PubMed Health⌕ Search

Biomedical subjects

J J Lemasters

Publications and source records attributed to J J Lemasters.

At least 145 records · Page 8Linked to original sources
PubMed1991

Suppression of Ca2+ oscillations in cultured rat hepatocytes by chemical hypoxia.

The model of "chemical hypoxia" with KCN plus iodoacetic acid mimics the ATP depletion and reductive stress of hypoxia. Here, we examined the effects of chemical hypoxia on cytosolic free Na+ and Ca2+ in single cultured rat hepatocytes by multiparameter digitized video microscopy and ratio imaging of sodium-binding furan indicator (SBFI) and Fura-2. Intracellular Na+ increased from about 10 mM to more than 100 mM after 20 min of chemical hypoxia, whereas cytosolic free Ca2+ remained virtually unchanged. In normoxic hepatocytes, phenylephrine (50 microM) and Arg-vasopressin (20-40 nM) induced Ca2+ oscillations in 70 and 40% of cells, respectively. These Ca2+ oscillations were suppressed after one spike following the onset of chemical hypoxia. Phenylephrine and vasopressin also increased inositol phosphate formation by 22 and 147%, respectively. This effect was suppressed by KCN plus iodoacetate. Intracellular acidosis is characteristic of chemical hypoxia. Intracellular acidosis induced by 40 mM Na-acetate suppressed Ca2+ oscillations but did not inhibit hormone-induced inositol phosphate formation. Cytosolic alkalinization also suppressed Ca2+ oscillations. However, prevention of intracellular acidosis with monensin (10 microM) did not prevent suppression of Ca2+ oscillations during chemical hypoxia. Mitochondrial depolarization with uncoupler did not change free Ca2+ levels during chemical hypoxia, indicating that mitochondria do not regulate free Ca2+ during chemical hypoxia. From these results, we conclude: 1) chemical hypoxia does not block Na+ influx across the plasma membrane; 2) Chemical hypoxia inhibits hormone-stimulated Ca2+ flux pathways across cellular membranes by two different mechanisms: (a) by ATP depletion, which disrupts hormone-myo-inositol 1,4,5-triphosphate coupling, and (b) by intracellular acidosis, which inhibits myo-inositol 1,4,5-triphosphate-stimulated Ca2+ release from intracellular stores; 3) during ATP depletion by chemical hypoxia, mitochondria do not take up Ca2+ to maintain cytosolic free Ca2+ at low concentrations.

Animals↗
PubMed1991

Protection by acidotic pH against anoxia/reoxygenation injury to rat neonatal cardiac myocytes.

We assessed the effect of acidosis on cell killing during anoxia and reoxygenation in cultured rat neonatal cardiac myocytes. After 4.5 hours of anoxia and glycolytic inhibition with 2-deoxyglucose, loss of viability was greater than 90% at pH 7.4. In contrast, at pH 6.2-7.0, viability was virtually unchanged. To model changes of pH and oxygenation during ischemia and reperfusion, myocytes were made anoxic at pH 6.2 for 4 hours, followed by reoxygenation at pH 7.4. Under these conditions, reoxygenation precipitated loss of viability to about half the cells. When pH was increased to 7.4 without reoxygenation, similar lethal injury occurred. No cell killing occurred after reoxygenation at pH 6.2. We conclude that acidosis protects against lethal anoxic injury, and that a rapid return from acidotic to physiologic pH contributes significantly to reperfusion injury to cardiac myocytes - a 'pH paradox'.

Acidosis↗
PubMed1991

A pH-dependent phospholipase A2 contributes to loss of plasma membrane integrity during chemical hypoxia in rat hepatocytes.

Previous studies have suggested that alterations in phospholipid composition of plasma membranes may underlie lethal cell injury due to hypoxic and ischemic injury. The present study was designed to determine if such alterations are due to the activation of a pH-dependent phospholipase A2. Loss of cell viability and phospholipase A2 activity measured by arachidonic acid release increased in parallel during metabolic inhibition with KCN and iodoacetate (chemical hypoxia). Acidosis (pH 6.5) and the phospholipase inhibitors, dibucaine and mepacrine, delayed loss of cell viability and release of arachidonic acid to a similar extent. These findings suggest that a pH-dependent phospholipase A2 causes alterations in plasma membrane phospholipid composition after ATP-depletion which contribute to lethal cell injury.

Animals↗
PubMed1991

Methyl palmitate prevents Kupffer cell activation and improves survival after orthotopic liver transplantation in the rat.

The purpose of this study was to determine whether prevention of Kupffer cell activation following orthotopic liver transplantation improves postoperative survival. First, particle phagocytosis by Kupffer cells was monitored continuously from the uptake of colloidal carbon by the perfused liver. Unstored livers took up carbon at rates of around 150 mg/g per hour, whereas storage for 24 h in Euro-Collins solution nearly doubled values to about 290 mg/g per hour. Treatment of rats with methyl palmitate, an inhibitor of phagocytosis by Kupffer cells, reduced carbon uptake to about one-third to one-half of control values in unstored and stored livers, respectively. Oxygen uptake, which was increased about 25% in stored and unstored livers by infusion of colloidal carbon, was only increased 5%-10% in both groups following treatment with methyl palmitate, suggesting that Kupffer cell activation was prevented by methyl palmitate. In livers transplanted after storage for 6 h in Euro-Collins solution (nonsurvival conditions), control rats survived only about 12 h, while treatment with methyl palmitate increased survival time significantly--more than threefold--to about 40 h. These data are consistent with the hypothesis that activation of Kupffer cells following cold ischemic storage and reperfusion is an early event involved in liver graft failure.

Animals↗
PubMed1991

Effect of hepatotoxic chemicals and hypoxia on hepatic nonparenchymal cells: impairment of phagocytosis by Kupffer cells and disruption of the endothelium in rat livers perfused with colloidal carbon.

Kupffer cells play an important role in liver function and phagocytosis of foreign particles in the hepatic portal tract. Therefore, the purpose of this study was to investigate the influence of several hepatotoxic chemicals (allyl alcohol, ethylhexanol, and menadione) and hypoxia on phagocytic activity of Kupffer cells in perfused rat liver. A recently developed optical method was used to determine rates of phagocytosis of carbon particles by Kupffer cells in periportal and pericentral regions of the liver lobule based on changes in reflected light from the liver surface (te Koppele, J.M., and Thurman, R.G. 1990. Am. J. Physiol. 259, G814-G821). With all chemicals studied, a rapid (10-30 min) decline in the rate of phagocytosis preceded parenchymal cell death as assessed from release of lactate dehydrogenase. These chemicals impaired parenchymal cell energy status as indicated by inhibition of O2 uptake and bile flow prior to cell death. Livers swell when they are damaged, a process which increases perfusion pressure and could theoretically damage the endothelium and lead to nonspecific uptake of carbon. In perfusions with a hepatotoxic concentration of allyl alcohol (350 microM), carbon particles accumulated in swollen livers after 70 min of perfusion. Histological studies revealed that carbon particles were localized predominantly in periportal regions of the liver lobule in perfusions with all hepatotoxicants studied. When perfusion pressure was elevated to 20 cm H2O in the absence of hepatotoxicants, carbon particles detected optically accumulated in upstream regions of the liver lobule (periportal or pericentral regions in perfusions in the anterograde or retrograde directions, respectively). In scanning electron microscopy of nonswollen livers, the endothelium remained intact. In swollen livers, however, the endothelium was disrupted and carbon was detected bound nonspecifically to parenchymal cells. Fifteen minutes after addition of allyl alcohol, bile canaliculi were dilated and endothelial fenestrations were enlarged. After 2 hr of perfusion with allyl alcohol, hepatic ultrastructure was severely disrupted. Thus, it is concluded that perfusion with hepatotoxic chemicals or hypoxia results in a rapid decrease of particle phagocytosis by Kupffer cells followed by changes in endothelial cell ultrastructure.

1-Propanol↗
PubMed1991

Protection by acidotic pH against anoxic cell killing in perfused rat liver: evidence for a pH paradox.

Reperfusion of ischemic tissues causes a paradoxical injury. Here, we measured lactate dehydrogenase (LDH) release as an indicator of tissue damage in perfused rat livers during anoxia and reoxygenation. During anoxia, LDH release was substantially reduced at acidotic pH (pH 6.1-6.9). Using anoxia at pH 6.1 followed by reoxygenation at pH 7.3 to model ischemia and reperfusion, an abrupt release of LDH occurred after reperfusion. A similar release of LDH occurred when pH of anoxic livers was increased to 7.3 without reoxygenation but LDH release did not occur after reoxygenation at pH 6.1. Thus, a rapid increase of pH rather than reoxygenation accounted for tissue injury after reperfusion of ischemic liver.

Animals↗
PubMed1991

Lipid order in hepatocyte plasma membrane blebs during ATP depletion measured by digitized video fluorescence polarization microscopy.

Low-light digitized video fluorescence polarization microscopy was used to measure lipid order parameters in plasma membrane blebs of single, cultured rat hepatocytes during ATP depletion with the metabolic inhibitors cyanide and iodoacetic acid. Hepatocytes were labeled on the microscope stage with the plasma membrane probe trimethylammoniumdiphenylhexatriene at successive stages of cell injury. A pair of fluorescence polarization ratio images was obtained from a series of four fluorescence images recorded with a polarizer in the emission path oriented first parallel and then perpendicular to each of two orthogonal excitation light polarization directions. From the polarization ratio images, the lipid order parameter S was determined in individual plasma membrane blebs. Results indicate that the plasma membrane becomes uniformly rigid within a few minutes of the addition of metabolic inhibitors when small surface blebs have formed and ATP levels have fallen by greater than 95%. The measured order parameter of S approximately 0.95 in plasma membrane blebs, compared with S approximately 0.75 in normoxic cell plasma membranes, remained unchanged throughout the course of bleb development and ultimate cell death. These findings demonstrate that significant alteration in hepatocyte plasma membrane structure occurs early in hypoxic cell injury.

Adenosine Triphosphate↗
PubMed1991

Leukocyte adhesion and cell death following orthotopic liver transplantation in the rat.

The purpose of these experiments was to employ video microscopy to identify early pathological changes after orthotopic liver transplantation in the rat. Liver transplantation was performed using the cuff technique. Survival was greater than 90% when livers were stored for 1 hr in Ringer's solution prior to transplantation (survival conditions), whereas all rats died following storage of grafts for 4 hr in cold Euro-Collins solution (nonsurvival conditions). Postoperatively, each recipient animal was anesthetized, the abdomen was opened, and the liver was placed on the stage of an inverted fluorescence microscope equipped with a low-light ISIT video camera. Precautions were taken to prevent the liver surface from drying. The fluorescent dyes fluorescein sodium (1.0 mumol/kg), acridine orange (2.5 mumol/kg), and propidium iodide (1 mumol/kg) were injected intravenously to label hepatocytes, polymorphonuclear leukocytes, and the nuclei of irreversibly damaged cells, respectively. In livers from untransplanted rats, movement of labeled leukocytes through the hepatic sinusoids was smooth and rapid (velocity, 500-550 microns/sec) and margination (adhesion) of cells was minimal (less than 1%). After transplantation, however, velocity was diminished 2-3-fold, and margination was increased to 10% of leukocytes in survival groups and to 40% in nonsurvival groups 4 hr postoperatively. In the nonsurvival groups, irreversible cell death detected by propidium iodide fluorescence was minimal 15 min after transplantation--however, massive cell damage was detected 4 hr postoperatively. In addition, serum transaminases and necrosis were higher at 4 hr than 15 min postoperatively. Taken together, these data demonstrate that leukocyte margination increases following orthotopic liver transplantation and is followed rapidly by cell death. These events likely play a role in the mechanism of early graft failure following transplantation.

Animals↗
PubMed1991

Increase in survival of liver grafts after rinsing with warm Ringer's solution due to improvement of hepatic microcirculation.

Temperature increases membrane fluidity and decreases vascular resistance in isolated organs. Therefore, these studies were designed to determine if a rinse with warm buffer could increase survival time in the rat model of orthotopic liver transplantation by improving hepatic microcirculation. Brief periods of warm ischemia (3-8 min) did not damage the liver as indexed by minimal release of LDH. Survival of rats for 30 days was greater than 90% in this model when livers were stored for 1 hr in Ringer's solution; yet grafts stored for 8 hr in Euro-Collins solution and rinsed with 20 ml of cold (0-4 degrees C) Ringer's solution survived postoperatively only around 3 days. However, livers stored for 8 hr in Euro-Collins and rinsed with 20 ml of warm (37 degrees C) Ringer's survived longer than 30 days (i.e., permanently). Serum transaminase levels reached peak values around 6000 U/L one day postoperatively in the cold-rinsed group, and liver injury assessed histologically was substantial. Under these conditions, pulmonary infiltration of inflammatory cells was observed in about 23% of lung tissue examined and was associated with massive bleeding. Following a warm rinse, however, maximal SGOT levels and injury to both liver and lung were reduced significantly by 80-90% 24 hr postoperatively. Moreover, the warm rinse improved hepatic microcirculation. It accelerated blood flow into the liver approximately two-fold, as indexed by the half-time of changes in hemoglobin reflectance from the liver surface, improved the distribution of colloidal carbon in the organ observed macroscopically, and decreased vascular resistance by over 50%. These data support the hypothesis that a brief rinse of liver grafts with warm buffer markedly improves the hepatic microcirculation, leading to dramatic improvement in graft survival. This work demonstrates clearly that a brief warm rinse may be useful clinically in liver transplantation.

Animals↗
PubMed1991

Carolina rinse solution--a new strategy to increase survival time after orthotopic liver transplantation in the rat.

Recently, we described a new solution, Carolina rinse, that prevents nonparenchymal cell injury in vitro after reperfusion of livers stored in University of Wisconsin cold solution (Currin RT, Toole JG, Thurman RG, Lemasters JJ. Transplantation 1990; 50: 1076). The present study was designed to examine the effect of Carolina rinse on graft survival in vivo. Unlike UW cold storage solution, which is high in potassium, Carolina rinse contains extracellular inorganic ions at levels similar to blood, a calcium channel blocker and a radical scavenger. Carolina rinse also contains fructose and mildly acidotic pH to reduce hypoxic cell death. Livers from Lewis rats were explanted, stored in UW cold storage solution under nonsurvival conditions, and rinsed with either 15 ml of Ringer's, UW solution, Carolina rinse, or Carolina rinse saturated with nitrogen prior to completion of implantation surgery. In the Ringer's rinse group, only 4% of recipients survived 30 days postoperatively. In this group, SGOT levels reached maximal values of about 5000 U/L. Survival was also poor (25%) when grafts were rinsed with UW solution. In the Carolina rinse group, however, 9 of 16 rats (56%) survived indefinitely, and maximal postoperative SGOT levels were reduced 3-fold. Liver injury indexed histologically was also decreased about 3-fold by Carolina rinse compared with the control group rinsed with Ringer's solution. Carolina rinse diminished postoperative sinusoidal endothelial cell damage assessed by electron microscopy and reduced carbon particle phagocytosis due to Kupffer cells significantly. Moreover, Carolina rinse diminished graft swelling and improved postoperative hepatic microcirculation compared with the Ringer's rinse group. Taken together, these results indicate that Carolina rinse is a superior alternative to Ringer's solution in vivo to protect liver grafts from reperfusion injury when removing high-potassium-containing cold storage solutions clinically prior to implantation.

Adenosine↗
PubMed1991

Evidence that adenosine is a key component in Carolina rinse responsible for reducing graft failure after orthotopic liver transplantation in the rat.

Recently, a rinse solution, Carolina rinse, designed to minimize reperfusion injury following liver transplantation in the rat has been developed. When used to rinse cold-stored grafts prior to completion of implantation surgery, Carolina rinse improved postoperative survival dramatically. Here we report the results of studies designed to determine the key components of Carolina rinse. Livers were explanted, stored for 12 hr in cold UW solution (0-4 degrees C), and rinsed with 15 ml of Ringer's solution immediately prior to completion of implantation surgery. In this group, 12/13 rats died within 2 days (nonsurvival conditions). In a second group, explants stored under identical conditions were rinsed with 15 ml of Carolina rinse. Carolina rinse increased average 30-day survival time significantly to over 75%. Furthermore, when grafts were rinsed with Carolina rinse lacking nicardipine or with the pH increased to 7.4, long-term survival of recipient rats was also about 75% (i.e., the modifications did not affect survival). However, Carolina rinse lost its efficacy (12% survival) when adenosine was omitted. In addition, when donor livers were rinsed with Ringer's solution containing adenosine (0.1 mM), average survival time was increased from 8% to 63%. Rinsing with Ringer's solution containing higher concentrations of adenosine (5 mM), however, did not improve survival significantly. Survival was also not improved by rinsing with Ringer's containing 0.1 mM ribose and 0.1 mM adenine, substrates for ATP synthesis that are not vasoactive. SGOT values were around 3000 U/L 1-3 days postoperatively in the nonsurviving group rinsed with Ringer's solution alone. Values were decreased over 6-fold by Carolina rinse but were not reduced significantly by Ringer's solution containing adenosine. Thus, adenosine improves survival following liver transplantation without preventing parenchymal cell injury, indicating that adenosine may work via nonhepatic mechanisms. Liver injury was also assessed by electron microscopy. After either adenosine or Ringer's rinse, sinusoidal thrombi and polymorphonuclear margination were observed together with a pattern of pericentral hepatocellular vacuolization and disruption of the sinusoidal lining characteristic of changes observed following hypoxia. With Ringer's rinse, Kupffer cells exhibited surface irregularity in pericentral regions indicative of activation. Following adenosine rinse, however, Kupffer cells appeared more flattened with less ruffling and reduced surface debris (i.e., they were less activated). Carbon uptake by Kupffer cells was also decreased significantly by Ringer's rinse when adenosine was present. Furthermore, adenosine lowered intracellular free calcium concentration in cultured Kupffer cells and improved hepatic microcirculation postoperatively. Adenosine rinse also affected extrahepatic systems: it reduced postoperative clotting time and diminished lung injury significantly.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine↗
PubMed1991

Cytosolic free Ca2+ and proteolysis in lethal oxidative injury in endothelial cells.

Oxygen free radicals (OFR) are thought to mediate ischemia-reperfusion injury to endothelium of heart, lung, brain, liver, and kidney and contribute to development of atherosclerosis, pulmonary O2 toxicity, and adult respiratory distress syndrome. Increased cytosolic free Ca2+ (Cai2+) has been proposed as a mechanism of injury from oxidative stress, yet the pathways by which an increase in Cai2+ may cause OFR-mediated endothelial cell injury remain unknown. Using multiparameter digitized video microscopy and the fluorescent probes, fura-2 acetoxymethyl ester and propidium iodide, we measured Cai2+ and cell viability in human umbilical endothelial cells during oxidative stress with xanthine (50 microM) plus xanthine oxidase (40 mU/ml). Oxidative stress caused a sustained increase in Cai2+ from a resting level of 90-100 nM to near 500 nM, which was preceded by formation of plasma membrane blebs. The increase in Cai2+ was prevented by removal of extracellular Ca2+ (Cao2+). Prevention of the increase in Cai2+ was associated with prolonged cell viability. Readdition of Cao2+ resulted in an immediate large increase in Cai2+ and rapid onset of cell death. The protease inhibitors, leupeptin and pepstatin, delayed the increase in Cai2+ and prolonged cell viability. The results are consistent with the hypothesis that endothelial cell injury due to oxidative stress may be the result of Cai2+ influx and resultant activation of Ca(2+)-dependent proteases.

Calcium↗
PubMed1991

Kupffer cell activation and endothelial cell damage after storage of rat livers: effects of reperfusion.

Reperfusion injury characterized by loss of endothelial cell viability occurs after cold ischemic storage of livers for transplantation surgery. Here, ultrastructural changes in stored rat livers were examined by scanning and transmission electron microscopy. With increasing times of storage in Euro-Collins solution (4 to 24 hr) followed by 15 min of reperfusion at 37 degrees C, a sequence of structural alterations was observed involving endothelial and Kupffer cells. Widening of endothelial fenestrations occurred after 4 hr and progressed over 8 to 24 hr to retraction of cellular processes, ball-like rounding, sinusoidal denudation and ultrastructural derangements consistent with loss of cell viability. Kupffer cells exhibited progressive rounding, ruffling of the cell surface, polarization, appearance of wormlike densities, vacuolization and degranulation over a similar time course. By contrast, the structures of parenchymal and fat-storing cells were relatively undisturbed by cold storage and reperfusion. Alterations to endothelial and Kupffer cells were also studied as a function of time of reperfusion. After 24 hr of storage, endothelial cells showed retraction of cytoplasm before reperfusion that progressed quickly to loss of viability and denudation during reperfusion. Kupffer cell activation (ruffling, degranulation) during reperfusion was slower and occurred after deterioration of endothelial cells. Livers stored in Euro-Collins solution were also compared with livers stored in University of Wisconsin cold storage solution, an improved preservation medium for transplantation. University of Wisconsin solution provided better preservation of endothelial structure and markedly reduced parenchymal cell blebbing and swelling before reperfusion. University of Wisconsin solution also reduced Kupffer cell activation and release of lysosomal enzymes. In conclusion, endothelial cell deterioration followed by Kupffer cell activation occurred after increasing times of cold ischemic storage and reperfusion of rat livers. Both changes may contribute to the pathophysiology of graft failure caused by reperfusion-mediated storage injury.

Acid Phosphatase↗
PubMed1991

A new method to monitor Kupffer cell phagocytosis continuously in perfused rat liver.

A new method was developed to monitor Kupffer cell phagocytosis continuously in perfused liver using a fluorescent probe, rhodamine-gelatin, synthesized from gelatin and rhodamine isothiocyanate. In perfused rat liver, phagocytosis by Kupffer cells was assessed both by uptake of the dye and from fluorescence measured from the liver surface. Uptake of rhodamine-gelatin and surface fluorescence (520----585 nm) increased as perfusate concentrations of rhodamine-gelatin were elevated. Histological examination revealed that fluorescence caused by rhodamine-gelatin was concentrated in cells lining the sinusoid. Furthermore, when hepatic parenchymal, endothelial and Kupffer cells were isolated by centrifugal elutriation after pretreatment in vivo with rhodamine-gelatin, the fluorescent label was detected nearly exclusively in the Kupffer cell fraction. Fluorescence of rhodamine-gelatin from the liver surface was directly proportional to uptake of rhodamine-gelatin, indicating that measurement of surface fluorescence could be used as an index of phagocytic activity of Kupffer cells. Uptake of rhodamine-gelatin and fluorescent labeling of sinusoidal cells were inhibited nearly completely by dextran sulfate, which decreases phagocytic activity, and were increased by about 50% by pretreatment with endotoxin, which stimulates phagocytosis. This new method was combined with standard procedures to monitor parenchymal cell function (e.g., oxygen uptake and release of lactate dehydrogenase), Kupffer cell phagocytic activity and parenchymal cell injury simultaneously during perfusion with the hepatotoxicant allyl alcohol. Activation of phagocytosis by Kupffer cells monitored by rhodamine-gelatin fluorescence occurred within 5 min of addition of allyl alcohol. On the other hand, parenchymal cell injury, assessed from release of lactate dehydrogenase, did not begin until 40 min and reached maximal values at around 90 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Recovery of cultured rat neonatal myocytes from hypercontracture after chemical hypoxia.

Cardiac myocytes exposed to anoxia hypercontract into a blebbed, rounded mass. Such hypercontraction is usually considered a manifestation of irreversible injury. Here, we studied functional recovery, long-term viability and ATP levels of cultured neonatal rat cardiac myocytes after metabolic inhibition with cyanide and 2-deoxyglucose, a model of 'chemical hypoxia' which mimics the ATP depletion and reductive stress of hypoxia. After addition of inhibitors, 5-day cultured myocytes ceased spontaneous contractions within 1-2 min, blebbed and hypercontracted after 35 min, and lost viability after 100 min as assessed by nuclear labelling with propidium iodide. 11-day cultured myocytes exhibited a similar progression of injury. When the metabolic inhibitors were removed, spontaneous contractions resumed after an average of 11 min in non-hypercontracted myocytes. In hypercontracted myocytes, weak contractions resumed after 35 min, regardless of the length of time spent in the hypercontracted state, but restoration of strong synchronous contractions took hours. Recovering cells remained viable and exhibited spontaneous contractions through 24 hours of observation, whereas contractility never returned in propidium iodide-labelled cells. ATP levels decreased rapidly after chemical hypoxia and were partially restored upon washout of inhibitors. ATP recovery was similar in non-hypercontracted and hypercontracted cells. Thus, contractile dysfunction during recovery in hypercontracted cells was not due to lack of regeneration of ATP. In conclusion, hypercontracture is not a manifestation of irreversible injury. Contractile dysfunction following recovery from hypercontracture observed here may represent 'in vitro stunning'.

Adenosine Triphosphate↗