Journal Logo

Original Article

Segmental Heterogeneity in the Mechanism of Sodium Nitroprusside-Induced Relaxation in Ovine Pulmonary Artery

Sathishkumar, K PhD; Ross, R Gracious PhD; Bawankule, Dnyaneshwar U PhD; Sardar, Kautuk K PhD; Prakash, Vellanki Ravi PhD; Mishra, Santosh K PhD

Author Information
Journal of Cardiovascular Pharmacology: June 2005 - Volume 45 - Issue 6 - p 491-498
doi: 10.1097/01.fjc.0000159043.50488.ac
  • Free

Abstract

Regional diversity is one of the key features in pulmonary circulation. For instance, hypoxia constricts the resistance pulmonary arteries but relaxes the conduit vessels.1,2 The disparate responses between vascular segments have been explained on the basis of cell diversity, which includes cell phenotype, heterogeneous distribution of K+ channel subtypes, and other cell signaling mechanisms.3 Heterogeneity in responses to vasoactive agents has also been reported in large, medium, and small pulmonary vessels.4-7 For example, nitric oxide, an important regulator of arterial tone, possessed variable potency in relaxing different segments of pulmonary vessels. NO was more potent in dilating medium-sized versus small pulmonary artery in rats.4 In sheep isolated pulmonary artery, endothelium-dependent relaxation responses to bradykinin and ionomycin decreased progressively along the pulmonary vascular tree.6 However, the mechanism of differential sensitivity of small and medium-sized vessels to NO is not understood. Further, in contrast to adult sheep, NO/NO donors were more potent in relaxing the small, as compared with the large, vessels in the newborn lambs, which has been explained on the basis of a greater NO-stimulated increase in cGMP content in small vessels.7

NO/NO donors induce vasodilation primarily through the activation of sGC, which results in an increase in cGMP levels.8-11 It has also been reported that NO activates a variety of K+ channels, such as KCa,12-14 KATP,15 and Kv16 channels in a cGMP-dependent/-independent manner to produce vasodilation. Plasma membrane Na+,K+-ATPase is another important target for NO-induced vasodilation.17 Considering the heterogeneity of signaling mechanisms along the pulmonary tree,2,7 we hypothesize that NO-mediated relaxation of small and medium-sized vessels may involve different mechanisms. To test this hypothesis, we used a prototype NO donor sodium nitroprusside to examine its mechanism of dilation of ovine medium-sized and small pulmonary arteries.

METHODS

Blood Vessel Preparation

Lungs from adult sheep were collected from the local slaughterhouse within 20-30 minutes of slaughter in cold (4-6°C) oxygenated modified Krebs-Henseleit solution of the following composition (in 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). The physiological solution contained penicillin (100 units/mL) and streptomycin (100 μg/mL).

The second- (medium) and fourth-generation (small) intralobar arteries were dissected from lungs, cleared of connective tissue, and cut into rings of about 2-3 mm length. The external diameters of second- and fourth-generation pulmonary arterial rings (henceforth called “medium” and “small” vessels) were 3-4 mm and 0.6-1.0 mm, respectively.

Tension Recording

Both medium and small pulmonary arterial rings were mounted between 2 stainless steel L-shaped hooks under resting tension of 2 g and 1 g, respectively, in 20 mL organ baths containing modified Krebs-Henseleit solution at 37°C and continuously aerated with room air. The arterial rings were equilibrated for 60 minutes before the muscle tension was recorded. The change in tension was measured by a high-sensitivity isometric force transducer (Model MLT0202/D, Powerlab, Australia) and recorded in a PC using the Chart v4.1.2 software program (Powerlab, Australia). The bath solution contained indomethacin (10 μM) to prevent the contribution of endogenous prostanoids to muscle tension.

The vessels were preconstricted with 1 μM of serotonin, and when the contraction reached equilibrium, ACh (100 μM) was added. A relaxant response to ACh confirmed the presence of a functional endothelium. The preparations were then washed with PSS to restore the resting force. The tissues were then contracted with either 0.3 or 1 μM serotonin, and when the contraction was stable, vasodilators such as SNP and SNAP were added cumulatively until maximal reversal of serotonin-induced contraction was obtained. In some experiments, 2 concentration-response curves were obtained with SNP in the same vascular ring, first in the absence and then in the presence of inhibitors. These were used as self-controls for the respective inhibitors in determining statistical significance. To study the role of sGC in mediating relaxation by SNP, some arteries were treated with ODQ (3 μM/L) as reported earlier.18 To study the role of different K+ channels in the mechanism of SNP-induced vasodilation, Kv channel blocker 4-aminopyridine13 (4-AP, 1 mM), KCa channel blocker paxilline (1 μM), and KATP channel blocker glibenclamide (1 μM) were used. To examine the role of Na+,K+-ATPase in SNP-induced relaxation, some arterial rings were treated with 1 μM ouabain.

Estimation of Na+,K+-ATPase activity

Isolation of sarcolemmal membranes from medium and small pulmonary arteries was performed as per the procedure described by Matlib and co-workers.19 Na+-K+-ATPase activity was determined by measuring the liberation of inorganic phosphate (Pi) from ATP in 1 mL of medium containing (mM) Tris HCl buffer, 50, pH 7.5; NaCl, 140; KCl, 14; MgCl2·6H2O, 5; EDTA, 0.5; ouabain, 1; and requisite volume (10 μL) of membrane homogenate in a final volume of 1 mL. This reaction mixture was preincubated for 5 minutes at 37°C. The reaction was started by the addition of 3 mM ATP solution. For total ATPase assay, ouabain was omitted from the reaction mixture, which was included for Mg2+-ATPase assay. After 1 hour of incubation at 37°C in both cases, the reaction was stopped by adding 0.1 mL of cold 5% sodium dodecyl sulfate, and color was developed with 3 mL of acidic ammonium molybdate and 0.1 mL of ANSA reagent (1-amino-2 naphthol-4-sulfonic acid, 25 mg; sodium metabisulfite-1.2 g; sodium sulfite 120 mg dissolved in 10 mL of distilled water). The inorganic phosphate in the reaction mixture was assayed according to the method of Yohatalou and co-workers.20 A standard phosphate (10 μg/mL) and blank were run simultaneously. The difference in the activity in the absence and presence of ouabain was taken as Na+,K+-ATPase activity. Protein content in the membrane fraction was determined by Lowry's method.21 Specific enzyme activity is expressed as nanomoles of Pi liberated/min/mg of protein. To determine the effect or SNP on Na+,K+-ATPase activity, tissues were pretreated with SNP (10 μM) for 30 minutes, and then the Na+,K+-ATPase activity was determined as described above. Basal activity of the enzyme was measured following incubation of the tissues for 30 minutes in Krebs-Henseleit solution.

Measurement of Ouabain-Sensitive 86Rb Uptake

86Rb uptake by the pulmonary artery was determined as per the method described by Gupta and co-workers.22 Endothelium-denuded arterial rings were equilibrated in PSS (37°C) for 2 hours while being aerated continuously with carbogen. After the equilibration, the test drugs were added for 30 minutes, followed by ouabain (0.2 mM) for 10 minutes. Then 86RbCl (2 μCi/mL) was added into the incubated vials for 10 minutes. The tissues were washed in cold (4°C) unlabeled PSS for 2 minutes to remove the radioisotope from extracellular compartments, blotted on the filter paper, and dried overnight in an oven maintained at 100°C. 86Rb content of the tissue was determined by γ counting. Ouabain-sensitive 86Rb uptake, which is known to be an index of Na+,K+-ATPase activity, was calculated by subtracting 86Rb-uptake in the presence of maximally effective concentration of ouabain (0.2 mM) from total 86Rb uptake.

Cyclic GMP (cGMP) Measurement

To assess the possibility that the cellular cGMP determines the differences in the potency and efficacy of SNP-induced relaxation, basal as well as SNP-stimulated intracellular cGMP was measured as described earlier by Tamaoki and co-workers.23 Briefly, endothelium-denuded pulmonary artery rings were weighed and equilibrated for 1.5 hours in PSS at 37°C, continuously aerated with carbogen. First 1 μM 5-HT was added to the medium for 10 minutes, and then either the vehicle or SNP (10 μM) was added; 30 minutes later the tissues were rapidly frozen in liquid nitrogen and ground in 1.07 N perchloric acid. The suspension was then sonicated and centrifuged at 10,000 × g for 1 minute, and the supernatant was collected for the assay. The amount of cGMP was assayed by radioimmunoassay with a [125I]cGMP RIA kit (Immunotech, Marseille Cedex 9, France), and cGMP levels were expressed as picomoles per milligram wet weight of tissue.

Drugs and Solutions

SNP, SNAP, serotonin, ODQ, ouabain, paxilline, 4-AP, TEA, and indomethacin were purchased from Sigma Chemicals (St Louis, MO). Solutions of SNP (10 mM), acetylcholine chloride (10 mM), TEA (1 mM), serotonin (10 mM), and 4-AP (1 mM) were prepared in double-distilled water. The pH of 4-AP was adjusted to 7.4 with HCl. Stock solutions of SNAP (10 mM), paxilline (1 mM), and ODQ (10 mM) were prepared in DMSO. Further dilutions of stock solution were made in modified Krebs-Henseleit solution.

Statistical Analysis

Relaxation responses are expressed as a percentage reversal of the serotonin precontraction. Results are expressed as mean ± SE with n equal to number of vascular rings. EC50 concentrations were determined by regression analysis and expressed as negative log molar concentration (pD2). Two-way ANOVA followed by Student paired/unpaired t test was used to determine the statistical significance, and P < 0.05 was considered statistically significant.

RESULTS

Role of Endothelium and Amplitude of Serotonin-Induced Contraction on SNP-Mediated Relaxation in Ovine Pulmonary Artery

Because endothelium was not removed routinely, we examined the influence of endothelium on the vasodilator response of SNP. Endothelial integrity was examined by demonstrating relaxation to ACh (100 μM) of serotonin (1 μM) precontracted arterial rings. The potency and efficacy of SNP in the presence and absence of endothelium was not significantly different in medium and small vessels, and the results are summarized in Table 1. Some pharmacological tools used to delineate the mechanism of SNP-induced relaxation also altered the absolute tension produced by serotonin, which was used to preconstrict the arterial rings. Therefore, we have determined the influence of the amplitude of serotonin-induced contraction on the vasodilator potency and efficacy of SNP. We found that increasing the concentration of serotonin from 0.3 μM to 1 μM consistently increased the amplitude of the contraction but did not significantly alter the potency or efficacy of relaxation induced by SNP (Table 2).

TABLE 1
TABLE 1:
Role of Endothelium on the Vasodilator Potency (pD2) and Efficacy (Emax) of SNP in Isolated Medium and Small Ovine Pulmonary Arteries
TABLE 2
TABLE 2:
Influence of Absolute Tension Generated by the Vasoconstrictor Serotonin (0.3/1.0 (M) on the Relaxant Potency and Efficacy of SNP in Medium and Small Ovine Pulmonary Arteries

Responses of SNP and SNAP in Ovine Medium and Small Pulmonary Arteries

Figure 1 illustrates the concentration-related relaxation responses of SNP (10−9-10−4 M) and SNAP (10−9-10−4 M) in ovine pulmonary arterial segments. Vascular rings were precontracted with serotonin (0.3/1 μM). The absolute tension produced by serotonin was 1.51 ± 0.06 g (n = 40) and 1.00 ± 0.04 g (n = 40) in medium and small vessels, respectively. The NO donors SNP and SNAP were more potent in dilating medium-sized vessels (SNP pD2 = 6.77 ± 0.08; Emax = 93.30 ± 0.20, n = 40; SNAP pD2 = 6.32 ± 0.12; Emax = 90.70 ± 1.60, n = 6) than the small (SNP pD2 = 5.44 ± 0.06; Emax = 77.70 ± 1.50, n = 40; SNAP pD2 = 5.27 ± 0.22; Emax = 73.59 ± 4.60,n = 6) vessels.

FIGURE 1
FIGURE 1:
A, Concentration-dependent relaxation caused by SNP (10−9-10−4 M; n = 40) and (B) SNAP (10−9-10−4 M; n = 6) in ovine isolated medium and small pulmonary arteries precontracted with serotonin (0.3/1.0 μM). The vertical bars represent standard error of mean. Asterisk shows significant (*P < 0.05) difference between medium and small vessels. Student unpaired t test was used for comparison after 2-way ANOVA.

Effect of ODQ on the Vasodilator Responses of SNP in Medium and Small Pulmonary Arteries

In a previous study, Homer and co-workers18 showed that 3 μM ODQ almost abolished the relaxant response of SNP in rat pulmonary artery and that 10 μM ODQ had no additional effect. In the present study, we therefore employed 3 μM ODQ as a selective inhibitor of sGC. Pretreatment of the tissues with ODQ (3 μM) for 30 minutes caused a small increase in the basal tone (180 ± 13 mg, n = 6 in medium and 97.6 ± 15 mg, n = 6 in small vessels) and enhancement of serotonin-induced contraction by 31% in medium-sized artery (1.63 ± 0.05 to 2.18 ± 0.16 g) and by 18% in small artery (1.83 ± 0.05 to 2.16 ± 0.16 g). ODQ partially attenuated the relaxation responses of SNP (10−9-10−4 M) with a decrease in the pD2 (4.07 ± 0.15, n = 6 versus control 6.62 ± 0.11) as well as the Emax (54.40 ± 4.10% versus control 95.30 ± 1.60, n = 6) in medium-sized vessels. In small vessels, however, ODQ caused a profound inhibition in the relaxant responses of SNP, thereby decreasing the Emax to 15.70 ± 4.10% (n = 6) from the control value of 86.20 ± 3.00% (n = 6). The results are depicted in Figure 2B.

FIGURE 2
FIGURE 2:
Effects of ODQ (3 μM), ouabain (1 μM), and ODQ (3 μM) plus ouabain (1 μM) on concentration-dependent relaxations produced by SNP (10−9-10−4 M) in ovine (A) medium (n = 6) and (B) small vessels (n = 6). Vertical bars represent standard error of mean. *P < 0.05. Student paired t test was used for comparison after 2-way ANOVA.

Effect of Ouabain on SNP-Induced Relaxation of Medium and Small Pulmonary Arteries

Figure 2 depicts the effect of ouabain on SNP (10−9-10−4 M)-induced relaxation of medium and small pulmonary vessels precontracted with serotonin (0.3 μM). Tissues were pretreated with ouabain (1 μM) for 30 minutes before SNP relaxation was elicited. Ouabain per se increased the basal tension by 182 ± 90 mg and 55 ± 37 mg in medium and small vessels, respectively. The absolute tension of serotonin-induced contractions in the presence of ouabain was increased by 72% (1.47 ± 0.39 to 2.5 ± 0.35 g) and by 39.7% (1.36 ± 0.13 to 1.90 ± 0.19 g) in medium and small vessels, respectively. Ouabain decreased the potency (pD2 = 5.13 ± 0.47 versus control pD2 = 6.62 ± 0.11, n = 6) and maximal relaxation (Emax = 67.47 ± 7.20% versus control 95.30 ± 1.60%, n = 6) in medium-sized vessels. In the smaller arteries, however, ouabain produced a stronger inhibition of the relaxation responses to SNP. The Emax was reduced to 31.50 ± 3.10% (n = 6) from the corresponding control value of 86.20 ± 3.00% (n = 6). In the medium-sized vessel, a combination of ODQ (3 μM) and ouabain (1 μM) markedly inhibited the dilator responses to SNP. Nevertheless, a residual relaxation response was still evident. On the other hand, both treatments abolished the vasodilator response of SNP in small vessels.

Effect of 4-AP on the Relaxant Response of SNP

Activation of Kv channel has been considered to be an important mechanism of dilation caused by SNP and authentic NO in rat small pulmonary arteries.13,16 We observed that pretreatment of ovine medium and small vessels with 4-AP (1 mM) for 30 minutes had variable effects. For instance, whereas 4-AP caused a significant contraction (636 ± 105 mg, n = 7) of medium-sized vessels, it failed to produce any change in the basal tension of small vessels. Representative raw tracings are shown in Figure 3A,B. Also, the serotonin contractions were increased by 20% (2.28 ± 0.31 to 2.73 ± 0.15 g) only in the medium-sized vessel with no significant change in the small vessel. Further, 4-AP (1 mM) caused a significant rightward shift (>0.5 log unit) in the concentration-response curve of SNP (10−9-10−4M) in the medium-sized vessel (n = 7). The pD2 and Emax values in the presence of 4-AP were 5.80 ± 0.06 and 83.70 ± 1.60% (n = 7), respectively, in comparison to the control (pD2 = 6.61 ± 0.16; Emax = 93.50 ± 2.70%, n = 7). On the other hand, 4-AP (1 mM) had no effect on the relaxation caused by SNP (10−9-10−4M) in the small vessels (control pD2 = 5.87 ± 0.05; Emax = 86.70 ± 1.80, n = 9 versus 4-AP pD2 = 5.74 ± 0.14; Emax = 82.80 ± 2.50%, n = 9; Fig. 3D). To examine the contribution of Kv channels to the heterogeneity in the vasodilator potency of SNP in medium and small vessels, we compared the pD2 and Emax values of SNP in the presence of 4-AP in both vessels. Interestingly, SNP was equipotent in both medium (pD2 = 5.80 ± 0.06; Emax = 83.73 ± 1.60%, n = 7) and small (pD2 = 5.74 ± 0.14, Emax = 86.70 ± 1.80%, n = 9) vessels in the presence of 4-AP.

FIGURE 3
FIGURE 3:
Effect of 4-AP (1 mM) on the basal tone of (A) medium and (B) small vessels. (C) Effect of 4-AP (1 mM) on SNP (10−9-10−4 M) induced dilation of ovine medium (n = 7) and (D) small pulmonary vessels (n = 9). Vertical bars represent standard error of mean. *P < 0.05, when compared with SNP. Student paired t test was used for comparison after 2-way ANOVA.

Figure 4A depicts the effect of 4-AP on ODQ-resistant relaxation elicited by SNP in the ovine medium-sized vessel. As shown in the figure, ODQ decreased but did not eliminate SNP-induced relaxation in medium-sized vessels. 4-AP (1 mM) further decreased the relaxant responses of SNP in the presence of the sGC inhibitor ODQ (3 μM). However, in the combined presence of ODQ (3 μM), ouabain (1 μM), and 4-AP (1 mM), the relaxation response to SNP was abolished (Fig. 4B). To specify the action of 4-AP on Kv channels, we tested the effect of 4-AP on sodium pump. 4-AP (1 mM) marginally increased the Na+,K+-ATPase activity to 71.40 ± 3.34 nM Pi/min/mg protein) from the basal value of 64.02 ± 1.68 nM Pi/min/mg protein in the medium-sized pulmonary artery (n = 4 for each group).

FIGURE 4
FIGURE 4:
A, Effect of 4-AP (1 mM) on SNP (10−9-10−4 M)-induced relaxation of the medium-sized pulmonary artery in the presence of ODQ (3 μM), n = 6. B, Effect of the combined pretreatment with ODQ (3 μM), ouabain (1 μM), and 4-AP (1 mM) on the relaxant response to SNP (10−9-10−4 M; n = 6). *P < 0.05 when compared with control; vertical bars represent SE. Student paired t test was used for comparison after 2-way ANOVA.

Effects of Paxilline and Glibenclamide on the Vasodilator Responses of SNP

Paxilline (1 μM), a highly selective KCa channel blocker,24 did not modify concentration-dependent relaxation produced by SNP (10−9-10−4 M) in medium (control pD2 = 6.82 ± 0.14, Emax = 94.20 ± 2.50%, n = 5; versus paxilline pD2 = 6.53 ± 0.20, Emax = 90.30 ± 3.50%, n = 5) and small (control pD2 = 5.14 ± 0.08, Emax = 71.60 ± 3.70%, n = 5; versus paxilline pD2 = 5.23 ± 0.20, Emax = 72.23 ± 4.50%, n = 5) pulmonary arteries. Similarly, glibenclamide (1 μM) a selective blocker of ATP-sensitive K+ channel, had negligible effect on the relaxation potency of SNP. The pD2 and Emax values of SNP, following pretreatment of the medium-sized vessel for 30 minutes with glibenclamide, were 6.68 ± 0.23 and 92.70 ± 3.30% (n = 3) in comparison to the control (pD2 = 6.75 ± 0.23, Emax = 94.13 ± 2.90%, n = 3). Similarly the vasodilator potency of SNP in the absence (pD2 = 5.48 ± 0.05, n = 3) and presence (pD2 = 5.62 ± 0.02, n = 3) of glibenclamide were comparable in small arteries.

Effect of SNP on Na+,K+-ATPase Activity and 86Rb Uptake

Figure 5A depicts the effect of SNP (10 μM) on the plasma membrane Na+,K+-ATPase activity of ovine pulmonary arterial strips. Treatment of the tissues for 30 minutes with SNP (10 μM) produced a significant (P < 0.05) increase in the sodium pump activity in both medium and small vessels to a similar extent. In addition to the determination of the sodium pump activity, we also examined the effect of SNP (10 μM) on ouabain-sensitive 86Rb uptake in the endothelium-denuded pulmonary arterial rings in the presence and absence of ODQ (3 μM). ODQ was without any effect on SNP-stimulated ouabain-sensitive 86Rb uptake in both vessels. Further, SNP-stimulated 86Rb uptake was almost identical in both vessels (Fig. 5B). To eliminate the nonspecificity of ODQ action, we also did some experiments to determine the effect of ODQ (10 μM) on both Na+,K+-ATPase activity and 86Rb uptake in ovine small pulmonary arteries. The Na+,K+-ATPase activity in basal and ODQ (10 μM)-treated vessels was 7.47 ± 0.38 (nM Pi/min/mg protein) and 8.45 ± 0.82 (nM Pi/min/mg protein), respectively. These values were not statistically significant. Similarly, ODQ (10 μM) had no effect on basal 86Rb uptake. Thus, the values of 86Rb uptake in the presence (0.26 ± 0.02 nM/mg tissue) and absence (0.21 ± 0.02 nM/mg tissue) were not statistically significant.

FIGURE 5
FIGURE 5:
A, Effect of SNP (10 μM) on Na+,K+-ATPase activity of endothelial-denuded pulmonary arterial strips (n = 6). Tissues were incubated for 30 minutes with Krebs-Henseleit solution (control) or SNP (10 μM/L). B, Effect of SNP (10 μM) on ouabain-sensitive 86Rb uptake in the presence and absence of ODQ (3 μM). For basal 86Rb uptake measurement, endothelium-denuded arterial rings were treated with DMSO, the vehicle that was used to dissolve ODQ (n = 6 for each determination). *P < 0.05, when compared with control; Vertical bars represent SE. Student unpaired t test was used for comparison after 2-way ANOVA.

Effect of SNP on cGMP

Under basal conditions, the intracellular content of cGMP of endothelium-denuded medium and small arteries was almost identical. Similarly, there was no significant difference in the increase in cGMP levels stimulated by SNP (10 μM), a concentration that produced submaximal relaxation of medium and small arterial segments (Fig. 6).

FIGURE 6
FIGURE 6:
Effect of SNP (10 μM) on intracellular cGMP in endothelium-denuded pulmonary artery rings. Tissues were incubated for 30 minutes with Krebs-Henseleit solution containing 1 μM serotonin (control) or 1 μM serotonin plus10 μM SNP. Serotonin was included in cGMP measurements because it was used to preconstrict the pulmonary vessels in tension experiments. The intracellular cGMP was determined by radioimmunoassay. Values are means ± SE. *P < 0.05 when compared with control; vertical bars represent SE, n = 6 for each determination. Student unpaired t test was used for comparison after 2-way ANOVA.

DISCUSSION

The most important observation of the present study is that the NO donor SNP was more potent in relaxing medium as compared with small ovine isolated pulmonary arteries through different mechanisms. The mechanisms involved in SNP-mediated relaxation of small arteries are (1) activation of a predominant sGC/cGMP pathway and (2) a secondary cGMP-independent stimulation of plasma membrane Na+,K+-ATPase. In the medium-sized vessel, however, an additional mechanism of cGMP-independent activation of Kv channel is evident, which may explain the basis of the greater reactivity of this segment to SNP. Interestingly, SNP was equipotent in dilating these 2 segments when the Kv channels were blocked by 4-AP. The pharmacological basis of the segmental heterogeneity is discussed below.

We showed that the endothelium and the amplitude of serotonin contraction do not affect the potency and efficacy of SNP-induced relaxation in ovine pulmonary arteries. Zhao and co-workers13 made similar observations in rat pulmonary artery.

It was observed that NO donors SNP and SNAP were more potent to relax medium as compared with small pulmonary arteries. In newborn lambs, however, NO-mediated relaxations were more potent in isolated small than large pulmonary arteries. Such heterogeneity in NO responses was attributed to a difference in the sGC activity of different vascular segments.7 To test this hypothesis and to explain the basis of differences in the responses of medium and small vessels to the NO donors, we measured the basal as well as SNP-stimulated intracellular cGMP content in these 2 vessels. The lack of difference in cGMP levels between medium and small vessels indicates that the intracellular cGMP is not the determinant of heterogeneity of SNP-mediated relaxation in the ovine pulmonary artery. Nevertheless, ODQ produced a greater inhibition of SNP response in small than medium-sized vessels. This phenomenon can be explained on the basis of differential potency of SNP to dilate these 2 segments. For example, SNP was about 10-fold more potent in relaxing medium-sized vessels in comparison to small arteries. We used ODQ (3 μM) to block sGC because it has recently been suggested that high concentrations of ODQ (>10 μM) may inhibit the enzymatic process responsible for the generation of NO from SNP.25 Further, according to a previous report, ODQ (3 μM) abolished relaxant responses of NO donors in rat pulmonary artery.18 The greater potency of SNP in medium versus small artery is not related to a differential activation of sGC/cGMP pathway in these vessels.

We then sought to examine the mechanisms other than cGMP contributing to the dilator responses of SNP in medium and small vessels. An increase in Na+,K+-ATPase activity may cause vasodilation by increasing Na+-Ca2+ exchange and a reduction in Ca2+ influx through voltage-dependent Ca2+ channels as a result of membrane hyperpolarization.26 In this study, we observed that inhibition of Na+,K+-ATPase by ouabain markedly inhibited dilations induced by SNP in ovine medium and small pulmonary arteries. It is, therefore, believed that SNP-induced relaxation is partly related to the activation of sarcolemmal Na+,K+-ATPase in these vessels. Our results with SNP are consistent with the observations made earlier on dog pulmonary artery17 but discordant with piglet pulmonary artery, wherein SNP did not activate Na+,K+-ATPase.27 In several vascular smooth muscles, it has been observed that NO/NO donors activate Na+,K+-ATPase through either cGMP-dependent17 or -independent mechanisms.22 We observed that the relaxant responses of SNP, resistant to ODQ, were further inhibited by ouabain in both arterial segments. It is quite possible that cGMP-independent activation of Na+,K+-ATPase occurs in ovine pulmonary vessels. The pharmacological observation of a cGMP-independent mechanism of sodium pump stimulation by SNP is evident from the observation that SNP-stimulated ouabain-sensitive 86Rb uptake in medium and small pulmonary arteries was not affected by ODQ. The contribution of sodium pump to the variable potency of SNP in relaxing medium and small arteries is ruled out because there was no difference either in plasma membrane Na+,K+-ATPase activity or ouabain-sensitive 86Rb uptake stimulated by SNP in these arteries. The use of cell-permeable analogues of cGMP is one of the pharmacological approaches in delineating the role of cGMP in cell signaling. However, 8-bromo-cGMP is a poor relaxant in ovine pulmonary artery. Further, this compound had no effect on ouabain-sensitive 86Rb uptake in this vessel (unpublished observation).

K+ channels control the resting membrane potential, which in turn is an important regulator of vascular tone. Previous studies have shown that NO/NO donors can activate vascular K+ channels12-16 in cGMP-dependent or -independent manner. Yuan and co-workers16 found that SNP activated Kv channels directly in rat pulmonary artery smooth muscle cells derived from small vessels. Surprisingly, we observed that 4-AP, a relatively selective Kv channel blocker, did not inhibit SNP-induced relaxations in ovine small pulmonary artery. On the other hand, SNP-mediated relaxation in medium artery was significantly inhibited by 4-AP. Interestingly, following inhibition of Kv channels by 4-AP in ovine medium-sized vessel, SNP was found to be equipotent in dilating both pulmonary artery segments. It is, therefore, believed that Kv channels may have a significant role in determining the segmental heterogeneity in the relaxant response of SNP in ovine pulmonary arteries. Whether SNP activates Kv channels directly or through a cGMP-dependent mechanism remains to be determined. However, our observation that a component of SNP relaxation resistant to ODQ was further inhibited by 4-AP indicates that NO released from SNP activates the Kv channels independent of cGMP. Our observation in sheep pulmonary artery is consistent with previous reports wherein NO was shown to activate Kv channels independent of cGMP in rat small pulmonary13 and mesenteric arteries.28 In the absence of patch-clamp studies, we can only suggest that the inhibitory effect of 4-AP on SNP-mediated relaxation in medium-sized vessel is indicative of the activation of the Kv channel by the NO donor. Molecular biology studies provide evidence for the presence of Kv2.1 channels in sheep pulmonary artery.29 However, there are no reports about their differential distribution in various segments of ovine pulmonary artery. In rat pulmonary vessels, however, Kv2.1 transcript levels were higher in conduit, compared with resistance artery.30 The finding that 4-AP caused a significant contraction of the medium-sized artery without having any effect on the basal tension of small ovine pulmonary artery indicates that the Kv channels in the medium-sized pulmonary artery are open in the resting state and may therefore regulate the resting membrane potential as well as the tone of this vascular segment. This observation is supported by the findings of an earlier study wherein 4-AP 1 mM was shown to increase [Ca2+]i in the adult pulmonary artery myocytes, possibly through the inhibition of Kv2.1 channels.29

KCa channels in vascular smooth muscles have been considered to be an important target for the activation by NO/NO donors.12-14,16 Indeed, regional variation in KCa channel distribution in pulmonary circulation has been considered to be the basis for variable potency of NO in causing dilation of conduit and resistance vessels. Archer and co-workers have shown that NO was more potent in relaxing KCa channel-enriched rat conduit versus resistance pulmonary artery.4 In the present study, it was found that paxilline, a selective blocker of KCa channels, had no effect on SNP-mediated relaxation in ovine pulmonary arteries. Therefore, it is suggested that KCa channels do not mediate SNP-induced relaxation of ovine medium and small pulmonary vessels. Reeve and co-workers showed that K+ channel expression is developmentally regulated in sheep pulmonary artery, with KCa channel predominance in the fetal and Kv channel in the adult.31 Further, sheep pulmonary artery KCa channel expression has been shown to decrease with maturation.32 It is, therefore, suggested that KCa channel has little contribution to SNP-mediated relaxation in adult sheep pulmonary artery. Similarly, KATP channels appear to have no role in SNP-induced relaxation in ovine medium and small pulmonary arteries, which is evident from the finding that glibenclamide was without effect on the relaxation caused by the NO donor in these vessels. This observation is in agreement with the observation made in the rat pulmonary artery.13

In conclusion, this study provides evidence for the involvement of different mechanisms in SNP-induced relaxation of ovine medium and small pulmonary arteries. In small vessels, SNP mediates relaxation through the activation of sGC and cGMP-independent sodium pump. In medium-sized vessels, however, Kv channel activation by the NO donor appears to be an additional mechanism in the relaxant response. Further, it is suggested that Kv channels are responsible for greater potency of SNP in dilating the medium-sized vessels. Such segmental heterogeneity may have an important bearing on the influence of NO in determining the vascular tone in ovine conduit and resistance vessels.

REFERENCES

1. McCulloch KM, Kempsill FEJ, Buchanan KJ, et al. Regional distribution of potassium currents in the rabbit pulmonary arterial circulation. Exp Physiol. 2000;85:487-496.
2. Coppock EA, Martens JR, Tamukun MM. Molecular basis of hypoxia-induced pulmonary vasoconstriction: role of voltage-gated K+ channels. Am J Physiol Lung Cell Mol Physiol. 2001;28:L1-L12.
3. Archer SL. Diversity of phenotype and function of vascular smooth muscle cells. J Lab Clin Med. 1996;127:525-529.
4. Archer SL, Huang JMC, Reeve HL. The differential distribution of electrophysiological distinct myocytes in conduit and resistance arteries determines their response to nitric oxide and hypoxia. Circ Res. 1996;78:431-442.
5. Ibe BO, Hillyard RM, Raj JU. Heterogeneity in prostacyclin and thromboxane synthesis in ovine pulmonary vascular tree: Effect of age and oxygen tension. Exp Lung Res. 1997;22:351-374.
6. Kemp BK, Smolich JJ, Cocks TM. Evidence for specific regional patterns of responses to different vasoconstrictors and vasodilators in sheep isolated pulmonary arteries and veins. Br J Pharmacol. 1997;121:441-450.
7. Gao Y, Tolsa JF, Raj JU. Heterogeneity endothelium-derived nitric oxide-mediated relaxation of different sized pulmonary arteries of newborn lambs. Pediatr Res. 1996;44:723-729.
8. Ignarro LJ, Burke TM, Wood KS, et al. Association between cyclic GMP accumulation and acetylcholine-elicited relaxation of bovine intrapulmonary artery. J Pharmacol Exp Ther. 1984;228:682-690.
9. Lincoln TM, Komalavilas P, Cornwell TL. Pleiotropic regulation of vascular smooth muscle tone by cyclic GMP-dependent protein kinase. Hypertension. 1994;23:1141-1147.
10. Barnes PJ, Liu SF. Regulation of pulmonary vascular tone. Pharmacol Res. 1995;47:87-131.
11. Tzao C, Nickerson PA, Russell JA, et al. Pulmonary hypertension alters soluble guanylate cyclase activity and expression in pulmonary arteries isolated from fetal lambs. Pediatr Pulmonol. 2001;31:97-105.
12. Archer SL, Huang JM, Daniel VH, et al. Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc Natl Acad Sci USA. 1994;91:7583-7587.
13. Zhao YJ, Wang J, Rubin LJ, et al. Inhibition of Kv and KCa channels antagonizes NO-induced relaxation in pulmonary artery. Am J Physiol Heart Circ Physiol. 1997;272:H904-H912.
14. Homer KL, Wanstall JC. Cyclic GMP-independent relaxation of rat pulmonary artery by spermine NONOate, a diazeniumdiolate nitric oxide donor. Br J Pharmacol. 2000;131:673-682.
15. Gambone LM, Murray PA. Sodium nitroprusside-induced pulmonary vasodilation is selectively attenuated by ATP-senstive K+-channel inhibition in conscious dogs. (Abstract) FASEB J. 1994;8:A25.
16. Yuan XJ, Tod ML, Rubin LJ, et al. NO hyperpolarizes pulmonary artery smooth muscle cells and decreases the intracellular Ca2+ concentration by activating voltage-gated K+ channels. Proc Natl Acad Sci USA. 1996;93:10489-10494.
17. Tamaoki J, Tagaya E, Nishimura K, et al. Role of Na+-K+ ATPase in cyclic GMP-mediated relaxation of canine pulmonary artery smooth muscle cells. Br J Pharmacol. 1997;122:112-116.
18. Homer KL, Fiore SA, Wanstall JC. Inhibition by 1H-[1,2,4]oxadiazolol[4,3-α]quinoxalin-1-one (ODQ) of response to nitric oxide donors in rat pulmonary artery: influence of the mechanism of nitric oxide generation. J Pharm Pharmacol. 1999;51:135-139.
19. Matlib MA, Crankshaw J, Garfield RE, et al. Characterization of membrane fractions and isolation of purified plasma membranes from rat myometrium. J Biol Chem. 1979;254:1834-1840.
20. Yohatalou T. Removal of protein inferference in Fiske-Subba Row method by sodium dodecyl sulphate. Anal Biochem. 1975;63:410-414.
21. Lowry OH, Rosebrough NJ, Farr AB, et al. Protein measurement with folin phenol reagent. J Biol Chem. 1951;193:265-275.
22. Gupta S, McArthur C, Grady C, et al. Role of endothelium-derived nitric oxide in stimulation of Na+-K+-ATPase activity by endothelium in rabbit aorta. Am J Physiol Heart Circ Physiol. 1994;266:H577-H582.
23. Tamaoki J, Togaya E, Yamawaki I, et al. Hypoxia impaired nitrovasodilator-induced pulmonary vasodilation: role of Na-K-ATPase activity. Am J Physiol. 1996;271:L172-L177.
24. Smirnov SV, Beck R, Tammaro P, et al. Electrophysiologically distinct smooth muscle cell subtypes in rat conduit and resistance pulmonary arteries. J Physiol. 2002;538:867-878.
25. Feelish M, Kotsonis P, Siebe J, et al. The soluble guanylyl cyclase inhibitor 1H- [1,2,4]oxadiazolo-[4,3-a]quinoxaline 1-one is a non selective haem protein inhibitor of nitric oxide synthase and other cytochorome P 450 enzymes involved in nitric oxide donor bioactivation. Mol Pharmacol. 1999;56:243-253.
26. Fleming WW. The electrogenic Na+-K+-pump in smooth muscle: physiologic and pharmacologic significance. Annu Rev Pharmacol Toxicol. 1980;20:129-149.
27. Cogolludo AL, Vizcaino FP, Zaragoza-Arnaez F, et al. Mechanisms involved in SNP-induced relaxation and [Ca2+]i reduction in piglet pulmonary and systemic arteries. Br J Pharmacol. 2001;132:959-967.
28. Irvine JC, Favaloro JL, Kemp-Harper BK. NO activates soluble guanylate cyclase and Kv channels to vasodilate resistance arteries. Hypertension. 2003;41:1301-1307.
29. Cornfield DN, Saqueton CB, Porter VA, et al. Voltage-gated K+-channel activity in ovine pulmonary vasculature is developmentally regulated. Am J Physiol Lung Cell Mol Physiol. 2000;278:L1297-L1304.
30. Patel AJ, Lazdunski M, Honore E. Kv2.1/Kv9.3, a novel ATP-dependent delayed-rectifier K+ channel in oxygen-sensitive pulmonary artery myocytes. EMBO J. 1997;22:6615-6625.
31. Reeve HL, Weir EK, Archer SL, et al. A maturational shift in pulmonary K+ channels, from Ca2+ sensitive to voltage dependent. Am J Physiol Lung Cell Mol Physiol. 1998;275:L1019-L1025.
32. Rhodes MT, Porter VA, Saqueton CB, et al. Pulmonary vascular response to normoxia and KCa channel activity is developmentally regulated. Am J Physiol Lung Cell Mol Physiol. 2001;280:L1250-L1257.
Keywords:

pulmonary artery; sodium nitroprusside; vasodilation; voltage-gated potassium channel; Na+; K+-ATPase; cGMP

© 2005 Lippincott Williams & Wilkins, Inc.