Hypoxia enhances a cGMP-independent nitric oxide relaxing mechanism in pulmonary arteries

Christopher J. Mingone,* Sachin A. Gupte,* Takafumi Iesaki, and Michael S. Wolin

Department of Physiology, New York Medical College, Valhalla, New York 10595

Submitted 29 October 2002 ; accepted in final form 9 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric oxide (NO) donors generally relax vascular preparations through cGMP-mediated mechanisms. Relaxation of endothelium-denuded bovine pulmonary arteries (BPA) and coronary arteries to the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) is almost eliminated by inhibition of soluble guanylate cyclase activation with 10 µM 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), whereas only a modest inhibition of relaxation is observed under hypoxia (PO2 = 8–10 Torr). This effect of hypoxia is independent of the contractile agent used and is also observed with NO gas. ODQ eliminated SNAP-induced increases in cGMP under hypoxia in BPA. cGMP-independent relaxation of BPA to SNAP was not attenuated by inhibition of K+ channels (10 mM tetraethylammonium), myosin light chain phosphatase (0.5 µM microcystin-LR), or adenylate cyclase (4 µM 2',5'-dideoxyadenosine). SNAP relaxed BPA contracted with serotonin under Ca2+-free conditions in the presence of hypoxia and ODQ, and contraction to Ca2+ readdition was also attenuated. The sarcoplasmic reticulum Ca2+-reuptake inhibitor cyclopiazonic acid (0.2 mM) attenuated SNAP-mediated relaxation of BPA in the presence of ODQ. Thus hypoxic conditions appear to promote a cGMP-independent relaxation of BPA to NO by enhancing sarcoplasmic reticulum Ca2+ reuptake.

calcium; guanylate cyclase; nitrovasodilator


IT IS WELL ACCEPTED that nitric oxide (NO) derived from the endothelium or nitrovasodilator drugs promotes vascular relaxation through stimulation of soluble guanylate cyclase (sGC) and generation of the mediator of smooth muscle relaxation cGMP (2, 12, 14). However, cGMP-independent mechanisms of smooth muscle relaxation have recently been reported (3, 5, 21, 26). Multiple mechanisms have been identified through which cGMP induces smooth muscle relaxation, where the dominant system mediating relaxation appears to vary between the vascular segments and the experimental conditions. In smooth muscle, relaxation through cGMP is generally associated with multiple processes regulated by activation of cGMP-dependent protein kinase that decrease intracellular Ca2+, the sensitivity of contraction to Ca2+, and the phosphorylation of myosin light chains (MLC) (17). NO can promote vascular relaxation through hyperpolarization by opening Ca2+-activated K+ channels through cGMP-dependent (1) and cGMP-independent (3) mechanisms. Recent studies also show that NO can cause vascular relaxation through stimulation of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) activity, thereby increasing Ca2+ uptake through the sarcoplasmic reticulum (5). The increase in sarcoplasmic reticulum filling with Ca2+ potentially influences additional systems associated with relaxation, including localized Ca2+ release involved in the activation of Ca2+-activated K+ channels and other processes, through which store-operated mechanisms inhibit extracellular Ca2+ influx elicited by contractile agents (15). Although it has been proposed that cGMP-dependent and cGMP-independent mechanisms activate the uptake of Ca2+ by SERCA in response to NO (5), the relative importance of these mechanisms remains to be investigated.

Previous studies (7) have shown that the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) causes a cGMP-dependent relaxation in bovine pulmonary arteries (BPA) and that 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) inhibits this response under 21% O2 (8, 10). Although the mechanism of sGC inhibition by ODQ is still under investigation, the original concept that ODQ is oxidizing the iron group of the sGC heme from Fe2+ -> Fe3+, preventing the binding of NO to sGC (23), appears to best explain its mechanism of action (8). The present study was developed on the basis of our initial observations that the inhibition of relaxation to NO by ODQ was markedly attenuated under a hypoxic atmosphere. First, it was necessary to confirm that ODQ blocked NO-induced increases in cGMP under hypoxia to verify that hypoxia was not functioning to prevent the actions of ODQ on sGC. Because 10 µM ODQ was observed to eliminate all detectable increases in cGMP caused by the NO donor SNAP, these conditions were used to probe the cGMP-independent mechanism of relaxation to NO. A nonspecific inhibitor of K+ channels [10 mM tetraethylammonium (TEA)] and blockade of cAMP production by adenylate cyclase [4 µM 2',5'-dideoxyadenosine (DDA)] were used to probe for the involvement of other vascular relaxing mechanisms, including hyperpolarization, caused by the opening of K+ channels and cAMP-mediated processes. Because the cGMP-independent mechanism of relaxation to NO appeared to be expressed in the presence of multiple contractile agents that are thought to function through a variety of different signaling systems, approaches were developed to probe some of the key control sites known to be influenced by relaxing mechanisms, including processes that lower intracellular Ca2+ through inhibition of intracellular release, enhancement of intracellular reuptake by SERCA, or attenuation of extracellular influx. In addition, the action of an inhibitor of MLC phosphatase (500 nM microcystin-LR) was examined to detect whether activation of this relaxing mechanism (9, 16, 27) was involved in the cGMP-independent actions of NO. Therefore, the objective of this study is to characterize the novel observation that hypoxia controls the expression of a cGMP-independent relaxation to NO and to investigate the role of signaling mechanisms that potentially contribute to the expression of vascular relaxation.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. ODQ, U-46619, and cGMP enzyme-linked immunoassay (ELISA) kits were purchased from Cayman Chemical (Ann Arbor, MI). DDA was obtained from Calbiochem (La Jolla, CA). SNAP was prepared in our laboratory according to previously published methods (13). All gasses were purchased from Tech Air (White Plains, NY). All salts were analyzed reagent grade and were obtained from Baker Chemical; all other chemicals were obtained from Sigma Chemical (St. Louis, MO).

Determination of changes in force in bovine arteries. Bovine lungs and hearts were obtained from a slaughterhouse and maintained in ice-cold oxygenated PBS solution during transport to our laboratory. Isolated endothelium-denuded pulmonary and coronary arterial rings were prepared by an adaptation of previously utilized methods. Briefly, the distal part of main lobar pulmonary arteries and left anterior descending coronary arteries was isolated and cleaned from surrounding tissue. The arteries were isolated and cut into rings ~4 mm in diameter and width, and the endothelium was removed by gentle rubbing of the lumen. The arterial rings were mounted on wire hooks attached to force displacement transducers (model FT03, Grass) for measurement of changes in isometric force. Tension was adjusted to 5 g, which is the optimal passive force for maximal contraction. Changes in force were recorded on a polygraph (model 7, Grass). Vessels were incubated in 10-ml baths (Metro Scientific, Farmingdale, NY) that were individually thermostated (37°C) in Krebs buffer gassed with 21% O2-5% CO2-balance N2. Krebs buffer contained (in mM) 118 NaCl, 4.7 KCl, 1.5 CaCl2, 25 NaHCO3, 1.1 MgSO4, 1.2 KH2PO4, and 5.6 glucose. Arteries were incubated for 1–2 h, during which passive tension was adjusted to maintain 5 g. The vessels were then depolarized with Krebs buffer containing 123 mM KCl (Hi-K+) in place of NaCl and subsequently reequilibrated with Krebs buffer before exposure to the experimental protocols. Control experiments were performed in the presence of the vehicle DMSO, which was previously shown to not affect relaxation to SNAP (10), to dissolve ODQ, cyclopiazonic acid (CPA), and thapsigargin.

Protocols for studies on mechanisms of NO-mediated relaxation. After the wash following the Hi-K+ treatment, the vessels that were to be subjected to hypoxia were switched to 95% N2-5% CO2 (PO2 = 8–10 Torr) and incubated under N2 for 20–30 min. The control vessels were incubated under 21% O2-5% CO2-balance N2 for a similar period of time. Those vessels that were to receive the sGC inhibitor ODQ (10-5 M dissolved in 0.1% DMSO) were incubated with the drug during this time. The vessels were then contracted with 30 mM KCl (K+), 0.1 µM U-46619, or 1–10 µM serotonin (5-HT). At the maximal point of contraction, the NO donor SNAP was added in an increasing cumulative dose-dependent manner. Drugs used for mechanistic studies were added during the 20- to 30-min period, in which the vessels were exposed to their respective gasses before contraction. None of the probes or hypoxia significantly altered force generation to contractile agents with this protocol, except the conditions that are mentioned in RESULTS. When arteries were exposed to NO, 200 ppm NO (balance N2) was delivered to the baths in a manner that generated the 50 nM concentration without changing the PO2, as previously described (10). In experiments with Ca2+-free conditions, arteries were exposed to an initial reference contraction with 30 mM K+ in the absence of probes (under 21% O2). The contracted rings were then washed with Krebs solution at the peak of the contraction. After 10 min, the Krebs solution was replaced with Ca2+-free Krebs solution containing 10-5 M EGTA, and some of the pulmonary arteries were exposed to hypoxic conditions, with or without ODQ (10-5 M), for 30 min before the experimental protocols described in RESULTS.

Measurement of tissue cGMP levels. At the appropriate time during force measurement experiments described in RESULTS, pulmonary artery rings were removed, swiftly blotted once on a paper towel to remove excess water, and then immediately submerged in liquid nitrogen to flash freeze the tissue sample. All samples were individually weighed and then homogenized, after pulverization with a mortar and pestle in the presence of liquid nitrogen, in 500 µl of phosphate-buffered 5% trichloroacetic acid solution. The precipitate was removed by centrifugation, and the supernatant was washed three times with water-saturated diethyl ether. Residual ether was removed by incubation at 70°C for 5 min before measurement of cGMP by ELISA, as described in the manual for the ELISA kit.

Statistical analysis. Values are means ± SE; n represents the number of arterial segments from different animals. Statistical analyses were performed by using a Student's t-test, and a one-way ANOVA with Bonferroni's correction was used for comparison between multiple groups.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypoxia increases cGMP-independent relaxation to the NO donor SNAP in BPA. The data in Fig. 1 show the dose-dependent relaxation to the NO donor SNAP of BPA precontracted with 30 mM K+. SNAP elicited an almost complete relaxation of BPA, with a similar concentration dependence under 21% O2 and hypoxia. Under 21% O2, the sGC inhibitor ODQ (10 µM) essentially eliminated relaxation (P < 0.05) of BPA to various doses of SNAP, except at the highest concentration. However, the inhibitory effects of ODQ were markedly reduced under hypoxia. Similar to 21% O2, ODQ essentially eliminates relaxation to 10-9–10-6 M SNAP, and the response to 10 µM SNAP was decreased from 90.6 ± 5.1 to 22.1 ± 4.0% relaxation (P < 0.01, n = 4) under 5% O2 (PO2 = 35 Torr). Although it is well documented that the NO donor SNAP increases cGMP in multiple vascular preparations, including BPA (7), and that 10 µM ODQ is generally observed to eliminate all detectable NO-mediated increases in cGMP in vascular preparations through oxidation of the heme of sGC under aerobic conditions (23), it is not known whether hypoxia alters the actions of ODQ. The influence of combinations of hypoxia and ODQ on cGMP levels elicited by 1 and 10 µM SNAP was examined because of the marked differences in functional responses that were observed at these doses of SNAP. The data in Fig. 2 indicate that SNAP increases cGMP levels in a concentration-dependent manner and that 10 µM ODQ completely inhibited all changes in cGMP levels observed in the presence of SNAP under 21% O2 and hypoxia. The levels of cGMP observed in the presence of all the SNAP + ODQ conditions shown in Fig. 2 were also not different from basal cGMP levels of 30 ± 15 pmol/g measured in BPA that were not exposed to SNAP. Interestingly, hypoxia appeared to enhance the increases in cGMP caused by SNAP without causing detectable increases in the relaxation responses that are shown in Fig. 1. Thus a cGMP-independent relaxation to the NO donor SNAP is observed in the presence of 10 µM ODQ.



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Fig. 1. Effects of inhibition of soluble guanylate cyclase (sGC) activation by 10 µM 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) on relaxation of endothelium-denuded bovine pulmonary artery (BPA) precontracted with 30 mM K+ to the nitric oxide (NO) donor S-nitroso-N-acetyl-penicillamine (SNAP) in the absence and presence of hypoxia. Values are means ± SE (n = 12).

 


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Fig. 2. Effects of inhibition of sGC activation by 10 µM ODQ on BPA cGMP levels in the presence of the NO donor SNAP [10-6 M (A) and 10-5 M (B)] in the absence and presence of combinations of hypoxia and ODQ. All increases in cGMP caused by 10-5 M SNAP (in the absence of ODQ) were significantly greater than cGMP levels in the presence of 10-6 M SNAP. Values are means ± SE (n = 8).

 

Inhibition of K+ channels and adenylate cyclase does not attenuate relaxation to the NO donor SNAP. Vasodilation resulting from hyperpolarization caused by the opening of K+ channels is a potential mechanism through which NO can promote relaxation (1, 3). Therefore, a nonspecificK+ channel blocker that inhibits almost all vascular K+ channels, TEA (10 mM), was used to examine the role of the K+ channel in the mechanism of relaxation elicited by NO (Fig. 3). TEA did not inhibit relaxation of 30 mM K+-precontracted BPA to SNAP under 21% O2 (Fig. 3A) or hypoxia (Fig. 3C). However, TEA significantly inhibited relaxation to SNAP under 21% O2 in BPA precontracted with 0.1 µM U-46619 (Fig. 3B), and it caused a smaller amount of inhibition under hypoxia (Fig. 3D) that did not reach statistical significance. As shown in Fig. 3, TEA did not inhibit relaxation of BPA contracted with 30 mM K+ or 0.1 µM U-46619 to the NO donor SNAP observed in the presence of the sGC inhibitor ODQ under 21% O2 (Fig. 3, A and B) or hypoxia (Fig. 3, C and D). Similar responses to arteries precontracted with U-46619 were seen in BPA contracted with 1 µM 5-HT. The largely cGMP-dependent relaxation to SNAP (e.g., 10 µM SNAP = 83 ± 11% relaxation, n = 4) under 21% O2 was significantly inhibited by 10 mM TEA (59 ± 6% relaxation). In the presence of 10 µM ODQ under hypoxia, the cGMP-independent relaxation to SNAP (e.g., 10 µM SNAP = 72 ± 11% relaxation, n = 4) was also not significantly altered by the presence of 10 mM TEA (78 ± 7% relaxation).



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Fig. 3. Effects of the K+ channel inhibitor tetraethylammonium (TEA, 10 mM) on BPA relaxation to the NO donor SNAP under 21% O2 (A and B) or hypoxia (C and D) in the presence or absence of ODQ in BPA precontracted with 30 mM K+ (n = 6–13; A and C) or 0.1 µM U-46619 (n = 4; B and D). Values are means ± SE.

 

The adenylate cyclase inhibitor DDA (4 µM) was employed to examine the role of cAMP in the cGMP-independent mechanism of relaxation to the NO donor SNAP. This concentration of DDA was chosen, because it significantly inhibits relaxation of BPA to isoproteronol (data not shown). DDA did not significantly alter the relaxation to SNAP under hypoxia in the presence or absence of ODQ (Fig. 4A). This observation is consistent with an absence of a role for increases in cAMP resulting from the stimulation of adenylate cyclase in the cGMP-independent mechanism of relaxation to NO.



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Fig. 4. Effects of the adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA; 4 µM, n = 6; A) and the myosin light chain phosphatase inhibitor 0.5 µM microcystin-LR (MC; n = 4; B) on BPA relaxation to the NO donor SNAP under hypoxia in the presence or absence of ODQ. Values are means ± SE.

 

Inhibition of MLC phosphatase does not alter relaxation to the NO donor SNAP. Because stimulation of MLC phosphatases is a potential site for the actions of vasodilators, including mechanisms that function through cGMP (16, 24), the actions of an inhibitor of MLC phosphatase (0.5 µM microcystin-LR) on relaxation to the NO donor SNAP were investigated. Because this probe was observed to cause the development of force associated with an increase in MLC phosphorylation of 30% by Western blot analysis (data not shown), it was added in the presence of 30 mM K+ contraction, and this prevented microcystin-LR from causing a significant enhancement of contraction. As shown in Fig. 4B, the MLC phosphatase inhibitor did not alter relaxation to the NO donor SNAP under hypoxia in the absence or presence of ODQ.

Influence of SNAP on contractile responses associated with Ca2+ release from intracellular stores and extracellular influx. When added under Ca2+-free conditions, 5-HT causes contraction of BPA, which decays to baseline over 25–30 min. The data in Fig. 5 indicate that when BPA are exposed to the NO donor SNAP after the initial contraction to 10 µM 5-HT under Ca2+-free conditions reaches maximal force, it causes concentration-dependent relaxation. The relaxation to 1 and 10 µM SNAP is markedly attenuated in the presence of ODQ under 21% O2. In contrast, ODQ had only modest inhibitory effects under N2 on the SNAP-induced relaxation of the contraction to 5-HT under Ca2+-free conditions. Because it has been shown (5) that NO can modulate relaxation through inhibition of Ca2+ influx across the plasma membrane, the effect of a subsequent concentration-dependent readdition of Ca2+ was investigated.



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Fig. 5. Effect of the NO donor SNAP on the initial transient contraction of BPA to 10 µM serotonin (5-HT) under Ca2+-free conditions in the absence and presence of combinations of hypoxia and ODQ [21% O2 force: 5-HT = 13.8 ± 1.9g(n = 10) and 5-HT + ODQ = 16 ± 2.4 g (n = 9); N2 force: 5-HT = 17.5 ± 1.4 g (n = 14) and 5-HT + ODQ = 16.7 ± 1.3 g (n = 14)]. Values are means ± SE.

 

The effects of Ca2+ addition to BPA pretreated with 5-HT under Ca2+-free conditions were examined under conditions of 21% O2 and hypoxia in the presence and absence of ODQ and/or SNAP. Ca2+ was added after the initial contraction to 5-HT had decayed to baseline. The addition of increasing doses of Ca2+ to the tissue bath elicited concentration-dependent contraction of BPA. As shown in Fig. 6, this contraction to Ca2+ was attenuated by SNAP under conditions of O2 and N2. The presence of ODQ reversed the effects of SNAP under 21% O2, but it did not enhance this contraction under hypoxia when SNAP was present.



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Fig. 6. Effect of the sGC inhibitor ODQ on attenuation by the NO donor SNAP of force generation elicited by readdition of Ca2+ to BPA (see Fig. 5) that were initially treated with 5-HT under Ca2+-free conditions in the presence of 21% O2 (A) or hypoxia (B). Values are means ± SE (n = 6).

 

Inhibition of Ca2+ reuptake by SERCA attenuates relaxation to the NO donor SNAP. CPA, an inhibitor of Ca2+ reuptake by SERCA, has been shown to inhibit relaxation to NO (5). This probe causes an initial transient contraction that decayed to baseline before the addition of contractile agents (KCl or 5-HT). BPA pretreated with 0.2 mM CPA were contracted to 30 mM K+ (Fig. 7, A and C) or 1 µM 5-HT (Fig. 7, B and D) under 21% O2 (Fig. 7, A and B) and hypoxia (Fig. 7, C and D) and then subjected to increasing cumulative concentrations of SNAP once steady-state force levels were observed. The relaxation of BPA precontracted with 30 mM K+ to SNAP in the absence of ODQ under 21% O2 and hypoxia was not significantly attenuated by CPA (Fig. 7, A and C). However, the cGMP-independent relaxation to SNAP observed under hypoxic conditions in the presence of ODQ was markedly attenuated by CPA (Fig. 7C). Interestingly, when the effects of 0.2 mM CPA were examined in the presence of 1 µM 5-HT as a contractile agent, it caused inhibition of the cGMP-dependent (21% O2) and cGMP-independent (hypoxia + ODQ) responses to SNAP (Fig. 7, B and D). To examine whether the TEA-inhibited component of relaxation to SNAP in the presence of U-46619 under 21% O2 shown in Fig. 3B originates from a cGMP-mediated stimulation of SERCA and the filling of capacitive Ca2+ stores that potentially activate relaxation through promoting the opening of K+ channels, we examined the influence of 10 mM TEA on relaxation to SNAP observed in the presence of CPA. As expected, CPA also inhibited relaxation to 10 µM SNAP from 82 ± 4 to 44 ± 4% (P < 0.05, n = 4) in BPA precontracted with 0.1 µM U-46619 under 21% O2. TEA also inhibited relaxation to 10 µM SNAP (47 ± 6% relaxation, n = 4, P < 0.05) in this experimental group under these conditions; however, TEA did not significantly alter relaxation observed in the presence of CPA (42 ± 5% relaxation), suggesting that the effects of TEA are dependent on a cGMP-mediated activation of the SERCA. The action of CPA was also confirmed with thapsigargin, another inhibitor of SERCA. A prolonged incubation of 60 min with a rather large dose of 100 µM thapsigargin was required to obtain reproducible results with this probe. However, under these conditions, thapsigargin decreased the 81.4 ± 9% relaxation (n = 4) to 10 µM SNAP under N2 in the presence of ODQ to 55 ± 10% relaxation (P < 0.05). Thus the cGMP-independent mechanism of relaxation to the NO donor SNAP appears to involve an acceleration of Ca2+ reuptake by the SERCA system.



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Fig. 7. Effects of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (CPA, 0.2 mM) on BPA relaxation to the NO donor SNAP under 21% O2 (A and B) or hypoxia (C and D) in the presence or absence of ODQ in BPA precontracted with 30 mM K+ (n = 7–8; A and C)or10 µM 5-HT (n = 4–7; B and D). Values are means ± SE.

 

Hypoxia also increases the importance of cGMP-in-dependent relaxation to NO in BCA. The relaxation of BCA precontracted with 30 mM K+ under 21% O2 and hypoxic atmospheres to 50 nM NO and SNAP was examined in the absence and presence of ODQ. Although hypoxia causes a decrease of ~40% in force generation to KCl in BCA (11), it did not significantly alter the response to NO or the concentration-dependent relaxation to the NO donor SNAP (Fig. 8). As shown in Fig. 8A, the sGC inhibitor ODQ eliminates relaxation to 50 nM NO under 21% O2, but it only partially attenuates relaxation to NO under hypoxia. Similar results were observed when relaxation of BCA to increasing cumulative does of the NO donor SNAP was examined (Fig. 8B). Thus hypoxia also appears to increase the expression of a cGMP-independent relaxation to NO in BCA.



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Fig. 8. Effects of inhibition of sGC activation by 10 µM ODQ on relaxation of endothelium-denuded bovine coronary artery precontracted with 30 mM KCl to 50 nM NO (A) or the NO donor SNAP (B) in the absence and presence of hypoxia. Values are means ± SE (n = 4).

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Relaxation to the NO donor SNAP in the presence of the sGC inhibitor ODQ was not associated with any detectable changes in cGMP, which indicates that this response occurs through processes that do not involve cGMP-mediated mechanisms. Enhancement of cGMP-independent relaxation to NO or the NO donor SNAP by hypoxia under a variety of conditions suggests that fundamental mechanisms of vascular regulation by NO and hypoxia are involved in the responses. On the basis of the absence of a detectable attenuation of the cGMP-independent relaxation to the NO donor SNAP by inhibition of K+ channels, adenylate cyclase, or MLC phosphatase, the primary mechanism of this response does not appear to be mediated through activation of vasodilator mechanisms involving hyperpolarization resulting from the opening of K+ channels, cAMP, or stimulation of MLC phosphatase activity. The observation of a cGMP-independent relaxation to the NO donor SNAP on contractions mediated through the release of intracellular Ca2+ or its influx across the sarcolemma and the prominent attenuation of this mechanism of relaxation by inhibition of Ca2+ reuptake by SERCA suggest that the primary site(s) of action of NO under these conditions is likely to involve processes that control intracellular Ca2+.

Detection of an enhancement by hypoxia of the cGMP-independent relaxation to NO under a variety of conditions provides some insight into processes that are involved. Because this effect is observed in the presence of force elicited by 30 mM KCl, the relaxing mechanism does not appear to be dependent on multiple components of receptor-elicited contractile mechanisms associated with G protein and/or phospholipase C activation and systems regulated through diacylglycerol and inositol triphosphate, because there is little evidence that these systems participate in the contractile response to KCl (20). Although hypoxia is a stimulus for contraction in BPA (4, 19) and relaxation in BCA (18), because the hypoxic enhancement of the cGMP-independent relaxation to NO is observed in BPA and BCA, the process involved in the effects of hypoxia is not likely to originate from signaling mechanisms directly contributing to PO2-elicited changes in force generation. It should also be noted that the data in Figs. 1 and 8 for BPA and BCA, respectively, indicate that hypoxia did not significantly alter the magnitude of relaxation to SNAP in the absence of ODQ in either vascular segment. However, hypoxia enhanced relaxation to NO in BCA and increased the levels of cGMP in the presence of SNAP in BPA, suggesting that hypoxia may actually function to increase the bioavail-ability of NO.

There is evidence that the opening of K+ channels in vascular smooth muscle is an important process involved in cGMP-dependent and cGMP-independent NO-mediated relaxation mechanisms (1, 3). Thus the opening of K+ channels and the production of relaxation through hyperpolarization could potentially contribute to the mechanisms involved in NO-mediated relaxation under hypoxia. However, blocking K+ channels with 10 mM TEA had minimal effects on the relaxation to SNAP under hypoxia in the presence and absence of ODQ. Because TEA blocks most K+ channels under the conditions employed, it appears that the NO donor SNAP does not induce a cGMP-independent relaxation through the activation of K+ channels in BPA. The role for NO in opening K+ channels via a cGMP mechanism may be an important part of smooth muscle relaxation in some systems (1), and evidence for the functional significance of this mechanism was detected in the present study when vascular segments were contracted with U-46619 under 21% O2. An increase in the level of membrane depolarization with 30 mM K+ and/or hypoxia (20) may have prevented the detection of cGMP-mediated relaxation in BPA through the opening of K+ channels under these conditions. Thus, after the stimulation of sGC is inhibited by ODQ, NO appears to activate vasodilation in BPA under hypoxia via mechanisms that are independent of cGMP and K+ channels.

Stimulation of MLC phosphatase activity is a potential mechanism of relaxation in vascular smooth muscle that could be activated by NO (16, 24). This system can be modulated through processes that control force generation in vascular smooth muscle independent of Ca2+ concentration or external stimuli (25). The cGMP system can phosphorylate MLC phosphatase and, in turn, induce relaxation through dephosphorylation of the MLC; however, the MLC phosphatase inhibitor microcystin-LR did not inhibit the relaxation of the BPA to SNAP. This suggests that relaxation may be occurring through a mechanism independent of the MLC phosphatase activation. Nevertheless, it should be noted that the absence of an effect of an MLC phosphatase inhibitor does not eliminate the possibility of an action of NO on the Ca2+ stimulation of the MLC phosphorylation by MLC kinase.

The effects of NO under experimental conditions that probe various mechanisms controlling intracellular Ca2+ were examined. Because 5-HT causes an initial rapid contraction under Ca2+-free conditions originating from the release of intracellular Ca2+ (11), which was observed to decay slowly over time in BPA, we studied the relaxant effects of NO on force generation to 5-HT under these conditions. SNAP-derived NO elicited relaxation of BPA contracted with 10 µM 5-HT in Ca2+-free Krebs solution, and this response was markedly inhibited by ODQ under 21% O2. However, the ODQ inhibitor of sGC activation caused only a modest inhibition of relaxation to SNAP under hypoxia. After decay of the initial contraction to 5-HT under Ca2+-free conditions, the addition of Ca2+ caused a contraction that is inhibited by SNAP under 21% O2 and hypoxia. The depression of this contraction to Ca2+ by SNAP is attenuated by ODQ under 21% O2 but not under hypoxia. The relaxation or depression of contraction by NO under these conditions and the inability of the sGC inhibitor ODQ to attenuate the actions of NO under hypoxia suggest that the cGMP-mediated and the cGMP-independent relaxing mechanisms observed under these conditions could originate from an inhibition of Ca2+ influx, as well as the enhancement of mechanisms that remove cytosolic Ca2+.

The potential role of an acceleration of Ca2+ removal by increased SERCA in relaxation elicited by NO was probed by inhibition of this pump with CPA. Although CPA did not inhibit relaxation by SNAP of the force generated by 30 mM K+ under conditions where cGMP mediation was the dominant mechanism (21% O2), CPA inhibited relaxation to this NO donor under conditions where the cGMP-independent relaxation was maximized (hypoxia + ODQ). However, CPA inhibited the SNAP-induced relaxation of BPA that were contracted with 5-HT under 21% O2 and hypoxia + ODQ. An observation that is difficult to explain is the absence in 30 mM K+-contracted BPA of inhibition of relaxation to SNAP under hypoxia (Fig. 7), because the probe inhibited relaxation under these conditions in the presence of ODQ. Perhaps cGMP may control processes under these conditions that regulate expression of cGMP-independent mechanisms of relaxation to NO. Overall, these data are consistent with an important role of stimulation of the SERCA system in the mechanism of cGMP-independent relaxation to the NO donor. However, the importance of this process in the cGMP-mediated relaxing mechanism appears to be determined by poorly understood aspects of the mechanisms involved in the generation of force. Processes such as the degree of filling of capacitive Ca2+ stores and the interactive nature of this store with systems that control influx, such as Ca2+-regulated K+ channels and cytosolic levels of Ca2+ (15, 22), may determine the importance of this mechanism in the observed relaxation to NO. Because TEA did not further attenuate cGMP-mediated relaxation to SNAP in the presence of CPA under conditions where TEA alone inhibited relaxation (Fig. 3B), the dominant mechanism through which cGMP regulates this K+ channel-dependent component of relaxation seems to originate from cGMP initially stimulating the filling of capacitive Ca2+ stores through the SERCA system. In the initial documentation of the role of SERCA in NO-mediated relaxation (5), it was suggested that cGMP-dependent and cGMP-independent mechanisms might be involved. The data in our study provide evidence that stimulation of SERCA has a role in both of these NO-mediated relaxing mechanisms.

This study reports evidence for the novel observation that hypoxia promotes expression of a cGMP-independent mechanism of relaxation to NO. Although NO and related nitrovasodilators have been reported to elicit responses through opening K+ channels (3) and by stimulating adenylate cyclase (6), these mechanisms and others, such as stimulating MLC phosphatase activity, do not seem to be contributing to the cGMP-independent mechanism of relaxation to NO examined in the present study. Although the cGMP-independent relaxing mechanisms observed under these conditions could originate from an inhibition of Ca2+ influx, the attenuation of this relaxation by the inhibitor CPA of the SERCA, which enhances the removal of cytosolic Ca2+, provides evidence that this previously identified (5) mechanism of relaxation of NO is a major contributor to the cGMP-independent response enhanced by hypoxia. The hypoxia-enhanced cGMP-independent relaxing mechanism to NO described in the present study is potentially an important protective vasodilator mechanism under hypoxic hypertensive conditions in the lung and systemic ischemic conditions that promote hypoxia and allow NO to accumulate. Because these studies were conducted in conduit-sized arteries at a low PO2, the physiological importance of the mechanisms examined need to be investigated in resistance-sized arteries under physiologically important hypoxic conditions that control blood flow.


    ACKNOWLEDGMENTS
 
DISCLOSURES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-31069, HL-43023, and HL-66331.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. S. Wolin, Dept. of Physiology, New York Medical College, Valhalla, NY 10595 (E-mail: mike_wolin{at}nymc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* Both authors contributed equally to this work. Back


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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