Endothelium-derived nitric oxide (NO) plays an important role in the regulation of vascular tone. The biosynthesis of NO in vascular endothelium occurs by the action of endothelial nitric oxide synthase (eNOS) on the amino acid L-arginine. NO thus released in response to stimuli, such as shear stress and endogenous neurohormones, acts on its receptor soluble guanylyl cyclase (sGC), giving rise to an increase in intracellular cGMP that mediates vascular smooth-muscle relaxation primarily through its action on protein kinase G (G-kinase). Consistent with other vasculature, NO and cGMP are reported to cause relaxation of rat pulmonary artery through the G-kinase pathway.1
However, the homeostatic mechanisms of adaptation of NO/cGMP signaling that involve different feedback loops are not well understood in pulmonary vasculature. The pulmonary vascular bed is a unique, low-pressure system, with a capacity to adapt to local changes in blood flow. It is significantly regulated by endothelium-derived NO,2 and there is growing evidence that endothelial dysfunction may lead to pulmonary hypertension.3,4 Accordingly, inhaled NO is used in the treatment of pulmonary hypertension.5,6 It is therefore of considerable significance to understand the influence of vascular endothelium/endogenous NO in modifying the vascular responses to exogenous NO. In a recent study,7 it has been demonstrated that the removal of vascular endothelium or the inhibition of nitric oxide synthase by NG-nitro-L-arginine (L-NA) increased the relaxant responses of exogenous NO in rat pulmonary artery rings. However, the mechanisms of sensitization have not been explained in this study. Although the phenomenon of supersensitivity has been described in some earlier studies in systemic arteries, the results are conflicting. For instance, NO deficiency caused by nitric oxide synthase inhibition/endothelium removal has been shown to either sensitize (rat aorta8,9) or cause no change (rabbit aorta10; rat aorta11) in the relaxant responses produced by nitrovasodilators. Regarding the mechanisms of sensitization, some reports suggest that specific supersensitivity to NO may occur at the level of its receptor sGC.9,12,13 However, in a recent report on rat aorta, it has been demonstrated that the enhanced SNP-induced cGMP response by long-term L-NAME treatment was not caused by increased sGC activity or by overexpression of sGC.14 In view of the controversies regarding the phenomenon of supersensitivity and signaling pathways associated with it, the present study was undertaken to elucidate the mechanisms by which endogenous NO deficiency modifies nitrovasodilator response in rat pulmonary artery. In a recent study, it was shown that continuous exposure to nitric oxide or cGMP decreased the G-kinase activity in pulmonary veins of newborn lambs, through a negative feedback mechanism.15 Conversely, it is predicted that deficiency of endogenous NO may sensitize vascular G-kinase to the relaxant action of cGMP. We have examined this hypothesis in the present study using 8-Br-cGMP, a G-kinase I activator to relax isolated rat pulmonary artery rings made deficient of NO, through either treatment with L-NAME or removal of endothelium.
Male Wistar rats (200-250 g) were used for the study, and all the procedures were approved by the Institute Animal Ethics Committee of the Indian Veterinary Research Institute. The animals were bled to death by excising abdominal aorta under pentobarbitone (60 mg/kg intraperitoneally) anesthesia. Heart and lungs en block were removed and transferred to ice-cold Krebs-Henseleit solution of the following composition (mM): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, NaHCO3 11.9, KH2PO4 1.2, and D-glucose 11.1 (pH 7.4). Both left and right pulmonary arteries were dissected carefully and cleaned of adhering connective tissues under a dissecting microscope.
Arterial rings of 2 to 3 mm in length were prepared and mounted in a 10-mL organ bath containing Krebs-Henseleit solution. The tissues were maintained at 37°C and continuously aerated with carbogen (95% O2 + 5% CO2) under a passive tension of 1.0 g for a period of 1 hour with intermittent changes in the bath fluid every 15 minutes. Endothelium was achieved by gentle rubbing of intima with the help of horse tail hair. Isometric tension was measured by the high-sensitivity force-displacement transducers (Model: MLT0202/D, Power Lab, Australia) and recorded on a PC using Chart Software V.5.2.2 (Power Lab, Australia).
After the equilibration period, each arterial ring was contracted twice with phenylephrine (1 μM). Endothelial integrity was examined by adding ACh at the plateau of phenylephrine contraction. The arterial rings were considered endothelium intact if ACh caused ≥80% reversal of phenylephrine-induced contraction. In the endothelium-denuded preparations, relaxation response to ACh was either absent or it was less than 10% of the maximal reversal of phenylephrine-induced contraction. To achieve inhibition of endogenous NO, tissues were treated with L-NAME (100 μM) for 30 minutes. In these tissues, a low concentration of phenylephrine (0.01-0.03 μM) was used to obtain a matching contraction comparable with that obtained in the absence of L-NAME. A marked decrease in the relaxation response to ACh indicated the inhibition of endogenous NO production. Concentration-dependent relaxations to different vasodilators were obtained by adding the drugs cumulatively at the plateau phase of phenylephrine contraction. For each vessel ring, only one vasodilator was tested under control conditions (endothelium intact), in the absence of endothelium (endothelium denuded), or in the presence of NO synthase inhibitor L-NAME.
Endothelium-intact pulmonary artery segments (main, left, and right branches), equilibrated in PSS for 30 minutes at 37°C under continuous carbogen bubbling, were exposed to 100 μM IBMX alone or with 100 μM L-NAME for the next 30 minutes. Next, these arteries were exposed to SNP (1.0 μM) for 5 minutes and were quickly transferred to liquid nitrogen. Arterial rings treated similarly in all respects except for SNP treatment were used to estimate basal cGMP concentrations. Liquid nitrogen snap-frozen arterial rings were homogenized in 1 mL of chilled trichloroacetic acid (6%) and centrifuged at 10000g for 10 minutes. Supernatant thus obtained was extracted five times with water-saturated diethyl ether and was then used for cGMP assay, using an EIA kit (Cayman Chemicals, Ann Arbor, Mich), following the manufacturer's instructions. Tissue pellets obtained after centrifugation were dissolved in 1 N NaOH for protein estimation by Lowry's method.16 Concentration of cGMP was expressed as picomoles per milligram of protein.
Drugs and Chemicals
SNP, 8-Br-cGMP, 3-isobutyl-1-methyl xanthine (IBMX), phenylephrine, acetylcholine (ACh) chloride, dibutyryl cAMP, NG-nitro-L-arginine methyl ester (L-NAME), levcromakalim, and glibenclamide were purchased from Sigma Chemicals (St. Louis, Mo). Stock solutions (10 mM) of SNP, 8-Br-cGMP, phenylephrine, ACh, dibutyryl cAMP, and L-NAME were prepared in double-distilled water. IBMX (10 mM) was dissolved in ethanol. Levcromakalim and glibenclamide stock solutions (10 mM) were prepared in DMSO. Further dilutions of stock solutions were made in Krebs-Henseleit solution.
Relaxation responses are expressed as the percent reversal of the phenylephrine contraction. Both Emax (the maximal response) and EC50 (the concentration producing 50% of the maximal response) were determined by nonlinear regression analysis (sigmoidal dose-response, with variable slope), using Graph Pad Prism version 4.00 (San Diego, Calif). Sensitivity/potency was expressed as pD2 = −log EC50. Efficacy of the vasodilator was referred to as Emax. Results are expressed as means ± SE, with n equal to the number of vascular rings. Data were analyzed by two-way ANOVA for multiple comparisons, followed by Bonferroni post hoc test. Comparison between two values was made by Student's t test. A value of P < 0.05 was considered statistically significant.
Effect of L-NAME/Endothelium Removal on ACh-Induced Relaxation of Rat Pulmonary Artery Rings
Figure 1 shows the effect of acute L-NAME (100 μM; endothelium-intact preparation) or endothelial removal on ACh (1 μM)-induced relaxation of rat pulmonary artery rings constricted with phenylephrine. In endothelium-intact rings, the tension achieved by phenylephrine (1 μM) was 0.34 ± 0.05 g (n = 30). In L-NAME-treated or endothelium-denuded preparations, a matching tension was achieved with phenylephrine (0.01-0.1 μM). ACh (1 μM) caused 83.0 ± 4.0% (n = 30) relaxation in endothelium-intact tissues. Pretreatment with L-NAME (100 μM) caused a 6.8 ± 0.75% (percentage of 1 μM PE contraction before L-NAME; n = 30) increase in the basal tension of pulmonary artery rings. In these rings, which were constricted to an identical level of tension with phenylephrine (10-30 nM), ACh (1 μM) produced 27.25 ± 3.45% relaxation (n = 10). L-NAME (1 mM) had no additional inhibitory effect on ACh-induced relaxation. Endothelium denudation almost abolished the vasodilator response to ACh (1 μM).
Effect of L-NAME/Endothelium Removal on SNP-Induced Relaxation
In endothelium-intact rings constricted with phenylephrine (1 μM), SNP (0.1 nM to 1 μM) caused concentration-dependent relaxation (pD2 7.61 ± 0.05, Emax104.4 ± 3.2%, n = 9). L-NAME (100 μM) significantly (P < 0.05) increased the potency (pD2 8.21 ± 0.04) but not the efficacy (Emax 107.1 ± 1.8%) of SNP (Fig. 2A; n = 8). Similarly, an increase in the potency (pD2 8.44 ± 0.11) but not the efficacy (Emax98.5 ± 4.0%) of SNP was evident in endothelium-denuded rings compared with the rings with intact endothelium (n = 4). L-NAME (100 μM), however, had no significant effect on the vasodilator potency (pD2 8.21 ± 0.09) and efficacy (Emax 98.6 ± 4.3%) of SNP in endothelium-denuded rings (Fig. 2C; n = 4).
Effect of L-NAME/Endothelium Removal on 8-Br-cGMP-Induced Relaxation
In rings with intact endothelium and constricted with phenylephrine (1 μM), 8-Br-cGMP (0.1-300 μM) caused concentration-dependent relaxation (pD2 4.22 ± 0.17; Emax 110.3 ± 17.5%; n = 7). As illustrated in Figure 3A, L-NAME (100 μM) significantly (P < 0.05, n = 6) increased the vasodilator potency (5.04 ± 0.09) of 8-Br-cGMP but had no significant effect on its efficacy (Emax 112.6 ± 9.7%). Similarly, endothelium removal significantly (P < 0.05; n = 5) increased the relaxant potency (pD2 5.28 ± 0.11) but not the efficacy (Emax109.4 ± 8.9%) of 8-Br-cGMP compared with that of endothelium-intact preparations (Fig. 3B).
Effect of L-NAME on the Vasodilator Response of Dibutyryl cAMP
Figure 4 illustrates the effect of L-NAME on the vasodilator response of dibutyryl cAMP. Dibutyryl cAMP (10-300 μM), added cumulatively at an interval of 0.5 log unit, produced concentration-dependent relaxation (pD2 4.14 ± 0.04; Emax 103.0 ± 6.2%; n = 5) of endothelium-intact pulmonary artery rings preconstricted with phenylephrine. In the presence of L-NAME (100 μM), there was a small increase in the relaxation response, without any significant change either in the potency (pD2 4.21 ± 0.05) or in the efficacy (Emax 114.5 ± 5.7%) of dibutyryl cAMP.
Effect of L-NAME on Levcromakalim-Induced Relaxation
In endothelium-intact rings, levcromakalim (1 nM to 10 μM), an opener of KATP channels, caused concentration-dependent relaxation (pD2 6.94 ± 0.04; Emax 95.6 ± 2.3%; n = 8). Glibenclamide (10 μM) caused about a 2-log-unit rightward shift in the concentration-response curve of levcromakalim, thereby significantly (P < 0.05) decreasing the potency (pD2 5.04 ± 0.14) but not the efficacy (Emax 108.9 ± 10.9%) of the potassium channel opener (Fig. 6A; n = 4). As shown in Figure 5B, pretreatment of the arterial rings with L-NAME (100 μM) significantly (P < 0.05) decreased the efficacy (Emax 78.4 ± 2.3%) but not the potency (pD2 6.79 ± 0.05 versus control; n = 4) of levcromakalim.
Effect of L-NAME on Basal and SNP-Stimulated Tissue cGMP
Figure 6 depicts the effect of L-NAME on basal and SNP-stimulated increase in cGMP in pulmonary artery rings with intact endothelium. L-NAME (100 μM) significantly (P < 0.05) decreased the basal cGMP (5.95 ± 1.6 pmol/mg protein, n = 9, versus control: 28.09 ± 1.0 pmol/mg protein, n = 6) levels in pulmonary artery rings with intact endothelium. However, L-NAME treatment significantly (P < 0.05) enhanced SNP (1 μM)-stimulated increase in cGMP levels (271.8 ± 39.93 pmol/mg protein, n = 5, versus control:, 66.19 ± 7.18 pmol/mg protein, n = 5).
In the present study, we provide evidence for the involvement of G-kinase in acute NO-deficiency-induced increases in the vasodilator potency to SNP in isolated rat pulmonary artery. This hypothesis is supported by the findings wherein we show that either L-NAME treatment or endothelium removal markedly increased the vasodilator potency of the nitrovasodilator SNP and the G-kinase activator 8-Br-cGMP. In conformity with an earlier study describing the role of sGC in the mechanism of supersensitivity,9 we also found that L-NAME significantly enhanced the increase in cGMP stimulated by SNP in endothelium-intact pulmonary artery rings treated with the phosphodiesterase inhibitor, IBMX. L-NAME treatment of endothelium-intact rings, however, had no significant effect either on the potency or the efficacy of dibutyryl-cAMP, an activator of PKA. Relaxations elicited with levcromakalim, an opener of KATP channels, were inhibited by L-NAME. These observations therefore suggest that the acute NO deficiency selectively increased the vasodilator potency of SNP through the increased sensitivity of G-kinase and sGC to stimulation by cGMP and NO, the specific ligands for the enzyme receptors, respectively. Chronic treatment (5 weeks) of rats with L-NAME (50 mg/kg in drinking water) significantly increased the vasodilator potency of SNP in rat pulmonary artery, but it had no effect on relaxant responses elicited with 8-Br-cGMP (unpublished observation).
NO deficiency after endothelium removal or L-NAME treatment was evident from a near abolition or marked decrease, respectively, of ACh-induced relaxation in rat pulmonary arteries. Under both these conditions, there was an almost identical increase in the sensitivity of relaxation produced by SNP and 8-Br-cGMP. This observation suggests that endothelium-derived NO determines the reactivity of the vascular responses to NO/cGMP. This is further confirmed by the observation that L-NAME had no significant effect on the vasodilator potency of SNP in endothelium-denuded rings. The observation that some part of relaxation (27.25 ± 3.45%, n = 10) induced by ACh was resistant to L-NAME in rat pulmonary artery is in accordance with previous reports on this vessel, wherein the role of EDHF has been emphasized.17,18
An increase in the sensitivity to SNP in the NO-deficient condition may involve different mechanisms, such as increased release of NO from the nitrovasodilator, decreased activity of PDE 5, sensitization of sGC, or increased sensitivity of PKG to cGMP. In a recent study on rat pulmonary artery, either endothelium removal or L-NA treatment was found to increase the relaxant potency of authentic NO,7 which is independent of the mechanism of NO release. It is therefore reasonable to predict that increased sensitivity of this vessel to the dilator response of SNP may not involve an increased NO release under the conditions of L-NAME treatment or endothelium removal. In the present investigation on pulmonary artery, as well as in an earlier study on rat aorta,9 L-NAME was found to potentiate SNP-stimulated increases in tissue cGMP in the presence of a phosphodiesterase inhibitor. These observations suggest that altered phosphodiesterase activity in NO deficiency may have little contribution in increasing the sensitivity of the vascular smooth muscle to the nitrovasodilators. This is further supported by an earlier report that enhanced NO-induced cGMP response from long-term treatment with L-NAME was not attributable to an alteration in the phosphodiesterase activity in rat aorta.14
As reported in many other vascular smooth muscles,19 G-kinase has been shown to be the key component in the NO-cGMP pathway in SNP-induced relaxation of rat pulmonary artery.20 The results of the present study showing increased sensitivity of pulmonary artery rings to 8-Br-cGMP after endothelium removal or L-NAME treatment suggest that G-kinase may be involved in the increased vasodilator potency to SNP in this tissue. However, there was no evidence for the involvement of the G-kinase pathway in the increased nitrovasodilator sensitivity in rat aorta.9 Their inference was based on the observation that endothelium removal, but not L-NAME treatment, increased the vasodilator potency of 8-Br-cGMP. They explain this finding in terms of endothelium to act as a diffusion barrier for the cell-permeable analogue. Therefore, the cGMP analogue was more potent in endothelium-denuded rings. But an identical increase in the relaxant potency of 8-Br-cGMP in rat pulmonary artery by either L-NAME treatment or endothelium denudation rules out the diffusion barrier mechanism in this vessel.
Although cAMP and cGMP activate their associated kinases, such as PKA and G-kinases, respectively,1,21 cross-activation of the protein kinases by the cyclic nucleotides is evident in some blood vessels.22-24 In rat pulmonary artery myocytes, cAMP was reported to increase BKCa currents through the activation of PKG, and there was no evidence for the involvement of PKA.23 Therefore, it is less likely that PKA is involved in the mechanism of supersensitivity to cGMP/8-Br-cGMP in rat pulmonary artery. Moreover, the affinity of cGMP to PKA is 50 times less in comparison with cAMP.25 Thus, a moderate but significant increase in vasodilator response to dibutyryl cAMP in L-NAME-treated pulmonary artery rings, as observed in the present study, may be attributed to cross-activation of G-kinase by the cAMP analogue.
In rat pulmonary artery rings, L-NAME significantly decreased cGMP levels, implying a decreased basal release of NO. However, the increase in cGMP stimulated by SNP was significantly enhanced by the NO synthase inhibitor. Our observation is consistent with the previous studies, which employed both acute and chronic models of NO deficiency to suggest the involvement of sGC in the nitrovasodilator supersensitivity mechanism.9,13
To prove that acute NO deficiency selectively potentiated the vasodilator responses to SNP and 8-Br-cGMP, we assessed the influence of L-NAME treatment on relaxation induced by the KATP channel opener levcromakalim, which is unrelated to the NO/cGMP pathway in rat pulmonary artery.20 We observed that L-NAME caused a small but significant decrease in the efficacy, but not the potency, of levcromakalim to relax pulmonary artery rings. The inhibitory effect of L-NAME is in agreement with a previous study demonstrating that either L-NAME or endothelium removal decreased the relaxant response of levcromakalim in rat aorta.26
In summary, we have demonstrated that endothelium removal or L-NAME treatment selectively increased the sensitivity of rat pulmonary artery to the vasodilator actions of SNP and 8-Br-cGMP. Further, L-NAME enhanced the SNP-stimulated increase in cGMP in this tissue in the presence of a phosphodiesterase inhibitor. These observations suggest that in addition to sGC, a downstream mechanism such as G-kinase is involved in the NO-deficiency-induced supersensitivity to the nitrovasodilator in rat pulmonary artery. The increase in the sensitivity of sGC/G-kinase in the acute NO-deficient condition may represent an adaptive mechanism of NO/cGMP signaling in the pulmonary vascular bed. Further studies are required to understand the molecular mechanisms that are associated with the acute regulation of sGC/G-kinase by the ambient NO/cGMP concentrations in vascular smooth-muscle cells.
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