced by Ang II (WKY-Ang II-300H2 S series, n = 6 and WKY-Ang II-Control series, n = 5). Hypertension in WKY rats was induced by chronic infusion of Ang II at 450 ng/1 min/kg bw for 10 days, using a subcutaneously implanted osmotic minipump (Alzet 2ML2).
104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131
Data analysis
132
The results are expressed as the mean SEM. For the evaluation of the effects of ICV infusions within series, the average over 5 min before the infusion was compared with the average over 5 min after the onset of infusion by one-way repeated-measures analysis of variance (ANOVA), followed by Tukey’s test. Comparisons between the series were evaluated by one- or two-way ANOVA for repeated measures when appropriate, followed by Tukey’s test. Two-sided P < 0.05 was considered significant. All analyses were conducted using STATISTICA 6.0 (StatSoft, Krakow, Poland).
133 134 135 136 137 138 139 140 141 142
Table 1 Mean arterial blood pressure (MABP) and heart rate (HR) at baseline in WKY, SHR and WKY-Ang II rats. Groups/series
MABP (mmHg)$,#,^
HR (beats/min)$,#,^
WKY rats WKY-Control (n = 6) WKY-30H2 S (n = 6) WKY-100H2 S (n = 6) WKY-300H2 S (n = 6) WKY-300H2 SþHex (n = 6)
114.8 0.5 114.3 0.6 113.8 0.6 113.7 0.4 114.3 0.5
323 1 328 2 328 2 325 2 329 2
SHR rats SHR-Control (n = 6) SHR-30H2 S (n = 6) SHR-100H2 S (n = 6) SHR-300H2 S (n = 6) SHR-300H2 SþHex (n = 6)
156.9 0.5 154.1 1.1 158.6 0.9 158.4 2.3 156.5 1.2
378 1 384 2 381 2 379 3 382 3
WKY-Ang II rats WKY-Ang II-Control (n = 5) WKY-Ang II-300H2 S (n = 6)
127.3 1.3 124.7 0.7
368 6 352 4
Means S.E.M. $ P < 0.05 WKY rats vs SHR rats. # P < 0.05 WKY rats vs WKY-Ang II rats. ^ P < 0.05 SHR rats vs WKY-Ang II rats.
Please cite this article in press as: Sikora M, et al. Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.004
G Model
PHAREP 113 1–8 M. Sikora et al. / Pharmacological Reports xxx (2014) xxx–xxx
143
Results
144 145 146
Baseline MABP was significantly higher in SHR and WKY-Ang II groups than in WKY group. There were no significant differences in MABP and HR between series within the groups (Table 1).
147
WKY group
148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163
In WKY-Control series ICV infusions of the buffer did not affect MABP and HR (Fig. 1A and B). ICV infusion of NaHS in WKY-30H2 S series decreased MABP [F(1,9) = 36.5, P < 0.001] and HR [F(1,9) = 15.6, P < 0.001] (Fig. 1A, 1B). Infusion of H2S donor in WKY-100H2 S series produced biphasic response, i.e. decrease in MABP and HR for the first 20 min of ICV infusion [F(1,4) = 10.6, P < 0.001 and F(1,4) = 6.7, P < 0.001, for MABP and HR respectively], followed by a significant increase in MABP and HR [F(1,5) = 7.8, P < 0.001 and F(1,5) = 7.5, P < 0.001, for MABP and HR respectively]. In WKY-300H2 S series we found significant increase in MABP and HR [F(1,9) = 34.7, P < 0.001 and F(1,9) = 28.3, P < 0.001, for MABP and HR respectively] (Fig. 1A and B). In WKY-300H2 SþHex series, ganglionic blockade with hexamethonium produced a significant decrease in MABP (mmHg) from 114.3 0.5 to 67.2 0.4 and HR (beats/min) from 329 2 to 250 3. The following ICV infusions of 300 nmol/h of NaHS did not affect MABP and HR
Fig. 1. Changes in mean arterial blood pressure (MABP) and heart rate (HR) in WKY rats during intracerebroventricular (ICV) infusions of: vehicle (WKY-control), NaHS at a dose of 30 nmol/h (WKY-30H2 S ), NaHS at a dose of 100 nmol/h (WKY-100H2 S ) and NaHS at a dose of 300 nmol/h (WKY-300H2 S ). (A) *P < 0.5 vs baseline; $P < 0.05 WKY-control vs WKY-30H2 S , ^P < 0.05 WKY-control vs WKY-100H2 S , #P < 0.05 WKY-control vs WKY-300H2 S . (B) Changes in HR (beats/min); *P < 0.05 vs baseline, $ P < 0.05 WKY-control vs WKY-30H2 S , ^P < 0.05 WKY-Control vs WKY-300H2 S .
3
(Fig. 3A and B). In WKY-Control series IV infusions of 300 nmol/h of 164 NaHS did not affect MABP and HR. 165 Two-way ANOVA series x time revealed significant differences 166 between series in WKY group in changes in MABP and HR during 167 ICV infusions [F(3,8) = 12.3, P < 0.001] for MABP and F(3,8) = 14.8, 168 P < 0.001 for HR) (Fig. 1A and B). Post hoc analysis revealed the Q2169 following significant differences between series in MABP changes: 170 WKY-Control vs WKY-30H2 S P < 0.001, WKY-Control vs 171 WKY-100H2 S P < 0.03, WKY-Control vs WKY-300H2 S P < 0.001 172 and WKY-30H2 S vs WKY-100H2 S P < 0.001, WKY-30H2 S vs 173 WKY-300H2 S P < 0.03 and WKY-100H2 S vs WKY-300H2 S P < 0.001 174 (Fig. 1A). There also were the following significant differences 175 between series in changes in HR: WKY-Control vs WKY-30H2 S 176 P < 0.001, WKY-Control vs WKY-300H2 S P < 0.001 and WKY-30H2 S 177 vs WKY-100H2 S P < 0.001, WKY-30H2 S vs WKY-300H2 S P < 0.03 and 178 WKY-100H2 S vs WKY-300H2 S P < 0.001 (Fig. 1B). 179 There was a significant difference in changes in MABP and HR 180 between WKY-300H2 SþHex and WKY-300H2 S series [F(1,8) = 17.8 181 P < 0.001 and F(1,8) = 21.8 P < 0.001, for MABP and HR 182 respectively] (Fig. 3A and B). Q3183 SHR group
184
In SHR-Control series ICV infusions of the buffer did not affect MABP and HR (Fig. 2A and B). ICV infusion of NaHS decreased MABP
185 186
Fig. 2. Changes in mean arterial blood pressure (MABP) and heart rate (HR) in SHR rats during intracerebroventricular (ICV) infusions of: vehicle (SHR-control), NaHS at a dose of 30 nmol/h (SHR-30H2 S ), NaHS at a dose of 100 nmol/h (SHR-100H2 S ) and NaHS at a dose of 300 nmol/h (SHR-300H2 S ). (A) Changes in MABP (mmHg); *P < 0.05 vs baseline. $ P < 0.05 SHR-control vs SHR-30H2 S , ^P < 0.05 SHR-control vs SHR-100H2 S , #P < 0.05 SHR-control vs SHR-300H2 S . (B) Changes in HR (beats/min); *P < 0.05 vs baseline, $P < 0.05 SHR-control vs SHR-100H2 S , #P < 0.05 SHR-control vs SHR-300H2 S .
Please cite this article in press as: Sikora M, et al. Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.004
G Model
PHAREP 113 1–8 4
M. Sikora et al. / Pharmacological Reports xxx (2014) xxx–xxx
Fig. 3. Changes in mean arterial blood pressure (MABP) and heart rate (HR) in WKY and SHR rats during intracerebroventricular (ICV) infusion of: NaHS at a dose of 300 nmol/h (WKY-300H2 S and SHR-300H2 S series), and concomitant ICV infusion of NaHS at a dose of 300 nmol/h and IV infusion of hexamethonium (WKY-300H2 SþHEX and SHR-300H2 SþHEX series). (A) Changes in MABP (mmHg); *P < 0.05 vs baseline, $ P < 0.05 WKY-300H2 SþHex vs WKY-300H2 S ; #P < 0.05 SHR-300H2 SþHex vs SHR-300H2 S . (B) Changes in HR (beats/min); *P < 0.05 vs baseline, $P < 0.05 WKY-300H2 SþHex vs WKY-300H2 S ; #P < 0.05 SHR-300H2 SþHex vs SHR-300H2 S .
187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210
and HR in all the experimental series in SHR rats: SHR-30H2 S [F(1,9) = 5.4, P < 0.001 and F(1,9) = 5.8, P < 0.001, for MABP and HR, respectively], SHR-100H2 S [F(1,9) = 10.4, P < 0.001] and [F(1,9) = 11.6, P < 0.001, for MABP and HR, respectively], SHR-300H2S [F(1,9) = 15.4, P < 0.001 and F(1,9) = 20.4, P < 0.001, for MABP and HR, respectively] (Fig. 2A and 2B). In SHR-300H2 SþHex series, ganglionic blockade with hexamethonium produced a significant decrease in MABP (mmHg) from 156.5 1.2 to 96.4 0.6 and HR (beats/min) from 382 3 to 322 1. The following ICV infusions of 300 nmol/h of NaHS did not affect MABP and HR (Fig. 3A and B). In SHR-Control series IV infusions of 300 nmol/h of NaHS did not affect MABP and HR. Two-way ANOVA series x time revealed significant difference in changes in MABP and HR between series in SHR group [F(3,8) = 1.7, P < 0.05 and F(3,8) = 1.9, P < 0.05 for MABP and HR, respectively] (Fig. 2A and B). Post hoc analysis revealed the following significant differences in changes in MABP: SHR-Control vs SHR-30H2 S P < 0.05, SHR-Control vs SHR-100H2 S P < 0.001, SHR-Control vs SHR-300H2 S P < 0.001 and SHR-30H2 S vs SHR-100H2 S P < 0.01, SHR-30H2 S vs SHR-300H2 S P < 0.001 and SHR-100H2 S vs SHR-300H2 S P < 0.05. There were also the following significant differences in HR changes between series: SHR-Control vs SHR-100H2 S P < 0.001, SHR-Control vs SHR-300H2 S P < 0.001 and SHR-30H2 S vs SHR-300H2 S P < 0.001.
Fig. 4. Changes in mean arterial blood pressure (MABP) and heart rate (HR) in WKY, SHR and WKY-Ang II rats during intracerebroventricular (ICV) infusion of NaHS at a dose of 300 nmol/h. (A) Changes in MABP (mmHg); *P < 0.05 vs baseline, $P < 0.05 WKY-300H2 S vs SHR-300H2 S , #P < 0.05 WKY-300H2 S vs WKY-Ang II-300H2 S ; ^ P < 0.05 SHR-300H2 S vs WKY-Ang II-300H2 S . (B) Changes in HR (beats/min), *P < 0.05 vs baseline, $P < 0.05 WKY-300H2 S vs SHR-300H2 S , #P < 0.05 WKY-300H2 S vs WKY-Ang II-300H2 S .
There was a significant difference in changes in MABP and HR between SHR-300H2 SþHex and SHR-300H2 S series [F(1,8) = 5.0, P < 0.001 and F(1,8) = 6.0, P < 0.001 for MABP and HR, respectively] (Fig. 3A and B).
211 212 213 214
WKY-Ang II group
215
ICV infusions of Krebs–Henseleit buffer in WKY-Ang II-Control group did not affect MABP and HR. ICV infusions of 300 nmol/h of NaHS in WKY-Ang II-300H2 S group decreased MABP and HR [F(1,9) = 4.8, P < 0.001 and F(1,9) = 9.2, P < 0.001, for MABP and HR, respectively] (Fig. 4A and B).
216 217 218 219 220
WKY vs SHR group
221
There were no significant differences in changes in MABP and HR between WKY-Control and SHR-Control ( Figs. 5A and 6A) as well as between WKY-30H2S and SHR-30H2S series ( Figs. 5B and 6B). There was a significant difference in MABP and HR between WKY and SHR groups infused ICV with 100 nmol/h of NaHS: WKY-100H2 S vs SHR-100H2 S series [F(1,8) = 18.1, P < 0.001 and F(1,8) = 14.8, P < 0.001, for MABP and HR respectively] (Figs. 5C and 6C). There was a significant difference in MABP and HR between WKY and SHR groups infused ICV with 300 nmol/h of NaHS:
222 223 224 225 226 227 228 229 230 231
Please cite this article in press as: Sikora M, et al. Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.004
G Model
PHAREP 113 1–8 M. Sikora et al. / Pharmacological Reports xxx (2014) xxx–xxx
5
Fig. 5. Changes in mean arterial blood pressure (MABP) in WKY and SHR rats during intracerebroventricular (ICV) infusions. (A) ICV infusion of vehicle in WKY rats (WKYcontrol) and SHR rats (SHR-control). (B) ICV infusion of NaHS at a dose of 30 nmol/h in WKY rats (WKY-30H2 S ) and SHR rats (SHR-30H2 S ). (C) ICV infusion of NaHS at a dose of 100 nmol/h in WKY rats (WKY-100H2 S ) and SHR rats (SHR-100H2 S ), $P < 0.05 WKY-100H2 S vs SHR-100H2 S series. (D) ICV infusion of NaHS at a dose of 300 nmol/h in WKY rats (WKY-300H2 S ) and SHR (SHR-300H2 S ). $P < 0.05 WKY-300H2 S vs SHR-300H2 S series.
232 233 234
WKY-300H2 S vs SHR-300H2 S series [F(1,8) = 20.5, P < 0.001 and F(1,8) = 27.4, P < 0.001 for MABP and HR, respectively] (Figs. 5D and 6D).
235
WKY vs SHR and WKY-Ang II groups
236 237 238 239 240 241 242 243 244 245
A significant difference was found in changes in MABP and HR between WKY, WKY-Ang II and SHR groups in series in which rats were ICV infused with 300 nmol/h of NaHS [F(2,8) = 12.0, P < 0.001 and F(2,8) = 14.75, P < 0.001 for MABP and HR, respectively] (Fig. 4A and B). Post hoc analysis revealed the following significant differences for MABP: WKY-300H2 S vs SHR-300H2 S P < 0.001, WKY-300H2 S vs WKY-Ang II-300H2 S P < 0.001, WKY-Ang II-300H2 S vs SHR-300H2 S (Fig. 4A and B) and for HR: WKY-300H2 S vs SHR-300H2 S P < 0.001, WKY-300H2 S vs WKY-Ang II-300H2 S P < 0.001 (Fig. 4A and B).
246
Discussion
247 248 249 250 251 252
The novel finding of this study is that there are significant differences between normotensive and hypertensive rats in hemodynamic responses to ICV infused NaHS, a H2S donor. We found that in contrast to normotensive WKY rats, hypertensive SHR and WKY-Ang II rats show hypotensive response to ICV infused NaHS. This implies that differences in
response to ICV infused NaHS between normotensive WKY and hypertensive SHR and WKY-Ang II rats are associated with high arterial blood pressure rather than interstrain differences. Similar patterns of hemodynamic response to NaHS in SHR and WKY-Ang II rats, suggest that hypertension independently on its origin is associated with alterations in H2S signaling in the brain. Therefore, we postulate that high arterial blood pressure changes H2S transmission in the brain rather than the other way round. However, primary disturbances in H2S homeostasis in the brain as a cofactor of the pathogenesis of hypertension cannot be excluded. In this line, decreased synthesis of endogenous H2S in the peripheral tissues has been suggested to be involved in the development of hypertension [20], and the treatment with H2S donors and precursors was found to decrease blood pressure in several experimental models of hypertension [21–23]. So far the effect of H2S on the brain regulation of the circulatory system has only been evaluated in a few studies, and their results were contradictory [9,26–28]. The present results are in line with our previous findings showing hypertensive response to ICV infusion of NaHS in WKY rats. However, in the current study the pressor response to NaHS at a dose of 100 nmol/h and higher was more pronounced than in our previous experiment [9]. This could be a result of longer baseline meassurments in our prevous study, which increased
Please cite this article in press as: Sikora M, et al. Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.004
253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277
G Model
PHAREP 113 1–8 6
M. Sikora et al. / Pharmacological Reports xxx (2014) xxx–xxx
Fig. 6. Changes in heart rate (HR) in WKY and SHR rats during intracerebroventricular (ICV) infusions. (A) ICV infusion of vehicle in WKY rats (WKY-control) and SHR rats (SHR-control). (B) ICV infusion of NaHS at a dose of 30 nmol/h in WKY rats (WKY-30H2 S ) and SHR rats (SHR-30H2 S ). (C) ICV infusion of NaHS at a dose of 100 nmol/h in WKY rats (WKY-100H2 S ) and SHR rats (SHR-100H2 S ); $P < 0.05 WKY-100H2 S vs SHR-100H2 S series. (D) ICV infusion of NaHS at a dose of 300 nmol/h in WKY rats (WKY-300H2 S ) and SHR rats (SHR-300H2 S ); $P < 0.05 WKY-300H2 S vs SHR-300H2 S .
278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303
time form the preparation of NaHS solution to the onset of ICV infusions up to aproximately 30–40 min. Whereas in the present study this time did not exceed 10 min. In addition, in the current study we found that ICV infused NaHS at a dose of 30 nmol/h decreases arterial blood pressure. The opposite hemodynamic effects of high and low doses of NaHS may be due to the following reasons. Neuroactive compounds infused ICV act on the circumventricular organs which lack the blood –brain barrier (BBB) as well as may penetrate behind the BBB to cardiovascular centers such as paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) [24,29]. As H2S easily permeates cell membranes and has a relatively long half-life it is likely that ICV infused H2S targets neurons of the circumventricular organs as well as neurons of the cardiovascular centers protected by the BBB. Therefore it is possible that lower doses of H2S caused hypotensive effects in WKY-30H2 S series acting on the circumventricular organs whereas higher doses of H2S produced hypertensive response in WKY-300H2 S series penetrating to the cardiovascular centers behind the BBB. In this context, biphasic response in WKY-100H2 S series could reflect an increase in H2S concentration in the CSF during continuous ICV infusion. Another possible explanation is that the opposite hemodynamic effects of H2S may depend on distinct mechanisms with a different stimulation thresholds. For example, it has been shown that H2S may exert both pro-nociceptive and
antinociceptive effects [30–32] and that it may act as a vasoconstrictor or a vasorelaxant in peripheral blood vessels [5]. To our knowledge, there are three other studies on hemodynamic effects of ICV infusions of H2S in rats [27,28,33]. Ren and collaborators reported that the ICV injection of H2S saturated buffer and H2S precursors ( L-cysteine and beta-mercaptopyruvate) significantly increase MABP after an initial transient hypotension [33]. In contrast, Liu and collaborators have found that the ICV infusion of NaHS, at the same doses we tested, causes a significant decrease in blood pressure [27]. The discrepancies between the results of Liu et al. and our findings seem to result from differences in experimental settings. Namely, Liu and collaborators dissolved NaHS in an artificial CSF without a buffer, whereas Ren et al. and us used buffered solutions of NaHS [27,33]. As NaHS is highly alkaline, findings by Liu et al. could partially result from pH changes in the CSF. The other three studies investigated the effect of NaHS infusions into specific cardiovascular centers in the brain. Streeter and collaborators demonstrated the presence of the H2S-producing enzyme, cystathionine-beta-synthase in the RVLM and the PVN, however they did not find significant hemodynamic effects of NaHS at a dose of 0.2–2000 pmol infused into the RVLM and PVN and 20 nmol of NaHS infused ICV [28]. In contrast, Guo et al. showed that bilateral microinjections of NaHS (4, 8, and 16 mM, 50 nl) into the RVLM decreases MABP, HR and renal sympathetic nerve activity in a dose-dependent
Please cite this article in press as: Sikora M, et al. Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.004
304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329
G Model
PHAREP 113 1–8 M. Sikora et al. / Pharmacological Reports xxx (2014) xxx–xxx
330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391
manner [34]. These studies, however, were conducted in anesthesia which strongly inhibits nervous reflexes and alters the neurogenic regulation of the circulatory system. The experiments on freely moving rats were conducted by Dawe and collaborators who found that infusion of 200 mmol of NaHS into the hypothalamus reduces blood pressure and heart rate [26]. However, it needs to be noted that infusion of neuroactive agents into a single cardiovascular center often produces different hemodynamic effects than ICV infusion which targets multiple cardiovascular centers [25]. An advantage of ICV infusions lies in similarity to real life conditions, in which compounds circulating in the blood and/or in the cerebrospinal fluid may target multiple cardiovascular centers in the brain. However, ICV infused agents may be cleared via the lymphatic system and enter the blood circulation. This may affect the function of peripheral tissues and cause the opposite hemodynamic effects to those produced by stimulation of the brain cardiovascular centers [35]. Therefore, to confirm that we are looking at the effects mediated by the brain mechanism, we performed two additional series of experiments. In the first experiment we checked the hemodynamic response after intravenous infusion of NaHS at the highest dose tested in ICV infusion experiments. In the second experiments during ICV infusions of NaHS we blocked the nervous control of the circulatory system with hexamethonium, a ganglionic blocker. The lack of hemodynamic response to NaHS in these two series provides strong evidence that the effects triggered by the ICV infusion of the H2S donor in the previous series were mediated by the brain mechanisms. A limitation of our study, is that increasing doses of NaHS achieved by infusing increasing volumes of NaHS solution at a rate 3 ml/h to 30 ml/h, could result in differences in ICV pressure between experimental series. This approach was imposed by a limited buffering capacity of Krebs –Henseleit buffer, which allow us to prepare a maximum 10 mM solution of NaHS to maintain pH within 7.4–7.45. Although we cannot exclude that there were differences in ICV pressure between experimental series, it seems unlikely that they had a significant effect on hemodynamic parameters. This is based on ours and others results showing that ICV infusions of a vehicle at the rate of up to 40 ml/h do not affect arterial blood pressure and heart rate [9,36]. In contrast, even moderate changes in pH of the cerebrospinal fluid cause significant changes in the neurogenic regulation of the circulatory and respiratory systems. Therefore, it seems that our approach is less likely to produce artifacts compared to infusing the same volumes of NaHS solutions but varying in pH. A key problem in studies on biological effects of H2S is a significant variation in reported physiological concentrations of H2S in tissues, particular in the brain. Dependent on the methods employed, the estimated concentration of H2S in the brain and the CSF has been reported to range from 0.1 to 200 mM [37–39]. A molecular form of H2S is about 20% of H2S in the NaHS solution (calculated for: pH 7.4; temperature 37 8C, pK = 6.755). This is why, the doses of NaHS donors tested in ours and other studies could increase H2S concentration in the brain and CSF with wide range, from two to several thousand times of estimated physiological level. In conclusion, exogenous H2S significantly affects the neurogenic control of arterial blood pressure and hypertension may be associated with disturbances in the H2S signaling in the brain. Further studies on the role of H2S in the circulatory system are needed as well as validation of technics used in measuring concentration of H2S in tissues.
392
Conflict of interest
393
No conflict of interest.
7
Funding
394
This work was supported by the National Science Center, grant Q4395 no. 2011/01/N/NZ4/03682 and Medical University of Warsaw, 396 grant nos. 1MA/PM11/12 and 1MA/PM11/13. 397 Acknowledgment
398
The authors are in debt to Dr. T. Zera for his critical comments on the manuscript.
399 400
References
401
[1] Abe K, Kimura H. The possible role of hydrogen sulfide as an endogenous neuromodulator. J Neurosci 1996;16(3):1066–71. [2] Wang R. Physiological implications of hydrogen sulfide: a whiff exploration that blossomed. Physiol Rev 2012;92(2):791–896. [3] Lowicka E, Beltowski J. Hydrogen sulfide (H2S) – the third gas of interest for pharmacologists. Pharmacol Rep 2007;59(1):4–24. [4] Kashfi K, Olson KR. Biology and therapeutic potential of hydrogen sulfide and hydrogen sulfide-releasing chimeras. Biochem Pharmacol 2013;85(5):689–703. [5] Olson KR, Dombkowski RA, Russell MJ, Doellman MM, Head SK, Whitfield NL, et al. Hydrogen sulfide as an oxygen sensor/transducer in vertebrate hypoxic vasoconstriction and hypoxic vasodilation. J Exp Biol 2006;209(Pt 20):4011–23. [6] Dombkowski RA, Russell MJ, Olson KR. Hydrogen sulfide as an endogenous regulator of vascular smooth muscle tone in trout. Am J Physiol Regul Integr Comp Physiol 2004;286(4):R678–85. [7] Bian JS, Yong QC, Pan TT, Feng ZN, Ali MY, Zhou S, et al. Role of hydrogen sulfide in the cardioprotection caused by ischemic preconditioning in the rat heart and cardiac myocytes. J Pharmacol Exp Ther 2006;316(2):670–8. [8] Hu X, Li T, Bi S, Jin Z, Zhou G, Bai C, et al. Possible role of hydrogen sulfide on the preservation of donor rat hearts. Transplant Proc 2007;39(10):3024–9. [9] Ufnal M, Sikora M, Dudek M. Exogenous hydrogen sulfide produces hemodynamic effects by triggering central neuroregulatory mechanisms. Acta Neurobiol Exp (Wars) 2008;68(3):382–8. [10] Xu M, Wu YM, Li Q, Wang X, He RR. Electrophysiological effects of hydrogen sulfide on pacemaker cells in sinoatrial nodes of rabbits. Sheng Li Xue Bao 2008;60(2):175–80. [11] Zhao W, Zhang J, Lu Y, Wang R. The vasorelaxant effect of H(2)S as a novel endogenous gaseous K(ATP) channel opener. EMBO J 2001;20(21):6008–16. [12] Li L, Whiteman M, Guan YY, Neo KL, Cheng Y, Lee SW, et al. Characterization of a novel, water-soluble hydrogen sulfide-releasing molecule (GYY4137): new insights into the biology of hydrogen sulfide. Circulation 2008;117(18):2351–60. [13] Dombkowski RA, Russell MJ, Schulman AA, Doellman MM, Olson KR. Vertebrate phylogeny of hydrogen sulfide vasoactivity. Am J Physiol Regul Integr Comp Physiol 2005;288(1):R243–52. [14] Cheng Y, Ndisang JF, Tang G, Cao K, Wang R. Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats. Am J Physiol Heart Circ Physiol 2004;287(5):H2316–23. [15] Siebert N, Cantre D, Eipel C, Vollmar B. H2S contributes to the hepatic arterial buffer response and mediates vasorelaxation of the hepatic artery via activation of K(ATP) channels. Am J Physiol Gastrointest Liver Physiol 2008;295(6):G1266–73. [16] d’Emmanuele di Villa Bianca R, Sorrentino R, Maffia P, Mirone V, Imbimbo C, Fusco F, et al. Hydrogen sulfide as a mediator of human corpus cavernosum smooth-muscle relaxation. Proc Natl Acad Sci U S A 2009;106(11):4513–8. [17] Ali MY, Ping CY, Mok YY, Ling L, Whiteman M, Bhatia M, et al. Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide? Br J Pharmacol 2006;149(6):625–34. [18] Kubo S, Doe I, Kurokawa Y, Nishikawa H, Kawabata A. Direct inhibition of endothelial nitric oxide synthase by hydrogen sulfide: contribution to dual modulation of vascular tension. Toxicology 2007;232(1–2):138–46. [19] Webb GD, Lim LH, Oh VM, Yeo SB, Cheong YP, Ali MY, et al. Contractile and vasorelaxant effects of hydrogen sulfide and its biosynthesis in the human internal mammary artery. J Pharmacol Exp Ther 2008;324(2):876–82. [20] Yang G, Wu L, Jiang B, Yang W, Qi J, Cao K, et al. H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine gammalyase. Science 2008;322(5901):587–90. [21] Zhong G, Chen F, Cheng Y, Tang C, Du J. The role of hydrogen sulfide generation in the pathogenesis of hypertension in rats induced by inhibition of nitric oxide synthase. J Hypertens 2003;21(10):1879–85. [22] Yan H, Du J, Tang C. The possible role of hydrogen sulfide on the pathogenesis of spontaneous hypertension in rats. Biochem Biophys Res Commun 2004;313(1):22–7. [23] Lu M, Liu YH, Goh HS, Wang JJ, Yong QC, Wang R, et al. Hydrogen sulfide inhibits plasma renin activity. J Am Soc Nephrol 2010;21(6):993–1002. [24] Dampney RA. Functional organization of central pathways regulating the cardiovascular system. Physiol Rev 1994;74(2):323–64.
402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469
Please cite this article in press as: Sikora M, et al. Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.004
G Model
PHAREP 113 1–8 8
470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492
M. Sikora et al. / Pharmacological Reports xxx (2014) xxx–xxx
[25] Ufnal M, Sikora M. The role of brain gaseous transmitters in the regulation of the circulatory system. Curr Pharm Biotechnol 2011;12(9):1322–33. [26] Dawe GS, Han SP, Bian JS, Moore PK. Hydrogen sulphide in the hypothalamus causes an ATP-sensitive K+ channel-dependent decrease in blood pressure in freely moving rats. Neuroscience 2008;152(1):169–77. [27] Liu WQ, Chai C, Li XY, Yuan WJ, Wang WZ, Lu Y. The cardiovascular effects of central hydrogen sulfide are related to K(ATP) channels activation. Physiol Res 2011;60(5):729–38. [28] Streeter E, Al-Magableh M, Hart JL, Badoer E. Hydrogen sulfide in the RVLM and PVN has no effect on cardiovascular regulation. Front Physiol 2011;2:55. [29] Cottrell GT, Ferguson AV. Sensory circumventricular organs: central roles in integrated autonomic regulation. Regul Pept 2004;117(1):11–23. [30] Cunha TM, Dal-Secco D, Verri Jr WA, Guerrero AT, Souza GR, Vieira SM, et al. Dual role of hydrogen sulfide in mechanical inflammatory hypernociception. Eur J Phar 2008;590(1–3):127–35. [31] Distrutti E, Sediari L, Mencarelli A, Renga B, Orlandi S, Russo G, et al. 5-Amino-2-hydroxybenzoic acid 4-(5-thioxo-5H-[1,2]dithiol-3yl)-phenyl ester (ATB-429), a hydrogen sulfide-releasing derivative of mesalamine, exerts antinociceptive effects in a model of postinflammatory hypersensitivity. J Pharmacol Exp Ther 2006;319(1):447–58. [32] Matsunami M, Tarui T, Mitani K, Nagasawa K, Fukushima O, Okubo K, et al. Luminal hydrogen sulfide plays a pronociceptive role in mouse colon. Gut 2009;58(6):751–61.
[33] Ren YS, Wu SY, Wang XJ, Yu F, Zhao J, Tang CS, et al. Multiple hemodynamic effects of endogenous hydrogen sulfide on central nervous system in rats. Chin Med J (Engl) 2011;124(21):3468–75. [34] Guo Q, Jin S, Wang XL, Wang R, Xiao L, He RR, et al. Hydrogen sulfide in the rostral ventrolateral medulla inhibits sympathetic vasomotor tone through ATP-sensitive K+ channels. J Pharmacol Exp Ther 2011;338(2):458–65. [35] Ufnal M, Skrzypecki J. Blood borne hormones in a cross-talk between peripheral and brain mechanisms regulating blood pressure, the role of circumventricular organs. Neuropeptides 2014;48(2):65–73. [36] Ye S, Mozayeni P, Gamburd M, Zhong H, Campese VM. Interleukin-1beta and neurogenic control of blood pressure in normal rats and rats with chronic renal failure. Am J Physiol Heart Circ Physiol 2000;279(6): H2786–96. [37] Furne J, Saeed A, Levitt MD. Whole tissue hydrogen sulfide concentrations are orders of magnitude lower than presently accepted values. Am J Physiol Regul Integr Comp Physiol 2008;295(5):R1479–85. [38] Leffler CW, Parfenova H, Basuroy S, Jaggar JH, Umstot ES, Fedinec AL. Hydrogen sulfide and cerebral microvascular tone in newborn pigs. Am J Physiol Heart Circ Physiol 2011;300(2):H440–7. [39] Goodwin LR, Francom D, Dieken FP, Taylor JD, Warenycia MW, Reiffenstein RJ, et al. Determination of sulfide in brain tissue by gas dialysis/ion chromatography: postmortem studies and two case reports. J Anal Toxicol 1989;13(2):105–9.
Please cite this article in press as: Sikora M, et al. Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.004
493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515
PHAREP 113 1–8 Pharmacological Reports xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep 1 2 3 4 5 6
Original research article
Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms Q1 Mariusz
Sikora, Adrian Drapala, Marcin Ufnal *
Department of Experimental and Clinical Physiology, Medical University of Warsaw, Warsaw, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 May 2013 Received in revised form 7 April 2014 Accepted 8 April 2014 Available online xxx
Background: Increasing evidence suggests that disturbances in H2S homeostasis may participate in the development of hypertension. In this study we compared hemodynamic responses to intracerebroventricular (ICV) infusions of sodium hydrosulfide (NaHS), a H2S donor, between normotensive rats (WKY), spontaneously hypertensive rats (SHR) and angiotensin II – induced hypertensive rats (WKY-Ang II). Methods: We tested the effects of NaHS on mean arterial blood pressure (MABP) and heart rate (HR) in 12–14-week-old, male rats. MABP and HR were continuously recorded at baseline and during ICV infusion of either vehicle (Krebs–Henseleit buffer) or NaHS. Results: ICV infusions of the vehicle did not affect MABP and HR. WKY rats infused with 30 nmol/h of NaHS showed a mild decrease in MABP and HR. ICV infusion of 100 nmol/h produced a biphasic response i.e. mild hypotension and bradycardia followed by an increase in MABP and HR, whereas, the infusion of 300 nmol/h of the H2S donor caused a monophasic increases in MABP and HR. In contrast, SHR rats as well as WKY-Ang II rats showed a decrease in MABP and HR during ICV infusions of NaHS. Conclusions: The results provide further evidence for the involvement of H2S in the neurogenic regulation of the circulatory system and suggest that alterations in H2S signaling in the brain could be associated with hypertension. ß 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Keywords: Angiotensin II Blood pressure Hydrogen sulphide Hypertension Brain
7 8
Introduction
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
The toxic effects of exposure to high concentrations of hydrogen sulfide (H2S) have been known for many centuries. However, only over the last two decades have the physiological functions of H2S as gaseous mediator been extensively investigated. This was triggered by Abe and Kimura describing the enzymatic production of H2S in the brain and showing its ability to influence long-term potentiation [1]. A growing body of data suggests that H2S is an important biological mediator involved in various physiological processes [2,3]. Recently, several H2S releasing compounds were developed; and their pharmacotherapeutic potential in cardiovascular, gastrointestinal and immunological diseases are being tested [4]. In the circulatory system H2S has been found to affect vascular tone [5,6] and cardiac functions [7–10]. Several studies have shown that peripherally administered H2S donors produce hypotensive effect [11,12]. It seems, however, that the effects of
* Corresponding author. E-mail addresses: [email protected], [email protected] (M. Ufnal).
H2S in the circulatory system depend on the dose as well as animal species [9,13]. In this context, H2S has been found to dilate mesenteric [14] and hepatic [15] arteries in rats as well as human corpus cavernosum [16] but contract rat and duck aorta [13]. Furthermore, it has been reported that H2S contracts rat and mouse aorta and human arteries at 10–100 mM concentrations while relaxes these vessels at 100–1000 mM concentrations [17–19]. Similarly, H2S may exert both positive as well as negative chronotropic effect on the heart [9,10]. It has been postulated that decreased synthesis of endogenous H2S plays a role in the development of hypertension. Yang and collaborators found that genetic deletion of cystathionine gamma-lyase (CSE), an enzyme which generates H2S, produces hypertension in mice [20]. Furthermore, it has been found that peripheral administration of H2S donors and precursors decreases blood pressure in several experimental models of hypertension such as hypertension induced by chronic inhibition of nitric oxide synthase, two-kidney-one-clip and in spontaneously hypertensive rats (SHR) [21–23]. Accumulating evidence highlights the crucial role of cardiovascular centers in the brain regulation of the circulatory system [24]. An increasing number of mediators, including
http://dx.doi.org/10.1016/j.pharep.2014.04.004 1734-1140/ß 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Please cite this article in press as: Sikora M, et al. Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.004
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
G Model
PHAREP 113 1–8 M. Sikora et al. / Pharmacological Reports xxx (2014) xxx–xxx
2
47 48 49 50 51 52 53 54 55 56 57
gaseous transmitters are being added to the group of mediators involved in the neurogenic control of arterial blood pressure [25]. In our previous studies we found that intracerebroventricular (ICV) infusions of NaHS, a H2S donor exerts significant hemodynamic effect in normotensive WKY rats [9], which imply that H2S plays a role in the brain control of arterial blood pressure. In this study we compared hemodynamic responses to ICV infusions of NaHS between normotensive rats (WKY), spontaneously hypertensive rats (SHR) and WKY rats with angiotensin II (Ang II) – induced hypertension.
58
Materials and methods
59 60 61 62 63
The study was carried out in accordance with domestic and European Union guidelines of animal welfare. The experimental design, animal care and procedures were approved by the Second Local Animal Research Ethics Committee at the Medical University of Warsaw.
64
Animals
65 66 67
We performed the experiments on 12–14 weeks old, conscious male Wistar Kyoto (WKY) and Spontaneous Hypertensive (SHR) rats.
68
Drugs
69 70 71 72 73 74 75 76 77 78
The following drugs were used: Hexamethonium (Hexamethonium bromide, Sigma–Aldrich, Switzerland), Angiotensin II (Sigma–Aldrich, USA), Krebs–Henseleit bicarbonate-buffer (Sigma–Aldrich, USA), sodium hydrosulfide (NaHS; Sigma–Aldrich, Germany). NaHS was used as a H2S donor and was prepared 10 min. before the onset of ICV infusion. Due to limited buffering capacity of the buffer, in order to maintain pH 7.45–7.5 the maximum 10 mM NaHS was prepared. Administration of increasing doses of NaHS was achieved by infusing increasing volumes of NaHS solution at a rate of 3–30 ml/h.
79
Surgical preparation
80 81 82 83 84 85 86 87 88
All rats were implanted with a stainless steel cannula into the lateral cerebral ventricle and week later an arterial catheter was inserted through the femoral artery into the abdominal aorta as previously described [9]. All surgical procedures were performed under general anesthesia with ketamine 100 mg/kg of bw IP (Bioketan, Vetoquinol Biowet, Poland) and xylazine 10 mg/kg of bw IP (Xylapan, Vetoquinol Biowet, Poland). After surgery the animals were given benzathine penicillin 30 000 IU IM (Debecylina, Polfa Tarchomin, Poland).
89
Hemodynamic measurements
90 91 92 93 94 95 96 97 98 99 100 101 102
Measurements were performed 2 days after the second surgery. Mean arterial blood pressure (MABP) was recorded on-line through the arterial catheter connected to the blood pressure recording system (Biopac MP100 unit; Biopac Systems, Goleta, CA). All measurements were done in animals freely moving in their living cages after stabilization of hemodynamic parameters (45 15 min). MABP and HR were recorded at baseline (10 min) and during ICV infusions (45 min). Heart rate (HR) was calculated from the consecutive systolic peaks on the blood pressure tracing by the AcqKnowledge v3.7 Biopac software. ICV infusions were performed through a stainless steel infusion tube which was inserted into the previously implanted cannula.
Experimental series
103
WKY and SHR rats were randomly assigned to experimental series. In control series normotensive WKY rats (WKY-Control; n = 6) and hypertensive SHR rats (SHR-Control; n = 6) were infused with the Krebs–Henseleit buffer at a rate of 30 ml/h. ICV infusions of 10 mM NaHS was administered to WKY and SHR rats either at a dose of 30 nmol/h (WKY-30H2 S series, n = 6 and SHR-30H2 S series, n = 6), 100 nmol/h (WKY-100H2 S series, n = 6 and SHR-100H2 S series, n = 6) or 300 nmol/h (WKY-300H2 S series, n = 6 and SHR-300H2 S series, n = 6). Additionally, to prove that hemodynamic effects of ICV infused NaHS are mediated by the brain mechanisms and not by peripheral action of NaHS, we did the two series of experiments. In the first experiment WKY and SHR rats were concomitantly infused ICV with 300 nmol/h of NaHS and IV with hexamethonium, a ganglionic blocker (10 mg bolus followed by continuous infusion at the dose of 0.5 mg/min/rat, WKY-300H2 SþHex series, n = 6 and SHR-300H2 SþHex series, n = 6, respectively). In the second experiment WKY and SHR rats were infused IV with 300 nmol/h of NaHS. Finally, to compare the pattern of hemodynamic response to NaHS in two different animal models of hypertension, we checked the response to ICV infusion of either: 300 nmol/h of NaHS or the Krebs–Henseleit buffer at a rate of 30 ml/h in SHR rats (SHR-300H2 S series, n = 6 and SHR-Control series, n = 6) and in WKY rats with hypertension induced by Ang II (WKY-Ang II-300H2 S series, n = 6 and WKY-Ang II-Control series, n = 5). Hypertension in WKY rats was induced by chronic infusion of Ang II at 450 ng/1 min/kg bw for 10 days, using a subcutaneously implanted osmotic minipump (Alzet 2ML2).
104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131
Data analysis
132
The results are expressed as the mean SEM. For the evaluation of the effects of ICV infusions within series, the average over 5 min before the infusion was compared with the average over 5 min after the onset of infusion by one-way repeated-measures analysis of variance (ANOVA), followed by Tukey’s test. Comparisons between the series were evaluated by one- or two-way ANOVA for repeated measures when appropriate, followed by Tukey’s test. Two-sided P < 0.05 was considered significant. All analyses were conducted using STATISTICA 6.0 (StatSoft, Krakow, Poland).
133 134 135 136 137 138 139 140 141 142
Table 1 Mean arterial blood pressure (MABP) and heart rate (HR) at baseline in WKY, SHR and WKY-Ang II rats. Groups/series
MABP (mmHg)$,#,^
HR (beats/min)$,#,^
WKY rats WKY-Control (n = 6) WKY-30H2 S (n = 6) WKY-100H2 S (n = 6) WKY-300H2 S (n = 6) WKY-300H2 SþHex (n = 6)
114.8 0.5 114.3 0.6 113.8 0.6 113.7 0.4 114.3 0.5
323 1 328 2 328 2 325 2 329 2
SHR rats SHR-Control (n = 6) SHR-30H2 S (n = 6) SHR-100H2 S (n = 6) SHR-300H2 S (n = 6) SHR-300H2 SþHex (n = 6)
156.9 0.5 154.1 1.1 158.6 0.9 158.4 2.3 156.5 1.2
378 1 384 2 381 2 379 3 382 3
WKY-Ang II rats WKY-Ang II-Control (n = 5) WKY-Ang II-300H2 S (n = 6)
127.3 1.3 124.7 0.7
368 6 352 4
Means S.E.M. $ P < 0.05 WKY rats vs SHR rats. # P < 0.05 WKY rats vs WKY-Ang II rats. ^ P < 0.05 SHR rats vs WKY-Ang II rats.
Please cite this article in press as: Sikora M, et al. Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.004
G Model
PHAREP 113 1–8 M. Sikora et al. / Pharmacological Reports xxx (2014) xxx–xxx
143
Results
144 145 146
Baseline MABP was significantly higher in SHR and WKY-Ang II groups than in WKY group. There were no significant differences in MABP and HR between series within the groups (Table 1).
147
WKY group
148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163
In WKY-Control series ICV infusions of the buffer did not affect MABP and HR (Fig. 1A and B). ICV infusion of NaHS in WKY-30H2 S series decreased MABP [F(1,9) = 36.5, P < 0.001] and HR [F(1,9) = 15.6, P < 0.001] (Fig. 1A, 1B). Infusion of H2S donor in WKY-100H2 S series produced biphasic response, i.e. decrease in MABP and HR for the first 20 min of ICV infusion [F(1,4) = 10.6, P < 0.001 and F(1,4) = 6.7, P < 0.001, for MABP and HR respectively], followed by a significant increase in MABP and HR [F(1,5) = 7.8, P < 0.001 and F(1,5) = 7.5, P < 0.001, for MABP and HR respectively]. In WKY-300H2 S series we found significant increase in MABP and HR [F(1,9) = 34.7, P < 0.001 and F(1,9) = 28.3, P < 0.001, for MABP and HR respectively] (Fig. 1A and B). In WKY-300H2 SþHex series, ganglionic blockade with hexamethonium produced a significant decrease in MABP (mmHg) from 114.3 0.5 to 67.2 0.4 and HR (beats/min) from 329 2 to 250 3. The following ICV infusions of 300 nmol/h of NaHS did not affect MABP and HR
Fig. 1. Changes in mean arterial blood pressure (MABP) and heart rate (HR) in WKY rats during intracerebroventricular (ICV) infusions of: vehicle (WKY-control), NaHS at a dose of 30 nmol/h (WKY-30H2 S ), NaHS at a dose of 100 nmol/h (WKY-100H2 S ) and NaHS at a dose of 300 nmol/h (WKY-300H2 S ). (A) *P < 0.5 vs baseline; $P < 0.05 WKY-control vs WKY-30H2 S , ^P < 0.05 WKY-control vs WKY-100H2 S , #P < 0.05 WKY-control vs WKY-300H2 S . (B) Changes in HR (beats/min); *P < 0.05 vs baseline, $ P < 0.05 WKY-control vs WKY-30H2 S , ^P < 0.05 WKY-Control vs WKY-300H2 S .
3
(Fig. 3A and B). In WKY-Control series IV infusions of 300 nmol/h of 164 NaHS did not affect MABP and HR. 165 Two-way ANOVA series x time revealed significant differences 166 between series in WKY group in changes in MABP and HR during 167 ICV infusions [F(3,8) = 12.3, P < 0.001] for MABP and F(3,8) = 14.8, 168 P < 0.001 for HR) (Fig. 1A and B). Post hoc analysis revealed the Q2169 following significant differences between series in MABP changes: 170 WKY-Control vs WKY-30H2 S P < 0.001, WKY-Control vs 171 WKY-100H2 S P < 0.03, WKY-Control vs WKY-300H2 S P < 0.001 172 and WKY-30H2 S vs WKY-100H2 S P < 0.001, WKY-30H2 S vs 173 WKY-300H2 S P < 0.03 and WKY-100H2 S vs WKY-300H2 S P < 0.001 174 (Fig. 1A). There also were the following significant differences 175 between series in changes in HR: WKY-Control vs WKY-30H2 S 176 P < 0.001, WKY-Control vs WKY-300H2 S P < 0.001 and WKY-30H2 S 177 vs WKY-100H2 S P < 0.001, WKY-30H2 S vs WKY-300H2 S P < 0.03 and 178 WKY-100H2 S vs WKY-300H2 S P < 0.001 (Fig. 1B). 179 There was a significant difference in changes in MABP and HR 180 between WKY-300H2 SþHex and WKY-300H2 S series [F(1,8) = 17.8 181 P < 0.001 and F(1,8) = 21.8 P < 0.001, for MABP and HR 182 respectively] (Fig. 3A and B). Q3183 SHR group
184
In SHR-Control series ICV infusions of the buffer did not affect MABP and HR (Fig. 2A and B). ICV infusion of NaHS decreased MABP
185 186
Fig. 2. Changes in mean arterial blood pressure (MABP) and heart rate (HR) in SHR rats during intracerebroventricular (ICV) infusions of: vehicle (SHR-control), NaHS at a dose of 30 nmol/h (SHR-30H2 S ), NaHS at a dose of 100 nmol/h (SHR-100H2 S ) and NaHS at a dose of 300 nmol/h (SHR-300H2 S ). (A) Changes in MABP (mmHg); *P < 0.05 vs baseline. $ P < 0.05 SHR-control vs SHR-30H2 S , ^P < 0.05 SHR-control vs SHR-100H2 S , #P < 0.05 SHR-control vs SHR-300H2 S . (B) Changes in HR (beats/min); *P < 0.05 vs baseline, $P < 0.05 SHR-control vs SHR-100H2 S , #P < 0.05 SHR-control vs SHR-300H2 S .
Please cite this article in press as: Sikora M, et al. Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.004
G Model
PHAREP 113 1–8 4
M. Sikora et al. / Pharmacological Reports xxx (2014) xxx–xxx
Fig. 3. Changes in mean arterial blood pressure (MABP) and heart rate (HR) in WKY and SHR rats during intracerebroventricular (ICV) infusion of: NaHS at a dose of 300 nmol/h (WKY-300H2 S and SHR-300H2 S series), and concomitant ICV infusion of NaHS at a dose of 300 nmol/h and IV infusion of hexamethonium (WKY-300H2 SþHEX and SHR-300H2 SþHEX series). (A) Changes in MABP (mmHg); *P < 0.05 vs baseline, $ P < 0.05 WKY-300H2 SþHex vs WKY-300H2 S ; #P < 0.05 SHR-300H2 SþHex vs SHR-300H2 S . (B) Changes in HR (beats/min); *P < 0.05 vs baseline, $P < 0.05 WKY-300H2 SþHex vs WKY-300H2 S ; #P < 0.05 SHR-300H2 SþHex vs SHR-300H2 S .
187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210
and HR in all the experimental series in SHR rats: SHR-30H2 S [F(1,9) = 5.4, P < 0.001 and F(1,9) = 5.8, P < 0.001, for MABP and HR, respectively], SHR-100H2 S [F(1,9) = 10.4, P < 0.001] and [F(1,9) = 11.6, P < 0.001, for MABP and HR, respectively], SHR-300H2S [F(1,9) = 15.4, P < 0.001 and F(1,9) = 20.4, P < 0.001, for MABP and HR, respectively] (Fig. 2A and 2B). In SHR-300H2 SþHex series, ganglionic blockade with hexamethonium produced a significant decrease in MABP (mmHg) from 156.5 1.2 to 96.4 0.6 and HR (beats/min) from 382 3 to 322 1. The following ICV infusions of 300 nmol/h of NaHS did not affect MABP and HR (Fig. 3A and B). In SHR-Control series IV infusions of 300 nmol/h of NaHS did not affect MABP and HR. Two-way ANOVA series x time revealed significant difference in changes in MABP and HR between series in SHR group [F(3,8) = 1.7, P < 0.05 and F(3,8) = 1.9, P < 0.05 for MABP and HR, respectively] (Fig. 2A and B). Post hoc analysis revealed the following significant differences in changes in MABP: SHR-Control vs SHR-30H2 S P < 0.05, SHR-Control vs SHR-100H2 S P < 0.001, SHR-Control vs SHR-300H2 S P < 0.001 and SHR-30H2 S vs SHR-100H2 S P < 0.01, SHR-30H2 S vs SHR-300H2 S P < 0.001 and SHR-100H2 S vs SHR-300H2 S P < 0.05. There were also the following significant differences in HR changes between series: SHR-Control vs SHR-100H2 S P < 0.001, SHR-Control vs SHR-300H2 S P < 0.001 and SHR-30H2 S vs SHR-300H2 S P < 0.001.
Fig. 4. Changes in mean arterial blood pressure (MABP) and heart rate (HR) in WKY, SHR and WKY-Ang II rats during intracerebroventricular (ICV) infusion of NaHS at a dose of 300 nmol/h. (A) Changes in MABP (mmHg); *P < 0.05 vs baseline, $P < 0.05 WKY-300H2 S vs SHR-300H2 S , #P < 0.05 WKY-300H2 S vs WKY-Ang II-300H2 S ; ^ P < 0.05 SHR-300H2 S vs WKY-Ang II-300H2 S . (B) Changes in HR (beats/min), *P < 0.05 vs baseline, $P < 0.05 WKY-300H2 S vs SHR-300H2 S , #P < 0.05 WKY-300H2 S vs WKY-Ang II-300H2 S .
There was a significant difference in changes in MABP and HR between SHR-300H2 SþHex and SHR-300H2 S series [F(1,8) = 5.0, P < 0.001 and F(1,8) = 6.0, P < 0.001 for MABP and HR, respectively] (Fig. 3A and B).
211 212 213 214
WKY-Ang II group
215
ICV infusions of Krebs–Henseleit buffer in WKY-Ang II-Control group did not affect MABP and HR. ICV infusions of 300 nmol/h of NaHS in WKY-Ang II-300H2 S group decreased MABP and HR [F(1,9) = 4.8, P < 0.001 and F(1,9) = 9.2, P < 0.001, for MABP and HR, respectively] (Fig. 4A and B).
216 217 218 219 220
WKY vs SHR group
221
There were no significant differences in changes in MABP and HR between WKY-Control and SHR-Control ( Figs. 5A and 6A) as well as between WKY-30H2S and SHR-30H2S series ( Figs. 5B and 6B). There was a significant difference in MABP and HR between WKY and SHR groups infused ICV with 100 nmol/h of NaHS: WKY-100H2 S vs SHR-100H2 S series [F(1,8) = 18.1, P < 0.001 and F(1,8) = 14.8, P < 0.001, for MABP and HR respectively] (Figs. 5C and 6C). There was a significant difference in MABP and HR between WKY and SHR groups infused ICV with 300 nmol/h of NaHS:
222 223 224 225 226 227 228 229 230 231
Please cite this article in press as: Sikora M, et al. Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.004
G Model
PHAREP 113 1–8 M. Sikora et al. / Pharmacological Reports xxx (2014) xxx–xxx
5
Fig. 5. Changes in mean arterial blood pressure (MABP) in WKY and SHR rats during intracerebroventricular (ICV) infusions. (A) ICV infusion of vehicle in WKY rats (WKYcontrol) and SHR rats (SHR-control). (B) ICV infusion of NaHS at a dose of 30 nmol/h in WKY rats (WKY-30H2 S ) and SHR rats (SHR-30H2 S ). (C) ICV infusion of NaHS at a dose of 100 nmol/h in WKY rats (WKY-100H2 S ) and SHR rats (SHR-100H2 S ), $P < 0.05 WKY-100H2 S vs SHR-100H2 S series. (D) ICV infusion of NaHS at a dose of 300 nmol/h in WKY rats (WKY-300H2 S ) and SHR (SHR-300H2 S ). $P < 0.05 WKY-300H2 S vs SHR-300H2 S series.
232 233 234
WKY-300H2 S vs SHR-300H2 S series [F(1,8) = 20.5, P < 0.001 and F(1,8) = 27.4, P < 0.001 for MABP and HR, respectively] (Figs. 5D and 6D).
235
WKY vs SHR and WKY-Ang II groups
236 237 238 239 240 241 242 243 244 245
A significant difference was found in changes in MABP and HR between WKY, WKY-Ang II and SHR groups in series in which rats were ICV infused with 300 nmol/h of NaHS [F(2,8) = 12.0, P < 0.001 and F(2,8) = 14.75, P < 0.001 for MABP and HR, respectively] (Fig. 4A and B). Post hoc analysis revealed the following significant differences for MABP: WKY-300H2 S vs SHR-300H2 S P < 0.001, WKY-300H2 S vs WKY-Ang II-300H2 S P < 0.001, WKY-Ang II-300H2 S vs SHR-300H2 S (Fig. 4A and B) and for HR: WKY-300H2 S vs SHR-300H2 S P < 0.001, WKY-300H2 S vs WKY-Ang II-300H2 S P < 0.001 (Fig. 4A and B).
246
Discussion
247 248 249 250 251 252
The novel finding of this study is that there are significant differences between normotensive and hypertensive rats in hemodynamic responses to ICV infused NaHS, a H2S donor. We found that in contrast to normotensive WKY rats, hypertensive SHR and WKY-Ang II rats show hypotensive response to ICV infused NaHS. This implies that differences in
response to ICV infused NaHS between normotensive WKY and hypertensive SHR and WKY-Ang II rats are associated with high arterial blood pressure rather than interstrain differences. Similar patterns of hemodynamic response to NaHS in SHR and WKY-Ang II rats, suggest that hypertension independently on its origin is associated with alterations in H2S signaling in the brain. Therefore, we postulate that high arterial blood pressure changes H2S transmission in the brain rather than the other way round. However, primary disturbances in H2S homeostasis in the brain as a cofactor of the pathogenesis of hypertension cannot be excluded. In this line, decreased synthesis of endogenous H2S in the peripheral tissues has been suggested to be involved in the development of hypertension [20], and the treatment with H2S donors and precursors was found to decrease blood pressure in several experimental models of hypertension [21–23]. So far the effect of H2S on the brain regulation of the circulatory system has only been evaluated in a few studies, and their results were contradictory [9,26–28]. The present results are in line with our previous findings showing hypertensive response to ICV infusion of NaHS in WKY rats. However, in the current study the pressor response to NaHS at a dose of 100 nmol/h and higher was more pronounced than in our previous experiment [9]. This could be a result of longer baseline meassurments in our prevous study, which increased
Please cite this article in press as: Sikora M, et al. Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.004
253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277
G Model
PHAREP 113 1–8 6
M. Sikora et al. / Pharmacological Reports xxx (2014) xxx–xxx
Fig. 6. Changes in heart rate (HR) in WKY and SHR rats during intracerebroventricular (ICV) infusions. (A) ICV infusion of vehicle in WKY rats (WKY-control) and SHR rats (SHR-control). (B) ICV infusion of NaHS at a dose of 30 nmol/h in WKY rats (WKY-30H2 S ) and SHR rats (SHR-30H2 S ). (C) ICV infusion of NaHS at a dose of 100 nmol/h in WKY rats (WKY-100H2 S ) and SHR rats (SHR-100H2 S ); $P < 0.05 WKY-100H2 S vs SHR-100H2 S series. (D) ICV infusion of NaHS at a dose of 300 nmol/h in WKY rats (WKY-300H2 S ) and SHR rats (SHR-300H2 S ); $P < 0.05 WKY-300H2 S vs SHR-300H2 S .
278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303
time form the preparation of NaHS solution to the onset of ICV infusions up to aproximately 30–40 min. Whereas in the present study this time did not exceed 10 min. In addition, in the current study we found that ICV infused NaHS at a dose of 30 nmol/h decreases arterial blood pressure. The opposite hemodynamic effects of high and low doses of NaHS may be due to the following reasons. Neuroactive compounds infused ICV act on the circumventricular organs which lack the blood –brain barrier (BBB) as well as may penetrate behind the BBB to cardiovascular centers such as paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) [24,29]. As H2S easily permeates cell membranes and has a relatively long half-life it is likely that ICV infused H2S targets neurons of the circumventricular organs as well as neurons of the cardiovascular centers protected by the BBB. Therefore it is possible that lower doses of H2S caused hypotensive effects in WKY-30H2 S series acting on the circumventricular organs whereas higher doses of H2S produced hypertensive response in WKY-300H2 S series penetrating to the cardiovascular centers behind the BBB. In this context, biphasic response in WKY-100H2 S series could reflect an increase in H2S concentration in the CSF during continuous ICV infusion. Another possible explanation is that the opposite hemodynamic effects of H2S may depend on distinct mechanisms with a different stimulation thresholds. For example, it has been shown that H2S may exert both pro-nociceptive and
antinociceptive effects [30–32] and that it may act as a vasoconstrictor or a vasorelaxant in peripheral blood vessels [5]. To our knowledge, there are three other studies on hemodynamic effects of ICV infusions of H2S in rats [27,28,33]. Ren and collaborators reported that the ICV injection of H2S saturated buffer and H2S precursors ( L-cysteine and beta-mercaptopyruvate) significantly increase MABP after an initial transient hypotension [33]. In contrast, Liu and collaborators have found that the ICV infusion of NaHS, at the same doses we tested, causes a significant decrease in blood pressure [27]. The discrepancies between the results of Liu et al. and our findings seem to result from differences in experimental settings. Namely, Liu and collaborators dissolved NaHS in an artificial CSF without a buffer, whereas Ren et al. and us used buffered solutions of NaHS [27,33]. As NaHS is highly alkaline, findings by Liu et al. could partially result from pH changes in the CSF. The other three studies investigated the effect of NaHS infusions into specific cardiovascular centers in the brain. Streeter and collaborators demonstrated the presence of the H2S-producing enzyme, cystathionine-beta-synthase in the RVLM and the PVN, however they did not find significant hemodynamic effects of NaHS at a dose of 0.2–2000 pmol infused into the RVLM and PVN and 20 nmol of NaHS infused ICV [28]. In contrast, Guo et al. showed that bilateral microinjections of NaHS (4, 8, and 16 mM, 50 nl) into the RVLM decreases MABP, HR and renal sympathetic nerve activity in a dose-dependent
Please cite this article in press as: Sikora M, et al. Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.004
304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329
G Model
PHAREP 113 1–8 M. Sikora et al. / Pharmacological Reports xxx (2014) xxx–xxx
330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391
manner [34]. These studies, however, were conducted in anesthesia which strongly inhibits nervous reflexes and alters the neurogenic regulation of the circulatory system. The experiments on freely moving rats were conducted by Dawe and collaborators who found that infusion of 200 mmol of NaHS into the hypothalamus reduces blood pressure and heart rate [26]. However, it needs to be noted that infusion of neuroactive agents into a single cardiovascular center often produces different hemodynamic effects than ICV infusion which targets multiple cardiovascular centers [25]. An advantage of ICV infusions lies in similarity to real life conditions, in which compounds circulating in the blood and/or in the cerebrospinal fluid may target multiple cardiovascular centers in the brain. However, ICV infused agents may be cleared via the lymphatic system and enter the blood circulation. This may affect the function of peripheral tissues and cause the opposite hemodynamic effects to those produced by stimulation of the brain cardiovascular centers [35]. Therefore, to confirm that we are looking at the effects mediated by the brain mechanism, we performed two additional series of experiments. In the first experiment we checked the hemodynamic response after intravenous infusion of NaHS at the highest dose tested in ICV infusion experiments. In the second experiments during ICV infusions of NaHS we blocked the nervous control of the circulatory system with hexamethonium, a ganglionic blocker. The lack of hemodynamic response to NaHS in these two series provides strong evidence that the effects triggered by the ICV infusion of the H2S donor in the previous series were mediated by the brain mechanisms. A limitation of our study, is that increasing doses of NaHS achieved by infusing increasing volumes of NaHS solution at a rate 3 ml/h to 30 ml/h, could result in differences in ICV pressure between experimental series. This approach was imposed by a limited buffering capacity of Krebs –Henseleit buffer, which allow us to prepare a maximum 10 mM solution of NaHS to maintain pH within 7.4–7.45. Although we cannot exclude that there were differences in ICV pressure between experimental series, it seems unlikely that they had a significant effect on hemodynamic parameters. This is based on ours and others results showing that ICV infusions of a vehicle at the rate of up to 40 ml/h do not affect arterial blood pressure and heart rate [9,36]. In contrast, even moderate changes in pH of the cerebrospinal fluid cause significant changes in the neurogenic regulation of the circulatory and respiratory systems. Therefore, it seems that our approach is less likely to produce artifacts compared to infusing the same volumes of NaHS solutions but varying in pH. A key problem in studies on biological effects of H2S is a significant variation in reported physiological concentrations of H2S in tissues, particular in the brain. Dependent on the methods employed, the estimated concentration of H2S in the brain and the CSF has been reported to range from 0.1 to 200 mM [37–39]. A molecular form of H2S is about 20% of H2S in the NaHS solution (calculated for: pH 7.4; temperature 37 8C, pK = 6.755). This is why, the doses of NaHS donors tested in ours and other studies could increase H2S concentration in the brain and CSF with wide range, from two to several thousand times of estimated physiological level. In conclusion, exogenous H2S significantly affects the neurogenic control of arterial blood pressure and hypertension may be associated with disturbances in the H2S signaling in the brain. Further studies on the role of H2S in the circulatory system are needed as well as validation of technics used in measuring concentration of H2S in tissues.
392
Conflict of interest
393
No conflict of interest.
7
Funding
394
This work was supported by the National Science Center, grant Q4395 no. 2011/01/N/NZ4/03682 and Medical University of Warsaw, 396 grant nos. 1MA/PM11/12 and 1MA/PM11/13. 397 Acknowledgment
398
The authors are in debt to Dr. T. Zera for his critical comments on the manuscript.
399 400
References
401
[1] Abe K, Kimura H. The possible role of hydrogen sulfide as an endogenous neuromodulator. J Neurosci 1996;16(3):1066–71. [2] Wang R. Physiological implications of hydrogen sulfide: a whiff exploration that blossomed. Physiol Rev 2012;92(2):791–896. [3] Lowicka E, Beltowski J. Hydrogen sulfide (H2S) – the third gas of interest for pharmacologists. Pharmacol Rep 2007;59(1):4–24. [4] Kashfi K, Olson KR. Biology and therapeutic potential of hydrogen sulfide and hydrogen sulfide-releasing chimeras. Biochem Pharmacol 2013;85(5):689–703. [5] Olson KR, Dombkowski RA, Russell MJ, Doellman MM, Head SK, Whitfield NL, et al. Hydrogen sulfide as an oxygen sensor/transducer in vertebrate hypoxic vasoconstriction and hypoxic vasodilation. J Exp Biol 2006;209(Pt 20):4011–23. [6] Dombkowski RA, Russell MJ, Olson KR. Hydrogen sulfide as an endogenous regulator of vascular smooth muscle tone in trout. Am J Physiol Regul Integr Comp Physiol 2004;286(4):R678–85. [7] Bian JS, Yong QC, Pan TT, Feng ZN, Ali MY, Zhou S, et al. Role of hydrogen sulfide in the cardioprotection caused by ischemic preconditioning in the rat heart and cardiac myocytes. J Pharmacol Exp Ther 2006;316(2):670–8. [8] Hu X, Li T, Bi S, Jin Z, Zhou G, Bai C, et al. Possible role of hydrogen sulfide on the preservation of donor rat hearts. Transplant Proc 2007;39(10):3024–9. [9] Ufnal M, Sikora M, Dudek M. Exogenous hydrogen sulfide produces hemodynamic effects by triggering central neuroregulatory mechanisms. Acta Neurobiol Exp (Wars) 2008;68(3):382–8. [10] Xu M, Wu YM, Li Q, Wang X, He RR. Electrophysiological effects of hydrogen sulfide on pacemaker cells in sinoatrial nodes of rabbits. Sheng Li Xue Bao 2008;60(2):175–80. [11] Zhao W, Zhang J, Lu Y, Wang R. The vasorelaxant effect of H(2)S as a novel endogenous gaseous K(ATP) channel opener. EMBO J 2001;20(21):6008–16. [12] Li L, Whiteman M, Guan YY, Neo KL, Cheng Y, Lee SW, et al. Characterization of a novel, water-soluble hydrogen sulfide-releasing molecule (GYY4137): new insights into the biology of hydrogen sulfide. Circulation 2008;117(18):2351–60. [13] Dombkowski RA, Russell MJ, Schulman AA, Doellman MM, Olson KR. Vertebrate phylogeny of hydrogen sulfide vasoactivity. Am J Physiol Regul Integr Comp Physiol 2005;288(1):R243–52. [14] Cheng Y, Ndisang JF, Tang G, Cao K, Wang R. Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats. Am J Physiol Heart Circ Physiol 2004;287(5):H2316–23. [15] Siebert N, Cantre D, Eipel C, Vollmar B. H2S contributes to the hepatic arterial buffer response and mediates vasorelaxation of the hepatic artery via activation of K(ATP) channels. Am J Physiol Gastrointest Liver Physiol 2008;295(6):G1266–73. [16] d’Emmanuele di Villa Bianca R, Sorrentino R, Maffia P, Mirone V, Imbimbo C, Fusco F, et al. Hydrogen sulfide as a mediator of human corpus cavernosum smooth-muscle relaxation. Proc Natl Acad Sci U S A 2009;106(11):4513–8. [17] Ali MY, Ping CY, Mok YY, Ling L, Whiteman M, Bhatia M, et al. Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide? Br J Pharmacol 2006;149(6):625–34. [18] Kubo S, Doe I, Kurokawa Y, Nishikawa H, Kawabata A. Direct inhibition of endothelial nitric oxide synthase by hydrogen sulfide: contribution to dual modulation of vascular tension. Toxicology 2007;232(1–2):138–46. [19] Webb GD, Lim LH, Oh VM, Yeo SB, Cheong YP, Ali MY, et al. Contractile and vasorelaxant effects of hydrogen sulfide and its biosynthesis in the human internal mammary artery. J Pharmacol Exp Ther 2008;324(2):876–82. [20] Yang G, Wu L, Jiang B, Yang W, Qi J, Cao K, et al. H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine gammalyase. Science 2008;322(5901):587–90. [21] Zhong G, Chen F, Cheng Y, Tang C, Du J. The role of hydrogen sulfide generation in the pathogenesis of hypertension in rats induced by inhibition of nitric oxide synthase. J Hypertens 2003;21(10):1879–85. [22] Yan H, Du J, Tang C. The possible role of hydrogen sulfide on the pathogenesis of spontaneous hypertension in rats. Biochem Biophys Res Commun 2004;313(1):22–7. [23] Lu M, Liu YH, Goh HS, Wang JJ, Yong QC, Wang R, et al. Hydrogen sulfide inhibits plasma renin activity. J Am Soc Nephrol 2010;21(6):993–1002. [24] Dampney RA. Functional organization of central pathways regulating the cardiovascular system. Physiol Rev 1994;74(2):323–64.
402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469
Please cite this article in press as: Sikora M, et al. Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.004
G Model
PHAREP 113 1–8 8
470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492
M. Sikora et al. / Pharmacological Reports xxx (2014) xxx–xxx
[25] Ufnal M, Sikora M. The role of brain gaseous transmitters in the regulation of the circulatory system. Curr Pharm Biotechnol 2011;12(9):1322–33. [26] Dawe GS, Han SP, Bian JS, Moore PK. Hydrogen sulphide in the hypothalamus causes an ATP-sensitive K+ channel-dependent decrease in blood pressure in freely moving rats. Neuroscience 2008;152(1):169–77. [27] Liu WQ, Chai C, Li XY, Yuan WJ, Wang WZ, Lu Y. The cardiovascular effects of central hydrogen sulfide are related to K(ATP) channels activation. Physiol Res 2011;60(5):729–38. [28] Streeter E, Al-Magableh M, Hart JL, Badoer E. Hydrogen sulfide in the RVLM and PVN has no effect on cardiovascular regulation. Front Physiol 2011;2:55. [29] Cottrell GT, Ferguson AV. Sensory circumventricular organs: central roles in integrated autonomic regulation. Regul Pept 2004;117(1):11–23. [30] Cunha TM, Dal-Secco D, Verri Jr WA, Guerrero AT, Souza GR, Vieira SM, et al. Dual role of hydrogen sulfide in mechanical inflammatory hypernociception. Eur J Phar 2008;590(1–3):127–35. [31] Distrutti E, Sediari L, Mencarelli A, Renga B, Orlandi S, Russo G, et al. 5-Amino-2-hydroxybenzoic acid 4-(5-thioxo-5H-[1,2]dithiol-3yl)-phenyl ester (ATB-429), a hydrogen sulfide-releasing derivative of mesalamine, exerts antinociceptive effects in a model of postinflammatory hypersensitivity. J Pharmacol Exp Ther 2006;319(1):447–58. [32] Matsunami M, Tarui T, Mitani K, Nagasawa K, Fukushima O, Okubo K, et al. Luminal hydrogen sulfide plays a pronociceptive role in mouse colon. Gut 2009;58(6):751–61.
[33] Ren YS, Wu SY, Wang XJ, Yu F, Zhao J, Tang CS, et al. Multiple hemodynamic effects of endogenous hydrogen sulfide on central nervous system in rats. Chin Med J (Engl) 2011;124(21):3468–75. [34] Guo Q, Jin S, Wang XL, Wang R, Xiao L, He RR, et al. Hydrogen sulfide in the rostral ventrolateral medulla inhibits sympathetic vasomotor tone through ATP-sensitive K+ channels. J Pharmacol Exp Ther 2011;338(2):458–65. [35] Ufnal M, Skrzypecki J. Blood borne hormones in a cross-talk between peripheral and brain mechanisms regulating blood pressure, the role of circumventricular organs. Neuropeptides 2014;48(2):65–73. [36] Ye S, Mozayeni P, Gamburd M, Zhong H, Campese VM. Interleukin-1beta and neurogenic control of blood pressure in normal rats and rats with chronic renal failure. Am J Physiol Heart Circ Physiol 2000;279(6): H2786–96. [37] Furne J, Saeed A, Levitt MD. Whole tissue hydrogen sulfide concentrations are orders of magnitude lower than presently accepted values. Am J Physiol Regul Integr Comp Physiol 2008;295(5):R1479–85. [38] Leffler CW, Parfenova H, Basuroy S, Jaggar JH, Umstot ES, Fedinec AL. Hydrogen sulfide and cerebral microvascular tone in newborn pigs. Am J Physiol Heart Circ Physiol 2011;300(2):H440–7. [39] Goodwin LR, Francom D, Dieken FP, Taylor JD, Warenycia MW, Reiffenstein RJ, et al. Determination of sulfide in brain tissue by gas dialysis/ion chromatography: postmortem studies and two case reports. J Anal Toxicol 1989;13(2):105–9.
Please cite this article in press as: Sikora M, et al. Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.004
493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515
