Dixon W. Wilde

Calcium current in smooth muscle cells from normotensive and genetically hypertensive rats.

Genetic hypertension results from numerous phenotypic expressions. We hypothesized that increased calcium current in vascular smooth muscle of genetically hypertensive animals is partly responsible for observed increases in agonist sensitivity, contractility, and calcium influx. Using adult, spontaneously hypertensive stroke-prone rats (SHRSP) and normotensive Wistar-Kyoto (WKY) controls from an inbred colony, we characterized calcium current in smooth muscle cells isolated from cerebral arteries. Calcium current in WKY cells reached a maximum of -27.7 +/- 2.7 pA (n = 32) at +20 mV. Peak inward current at +20 mV in SHRSP cells had a mean amplitude of -44.4 +/- 3.0 pA (n = 72, P < .05). SHRSP cells exhibited a higher calcium current density. Maximal inward current normalized to cell capacitance yielded mean values of 2.07 +/- 0.11 pA/pF for WKY (n = 32) and 2.80 +/- 0.12 pA/pF (n = 79) for SHRSP (P < .05) cells. Transient-type Ca2+ channel current had the same magnitude and current-voltage relation in both cell types, giving an L-type/T-type ratio of 3.85 for WKY and 6.25 for SHRSP cells. The voltage-dependent inactivation curve for SHRSP calcium current was shifted to the right only over the range of -50 to -30 mV, but the half-maximal inactivation voltages and Boltzmann coefficients were not significantly different between cell types. Increased calcium inward current in this model of genetic hypertension could account in part for altered calcium homeostasis and increased vascular reactivity, contributing to hypertension and vasospasm.

High-Fat Diet Elevates Blood Pressure and Cerebrovascular Muscle Ca2+ Current

Abstract —Dietary fat contributes to the elevation of blood pressure and increases the risk of stroke and coronary artery disease. Previous observations have shown that voltage-gated Ca2+ current density is significantly increased in hypertension and can be affected by free fatty acids (FAs). We hypothesized that a diet of elevated fat level would lead to an increase in blood pressure, an elevation of L-type Ca2+ current, and an increase in saturated FA content in vascular smooth muscle cell membranes. Male Osborne-Mendel rats were fed normal rat chow or a high-fat diet (Ob/HT group) for 8 weeks. Blood pressures in the Ob/HT group increased moderately from 122.5±0.7 to 134.4±0.8 mm Hg ( P <0.05, n=26). Voltage-clamp examination of cerebral arterial cells revealed significantly elevated L-type Ca2+ current density in the Ob/HT group. Voltage-dependent inactivation of the Ob/HT L-type channels was significantly delayed. Total serum FA contents were significantly elevated in the Ob/HT group, and HPLC analyses of fractional pools of FAs from segments of abdominal aorta revealed that arachidonic acid levels were elevated in the phospholipid fraction in Ob/HT. No differences in vascular membrane cholesterol contents were noted. Plasma cholesterol was significantly elevated in portal venous and cardiac blood samples from Ob/HT rats. These findings suggest that an elevation of plasma FAs may contribute to the development of hypertension via a process involving the elevation of Ca2+ current density and an alteration of channel kinetics in the vascular smooth muscle membrane.

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.

Halothane Alters Control of Intracellular Ca2+ Mobilization in Single Rat Ventricular Myocytes

In an attempt to understand the cellular mechanisms underlying volatile anesthetic-induced myocardial depression, halothane-induced negative inotropy was investigated in an animal model through continuous monitoring of intracellular Ca2+ concentration [( Ca2+]i) in rat ventricular myocytes loaded with fura-2. Single cells were stimulated with 15 mM caffeine or 15 mM extracellular K+ (K+O) or were paced by extracellular glass suction pipette electrode. With each stimulus modality, halothane (0.6-1.5%) caused a significant (P less than 0.05) and dose-dependent depression of the Ca2+ transient. Caffeine and electrically stimulated Ca2+ transients were reduced, in 1.5% halothane, to 35 +/- 14 and 42 +/- 8% of control, respectively. Resting or basal [Ca2+]i was unaffected by halothane. Halothane did not elicit spontaneous Ca2+ transients in these cells. Single cells stimulated by trains of electrical stimuli at 1.0, 1.5, and 2.0 Hz showed a change in [Ca2+]i from prestimulus levels to a stimulated baseline steady state that appeared to increase with stimulus frequency. Halothane at 0.7% increased the change in resting to stimulated baseline [Ca2+]i and depressed net transients (P less than 0.05) at 1.0 and 1.5 Hz. In contrast, 0.1 microM ryanodine depressed the Ca2+ transients in myocytes stimulated by trains of stimuli, but did not potentiate the change in stimulated baseline [Ca2+]i at any pacing rate. The results are consistent with the hypothesis that halothane reduces Ca2+i availability by causing a net loss of Ca2+ from the sarcoplasmic reticulum. The results from experiments using onset of pacing to induce a sudden increase in Ca2+i load in previously quiescent myocytes suggest that halothane may act to limit sarcoplasmic reticulum and/or sarcolemmal uptake/extrusion mechanisms, as compared to ryanodine, which depletes sarcoplasmic reticulum Ca2+ stores without affecting reuptake and extrusion.

Effects of isoflurane and enflurane on intracellular Ca2+ mobilization in isolated cardiac myocytes.

BackgroundEnflurane and isoflurane may reduce cardiac contractility by altering mobilization and clearance of intracellular Ca2+ (Ca2+). It was hypothesized that the negative inotropic actions of these agents involve limiting both membrane Ca2+ entry and altering intracellular Ca2+ release. MethodsThe Ca2+1 transients in rat ventricular myocytes loaded with fura-2 were recorded from a fluorescence microscope. Transients stimulated by membrane depolarization (suction electrode or elevated [K+]o) or 15 mM caffeine to release Ca2+ from the sarcoplasmic reticulum (SR) were analyzed for net amplitude, maximal rate of rise (VR), average rate of decline (VR) in [Ca2+]1, and duration. ResultsEnflurane and isoflurane reduced electrically stimulated Ca2+1 transients in a dose-dependent manner. Enflurane depressed the Ca2+1 transient amplitude more than isoflurane. Enflurane was more effective than isoflurane in reducing VR and VF in a concentration-dependent manner. At similar concentrations, both enflurane and isoflurane reduced the steady state elevation of [Ca2+]1 by 50 mM K+o. Similarly, enflurane and isoflurane depressed caffeine-sensitive release of Ca2+ from the SR. The reduction in the Ca2+1 transient because of SR Ca2+ release was greater in enflurane than in equal concentrations of isoflurane. Rates of elevation and decline in [Ca2+]1 were also reduced in enflurane and isoflurane. ConclusionsThe negative inotropic actions of enflurane and isoflurane involve a depression of Ca2+ influx during membrane excitation, as well as a reduction in SR Ca2+ release. Slowed rates of elevation in [Ca2+]1 indicate that the latter mechanism may, in part, be caused by alterations in the kinetics of SR Ca2+ release.

Ceramide-induced vasorelaxation: An inhibitory action on protein kinase C.

Experiments were designed to examine the role of sphingosine, PP2A phosphatases, and protein kinase C (PKC) inhibition in mediating the vasodilatory effects of ceramide in rat thoracic aorta. Sphingosine did not cause vasorelaxation, and oleoylethanol-amine, a ceramidase inhibitor, did not affect sphingomyelinase-induced relaxation. Okadaic acid potentiated the relaxation response to ceramide. These observations rule out involvement of sphingosine and PP2A phosphatases in mediating ceramide-induced relaxation. Sphingomyelinase attenuated contractile and single-cell intracellular calcium responses to phorbol ester. Chelerythrine incubation potentiated the relaxation response to ceramide. These observations support a role for PKC inhibition in mediating the vasodilatory effects of ceramide.

Effects of Volatile Anesthetics on the Intracellular Ca2+ Concentration in Cardiac Muscle Cells

The myocardial depressant effects of the three halogenated volatile anesthetics, halothane, enflurane and isoflurane, have been well documented clinically and experimentally.1–4 In recent years multiple mechanisms of action of these agents have been demonstrated. The results of a number of studies have pointed to anesthetic effects on transmembrane signalling in a variety of cell types as a central facet of anesthetic action (for brief review see Maze5). Many efforts have been made to elucidate the mechanisms by which these agents alter excitation-contraction coupling in the heart to produce negative inotropy. Halothane, for example, has been observed to depress myocardial contractility4 through depletion of the sarcoplasmic reticulum (SR) stores of calcium (Ca2+).6–9 The evidence suggests that the negative inotropic effect of halothane is mediated by enhancement of Ca2+ efflux through SR Ca2+ release channels.10 Halothane’s effects to limit availability of Ca2+ for contraction do not appear restricted to actions on SR Ca2+ release, however. Direct effects of halothane on myocardial Ca2+ channels have also been proposed,11 although it remains unclear whether this is a direct effect on the L-type Ca2+ channels, or instead the indirect effect of a change in some intracellular mediator. Halothane may also alter Ca2+ uptake by the myocardial SR12,13 and have effects on mitochondrial Ca2+ metabolism and storage. These various putative mechanisms may act alone or in combination to limit Ca2+ availability for contraction in the heart.

Interactions of Ethanol and Quinidine on Contractility and Myocyte Action Potential in the Rat Ventricle

The combined effects of ethanol and quinidine on cardiac electromechanical coupling are unknown, but both drugs affect cardiac conduction and can cause myocardial depression. Isolated left ventricular papillary and ventricular myocytes were used to assess the combined effects of quinidine and ethanol on the electrophysiologic and mechanical properties of rat myocardium. The combination of quinidine (1-300 microM) and ethanol (120-240 mg/dL) depressed active papillary muscle tension within the clinically useful concentration range. In electrophysiologic studies of isolated ventricular myocytes, quinidine prolonged the action potential duration at 50% (APD50) and 90% (APD90) repolarization, the absolute refractory period, and the relative refractory period, but decreased the maximum rate of change of depolarization in phase 0 (Vmax). When cells were exposed to ethanol (240 mg/dL) and quinidine (1.5 microM) together, a significant decrease in the quinidine-induced prolongation of the absolute refractory and relative refractory periods was seen. Additional changes in action potential parameters from the quinidine values included slight reductions in Vmax and in APD50 and APD90, but these reductions were not consistently displayed, nor were they statistically significant.