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Biomedical subjects

T J Blanck

Publications and source records attributed to T J Blanck.

At least 19 recordsLinked to original sources
PubMed1992

Spectral analysis of cytochromes in rat heart myocytes: transient and steady-state photodiode array spectrophotometry measurements.

Myocytes prepared from rat heart have been studied by optical spectroscopy using a photodiode array spectrophotometer adapted to a stopped flow apparatus (PASF). The isolated cells were viable for 3-4 h (i.e., over the total time of the experiments), as tested employing morphological parameters of cell damage, reactivity toward trypan blue, and the ability to use succinate in the absence and presence of digitonin. Respiration was activated by addition of sodium ascorbate and tetramethyl-para-phenylenediamine (TMPD) as exogenous reductants, in order to single out the contributions of cytochrome c and cytochrome c oxidase among the complexes of the mitochondrial respiratory chain. TMPD was shown to be freely permeable across cytoplasmic and mitochondrial membranes, with a measured KD = 0.9 mM. The use of singular value decomposition analysis coupled to PASF acquisition proved very powerful in resolving statically and kinetically, in the millisecond time region, the spectral contributions of the cytochromes. Spectral analysis was improved by adding carbon monoxide at concentrations which did not affect cytochrome c oxidase activity, but kept myoglobin fully saturated (and thus uninfluential to absorbance changes).

Animals↗
PubMed1992

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 greater than 250% of control values (control Bmax 11.16 +/- 0.98; ischemic 28.35 +/- 2.78 fmol/mg protein; p less than 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

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)

Animals↗
PubMed1992

Halothane, enflurane, and isoflurane stimulate calcium leakage from rabbit sarcoplasmic reticulum.

The sarcoplasmic reticulum (SR) controls uptake and release of Ca2+ in muscle. Little information is available regarding the effect of volatile anesthetics on Ca2+ release from SR isolated from normal skeletal muscle, even though an abnormality of Ca2+ handling is implicated in malignant hyperthermia. In this study we used a Ca2+ electrode to monitor continuously the release of Ca2+ from SR and the effect of volatile anesthetics on this process. We found that halothane, enflurane, and isoflurane at 0.6, 0.7, and 0.8 vol%, respectively, each increased the velocity of Ca2+ leakage by at least 150% when compared to control. Ruthenium red, a blocker of the SR Ca(2+)-release channel, was shown to have no effect on the velocity of Ca2+ leakage. Halothane and isoflurane both shortened the time at which Ca2+ leakage began (T) in a dose-dependent fashion. Halothane at 4.8 vol% decreased T from 293 +/- 21 s to 149 +/- 20 s. Isoflurane (4.8 vol%) decreased T to 203 +/- 16 s, and enflurane at 5 vol% had little effect, decreasing T to 259 +/- 19 s. We noted a marked stimulation in the ATPase activity of the SR by all three volatile anesthetics. Halothane at 0.63 vol%, isoflurane at 0.42 vol%, and enflurane at 0.62 vol% each increased ATPase activity by at least 300%. We conclude that the stimulation of the velocity of Ca2+ leakage by the volatile anesthetics is related to the more rapid depletion of ATP, but that the shortening of the onset of Ca2+ leakage is a independent phenomenon with a markedly different dose dependence.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1992

Depression of calcium channel blocker binding to rat brain membranes by halothane.

The present study evaluates the action of volatile anesthetics on the voltage-dependent Ca2+ channels in isolated rat brain membranes, measured as changes in binding of the Ca2+ channel blocker [3H]isradipine to these membranes. Equilibrium binding studies with increasing concentrations of [3H]isradipine (0.01-1 nM) in the presence of halothane (1.9%), isoflurane (2.3%), and enflurane (4.8%) at 25 degrees C were performed. Only halothane produced a significant depression in the specific binding of isradipine to the brain membranes at 0.5 and 1.0 nM [3H]isradipine (P = 0.028 and 0.018, respectively). Isoflurane and enflurane had such inconsistent effects that the data were inconclusive. Halothane produced a significant dose-dependent inhibition of binding, the maximum inhibition being 44% (P less than 0.005). Nonlinear regression analysis fit of the binding data indicates halothane produced a 48% decrease (P less than 0.05) in the maximal number of binding sites (Bmax) with no effect on the dissociation constant (Kd). As voltage-dependent Ca2+ channels are important in mediating neurotransmission, the marked decrease in channel number (Bmax) associated with halothane exposure suggests that this phenomenon might be related to the mechanism of general anesthesia.

Animals↗
PubMed1991

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)

Adult↗
PubMed1991

Volatile anesthetics depress calcium channel blocker binding to bovine cardiac sarcolemma.

Volatile anesthetics produce their negative inotropic effect on the heart mainly by interference with calcium homeostasis in the myocardial cell. In order to elucidate the mechanism of the depression, we have evaluated the effect of the volatile anesthetics on the binding of the calcium channel blocker [3H]nitrendipine to purified bovine cardiac sarcolemma. The radioligand binding studies were carried out at 25 degrees C, with increasing concentrations of [3H]nitrendipine (0.01-1 nM), in the presence or absence of unlabeled nitrendipine to determine specific binding, and with or without 1.9% halothane, 2.3% isoflurane, and 4.8% enflurane. Separately, [3H]nitrendipine was measured in the presence of increasing doses of halothane (0.78, 1.33, 1.90, and 2.57%). Kinetic studies of association and dissociation rate were performed with 1.90% halothane and 1 nM [3H]nitrendipine at different time intervals. All three volatile anesthetics produced depression of [3H]nitrendipine binding to the isolated cardiac sarcolemma. Only halothane produced a significant depression in binding, ranging between 59 and 66% (P less than 0.05), depending on the concentration of [3H]nitrendipine used. Isoflurane produced 29-38% depression, and enflurane produced 5-22% depression. Halothane also produced a significant (P less than 0.01) dose-dependent decrease in [3H]nitrendipine-specific binding. The kinetic binding experiments demonstrated that the time course for halothane's effect on association and dissociation of [3H]nitrendipine was 5 min for the half-maximum effect; the maximal reduction in binding capacity was at 15-30 min (P less than 0.05). Scatchard analysis revealed that all three volatile anesthetics produced reduction in the maximal number of binding sites; however, they varied in their effect on binding affinity. Only halothane produced a homogenous increase in the dissociation constant, signifying reduced affinity of the Ca2+ blocker to the channel. We suggest that the volatile anesthetics produce conformational changes in these channels consistent with their ability to depress channel-mediated Ca2+ influx into myocytes.

Anesthetics↗
PubMed1991

Halothane depresses D600 binding to bovine heart sarcolemma.

Volatile anesthetics exert their negative inotropic effects by interfering with Ca2+ homeostasis in the myocardial cell. The mechanism of this dose-dependent action is uncertain. 3H-D600 (3H-Gallopamil), a Ca(2+)-channel antagonist, binds to the voltage-dependent Ca2+ channels (VDCC) in a specific, saturable, and reversible manner. We used this ligand to study the effect of halothane on the binding characteristics of the VDCC in purified bovine heart sarcolemma. Cardiac sarcolemmal vesicles were isolated from fresh bovine heart by differential centrifugation and filtration. 3H-D600 equilibrium binding assays were performed in the presence or absence of 1.0 mM unlabeled D600 to determine total and nonspecific binding in room air and at 0.7, 1.3, and 2.5% (vol/vol) halothane. Halothane produced a significant dose-dependent and reversible depression of 3H-D600 specific binding in bovine heart sarcolemma. Depression was completely reversed when halothane had evaporated from the samples prior to filtration. Halothane 1.3% (vol/vol) produced a 40% reduction in the maximum binding capacity. The dissociation constant was not affected by any concentration of halothane. One mechanism by which the volatile anesthetics may induce negative inotropism is through the reduction of functional VDCCs in the heart, leading to reduction of Ca2+ entry. The results of this study support this hypothesis.

Animals↗
PubMed1990

Volatile anesthetic effects on left ventricular relaxation in swine.

The effects of halothane (0.5, 1.0, and 1.5%; n = 10), enflurane (1.0, 2.0, and 3.0%; n = 8), and isoflurane (0.75, 1.5, and 2.25%; n = 8) on isovolumic relaxation were studied in open-chest swine. The time constant for isovolumic left ventricular pressure decline, T, was determined at each anesthetic concentration at the intrinsic heart rate and during atrial pacing to 150 beats per min. The effect of increased left ventricular afterload on T was investigated by partial occlusion of the thoracic aorta to raise the left ventricular systolic pressure to baseline in the presence of volatile anesthetics, and 20% above baseline in the absence of volatile anesthetics. Heart rate and left ventricular systolic pressure decreased substantially with all three anesthetics, whereas left ventricular end-diastolic pressure increased (by 3-4 mmHg). Relaxation time constants increased with all three anesthetics at the intrinsic heart rate; when the heart rate was controlled by pacing, T increased in the halothane and enflurane, but not in the isoflurane, experiments. T was significantly prolonged (by 30-100%) by partial aortic occlusion in the presence of anesthetic, but not in the control measurements. T did not change significantly in the isoflurane experiments when atrial pacing was employed with partial aortic occlusion. The volatile anesthetics, particularly halothane, seem to impair the relaxation process of the left ventricle; further investigation of the mechanisms of this interference, such as anesthetic effects on intracellular calcium movement and total left ventricular load, is warranted.

Animals↗
PubMed1989

The effect of halothane, enflurane, and isoflurane on the dynamic stiffness of rabbit papillary muscle.

The authors examined the effect of halothane, enflurane, and isoflurane on the dynamic stiffness of rabbit papillary muscles in Ba2+ contracture. Ca2+ was replaced by Ba2+ in order to constantly activate myofibrils. The dynamic stiffness of the contractured muscle was examined by exposing the muscle to sinusoidal length perturbations at frequencies of 0.05-30 Hz under two concentrations of anesthetic, approximately 0.5, and 1.5-2 mM, and at two Ba2+ concentrations, 0.5 and 1.5-2 mM. The anesthetics had no effect on the frequency (fmin) at which minimum stiffness occurred, but markedly decreased the stiffness modulus at high frequencies (Khi). The decrease in Khi was significant for all anesthetics at the P less than 0.05 level. Increasing the Ba2+ concentration from 0.5 to 1.5-2 mM in the presence of 0.5 mM of anesthetic resulted in a return of Khi to control levels. The authors conclude that halothane, enflurane, and isoflurane did not alter actin-myosin ATPase kinetics, because fmin was unchanged, but decreased the number of crossbridge interactions, because Khi was significantly decreased by all three anesthetics.

Animals↗
PubMed1989

Depression of myocardial force and stiffness without change in crossbridge kinetics: effects of volatile anesthetics reproduced by nifedipine.

The authors examined the effects of nifedipine, a sarcolemmal slow Ca2+ channel blocker, on dynamic stiffness and force of rabbit right ventricular trabeculum and papillary muscle in Ba2+ contracture, in an attempt to reproduce the effects of halothane, enflurane, and isoflurane on a similar preparation as reported by Shibata et al. Once barium contracture force was established, muscle length was perturbed with small amplitude sinusoidal oscillations in the frequency range of 0.1-100 Hz. Nifedipine 1 microM was then added to the superfusate and dynamic stiffness was again measured. Additional barium was used to determine restoration of contracture force to and beyond control levels. Nifedipine produced a significant decrease in contracture force and high-frequency stiffness with no effect on the frequency (fmin) at which stiffness amplitude exhibited a minimum (P less than 0.005). Contracture force and stiffness could be restored by adding additional barium to the nifedipine-treated muscles. These results are similar to those reported by Shibata et al. using volatile anesthetics. Since nifedipine, which acts specifically at the sarcolemmal slow Ca2+ channel, affects contracture force and dynamic stiffness in this preparation in a manner similar to the volatile anesthetics, the authors suggest that the anesthetics studied by Shibata et al. may well exert a significant component of their negative inotropic activity via their action on the sarcolemmal slow Ca2+ channel.

Anesthetics↗
PubMed1988

Halothane decreases calcium channel antagonist binding to cardiac membranes.

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 less than 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.

Animals↗
PubMed1988

Thiopental does not alter Ca2+ uptake by cardiac sarcoplasmic reticulum.

The effect of thiopental on Ca2+ uptake by cardiac sarcoplasmic reticulum (SR) isolated from the rabbit was examined to clarify the role of the sarcoplasmic reticulum in the negative inotropic action of thiopental. Thiopental, from 0 to 378 microM, did not alter the rate of Ca2+ uptake by the SR. We also compared the ATP dependence of Ca2+ uptake in the presence and absence of 284 microM thiopental. The Km for ATP and the Vmax of Ca+ uptake were unaffected by thiopental. It is concluded that thiopental does not alter Ca2+ uptake by the SR and that the negative inotropic effects of thiopental occur at other sites in the myocardial cell.

Animals↗
PubMed1987

The effect of volatile anesthetics on the pH dependence of calcium uptake by cardiac sarcoplasmic reticulum.

The effect of volatile anesthetics (VA) on the pH dependence of calcium uptake by cardiac sarcoplasmic reticulum (SR) was studied. SR was incubated at 37 degrees C with 45CaCl2 in the control state (no anesthetic) and in the presence of each of the VA from pH 6.6-7.6. The VA used were: halothane, 1.3%; enflurane, 1.8%; and isoflurane, 1.2%. In the control state, the initial rate of calcium uptake, measured after a 2-min incubation, was maximal at pH 6.8 (mean +/- SEM: 665 +/- 37 nmoles/mg) and markedly inhibited at pH 7.6 (107 +/- 9 nmoles/mg). In the presence of the VA, the calcium uptake rate was mildly depressed (7-32%) at pH 6.6-6.8, unchanged at pH 7.0, and greatly enhanced (52-78%) at pH 7.2-7.6, when compared to control. The maximal uptake of calcium by the SR at a calcium concentration of 10(-6)M, measured by a 20-min incubation, had a similar pH dependence in the control state, with a decline first evident at pH 7.2 and a 50% drop in the maximal uptake of calcium from pH 7.0-7.6. The presence of the VA was associated with a uniform depression of the maximal uptake of calcium by the SR at all pH levels measured. In view of these findings, it appears that pH does affect SR function in the presence of VA. This alteration of the pH effect by VA may be a factor responsible for discrepancies in results previously reported by investigators studying the effects of VA on the uptake of calcium by the SR.

Anesthetics↗
PubMed1987

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 less than 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 less than 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 less than 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 less than 0.01) and beta ATP (12 +/- 4%; P less than 0.05), and an increase in Pi (18 +/- 6%; P less than 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.

Adenosine Triphosphate↗