Stephen J. Contney

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.

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)

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.

Reversal of minimum alveolar concentrations of volatile anesthetics by chromosomal substitution

GENETIC susceptibility to pharmacological agents, termed “pharmacogenomics,” is an emerging field focused on genetic differences underlying alterations of physiologic responses to pharmacological agents. The objective of this study was to systematically compare differences in the minimum alveolar concentration (MAC) (defined as a movement response to a noxious sensory stimulus by volatile anesthetics) in two genotypically distinct parental strains of rats (Dahl Salt Sensitive [SS] and control Brown Norway [BN]) and in two consomic (chromosomal transfer) strains (SS.BN.13 and SS.BN.16). The latter were two strains available from a larger panel of consomics in which single chromosomes from the BN strain have been introgressed into an otherwise unchanged SS genetic background. The underlying hypothesis of this study is that SS animals have a greater overall sensitivity to the action of volatile anesthetics that is attributable to specific pharmacogenomic mechanisms related to one or several chromosomes.

Mechanism of Differential Cardiovascular Response to Propofol in Dahl Salt-Sensitive, Brown Norway, and Chromosome 13-Substituted Consomic Rat Strains: Role of Large Conductance Ca2+ and Voltage-Activated Potassium Channels

Cardiovascular sensitivity to general anesthetics is highly variable among individuals in both human and animal models, but little is known about the genetic determinants of drug response to anesthetics. Recently, we reported that propofol (2,6-diisopropylphenol) causes circulatory instability in Dahl salt-sensitive SS/JRHsdMcwi (SS) rats but not in Brown Norway BN/NHsdMcwi (BN) rats and that these effects are related to genes on chromosome 13. Based on the hypothesis that propofol does target mesenteric circulation, we investigated propofol modulation of mesenteric arterial smooth muscle cells (MASMC) in SS and BN rats. The role of chromosome 13 was tested using SS-13BN/Mcwi and BN-13SS/Mcwi consomic strains with chromosome 13 substitution. Propofol (5 μM) produced a greater in situ hyperpolarization of MASMC membrane potential in SS than BN rats, and this effect was abrogated by iberiotoxin, a voltage-activated potassium (BK) channel blocker. In inside-out patches, the BK channel number, Po, and apparent Ca2+ sensitivity, and propofol sensitivity all were significantly greater in MASMC of SS rats. The density of whole-cell BK current was increased by propofol more in SS than BN myocytes. Immunolabeling confirmed higher expression of BK α subunit in MASMC of SS rats. Furthermore, the hyperpolarization produced by propofol, the BK channel properties, and propofol sensitivity were modified in MASMC of SS-13BN/Mcwi and BN-13SS/Mcwi strains toward the values observed in the background SS and BN strains. We conclude that differential function and expression of BK channels, resulting from genetic variation within chromosome 13, contribute to the enhanced propofol sensitivity in SS and BN-13SS/Mcwi versus BN and SS-13BN/Mcwi strains.

Chromosomal substitution-dependent differences in cardiovascular responses to sodium pentobarbital.

In this study we addressed initial laboratory observations of enhanced cardiovascular sensitivity to sodium pentobarbital (PTB) in normotensive Dahl Salt Sensitive rats (SS) compared to Brown Norway (BN) rats. We also used unique consomic (chromosomal substitution) strains to confirm preliminary observations that such differences were related to chromosome 13. Increasing concentrations of PTB were administered sequentially to SS, BN, and SS strains with BN chromosomal substitutions until the point of cardiovascular collapse. Both spontaneous and controlled ventilation were studied. The effect of large (450 &mgr;g/mL) and small (35 &mgr;g/mL) concentrations of PTB on in situ transmembrane potential of mesenteric arterial vascular smooth muscle (VSM) cells was also measured in these animals with local sympathetic innervation both intact and eliminated. An analysis of variance was used to identify significant differences among groups. Despite virtually identical plasma clearance of PTB, cardiovascular collapse occurred at approximately 35%–45% smaller cumulative doses of administered PTB in SS and other strains compared with BN and SS.13BN (introgression of BN chromosome 13 into an SS) in both spontaneous and controlled ventilation. In neurally intact preparations, large dose PTB-induced VSM hyperpolarization was 4–5 times greater than the small dose in SS and SS.16BN but not in BN and SS.13BN strains. Denervation eliminated this strain difference. These results suggest that enhanced cardiovascular sensitivity to PTB in SS rats is related to greater hyperpolarization of VSM transmembrane potential in resistance vessels and this effect is associated with chromosome 13.

Multiple Agents Potentiate α1-Adrenoceptor–induced Conduction Depression in Canine Cardiac Purkinje Fibers

Background Halothane more so than isoflurane potentiates an &agr;1-adrenoceptor (&agr;1-AR)-mediated action of epinephrine that abnormally slows conduction in Purkinje fibers and may facilitate reentrant arrhythmias. This adverse drug interaction was further evaluated by examining conduction responses to epinephrine in combination with thiopental and propofol, which “sensitize” or reduce the dose of epinephrine required to induce arrhythmias in the heart, and with etomidate, which does not, and responses to epinephrine with verapamil, lidocaine, and l-palmitoyl carnitine, a potential ischemic metabolite. Methods Action potentials and conduction times were measured in vitro using two microelectrodes in groups of canine Purkinje fibers stimulated at 150 pulses/min. Conduction was evaluated each minute after exposure to 5 &mgr;m epinephrine (or phenylephrine) alone or with the test drugs. Changes in the rate of phase 0 depolarization (Vmax) and the electrotonic spread of intracellular current were measured during exposure to epinephrine with octanol to evaluate the role of inhibition of active and passive (intercellular coupling) membrane properties in the transient depression of conduction velocity. Results Lidocaine (20 &mgr;m) and octanol (0.2 mm) potentiated &agr;1-AR–induced conduction depression like halothane (0.4 mm), with maximum depression at 3–5 min of agonist exposure, no decrease of Vmax, and little accentuation at a rapid (250 vs. 150 pulses/min) stimulation rate. Thiopental (95 &mgr;m), propofol (50 &mgr;m), and verapamil (2 &mgr;m) similarly potentiated epinephrine responses, whereas etomidate (10 &mgr;m) did not. Between groups, the decrease of velocity induced by epinephrine in the presence of (10 &mgr;m) l-palmitoyl carnitine (−18%) was significantly greater than that resulting from epinephrine alone (−6%; 0.05 ≤P ≤ 0.10). Current injection experiments were consistent with marked transient inhibition of cell-to-cell coupling correlating with &agr;1-AR conduction depression in fibers exposed to octanol. Conclusions Anesthetic “sensitization” to the arrhythmogenic effects of catecholamines may be a special case of a more general phenomenon by which not only some anesthetics and antiarrhythmic drugs but also possible ischemic fatty acid metabolites potentiate conduction depression due to acute &agr;1-AR–mediated cell-to-cell uncoupling.