Accepted Manuscript Role of endothelial nitric oxide in pulmonary and systemic arteries during hypoxia Cristina Nuñez, Victor M. Victor, Miguel Martí, Pilar D’Ocon PII: DOI: Reference:
S1089-8603(13)00347-9 http://dx.doi.org/10.1016/j.niox.2013.12.008 YNIOX 1340
To appear in:
Nitric Oxide
Received Date: Revised Date:
1 July 2013 12 November 2013
Please cite this article as: C. Nuñez, V.M. Victor, M. Martí, P. D’Ocon, Role of endothelial nitric oxide in pulmonary and systemic arteries during hypoxia, Nitric Oxide (2013), doi: http://dx.doi.org/10.1016/j.niox.2013.12.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
ROLE OF ENDOTHELIAL NITRIC OXIDE IN PULMONARY AND SYSTEMIC
2
ARTERIES DURING HYPOXIA
3 4 5 6
Cristina Nuñez1* Victor M Victor1,2,3*, Miguel Martí1 and Pilar D’Ocon1
7 8
1
9
Valencia, Spain
Departamento de Farmacología and CIBERehd, Facultad de Medicina, Universidad de
10
2
11
Valencia, Spain
12
3
FISABIO- Hospital Universitario Doctor Peset, Av Gaspar Aguilar 90, 46017,
Department of Physiology, University of Valencia, Valencia, Spain
13 14
* These authors contributed equally to the work
15 16 17
RUNNING HEAD: NO and vascular response to hypoxia
18 19
Corresponding author: Pilar D´Ocon. Departamento de Farmacología. Facultat de
20
Farmàcia. Universitat de València. Avda. Vicent Andrés Estelles s/n, Burjassot, 46100
21
València SPAIN.
22
Telephone: 34-963544828
23
E-mail: [email protected]
Fax 34-963544943
24 25 26 27
1
28
ABSTRACT
29
Our aim was to investigate the role played by endothelial nitric oxide (NO) during acute
30
vascular response to hypoxia, as a modulator of both vascular tone (through guanylate
31
cyclase (sGC) activation) and mitochondrial O2 consumption (through competitive
32
inhibition of cytochrome-c-oxydase (CcO)). Organ bath experiments were performed
33
and O2 consumption (Clark electrode) was determined in isolated aorta, mesenteric and
34
pulmonary arteries of rats and eNOS–knockout mice. All pre-contracted vessels
35
exhibited a triphasic hypoxic response consisting of an initial transient contraction (not
36
observed in vessels from eNOS–knockout mice) followed by relaxation and subsequent
37
sustained contraction. Removal of the endothelium, inhibition of eNOS (by L-NNA)
38
and inhibition of sGC (by ODQ) abolished the initial contraction without altering the
39
other two phases. The initial hypoxic contraction was observed in the presence of L-
40
NNA+NO-donors. L-NNA and ODQ increases O2 consumption in hypoxic vessels and
41
increases the arterial tone in normoxia but not in hypoxia. When L-NNA+mitochondrial
42
inhibitors (cyanide, rotenone or myxothiazol) were added, the increase in tone was
43
similar in normoxic and hypoxic vessels, which suggests that inhibition of the binding
44
of NO to reduced CcO restored the action of NO on sGC. CONCLUSION: A complex
45
equilibrium is established between NO, sGC and CcO in vessels in function of the
46
concentration of O2 : as O2 falls, NO inhibition of mitochondrial O2 consumption
47
increases and activation of sGC decreases, thus promoting a rapid increase in tone in
48
both pulmonary and systemic vessels, which is followed by the triggering of NO-
49
independent vasodilator/vasoconstrictor mechanisms.
50 51
KEY WORDS: NITRIC OXIDE, MITOCHONDRIA, BLOOD VESSELS,
52
HYPOXIA
2
53
INTRODUCTION
54
It is well known that endothelial NO modulates O2 concentration through activation of
55
soluble guanylate cyclase (sGC) and production of cyclic GMP, which mediates
56
vasodilatation and increases the availability of O2 in the surrounding tissues [1]. In
57
addition to the role it plays in vascular tone, a considerable body of evidence points to
58
NO being a local modulator of O2 availability through its inhibition of cytochrome c
59
oxidase (CcO), the terminal enzyme in the mitochondrial electron transport chain [2-6].
60
The inhibition of CcO by NO is reversible, competes with O2 and depends totally on the
61
concentration of O2, which makes it highly relevant in hypoxic conditions [6-8]. In fact,
62
an immediate effect of the reduced supply of O2 during hypoxia is that endogenous NO
63
becomes a more effective inhibitor of CcO. Subsequently, the consumption of
64
mitochondrial O2 by the vessel is reduced, the depletion of local O2 by the mitochondria
65
is impeded and the availability of O2 in hypoxic tissues increases [6,7]. However, the
66
functional consequence of the inhibition of O2 consumption in hypoxic vessels has not
67
been explored previously.
68
These evidences suggest that the release of endothelial NO promotes and increases the
69
availability of O2 by two mechanisms: activation of sGC, which leads to vasodilatation,
70
and inhibition of CcO, with a consequent reduction in O2 consumption. This double
71
activity of NO could be especially relevant in hypoxic conditions, although, precisely in
72
hypoxia, a decrease in NOS activity would be expected due to the reduction in the
73
substrate (O2) [9,10]. This, in turn, would undermine NO synthesis, affecting the
74
activation of sGC by NO and enhancing vascular tone. In fact, it has been reported that
75
NO is involved in the vasoconstriction observed in pulmonary vessels [11-15].
76
However, this is a controversial issue, as evidence also points to NO being a mediator of
3
77
hypoxic vasodilatation [16-21], while some studies have found the change in vascular
78
tone observed in hypoxia not to be modulated by NO [22,23].
79
These discrepancies could be attributable to experimental procedure (in vitro / in vivo
80
models), variable NO levels during hypoxia, the undermining of NO metabolism by
81
CcO in the mitochondria [19], or the augmentation of nitrite reductase activity of
82
different proteins [20,21,24].
83
Therefore, in spite of the general reduction in NOS activity that takes place during
84
hypoxia, the activity of NO in hypoxic vessels would seem to be of a heterogeneous
85
nature and remains a matter of controversy. Our hypothesis is that the vascular response
86
to hypoxia is conditioned largely by a complex equilibrium between NO availability
87
and activity on sGC and CcO. Thus, the aim of the present work was to investigate the
88
exact role that endothelial NO plays in the acute vascular response to hypoxia, taking
89
into consideration not only its action on sGC but also its role as a modulator of O2
90
consumption, aspects not previously studied in parallel. For this purpose, we have
91
analysed the action of NO on CcO (by determining O2 consumption) and changes in
92
vascular tone as a consequence of sGC modulation in vessels submitted to low
93
concentrations of O2.
94
In order to rule out extravascular influences, such as circulating mediators and neural
95
activity, and to evaluate effects localized at the vessel wall, we performed parallel
96
experiments in isolated vessels representative of the systemic (aorta and mesenteric
97
artery) and pulmonary (proximal pulmonary artery) beds, which exhibit opposing
98
responses to hypoxia in vivo [23]. Our results have allowed us to compare the hypoxic
99
response in these three isolated vessels and to analyse the role of endothelial NO during
100
hypoxia in both pulmonary and non-pulmonary vascular beds. Moreover, our findings 4
101
highlight that the vital balance between the two physiological targets of NO, sGC and
102
CcO, depends on the concentration of O2.
103 104
MATHERIALS AND METHODS
105
Preparation of arterial rings
106
Male Sprague-Dawley rats (250-300 g) or wild-type (WT) and eNOS knockout (eNOS
107
(-/-) mice (C57BL/6Jx129, 20-25 g, UCL, London, UK) were decapitated under brief
108
anaesthesia with inhaled isoflurane. Subsequently, their thoracic aortas and main
109
pulmonary and mesenteric arteries were removed, cleaned of adhering fat and
110
connective tissue in Krebs solution and cut into 5 mm (rat vessels) or 1.5 mm (mouse
111
vessels) rings. When necessary, the endothelium was disrupted by gently rubbing the
112
luminal surface. All protocols complied with European Community guidelines for the
113
use of experimental animals and were approved by the Ethics Committee of the
114
University of Valencia.
115 116
Contractility Studies
117
Rings obtained from rat vessels were suspended in a 5 mL organ bath (37ºC) containing
118
Krebs solution (in 10 -3 mol/L) (NaCl 118, KCl 4.75, CaCl2 1.9, MgSO4 1.2, KH2PO4
119
1.2, NaHCO3 25 and glucose 10.1) and gassed with 12% O2, 5% CO2 and 83% N2,
120
which produced an O2 concentration of ≈ 130x10 -6 mol/L similar to that present in
121
aortic blood [5,6]. The rings were monitored with a dissolved O2 meter (ISO2, World
122
Precision Instruments, Stevenage, Herts, UK). An initial load of 2 g (aorta) or 1 g
123
(pulmonary and mesenteric arteries) was applied to each preparation and maintained
124
throughout a 75-90 min equilibration period. Tension was recorded isometrically by
5
125
Grass FTO3 force-displacement transducers and data were recorded on a disc (Power
126
Lab).
127
Selected mouse arterial rings were mounted in a myograph (J.P. Trading, Aarhus,
128
Denmark) with separate 5 mL organ baths in similar conditions. Following a 60 min
129
stabilization period, a tension of 1.5 g (aorta) or 0.5 g (pulmonary and mesenteric
130
arteries) was applied to each vessel. This tension was previously determined as optimal
131
for each vessel using the contractile response to a depolarizing solution as a reference
132
(6x10 -2 mol/L KCl-Krebs obtained by isotonic replacement of NaCl by KCl; results not
133
shown). The rings were stimulated with phenylephrine (PHE, 10-9 - 10 -5 mol/l) or KCl
134
(1.5, 3 or 8X10-2 mol/L) in order to determine the range of response to both stimuli. The
135
presence (>90%) or absence (
S1089-8603(13)00347-9 http://dx.doi.org/10.1016/j.niox.2013.12.008 YNIOX 1340
To appear in:
Nitric Oxide
Received Date: Revised Date:
1 July 2013 12 November 2013
Please cite this article as: C. Nuñez, V.M. Victor, M. Martí, P. D’Ocon, Role of endothelial nitric oxide in pulmonary and systemic arteries during hypoxia, Nitric Oxide (2013), doi: http://dx.doi.org/10.1016/j.niox.2013.12.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
ROLE OF ENDOTHELIAL NITRIC OXIDE IN PULMONARY AND SYSTEMIC
2
ARTERIES DURING HYPOXIA
3 4 5 6
Cristina Nuñez1* Victor M Victor1,2,3*, Miguel Martí1 and Pilar D’Ocon1
7 8
1
9
Valencia, Spain
Departamento de Farmacología and CIBERehd, Facultad de Medicina, Universidad de
10
2
11
Valencia, Spain
12
3
FISABIO- Hospital Universitario Doctor Peset, Av Gaspar Aguilar 90, 46017,
Department of Physiology, University of Valencia, Valencia, Spain
13 14
* These authors contributed equally to the work
15 16 17
RUNNING HEAD: NO and vascular response to hypoxia
18 19
Corresponding author: Pilar D´Ocon. Departamento de Farmacología. Facultat de
20
Farmàcia. Universitat de València. Avda. Vicent Andrés Estelles s/n, Burjassot, 46100
21
València SPAIN.
22
Telephone: 34-963544828
23
E-mail: [email protected]
Fax 34-963544943
24 25 26 27
1
28
ABSTRACT
29
Our aim was to investigate the role played by endothelial nitric oxide (NO) during acute
30
vascular response to hypoxia, as a modulator of both vascular tone (through guanylate
31
cyclase (sGC) activation) and mitochondrial O2 consumption (through competitive
32
inhibition of cytochrome-c-oxydase (CcO)). Organ bath experiments were performed
33
and O2 consumption (Clark electrode) was determined in isolated aorta, mesenteric and
34
pulmonary arteries of rats and eNOS–knockout mice. All pre-contracted vessels
35
exhibited a triphasic hypoxic response consisting of an initial transient contraction (not
36
observed in vessels from eNOS–knockout mice) followed by relaxation and subsequent
37
sustained contraction. Removal of the endothelium, inhibition of eNOS (by L-NNA)
38
and inhibition of sGC (by ODQ) abolished the initial contraction without altering the
39
other two phases. The initial hypoxic contraction was observed in the presence of L-
40
NNA+NO-donors. L-NNA and ODQ increases O2 consumption in hypoxic vessels and
41
increases the arterial tone in normoxia but not in hypoxia. When L-NNA+mitochondrial
42
inhibitors (cyanide, rotenone or myxothiazol) were added, the increase in tone was
43
similar in normoxic and hypoxic vessels, which suggests that inhibition of the binding
44
of NO to reduced CcO restored the action of NO on sGC. CONCLUSION: A complex
45
equilibrium is established between NO, sGC and CcO in vessels in function of the
46
concentration of O2 : as O2 falls, NO inhibition of mitochondrial O2 consumption
47
increases and activation of sGC decreases, thus promoting a rapid increase in tone in
48
both pulmonary and systemic vessels, which is followed by the triggering of NO-
49
independent vasodilator/vasoconstrictor mechanisms.
50 51
KEY WORDS: NITRIC OXIDE, MITOCHONDRIA, BLOOD VESSELS,
52
HYPOXIA
2
53
INTRODUCTION
54
It is well known that endothelial NO modulates O2 concentration through activation of
55
soluble guanylate cyclase (sGC) and production of cyclic GMP, which mediates
56
vasodilatation and increases the availability of O2 in the surrounding tissues [1]. In
57
addition to the role it plays in vascular tone, a considerable body of evidence points to
58
NO being a local modulator of O2 availability through its inhibition of cytochrome c
59
oxidase (CcO), the terminal enzyme in the mitochondrial electron transport chain [2-6].
60
The inhibition of CcO by NO is reversible, competes with O2 and depends totally on the
61
concentration of O2, which makes it highly relevant in hypoxic conditions [6-8]. In fact,
62
an immediate effect of the reduced supply of O2 during hypoxia is that endogenous NO
63
becomes a more effective inhibitor of CcO. Subsequently, the consumption of
64
mitochondrial O2 by the vessel is reduced, the depletion of local O2 by the mitochondria
65
is impeded and the availability of O2 in hypoxic tissues increases [6,7]. However, the
66
functional consequence of the inhibition of O2 consumption in hypoxic vessels has not
67
been explored previously.
68
These evidences suggest that the release of endothelial NO promotes and increases the
69
availability of O2 by two mechanisms: activation of sGC, which leads to vasodilatation,
70
and inhibition of CcO, with a consequent reduction in O2 consumption. This double
71
activity of NO could be especially relevant in hypoxic conditions, although, precisely in
72
hypoxia, a decrease in NOS activity would be expected due to the reduction in the
73
substrate (O2) [9,10]. This, in turn, would undermine NO synthesis, affecting the
74
activation of sGC by NO and enhancing vascular tone. In fact, it has been reported that
75
NO is involved in the vasoconstriction observed in pulmonary vessels [11-15].
76
However, this is a controversial issue, as evidence also points to NO being a mediator of
3
77
hypoxic vasodilatation [16-21], while some studies have found the change in vascular
78
tone observed in hypoxia not to be modulated by NO [22,23].
79
These discrepancies could be attributable to experimental procedure (in vitro / in vivo
80
models), variable NO levels during hypoxia, the undermining of NO metabolism by
81
CcO in the mitochondria [19], or the augmentation of nitrite reductase activity of
82
different proteins [20,21,24].
83
Therefore, in spite of the general reduction in NOS activity that takes place during
84
hypoxia, the activity of NO in hypoxic vessels would seem to be of a heterogeneous
85
nature and remains a matter of controversy. Our hypothesis is that the vascular response
86
to hypoxia is conditioned largely by a complex equilibrium between NO availability
87
and activity on sGC and CcO. Thus, the aim of the present work was to investigate the
88
exact role that endothelial NO plays in the acute vascular response to hypoxia, taking
89
into consideration not only its action on sGC but also its role as a modulator of O2
90
consumption, aspects not previously studied in parallel. For this purpose, we have
91
analysed the action of NO on CcO (by determining O2 consumption) and changes in
92
vascular tone as a consequence of sGC modulation in vessels submitted to low
93
concentrations of O2.
94
In order to rule out extravascular influences, such as circulating mediators and neural
95
activity, and to evaluate effects localized at the vessel wall, we performed parallel
96
experiments in isolated vessels representative of the systemic (aorta and mesenteric
97
artery) and pulmonary (proximal pulmonary artery) beds, which exhibit opposing
98
responses to hypoxia in vivo [23]. Our results have allowed us to compare the hypoxic
99
response in these three isolated vessels and to analyse the role of endothelial NO during
100
hypoxia in both pulmonary and non-pulmonary vascular beds. Moreover, our findings 4
101
highlight that the vital balance between the two physiological targets of NO, sGC and
102
CcO, depends on the concentration of O2.
103 104
MATHERIALS AND METHODS
105
Preparation of arterial rings
106
Male Sprague-Dawley rats (250-300 g) or wild-type (WT) and eNOS knockout (eNOS
107
(-/-) mice (C57BL/6Jx129, 20-25 g, UCL, London, UK) were decapitated under brief
108
anaesthesia with inhaled isoflurane. Subsequently, their thoracic aortas and main
109
pulmonary and mesenteric arteries were removed, cleaned of adhering fat and
110
connective tissue in Krebs solution and cut into 5 mm (rat vessels) or 1.5 mm (mouse
111
vessels) rings. When necessary, the endothelium was disrupted by gently rubbing the
112
luminal surface. All protocols complied with European Community guidelines for the
113
use of experimental animals and were approved by the Ethics Committee of the
114
University of Valencia.
115 116
Contractility Studies
117
Rings obtained from rat vessels were suspended in a 5 mL organ bath (37ºC) containing
118
Krebs solution (in 10 -3 mol/L) (NaCl 118, KCl 4.75, CaCl2 1.9, MgSO4 1.2, KH2PO4
119
1.2, NaHCO3 25 and glucose 10.1) and gassed with 12% O2, 5% CO2 and 83% N2,
120
which produced an O2 concentration of ≈ 130x10 -6 mol/L similar to that present in
121
aortic blood [5,6]. The rings were monitored with a dissolved O2 meter (ISO2, World
122
Precision Instruments, Stevenage, Herts, UK). An initial load of 2 g (aorta) or 1 g
123
(pulmonary and mesenteric arteries) was applied to each preparation and maintained
124
throughout a 75-90 min equilibration period. Tension was recorded isometrically by
5
125
Grass FTO3 force-displacement transducers and data were recorded on a disc (Power
126
Lab).
127
Selected mouse arterial rings were mounted in a myograph (J.P. Trading, Aarhus,
128
Denmark) with separate 5 mL organ baths in similar conditions. Following a 60 min
129
stabilization period, a tension of 1.5 g (aorta) or 0.5 g (pulmonary and mesenteric
130
arteries) was applied to each vessel. This tension was previously determined as optimal
131
for each vessel using the contractile response to a depolarizing solution as a reference
132
(6x10 -2 mol/L KCl-Krebs obtained by isotonic replacement of NaCl by KCl; results not
133
shown). The rings were stimulated with phenylephrine (PHE, 10-9 - 10 -5 mol/l) or KCl
134
(1.5, 3 or 8X10-2 mol/L) in order to determine the range of response to both stimuli. The
135
presence (>90%) or absence (
