William J. Stekiel

Effects of Volatile Anesthetic Agents on In Situ Vascular Smooth Muscle Transmembrane Potential in Resistance- and Capacitance-regulating Blood Vessels

Introduction This study was designed to compare the inhibitory effect of inhaled volatile anesthetic agents on in situ sympathetic neural versus nonneural regulation of vascular smooth muscle transmembrane potentials as correlates of vascular smooth muscle tone in resistance‐ and capacitance‐regulating blood vessels. Methods Vascular smooth muscle transmembrane potentials were measured in situ with glass microelectrodes in neurally intact, small (200–300 m OD) mesenteric arteries and veins of rats before, during, and after inhaled halothane, isoflurane, or sevoflurane (0.5 or 1.0 minimum alveolar concentration [MAC]). Such transmembrane potentials and their anesthetically induced changes were compared, respectively, with those measured in similar vessel preparations after local sympathetic neural denervation with 6‐hydroxydopamine. Results In neurally intact vessels, transmembrane potentials (in millivolts, mean +/‐ SD) before inhalation of the anesthetic agent were ‐39 +/‐ 2.8 (artery) and ‐43 +/‐ 4.6 (vein). At 1.0 MAC, halothane, isoflurane, and sevoflurane induced respective hyperpolarizations (in millivolts, mean +/‐ SD) of 9 +/‐ 3.1, 6 +/‐ 2.7, and 9 +/‐ 4.0 in arteries and 6 +/‐ 4.4, 2.8 +/‐ 3.0, and 8.7 +/‐ 5.6 in veins. Sympathetic denervation significantly attenuated these hyperpolarizations (except for venous response to isoflurane). At 0.5 MAC, transmembrane potential responses to all three volatile anesthetic agents were small and not consistently significant in either the intact or denervated vessels. Conclusions In resistance‐regulating arteries in situ, inhaled halothane, isoflurane, and sevoflurane (1.0 MAC) attenuate both sympathetic neural and nonneural regulation of vascular smooth muscle transmembrane potentials (and tone). In capacitance‐regulating veins in situ, sevoflurane (1.0 MAC) also attenuates both regulatory mechanisms, whereas halothane and isoflurane primarily attenuate nonneural mechanisms. At 0.5 MAC, none of these agents significantly affected either mode of regulation of vascular smooth muscle transmembrane potentials in arteries or veins.

Pressure-induced activation of membrane K+ current in rat saphenous artery

Pressurization of isolated arteries may result in Ca2+-dependent contraction and membrane depolarization. Because the open state probability of some vascular muscle K+ channels is augmented by rises in cytosolic Ca2+ and membrane depolarization, we investigated the possibility that increases in intraluminal pressure activate K+ channels in isolated, perfused rat saphenous arteries. Stepwise increases in intraluminal pressure from 5 to 205 mm Hg resulted in increasing, active arterial contraction, measured as smaller diameters in physiological salt solution than in Ca2+-free solution. Addition of 10 mM tetraethylammonium to the physiological salt solution to block arterial muscle K+ channels caused progressively greater diameter reductions at pressures above 25 mm Hg. Microelectrode measurements of membrane potential showed that tetraethylammonium depolarized arterial muscle more at 105 mm Hg (16±1 mV) than at 25 mm Hg (10±1 mV). The sensitivity of K+ current to tetraethylammonium was also demonstrated in patch-clamped vascular muscle cells from the same arteries. Peak whole-cell K+ current was suppressed 47% and 79% by 1 and 10 mM tetraethylammonium, respectively. This same current was enhanced 3.6-fold by the Ca2+ ionophore A23187 (10 μM), suggesting a Ca2+ dependence. We conclude that increases in intraluminal pressure progressively activate tetraethylammonium-sensitive K+ channels in the arterial muscle membrane. This can serve as a negative feedback mechanism to limit pressure-induced arterial constriction.

Neural and local control of arterioles in SHR.

This study sought to determine if neural influences and/or alterations in arteriolar responses to oxygen could contribute to an elevated microvascular resistance in spontaneously hypertensive rats (SHR). Diameters of third-order arterioles (3A) and fourth-order arterioles (4A) were measured in the cremaster muscle of 12- to 15-week-old SHR and normotensive Wistar-Kyoto (WKY) controls anesthetized with pentobarbital. The preparation was suffused with physiological salt solution (PSS) equilibrated with various concentrations of oxygen (0% O2, 5% O2, or 10% O2) with and without local neural blockade with 10(-7) g/ml tetrodotoxin (TTX). Total active tone was assessed with 10(-4) M adenosine. SHR 3A (but not 4A) exhibited a smaller resting diameter than WKY, and larger dilations in response to TTX and adenosine. When suffusion solution PO2 was elevated in the presence or absence of TTX, SHR arterioles constricted more than did those of WKY, and SHR 4A exhibited a higher incidence of complete closure. Therefore, both neural influences and local vascular control mechanisms may contribute to an elevated microvascular resistance in SHR.

Altered beta-receptor control of in situ membrane potential in hypertensive rats.

Sympathetic neural activation of vascular smooth muscle beta-receptors induces membrane hyperpolarization and arterial relaxation. This response, which likely is mediated by the Gs protein-adenylyl cyclase-cyclic AMP signaling cascade, is reduced in some hypertensive animal models and in human essential hypertension. Since reduced beta-receptor-mediated vasodilation is a potential mechanism for enhanced arterial resistance, this study was designed to identify which step (or steps) in the beta-receptor signaling cascade is altered in hypertension. Transmembrane potentials were recorded in situ in small first-order arterioles and venules of cremaster muscle from hypertensive, reduced renal mass rats and normotensive, sham-operated controls. Vascular muscle cells in arterioles and venules of hypertensive rats were 5-7 mV more depolarized than in respective vessels of control rats during superfusion with physiological salt solution. Hyperpolarization and depolarization responses were reduced in hypertensive rats during superfusion with a beta-receptor agonist and antagonist, respectively, suggesting attenuated beta-receptor responsiveness compared with normotensive rats. Furthermore, direct activation of Gs protein by 10 ng/mL cholera toxin did not affect arterial or venous transmembrane potential in hypertensive rats, but hyperpolarized arterial and venous vascular muscle in normotensive controls by 17 mV. However, when the Gs protein-adenylate cyclase coupling step of the beta-receptor cascade was bypassed by using 10(-5) M forskolin to directly activate adenylate cyclase, arterial and venous vascular muscle of hypertensive rats hyperpolarized by 25-27 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Potassium channel-mediated hyperpolarization of mesenteric vascular smooth muscle by isoflurane

BACKGROUND A primary source of calcium (Ca2+) necessary for excitation contraction in vascular smooth muscle (VSM) is influx via voltage-dependent Ca2+ channels. Thus, force generation in VSM is coupled closely to resting transmembrane potential, which itself is primarily a function of potassium conductance. Previously, the authors reported that volatile anesthetics hyperpolarize VSM of small mesenteric resistance arteries and capacitance veins. The current study was designed to determine whether isoflurane-mediated hyperpolarization is the result of specific effects on one or more of four types of potassium channels known to exist in VSM. METHODS Transmembrane potentials (Em) were recorded from in situ mesenteric capacitance and resistance vessels in Sprague-Dawley rats weighing 250-300 g. In separate experiments, selective inhibitors of each of four types of potassium channels known to exist in VSM were administered in the superfusate of the vessel preparations to assess their effects on isoflurane-mediated hyperpolarization. RESULTS Resting VSM Em ranged from -38 to -43 mV after local sympathetic denervation. Isoflurane produced a significant hyperpolarization (2.7-4.3 mV), whereas each potassium channel inhibitor significantly depolarized (2.8-8.5 mV) the VSM. Both 100 nM iberiotoxin (inhibitor of high conductance calcium-activated potassium channels) and 1 microM glybenclamide (inhibitor of adenosine triphosphatase-sensitive potassium channels) significantly inhibited VSM hyperpolarization induced by 1 MAC (minimum alveolar concentration) levels of inhaled isoflurane (0.1-0.9 mV Em change, which was not significant). In contrast, isoflurane hyperpolarized the VSM significantly despite the presence of 3 mM 4 aminopyridine (inhibitor of voltage-dependent potassium channels) or 10 microM barium chloride (an inhibitor of inward rectifier potassium channels) (3.7-8.2 mV change in VSM Em). CONCLUSIONS These results suggest that isoflurane-mediated hyperpolarization (and associated relaxation) of VSM can be attributed in part to an enhanced (or maintained) opening of calcium-activated and adenosine triphosphate-sensitive potassium channels but not voltage-dependent or inward rectifier potassium channels.

Sympathetic neural control of vascular muscle in reduced renal mass hypertension.

Vascular smooth muscle (VSM) transmembrane potentials (EM) were measured in situ in small branch arteries (150-300-μ;m o.d.), small branch veins (300-400-μ;m o.d.), arterioles (90- 150μm o.d.), and venules (80-250-μ o.d.) in the mesenteric and gracilis muscle and the arterioles and venules of cremaster muscle vascular beds in anesthetized rats with reduced renal mass hypertension (HT-RRM) and normotensive sham-operated RRM control rats. All rats were given a 4% NaCl diet for 2 weeks with water ad libitum. Relative to sham, HT-RRM mesenteric and gracilis arterial and venous vessels, but not the microvessels of the cremaster muscle bed, were less polarized during superfusion with normal physiological salt solution. Also relative to sham, hyperpolarization responses to local sympathetic neural (SNS) denervation with 6-hydroxydopamine were greater in mesenteric and gracilis small arteries, arterioles, veins, and venules but not in cremaster microvessels. The immediate (less than 5-minute) electrogenic depolarization response to local blockade of VSM Na+-K+ pump activity with 10−3 M ouabain was similar between each respective HT-RRM and sham vessel pair in each vascular bed. These results suggest that in all three vascular beds: 1) significant SNS control of VSM Em (and active tone) exists all the way to the arterial and venous microvasculature (except cremaster venules); 2) in HT-RRM, such SNS control is elevated relative to sham in both arterial resistance and venous capacitance vessels in mesenteric and gracilis vascular beds but not in the cremaster microvessels; and 3) any circulating Na+-K+ pump inhibitors in the circulation of this volume-expanded model of hypertension do not appear to affect VSM tone in the vessels studied.

Mechanisms of isoflurane-mediated hyperpolarization of vascular smooth muscle in chronically hypertensive and normotensive conditions.

Background The purpose of this study was to compare the effects of isoflurane on membrane and intracellular mechanisms that regulate vascular smooth muscle (VSM) transmembrane potential (Em; which is related to VSM tone) in the spontaneously hypertensive rat (SHR) model of essential hypertension and its normotensive Wistar-Kyoto (WKY) control. Methods Vascular smooth muscle Em values were measured in situ in locally denervated, superfused, intact, small (200–300-&mgr;m OD) mesenteric arteries and veins in anesthetized 9– 12-week-old SHR and WKY. Effects of 1.0 minimum alveolar concentration (0.60 mm) superfused isoflurane on VSM Em were measured before and during superfusion with specific inhibitors of VSM calcium-activated (KCa) and adenosine triphosphate–regulated (KATP) potassium channels, and with endogenous mediators of vasodilatation (nitric oxide, cyclic guanosine monophosphate, protein kinase G, cyclic adenosine monophosphate, and protein kinase A). Results Isoflurane significantly hyperpolarized small arteries (5 ± 3.4 mV) and veins (6 ± 4.7 mV) (pooled SHR and WKY, mean ± SD). Inhibition of KCa and KATP channels, cyclic adenosine monophosphate, and protein kinase A, but not nitric oxide, cyclic guanosine monophosphate, and protein kinase G, abolished such hyperpolarization equally in SHR and WKY vessels. Conclusions Isoflurane-induced in situ VSM hyperpolarization in denervated, small mesenteric vessels involves a similar activation of KCa and KATP channels and cyclic adenosine monophosphate, but not nitric oxide or cyclic guanosine monophosphate, second messenger pathways in both SHR and WKY. A greater isoflurane-induced VSM hyperpolarization (observed previously in neurally intact SHR vessels) suggests enhanced inhibition of elevated sympathetic neural input as a major mechanism underlying such hyperpolarization (and coupled relaxation) in this neurogenic model of hypertension.

Responses of cremasteric arterioles of spontaneously hypertensive rats to changes in extracellular K+ concentration.

Objective: The goal of this study was to determine the effect of changes in extracellular K+ concentration ([K+]0) on active tone in cremasteric arterioles of spontaneously hypertensive rats (SHR) and their normotensive Wistar‐Kyoto (WKY) and Wistar controls.

The Inhibitory Action of Halothane on Reflex Constriction in Mesenteric Capacitance Veins

Potent inhalational anesthetics depress autonomic reflex responses at multiple sites. Most studies emphasize cardiac chronotropic changes and changes in systemic blood pressure. Recently, active reflex venoconstriction of 500-1,000 microns O.D. mesenteric veins has been demonstrated. In the current study, the effects of halothane on the reflex responses of similar mesenteric veins were measured. Mesenteric vein diameter and intravenous pressure were measured in 500-1,000 microns O.D. veins from the mesentery of segments of terminal ileum externalized in situ from 27 New Zealand white rabbits anesthetized with alpha-chloralose. Mean arterial pressure was measured with femoral arterial cannulation, and heart rate was determined from the arterial pressure signal. In a separate group of six animals, sympathetic efferent nerve activity was measured from a postganglionic splanchnic nerve. Reflex venoconstriction and increases in mean arterial pressure and mesenteric vein pressure in response to bilateral carotid occlusion were attenuated by 0.5% and 1% inhaled halothane but not by superfusate equilibrated with 3% halothane. Decreases in mesenteric vein diameter and increases in mesenteric vein pressure in response to celiac ganglion stimulation were unaffected by both 0.5% inhaled halothane and superfusate equilibrated with 5% halothane. The bilateral carotid occlusion reflex-mediated increase in sympathetic efferent nerve activity was depressed by both 0.5% and 1% inhaled halothane. The effect of inhaled halothane on prestimulation baseline vein diameter was inconsistent. Superfusate equilibrated with 5% but not 3% halothane caused baseline venodilation. These results suggest a mechanism whereby control of venous tone is inhibited by halothane proximal to the postganglionic neuron. This could involve central or ganglionic inhibition.

Biomechanical and Electrical Responses of Normal and Hypertensive Veins to Short-Term Pressure Increases

Observation of elevated central venous pressure or systemic filling pressure in various forms of arterial hypertension strongly suggests that systemic veins may participate in inducing and/or maintaining the hypertensive state [1,12]. However, sufficient information is not available to identify the mechanism(s) by which veins could contribute to the development and/or maintenance of chronic arterial pressure elevation [6]. It is plausible to hypothesize that some basic properties of the smooth muscle — such as intrinsic myogenic reactivity — in the systemic veins are altered in the hypertensive state, for then even a relatively small enhancement of stretch-induced intrinsic tone of the vessel wall could contribute significantly to the hemodynamic changes observed in arterial hypertension. Augmentation of this response may lead to the increase of central cardiopulmonary blood volume, postcapillary resistance, and the reduction of the pressure-buffer capacity of the venous system. This hypothesis is encouraged by our recent data demonstrating that there is an enhanced pressure-induced myogenic tone in isolated, small (100–150 μm internal diameter) gracilis arteries from reduced renal mass (RRM) rats — a model of volume-expanded hypertension — relative to non-RRM controls [13]. In accordance with these data, results recently published by Mulvany [10] suggest that increased intrinsic oscillatory activity of the mesenteric resistance vessels of spontaneous hypertensive rats plays a part in the development of high blood pressure.