Thomas J. J. Blanck

Alteration of Voltage-Dependent Calcium Channels in Canine Brain during Global Ischemia and Reperfusion

Elevated intracellular calcium (iCa2+) plays an important role in the pathophysiology of ischemic brain damage. The mechanisms by which iCa2+ increases are uncertain. Recent evidence implicates the voltage-dependent calcium channel (VDCC) as a likely site for the alteration in Ca2+ homeostasis during ischemia. The purpose of this study was to determine whether VDCCs are altered by global ischemia and reperfusion in a canine cardiac arrest, resuscitation model. We employed the radioligand, [3H]PN200-110, to quantitate the equilibrium binding characteristics of the VDCCs in the cerebral cortex. Twenty-five adult beagles were separated into four experimental groups: (a) nonischemic controls, (b) those undergoing 10-min ventricular fibrillation and apnea, (c) those undergoing 10-min ventricular fibrillation and apnea followed by spontaneous circulation and controlled respiration for 2 and (d) 24 h. Brain cortex samples were taken prior to killing of the animal, frozen immediately in liquid nitrogen, and crude synaptosomal membranes isolated by differential centrifugation/filtration. After 10 min of ischemia the maximal binding (Bmax) of [3H]PN200-110 increased to >250% of control values (control Bmax 11.16 ± 0.98; ischemic 28.35 ± 2.78 fmol/mg protein; p < 0.05). Bmax returned to near control values after 2 h of reperfusion but remained significantly greater than the control at 24 h. Although the affinity constant (Kd) (control = 0.12 ± 0.03 nM) appeared to increase with ischemia and normalize with reperfusion, the changes were not statistically significant. We conclude that the binding of [3H]PN200-110 to L-type VDCCs is increased after 10 min of global ischemia/anoxia produced by ventricular fibrillation and apnea in the dog. This change is only partially reversible after 24 h of reperfusion. This study supports the hypothesis that ischemia increases the number of VDCCs in the cell membrane which may allow increased entry of Ca2+ into the cell during ischemia and early reperfusion.

Increased activation of L-type voltage-dependent calcium channels is associated with glycine enhancement of N-methyl-D-aspartate-stimulated dopamine release in global cerebral ischemia/reperfusion

Abstract: We investigated the relationships among N‐methyl‐d‐aspartate, glycine, L‐type voltage‐dependent calcium channels, and [3H]dopamine release in a canine model of global cerebral ischemia/reperfusion. The binding of [3H]PN200‐110 ([3H]isradipine) to L‐type voltage‐dependent calcium channels, that open as a consequence of N‐methyl‐d‐aspartate‐induced changes in membrane potential, was approximately doubled in striatal membranes prepared from ischemic animals relative to controls, and remained significantly elevated at 30 min and 2 h of reperfusion. These changes coincided temporally with changes in the ability of the voltage‐sensitive calcium channel blocker nitrendipine to inhibit glycine enhancement of N‐methyl‐d‐aspartate‐stimulated [3H]dopamine release in striatal slices prepared from the same animals. Compared with nonischemic controls, N‐methyl‐d‐aspartate‐stimulated [3H]dopamine release was increased in ischemic animals and remained increased throughout reperfusion up to at least 24 h. Glycine enhanced N‐methyl‐d‐aspartate‐stimulated release in all treatment groups. The enhancement of N‐methyl‐d‐aspartate‐stimulated dopamine release by glycine was reduced by the inclusion of nitrendipine in striatal slices from ischemic and 30‐min reperfused animals. These data suggest that glycine may facilitate opening of the voltage‐dependent calcium channels activated by N‐methyl‐d‐aspartate and that this facilitation is blocked by the antagonist nitrendipine.

Halothane Does Not Alter Ca2+Affinity of Troponin C

Troponin C has been suggested as a possible target for the negative inotropic action of volatile anesthetics. This study has examined the effect of halothane on the structure and response of isolated cardiac troponin C to Ca2+ and the response of skinned soleus and cardiac muscle fibers to Ca2+. The high-affinity Ca(2+)-binding sites of cardiac troponin C were assessed by measurement of the change in intrinsic tyrosine fluorescence and ultraviolet circular dichroism in response to Ca2+ in the presence and absence of halothane. Halothane (0.9 mM, 1.4%) did not alter the 45% enhancement in intrinsic tyrosine fluorescence that occurs with saturation of the high-affinity sites or change the Ca2+ concentration at which half-maximal enhancement occurred. The molar ellipticity in the far ultraviolet region, a measure of the secondary structure, increased to a similar extent with addition of 10(-6) M Ca2+ in the absence and presence of 1.0 mM (1.6%) halothane. The binding rate of the sulfhydryl reagent, 5,5-dithiobis (2-nitrobenzoic acid), to troponin C in response to Ca2+ titration was used as a measure of the integrity of the low-affinity Ca(2+)-binding site in troponin C in the presence and absence of 1.0 mM (1.6%) halothane. The rate of reaction was stimulated twofold, and the half maximal effect was observed at pCa 4.8 +/- 0.2 in both control and halothane-treated samples. Halothane (5 mM; 7.8%) did not change the pCa/tension response of skinned soleus fibers; the data were fit to the Hill equation and yielded dissociation constants of 6.2 x 10(-7) M for control and halothane-treated specimens.(ABSTRACT TRUNCATED AT 250 WORDS)

Halothane decreases calcium channel antagonist binding to cardiac membranes

&NA; BLANCK TJJ, RUNGE S, STEVENSON RL. Halothane decreases calcium channel antagonist binding to cardiac membranes. Anesth Analg 1988;67:1032‐5. The effect of halothane concentration on the binding of the calcium antagonist, [3H] nitrendipine (3HNTP), to rat and rabbit heart membranes was examined in vitro because it has been hypothesized that one mechanism by which halothane depresses cardiac contractility is by interfering with Ca2+ channel function. Membranes were incubated for 90 minutes in a closed system with 3HNTP and increasing concentrations of halothane. The amount of 3HNTP bound to membranes was quantified by radioligand binding technique and liquid scintillation counting. It was found in both the rat and rabbit cardiac membranes that halothane (0.4‐2.0%) caused a dose‐dependent decrease in specific 3HNTP binding (P < 0.0001). The decrease in 3HNTP binding caused by halothane was also found to be reversible. These results indicate that halothane interferes with one property of the Ca2+ channel and suggest that this may be one possible mechanism for the negative inotropic action of halothane.

Effects of halothane on myocardial high-energy phosphate metabolism and intracellular pH utilizing 31P NMR spectroscopy

Utilizing 31phosphorus nuclear magnetic resonance (NMR) spectroscopy, the authors tested the two hypotheses that the negative inotropic action of halothane is the result of: 1) myocardial intracellular acidosis, and 2) a decrease in myocardial high-energy phosphates. In isolated, paced, Langendorff-perfused rabbit hearts, halothane (1.5 vol %) dissolved in the coronary perfusate produced a 48 ± 2% decrease (P < 0.01) in left ventricular developed pressure. In contrast, halothane administration had no significant effect on myocardial intracellular pH (7.18 ± 0.04 at control vs. 7.21 ± 0.02 during halothane). Halothane exposure decreased (P < 0.01) the forward rate constant of the creatine kinase reaction by 32 ± 6%, as measured using saturation transfer NMR, suggesting a decline in the rate of high-energy phosphate metabolism. This was further indicated by a concomitant decrease (P < 0.05) in myocardial oxygen consumption (20 ± 5%). During the halothane-induced reduction in left ventricular developed pressure, only small decreases in the myocardial steady state concentrations of phosphocreatine (7 ± 1%; P < 0.01) and βATP (12 ± 4%; P < 0.05), and an increase in Pi (18 ± 6%; P < 0.05) were observed. However, similar changes in steady-state high-energy phosphate metabolites were also measured in time-control hearts not exposed to halothane. These results indicate that the negative inotropic action of halothane is not mediated by myocardial intracellular acidosis. Moreover, these findings do not support the concept that the negative inotropic action of halothane is the result of a reduction in myocardial high-energy phosphates.

Hemodynamic effects and onset time of increasing doses of vecuronium in patients undergoing myocardial revascularization

Study objectives were (1) to compare the hemodynamic effects of increasing doses of vecuronium, given as a bolus during induction of anesthesia using high-dose fentanyl, in patients undergoing myocardial revascularization; and (2) to determine whether increasing the dose of vecuronium would decrease the onset time to maximal depression of twitch response. Forty patients scheduled for elective coronary artery bypass surgery were randomly assigned to four equal groups to receive either 0.1, 0.2, 0.3, or 0.4 mg/kg of vecuronium. Hemodynamic measurements and neuromuscular blockade were recorded at five time points: A, awake state; B, anesthetized state after the administration of fentanyl, 10 micrograms/kg; C, 2 minutes after vecuronium bolus; D, 5 minutes after vecuronium bolus; and E, after intubation. Increasing the dose of vecuronium from 0.1 to 0.2 mg/kg decreased the onset time from 3.8 +/- 0.3 minutes to 1.8 +/- 0.2 minutes (P less than 0.05). However, higher doses of vecuronium (0.3 or 0.4 mg/kg) did not result in further decreases in onset time. There were no significant differences in any hemodynamic parameter measured among the four groups in the anesthetized baseline state. Compared with the anesthetized state, the administration of vecuronium resulted in few alterations in hemodynamics within the groups studied. There were no changes in any hemodynamic parameter at 2 and 5 minutes following administration of 0.4 mg/kg of vecuronium. There were also no dose-related changes in any hemodynamic parameter. Thus, high doses of vecuronium of up to 0.4 mg/kg may be administered to patients with coronary artery disease with few hemodynamic changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a Halothane-Induced Reduction in Maximal Calcium-Activated Force in Mammalian Myocardium

In addition to their general anesthetic effects, the halogenated volatile anesthetics (e.g., halothane, enflurane, isoflurane) are known to significantly depress cardiac contractility. In the late 1960’s, Goldberg and Ullrick1 reported a dose-dependent and reversible depression of twitch force by halothane (as delivered by a calibrated vaporizer) in the range of 0.1% to 2.35%. They argued that “… halothane seems to exert its cardiac effects primarily by decreasing the intensity of the active state. …” In a comparative study of the effects of several general anesthetic agents on cardiac contractility, Brown and Crout2 reported similar results for halothane as well as for methoxyflurane. They, too, suggested that these agents exerted their cardio-depressive effects via a diminution of the active state of cardiac muscle. Although the concept of “active state” is no longer useful, it is known to be associated with the calcium transient; that is, the rapid rise and fall of intracellular free calcium concentration seen subsequent to electrical stimulation.