Ethanol-induced Vasoconstriction Research Articles

Identification of an Ethanol Recognition Site in BK Beta1 Subunit that Mediates Ethanol-Induced Cerebral Artery Myocyte BK Channel Inhibition and the Resulting Artery Constriction

Ethanol at concentrations obtained in circulation during moderate-heavy episodic drinking (30-80 mM) decreases the activity of voltage/calcium-gated, large conductance potassium (BK) channels in smooth muscle (SM), which leads to cerebral artery constriction (Liu et al., 2004). The SM-abundant BK beta1 subunit (encoded by KCNMB1) is necessary for ethanol to inhibit cerebral artery SM BK channels under physiological voltages and calcium (Bukiya et al., 2009, Kuntamallappanavar and Dopico, 2016). Moreover, beta1 transmembrane domain 2 (TM2) is required for such action of ethanol (Kuntamallappanavar and Dopico, 2017). The precise ethanol recognition site(s) in beta1-TM2 that mediates ethanol-induced BK channel inhibition and associated cerebral artery constriction has remained unknown. Here, we address this question by combining computational modeling, mutagenesis, patch-clamp electrophysiology, in vitro artery electroporation and pressurization. Computational dynamics revealed two potential ethanol-recognition residues in beta1-TM2: Ser160 and Thr165. Macroscopic currents evoked from BK channel-forming cbv1 subunits co-expressed with beta1T165A displayed an ethanol response similar to that of cbv1+beta1, allowing ethanol inhibition of current at physiological, internal calcium>2 microM. In contrast, currents from cbv1+beta1S160A,T165A and cbv1+beta1S160A channels displayed an ethanol response identical to that of cbv1 alone: ethanol inhibition was blunted. Collectively, our data suggest that hydrogen bonding between Ser160 and ethanol's hydroxyl group is essential for ethanol-mediated inhibition of slo1+beta1 current. Consistent with in vitro electrophysiology findings, KCNMB1 K/O mouse arteries electroporated with beta1S160A cDNA failed to constrict in response to ethanol whereas electroporation with wt beta1 cDNA evoked ethanol-induced vasoconstriction. In conclusion, Ser160 in the beta1-TM2 region is required for ethanol-induced inhibition of beta1-containing BK channels and the resulting cerebral artery constriction. Support: R37-AA11560 (AMD) and AHA Predoctoral Fellowship (GK).

Ethanol-induced vasoconstriction is mediated via redox-sensitive cyclo-oxygenase-dependent mechanisms

The present study investigated the role of ROS (reactive oxygen species) and COX (cyclo-oxygenase) in ethanol-induced contraction and elevation of [Ca2+]i (intracellular [Ca2+]). Vascular reactivity experiments, using standard muscle bath procedures, showed that ethanol (1-800 mmol/l) induced contraction in endothelium-intact (EC50: 306+/-34 mmol/l) and endothelium -denuded (EC50: 180+/-40 mmol/l) rat aortic rings. Endothelial removal enhanced ethanol-induced contraction. Preincubation of intact rings with L-NAME [NG-nitro-L-arginine methyl ester; non-selective NOS (NO synthase) inhibitor, 100 micromol/l], 7-nitroindazole [selective nNOS (neuronal NOS) inhibitor, 100 micromol/l], oxyhaemoglobin (NO scavenger, 10 micromol/l) and ODQ (selective inhibitor of guanylate cyclase enzyme, 1 micromol/l) increased ethanol-induced contraction. Tiron [O2- (superoxide anion) scavenger, 1 mmol/l] and catalase (H2O2 scavenger, 300 units/ml) reduced ethanol-induced contraction to a similar extent in both endothelium-intact and denuded rings. Similarly, indomethacin (non-selective COX inhibitor, 10 micromol/l), SC560 (selective COX-1 inhibitor, 1 micromol/l), AH6809 [PGF2alpha (prostaglandin F2alpha)] receptor antagonist, 10 micromol/l] or SQ29584 [PGH2(prostaglandin H2)/TXA2 (thromboxane A2) receptor antagonist, 3 micromol/l] inhibited ethanol-induced contraction in aortic rings with and without intact endothelium. In cultured aortic VSMCs (vascular smooth muscle cells), ethanol stimulated generation of O2- and H2O2. Ethanol induced a transient increase in [Ca2+]i, which was significantly inhibited in VSMCs pre-exposed to tiron or indomethacin. Our data suggest that ethanol induces vasoconstriction via redox-sensitive and COX-dependent pathways, probably through direct effects on ROS production and Ca2+ signalling. These findings identify putative molecular mechanisms whereby ethanol, at high concentrations, influences vascular reactivity. Whether similar phenomena occur in vivo at lower concentrations of ethanol remains unclear.

Importance of PKC and PI3Ks in ethanol-induced contraction of cerebral arterial smooth muscle.

We investigated the relationships of two potential intracellular signaling pathways, protein kinase C (PKC) and phosphatidylinositol 3-kinases (PI3Ks), to ethanol-induced contractions in cerebral arteries. Ethanol (20-200 mM) induces concentration-dependent constriction in isolated canine basilar arteries that is inhibited in a concentration-dependent manner by pretreatment of these vessels with 10(-9)-10(-3) M Gö-6976 (an antagonist selective for PKC-alpha and PKC-betaI), 10(-10)-10(-4) M bisindolylmaleimide I (a specific antagonist of PKC), and 10(-10)-10(-4) M wortmannin or 10(-8)-10(-2) M LY-294002 (selective antagonists of PI3Ks). Ethanol-induced increases in intracellular Ca(2+) concentration (from approximately 100 to approximately 500 nM) in canine basilar smooth muscle cells are also suppressed markedly (approximately 20-70%) in the presence of a similar concentration range of Gö-6976, bisindolymaleimide I, wortmannin, or LY-294002. This study suggests that activation of PKC isoforms and PI3Ks appears to be an important signaling pathway in ethanol-induced vasoconstriction of cerebral blood vessels.

Endogenous nitric oxide attenuates ethanol-induced vasoconstriction in the human placenta.

The purpose of this study was to clarify the role of endogenous nitric oxide and prostanoids in ethanol-induced perturbation of microcirculation in perfused human placenta. Infusion of ethanol into chorionic plate vessels at 10-65 mM increases perfusion pressure in a concentration-dependent fashion, and is an indicator of fetal-placental vasoconstriction. Simultaneous infusion of N(omega)-nitro-L-arginine, methylene blue and endothelial cell removal significantly enhances the ethanol-induced increase in perfusion pressure. In contrast, sodium nitroprusside attenuates this effect. Indomethacin did not significantly modify the ethanol-induced response. In conclusion, inhibition of the action of endogenous nitric oxide is associated with an increase in fetal-placental vasoconstriction. These results suggest that endogenous nitric oxide acts as a vasodilator that reduces ethanol-induced vasoconstriction, thus improving microcirculation, and leads to decreased placental damage.

Role of endothelium-derived hyperpolarizing factor in ethanol-induced vasoconstriction

Role of endothelium-derived hyperpolarizing factor in ethanol-induced vasoconstriction

VIOLENT CRIME: THE ROLE OF ALCOHOL AND NEW APPROACHES TO THE PREVENTION OF INJURY

This study investigated the role of endogenous nitric oxide in the regulation of hepatic vascular tone in the presence of ethanol. In the perfused rat liver, upon the initiation of ethanol infusion into the liver, portal pressure was increased in a dose-dependent manner, reaching maximal levels in 2-5 min, then decreasing gradually. Simultaneous infusion of N(G)-monomethy 1-L-arginine, a nitric oxide synthesis inhibitor, enhanced this ethanol-induced increase in portal pressure. This enhancement was reversed by simultaneous infusion of a precursor of nitric oxide, L-arginine. These results suggest that endogenous nitric oxide acts as a vasodilator which reduces ethanol-induced vasoconstriction, thus improving the perturbation of hepatic microcirculation by ethanol.

Roles of endothelin-1 and nitric oxide in the mechanism for ethanol-induced vasoconstriction in rat liver.

This study was designed to investigate the mechanism for ethanol-induced hepatic vasoconstriction in isolated perfused rat liver. Upon initiation of ethanol infusion into the portal vein at concentrations ranging from 25 to 100 mM, portal pressure began to increase in a concentration-dependent manner and reached maximal levels in 2-5 min (initial phase), followed by a gradual decrease over the period of ethanol infusion (escape phenomenon). Endothelin-1 antiserum significantly inhibited this ethanol-induced hepatic vasoconstriction by 45-80%. Cessation of infusion of endothelin-1 antiserum was followed by a subsequent increase in portal pressure. On the other hand, when a nitric oxide synthesis inhibitor, NG-monomethyl-L-arginine (L-NMMA), was infused into the portal vein simultaneously with ethanol, the initial phase of the response of portal pressure to ethanol was not altered and the peak values of portal pressure remained unchanged. However, after the peak increase in portal pressure, the rate of decrease was less than in the absence of L-NMMA. Thus, L-NMMA diminished the escape phenomenon and sustained the vasoconstriction. This study supports the hypothesis that two endothelium-derived vasoactive factors, endothelin-1 and nitric oxide, regulate hepatic vascular tone in the presence of ethanol.

Ethanol-induced vasoconstriction causes focal hepatocellular injury in the isolated perfused rat liver.

The role of microcirculation in the pathogenesis of alcoholic liver injury was investigated in isolated perfused livers from fed rats. Infusion of ethanol into the portal vein at concentrations ranging from 25 to 200 mmol/L increased portal pressure, which is an indicator of hepatic vasoconstriction, in a concentration-dependent fashion. Portal pressure started to rise immediately on initiation of ethanol load and remained at higher than basal levels throughout the period of ethanol infusion. Release of lactate dehydrogenase, an indicator of cell injury, into the effluent perfusate began to increase after 20 to 30 min of ethanol infusion and continued to increase until the end of the experiment (60 min after the initiation of ethanol infusion). The lactate dehydrogenase level in the effluent perfusate at 60 min was dependent on the ethanol concentration (0 mmol/L, 8 +/- 3 IU/L; 25 mmol/L, 22 +/- 3 IU/L; 50 mmol/L, 51 +/- 11 IU/L; 100 mmol/L, 60 +/- 7 IU/L; 200 mmol/L, 120 +/- 7 IU/L). Simultaneous infusion of sodium nitroprusside (100 mumol/L), a known vasodilator, inhibited significantly the ethanol-induced increases in portal pressure and lactate dehydrogenase release by abolishing hepatic vasoconstriction. In histological examinations focal hepatocellular necrosis, evidenced by trypan blue staining of cell nuclei, was detected predominantly in midzonal and pericentral areas of the liver lobule after 60 min of ethanol infusion. Change in portal pressure during 60 min of ethanol infusion correlated significantly with levels of lactate dehydrogenase after ethanol infusion (r = 0.82; p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of intravascular ethanol on modulation of gastric mucosal integrity: possible role of endothelin-1.

The influences of intravascular ethanol on gastric mucosal integrity and its relation to gastric circulation were investigated in rats. Ulcer formation of the gastric mucosa correlated with the blood ethanol concentration in the presence of 150 mM HCl in the rat stomach. Furthermore, the gastric mucosal hemoglobin concentration (IHb) and hemoglobin oxygen saturation (ISO2), estimated using organ reflectance spectrophotometry, decreased in a concentration-dependent manner with blood ethanol. In isolated, vascularly perfused rabbit stomach, various concentrations (10-400 mM) of ethanol infused into the celiac artery increased the perfusion pressure and released endothelin-1 (ET-1) from the gastric vasculature in a concentration-dependent manner. Moreover, a significant correlation existed between changes in the perfusion pressure and ET-1 concentration in effluents from the gastric vasculature. Furthermore, anti-ET-1 antibody reduced 100 mM ethanol-induced vasoconstriction in a concentration-dependent manner. The results indicate that intravascular ethanol increases the susceptibility of gastric mucosa to injury induced by intraluminal HCl by causing gastric vasoconstriction mediated by ET-1. Thus intravascular ethanol may play an important role in the mechanism of ethanol-induced gastric mucosal injury.

Ethanol causes epicardial coronary artery vasoconstriction in the intact dog.

Ethanol produces in vitro vasoconstriction of coronary arteries and can precipitate angina in patients with coronary obstructive disease. To demonstrate the in vivo effect of ethanol on coronary dynamics, baseline measurements of left anterior descending (LAD) coronary artery dimension by quantitative angiography, hemodynamics, arterial and coronary sinus blood gases, and blood ethanol levels were obtained in 14 closed-chest mongrel dogs. Three ethanol levels were established by intravenous bolus followed by 1-hour maintenance infusions. All measurements made at baseline were recorded every 30 minutes. Phentolamine (5 mg i.v.) and nicardipine (0.15 mg/kg i.v.) were given to evaluate constrictor mechanisms. Blood ethanol levels achieved at 60, 120, and 180 minutes were 649 +/- 48, 1,285 +/- 81, and 2,546 +/- 130 micrograms/ml, respectively. LAD cross-sectional area was reduced significantly from control at the end of each of the three dosing periods (-24 +/- 5%, -40 +/- 3%, and -53 +/- 3%; p less than 0.004). alpha-Adrenergic blockade had no effect on LAD cross-sectional area, while nicardipine partially reversed the ethanol-induced vasoconstriction. No significant change in vessel cross-sectional area took place in control dogs. These data suggest that ethanol induces epicardial coronary artery vasoconstriction in dogs at clinically important blood levels. alpha-Adrenergic blockade does not alter or reverse ethanol-induced vasoconstriction, while calcium channel blockade appears to be an effective vasodilator of ethanol-constricted vessels.