Inhibitory effects of L-NG-nitro-arginine on the synthesis of EDRF and the cerebroarterial response to vasodilator nerve stimulation

Treatment with L-NG-nitro-arginine (L-NA) inhibited the brady-kinin-induced relaxation, mediated via EDRF, in dog coronary artery strips partially contracted with prostaglandin F2 alpha; the inhibition was prevented by L-, but not D-, arginine. Relaxation caused by nitroglycerin was not altered by L-NA. The release of EDRF, as assayed using dog coronary artery strips without endothelium, from perfused femoral artery segments with endothelium in response to acetylcholine and substance P was significantly reduced by treatment of the femoral artery with L-NA. The inhibitory effect was reversed by L-arginine. Relaxant responses of dog cerebral artery strips with and without endothelium to electrical stimulation of non-adrenergic, non-cholinergic nerves were suppressed by L-NA, whereas relaxation of coronary arteries with and without endothelium by the stimulation of adrenergic nerves was not influenced. The L-NA-induced inhibition was reversed by L-arginine. It is speculated that L-NA inhibits the synthesis of EDRF, as does L-NG-monomethyl arginine, and NO-like substance(s) produced plays an important role in transferring information from vasodilator nerves to smooth muscle in cerebral arteries.     

Prostacyclin increases cAMP in coronary arteries.

This study was designed to determine whether prostacyclin (PGI2)-induced relaxation in circular strips of coronary arteries might be mediated by cAMP. Partially depolarized circular strips of bovine coronary arteries were used and PGI2-induced changes in length were compared with tissue concentrations of cAMP and cGMP, measured by RIA. It was found that PGI2 produced significant and concentration dependent increases in cAMP levels which were closely associated with the relaxant effects produced by the same concentrations (0.3 - 26.7 muM). Cyclic GMP was not changed by these concentrations. The relaxant effects of PGI2 were not antagonized by propranolol. There was a significant linear correlation between log increases in cAMP and percent relaxation produced by PGI2 which was almost identical with similar correlations obtained with either isoprenaline or adenosine, indicating that the relaxant effects of PGI2 are in analogy to those of isoprenaline and adenosine likely to be mediated by cAMP.     

The relaxant effect of nociceptin on porcine coronary arterial ring segments

Nociceptin (NC), alias orphanin FQ, has been identified as the endogenous ligand of the opioid receptor-like 1 receptor (ORL1). The purpose of this study was to assess the effect of nociceptin on porcine coronary arteries and to investigate the mechanism of its action, if any. Rings of coronary arteries from porcine hearts were suspended in baths containing Krebs solution, and isometric tension was measured. The response to nociceptin (10(-1)2-10(-5) mol/L) was investigated in porcine coronary arterial rings and also in such rings contracted with prostaglandin F2alpha (PGF2alpha). The effects of endothelium, nitroxide (NO), methylene blue, cyclic GMP (cGMP), naloxone, [Nphe1]NC(1-13)NH2, and propranolol on nociceptin-induced relaxation were also assessed. Our study showed nociceptin relaxed the porcine coronary arterial rings and inhibited the vasocontractivity to PGF2alpha. The relaxing response of nociceptin in coronary arteries was significantly reduced by removal of endothelium and by the presence of L-NNA, cGMP, and [Nphe1]NC(1-13)NH2, the selective nociceptin receptor antagonist, but not by naloxone, the nonselestive opioid receptor blocker or propranolol, which blocks the adrenergic beta-receptor. Our results suggest that nociceptin induces relaxation of isolated coronary artery through NO, cGMP, and ORL1.     

Glutathione attenuates coronary constriction to acetylcholine in patients with coronary spastic angina

This study examined the effect of reduced glutathione (GSH), an important antioxidant that restores intracellular redox imbalance and prevents inactivation of endothelial-derived nitric oxide, on the abnormal vasomotor reactivity in spastic coronary arteries. The responses of epicardial diameter of the left coronary arteries to intracoronary infusion of acetylcholine (ACh; 50 microg/min) were measured by quantitative coronary angiography before and during combined intracoronary infusion of GSH (50 mg/min for 6 min) or saline as a placebo in 24 patients with coronary spastic angina and in 28 control patients. All of the spastic coronary arteries showed constrictor response to ACh, whereas the control coronary arteries as a whole showed only minimal diameter changes to ACh. GSH infusion suppressed constrictor response of epicardial diameter to ACh in patients with coronary spastic angina, whereas it had no significant effect in control subjects. Saline infusion did not have any effects. The results indicate that GSH attenuated the constrictor response to ACh in epicardial coronary arteries of patients with coronary spastic angina. GSH may have an important role in the regulation of coronary vasomotor function in patients with coronary spastic angina.     

Differences in nitric oxide production in porcine resistance arteries and epicardial conduit coronary arteries

The purpose of this study was to test the hypothesis that endothelial cells from resistance arteries and epicardial conduit coronary arteries differ in their expression of nitric oxide synthase (NOS) and calcium metabolism, and that these differences contribute to the mechanism underlying disparate physiological vasodilator responses observed between the two populations of vessels. The functional vasodilator responses of isolated resistance arteries and epicardial conduit coronary arteries were compared in vitro using both the receptor-independent agonist A23187 ionophore to increase intracellular calcium and the receptor-dependent agonist bradykinin. Constitutive NOS (cNOS) activity in monocultures of endothelial cells derived from resistance arteries and conduit arteries was assayed using a fibroblast-reporter cell method. Intracellular calcium concentration was assessed using fura-2 microfluorometry. Nitric oxide production was determined using a chemiluminescence technique, while cNOS protein was quantitated by Western blot analysis. A23187 was a less potent vasodilator of resistance arteries studied in vitro, compared to epicardial conduit arteries (EC₅₀ = 1.6 μM, resistance artery vs. EC₅₀ = 0.03 μM, conduit artery); however, bradykinin was more potent in resistance arteries (EC₅₀ = 0.3 nM, resistance artery vs. EC₅₀ = 2 nM, conduit artery). In pure monocultures of endothelium, nitric oxide production measured by chemiluminescence both basally and in response to A23187 was significantly less in resistance arteries (6.1 ± 0.5, basal vs. 10.80 ± 0.55, stimulated nmol/μg protein), compared to conduit arteries (7.7 ± 0.5, basal vs. 17.00 ± 1.52, stimulated nmol/μg protein; P < 0.05 resistance artery endothelium vs. conduit artery endothelium). cNOS enzyme activity assessed by cGMP production in reporter cell fibroblasts was also lower in resistance arteries compared to conduit arteries (0.17 ± 0.03 vs. 0.33 ± 0.05 fmol cGMP/μg protein, respectively; P < 0.05 resistance artery endothelium vs. conduit artery endothelium). Conduit arteries expressed 2.1 × more cNOS protein than resistance arteries, as assessed by Western blotting of cellular homogenates. No significant differences were found with microfluorimetry in either basal or ionophore-stimulated intracellular calcium concentrations. The results signified that porcine resistance arteries expressed less NOS and produced less nitric oxide than epicardial conduit arteries both basally and in response to an increase in intracellular calcium. This difference was reflected functionally as a decreased vasodilatory response to increased intracellular calcium in resistance arteries that could not be explained on the basis of differences in the metabolism of intracellular calcium. In contrast, the functional vasodilator response of intact vessels to a receptor-mediated agonist was enhanced in resistance arteries compared to conduit arteries, suggesting an important role of signal transduction mechanisms in specific physiological responses. Thus, the ability of the endothelium to regulate on a regional basis the expression of NOS and integrate receptor-mediated responses with these differences may provide a mechanism for diverse vasomotor responses in different populations of vessels. © 1996 Wiley-Liss, Inc.     

Role of smooth muscle cells on endothelial cell cytosolic free calcium in porcine coronary arteries

We tested the hypothesis that the cytosolic free calcium concentration in endothelial cells is under the influence of the smooth muscle cells in the coronary circulation. In the left descending branch of porcine coronary arteries, cytosolic free calcium concentration ([Ca(2+)](i)) was estimated by determining the fluorescence ratio of two calcium probes, fluo 4 and fura red, in smooth muscle and endothelial cells using confocal microscopy. Acetylcholine and potassium, which act directly on smooth muscle cells to increase [Ca(2+)](i), were found to indirectly elevate [Ca(2+)](i) in endothelial cells; in primary cultures of endothelial cells, neither stimulus affected [Ca(2+)](i), yet substance P increased the fluorescence ratio twofold. In response to acetylcholine and potassium, isometric tension developed by arterial strips with intact endothelium was attenuated by up to 22% (P < 0.05) compared with strips without endothelium. These findings suggest that stimuli that increase smooth muscle [Ca(2+)](i) can indirectly influence endothelial cell function in porcine coronary arteries. Such a pathway for negative feedback can moderate vasoconstriction and diminish the potential for vasospasm in the coronary circulation.     

Effects of atrial natriuretic factor, nitroprusside and acetylcholine on cGMP immunostaining in the isolated perfused rat kidney.

In the present study the localization of the cGMP production in response to the vasodilators acetylcholine (ACh) and sodium nitroprusside (SNP) and to atrial natriuretic factor (ANF) was studied in the isolated perfused rat kidney using cGMP immunocytochemistry. After ACh (0.3 microM) infusion increased cGMP immunoreactivity was found in kidney interlobar and segmental arteries and in glomeruli. SNP (1 microM) and ANF (0.01 microM) elevated cGMP staining in the same elements of the kidney as ACh. In the glomeruli ACh and SNP stimulated cGMP production in mesangial cells whereas ANF stimulated cGMP production in mesangial cells whereas ANF stimulated cGMP production in epithelial cells (podocytes). However, SNP at higher doses (10 microM) stimulated cGMP production not only in glomeruli, but also in interstitial cells throughout the cortex. In addition SNP and ANF increased cGMP production in the medulla.     

EDHF contributes to strain-related differences in pulmonary arterial relaxation in rats

Pulmonary arteries from the Madison (M) strain relax more in response to acetylcholine (ACh) than those from the Hilltop (H) strain of Sprague-Dawley rats. We hypothesized that differences in endothelial nitric oxide (NO) synthase (eNOS) expression and function, metabolism of ACh by cholinesterases, release of prostacyclin, or endothelium-derived hyperpolarizing factor(s) (EDHF) from the endothelium would explain the differences in the relaxation response to ACh in isolated pulmonary arteries. eNOS mRNA and protein levels as well as the NO-dependent relaxation responses to thapsigargin in phenylephrine (10(-6) M)-precontracted pulmonary arteries from the M and H strains were identical. The greater relaxation response to ACh in M compared with H rats was also observed with carbachol, a cholinesterase-resistant analog of ACh, a response that was not modified by pretreatment with meclofenamate (10(-5) M). N(omega)-nitro-L-arginine (10(-4) M) completely abolished carbachol-induced relaxation in H rat pulmonary arteries but not in M rat pulmonary arteries. Precontraction with KCl (20 mM) blunted the relaxation response to carbachol in M rat pulmonary arteries and eliminated differences between the M and H rat pulmonary arteries. NO-independent relaxation present in the M rat pulmonary arteries was significantly reduced by 17-octadecynoic acid (2 microM) and was completely abolished by charybdotoxin plus apamin (100 nM each). These findings suggest that EDHF, but not NO, contributes to the strain-related differences in pulmonary artery reactivity. Also, EDHF may be a metabolite of cytochrome P-450 that activates Ca(2+)-dependent K(+) channels.     

Role of ceramide in TNF-alpha-induced impairment of endothelium-dependent vasorelaxation in coronary arteries

The present study tested the hypothesis that ceramide, a sphingomylinase metabolite, serves as an second messenger for tumor necrosis factor-alpha (TNF-alpha) to stimulate superoxide production, thereby decreasing endothelium-dependent vasorelaxation in coronary arteries. In isolated bovine small coronary arteries, TNF-alpha (1 ng/ml) markedly attenuated vasodilator responses to bradykinin and A-23187. In the presence of N(G)-nitro-L-arginine methyl ester, TNF-alpha produced no further inhibition on the vasorelaxation induced by these vasodilators. With the use of 4,5-diaminofluorescein diacetate fluorescence imaging analysis, bradykinin was found to increase nitric oxide (NO) concentrations in the endothelium of isolated bovine small coronary arteries, which was inhibited by TNF-alpha. Pretreatment of the arteries with desipramine (10 microM), an inhibitor of acidic sphingomyelinase, tiron (1 mM), a superoxide scavenger, and polyethylene glycol-superoxide dismutase (100 U/ml) largely restored the inhibitory effect of TNF-alpha on bradykinin- and A-23187-induced vasorelaxation. In addition, TNF-alpha activated acidic sphingomyelinase and increased ceramide levels in coronary endothelial cells. We conclude that TNF-alpha inhibits NO-mediated endothelium-dependent vasorelaxation in small coronary arteries via sphingomyelinase activation and consequent superoxide production in endothelial cells.     

The role of lysolecithin in the relaxation of vascular smooth muscle

The effect of lysolecithin (lysophosphatidylcholine) on the relaxation of rabbit aortic strip closely resembled that produced by acetylcholine (ACh) which releases the endothelium-derived relaxing factor (EDRF). Relaxation induced by lysolecithin depended on the presence of endothelium and was inhibited by hemoglobin and methylene blue. It appeared to be mediated by the second messenger, c-GMP. Lysolecithin induced relaxation was slower but more persistent than that resulting from the endothelium-derived relaxing factor (EDRF) produced by acetylcholine (ACh). Like lysolecithin, Triton X-100, a non-ionic detergent, also preferentially relaxed aortic strips with intact endothelium. The results demonstrate the importance of phospholipids derived from cell membranes in vascular smooth muscle relaxation. Endothelium-derived relaxing factors appear as a group of heterogeneous substances.     

Influence of pyridoxal-5'-phosphate on responses of isolated coronary arteries to noradrenaline, in the presence of pyrogallol

The effect of PLP on the noradrenaline-induced relaxation of coronary arteries was studied in vitro, after known inhibitor of COMT, Pyrogallol. Relaxation of response to noradrenaline were increased by PLP. Pyrogallol potentiated responses of coronary strips to noradrenaline and also reduced or abolished the enhancing effects of PLP. It is concluded that PLP enhances the response of coronary smooth muscle to noradrenaline by inhibiting a enzymatic pathway for the inactivation of catecolamines.     

Prenatal lipopolysaccharide exposure causes mesenteric vascular dysfunction through the nitric oxide and cyclic guanosine monophosphate pathway in offspring

Cardiovascular diseases, such as hypertension, could be programmed in fetal life. Prenatal lipopolysaccharide (LPS) exposure in utero results in increased blood pressure in offspring, but the vascular mechanisms involved are unclear. Pregnant Sprague–Dawley rats were intraperitoneally injected with LPS (0.79 mg/kg) or saline (0.5 ml) on gestation days 8, 10, and 12. The offspring of LPS-treated dams had higher blood pressure and decreased acetylcholine (ACh)-induced relaxation and increased phenylephrine (PE)-induced contraction in endothelium-intact mesenteric arteries. Endothelium removal significantly enhanced the PE-induced contraction in offspring of control but not LPS-treated dams. The arteries pretreated with l-NAME to inhibit nitric oxide synthase (eNOS) in the endothelium or ODQ to inhibit cGMP production in the vascular smooth muscle had attenuated ACh-induced relaxation but augmented PE-induced contraction to a larger extent in arteries from offspring of control than those from LPS-treated dams. In addition, the endothelium-independent relaxation caused by sodium nitroprusside was also decreased in arteries from offspring of LPS-treated dams. The functional results were accompanied by a reduction in the expressions of eNOS and soluble guanylate cyclase (sGC) and production of NO and cGMP in arteries from offspring of LPS-treated dams. Furthermore, LPS-treated dam’s offspring arteries had increased oxidative stress and decreased antioxidant capacity. Three-week treatment with TEMPOL, a reactive oxygen species (ROS) scavenger, normalized the alterations in the levels of ROS, eNOS, and sGC, as well as in the production of NO and cGMP and vascular function in the arteries of the offspring of LPS-treated dams. In conclusion, prenatal LPS exposure programs vascular dysfunction of mesenteric arteries through increased oxidative stress and impaired NO–cGMP signaling pathway.     

Statins as coronary vasodilators in isolated bovine coronary arteries--involvement of PGI2 and NO

Here we studied direct vasodilation induced by statins in isolated bovine coronary arteries. In rings of coronary bovine arteries preconstricted with prostaglandin F(2 alpha) (3 x 10(-8) - 10(-5)), lovastatin, simvastatin, atorvastatin and cerivastatin (3-30 microM) but not pravastatin induced concentration-dependent vasodilation. Removal of endothelium diminished response to simvastatin, cerivastatin and atorvastatin (30 microM) (67.4+/-4.56 vs. 22.7+/-8.14%, 96.9+/-2.27% vs. 54.5+/-6.86%, 67.4+/-4.01% vs. 34.6+/-5.66%, respectively). In presence of L-NAME (300 microM) or indomethacin (5 microM) responses to simvastatin, atorvastatin and cerivastatin, were also partially diminished. In contrast, lovastatin-induced vasorelaxation was not significantly affected by removal of endothelium (35.6+/-4.19% vs. 28.8+/-5.24%) or by pretreatment with L-NAME or indomethacin. In summary, with the exception of pravastatin, statins act as coronary vasodilators. Simvastatin, cerivastatin and atorvastatin but not lovastatin induced vasodilation displayed endothelium dependent- and endothelium-independent components. The endothelium-dependent effect of statins was mediated by NO and PGI(2), while the mechanism of smooth muscle cells-dependent component remains to be determined.     

Extracellular calcium-dependent and -independent effects of adenosine in bovine coronary artery

Adenosine relaxes the coronary arteries of various species through A2 receptors. The aim of the present investigation was to evaluate the relaxing effects of adenosine in relation to the role of calcium in bovine coronary arteries by studying the vasodilatory effect of adenosine in normal and calcium-free medium and on calcium-45 efflux into calcium-free medium. Acetylcholine (ACh) and norepinephrine (NE) were used to induce tone in coronary artery rings. Adenosine, 5'-(N-ethylcarboxamido)adenosine (NECA), and N6-(L-phenylisopropyl)adenosine (L-PIA) produced concentration-dependent relaxations of the coronary artery rings. Both in normal and calcium-free medium, the order of potency for adenosine analogs (NECA greater than L-PIA greater than adenosine) was similar and 8-phenyltheophylline antagonized the relaxation responses to adenosine and its analogs. Removal of extracellular calcium shifted the concentration-response curves to the right in a parallel fashion, slowed the rate of relaxation, and in NE contracted rings reduced the maximum responses for adenosine and its analogs. In calcium-free medium, adenosine was without an effect on calcium-45 efflux in the presence of ACh. However, adenosine inhibited the stimulated calcium-45 efflux induced by NE. The data suggest that the vasodilatory action of adenosine in bovine coronary smooth muscle has both extracellular calcium-dependent and -independent components.     

Effect of length changes on sensitivity of helical artery strips to adenosine

The relationship between length and the responses of helical strips of rabbit femoral and coronary arteries to vasoactive agents was investigated by conducting concentration--response determinations at the length for maximum active force (Lmax) and a shorter length. The mean effective dose (ED50) values for norepinephrine (NE) were smaller when femoral artery strips were set at Lmax, in comparison to the values at the shorter length. Maximal relaxation of femoral artery strips by adenosine was greater when the strips were set at Lmax. However, adenosine ED50 values were smaller at Lmax only in groups of strips in which responses at Lmax were obtained prior to those at the shorter length. Experiments with coronary artery strips did not demonstrate consistent relationships between strip length and ED50 values for acetylcholine (ACh) or adenosine. The results of experiments with artery strips from normotensive and those from one-kidney, one-clip hypertensive rabbits were similar. Thus, the femoral artery data indicate that helical artery strip preparations may exhibit length-dependent sensitivity to a vasodilator agent as well as vasoconstrictor agents under certain experimental conditions. However, the coronary artery data suggest that length may not affect sensitivity of isolated artery preparations from all vascular beds in the same manner.     

NF-kappaB activates fibronectin gene expression in rat hepatocytes

Resveratrol (RSVL), an edible polyphenolic stilbene, claims a myriad of cardiovascular benefits. However, the molecular underpinnings of such actions are poorly understood. Currently, in sheep coronary arteries (SCA), RSVL markedly (threefold) enhanced cGMP formation (t(1/2): 6.5 min; EC(50): 3 microM). This response was not abrogated by the phosphodiesterase inhibitor (IBMX, 0.5 mM), but was partly sensitive (20-30%) to either removal of the endothelium, treatment with the nitric oxide synthase-inhibitor (L-NMMA, 10 microM), or with the soluble GC (sGC)-inhibitor (ODQ, 10 microM). In membrane preparations from denuded SCA, either RSVL or the pGC agonist atrial natriuretic peptide (ANP, 0.1-1 microM) activated GC in the particulate, but not in the soluble, membrane fraction. By contrast, the nitric oxide donor, sodium nitroprusside (SNP, 1-10 microM), stimulated GC only in the soluble fraction. Further, pretreatment with RSVL partly desensitized the ANP response, but was additive to that of SNP. In arterial tension studies, RSVL relaxed PGF(2alpha)-precontracted denuded rings in a concentration-dependent manner, a response that was markedly enhanced (approximately 18 fold) in the presence of IBMX. Conversely, precontraction with phorbol ester, which also desensitizes pGC, blunted relaxations to RSVL but not to forskolin or SNP. These findings demonstrate that RSVL increases cGMP in coronary arteries, mostly by activation of pGC. This pathway triggers vasorelaxant responses that remain effective in endothelium-disrupted arteries.     

Heterogeneous cholinergic reactions of ringed seal coronary arteries

Coronary blood flow of some seal species is unusual in that it is highly variable in both non-diving and diving conditions and shows intermittent fluctuations, especially during dives when it frequently ceases for brief periods. We sought regulatory mechanisms governing these reactions by studying isometric tension recordings of isolated left circumflex (LC) and left anterior descending (LAD) coronary arteries of ringed seals, Phoca hispida, during reactions to a variety of agents for stimulating or blocking autonomic responses of the vascular smooth muscle. Micromolar acetylcholine (ACh) produced constriction of the small diameter segments of the LAD, but relaxation of the LC and larger segments of LAD. Both constrictions and dilations were prevented by atropine. Small vessel constriction by ACh was prevented by micromolar indomethacin and by a thromboxane receptor antagonist. Large vessel ACh dilations were prevented or reduced by rubbing off the endothelium and by the -arginine analog, -NG-nitro-arginine, an inhibitor of nitric oxide synthesis. We conclude that cholinergic, muscarinic, dilation of ringed seal large coronary arteries is mediated by endothelial-derived relaxing factor (EDRF), whereas ACh constriction of small arteries is mediated by a prostaglandin.     

Role of brain-derived neurotrophic factor in hyperoxia-induced enhancement of contractility and impairment of relaxation in lung parenchyma

Prolonged hyperoxic exposure contributes to neonatal lung injury, and airway hyperreactivity is characterized by enhanced contraction and impaired relaxation of airway smooth muscle. Our previous data demonstrate that hyperoxia in rat pups upregulates expression of brain-derived neurotrophic factor (BDNF) mRNA and protein, disrupts NO-cGMP signaling, and impairs cAMP production in airway smooth muscle. We hypothesized that BDNF-tyrosine kinase B (TrkB) signaling plays a functional role in airway hyperreactivity via upregulation of cholinergic mechanisms in hyperoxia-exposed lungs. Five-day-old rat pups were exposed to >or=95% oxygen or room air for 7 days and administered daily tyrosine kinase inhibitor K-252a (50 microg x kg(-1) x day(-1) i.p.) to block BDNF-TrkB signaling or vehicle. Lungs were removed for HPLC measurement of ACh or for in vitro force measurement of lung parenchymal strips. ACh content doubled in hyperoxic compared with room air-exposed lungs. K-252a treatment of hyperoxic pups restored ACh content to room air levels. Hyperoxia increased contraction and impaired relaxation of lung strips in response to incremental electrical field stimulation. K-252a administration to hyperoxic pups reversed this increase in contraction and decrease in relaxation. K-252a or TrkB-Fc was used to block the effect of exogenous BDNF in vitro. Both K-252a and TrkB-Fc blocked the effects of exogenous BDNF. Hyperoxia decreased cAMP and cGMP levels in lung strips, and blockade of BDNF-TrkB signaling restored cAMP but not cGMP to control levels. Therefore, hyperoxia-induced increase in activity of BDNF-TrkB receptor signaling appears to play a critical role in enhancing cholinergically mediated contractile responses of lung parenchyma.     

Functional expression of NOS 1 in vascular smooth muscle

Substances that increase intracellular calcium concentration ([Ca(2+)](i)), such as serotonin, are known to induce vascular smooth muscle (VSM) contraction. However, increases in [Ca(2+)](i) also activate Ca(2+)/calmodulin-dependent nitric oxide synthases (NOS), which leads to increases in cGMP and activation of cGMP-dependent protein kinase (PKG). One recently identified substrate protein of PKG is the small heat shock protein, HSP20. The purpose of this study was to determine if serotonin activates a Ca(2+)-dependent NOS in VSM. Strips of bovine carotid arterial smooth muscle denuded of endothelium were stimulated with serotonin in the presence and absence of the nonspecific NOS inhibitor N-monomethyl-L-arginine (L-NMMA). Activation of NOS was determined by increases in cGMP and in the phosphorylation of HSP20. Immunohistochemical and Western blotting techniques were performed to identify specific NOS isoforms in bovine carotid arterial smooth muscle preparations. Serotonin stimulation led to significant increases in cGMP and in the phosphorylation of HSP20, which were inhibited by pretreatment with L-NMMA. Antibodies against NOS 1 stained the media of bovine carotid and human renal arteries, whereas antibodies against NOS 3 stained only the endothelium. Additionally, the conversion of radiolabeled L-arginine to L-citrulline NOS activity demonstrated a consistent amount of activity present in the endothelium-denuded smooth muscle preparations that was reduced by 99% with an NOS 1 specific inhibitor. Finally, an NOS 1 specific inhibitor, 7-nitroindazole, augmented contractions induced by high extracellular KCl. This study demonstrates that NOS 1 is present in VSM and may effect physiological contractile responses.     

Coronary endothelium-derived vasodilation during cooling and rewarming of the in situ heart.

The integrity of coronary vascular endothelial vasodilator function during core cooling and rewarming was investigated in a pentobarbital-anesthetized open-chest dog model. Vasodilator response was assessed as the change from baseline blood flow by injecting the endothelial-dependent vasodilator acetylcholine (ACh) (1.0 microg) or the endothelial-independent vasodilator nitroglycerin (NTG) (50 microg) into the left anterior descending (LAD) coronary artery. Change in blood flow was measured using a transit time ultrasonic volume flowmeter technique. During cooling and rewarming LAD blood flow was significantly decreased. After rewarming, aortic pressure was artificially elevated to reach control. This procedure restored heart work (LV-RPP, left ventricular rate pressure product) and coronary perfusion pressure, but LAD blood flow remained lowered. Ability to dilate the vascular bed supplied by LAD, after injections of ACh or NTG, was present both during cooling and rewarming. At 25 degrees C coronary blood flow (LAD) increased from 3 +/- 1 to 9 +/- 1 mL x min(-1) in response to both ACh and NTG. Posthypothermic blood flow increased from 7 +/- 1 to 19 +/- 2 and 20 +/- 3 mL x min(-1) in response to ACh and NTG, respectively. Measured as the percent change from baseline LAD blood flow, the response was not significantly different from the one obtained in prehypothermic hearts. In conclusion, coronary vasodilator function, both endothelium dependent and endothelium independent, is present but not maintained at the same level during cooling to 25 degrees C and rewarming. In spite of the deterioration of cardiac function, no selective defect in the endothelium-dependent response was detected, either during hypothermia or after rewarming.