Cannabinoid CB1 receptor and endothelium-dependent hyperpolarization in guinea-pig carotid, rat mesenteric and porcine coronary arteries

1. The purpose of these experiments was to determine whether or not the endothelium-dependent hyperpolarizations of the vascular smooth muscle cells (observed in the presence of inhibitors of nitric oxide synthase and cyclo-oxygenase) can be attributed to the production of an endogenous cannabinoid. 2. Membrane potential was recorded in the guinea-pig carotid, rat mesenteric and porcine coronary arteries by intracellular microelectrodes. 3. In the rat mesenteric artery, the cannabinoid receptor antagonist, SR 141716 (1 microM), did not modify either the resting membrane potential of smooth muscle cells or the endothelium-dependent hyperpolarization induced by acetylcholine (1 microM) (17.3 +/- 1.8 mV, n = 4 and 17.8 +/- 2.6 mV, n = 4, in control and presence of SR 141716, respectively). Anandamide (30 microM) induced a hyperpolarization of the smooth muscle cells (12.6 +/- 1.4 mV, n = 13 and 2.0 +/- 3.0 mV, n = 6 in vessels with and without endothelium, respectively) which could not be repeated in the same tissue, whereas acetylcholine was still able to hyperpolarize the preparation. The hyperpolarization induced by anandamide was not significantly influenced by SR 141716 (1 microM). HU-210 (30 microM), a synthetic CB1 receptor agonist, and palmitoylethanolamide (30 microM), a CB2 receptor agonist, did not influence the membrane potential of the vascular smooth muscle cells. 4. In the rat mesenteric artery, the endothelium-dependent hyperpolarization induced by acetylcholine (1 microM) (19.0 +/- 1.7 mV, n = 6) was not altered by glibenclamide (1 microM; 17.7 +/- 2.3 mV, n = 3). However, the combination of charybdotoxin (0.1 microM) plus apamin (0.5 microM) abolished the acetylcholine-induced hyperpolarization and under these conditions, acetylcholine evoked a depolarization (7.7 +/- 2.7 mV, n = 3). The hyperpolarization induced by anandamide (30 microM) (12.6 +/- 1.4 mV, n = 13) was significantly inhibited by glibenclamide (4.0 +/- 0.4 mV, n = 4) but not significantly affected by the combination of charybdotoxin plus apamin (17.3 +/- 2.3 mV, n = 4). 5. In the guinea-pig carotid artery, acetylcholine (1 microM) evoked endothelium-dependent hyperpolarization (18.8 +/- 0.7 mV, n = 15). SR 141716 (10 nM to 10 microM), caused a direct, concentration-dependent hyperpolarization (up to 10 mV at 10 microM) and a significant inhibition of the acetylcholine-induced hyperpolarization. Anandamide (0.1 to 3 microM) did not influence the membrane potential. At a concentration of 30 microM, the cannabinoid agonist induced a non-reproducible hyperpolarization (5.6 +/- 1.3 mV, n = 10) with a slow onset. SR 141716 (1 microM) did not affect the hyperpolarization induced by 30 microM anandamide (5.3 +/- 1.5 mV, n = 3). 6. In the porcine coronary artery, anandamide up to 30 microM did not hyperpolarize or relax the smooth muscle cells. The endothelium-dependent hyperpolarization and relaxation induced by bradykinin were not influenced by SR 141716 (1 microM). 7. These results indicate that the endothelium-dependent hyperpolarizations, observed in the guinea-pig carotid, rat mesenteric and porcine coronary arteries, are not related to the activation of cannabinoid CB1 receptors.     

Time dependence of endothelium-mediated vasodilation by intermittent antegrade warm blood cardioplegia

BACKGROUND: The technique of intermittent antegrade warm blood cardioplegia (IAWBC) exposes the heart to brief periods of normothermic ischemia. This may impair endothelial function in coronary arteries. METHODS: Three cardioplegic technique were tested in porcine hearts arrested for 32 to 36 minutes and reperfused for 30 minutes: IAWBC, antegrade cold blood cardioplegia (ACBC), and antegrade cold crystalloid cardioplegia (ACCC). In the hearts arrested with IAWBC, three different intervals of ischemia were used: three 10-minute intervals (IAWBC1), two 15-minute intervals (IAWBC2), and one 30-minute interval (IAWBC3). Rings from the coronary arteries were used to evaluate in vitro the contractile responses to U46619 and the relaxant responses to bradykinin, A23187, and sodium nitroprusside. RESULTS: All six groups (treatment groups and control group) displayed similar responses to U46619 (30 nmol/L) and nitroprusside. In the IAWBC1, IAWBC2, AND ACBC groups, endothelium-dependent relaxations to bradykinin and A23187 were preserved compared with controls, whereas those of the ACCC and IAWBC3 groups were significantly impaired (bradykinin: control, 8.72 +/- 0.07; IAWBC1, 8.73 +/- 0.03; IAWBC2, 8.65 +/- 0.05; IAWBC3, 8.30 +/- 0.07 [p < 0.05]; ACBC, 8.50 +/- 0.03; ACCC, 8.25 +/- 0.09 [p < 0.05]; A23187: control, 7.07 +/- 0.08; IAWBC1, 7.07 +/- 0.06; IAWBC2, 7.04 +/- 0.03; IAWBC3, 6.64 +/- 0.01 [p < 0.05]; ACBC, 6.80 +/- 0.05; ACCC, 6.60 +/- 0.08 [p < 0.05]; nitroprusside: control, 6.19 +/- 0.1; IAWBC1, 6.19 +/- 0.07; IAWBC2, 6.03 +/- 0.03; IAWBC3, 6.08 +/- 0.05; ACBC, 6.04 +/- 0.2; ACCC, 6.05 +/- 0.03; all values are expressed as the negative logarithm of the concentration producing 50% of the maximal response). CONCLUSIONS: Myocardial preservation with IAWBC with ischemic intervals of 15 minutes or shorter does not alter the endothelium-dependent relaxation to bradykinin or A23187 in porcine coronary arteries, but these responses are significantly impaired by ACCC and IAWBC with an ischemic interval of 30 minutes.     

Bioassay of an endothelium-derived hyperpolarizing factor from bovine coronary arteries: role of a cytochrome P450 metabolite

An endothelium-derived hyperpolarizing factor (EDHF) mediates a part of the vasodilatory action of bradykinin. A bioassay method was developed to investigate the properties of EDHF on bovine coronary arterial smooth muscle cells. Cannulated bovine coronary arteries with an intact endothelium that were treated with indomethacin and NG-nitro-L-arginine methyl ester served as the EDHF donor. The effect of the donor vessel perfusate was examined on a 240 pS single-channel calcium (Ca2+)-activated potassium (K+) current (KCa) and resting membrane potential in recipient coronary arterial smooth muscle cells. The open state probability (NPo) of the channel averaged 0.011 +/- 0.003 during basal perfusate flow. After stimulation of the donor vessels with bradykinin (10(-10)-10(-6) M), the perfusate induced a 1.2- to 5-fold increase in the NPo (n = 7, p < 0.001). This increase in channel activity was attenuated by either removing the endothelium of the donor arterial segment or upon inhibition of cytochrome P450 in the donor arterial segment with the combination of 17-octadecynoic acid and miconazole. The resting cell membrane averaged -60 +/- 2 mV, and hyperpolarized to -69 +/- 1.5 mV (n = 6, p < 0.05) in response to the perfusate following stimulation of the donor vessel with bradykinin. Addition of 14, 15-epoxyeicosatrienoic acid mimicked the effects of the perfusate and increased the NPo of the KCa channel from 0.01 +/- 0.001 to 0.05 +/- 0.001. These findings suggest that bradykinin stimulates the release of a transferable endothelial factor that activates KCa channels and hyperpolarizes coronary arterial smooth muscle cell membranes. These findings support the hypothesis that coronary arteries release an EDHF which is a cytochrome P450 metabolite of arachidonic acid.     

Effects of timolol and betaxolol on choroidal blood flow in the rabbit

Endothelium-dependent hyperpolarization of vascular smooth muscle cells (VSMCs) plays a crucial role in regulating vascular tone, especially in resistance vessels. It has been proposed that metabolites of arachidonic acid (AA), formed by cytochrome P-450 monooxygenase (P450), are endothelium-derived hyperpolarizing factors (EDHFs). These metabolites have been reported to mediate dilation to endogenous vasoactive compounds, such as bradykinin and acetylcholine. However, it is not known whether these metabolites of AA contribute to dilation of human resistance vessels. This is important since it has been proposed that EDHF serves as a compensatory mechanism to maintain dilation in disease states. Therefore, we studied the effect of AA on vessel diameter and VSMC membrane potential in isolated human coronary microvessels. Arterioles (81+/-5 microm, n=70) were dissected from right atrial appendages at the time of cardiac surgery and cannulated at a distending pressure of 60 mm Hg and zero flow. Changes in internal diameter were recorded with videomicroscopy. Some vessels were impaled with glass microelectrodes to measure membrane potential of VSMCs while internal diameters were simultaneously recorded. After constriction (47+/-2%) with endothelin-1, AA (10(-10)to 10(-5)mol/L) induced substantial dilation of human coronary microvessels, which was abolished by removal of the endothelium. Treatment with 17-octadecynoic acid (17-ODYA, 10(-5) mol/L; a P450 inhibitor) attenuated maximal dilation to AA (49+/-9% versus 91+/-4% [control]; P<0.05 versus control), whereas indomethacin (INDO, 10(-5) mol/L; a cyclooxygenase inhibitor) and N omega-nitro-L-arginine methyl ester (L-NAME, 10(-4) mol/L; a NO synthase inhibitor) were without effect. Both 17-ODYA and miconazole (10(-5) mol/L, a chemically distinct P450 inhibitor) further reduced the dilation to AA in the presence of INDO. The presence of 40 mmol/L KCl or charybdotoxin (10(-8) mol/L, a blocker of large-conductance Ca2+-activated K+ channels) impaired dilation to AA (19+/-9% [KCI] versus 76+/-5% [control] and 47+/-6% [charybdotoxin] versus 91+/-3% [control]; P<0.05 for both). After depolarization with endothelin-1 (-26+/-1 mV from -48+/-3 mV [before endothelin]), AA (10(-5)mol/L) in the presence of INDO and L-NAME induced hyperpolarization of VSMCs (-57+/-5 mV). In the presence of 17-ODYA together with INDO and L-NAME, endothelin produced similar depolarization (-26+/-2 mV from - 48+/- 3 mV), but hyperpolarization to AA was reduced (-33+/-2 mV; P<0.05 versus absence of 17-ODYA). AA metabolites formed primarily by P450 produce potent endothelium-dependent dilation of human coronary arterioles via opening of Ca2+-activated K+ channels and hyperpolarization of VSMCs. These findings support an important role for P450 metabolites in the regulation of human coronary arteriolar tone.     

Effect of mycophenolic acid on TNF alpha-induced expression of cell adhesion molecules in human venous endothelial cells in vitro

1. The characteristic features of the endothelium-mediated regulation of the electrical and mechanical activity of the smooth muscle cells of cerebral arteries were studied by measuring membrane potential and isometric force in endothelium-intact and -denuded strips taken from the rabbit middle cerebral artery (MCA). 2. In endothelium-intact strips, histamine (His, 3-10 microM) and high K+ (20-80 mM) concentration-dependently produced a transient contraction followed by a sustained contraction. Noradrenaline (10 microM), 5-hydroxytryptamine (10 microM) and 9,11-epithio-11, 12-methano-thromboxane A2 (10 nM) each produced only a small contraction (less than 5% of the maximum K+-induced contraction). 3. N(G)-nitro-L-arginine (L-NOARG, 100 microM), but not indomethacin (10 microM), greatly enhanced the phasic and the tonic contractions induced by His (1-10 microM) in endothelium-intact, but not in endothelium-denuded strips, suggesting that spontaneous or basal release of nitric oxide (NO) from endothelial cells potently attenuates the His-induced contractions. Acetylcholine (ACh, 0.3-3 microM) caused concentration-dependent relaxation (maximum relaxation by 89.7 +/- 7.5%, n=4, P<0.05) when applied to endothelium-intact strips precontracted with His. L-NOARG had little effect on this ACh-induced relaxation (n=4; P<0.05). Apamin (0.1 microM), but not glibenclamide (3 microM), abolished the relaxation induced by ACh (0.3-3 microM) in L-NOARG-treated strips (n=4, P<0.05). 4. In endothelium-intact tissues, His (3 microM) depolarized the smooth muscle membrane potential (by 4.4 +/- 1.8 mV, n = 12, P < 0.05) whereas ACh (3 microM) caused membrane hyperpolarization (-20.9 +/- 3.0 mV, n = 25, P< 0.05). The ACh-induced membrane hypepolarization persisted after application of L-NOARG (-23.5 +/- 5.9 mV, n=8, P<0.05) or glibenclamide (-20.6 +/- 5.4 mV, n=5, P<0.05) but was greatly diminished by apamin (reduced to - 5.8 +/- 3.2 mV, n = 3, P< 0.05). 5. Sodium nitroprusside (0.1-10 microM) did not hyperpolarize the smooth muscle cell membrane potential (0.2 +/- 0.3 mV, n=4, P>0.05) but it greatly attenuated the His-induced contraction in endothelium-denuded strips (n-4, P<0.05). 6. These results suggest that, under the present experimental conditions: (i) spontaneous or basal release of NO from endothelial cells exerts a significant negative effect on agonist-induced contractions in rabbit MCA, and (ii) ACh primarily activates the release of endothelium-derived hyperpolarizing factor (EDHF) in rabbit MCA.     

Linoleic acid induces relaxation and hyperpolarization of the pig coronary artery

Linoleic acid, a polyunsaturated C18 fatty acid, is one of the major fatty acids in the coronary arterial wall. Although diets rich in linoleic acid reduce blood pressure and prevent coronary artery disease in both humans and animals, very little is known about its mechanism of action. We believed that its beneficial effects might be mediated by changes in vascular tone. We investigated whether linoleic acid induces relaxation of porcine coronary artery rings and the mechanism involved in this process. Linoleic acid and two of its metabolites, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-hydroperoxyoctadecadienoic acid (13-HPODE), induced dose-dependent relaxation of prostaglandin (PG) F2alpha-precontracted rings that was not affected by indomethacin (10[-5] mol/L), a cyclooxygenase inhibitor, or cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC; 10[-5] mol/L), a lipoxygenase inhibitor. Removal of endothelial cells had no effect on vasorelaxation, suggesting a direct effect on the vascular smooth muscle cells (VSMC). When rings were contracted with KCl, linoleic acid failed to induce relaxation. Although tetrabutylammonium (5 x 10[-3] mol/L), a nonselective K+ channel blocker, slightly inhibited the relaxation caused by linoleic acid, glibenclamide (10[-6] mol/L), an ATP-sensitive K+ channel blocker, and charybdotoxin (7.5x10[-8] mol/L) or tetraethylammonium (5x10[-3] mol/L), two different Ca2+-activated K+ channel blockers, had no effect. However, relaxation was completely blocked by ouabain (5x10[-7] mol/L), a Na+/K+-ATPase inhibitor, or by a K+-free solution. In addition, linoleic acid (10[-6] mol/L) caused sustained hyperpolarization of porcine coronary VSMC (from -49.5+/-2.0 to -60.7+/-4.2 mV), which was also abolished by ouabain. We concluded that linoleic acid induces relaxation and hyperpolarization of porcine coronary VSMC via a mechanism that involves activation of the Na+/K+-ATPase pump.     

Binding of the novel nonxanthine A2A adenosine receptor antagonist [3H]SCH58261 to coronary artery membranes

This study demonstrates quantification of A2A adenosine receptors (A2AAdoRs) in membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells. Radioligand binding assays were performed using the new selective A2AAdoR antagonist radioligand [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-epsilon]-1,2,4-triazolo[1,5-c)pyrimidine ([3H]SCH58261). Binding of the radioligand to membranes was rapid, reversible, and saturable. The densities of A2AAdoRs in membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells were 900 +/- 61, 892 +/- 35, and 959 +/- 76 fmol/mg protein, respectively. Equilibrium dissociation constants (Kd values) calculated from results of saturation binding assays were 2.19, 1.20, and 0.81 nmol/L, and Kd values calculated from results of association and dissociation assays were 2.42, 1.01, and 0.40 nmol/L for [3H]SCH58261 binding to membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells, respectively. The specific binding of [3H]SCH58261 as a percentage of total binding at a radioligand concentration equal to the Kd value was 65% to 90% in the three membrane preparations. The order of ligand potencies determined by assay of competition binding to sites in porcine coronary membranes using [3H]SCH58261, unlabeled antagonists (SCH58261, 8-(3-chlorostyryl)caffeine [CSC], and xanthine amine congener [XAC]), and unlabeled agonists ([3H]2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoaden osine [CGS 21680], 2-hexynyl-5'-N-ethylcarboxamidoadenosine [HE-NECA], [3H]5'-N-ethylcarboxamidoadenosine [NECA], and R(-)N6-(2-phenylisopropyl)adenosine [R-PIA]) was SCH58261 > HE-NECA = CSC = CGS 21680 = XAC > NECA = R-PIA. The Hill coefficients of displacement by A2AAdoR ligands of [3H]SCH58261 binding were not significantly different from unity, indicating that [3H]SCH58261 bound to a group of homogeneous noninteracting sites in all membrane preparations. The order of ligand potencies to compete for [3H]SCH58261 binding sites in porcine striatal and PC12 cell membranes was, in part, different from that for porcine coronary arterial membranes. The different rank orders of potencies for agonists and antagonists at A2A receptors of porcine coronary arteries, striatum, and PC12 cells and significant differences in absolute values of potency of ligands in the three preparations may indicate the existence of different subtypes of A2AAdoRs. The antagonist radio-ligand [3H]SCH58261 should be of value for pharmacological characterization of A2A adenosine receptors in other preparations.     

Angiotensin-(1-7) and the rat aorta: modulation by the endothelium

Peptide metabolites of angiotensin I and II are active components of the renin-angiotensin system. One such peptide is angiotensin-(1-7), which has been shown to be present in various tissues and has properties distinct from those of angiotensin II. We examined the effects of angiotensin-(1-7) on endothelium-intact and denuded rat aorta. Second, we evaluated whether an interaction occurred between angiotensin-(1-7) and angiotensin peptides, as well as noradrenaline. Finally, we addressed whether the responses to angiotensin-(1-7) were mediated by an AT1 receptor. Angiotensin-(1-7) produced concentration-dependent relaxations of the rat aorta that were significantly greater in endothelium-intact preparations (81.1 +/- 18.9% and 29.6 +/- 2.9% for intact and denuded, respectively). Angiotensin-(1-7) inhibited responses generated to angiotensin I, II, III, and noradrenaline. In endothelium-denuded preparations, angiotensin-(1-7) produced a rightward shift of the concentration-effect curves to angiotensin II and noradrenaline. In addition, the inhibition against angiotensin I and II was significantly greater in endothelium-intact preparations [mean median inhibitory concentration (IC50) values for endothelium-intact preparations, 1.25 x 10(-9) M and 1.57 x 10(-9) M for angiotensin I and II, respectively; and for endothelium-denuded preparations, 1.77 x 10(-8) M and 1.17 x 10(-8) M for angiotensin I and II, respectively). Losartan did not affect relaxations in endothelium-intact preparations but caused a significant potentiation of the relaxation by angiotensin-(1-7) in denuded preparations. We conclude that angiotensin-(1-7) is a component of the renin-angiotensin system that acts to modulate the pressor effects of angiotensin II and noradrenaline.     

Effects of short-term treatment with trandolapril on vasodilatator responses mediated by receptors or on blood flow in the coronary arteries in SHR

The aim of the study was to assess the effects of a one-month treatment period with the ACE inhibitor trandolapril (0.3 mg/kg/day) on the endothelial reactivity in epicardial right coronary arteries (CA) of 26-30 week-old SHRs. For this purpose, segments of CA were mounted in an arteriograph where wall thickness and internal diameter (ID) were continuously monitored while intraluminal pressure (IP) was controlled. In the absence of flow and under an IP of 30 mmHg, IDs were not significantly different in control compared to those of treated SHR arteries (microns, 250 +/- 8 vs 240 +/- 7). In preconstricted preparations (5HT, 10 microns extraluminally) C/E curves were constructed by adding acetylcholine (AC, 0.01-10 microM) or bradykinin (BK, 0.01-10 microM) in the bath. On the other hand, the effect of a stepwise increase in intraluminal flow (50-450 microliters/min; IP = 30 mmHg) of perfusion solution was observed. The effects of subsequent additions of sodium nitroprusside (SNP) were assessed. Maximal relaxations were expressed as percent of maximal contractions. Results were as follows: [table: see text] These results show that the endothelium-dependent relaxation induced by AC and BK were significantly increased in coronary arteries of treated compared to control SHRs whereas the flow-induced relaxation seemed to remain unaffected in our experimental conditions. From these data, it can be concluded that a short period of ACE inhibition in SHRs is able to improve the endothelium-dependent vasodilation induced by agonists in the coronary arterial bed.     

Potentiation of endothelium-dependent relaxation by epoxyeicosatrienoic acids

Epoxyeicosatrienoic acids (EETs) are potent endothelium-derived vasodilators formed from cytochrome P-450 metabolism of arachidonic acid. EETs and their diol products (DHETs) are also avidly taken up by endothelial cells and incorporated into phospholipids that participate in signal transduction. To investigate the possible functional significance of EET and DHET incorporation into cell lipids, we examined the capacity of EETs and DHETs to relax porcine coronary arterial rings and determined responses to bradykinin (which potently activates endothelial phospholipases) before and after incubating the rings with these eicosanoids. 14,15-EET and 11,12-EET (5 mumol/L) produced 75 +/- 9% and 52 +/- 4% relaxation, respectively, of U46619-contracted rings, whereas 8,9-EET and 5,6-EET did not produce significant relaxation. The corresponding DHET regioisomers produced comparable relaxation responses. Preincubation with 14,15-EET, 11,12-EET, 14,15-DHET, and 11,12-DHET augmented the magnitude and duration of bradykinin-induced relaxation, whereas endothelium-independent relaxations to aprikalim and sodium nitroprusside were not potentiated. Pretreatment with 2 mumol/L triacsin C (an inhibitor of acyl coenzyme A synthases) inhibited [3H]14,15-EET incorporation into endothelial phospholipids and blocked 11,12-EET- and 14,15-DHET-induced potentiation of relaxation to bradykinin. Exposure of [3H]14,15-EET-labeled endothelial cells to the Ca2+ ionophore A23187 (2 mumol/L) resulted in a 4-fold increased release of EET and DHET into the medium. We conclude that incorporation of EETs and DHETs into cell lipids results in potentiation of bradykinin-induced relaxation in porcine coronary arteries, providing the first evidence that incorporated EETs and DHETs are capable of modulating vascular function.     

Adventitial expression of recombinant eNOS gene restores NO production in arteries without endothelium

The current study was designed to determine the effect of recombinant endothelial nitric oxide synthase (eNOS) gene expression on endothelium-dependent relaxations to bradykinin in isolated canine basilar, coronary, or femoral arteries. Arterial rings were exposed ex vivo (30 minutes at 37 degrees C) to an adenoviral vector encoding either the eNOS gene (AdCMVeNOS) or the beta-galactosidase reporter gene (AdCMVbeta-Gal). Twenty-four hours after transduction, transgene expression was evident mainly in the adventitia. Expression of recombinant proteins was much higher in basilar arteries than in coronary or femoral arteries. Rings of control, AdCMVbeta-Gal, and AdCMVeNOS arteries with and without endothelium were suspended for isometric tension recording. Levels of cGMP were measured by radioimmunoassay. In AdCMVeNOS basilar arteries with endothelium, relaxations to low concentrations of bradykinin (3 x 10(-11) to 10(-9) mol/L) were significantly augmented. In contrast, in coronary and femoral arteries with endothelium, AdCMVeNOS transduction did not affect relaxations to bradykinin. Removal of the endothelium abolished bradykinin-induced relaxations in control and AdCMVbeta-Gal basilar arteries. However, in basilar arteries transduced with AdCMVeNOS even when the endothelium was removed, stimulation with bradykinin (3 x 10(-11) to 10(-9) mol/L) caused relaxations as well as increases in cGMP production. The relaxations to bradykinin were completely blocked by an NOS inhibitor, NG-nitro-L-arginine methyl ester. Electron microscopic analysis revealed that recombinant eNOS protein was expressed in fibroblasts of the basilar artery adventitia. These results suggest that genetically modified adventitial fibroblasts may restore production of NO in cerebral arteries without endothelium. Our findings support a novel concept in vascular biology that fibroblasts in the adventitia may play a role in the regulation of vascular tone after successful transfer and expression of recombinant eNOS gene.     

Metalloregulated expression of the ars operon

Myocardial dysfunction after cardiac operations might be influenced by altered myocardial perfusion in the postoperative period. To investigate possible alterations in vascular reactivity, in vitro coronary microvascular responses were examined after ischemic cardioplegia with use of a porcine model of cardiopulmonary bypass. Since myocardial perfusion is primarily regulated by arteries less than 200 microns in diameter, these vascular segments were examined. After 1 hour of ischemic arrest with cold crystalloid cardioplegia and 1 hour of reperfusion, microvessels (100 to 190 microns in diameter) were pressurized in a no-flow state, preconstricted by 30% to 60% of the baseline diameter with acetylcholine, and examined with video microscopic imaging and electronic dimension analysis. Endothelium-dependent relaxations to bradykinin (55% +/- 13% versus 99% +/- 1% = maximum relaxation of the preconstricted diameter in cardioplegia-reperfusion vessels versus control vessels, respectively; p < 0.05) and the calcium ionophore A 23187 (33% +/- 6% versus 90% +/- 4%; p < 0.05) were markedly impaired while endothelium-independent relaxation to sodium nitroprusside was similar to control value. After 1 hour of ischemic cardioplegia without reperfusion, endothelium-dependent relaxation was only slightly affected. Transmission electron microscopy showed minimal endothelial damage after ischemic cardioplegia and reperfusion. These findings have important implications regarding coronary spasm and cardiac dysfunction after cardiac operations.     

Evidence that mechanisms dependent and independent of nitric oxide mediate endothelium-dependent relaxation to bradykinin in human small resistance-like coronary arteries

1. The effects of the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine (L-NOARG), the NO scavenger, oxyhaemoglobin (HbO) and high extracellular K+ upon endothelium-dependent relaxation to bradykinin were investigated in human isolated small coronary arteries. 2. Endothelium-dependent relaxations to bradykinin were compared in vessels contracted to approximately 50% of their maximum contraction to 124 mM KCl Krebs solution, regardless of treatments, with the thromboxane A2 mimetic, U46619 and acetylcholine. All relaxations were expressed as percentage reversal of the initial level of active force. 3. L-NOARG (100 microM) caused a small but significant, 12% (P < 0.01), decrease in the maximum relaxation (Rmax: 91.5 +/- 5.4%) to bradykinin but did not significantly affect the sensitivity (pEC50: 8.08 +/- 0.17). Increasing the concentration of L-NOARG to 300 microM had no further effect on the pEC50 or Rmax to bradykinin. HbO (20 microM) and a combination of HbO (20 microM) and L-NOARG (100 microM) reduced Rmax to bradykinin by 58% (P < 0.05) and 54% (P < 0.05), respectively. HbO (20 microM) and L-NOARG (100 microM, combined but not HbO (20 microM) alone, caused a significant 11 fold (P < 0.05) decrease in sensitivity to bradykinin. HbO (20 microM) decreased the sensitivity to the endothelium-independent NO donor, S-nitroso-N-acetylpenicillamine (SNAP), approximately 17 fold (P < 0.05). 4. Raising the extracellular concentration of K+ isotonically to 30 mM, reduced the Rmax to bradykinin from 96.6 +/- 3.1% to 43.9 +/- 10.1% (P < 0.01) with no significant change in sensitivity. A combination of HbO, L-NOARG and high K+ (30 mM) abolished the response to bradykinin. High K+ did not change either the sensitivity or maximum relaxation to SNAP. 5. In conclusion, L-NOARG does not completely inhibit endothelial cell NO synthesis in human isolated small coronary arteries. By comparison, HbO appeared to block all the effects of NO in this tissue and revealed that most of the relaxation to bradykinin was due to NO. The non-NO -dependent relaxation to bradykinin in the human isolated small coronary arteries appeared to be mediated by a K(+)-sensitive vasodilator mechanism, possibly endothelium-derived hyperpolarizing factor (EDHF).     

Expression of Ly-6, a marker for highly malignant murine tumor cells, is regulated by growth conditions and stress

Bradykinin-induced responses were studied in isolated porcine iliac arteries. Relaxation was endothelium dependent and seen at low concentrations (10(-10)-10(-8) M) of bradykinin. It was inhibited by the bradykinin B2-receptor antagonist icatibant (HOE-140) and by the nitric oxide synthase inhibitor Nomega-nitro-L-arginine. Bradykinin-induced relaxation was significantly potentiated by the kininase I carboxypeptidase inhibitor mergepta (10(-6) M). Bradykinin (>10(-7) M) elicited contraction of preparations with or without endothelium. The contraction was abolished by indomethacin but was not affected by the thromboxane A2/prostaglandin H2-receptor antagonist SQ 29,548. Icatibant and the bradykinin B1-receptor antagonist desArg9[Leu8]bradykinin significantly decreased bradykinin-induced contraction regardless of endothelial function. The contraction also was decreased by treatment with mergepta. The bradykinin B1-receptor agonist desArg9-bradykinin contracted endothelium-denuded arterial strips. This contraction was significantly decreased by desArg9[Leu8]bradykinin but not by icatibant. The desArg9-bradykinin-induced contraction also was inhibited by the protein-synthesis inhibitor cycloheximide. Neither bradykinin-induced relaxation nor contraction was affected by the ACE inhibitors enalaprilat or cilazaprilat. In conclusion, bradykinin-induced relaxation of isolated porcine iliac arteries was mediated by endothelial bradykinin B2 receptors and mainly nitric oxide. Bradykinin-induced contraction was endothelium independent, indomethacin sensitive, and probably mediated by bradykinin B1 (inducible) and B2 receptors located in the vascular smooth-muscle layer. Kininase I carboxypeptidase, and not ACE, is the main enzyme responsible for bradykinin degradation in these vessels.     

Potentiation of endothelium-dependent hyperpolarization to serotonin by dietary intake of NC 020, a defined fish oil, in the porcine coronary artery

The membrane potential of vascular smooth muscle cells of the porcine coronary artery was measured to examine whether serotonin evokes endothelium-dependent hyperpolarization, and if it does, whether the electrical responses are modulated by the chronic dietary intake of NC 020, a defined fish oil. Serotonin induced transient, concentration-dependent hyperpolarizations of coronary arterial smooth muscle cells. The hyperpolarization was observed in tissues with, but not in those without endothelium. In coronary arteries obtained from pigs fed chronically with NC 020, serotonin induced significantly larger hyperpolarizations than those observed in control arteries. These results suggest that endothelium-dependent hyperpolarization may contribute to the endothelium-dependent relaxation evoked by serotonin and to its potentiation by the dietary intake of fish oil (NC 020).     

Different populations of progesterone receptor-steroid complexes in binding to specific DNA sequences: effects of salts on kinetics and specificity

The endothelium plays an obligatory role in a number of relaxations of isolated arteries. These endothelium-dependent relaxations are due to the release by the endothelial cells of potent vasodilator substances [endothelium-derived relaxing factors (EDRF)]. The best characterized EDRF is nitric oxide (NO). Nitric oxide is formed by the metabolism of L-arginine by the constitutive NO synthase of endothelial cells. In arterial smooth muscle, the relaxations evoked by EDRF are explained best by the stimulation by NO of soluble guanylate cyclase that leads to the accumulation of cyclic GMP. The endothelial cells also release an unidentified substance that causes hyperpolarization of the cell membrane (endothelium-derived hyperpolarizing factor, EDHF). The release of EDRF from the endothelium can be mediated by both pertussis toxin-sensitive (alpha2-adrenergic activation, serotonin, thrombin, aggregating platelets) and insensitive (adenosine diphosphate, bradykinin) G-proteins. In blood vessels from animals with regenerated endothelium, and/or atherosclerosis, there is a selective loss of the pertussis-toxin sensitive mechanism of EDRF-release which favors the occurrence of vasospasm, thrombosis and cellular growth.     

Endothelium-dependent hyperpolarization in isolated arteries taken from animals treated with NO-synthase inhibitors

To study the effects of chronic in vivo inhibition of NO synthase on endothelium-dependent hyperpolarization, cell-membrane potential (in individual vascular smooth-muscle cells) and changes in tension (in isolated rings) were recorded from isolated canine coronary arteries and guinea-pig carotid arteries and aortas. In coronary arteries taken from control dogs and contracted with U46619, acetylcholine- and bradykinin-induced endothelium-dependent relaxations, which were unaffected by short-term in vitro exposure to indomethacin but were inhibited partially by L-nitro-arginine (LNA). In coronary arteries taken from dogs treated over the long term in vivo with LNA (30 mg/kg on the first day and 20 mg/kg the 7 following days, i.v.), the response to acetylcholine and bradykinin was inhibited when compared with arteries from control dogs. Short-term in vitro exposure to LNA or indomethacin or both did not influence the effects of either agonist. In these arteries, the hyperpolarizing response to acetylcholine, observed in the presence of LNA and indomethacin, was enhanced, whereas that to bradykinin was partially inhibited. In the guinea pig isolated aorta, the relaxation to bradykinin was abolished by long-term in vivo treatment with L-nitro-arginine-methyl-ester (L-NAME; 1.5 mg/ml, in the drinking water for > or =4 days). In the isolated guinea pig carotid artery studied in the presence of LNA and indomethacin, acetylcholine induced a hyperpolarization that was not significantly affected by long-term in vivo treatment with L-NAME. These findings indicate that endothelium-dependent hyperpolarizations are maintained during long-term inhibition of NO synthase and probably act as a back-up mechanism to elicit endothelium-dependent relaxations.     

Changes in coronary vasodilation in hypertensive patients with angiographically normal coronary arteries

In normal subjects, coronary arteries dilate in response to sympathetic stimulation evoked by the cold pressor test. Similarly, in normal coronary arteries the increase in blood flow velocity induced by papaverine results in flow-dependent coronary dilation. In order to assess the coronary responses to both stimuli in hypertensive patients, variations of proximal left anterior descending coronary artery diameters and coronary blood flow velocity have been measured using quantitative coronary angiography and intracoronary Doppler in 10 control subjects and in 12 hypertensive patients. All the patients had angiographically normal coronary arteries. Total serum cholesterol, triglycerides, HDL- and LDL-cholesterol were within normal range in all patients. All patients were nonsmokers and none of them had diabetes mellitus. During the cold pressor test (hands immersed in ice water for 120 s), the rate-pressure product and coronary blood flow velocity increased respectively by 33 +/- 9% (p < 0.001) and 51 +/- 26% (p < 0.05) in control subjects, by 28 +/- 18% (p < 0.001) and 68 +/- 52% (p < 0.05) in hypertensive patients. In control subjects, coronary arteries dilated by + 12.0 +/- 4.4% (p < 0.001), and constricted by -10.3 +/- 8.5% (p < 0.001) in hypertensive patients. After injection of 10 mg of papaverine into the distal left anterior descending coronary artery, proximal left anterior descending coronary artery dilated by + 17.0 +/- 10.6% (p < 0.001) in control subjects, and did not vary (-0.7% +/- 10.6%) in hypertensive patients, when blood flow velocity was increased respectively by 449 +/- 97% and 383 +/- 103% (p < 0.001 in both groups).(ABSTRACT TRUNCATED AT 250 WORDS)     

MSX-1 gene expression and regulation in embryonic palatal tissue

1. During cardiac surgery, the heart is arrested and protected by hyperkalaemic cardioplegia. The coronary endothelium may be damaged by ischaemia-reperfusion and cardioplegia. Subsequently, this may affect cardiac function immediately after cardiac surgery and cause mortality and morbidity. 2. We investigated coronary endothelium-smooth muscle interaction after exposure to depolarizing (hyperkalaemic; K+ 20 or 50 mmol/L) and hyperpolarizing (the K+ channel opener aprikalim) cardioplegia and organ preservation solution (University of Wisconsin (UW) solution). Endothelium-dependent relaxation and hyperpolarization of the coronary smooth muscle were studied in the porcine and human large conductance and micro-coronary arteries. Intracellular free calcium concentration in endothelial cells was also measured. 3. The endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation to A23187, bradykinin, and substance P in arteries contracted by either U46619 (10 nmol/L) or K+ (25 mmol/L) was reduced after exposure to either high K+ or UW solution, but was maximally preserved after exposure to aprikalim. The hyperpolarization of the membrane potential in response to the above endothelium-derived relaxing factor stimuli was also reduced by exposure to depolarizing cardioplegia. Studies in microcoronary arteries are in accordance with findings in large arteries. The intracellular free calcium concentration remained unchanged after exposure to hyperkalaemia. 4. We concluded that: (i) during cardiac surgery, the function of coronary circulation may be changed due to exposure to depolarizing cardioplegia or preservation solutions; (ii) the functional change in the coronary circulation is related to the altered interaction between the endothelium and smooth muscle; (iii) depolarizing (hyperkalaemia) cardioplegia or hyperkalaemic organ preservation solutions affect endothelium-smooth muscle interaction through the EDHF pathway; (iv) EDHF relaxes the porcine large and microcoronary arteries through multiple K+ channels; and (v) that hyperpolarizing vasodilators (K+ channel openers) may protect EDHF-mediated endothelial function when used as cardioplegia.     

Contraction and endothelium dependent relaxation via alpha adrenoceptors are variable in various pig arteries

OBJECTIVE: The aim was to examine differences in the vascular response to alpha adrenoceptor stimulation on both smooth muscle and endothelium among large arteries perfusing various organs. METHODS: In ring preparations of coronary, carotid, iliac, mesenteric, and renal arteries from pigs (n = 24), the magnitudes of contraction to noradrenaline (with beta blocker), with or without endothelium, and endothelium dependent relaxation to noradrenaline (with alpha 1 and beta blocker) were compared in vitro. RESULTS: In both endothelium-intact and denuded groups, the contractions to noradrenaline were significantly smaller in coronary and carotid arteries than in iliac, mesenteric, and renal arteries. The presence of endothelium significantly suppressed the contractions in coronary and carotid arteries, but did not affect the contractions in iliac, mesenteric, and renal arteries. The endothelium dependent relaxations to noradrenaline, which were abolished by LNMMA, were more prominent in coronary and carotid arteries than in iliac, mesenteric, and renal arteries. CONCLUSIONS: The contractions via alpha adrenoceptors were small, and endothelium dependent relaxations via alpha 2 adrenoceptors were large, in the coronary and carotid arteries, which perfuse the vital organs (heart and brain), as compared with those in iliac, mesenteric, and renal arteries. These results suggest that in the state of activation of the sympathetic nervous system, arterial tone in response to alpha adrenoceptor stimulation may be regulated not only by alpha adrenoceptors on vascular smooth muscle but also by those on endothelium, through release of endothelium derived relaxing factor (EDRF) via alpha 2 adrenoceptors.