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H S Silverman

Publications and source records attributed to H S Silverman.

At least 37 records · Page 2Linked to original sources
PubMed1994

Sodium channel blockade reduces hypoxic sodium loading and sodium-dependent calcium loading.

BACKGROUND: Studies have shown that the rise in intracellular ionized calcium, [Ca2+]i, in hypoxic myocardium is driven by an increase in sodium, [Na+]i, but the source of Na+ is not known. METHODS AND RESULTS: Inhibitors of the voltage-gated Na+ channel were used to investigate the effect of Na+ channel blockade on hypoxic Na+ loading, Na(+)-dependent Ca2+ loading, and reoxygenation hypercontracture in isolated adult rat cardiac myocytes. Single electrically stimulated (0.2 Hz) cells were loaded with either SBFI (to index [Na+]i) or indo-1 (to index [Ca2+]i) and exposed to glucose-free hypoxia (PO2 < 0.02 mm Hg). Both [Na+]i and [Ca2+]i increased during hypoxia when cells became inexcitable following ATP-depletion contracture. The hypoxic rise in [Na+]i and [Ca2+]i was significantly attenuated by 1 mumol/L R 56865. Tetrodotoxin (60 mumol/L), a selective Na(+)-channel blocker, also markedly reduced the rise in [Ca2+]i during hypoxia and reoxygenation. Reoxygenation-induced cellular hypercontracture was reduced from 83% (45 of 54 cells) under control conditions to 12% (4 of 32) in the presence of R 56865 (P < .05). Lidocaine reduced hypercontracture dose dependently with 13% of cells hypercontracting in 100 mumol/L lidocaine, 42% in 50 mumol/L lidocaine, and 93% in 25 mumol/L lidocaine. The Na(+)-H+ exchange blocker, ethylisopropylamiloride (10 mumol/L) was also effective, limiting hypercontracture to 12%. R 56865, lidocaine, and ethylisopropylamiloride were also effective in preventing hypercontracture in normoxic myocytes induced by 75 mumol/L veratridine, an agent that impairs Na+ channel inactivation. Ethylisopropylamiloride prevented the veratridine-induced rise in [Ca2+]i without affecting Na(+)-Ca2+ exchange, suggesting that amiloride derivatives can reduce Ca2+ loading by blocking Na+ entry through Na+ channels, an action that may in part underlie their ability to prevent hypoxic Na+ and Ca2+ loading. CONCLUSIONS: Na+ influx through the voltage-gated Na+ channel is an important route of hypoxic Na+ loading, Na(+)-dependent Ca2+ loading, and reoxygenation hypercontracture in isolated rat cardiac myocytes. Importantly, the Na+ channel appears to serve as a route for hypoxic Na+ influx after myocytes become inexcitable.

Amiloride↗
PubMed1993

Impaired posthypoxic relaxation in single cardiac myocytes: role of intracellular pH and inorganic phosphate.

OBJECTIVE: The aim was to examine the effects of alterations in intracellular pH and inorganic phosphate concentration (known to influence myofilament kinetics and to change rapidly during hypoxia) on cell contraction, relaxation, and the Ca2+ transient in normoxic and hypoxic myocytes. METHODS: Single adult rat ventricular myocytes were electrically stimulated (0.2 Hz) and cell length (photodiode array), intracellular Ca2+ (indo-1 fluorescence), or intracellular pH (SNARF-1 fluorescence) measured. Hypoxia was induced in a special open chamber in which a laminar layer of argon prevented the back diffusion of atmospheric oxygen. RESULTS: Electrically stimulated contraction was preserved during exposure to hypoxia. At reoxygenation 10 minutes later the time from the stimulus to the peak of contraction (TPK) increased by 30(SEM 9)% and the time from the peak of contraction to 50% recovery of cell length (RT50) increased by 59(13)% relative to prehypoxic values (n = 8). These changes were not accompanied by a change in the kinetics of the Ca2+ transient. pHi fell from a baseline of 7.33(0.04) to 7.25(0.03) during hypoxia and then overshot to 7.44(0.03) at reoxygenation (n = 5). Since an intracellular alkalosis can slow myofilament relaxation, proton extrusion routes were blocked to examine posthypoxic relaxation in the absence of an alkalosis. Despite inhibition of the pHi overshoot, posthypoxic relaxation remained impaired. Intracellular inorganic phosphate levels were manipulated in two protocols (2-deoxyglucose to "trap" phosphate and Tris(hydroxymethyl)-aminomethane to buffer phosphate) and both TPK and RT50 increased in normoxic cells. Having established that these two interventions, which would be expected to decrease intracellular inorganic phosphate, result in a slowing of relaxation, myocytes were first phosphate loaded (exposed to 5.0 mM phosphate) and then made hypoxic and reoxygenated after 10 min to blunt the expected fall in phosphate accompanying reoxygenation. This led to a reduction in the slowing of contraction and relaxation following reoxygenation [TPK increased by 7(5)% and RT50 by 17(9)%, n = 8; p < 0.05 v cells studied in control buffer]. CONCLUSIONS: Impaired posthypoxic relaxation is not the result of changes in pHi but is attenuated by phosphate loading of cells and may be due to a rapid decrease in intracellular phosphate accompanying the resynthesis of high energy phosphates at reoxygenation.

Animals↗
PubMed1993

Repletion of sarcoplasmic reticulum Ca after ryanodine in rat ventricular myocytes.

The ryanodine (R)-induced loss of sarcoplasmic reticulum (SR) Ca2+ and the abilities of the SR to accumulate Ca2+ and participate in contractile activation after R were studied in rat ventricular myocytes. Indo 1 fluorescence (IF) indexed cytosolic Ca2+, and caffeine assayed SR Ca2+. Before R, there was a negative staircase, and the SR accumulated Ca2+ at rest. During stimulation (0.5 Hz), R decreased IF and contraction, converting the negative staircase to positive. When R was pulsed onto resting cells, IF increased and cells shortened, subsequently behaving as if stimulated in R. After R, there was no caffeine-releasable Ca2+ at rest, and little accumulated during 0.5-Hz stimulation. At high rates, caffeine-releasable Ca2+ and diastolic IF increased. In isoproterenol and R, IF transients and contractions recovered at 0.5 Hz with a marked positive staircase and little diastolic IF increase. Within 10 beats, SR Ca2+ accumulated to pre-R levels. R eliminated the positive inotropic effect of paired-pulse stimulation, but isoproterenol temporarily restored it. Twitch contractions in thapsigargin, an SR Ca2+ pump blocker, and isoproterenol were slow compared with control or R + isoproterenol. R leaks SR Ca2+ into the cytosol. SR Ca2+ can be repleted in R by high-rate stimulation or by low-rate stimulation with a beta-adrenergic agonist. SR Ca2+ release in R can be temporarily restored if Ca2+ influx and SR Ca2+ pumping are increased enough to overcome the SR Ca2+ leak.

Animals↗
PubMed1992

Cytosolic calcium and myofilaments in single rat cardiac myocytes achieve a dynamic equilibrium during twitch relaxation.

1. Single isolated rat cardiac myocytes were loaded with either the pentapotassium salt form or the acetoxymethyl ester (AM) form of the calcium-sensitive fluorescent probe, Indo-1. The relationship of the Indo-1 fluorescence transient, an index of the change in cytosolic calcium [Ca2+]i concentration, to the simultaneously measured cell length during the electrically stimulated twitch originating from slack length at 23 degrees C was evaluated. It was demonstrated that even if the Ca2+ dissociation rate from Indo-1 was assumed to be as slow as 10 s-1, the descending limb ('relaxation phase') of the Indo-1 fluorescence transient induced by excitation under these conditions is in equilibrium with the [Ca2+]i transient. Additionally, the extent of Indo-1 loading employed did not substantially alter the twitch characteristics. 2. A unique relationship between the fluorescence transient and cell length was observed during relaxation of contractions that varied in amplitude. This was manifest as a common trajectory in the cell length vs. [Ca2+]i phase-plane diagrams beginning at the time of cell relengthening. The common trajectory could also be demonstrated in Indo-1 AM-loaded cells. The Indo-1 fluorescence-length relation defined by this common trajectory is steeper than that described by the relation of peak contraction amplitude and peak fluorescence during the twitch contractions. 3. The trajectory of the [Ca2+]i-length relation elicited via an abrupt, rapid, brief (200 ms) pulse of caffeine directly onto the cell surface or by 'tetanization' of cells in the presence of ryanodine is identical to the common [Ca2+]i-length trajectory formed by electrically stimulated contractions of different magnitudes. As the [Ca2+]i and length transients induced by caffeine application or during tetanization in the presence of ryanodine develop with a much slower time course than those elicited by electrical stimulation, the common trajectory is not fortuitous, i.e. it cannot be attributed to equivalent rate-limiting steps for the decrease of [Ca2+]i and cell relengthening. 4. The [Ca2+]i-length relation defined by the common trajectory shifts appropriately in response to perturbations that have previously been demonstrated to alter the steady-state myofilament Ca2+ sensitivity in skinned cardiac fibres. Specifically, the trajectory shifts leftward in response to an acute increase in pH or following the addition of novel myofilament calcium-sensitizing thiadiazinone derivatives; a rightward shift occurs in response to an acute reduction in pH or following the addition of butanedione monoxime.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Cytosolic pH measurements in single cardiac myocytes using carboxy-seminaphthorhodafluor-1.

This study examines the use of carboxy-seminaphthorhodafluor-1 (C-SNARF-1) as an indicator of cytosolic pH in isolated rat cardiac myocytes. The emission spectrum of C-SNARF-1 when excited at 530 nm contains two well-separated peaks at approximately 590 and 640 nm, corresponding to the acidic and basic forms of the indicator. This spectral feature allows the indicator to be used in the single excitation, dual emission ratio mode. When C-SNARF-1 is loaded into rat cardiac myocytes as the membrane permeant ester derivative, C-SNARF-1/AM, the indicator localizes within the cytosol with virtually no partitioning into the mitochondria. C-SNARF-1 does not load into isolated mitochondria in suspension. There was no evidence for the presence of non-deesterified C-SNARF-1 within the cells. C-SNARF-1 can be calibrated in situ using a technique that abolishes all transsarcolemmal pH gradients. A 0.7-unit shift in the apparent pK (pKapp = pK-log10) between the in vitro calibration and the in situ calibration is consistent with a change in beta (I640 to pH 9/I640 at pH 5) in the cytosolic environment (beta in situ/beta in vitro = 0.21) and not a change in the true pK of the indicator. The contribution of cellular autofluorescence to the total signal can be made negligible. There is no effect of C-SNARF-1 on the contractile properties of rat cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Dimethylthiourea, an oxygen radical scavenger, protects isolated cardiac myocytes from hypoxic injury by inhibition of Na(+)-Ca2+ exchange and not by its antioxidant effects.

Myocardial reoxygenation injury may be attenuated by oxygen free radical scavengers, arguing for a role of oxygen radicals in this process. To determine whether free radical scavengers affect reoxygenation injury in isolated cardiac myocytes, resting rat ventricular myocytes were exposed to hypoxic (PO2 less than 0.02 mm Hg) glucose-free buffer alone (n = 50) or with the addition of the oxygen radical scavengers 1,3-dimethyl-2-thiourea (DMTU, 25 mM, n = 46), human recombinant superoxide dismutase (SOD, 1,000 units/ml, n = 40), or the combination of these agents (n = 41). All cells responded by undergoing contracture to a rigor form. Hypoxia was then continued for a second period (T2), the duration of which correlates inversely with survival. After reoxygenation, cells either retained their rectangular shape (survival) or hypercontracted to a rounded form (death). For the group of cells with a T2 period greater than 30 minutes, no cell exposed to buffer alone (n = 20) or to SOD (n = 16) survived, in contrast to 15 of 24 (63%) cells exposed to DMTU. The addition of SOD to DMTU offered no advantage to DMTU alone. The protective effect of DMTU was not observed when it was added at reoxygenation, suggesting that this agent has an important effect during the hypoxic period when intracellular Ca2+ is known to rise, most likely because of the reversal of Na(+)-Ca2+ exchange. Therefore, the effects of DMTU on Ca2+ regulation (indexed by indo-1 fluorescence) during hypoxia were studied. DMTU significantly blunted the [Ca2+] rise during the hypoxic period.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Dependence of hypoxic cellular calcium loading on Na(+)-Ca2+ exchange.

Na(+)-Ca2+ exchange has been shown to contribute to reperfusion- and reoxygenation-induced cellular Ca2+ loading and damage in the heart. Despite the fact that both [Na+]i and [Ca2+]i have been documented to rise during ischemia and hypoxia, it remains unclear whether the rise in [Ca2+]i occurring during hypoxia is linked to the rise in [Na+]i via Na(+)-Ca2+ exchange before reoxygenation and how this relates to cellular injury. Single electrically stimulated (0.2 Hz) adult rat cardiac myocytes loaded with Na(+)-sensitive benzofuran isophthalate (SBFI), the new fluorescent probe, were exposed to glucose-free hypoxia (PO2 less than 0.02 mm Hg), and SBFI fluorescence was monitored to index changes in [Na+]i. Parallel experiments were performed with indo-1-loaded cells to index [Ca2+]i. The SBFI fluorescence ratio (excitation, 350/380 nm) rose significantly during hypoxia after the onset of ATP-depletion contracture, consistent with a rise in [Na+]i. At reoxygenation, the ratio fell rapidly toward baseline levels. The indo-1 fluorescence ratio (emission, 410/490 nm) also rose only after the onset of rigor contracture and then often showed a secondary rise early after reoxygenation at a time when [Na+]i fell. The increase in both [Na+]i and [Ca2+]i, seen during hypoxia, could be markedly reduced by performing experiments in Na(+)-free buffer. These experiments suggested that hypoxic Ca2+ loading is linked to a rise in Na+i via Na(+)-Ca2+ exchange. To show that Na(+)-Ca2+ exchange activity was not fully inhibited by profound intracellular ATP depletion, cells were exposed to cyanide, and then buffer Na+ was abruptly removed after contracture occurred. The sudden removal of buffer Na+ would be expected to stimulate cell Ca2+ entry via Na(+)-Ca2+ exchange. A large rapid rise in the indo-1 fluorescence ratio ensued, which was consistent with abrupt cell Ca2+ loading via the exchanger. The effect of reducing hypoxic buffer [Na+] on cell morphology after reoxygenation was examined. Ninety-five percent of cells studied in a normal Na(+)-containing buffer (144 mM NaCl, n = 38) and reoxygenated 30 minutes after the onset of hypoxic rigor underwent hypercontracture. Only 12% of cells studied in Na(+)-free buffer (144 mM choline chloride, n = 17) hypercontracted at reoxygenation (p less than 0.05). Myocytes were also exposed to hypoxia in the presence of R 56865, a compound that blocks noninactivating components of the Na+ current. R 56865 blunted the rise in [Na+]i typically seen after the onset of rigor, suggesting that Na+ entry may occur, in part, through voltage-gated Na+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Relation of mitochondrial and cytosolic free calcium to cardiac myocyte recovery after exposure to anoxia.

Mitochondrial calcium overload has been suggested as a marker for irreversible injury in the ischemic heart. A new technique is used to measure dynamic changes in mitochondrial free calcium concentration ([Ca2+]m) in electrically stimulated (0.2 Hz) adult rat cardiac myocytes during exposure to anoxia and reoxygenation. Cells were incubated with indo-1 AM, which distributes in both the cytosol and mitochondria. After Mn2+ quenching of the cytosolic signal, cells were exposed to anoxia, and the residual fluorescence was monitored. [Ca2+]m averaged 94 +/- 3 nM (n = 16) at baseline, less than the baseline diastolic cytosolic free calcium concentration ([Ca2+]c, 124 +/- 4 nM, n = 12), which was measured in cells loaded with the pentapotassium salt of indo-1. [Ca2+]m and [Ca2+]c rose steadily only after the onset of ATP-depletion rigor contracture. At reoxygenation 35 minutes later, [Ca2+]c fell rapidly to preanoxic levels and then often showed a transient further rise. In contrast, [Ca2+]m showed only a slight transient fall and a secondary rise at reoxygenation. At reoxygenation, cells immediately either recovered, demonstrating partial relengthening and retaining their rectangular shape and response to stimulation, or they hypercontracted to rounded dysfunctional forms. Recovery occurred only in cells in which [Ca2+]m or [Ca2+]c remained below 250 nM before reoxygenation. Early during reoxygenation, [Ca2+]m remained higher in cells that hypercontracted (305 +/- 36 nM) than in cells that recovered (138 +/- 9 nM, p less than 0.05), whereas [Ca2+]c did not differ between the two groups (156 +/- 10 versus 128 +/- 10 nM, respectively; p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Contrasting effects of anoxia and graded hypoxia on single cardiac myocyte function.

The hypoxic cardiac myocyte has been used as a simplified model of myocardial ischemia. Precise control of oxygen tension is possible in a system with limited diffusion barriers offering an advantage over tissue preparations. This system was employed to study the effects of graded reductions in oxygen tension on cell morphology, spontaneous and electrically stimulated mechanical activity, and [Ca2+]i in single adult rat cardiac myocytes. All of 10 resting myocytes exposed to glucose-free anoxia (pO2 < .02 torr) abruptly underwent rigor contracture, retaining a clear sarcomere pattern, following a lag period of 22.6 +/- 2.8 minutes. These cells relengthened at reoxygenation 5 minutes following rigor onset. In contrast, 5 of 12 cells exposed to graded hypoxia (1-3 torr) were partially rounded and displayed a disorganized sarcomere pattern during hypoxic exposure and further shortened at reoxygenation (p = .03). Spontaneous mechanical oscillations thought to result from spontaneous sarcoplasmic reticulum calcium cycling only developed in those cells exposed to hypoxia, and their frequency was markedly enhanced in 6 of the 12 exposed cells. None of the 10 cells exposed to anoxia showed an increase in spontaneous activity. When spontaneous mechanical activity and underlying Ca2+i oscillations were induced by raising buffer [Ca2+] in 3 indo-1 loaded myocytes, anoxia abolished these as well. Cells stimulated at 0.2 Hz demonstrated spontaneous calcium oscillations and a significant rise in [Ca2+]i (indo-1 fluorescence ratio) prior to rigor onset only when exposed to graded hypoxia. Thus moderate hypoxia may cause earlier calcium loading and more progressive cell destruction than occurs during anoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Study of the mechanisms of hydrogen peroxide and hydroxyl free radical-induced cellular injury and calcium overload in cardiac myocytes.

There is evidence that myocardial injury, as would occur on post-ischemic reperfusion, may be caused by the generation of oxygen radicals, as well as by the induction of intracellular calcium overload; however, the relationship between these two mechanisms of injury is not known. To test the hypothesis that oxidants and oxygen radicals can cause cardiac myocyte injury and intracellular calcium overload, isolated adult rat ventricular myocytes were exposed to H2O2 (1-10 mM) and Fe3(+)-nitrilotriacetate. EPR measurements confirmed the production of the highly reactive .OH radical by this system. The oxygen radical generating system initially caused a transient augmentation of twitch amplitude in single field stimulated myocytes. This was followed by contractile oscillations occurring during the twitch prior to full cell relaxation, and spontaneous mechanical oscillations occurring between electrically stimulated contractions. Eventually, cells became inexcitable and abruptly underwent contracture. In the presence of lower bathing calcium concentrations, these oxidant-induced alterations were prevented or delayed. However, cells exposed to the radical generating system in the absence of extracellular calcium still eventually underwent contracture but stimulated contractions or mechanical oscillations were not seen. Measurements in single myocytes loaded with the fluorescent probe of intracellular calcium, Indo-1, demonstrated a rise in both systolic and diastolic fluorescence ratio, as well as oscillations and widening of the fluorescence transient, suggestive of cellular calcium loading, following exposure to the radical generating system. Injured myocytes did not take up trypan blue dye. Contractile dysfunction and calcium channel blocker, nitrendipine. NMR measurements of cellular [ATP] demonstrated that these alterations in cellular calcium preceded the depletion of ATP. Subsequent depletion of ATP was accompanied by the appearance of increased concentrations of sugar phosphates indicative of a block in glycolysis and ATP depletion correlated with cellular rigor. Thus, oxygen free radicals can cause cardiac myocyte injury with contractile abnormalities which occur due to myocyte calcium loading. The mechanism of oxidant-induced calcium loading is not due to nonspecific membrane damage, or energy depletion, but rather due to increased calcium influx through voltage gated calcium channels. This early calcium overload state as well as oxidant induced block of glycolysis result in cellular energy depletion and cell death with the induction of contracture.

Adenosine Triphosphate↗
PubMed1991

Measurement of mitochondrial free Ca2+ concentration in living single rat cardiac myocytes.

A technique that allows the continuous measurement of mitochondrial free Ca2+ ([Ca2+]m) in a single living cardiac myocyte is described. It involves the introduction of the fluorescent chelating agent indo-1 into the cell by exposure to the acetoxymethyl ester, followed by selective quenching of the fluorescence of indo-1 in the cytosol by Mn2+. The identity of the remaining fluorescence due to intramitochondrial indo-1 is established by its resistance to treatment of the cell with digitonin at concentrations that release cytosolic but not mitochondrial enzymes and by the finding that ruthenium red and carbonyl cyanide p-trifluoromethoxyphenylhydrazone prevent its response to elevated cytosolic free Ca2+ ([Ca2+]c). [Ca2+]m is found to be low (less than 100 nM) in unstimulated cells and to rise in procedures that chronically elevate [Ca2+]c, such as Na+ replacement. The gradient [Ca2+]m/[Ca2+]c is less than unity at values of [Ca2+]c of less than 500 nM but rapidly increases at higher values of [Ca2+]c. Although there is no detectable increase in [Ca2+]m during a single electrical stimulation, [Ca2+]m increases up to 600 nM as the pacing frequency is raised to 4 Hz in the presence of norepinephrine; this increase occurs over the course of many contractions. It is concluded that these findings are consistent with an increase in [Ca2+]m acting as a signal to increase dehydrogenase activity, and hence flux through oxidative phosphorylation, in response to increased work loads.

Animals↗
PubMed1991

A cellular mechanism for impaired posthypoxic relaxation in isolated cardiac myocytes. Altered myofilament relaxation kinetics at reoxygenation.

Single, isolated rat ventricular myocytes were made hypoxic for 10 minutes and then reoxygenated. During hypoxia, there was a marked abbreviation of the mechanical twitch, without a decrease in its amplitude. Immediately after reoxygenation, both the time to peak shortening and the duration of relaxation were markedly prolonged, and they remained prolonged for 10-50 minutes. The alterations in contraction and relaxation were not associated with any change in the time course of either the transmembrane action potential or the cytosolic calcium transient, as recorded with the fluorescent probe indo 1. Intracellular pH, measured with a fluorescent probe (carboxyseminaphthorhodofluor), showed an acid shift during hypoxia and an alkaline rebound immediately after reoxygenation. The time courses of intracellular pH and contraction duration were not parallel during hypoxia or reoxygenation, and simulation of the alkaline pH shift by lowering PCO2 or superfusing NH4Cl (in the absence of exposure to hypoxia) did not quantitatively reproduce the prolongation of relaxation seen after reoxygenation. The prolongation of contraction after reoxygenation could be overridden by the beta-adrenergic agonist isoproterenol or the nonenzymatic phosphatase butanedione monoxime. We conclude that delayed relaxation after reoxygenation exists at the single cell level and is due to an alteration of the properties of the myofilaments. Intracellular pH is not the primary mediator of this alteration. We speculate that alteration of intracellular inorganic phosphate or covalent modification of the myofilaments might be involved.

Actin Cytoskeleton↗
PubMed1991

Nicardipine prevents calcium loading and "oxygen paradox" in anoxic single rat myocytes by a mechanism independent of calcium channel blockade.

The protective effect of nicardipine (1 and 4 microM) against reoxygenation injury was studied in an unstimulated rat single myocyte oxygen paradox model in comparison with control (no drug) or nifedipine (1 microM). Either concentration of nicardipine was strongly protective, approximately doubling the duration of ATP depletion (rigor) that cells could withstand without undergoing hypercontracture when reoxygenated. Nifedipine (1 microM), which matched the negative inotropic effect of nicardipine (4 microM) (as measured by extent of shortening when stimulated), had no protective effect against reoxygenation injury. Neither drug affected the time to rigor, which is a measure of the rate at which the resting cell consumes its endogenous glycogen stores during anaerobic metabolism. Intracellular calcium, measured with the fluorescent probe indo-1, which partitions into both cytosol and mitochondria, rose progressively throughout the rigor period. This rise in calcium was almost totally suppressed by nicardipine (1 microM) but was unaffected by nifedipine. We conclude that nicardipine possesses a direct protective effect on the myocardium not shared by all dihydropyridines. This effect is associated with the prevention of intracellular, and probably mitochondrial, calcium loading but is probably not due to blockade of the L-type calcium channel or reduction of metabolic rate.

Animals↗
PubMed1990

Pharmacokinetics of recombinant human superoxide dismutase.

Superoxide dismutase (SOD) disposition was studied in order to design a rational approach for drug administration in the setting of acute myocardial infarction. Four chronically instrumented conscious dogs received the following dosage regimens of recombinant human SOD (rhSOD) on successive days: (a) 5 mg/kg left atrial (LA) bolus, (b) 5 mg/kg central vein (CV) bolus, (c) 15 mg/kg CV bolus, and (d) 5 mg/kg CV infusion over 60 min; additionally, all dogs received (e) a 5 mg/kg CV bolus under pentobarbital anesthesia. Serial serum samples were obtained after each dose and serial myocardial samples were obtained after dose (e). The serum rhSOD concentration was measured by radioimmunoassay and the data were fit to a two-compartment model. The distribution half-life was 7.8 +/- 1.7 min (mean +/- SEM), and the elimination half-life was 51.1 +/- 5.9 min; the central compartment volume of distribution (Vc) was 81 +/- 26 ml/kg and the steady-state volume of distribution was 156 +/- 20 ml/kg. The dosage regimen had no influence on clearance rates. Peak plasma concentrations (micrograms/ml) for the dosage regimens were (a) 65 +/- 28, (b) 89 +/- 19, (c) 214 +/- 61, (d) 20 +/- 5, and (e) 86 +/- 9. The peak level following continuous infusion did not occur until 50 min of infusion and was only one-fourth of the level achieved with a bolus of the same dose. Myocardial levels were less than 1% of serum levels, suggesting negligible rhSOD penetration into the myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Anoxic contractile failure in rat heart myocytes is caused by failure of intracellular calcium release due to alteration of the action potential.

Anoxia of the heart causes failure of contraction before any irreversible injury occurs; the mechanism by which anoxia blocks cardiac excitation-contraction coupling is unknown. Studies in whole muscle are confounded by heterogeneity; however, achieving the low oxygen tensions required to study anoxia in a single myocyte during electrophysiological recording has been a barrier in experimental design. Guided by calculations of oxygen transport, we developed a system to insulate myocytes in an open dish from oxygen by a laminar counterflowing argon column, permitting free access to the cell by microelectrodes while maintaining a PO2 less than 0.02 torr (1 torr = 133 Pa). In the absence of glucose, the amplitude of stimulated contraction of anoxic ventricular myocytes fell to zero over 2 min after a lag period attributable to the consumption of endogenous glycogen. The cytosolic calcium concentration transient, measured by indo-1 fluorescence, fell to zero simultaneously with contraction. After the twitch had failed, microinjection of caffeine around the cell still caused a large calcium release and contraction, indicating that sarcoplasmic reticular calcium stores were not depleted. Twitch failure was accompanied by shortening and then failure of the action potential; under voltage clamp, large outward currents, reversing at the resting potential, developed during contractile failure. After failure of action potential-mediated contraction, voltage-clamp depolarization, with a large command voltage to compensate for the series-resistance error due to outward currents, restored normal twitch contraction. We conclude that anoxic contractile failure in the rat myocyte is due to alteration of the action potential and the distal pathways of excitation-contraction coupling remain essentially intact.

Action Potentials↗