Journal of Neurochemrslry Raven Press, Ltd., New York 0 1992 International Society for Neurochemstry
Drug-Induced Changes in Blood Pressure Lead to Changes in Extracellular Concentrations of Epinephrine, Dihydroxyphenylacetic Acid, and 5-Hydroxyindoleacetic Acid in the Rostra1 Ventrolateral Medulla of the Rat *Bhaskar R. Dev, Patrick A. Mason, and Curt R. Freed Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado, U.S.A.
Abstract: Neurochemical changes in the extracellular fluid of the rostral ventrolateral medulla (RVLM) were produced by changes in arterial blood pressure. Blood pressure was raised or lowered with systemic infusions of phenylephrine or nitroprusside and neurochemicals were recovered from RVLM by in vivo microdialysis. A dialysis probe 300 pm in diameter and 500 pm in length was stereotaxically implanted in the RVLM of the urethane-anesthetized rat. Sterile physiological Ringer’s solution was perfused at a rate of 1.5 pl/ min. The perfusate was collected under ice-cold conditions every I5 min for the assay of epinephrine, dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (~-HIAA), ascorbic acid, and uric acid. After stable baseline neurochemical concentrations were achieved, animals were infused with phenylephrine or nitroprusside intravenously to raise or lower the blood pressure. Increasing blood pressure 50 mm Hg above the baseline value by phenylephrine led to a significant reduction in heart rate and a reduction in extracellular epinephrine and DOPAC concentrations. The 5HIAA concentration was increased during the hypertensive drug infusion. There were no changes in the concentrations of ascorbic acid or uric acid. Hypotension produced by ni-
troprusside (-20 mm Hg) led to neurochemical changes which were the reciprocal of those seen during hypertension. During hypotension, heart rate increased as did the extracellular fluid epinephrine concentration. The 5-HIAA concentration fell with hypotension and remained depressed following the nitroprusside infusion. Ascorbic acid and uric acid concentrations did not change during hypotension but ascorbic acid did increase after the nitroprusside infusion stopped. These data provide direct evidence that epinephrine release in RVLM is linked to changes in systemic blood pressure. Furthermore, because changes in epinephrine concentration parallel changes in sympathetic activity as measured by heart rate, epinephrine in the extracellular fluid space of the RVLM may be a marker for sympathetic nerve activity. Key Words: In vivo microdialysis-Rostra1 ventrolateral medulla-Epinephrine-Ascorbic acid-Uric acid-Blood pressure. Dev B. R. et al. Drug-induced changes in blood pressure lead to changes in extracellular concentrations of epinephrine, dihydroxyphenylacetic acid, and 5-hydroxyindoleacetic acid in the rostral ventrolateral medulla of the rat. J. Neurochern. 58, 1386- 1394 (1 992).
The ventrolateral medulla has been known to be an important site for regulation of sympathetic vasomotor activity since Alexander demonstrated separate pressor and depressor regions in the medulla in 1946 (Alexander, 1946). Noradrenergic neurons are found in the caudal ventrolateral medulla, the A1 region (Dahlstrom and Fuxe, 1964). These cells are believed to be the inhibitory vasomotor neurons (Blessing and Reis, 1982). The rostral ventrolateral medulla (RVLM), also
called the C1 region, has adrenergic neurons identified by the presence of the enzyme phenylethanolamineN-methyltransferase (PNMT) (Hokfelt et al., 1974). The PNMT-containing neurons of the RVLM area project widely both rostral to the hypothalamus and caudal to the spinal cord. These caudal projections to the intermediolateral and intermediomedial cell columns of the thoracic spinal cord are likely to have a role in controlling sympathetic outflow (Ross et al.,
Received July 12, 1991; accepted September 13, 1991. Address correspondence and reprint requests to Dr. C. R. Freed at Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, C-237, Denver, CO 80262, U.S.A.
*Dr. B. R. Dev has changed his name from D. Bhaskaran.
Abbreviations used: DOPAC, dihydroxflhenylacetic acid 5-HIAA, 5-hydroxyindoleacetic acid; PNMT, phenylethanolamine-N-methyltransferase; RVLM, rostral ventrolateral medulla.
I386
1387
EPINEPHRINE AND BLOOD PRESSURE 1981, 1984, 1985). Supporting the importance of the PNMT-containing neurons in regulating blood pressure is the observation that PNMT activity is changed i n the C1 nucleus in spontaneously hypertensive rats (Saavedra, 1979). T h e noradrenergic vasodepressor neurons of the A1 region are said to exert their sympathoinhibitory effect by projecting into the RVLM and inhibiting the adrenergic neurons located there (Granata et al., 1985, 1986; Ross et al., 1985). However, this explanation has been challenged by other laboratories (Day et al., 1983; Sun and Guyenet, 1986; Blessing et al., 1987; Head et al., 1987). Previously, we have reported blood pressure-induced changes in neurotransmitter metabolism in RVLM using in vivo electrochemical methods (Bhaskaran and Freed, 1989). In that study, we found that the electrochemical peak measured at low oxidation potential (the catechol peak) was reduced during phenylephrine hypertension and rose during nitroprusside hypotension. A second electrochemical peak measured a t high oxidation potential (the hydroxyindole peak) showed the opposite changes. During hypertension the peak was increased, and during hypotension the peak was re-
duced. In these previous in vivo electrochemical studies, we used a P N M T inhibitor t o show that the catechol peak was composed in part of epinephrine. However, because electrochemical electrodes do not measure single specific chemical species, measurements made by electrochemical methods are intrinsically ambiguous. To relate more precisely the neurochemical changes occurring in RVLM to changes in systemic blood pressure, we have used the technique of in vivo dialysis to measure extracellular fluid catechols and indoles.
MATERIALS AND METHODS Male Sprague-Dawley rats weighing 300-350 g were anesthetized with urethane (1.25 g/kg, i.p.) and had the femoral artery and vein catheterized for blood pressure measurement and drug infusion, respectively. A microdialysis probe was lowered into the RVLM area using coordinates from Paxinos and Watson (1986). The upper incisor bar of the stereotaxic frame (Narishige Scientific Instrument, Japan) was lowered 3.4 mm below the horizontal zero. The interaural line was used as the rostral-caudal zero point. Coordinates used were: A, -3.8 mm; L, +2.0 mm; and V, -10.2 mm from the skull surface. The microdialysis probe was made from concentric stainless steel tubing. A 32-gauge inner tube was placed inside a 22-gauge outer tube. A tubular dialysis membrane 300 pm diameter with a molecular mass cut-off of 12,500 daltons (Enka AG, F.R.G.) was glued to the outer tube. The length ofthe dialysis membrane tip was 0.5 mm. When these probes were tested in vitro at 25"C, recovery of catecholamines averaged 7%. The dialysis probe was perfused with Ringer's solution (Travenol Laboratory, Deerfield, IL, U.S.A.) at a rate of 1.5 pl/min and the perfusate was collected under ice-cold conditions every 15 min. Epinephrine, dihydroxyphenylacetic
acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), ascorbic acid, and uric acid were measured. Although we had no reason to predict either ascorbic acid or uric acid would change in RVLM with changes in blood pressure, these compounds have been shown to interfere with the interpretation of electrochemical studies. As the purpose of the present experiments was to validate and amplify our previous electrochemical study, measuring ascorbate and urate levels made it possible to associate or dissociate the epinephrine measurements made by dialysis to the catechol and indole estimates by in vivo electrochemistry. Epinephrine, DOPAC, and 5-HIAA were assayed using a 1 X 100 mm microbore HPLC column with electrochemical detection. The HPLC system consisted of an ISCO pLC-500 microflow pump with a Rheodyne injector (7413) with a sample injection loop volume of 5 pl. The column was 100 X 1 mm packed with Spherisorb ODS 2 with particle size of 3 pm (Keystone Scientific, State College, PA, U.S.A.). The electrochemical detector featured a dual glassy carbon electrode (Bioanalytical Systems, West Lafayette, IN, U.S.A.) set at a potential of 0.7 V. The mobile phase consisted of 0.1 M sodium dihydrogen phosphate buffer with sodium octyl sulfate (0.1 mM) and EDTA. The pH was 4.3 (Routledge and Marsden, 1987). The flow rate was I20 pl/min. Under these conditions, retention times for the compounds ofinterest were: epinephrine, 3.4 min; DOPAC, 5.0 min; and 5-HIAA, 13.7 min. Norepinephrine retention time was 1.9 min and eluted with the solvent front, so norepinephrine was not measured in these experiments. Whereas experimental values are expressed as percent of baseline concentrations, actual concentrations were estimated by external standards injected into the HPLC column. With these conditions, the method is sensitive enough to measure the epinephrine, DOPAC, and 5-HIAA levels as low as 10 fmol/5 pl injected. Because of the potential ambiguity of HPLC retention time as the sole identifier of epinephrine, we repeated the entire experiment using a different chromatographic system. New groups of animals were infused with phenylephrine and nitroprusside and dialysis samples were once again collected as described above. Instead of sodium octyl sulfate, we used the waxy detergent Igepon as described by Mefford et al. (1987). The mobile phase consisted of 0.15 M sodium acetate and methanol (85:1 3 , 150 mg/L Igepon T-77 (GAF, Wayne, NJ, U.S.A.), and 500 pi of 0.2 M EDTA/L, pH 7.56. The flow rate was 80 pl/min. Under these conditions, the retention time for norepinephrine was 2.5 min, for epinephrine 3.8 min, for DOPAC 5.0 min, and for 5-HIAA 13.0 min. Using a mobile phase containing Igepon but a different brand of HPLC column, Mefford et al. (1987) found that norepinephrine eluted after epinephrine. With the Keystone Spherisorb column described above, norepinephrine has eluted before epinephrine, as it did with the sodium octyl sulfate mobile phase described above. For additional characterization of the chromatographic peak corresponding to epinephrine, the PNMT inhibitor LY 134046 was given at a dose of 30 mg/kg. This drug has been shown to reduce the tissue concentration of epinephrine in the hypothalamus by 37% 1 h after drug administration (Fuller et al., 1981). In our previous study of RVLM using the in vivo electrochemical electrode, we found that LY 134046 reduced the catechol electrochemical peak by 25% after 1 h (Bhaskaran and Freed, 1989). Ascorbate and urate were measured on a second HPLC system. This consisted of a Waters model 5 10 pump and a reverse-phase column (Hypersil C18, 100 X 2.1 mm, 5 pm
J.
Neurochem., Vol. 58, No. 4, 1992
1388
B. R. DEV ET AL.
particle size, Hewlett-Packard). The mobile phase was made from 80 mM sodium acetate anhydrous, 80 mMglacial acetic acid, 0.75 mM tridecylamine, and 30% methanol (Pachia and Kissinger, 1979). The pH was 4.3. The flow rate was 0.4 ml/min. The first 90-min perfusate following the implantation of the microdialysis probe was discarded. Then a 2-h baseline recording was done before altering the arterial blood pressure. One group of animals was infused with L-phenylephrine hydrochloride at a dose of 1- 10 pg/min to raise the blood pressure 50 mm Hg above the baseline for 60 min. The infusion volume was 0.8 f 0.2 rnl/h. Another group of animals was infused with sodium nitroprusside dihydrate at a dose of0.251.25 &min to lower the blood pressure by 20 mm Hg for 60 min. This infusion volume was 0.6 0.2 ml/h. Since urethane anesthetized rats had a baseline blood pressure of about 75 mm Hg, they could tolerate only the 20 mm Hg reduction. A control group of animals was infused with 1 ml of normal saline over a 60-min period and blood pressure, heart rate, and brain neurochemistry were monitored. All groups consisted of at least six rats. Phenylephrine and nitroprusside were continuously infused using a peristaltic infusion pump (Gilson “minipuls 2”) at a rate to maintain the desired blood pressure. The blood pressure and heart rate were monitored using a physiological pressure transducer (Bell and Howell) attached to a Gilson physiograph. The dialysis perfusate was assayed immediately for epinephrine, DOPAC, 5-HIAA, ascorbic acid, and uric acid every 15 min throughout the baseline period, during the drug infusion, and for 2 h after the discontinuation of the infusion. After each experiment the rats were decapitated, and the brains were removed and stored in 10%formalin for at least a week. These brains were frozen and 40-pm sections were sliced on a cryostat (Histostat microtome, A 0 Scientific Instruments). These histological sections were stained with
*
FIG. 1. Placement
J Kiwochem.. Vol 58, No 4, 1992
thionin to identify the placement of the microdialysis probe with reference to the rat stereotaxic atlas ofPaxinos and Watson (1986). If probes were misplaced, neurochemical data were excluded from the analysis. Perfusate concentrations at each time point were standardized to the mean baseline concentrations and expressed as a percent of the baseline value. Statistical analysis was done using analysis of variance for repeated measures followed by a post-hoc Dunnett test (Keppel, 1982).
RESULTS The placement of the in vivo microdialysis probe in the RVLM area is shown in Fig. 1. Probes placed in the region of the nucleus ambiguous showed higher DOPAC and smaller 5-HIAA levels with no detectable epinephrine. Probes placed more medially showed high levels of DOPAC, 5-HIAA, and ascorbic and uric acids with a smaller epinephrine level. If probes were found to be outside the region of the RVLM, the data for the animal were discarded. Typical chromatograms of standard solutions of catecholamines and of the microdialysis samples from the RVLM area are shown in Fig. 2. Catecholamine standards show norepinephrine as well as epinephrine, dopamine, and DOPAC. The tissue eluates show the presence of epinephrine, DOPAC, 5-HIAA, and uric and ascorbic acids. As can be seen, the large solvent front in the tissue eluate prevented detection of norepinephrine in the experimental samples. To provide additional evidence that epinephrine was the peak being measured in situ, the PNMT inhibitor LY 134046 was given at a dose of 30 mg/kg intrave-
of the in vivo microdialysis probe in the RVLM of rat brain. The arrow shows the probe track
EPINEPHRINE AND BLOOD PRESSURE
1389
C
P
”
FIG. 2. Chromatograms of the in vivo microdialysis perfusate from the RVLM of rat brain. a: Chromatogram of perfusateusing the sodium octyl sulfate mobile phase. b: Chromatogram of perfusate using the lgepon mobile phase. Only epinephrine was measured from the perfusates with this column. The peaks shown correspond to approximately 25 fmol of epinephrine and DOPAC injected. c: Uric acid and ascorbic acid measured using mobile phase described in Materials and Methods. The peaks shown represent about 400 fmol of ascorbic acid and uric acid injected. d: Chrornatogram of neurochemical standards using the sodium octyl sulfate mobile phase. e: Standard chrornatogram using the lgepon mobile phase. DA, dopamine; Epi, epinephrine; NE,norepinephrine.
,
l
0
l
.
,
.
l
.
,
12
8
4
)
,
,
,
0
10
L -
,
4
min
0
4
8
min
min
d
4
I
0
1
1
4
1
1
1
8
man
nously in a series of six animals. This dose led to a 44 k 3% reduction in the HPLC epinephrine concentration peak over 2 h (n = 6). This compound has been shown to cause some reduction in blood pressure and heart rate in spontaneously hypertensive animals but does not antagonize vasoconstriction responses induced by nerve stimulation or norepinephrine infusion (Hahn et al., 1983). Thus, the drug appears to act on central neurotransmission and not as an a receptor antagonist. Figure 3 presents the changes in blood pressure and heart rate during drug-induced hypertension and hy-
I
I2
I
,
,
16
0
I
I
4
I
,-,
8
min
,
,
12
potension. Phenylephrine hypertension led to a significant reflex reduction in heart rate during the time of the infusion. Both heart rate and blood pressure had returned to basal levels within 1 h after the discontinuation of the infusion. Hypotension produced by nitroprusside led to a reflex increase in heart rate. One hour after the infusion, blood pressure had rebounded above control levels and heart rate remained persistently elevated. These data are those obtained during the first running of the experiment. Dialysis data from these first experiments are shown in Fig. 4 (SOS results). .I Neurochem., Vol 58. No 4, 1992
B. R. DEV ET AL.
1390
550-
Heart rate
,
250
,
Blood pressure 110
-
?t””.:’.
90 -
E
6$ -120
.
, -60
-
PHENYL ,
, 0
.
, .
60
, 120
,
, 180
,
50
-120
-60
0
60
.
120
,
.
100
Time (min) FIG. 3. Phenylephrine (PHENYL)- and nitroprusside (NITRO)-induced changes in blood pressure and heart rate. Phenylephrine-induced hypertension sustained for a 1-h period led to a reduction in heart rate. Both blood pressure and heart rate returned to normal following phenylephrine infusion. Nitroprusside-induced hypotension led to an increase in heart rate which remained elevated during the reflex hypertensive phase. Results are means f SEM (n = 6).
Nearly identical cardiovascular results were observed in the repeat of the experiment. Dialysis data for epinephrine from the repeat experiment are also presented in Fig. 4 (Igepon). As shown in Fig. 4, phenylephrine-induced hypertension reduced the extracellular fluid concentration of epinephrine by about 30% from the baseline concentration. In one experiment, the epinephrine concentration remained depressed after the period of hypertension whereas in the second, the concentration tended to rebound. Nitroprusside-induced hypotension produced an increase in epinephrine concentration which also persisted beyond the period of the drug infusion. The changes in 5-HIAA levels were opposite to the changes seen in epinephrine. Hypertension led to a 30% increase in 5-HIAA whereas hypotension was associated with a 30%reduction in 5-HIAA. Figure 5 shows the blood pressure-mediated changes in the extracellular fluid concentrations of DOPAC, ascorbic acid, and uric acid. DOPAC concentrations fell during hypertension and rose during hypotension with a pattern similar to that seen with epinephrine. Changes in DOPAC during nitroprusside hypotension did not reach statistical significance because of the variability of the data. Ascorbic acid did not change significantly during either hypertension or hypotension. However, there was an increase in extracellular fluid J Neurveliem , Pol 58, tvo 4, 1992
ascorbic acid concentrations after the nitroprusside infusion was stopped. Uric acid did not change in response to any of the blood pressure manipulations. The saline-infused control group did not show any significant change in blood pressure, heart rate, or neurochemical profiles.
DISCUSSION This article has provided the first demonstration that epinephrine release from the RVLM changes with changing systemic blood pressure. We have earlier presented in vivo electrochemical results that were compatible with blood pressure related changes in epinephrine release (Bhaskaran and Freed, 1989). Because electrochemical measurements are inherently ambiguous, we have used in vivo dialysis and HPLC methods with two independent chromatographic systems to firmly establish that epinephrine release is altered during blood pressure changes. We have found that epinephrine release is directly correlated with changes in heart rate. During phenylephrine hypertension there was a reflex reduction in heart rate and a fall in epinephrine concentration in the extracellular fluid space. Phenylephrine hypertension is known to cause baroreceptor-mediated sympathoinhibition (Guyton, 198l), and the reduction in
1391
EPINEPHRINE AND BLOOD PRESSURE Nitroprusside Hypotension
Pti e ny rep h r i ne Hypertension a. Epinephrine 140-
120 -
120 -
100.
100
80
c
0 .c
b.Epinephrine 140-
Itgeponl
-
Baaeltne level 2 lt5 Irnolel5ul
80 -
-I*-
llgeponi
'yu.!v;
80
-
60
-
t
iu/h 1
Badelhe level
fmoielsui
3-
-* *-
z
c C
I so5 I
is 0 s )
0)
0 C
0
120-
120-
0 Q)
-.-C al Q)
Q
m
80 -
2622 W I W
80 -
2W.3
mWlsI5ul
dp
L
60 -
-* *-
80
*d
-
IsosJ
120-
120-
.
Baaelha level
-
210W5 tmcc/MI
60
60
c
. 80 -
YHENYI,
.
j
,
1
.
1
epinephrine concentration we have observed has occurred coincident with this inhibitory phase. The fact that the changes in epinephrine release parallel the changes in heart rate supports the possibility that epinephrine release may be linked to changes in central sympathetic activity. During nitroprusside hypotension when blood pressure fell, there was an increase in heart rate and an increase in epinephrine concentration in the extracel-
60
-
Baseline level 213t28 mwle1W
-**-----a
NITRO
1
,
lular fluid space of the RVLM. Thus, epinephrine rose during a period of increased sympathetic outflow. After the cessation of nitroprusside or phenylephrine infusions, blood pressure returned toward baseline values more rapidly than did the epinephrine concentrations. As we and others have observed for the past decade, the offset time of extracellular neurochemical measurements is usually slow relative to the onset time (Quintin et al., 1987; Bhaskaran and Freed, 1989). J . Neurochem.. Val. 58, No. 4, 1992
B. R. DEV ET AL.
1392
Nitroprusside Hypotension
Phenylephrine Hypertension
I2Ot
Ascorbic a c i d
c
C
C 100 Bssellne level 4 4 0 t 2 8 tmoW5ul
120r
Uric a c i d
100
100 Basellne lev&
80
PHENY!
-120
, -80
0
h+r.
’
380+42 lmobl5ul
60
Uric a c l d
‘2or
,
,
80
1
,
120
,
, 180
,
-
6 4
Baneme level 3 7 7 i30 tmolel5ul
,
-120
, -60
,
-
, NITRO . , 0
60
I
, . , , 120
180
Time (min) FIG. 5. The changes in the extracellular fluid concentration of DOPAC (upper panel), ascorbic acid (middle panel), and uric acid (lower panel) in RVLM in response to phenylephrine (PHENYL)-inducedhypertension and nitroprusside (NITRO)-inducedhypotension. The DOPAC level fell in response to hypertension and there was no significant change during hypotension. Ascorbic acid and uric acid showed no significant change with changing blood pressure. The ascorbic acid concentration did rise following nitroprusside infusion. Significance values are changes from baseline. ‘p < 0.05. Results are means _t SEM (n = 6).
For over a decade, brain epinephrine has been implicated in blood pressure regulation. Saavedra ( 1979) found that spontaneously hypertensive rats had elevated PNMT activity in the RVLM. In other areas of brain, epinephrine release has been correlated with changing blood pressure. Tuomisto et al. (1 983), using a push-pull cannula system in the freely moving spontaneously hypertensive rat, found low epinephrine release from the posterior hypothalamus. More recently, Routledge and Marsden ( 1987) observed that electrical stimulation of the C 1 nucleus led to epinephrine release J. Neurochem., Val. 58. No. 4. 1992
from posterior hypothalamus and an increase in systemic blood pressure. We are uncertain of the cells of origin of the epinephrine released in RVLM. Adrenergic neurons in A2 and A5 (Van Der Gugten et al., 1976) are known to project to the RVLM. It is also possible that recurrent collaterals of the C1 neurons themselves are the source of extracellular epinephrine seen in this study (Sun and Guyenet, 1986; Blessing et al., 1987). Also unknown are the neuronal events leading up to epinephrine release. Noradrenergic neurons from nucleus tractus so-
1393
EPINEPHRINE AND BLOOD PRESSURE litarius (Ross et al., 1985), caudal ventrolateral medulla (Granata, 1985), A5 (Sun and Guyenet, 1986), and area postrema (Blessing et al., 1987) have been reported to project into the RVLM. It is possible that these neurons have a role in regulating epinephrine release. Norepinephrine release during blood pressure change has been measured in hypothalamus. Using push-pull cannulae in the awake rat, Qualy and Westfall (1988) found that phenylephrine hypertension reduced norepinephrine release from the paraventricular nucleus whereas nitroprusside hypotension increased it. In the present study, we were unable to measure changes in norepinephrine release because of chromatographic limitations. Using mobile phases containing both sodium octyl sulfate and Igepon, we found that norepinephrine eluted during the solvent front and was not in sufficiently high concentration to be measured in the solvent front signal. We have previously reported blood pressure-related changes in catechol and indole release from a number of brain nuclei. These have included the dorsal raphe (Echizen and Freed, 1984), the locus ceruleus (Bhaskaran and Freed, 1988b), and the nucleus tractus solitarius (Bhaskaran and Freed, 1988a). The patterns of neurochemical change in each nucleus have been different. Only in the RVLM have opposite neurochemical responses been seen for hypertension and hypotension. Furthermore, changes in epinephrine concentration have been reciprocal to the change in blood pressure. In the present experiment, extracellular fluid 5HIAA concentration was also affected by blood pressure. Thus, there was an increase in endogenous 5HIAA levels during phenylephrine-induced hypertension and a reduction in 5-HIAA when blood pressure fell. Serotonin-containingneurons of the cardiovascular regulatory regions of the brainstem have been well documented (Dahlstrom and Fuxe, 1964; Bowker et al., 1981; Loewy and McKellar, 1981). Both electrical (Howe et al., 1983)and chemical (Pilowsky et al., 1986) stimulation of these neurons produced an increase in arterial pressure. Young spontaneously hypertensive rats were shown to have increased serotonin and 5HIAA in the ventrolateral medulla compared to normotensive rats of similar age group (Koulu et al., 1986). Smith et a]. (1979) also observed an 80% greater serotonin level in the medulla of young spontaneously hypertensive rats. Nicholas and Hancock (1988) have suggested that serotonin may modify sympathetic preganglionic activity by acting on epinephrine containing neurons of the RVLM. In every nucleus we have studied, 5-HIAA has increased with increased blood pressure. Presumably, the raphe nuclei contain the cell bodies for the serotonergic nerve terminals releasing serotonin and generating 5HIAA in the extracellular fluid space of the RVLM and other nuclei. In addition to epinephrine and SHIAA, we have found that DOPAC release changes with changing
blood pressure. Changes in DOPAC release paralleled the epinephrinechanges but were smaller in magnitude. The neuronal source of DOPAC is uncertain. DOPAC is not ordinarily considered to be a significant metabolite in norepinephrine or epinephrine cells. However, Quintin et a]. (1986) have provided evidence that the DOPAC concentration measured by an electrochemical electrode implanted in locus ceruleus changed with cell firing in locus ceruleus. They concluded that DOPAC was a byproduct of norepinephrine synthesis. Sved (1989) has argued that the PNMT-containing neurons may not be important in blood pressure regulation because measured tissue concentrations of epinephrine are very low in brain. The basal concentrations of epinephrine we have measured in the extracellular fluid of RVLM is about 5 fmol/pl. This corresponds to a concentration of about 5 nM. Assuming 10%recovery by the dialysis probe in vivo, the total extracellular fluid concentration would be 50 nA4. The tissue content of epinephrine in this region has been reported to be about 250 nM (Van Der Gugten et al., 1976). Our results suggest that the intracellular and extracellular concentrations of epinephrine may be within an order of magnitude of each other. The epinephrine result contrasts with dopamine in the striatum which has a tissue concentration of 50 pM and an extracellular concentration of about 100 nM (Zetterstrom et a]., 1983). Thus, the epinephrine concentration in the extracellular fluid of the RVLM is similar to the extracellular fuid dopamine concentration in the striatum, but [he storage pools are very different in size. Regardless of the sequence of regulatory steps leading to epinephrine release in RVLM, changes in epinephrine concentration can be directly correlated to changes in heart rate and inversely related to changes in blood pressure. DOPAC concentration changes paralleled the changes in epinephrine concentration. By contrast, 5HIAA concentration was directly correlated with the change in blood pressure and inversely with heart rate. We have seen this reciprocal symphony of neurochemical changes only in the C1 nucleus. In this structure, it appears that epinephrine and serotonin metabolism are closely linked to changes in sympathetic nerve activity. In particular, epinephrine in the RVLM appears to be a marker for sympathetic nerve activity. Acknowledgment: We thank Lee Phebus of Eli Lilly Company and John Atkin of ENKA AG for supplying the 300grn dialysis fiber. LY 134046 was a generous gift of the Eli Lilly Company. This work was supported by U.S. Public Health Service Grants R01 HL30722, NS18639, and NS09 199.
REFERENCES Alexander R. S. (1946) Tonic and reflex functions of medullary sympathetic cardiovascular centers. J. Neuruphysiol. 9, 205-2 I 7. Bhaskaran D. and Freed C. R. ( 1 9 8 8 ~ Changes ) in arterial blood pressure lead to baroreceptor-mediated changes in norepinephrine and 5-hydroxyindoleaceticacid in rat nucleus tractus solitarius. J. Pharmucol. Exp. T h r . 245, 356-363.
J. Neurochem.. Vol. 58, No. 4. 1992
1394
B. R . DEV E T AL.
Bhaskaran D. and Freed C. R. (19886) Changes in neurotransmitter turnover in locus coeruleus produced by changes in arterial blood pressure. Bruin Res. Bull. 21, 19 1 - 199. Bhaskaran D. and Freed C. R. ( 1989)Catechol and indole metabolism in rostral ventrolateral medulla change synchronously with changing blood pressure. J. Pharmacol. Exp. Ther. 249, 660666. Blessing W. W. and Reis D. J. (1982) Inhibitory cardiovascular function of neurons in the caudal ventrolateral medulla of the rabbit: relationship to the area containing Al noradrenergic cells. Bruin Re.y. 253, 161-171. Blessing W. W., Hedger S. C., Joh T. H., and Willoughby J. 0.(1987) Neurons in the area postrema are the only catecholamine-synthesizing cells in the medulla or pons with projections to the rostral ventrolateral medulla (CI-area) in the rabbit. Brain Res. 419, 336-340. Bowker R. M., Westlund K. N., and Coulter J. D. (1981) Origins of serotonergic projections to the spinal cord in rat: an immunocytochemical-retrograde transport study. Brain Res. 226, 187199. Dahlstrom A. and Fuxe K. (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system. 1. Demonstration of monoamines in the cell bodies of brain stem neurons. Acru Physiol. Scand. 62, 1-55. Day T. A,, Ro A., and Renaud L. P. (1983) Depressor area within caudal ventrolateral medulla of the rat does not correspond to the Al catecholamine cell group. Bruin Res. 279, 299-302. Echizen H. and Freed C. R. ( 1984) Altered serotonin and norepinephrine metabolism in rat dorsal raphe nucleus after drug-induced hypertension. Life Sci. 34, I58 1- 1589. Fuller R. W., Luecke S. H., Toomey R. E.. Horng J. S.. Ruff010 R. R., and Molloy B. B. (1981) Properties of 8,9dichloro-2,3,4,5tetrahydro-lH-2-benzazepine,an inhibitor of norepinephrine Nmethyltransferase. Biochem. Pharmacol. 30, 1345-1 352. Granata A. R., Kumada M., and Reis D. J. (1985) Sympathoinhibition by Al-noradrenergic neurons is mediated by neurons in the CI area of the rostral medulla. J . .4uton. Nerv. Sy.xt. 14,387395. Granata A. R., Numao Y., Kumada M.. and Reis D. J. (1986) Al noradrenergic neurons tonically inhibit sympathoexcitatory neurons of CI area in rat brainstem. Bruin Res. 377, 127- 146. Guyton A. C. (1981) Textbook ofMedicul Physiology. 6th ed. W. B. Saunders, Philadelphia. Hahn R. A,, Fuller R. W., and Luecke S. K. H. (1983) Cardiovascular effects of LY 134046, an inhibitor of norepinephrine N-methyltransferase in spontaneously hypertensive rats. J. Pharmacol. Exp. Ther. 226, 39-45. Head G. A,, Badoer E., and Korner P. 1. (1987) Cardiovascular role of Al catecholaminergic neurons in the rabbit. Effect of chronic lesions on responses to methyldopa, clonidine and 6-OHDA induced transmitter release. Bruin Res. 412, 18-28. Hokfelt T., Fuxe K., Goldstein M.. and Johansson 0. (1974) Imrnunohistochemical evidence for the existence of adrenaline neurons in the rat brain. Bruin Res. 66, 235-25 I . Howe P. R. C., Kuhn D. M., Minson J. B., Stead B. H., and Chalmers J. P. (1983) Evidence for a bulbospinal serotonergic pressor pathway in the rat brain. Bruin Res. 270, 29-36. Keppel G. (1982) Design and Analvsis: A Reseurcher's Handbook. 2nd ed. Prentice-Hall, Englewood Cliffs, N.J. Koulu M., Saavedra J. M., Niwa M., Scheinin M.. and Linnoila M. ( I 986) Association between increased serotonin metabolism in rat brainstem nuclei and development of spontaneous hypertension. Brain Res. 371, 177-181. Loewy A. D. and McKellar S. ( I 98 1) Serotonergic projections from the ventral medulla to the intermediolateral cell column in the rat. Brain Res. 211, 146-152. Mefford 1. N., Ota M., Stipetic M.. and Singleton W. (1987) Appli-
J Neurochem.. Vol. 58, No. 4, 1992
cation of a novel cation-exchangereagent, Igepon T-77 (n-methyl oleoyl taurate), to microbore separations of alumina extracts of catecholamines from cerebrospinal fluid, plasma, urine and brain tissue with amperometric detection. J. Chromaiogr. 420, 241251. Nicholas A. P. and Hancock M. B. (1988) Immunocytochemical evidence for substance P and serotonin input to medullary bulbospinal adrenergic neurons. Synapse 2, 569-576. Pachla L. A. and Kissinger P. T. (1979) Analysis of ascorbic acid by liquid chromatography with amperometric detection. Methods Enzymol. 62, 15-24. Paxinos G. and Watson C. (1986) The Rat Brain in Stereataxic Coordinures. 2nd ed. Academic Press, Sydney, Australia. Pilowsky P. M., Kapoor V., Minson J. B., West M. J., and Chalmers J. P. ( I 986) Spinal cord serotonin release and raised blood pressure after brainstem kainic acid injection. Bruin Res. 366, 354357. Qualy J. M. and Westfall T. C. (1988) Release of norepinephrine from the paraventricular hypothalamic nucleus of hypertensive rats. Am. J. Physiol. 254, H993-H1003. Quintin L., Hilaire G., and Pujol J.-F. (1986) Variations in 3,4-dihydroxyphenylacetic acid concentration are correlated to single cell firing changes in the rat locus coeruleus. Neuroscience 18, 889-899. Quintin L., Gillon J.-Y., Ghignome M., Renaud B., and Pujol J.-F. ( 1987) Baroreflex-linked variations of catecholamine metabolism in the caudal ventrolateral medulla: an in vivo electrochemical study. Bruin Res. 425, 319-336. Ross C. A,, Armstrong D. M., Ruggiero D. A., Pickel V. M., Joh T. H., and Reis D. J. (1981) Adrenaline neurons in the rostral ventrolateral medulla innervate thoracic spinal cord a combined immunocytochemical and retrograde transport demonstration. Neurosci. Lett. 25, 257-262. Ross C . A., Ruggiero D. A., Joh T. H., Park D. H., and Reis D. J. (1 984) Rostra1 ventrolateral medulla: selective projections to the thoracic autonomic cell column from the region containing C1 adrenaline neurons. J. Comp. Neurol. 228, 168-185. Ross C. A., Ruggiero D. A,, and Reis D. J. (1985) Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla. J. Comp. Neurol. 242, 5 I 1-534. Routledge C. and Marsden C. A. (1987) Electrical stimulation ofthe C1 region of the rostral ventrolateral medulla ofthe rat increases mean arterial pressure and adrenaline release in the posterior hypothalamus. NLwroscience 20, 457-466. Saavedra J. M. (1979) Adrenaline levels in brain stem nuclei and effects of a PNMT inhibitor on spontaneously hypertensive rats. Bruin Res. 166, 283-292. Smith M. L., Browning R. A,. and Myers J. H. (1979) In vivo rate of serotonin synthesis in brain and spinal cord of young, spontaneously hypertensive rats. Eur. J. Pharmacol. 53, 301-305. Sun M. K. and Guyenet P. G. (1986) Effect of clonidine and gamma aminobutyric acid on the discharges of medullo-spinal sympathoexcitatory neurons in the rat. Bruin Rex 368, 1-17. Sved A. F. ( 1 989) PNMT-containing catecholaminergic neurons are not necessarily adrenergic. Brain Res. 481, 113-1 18. Tuomisto L.. Yamatodani A,, Diet1 H.. Waldmann U., and Philippu A. (1983) In vivo release of endogenous catecholamines, histamine and GABA in the hypothalamus of Wistar Kyoto and spontaneously hypertensive rats. Naunyn Schmiedebergs Arch. Pharmucol. 323, 183-187. Van Der Gugten J., Palkovits M., Wijnen H. L. J . M., and Versteeg D. H. G. (1976) Regional distribution of adrenaline in rat brain. Brain Rex 107, 171-175. Zetterstrom T., Sharp T., Marsden C. A,, and Ungerstedt U. (1983) In vivo measurement of dopamine and its metabolites by intracerebral dialysis: changes after &hetamine. J. Neurochem. 41, 1769-1773.
Drug-Induced Changes in Blood Pressure Lead to Changes in Extracellular Concentrations of Epinephrine, Dihydroxyphenylacetic Acid, and 5-Hydroxyindoleacetic Acid in the Rostra1 Ventrolateral Medulla of the Rat *Bhaskar R. Dev, Patrick A. Mason, and Curt R. Freed Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado, U.S.A.
Abstract: Neurochemical changes in the extracellular fluid of the rostral ventrolateral medulla (RVLM) were produced by changes in arterial blood pressure. Blood pressure was raised or lowered with systemic infusions of phenylephrine or nitroprusside and neurochemicals were recovered from RVLM by in vivo microdialysis. A dialysis probe 300 pm in diameter and 500 pm in length was stereotaxically implanted in the RVLM of the urethane-anesthetized rat. Sterile physiological Ringer’s solution was perfused at a rate of 1.5 pl/ min. The perfusate was collected under ice-cold conditions every I5 min for the assay of epinephrine, dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (~-HIAA), ascorbic acid, and uric acid. After stable baseline neurochemical concentrations were achieved, animals were infused with phenylephrine or nitroprusside intravenously to raise or lower the blood pressure. Increasing blood pressure 50 mm Hg above the baseline value by phenylephrine led to a significant reduction in heart rate and a reduction in extracellular epinephrine and DOPAC concentrations. The 5HIAA concentration was increased during the hypertensive drug infusion. There were no changes in the concentrations of ascorbic acid or uric acid. Hypotension produced by ni-
troprusside (-20 mm Hg) led to neurochemical changes which were the reciprocal of those seen during hypertension. During hypotension, heart rate increased as did the extracellular fluid epinephrine concentration. The 5-HIAA concentration fell with hypotension and remained depressed following the nitroprusside infusion. Ascorbic acid and uric acid concentrations did not change during hypotension but ascorbic acid did increase after the nitroprusside infusion stopped. These data provide direct evidence that epinephrine release in RVLM is linked to changes in systemic blood pressure. Furthermore, because changes in epinephrine concentration parallel changes in sympathetic activity as measured by heart rate, epinephrine in the extracellular fluid space of the RVLM may be a marker for sympathetic nerve activity. Key Words: In vivo microdialysis-Rostra1 ventrolateral medulla-Epinephrine-Ascorbic acid-Uric acid-Blood pressure. Dev B. R. et al. Drug-induced changes in blood pressure lead to changes in extracellular concentrations of epinephrine, dihydroxyphenylacetic acid, and 5-hydroxyindoleacetic acid in the rostral ventrolateral medulla of the rat. J. Neurochern. 58, 1386- 1394 (1 992).
The ventrolateral medulla has been known to be an important site for regulation of sympathetic vasomotor activity since Alexander demonstrated separate pressor and depressor regions in the medulla in 1946 (Alexander, 1946). Noradrenergic neurons are found in the caudal ventrolateral medulla, the A1 region (Dahlstrom and Fuxe, 1964). These cells are believed to be the inhibitory vasomotor neurons (Blessing and Reis, 1982). The rostral ventrolateral medulla (RVLM), also
called the C1 region, has adrenergic neurons identified by the presence of the enzyme phenylethanolamineN-methyltransferase (PNMT) (Hokfelt et al., 1974). The PNMT-containing neurons of the RVLM area project widely both rostral to the hypothalamus and caudal to the spinal cord. These caudal projections to the intermediolateral and intermediomedial cell columns of the thoracic spinal cord are likely to have a role in controlling sympathetic outflow (Ross et al.,
Received July 12, 1991; accepted September 13, 1991. Address correspondence and reprint requests to Dr. C. R. Freed at Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, C-237, Denver, CO 80262, U.S.A.
*Dr. B. R. Dev has changed his name from D. Bhaskaran.
Abbreviations used: DOPAC, dihydroxflhenylacetic acid 5-HIAA, 5-hydroxyindoleacetic acid; PNMT, phenylethanolamine-N-methyltransferase; RVLM, rostral ventrolateral medulla.
I386
1387
EPINEPHRINE AND BLOOD PRESSURE 1981, 1984, 1985). Supporting the importance of the PNMT-containing neurons in regulating blood pressure is the observation that PNMT activity is changed i n the C1 nucleus in spontaneously hypertensive rats (Saavedra, 1979). T h e noradrenergic vasodepressor neurons of the A1 region are said to exert their sympathoinhibitory effect by projecting into the RVLM and inhibiting the adrenergic neurons located there (Granata et al., 1985, 1986; Ross et al., 1985). However, this explanation has been challenged by other laboratories (Day et al., 1983; Sun and Guyenet, 1986; Blessing et al., 1987; Head et al., 1987). Previously, we have reported blood pressure-induced changes in neurotransmitter metabolism in RVLM using in vivo electrochemical methods (Bhaskaran and Freed, 1989). In that study, we found that the electrochemical peak measured at low oxidation potential (the catechol peak) was reduced during phenylephrine hypertension and rose during nitroprusside hypotension. A second electrochemical peak measured a t high oxidation potential (the hydroxyindole peak) showed the opposite changes. During hypertension the peak was increased, and during hypotension the peak was re-
duced. In these previous in vivo electrochemical studies, we used a P N M T inhibitor t o show that the catechol peak was composed in part of epinephrine. However, because electrochemical electrodes do not measure single specific chemical species, measurements made by electrochemical methods are intrinsically ambiguous. To relate more precisely the neurochemical changes occurring in RVLM to changes in systemic blood pressure, we have used the technique of in vivo dialysis to measure extracellular fluid catechols and indoles.
MATERIALS AND METHODS Male Sprague-Dawley rats weighing 300-350 g were anesthetized with urethane (1.25 g/kg, i.p.) and had the femoral artery and vein catheterized for blood pressure measurement and drug infusion, respectively. A microdialysis probe was lowered into the RVLM area using coordinates from Paxinos and Watson (1986). The upper incisor bar of the stereotaxic frame (Narishige Scientific Instrument, Japan) was lowered 3.4 mm below the horizontal zero. The interaural line was used as the rostral-caudal zero point. Coordinates used were: A, -3.8 mm; L, +2.0 mm; and V, -10.2 mm from the skull surface. The microdialysis probe was made from concentric stainless steel tubing. A 32-gauge inner tube was placed inside a 22-gauge outer tube. A tubular dialysis membrane 300 pm diameter with a molecular mass cut-off of 12,500 daltons (Enka AG, F.R.G.) was glued to the outer tube. The length ofthe dialysis membrane tip was 0.5 mm. When these probes were tested in vitro at 25"C, recovery of catecholamines averaged 7%. The dialysis probe was perfused with Ringer's solution (Travenol Laboratory, Deerfield, IL, U.S.A.) at a rate of 1.5 pl/min and the perfusate was collected under ice-cold conditions every 15 min. Epinephrine, dihydroxyphenylacetic
acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), ascorbic acid, and uric acid were measured. Although we had no reason to predict either ascorbic acid or uric acid would change in RVLM with changes in blood pressure, these compounds have been shown to interfere with the interpretation of electrochemical studies. As the purpose of the present experiments was to validate and amplify our previous electrochemical study, measuring ascorbate and urate levels made it possible to associate or dissociate the epinephrine measurements made by dialysis to the catechol and indole estimates by in vivo electrochemistry. Epinephrine, DOPAC, and 5-HIAA were assayed using a 1 X 100 mm microbore HPLC column with electrochemical detection. The HPLC system consisted of an ISCO pLC-500 microflow pump with a Rheodyne injector (7413) with a sample injection loop volume of 5 pl. The column was 100 X 1 mm packed with Spherisorb ODS 2 with particle size of 3 pm (Keystone Scientific, State College, PA, U.S.A.). The electrochemical detector featured a dual glassy carbon electrode (Bioanalytical Systems, West Lafayette, IN, U.S.A.) set at a potential of 0.7 V. The mobile phase consisted of 0.1 M sodium dihydrogen phosphate buffer with sodium octyl sulfate (0.1 mM) and EDTA. The pH was 4.3 (Routledge and Marsden, 1987). The flow rate was I20 pl/min. Under these conditions, retention times for the compounds ofinterest were: epinephrine, 3.4 min; DOPAC, 5.0 min; and 5-HIAA, 13.7 min. Norepinephrine retention time was 1.9 min and eluted with the solvent front, so norepinephrine was not measured in these experiments. Whereas experimental values are expressed as percent of baseline concentrations, actual concentrations were estimated by external standards injected into the HPLC column. With these conditions, the method is sensitive enough to measure the epinephrine, DOPAC, and 5-HIAA levels as low as 10 fmol/5 pl injected. Because of the potential ambiguity of HPLC retention time as the sole identifier of epinephrine, we repeated the entire experiment using a different chromatographic system. New groups of animals were infused with phenylephrine and nitroprusside and dialysis samples were once again collected as described above. Instead of sodium octyl sulfate, we used the waxy detergent Igepon as described by Mefford et al. (1987). The mobile phase consisted of 0.15 M sodium acetate and methanol (85:1 3 , 150 mg/L Igepon T-77 (GAF, Wayne, NJ, U.S.A.), and 500 pi of 0.2 M EDTA/L, pH 7.56. The flow rate was 80 pl/min. Under these conditions, the retention time for norepinephrine was 2.5 min, for epinephrine 3.8 min, for DOPAC 5.0 min, and for 5-HIAA 13.0 min. Using a mobile phase containing Igepon but a different brand of HPLC column, Mefford et al. (1987) found that norepinephrine eluted after epinephrine. With the Keystone Spherisorb column described above, norepinephrine has eluted before epinephrine, as it did with the sodium octyl sulfate mobile phase described above. For additional characterization of the chromatographic peak corresponding to epinephrine, the PNMT inhibitor LY 134046 was given at a dose of 30 mg/kg. This drug has been shown to reduce the tissue concentration of epinephrine in the hypothalamus by 37% 1 h after drug administration (Fuller et al., 1981). In our previous study of RVLM using the in vivo electrochemical electrode, we found that LY 134046 reduced the catechol electrochemical peak by 25% after 1 h (Bhaskaran and Freed, 1989). Ascorbate and urate were measured on a second HPLC system. This consisted of a Waters model 5 10 pump and a reverse-phase column (Hypersil C18, 100 X 2.1 mm, 5 pm
J.
Neurochem., Vol. 58, No. 4, 1992
1388
B. R. DEV ET AL.
particle size, Hewlett-Packard). The mobile phase was made from 80 mM sodium acetate anhydrous, 80 mMglacial acetic acid, 0.75 mM tridecylamine, and 30% methanol (Pachia and Kissinger, 1979). The pH was 4.3. The flow rate was 0.4 ml/min. The first 90-min perfusate following the implantation of the microdialysis probe was discarded. Then a 2-h baseline recording was done before altering the arterial blood pressure. One group of animals was infused with L-phenylephrine hydrochloride at a dose of 1- 10 pg/min to raise the blood pressure 50 mm Hg above the baseline for 60 min. The infusion volume was 0.8 f 0.2 rnl/h. Another group of animals was infused with sodium nitroprusside dihydrate at a dose of0.251.25 &min to lower the blood pressure by 20 mm Hg for 60 min. This infusion volume was 0.6 0.2 ml/h. Since urethane anesthetized rats had a baseline blood pressure of about 75 mm Hg, they could tolerate only the 20 mm Hg reduction. A control group of animals was infused with 1 ml of normal saline over a 60-min period and blood pressure, heart rate, and brain neurochemistry were monitored. All groups consisted of at least six rats. Phenylephrine and nitroprusside were continuously infused using a peristaltic infusion pump (Gilson “minipuls 2”) at a rate to maintain the desired blood pressure. The blood pressure and heart rate were monitored using a physiological pressure transducer (Bell and Howell) attached to a Gilson physiograph. The dialysis perfusate was assayed immediately for epinephrine, DOPAC, 5-HIAA, ascorbic acid, and uric acid every 15 min throughout the baseline period, during the drug infusion, and for 2 h after the discontinuation of the infusion. After each experiment the rats were decapitated, and the brains were removed and stored in 10%formalin for at least a week. These brains were frozen and 40-pm sections were sliced on a cryostat (Histostat microtome, A 0 Scientific Instruments). These histological sections were stained with
*
FIG. 1. Placement
J Kiwochem.. Vol 58, No 4, 1992
thionin to identify the placement of the microdialysis probe with reference to the rat stereotaxic atlas ofPaxinos and Watson (1986). If probes were misplaced, neurochemical data were excluded from the analysis. Perfusate concentrations at each time point were standardized to the mean baseline concentrations and expressed as a percent of the baseline value. Statistical analysis was done using analysis of variance for repeated measures followed by a post-hoc Dunnett test (Keppel, 1982).
RESULTS The placement of the in vivo microdialysis probe in the RVLM area is shown in Fig. 1. Probes placed in the region of the nucleus ambiguous showed higher DOPAC and smaller 5-HIAA levels with no detectable epinephrine. Probes placed more medially showed high levels of DOPAC, 5-HIAA, and ascorbic and uric acids with a smaller epinephrine level. If probes were found to be outside the region of the RVLM, the data for the animal were discarded. Typical chromatograms of standard solutions of catecholamines and of the microdialysis samples from the RVLM area are shown in Fig. 2. Catecholamine standards show norepinephrine as well as epinephrine, dopamine, and DOPAC. The tissue eluates show the presence of epinephrine, DOPAC, 5-HIAA, and uric and ascorbic acids. As can be seen, the large solvent front in the tissue eluate prevented detection of norepinephrine in the experimental samples. To provide additional evidence that epinephrine was the peak being measured in situ, the PNMT inhibitor LY 134046 was given at a dose of 30 mg/kg intrave-
of the in vivo microdialysis probe in the RVLM of rat brain. The arrow shows the probe track
EPINEPHRINE AND BLOOD PRESSURE
1389
C
P
”
FIG. 2. Chromatograms of the in vivo microdialysis perfusate from the RVLM of rat brain. a: Chromatogram of perfusateusing the sodium octyl sulfate mobile phase. b: Chromatogram of perfusate using the lgepon mobile phase. Only epinephrine was measured from the perfusates with this column. The peaks shown correspond to approximately 25 fmol of epinephrine and DOPAC injected. c: Uric acid and ascorbic acid measured using mobile phase described in Materials and Methods. The peaks shown represent about 400 fmol of ascorbic acid and uric acid injected. d: Chrornatogram of neurochemical standards using the sodium octyl sulfate mobile phase. e: Standard chrornatogram using the lgepon mobile phase. DA, dopamine; Epi, epinephrine; NE,norepinephrine.
,
l
0
l
.
,
.
l
.
,
12
8
4
)
,
,
,
0
10
L -
,
4
min
0
4
8
min
min
d
4
I
0
1
1
4
1
1
1
8
man
nously in a series of six animals. This dose led to a 44 k 3% reduction in the HPLC epinephrine concentration peak over 2 h (n = 6). This compound has been shown to cause some reduction in blood pressure and heart rate in spontaneously hypertensive animals but does not antagonize vasoconstriction responses induced by nerve stimulation or norepinephrine infusion (Hahn et al., 1983). Thus, the drug appears to act on central neurotransmission and not as an a receptor antagonist. Figure 3 presents the changes in blood pressure and heart rate during drug-induced hypertension and hy-
I
I2
I
,
,
16
0
I
I
4
I
,-,
8
min
,
,
12
potension. Phenylephrine hypertension led to a significant reflex reduction in heart rate during the time of the infusion. Both heart rate and blood pressure had returned to basal levels within 1 h after the discontinuation of the infusion. Hypotension produced by nitroprusside led to a reflex increase in heart rate. One hour after the infusion, blood pressure had rebounded above control levels and heart rate remained persistently elevated. These data are those obtained during the first running of the experiment. Dialysis data from these first experiments are shown in Fig. 4 (SOS results). .I Neurochem., Vol 58. No 4, 1992
B. R. DEV ET AL.
1390
550-
Heart rate
,
250
,
Blood pressure 110
-
?t””.:’.
90 -
E
6$ -120
.
, -60
-
PHENYL ,
, 0
.
, .
60
, 120
,
, 180
,
50
-120
-60
0
60
.
120
,
.
100
Time (min) FIG. 3. Phenylephrine (PHENYL)- and nitroprusside (NITRO)-induced changes in blood pressure and heart rate. Phenylephrine-induced hypertension sustained for a 1-h period led to a reduction in heart rate. Both blood pressure and heart rate returned to normal following phenylephrine infusion. Nitroprusside-induced hypotension led to an increase in heart rate which remained elevated during the reflex hypertensive phase. Results are means f SEM (n = 6).
Nearly identical cardiovascular results were observed in the repeat of the experiment. Dialysis data for epinephrine from the repeat experiment are also presented in Fig. 4 (Igepon). As shown in Fig. 4, phenylephrine-induced hypertension reduced the extracellular fluid concentration of epinephrine by about 30% from the baseline concentration. In one experiment, the epinephrine concentration remained depressed after the period of hypertension whereas in the second, the concentration tended to rebound. Nitroprusside-induced hypotension produced an increase in epinephrine concentration which also persisted beyond the period of the drug infusion. The changes in 5-HIAA levels were opposite to the changes seen in epinephrine. Hypertension led to a 30% increase in 5-HIAA whereas hypotension was associated with a 30%reduction in 5-HIAA. Figure 5 shows the blood pressure-mediated changes in the extracellular fluid concentrations of DOPAC, ascorbic acid, and uric acid. DOPAC concentrations fell during hypertension and rose during hypotension with a pattern similar to that seen with epinephrine. Changes in DOPAC during nitroprusside hypotension did not reach statistical significance because of the variability of the data. Ascorbic acid did not change significantly during either hypertension or hypotension. However, there was an increase in extracellular fluid J Neurveliem , Pol 58, tvo 4, 1992
ascorbic acid concentrations after the nitroprusside infusion was stopped. Uric acid did not change in response to any of the blood pressure manipulations. The saline-infused control group did not show any significant change in blood pressure, heart rate, or neurochemical profiles.
DISCUSSION This article has provided the first demonstration that epinephrine release from the RVLM changes with changing systemic blood pressure. We have earlier presented in vivo electrochemical results that were compatible with blood pressure related changes in epinephrine release (Bhaskaran and Freed, 1989). Because electrochemical measurements are inherently ambiguous, we have used in vivo dialysis and HPLC methods with two independent chromatographic systems to firmly establish that epinephrine release is altered during blood pressure changes. We have found that epinephrine release is directly correlated with changes in heart rate. During phenylephrine hypertension there was a reflex reduction in heart rate and a fall in epinephrine concentration in the extracellular fluid space. Phenylephrine hypertension is known to cause baroreceptor-mediated sympathoinhibition (Guyton, 198l), and the reduction in
1391
EPINEPHRINE AND BLOOD PRESSURE Nitroprusside Hypotension
Pti e ny rep h r i ne Hypertension a. Epinephrine 140-
120 -
120 -
100.
100
80
c
0 .c
b.Epinephrine 140-
Itgeponl
-
Baaeltne level 2 lt5 Irnolel5ul
80 -
-I*-
llgeponi
'yu.!v;
80
-
60
-
t
iu/h 1
Badelhe level
fmoielsui
3-
-* *-
z
c C
I so5 I
is 0 s )
0)
0 C
0
120-
120-
0 Q)
-.-C al Q)
Q
m
80 -
2622 W I W
80 -
2W.3
mWlsI5ul
dp
L
60 -
-* *-
80
*d
-
IsosJ
120-
120-
.
Baaelha level
-
210W5 tmcc/MI
60
60
c
. 80 -
YHENYI,
.
j
,
1
.
1
epinephrine concentration we have observed has occurred coincident with this inhibitory phase. The fact that the changes in epinephrine release parallel the changes in heart rate supports the possibility that epinephrine release may be linked to changes in central sympathetic activity. During nitroprusside hypotension when blood pressure fell, there was an increase in heart rate and an increase in epinephrine concentration in the extracel-
60
-
Baseline level 213t28 mwle1W
-**-----a
NITRO
1
,
lular fluid space of the RVLM. Thus, epinephrine rose during a period of increased sympathetic outflow. After the cessation of nitroprusside or phenylephrine infusions, blood pressure returned toward baseline values more rapidly than did the epinephrine concentrations. As we and others have observed for the past decade, the offset time of extracellular neurochemical measurements is usually slow relative to the onset time (Quintin et al., 1987; Bhaskaran and Freed, 1989). J . Neurochem.. Val. 58, No. 4, 1992
B. R. DEV ET AL.
1392
Nitroprusside Hypotension
Phenylephrine Hypertension
I2Ot
Ascorbic a c i d
c
C
C 100 Bssellne level 4 4 0 t 2 8 tmoW5ul
120r
Uric a c i d
100
100 Basellne lev&
80
PHENY!
-120
, -80
0
h+r.
’
380+42 lmobl5ul
60
Uric a c l d
‘2or
,
,
80
1
,
120
,
, 180
,
-
6 4
Baneme level 3 7 7 i30 tmolel5ul
,
-120
, -60
,
-
, NITRO . , 0
60
I
, . , , 120
180
Time (min) FIG. 5. The changes in the extracellular fluid concentration of DOPAC (upper panel), ascorbic acid (middle panel), and uric acid (lower panel) in RVLM in response to phenylephrine (PHENYL)-inducedhypertension and nitroprusside (NITRO)-inducedhypotension. The DOPAC level fell in response to hypertension and there was no significant change during hypotension. Ascorbic acid and uric acid showed no significant change with changing blood pressure. The ascorbic acid concentration did rise following nitroprusside infusion. Significance values are changes from baseline. ‘p < 0.05. Results are means _t SEM (n = 6).
For over a decade, brain epinephrine has been implicated in blood pressure regulation. Saavedra ( 1979) found that spontaneously hypertensive rats had elevated PNMT activity in the RVLM. In other areas of brain, epinephrine release has been correlated with changing blood pressure. Tuomisto et al. (1 983), using a push-pull cannula system in the freely moving spontaneously hypertensive rat, found low epinephrine release from the posterior hypothalamus. More recently, Routledge and Marsden ( 1987) observed that electrical stimulation of the C 1 nucleus led to epinephrine release J. Neurochem., Val. 58. No. 4. 1992
from posterior hypothalamus and an increase in systemic blood pressure. We are uncertain of the cells of origin of the epinephrine released in RVLM. Adrenergic neurons in A2 and A5 (Van Der Gugten et al., 1976) are known to project to the RVLM. It is also possible that recurrent collaterals of the C1 neurons themselves are the source of extracellular epinephrine seen in this study (Sun and Guyenet, 1986; Blessing et al., 1987). Also unknown are the neuronal events leading up to epinephrine release. Noradrenergic neurons from nucleus tractus so-
1393
EPINEPHRINE AND BLOOD PRESSURE litarius (Ross et al., 1985), caudal ventrolateral medulla (Granata, 1985), A5 (Sun and Guyenet, 1986), and area postrema (Blessing et al., 1987) have been reported to project into the RVLM. It is possible that these neurons have a role in regulating epinephrine release. Norepinephrine release during blood pressure change has been measured in hypothalamus. Using push-pull cannulae in the awake rat, Qualy and Westfall (1988) found that phenylephrine hypertension reduced norepinephrine release from the paraventricular nucleus whereas nitroprusside hypotension increased it. In the present study, we were unable to measure changes in norepinephrine release because of chromatographic limitations. Using mobile phases containing both sodium octyl sulfate and Igepon, we found that norepinephrine eluted during the solvent front and was not in sufficiently high concentration to be measured in the solvent front signal. We have previously reported blood pressure-related changes in catechol and indole release from a number of brain nuclei. These have included the dorsal raphe (Echizen and Freed, 1984), the locus ceruleus (Bhaskaran and Freed, 1988b), and the nucleus tractus solitarius (Bhaskaran and Freed, 1988a). The patterns of neurochemical change in each nucleus have been different. Only in the RVLM have opposite neurochemical responses been seen for hypertension and hypotension. Furthermore, changes in epinephrine concentration have been reciprocal to the change in blood pressure. In the present experiment, extracellular fluid 5HIAA concentration was also affected by blood pressure. Thus, there was an increase in endogenous 5HIAA levels during phenylephrine-induced hypertension and a reduction in 5-HIAA when blood pressure fell. Serotonin-containingneurons of the cardiovascular regulatory regions of the brainstem have been well documented (Dahlstrom and Fuxe, 1964; Bowker et al., 1981; Loewy and McKellar, 1981). Both electrical (Howe et al., 1983)and chemical (Pilowsky et al., 1986) stimulation of these neurons produced an increase in arterial pressure. Young spontaneously hypertensive rats were shown to have increased serotonin and 5HIAA in the ventrolateral medulla compared to normotensive rats of similar age group (Koulu et al., 1986). Smith et a]. (1979) also observed an 80% greater serotonin level in the medulla of young spontaneously hypertensive rats. Nicholas and Hancock (1988) have suggested that serotonin may modify sympathetic preganglionic activity by acting on epinephrine containing neurons of the RVLM. In every nucleus we have studied, 5-HIAA has increased with increased blood pressure. Presumably, the raphe nuclei contain the cell bodies for the serotonergic nerve terminals releasing serotonin and generating 5HIAA in the extracellular fluid space of the RVLM and other nuclei. In addition to epinephrine and SHIAA, we have found that DOPAC release changes with changing
blood pressure. Changes in DOPAC release paralleled the epinephrinechanges but were smaller in magnitude. The neuronal source of DOPAC is uncertain. DOPAC is not ordinarily considered to be a significant metabolite in norepinephrine or epinephrine cells. However, Quintin et a]. (1986) have provided evidence that the DOPAC concentration measured by an electrochemical electrode implanted in locus ceruleus changed with cell firing in locus ceruleus. They concluded that DOPAC was a byproduct of norepinephrine synthesis. Sved (1989) has argued that the PNMT-containing neurons may not be important in blood pressure regulation because measured tissue concentrations of epinephrine are very low in brain. The basal concentrations of epinephrine we have measured in the extracellular fluid of RVLM is about 5 fmol/pl. This corresponds to a concentration of about 5 nM. Assuming 10%recovery by the dialysis probe in vivo, the total extracellular fluid concentration would be 50 nA4. The tissue content of epinephrine in this region has been reported to be about 250 nM (Van Der Gugten et al., 1976). Our results suggest that the intracellular and extracellular concentrations of epinephrine may be within an order of magnitude of each other. The epinephrine result contrasts with dopamine in the striatum which has a tissue concentration of 50 pM and an extracellular concentration of about 100 nM (Zetterstrom et a]., 1983). Thus, the epinephrine concentration in the extracellular fluid of the RVLM is similar to the extracellular fuid dopamine concentration in the striatum, but [he storage pools are very different in size. Regardless of the sequence of regulatory steps leading to epinephrine release in RVLM, changes in epinephrine concentration can be directly correlated to changes in heart rate and inversely related to changes in blood pressure. DOPAC concentration changes paralleled the changes in epinephrine concentration. By contrast, 5HIAA concentration was directly correlated with the change in blood pressure and inversely with heart rate. We have seen this reciprocal symphony of neurochemical changes only in the C1 nucleus. In this structure, it appears that epinephrine and serotonin metabolism are closely linked to changes in sympathetic nerve activity. In particular, epinephrine in the RVLM appears to be a marker for sympathetic nerve activity. Acknowledgment: We thank Lee Phebus of Eli Lilly Company and John Atkin of ENKA AG for supplying the 300grn dialysis fiber. LY 134046 was a generous gift of the Eli Lilly Company. This work was supported by U.S. Public Health Service Grants R01 HL30722, NS18639, and NS09 199.
REFERENCES Alexander R. S. (1946) Tonic and reflex functions of medullary sympathetic cardiovascular centers. J. Neuruphysiol. 9, 205-2 I 7. Bhaskaran D. and Freed C. R. ( 1 9 8 8 ~ Changes ) in arterial blood pressure lead to baroreceptor-mediated changes in norepinephrine and 5-hydroxyindoleaceticacid in rat nucleus tractus solitarius. J. Pharmucol. Exp. T h r . 245, 356-363.
J. Neurochem.. Vol. 58, No. 4. 1992
1394
B. R . DEV E T AL.
Bhaskaran D. and Freed C. R. (19886) Changes in neurotransmitter turnover in locus coeruleus produced by changes in arterial blood pressure. Bruin Res. Bull. 21, 19 1 - 199. Bhaskaran D. and Freed C. R. ( 1989)Catechol and indole metabolism in rostral ventrolateral medulla change synchronously with changing blood pressure. J. Pharmacol. Exp. Ther. 249, 660666. Blessing W. W. and Reis D. J. (1982) Inhibitory cardiovascular function of neurons in the caudal ventrolateral medulla of the rabbit: relationship to the area containing Al noradrenergic cells. Bruin Re.y. 253, 161-171. Blessing W. W., Hedger S. C., Joh T. H., and Willoughby J. 0.(1987) Neurons in the area postrema are the only catecholamine-synthesizing cells in the medulla or pons with projections to the rostral ventrolateral medulla (CI-area) in the rabbit. Brain Res. 419, 336-340. Bowker R. M., Westlund K. N., and Coulter J. D. (1981) Origins of serotonergic projections to the spinal cord in rat: an immunocytochemical-retrograde transport study. Brain Res. 226, 187199. Dahlstrom A. and Fuxe K. (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system. 1. Demonstration of monoamines in the cell bodies of brain stem neurons. Acru Physiol. Scand. 62, 1-55. Day T. A,, Ro A., and Renaud L. P. (1983) Depressor area within caudal ventrolateral medulla of the rat does not correspond to the Al catecholamine cell group. Bruin Res. 279, 299-302. Echizen H. and Freed C. R. ( 1984) Altered serotonin and norepinephrine metabolism in rat dorsal raphe nucleus after drug-induced hypertension. Life Sci. 34, I58 1- 1589. Fuller R. W., Luecke S. H., Toomey R. E.. Horng J. S.. Ruff010 R. R., and Molloy B. B. (1981) Properties of 8,9dichloro-2,3,4,5tetrahydro-lH-2-benzazepine,an inhibitor of norepinephrine Nmethyltransferase. Biochem. Pharmacol. 30, 1345-1 352. Granata A. R., Kumada M., and Reis D. J. (1985) Sympathoinhibition by Al-noradrenergic neurons is mediated by neurons in the CI area of the rostral medulla. J . .4uton. Nerv. Sy.xt. 14,387395. Granata A. R., Numao Y., Kumada M.. and Reis D. J. (1986) Al noradrenergic neurons tonically inhibit sympathoexcitatory neurons of CI area in rat brainstem. Bruin Res. 377, 127- 146. Guyton A. C. (1981) Textbook ofMedicul Physiology. 6th ed. W. B. Saunders, Philadelphia. Hahn R. A,, Fuller R. W., and Luecke S. K. H. (1983) Cardiovascular effects of LY 134046, an inhibitor of norepinephrine N-methyltransferase in spontaneously hypertensive rats. J. Pharmacol. Exp. Ther. 226, 39-45. Head G. A,, Badoer E., and Korner P. 1. (1987) Cardiovascular role of Al catecholaminergic neurons in the rabbit. Effect of chronic lesions on responses to methyldopa, clonidine and 6-OHDA induced transmitter release. Bruin Res. 412, 18-28. Hokfelt T., Fuxe K., Goldstein M.. and Johansson 0. (1974) Imrnunohistochemical evidence for the existence of adrenaline neurons in the rat brain. Bruin Res. 66, 235-25 I . Howe P. R. C., Kuhn D. M., Minson J. B., Stead B. H., and Chalmers J. P. (1983) Evidence for a bulbospinal serotonergic pressor pathway in the rat brain. Bruin Res. 270, 29-36. Keppel G. (1982) Design and Analvsis: A Reseurcher's Handbook. 2nd ed. Prentice-Hall, Englewood Cliffs, N.J. Koulu M., Saavedra J. M., Niwa M., Scheinin M.. and Linnoila M. ( I 986) Association between increased serotonin metabolism in rat brainstem nuclei and development of spontaneous hypertension. Brain Res. 371, 177-181. Loewy A. D. and McKellar S. ( I 98 1) Serotonergic projections from the ventral medulla to the intermediolateral cell column in the rat. Brain Res. 211, 146-152. Mefford 1. N., Ota M., Stipetic M.. and Singleton W. (1987) Appli-
J Neurochem.. Vol. 58, No. 4, 1992
cation of a novel cation-exchangereagent, Igepon T-77 (n-methyl oleoyl taurate), to microbore separations of alumina extracts of catecholamines from cerebrospinal fluid, plasma, urine and brain tissue with amperometric detection. J. Chromaiogr. 420, 241251. Nicholas A. P. and Hancock M. B. (1988) Immunocytochemical evidence for substance P and serotonin input to medullary bulbospinal adrenergic neurons. Synapse 2, 569-576. Pachla L. A. and Kissinger P. T. (1979) Analysis of ascorbic acid by liquid chromatography with amperometric detection. Methods Enzymol. 62, 15-24. Paxinos G. and Watson C. (1986) The Rat Brain in Stereataxic Coordinures. 2nd ed. Academic Press, Sydney, Australia. Pilowsky P. M., Kapoor V., Minson J. B., West M. J., and Chalmers J. P. ( I 986) Spinal cord serotonin release and raised blood pressure after brainstem kainic acid injection. Bruin Res. 366, 354357. Qualy J. M. and Westfall T. C. (1988) Release of norepinephrine from the paraventricular hypothalamic nucleus of hypertensive rats. Am. J. Physiol. 254, H993-H1003. Quintin L., Hilaire G., and Pujol J.-F. (1986) Variations in 3,4-dihydroxyphenylacetic acid concentration are correlated to single cell firing changes in the rat locus coeruleus. Neuroscience 18, 889-899. Quintin L., Gillon J.-Y., Ghignome M., Renaud B., and Pujol J.-F. ( 1987) Baroreflex-linked variations of catecholamine metabolism in the caudal ventrolateral medulla: an in vivo electrochemical study. Bruin Res. 425, 319-336. Ross C. A,, Armstrong D. M., Ruggiero D. A., Pickel V. M., Joh T. H., and Reis D. J. (1981) Adrenaline neurons in the rostral ventrolateral medulla innervate thoracic spinal cord a combined immunocytochemical and retrograde transport demonstration. Neurosci. Lett. 25, 257-262. Ross C . A., Ruggiero D. A., Joh T. H., Park D. H., and Reis D. J. (1 984) Rostra1 ventrolateral medulla: selective projections to the thoracic autonomic cell column from the region containing C1 adrenaline neurons. J. Comp. Neurol. 228, 168-185. Ross C. A., Ruggiero D. A,, and Reis D. J. (1985) Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla. J. Comp. Neurol. 242, 5 I 1-534. Routledge C. and Marsden C. A. (1987) Electrical stimulation ofthe C1 region of the rostral ventrolateral medulla ofthe rat increases mean arterial pressure and adrenaline release in the posterior hypothalamus. NLwroscience 20, 457-466. Saavedra J. M. (1979) Adrenaline levels in brain stem nuclei and effects of a PNMT inhibitor on spontaneously hypertensive rats. Bruin Res. 166, 283-292. Smith M. L., Browning R. A,. and Myers J. H. (1979) In vivo rate of serotonin synthesis in brain and spinal cord of young, spontaneously hypertensive rats. Eur. J. Pharmacol. 53, 301-305. Sun M. K. and Guyenet P. G. (1986) Effect of clonidine and gamma aminobutyric acid on the discharges of medullo-spinal sympathoexcitatory neurons in the rat. Bruin Rex 368, 1-17. Sved A. F. ( 1 989) PNMT-containing catecholaminergic neurons are not necessarily adrenergic. Brain Res. 481, 113-1 18. Tuomisto L.. Yamatodani A,, Diet1 H.. Waldmann U., and Philippu A. (1983) In vivo release of endogenous catecholamines, histamine and GABA in the hypothalamus of Wistar Kyoto and spontaneously hypertensive rats. Naunyn Schmiedebergs Arch. Pharmucol. 323, 183-187. Van Der Gugten J., Palkovits M., Wijnen H. L. J . M., and Versteeg D. H. G. (1976) Regional distribution of adrenaline in rat brain. Brain Rex 107, 171-175. Zetterstrom T., Sharp T., Marsden C. A,, and Ungerstedt U. (1983) In vivo measurement of dopamine and its metabolites by intracerebral dialysis: changes after &hetamine. J. Neurochem. 41, 1769-1773.
