Preservation of metabolic reserves and function after storage of myocytes in hypothermic UW solution.

Isolated rat myocytes cold stored anaerobically up to 24 h in University of Wisconsin solution lost 95% of their ATP and 100% of their glycogen. They underwent contracture when rewarmed in a Krebs-Henseleit (KH) medium that contained Ca unless Ca addition was delayed. In the latter case, cell function, measured by stimulation-induced cell shortening, was surprisingly well retained. Aerobically stored cells were resistant to Ca on rewarming, although 96% of glycogen was still lost, along with 46% of ATP. Cells that were incubated for 48 h aerobically with the substrates glucose and pyruvate at pH 6.2 retained 77% of their ATP and 59% of their glycogen, with good cell morphology. At pH 6.2, the demand for ATP was only 55% of that at pH 7.4. However, after rewarming, these cells functioned no better than anaerobically stored cells, although their inotropic response to isoproterenol was improved. We conclude that 1) aerobic conditions with substrates at low pH preserve myocyte metabolic reserves well for 48 h, partly by reducing the demand for ATP; 2) rewarming conditions are critical for anaerobically stored cells with metabolic stores that are severely depleted; and 3) unloaded cell function is surprisingly insensitive to a period of severe metabolic deprivation.

Stimulation of Na-K-2Cl cotransporter in neurons by activation of Non-NMDA ionotropic receptor and group-I mGluRs.

In a previous study, we found that Na(+)-K(+)-2Cl(-) cotransporter in immature cortical neurons was stimulated by activation of the ionotropic N-methyl-D-aspartate (NMDA) glutamate receptor in a Ca(2+)-dependent manner. In this report, we investigated whether the Na(+)-K(+)-2Cl(-) cotransporter in immature cortical neurons is stimulated by non-NMDA glutamate receptor-mediated signaling pathways. Expression of the Na(+)-K(+)-2Cl(-) cotransporter and metabotropic glutamate receptors (mGluR1 and 5) was detected in cortical neurons via immunoblotting and immunofluorescence staining. Significant stimulation of cotransporter activity was observed in the presence of both trans-(+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) (10 microM), a metabotropic glutamate receptor (mGluR) agonist, and (RS)-3,5-dihydroxyphenylglycine (DHPG) (20 microM), a selective group-I mGluR agonist. Both trans-ACPD and DHPG-mediated effects on the cotransporter were eradicated by bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM, a Ca(2+) chelator. In addition, DHPG-induced stimulation of the cotransporter activity was inhibited in the presence of mGluRs antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) (1 mM) and also with selective mGluR1 antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) (100 microM). A DHPG-induced rise in intracellular Ca(2+) in cortical neurons was detected with Fura-2. Moreover, DHPG-mediated stimulation of the cotransporter was abolished by inhibition of Ca(2+)/CaM kinase II. Interestingly, the cotransporter activity was increased by activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. These results suggest that the Na(+)-K(+)-2Cl(-) cotransporter in immature cortical neurons is stimulated by group-I mGluR- and AMPA-mediated signal transduction pathways. The effects are dependent on a rise of intracellular Ca(2+).

Reduction in density of transverse tubules and L-type Ca(2+) channels in canine tachycardia-induced heart failure.

OBJECTIVE: Persistent supraventricular tachycardia leads to the development of a dilated cardiomyopathy with impairment of excitation-contraction (EC) coupling. Since the initial trigger for EC coupling in ventricular muscle is the influx of Ca(2+) through L-type Ca(2+) channels (I(Ca)) in the transverse tubules (T-tubules), we determined if the density of the T-tubule system and L-type Ca(2+) channels change in canine tachycardia pacing-induced cardiomyopathy. METHODS: Confocal imaging of isolated ventricular myocytes stained with the membrane dye Di-8-ANEPPS was used to image the T-tubule system, and standard whole-cell patch clamp techniques were used to measure I(Ca) and intramembrane charge movement. RESULTS: A complex staining pattern of interconnected tubules including prominent transverse components spaced every approximately 1.6 microm was present in control ventricular myocytes, but failing cells demonstrated a far less regular T-tubule system with a relative loss of T-tubules. In confocal optical slices, the average % of the total cell area staining for T-tubules decreased from 11.5+/-0.4 in control to 8.7+/-0.4% in failing cells (P<0.001). Whole-cell patch clamp studies revealed that I(Ca) density was unchanged. Since whole-cell I(Ca) is due to both the number of channels as well as the functional properties of those channels, we measured intramembrane charge movement as an assay for changes in channel number. The saturating amount of charge that moves due to gating of L-type Ca(2+) channels, Q(on,max), was decreased from 6.5+/-0.6 in control to 2.8+/-0.3 fC/pF in failing myocytes (P<0.001). CONCLUSIONS: Cellular remodeling in heart failure results in decreased density of T-tubules and L-type Ca(2+) channels, which contribute to abnormal EC coupling.

Regulation of Na(+)-K(+)-Cl(-) cotransporter in primary astrocytes by dibutyryl cAMP and high [K(+)](o).

In this study, we examined the Na(+)-K(+)-Cl(-) cotransporter activity and expression in rat cortical astrocyte differentiation. Astrocyte differentiation was induced by dibutyryl cAMP (DBcAMP, 0. 25 mM) for 7 days, and cells changed from a polygonal to process-bearing morphology. Basal activity of the cotransporter was significantly increased in DBcAMP-treated astrocytes (P < 0.05). Expression of an approximately 161-kDa cotransporter protein was increased by 91% in the DBcAMP-treated astrocytes. Moreover, the specific [(3)H]bumetanide binding was increased by 67% in the DBcAMP-treated astrocytes. Inhibition of protein synthesis by cyclohexamide (2-3 microgram/ml) significantly attenuated the DBcAMP-mediated upregulation of the cotransporter activity and expression. The Na(+)-K(+)-Cl(-) cotransporter in astrocytes has been suggested to play a role in K(+) uptake. In 75 mM extracellular K(+) concentration, the cotransporter-mediated K(+) influx was stimulated by 147% in nontreated cells and 79% in DBcAMP-treated cells (P < 0.05). To study whether this high K(+)-induced stimulation of the cotransporter is attributed to membrane depolarization and Ca(2+) influx, the role of the L-type voltage-dependent Ca(2+) channel was investigated. The high-K(+)-mediated stimulation of the cotransporter activity was abolished in the presence of either 0.5 or 1.0 microM of the L-type channel blocker nifedipine or Ca(2+)-free HEPES buffer. A rise in intracellular free Ca(2+) in astrocytes was observed in high K(+). These results provide the first evidence that the Na(+)-K(+)-Cl(-) cotransporter protein expression can be regulated selectively when intracellular cAMP is elevated. The study also demonstrates that the cotransporter in astrocytes is stimulated by high K(+) in a Ca(2+)-dependent manner.

Control of the mitochondrial permeability transition pore by high-affinity ADP binding at the ADP/ATP translocase in permeabilized mitochondria.

Low levels of ADP binding at the ADP/ATP translocase caused inhibition of the Ca2+-induced permeability transition of the mitochondrial inner membrane, when measured using the shrinkage assay on mitochondria, which have already undergone a transition. Inhibition was prevented by carboxyatractyloside, but potentiated by bongkrekic acid, which increased the affinity for inhibition by ADP. This suggests that inhibition was related to the conformation of the translocase. Ca2+ addition was calculated to remove most of the free ADP. Ca2+ added after ADP induced a slow decay of the inhibition, which probably reflected the dissociation of ADP from the translocator. We conclude that the probability of forming a permeability transition pore (PTP) is much greater when the translocase is in the CAT conformation than in the BKA conformation, and, in the absence of CAT and BKA, the translocator is shifted between the BKA and CAT conformations by ADP binding and removal, even in deenergized mitochondria with no nucleotide gradients.

Biphasic mechanism for hypothermic induced loss of protein synthesis in hepatocytes.

BACKGROUND: A complication in liver transplantation is increased clotting times due to inhibition of protein synthesis resulting from prolonged hypothermic preservation. Protein synthesis is also blocked in cold preserved hepatocytes. In this study, the mechanism of inhibition of protein synthesis in cold preserved hepatocytes was investigated. METHODS: Hepatocytes prepared from rat liver were cold preserved in University of Wisconsin solution for 4, 24, and 48 hr. Protein synthesis was measured as incorporation of radiolabeled leucine into acid precipitable proteins. Hepatocytes were treated with antioxidants (dithiothreitol, trolox or deferoxamine, nitric oxide synthase inhibitor (N(G)-monomethyl-L-arginine monoacetate), steroids (dexamethasone or methylprednisolone), methods to keep adenosine triphosphate high (aerobic storage), and cytoskeletal disrupting agents (cytochalasin D or colchicine). RESULTS: There was a 26% decrease in protein synthesis after only 4 hr of cold storage and a further 25% decrease at 24 hr. Antioxidants, elevated adenosine triphosphate, and N(G)-monomethyl-L-arginine monoacetate did not affect the rate of loss of protein synthesis. Protein synthesis was not due to inhibition of amino acid transport or lack of amino acids in the storage medium. Steroid pretreatment of hepatocytes had no effect on the loss of protein synthesis occurring in the first 4 hr of storage but did suppress the loss occurring during the next 44 hr of storage. Cytoskeletal disrupting agents, added to freshly isolated cells, inhibited protein synthesis. CONCLUSION: The mechanism of loss of protein synthesis in cold preserved liver cells is not mediated by: (1) oxygen free radical generation or improved by antioxidant therapy, (2) nitric oxide generation in hepatocytes, (3) an adenosine triphosphate-sensitive destruction of cell viability, and (4) decreased permeability of amino acids or loss of amino acids from the cells. Loss of protein synthesis due to hypothermic storage appears biphasic. The first phase, occurring within 4 hr of storage, may be the result of the effects of hypothermia on the cell cytoskeletal system and may be untreatable. The second phase, which occurs during the next 24 to 48 hr is sensitive to steroid pretreatment. This phase may be amenable to improved preservation methodology. Improved preservation of the liver may require the use of steroids to conserve protein synthetic capabilities.

Ca uptake by heart cells: I. Ca uptake by the sarcoplasmic reticulum of intact heart cells in suspension.

Electric field stimulation of adult rat heart cells suspended in medium with 0.2 mM Ca and isoproterenol caused 45Ca uptake at a rate (5.25 pmol/mg/beat) proportional to stimulation frequency. Uptake was strongly inhibited by verapamil or thapsigargin. 45Ca autoradiography showed that stimulation dependent verapamil sensitive uptake was associated with the rod shaped cells, while the uptake by round cells was unaffected by stimulation and was verapamil-insensitive. 45Ca efflux measurements revealed a caffeine-sensitive component of uptake which was abolished by thapsigargin, and a caffeine-insensitive component. Part of the latter was sensitive to thapsigargin but not to 30 s of stimulation; another part was sensitive to such stimulation but not to thapsigargin. With longer times of stimulation, the caffeine-insensitive pool increased in size, part of which appeared to be mitochondrial Ca uptake via a thapsigargin-sensitive pool. The caffeine-sensitive pool labelled quickly in stimulated cells and its size and rate of labelling was increased by stimulation frequency (3.87 pmol/mg/beat), while the caffeine-insensitive pool labelled more slowly and was relatively insensitive to stimulation (0.77 pmol/mg/beat). We conclude that essentially all of the SR Ca pool, as defined by its involvement in excitation-contraction coupling, is released by caffeine.

Ca uptake by heart cells: II. Most entering Ca appears to leave without mixing with the sarcoplasmic reticulum Ca pool.

The rate of verapamil-sensitive uptake of 45Ca by rat heart cells stimulated to beat in suspension with 0.2 mM Ca and isoproterenol was increased > 2-fold by cell loading with the chelator Quin-2. No effect of Quin-2 loading was observed on the rate of uptake of trace levels of 54Mn, present in addition to Ca, which was used as an index of Ca channel activity. Quin-2 loading also had little effect on the rate of 45Ca uptake by cells diluted into a high K/low Na medium, where Ca uptake was primarily by Na/Ca exchange. The fast chelator 1,2-bis(o-aminophenoxy)ethane-N,N,-N',N'-tetraacetic acid (BAPTA) was 3-fold more effective than the slow chelator EGTA at preventing Ca efflux. BAPTA loading also caused an increase in sarcoplasmic reticulum (SR) Ca content. These results suggest that chelator loading had little effect on the rate of Ca influx by Ca channels or by Na/Ca exchange, and that the increased rate of 45Ca uptake seen with Quin-2 loading was caused by an inhibition of Ca efflux, either directly by chelation or by increased Ca uptake by the SR or by other intracellular organelles. This further suggests that most of the Ca entering the cell without chelator leaves again within the same beat, and that this may result from Ca efflux from a kinetically limited Ca pool in or around the diad cleft.

Calibration of intracellular Ca transients of isolated adult heart cells labelled with fura-2 by acetoxymethyl ester loading.

A procedure for calibration of fluorescence signals from adult rat heart cells loaded with the -AM ester of fura-2 is described. Calibration is complicated by dye compartmentation and potentially incomplete dye hydrolysis. These problems were overcome by subtracting from fluorescence transients the non-cytosolic (mitochondrial) component of fura-2 fluorescence plus any Ca-insensitive component of dye fluorescence, after selectively and sequentially quenching cytosolic and non-cytosolic dye with Mn. The Kd of fura-2 in cells loaded by the -AM ester, in cells depleted of ATP and equilibrated with Ca buffers, was found to be 371 +/- 39 nM at 37 degrees C. We found that calibration values for RMAX and RMIN derived from previously measured cells were of general validity, removing the need to measure RMAX and RMIN on every cell. Once these calibration values are determined, the calibration procedure to measure cytosolic Ca on any cell is a five minute procedure to determine compartmentation, using just one non-toxic and inexpensive solution. Finally, we have calculated how the errors intrinsic to the measurements translate into errors of the calculated Ca concentration and transient peak heights. These calculations allow reasonable parameters for data acquisition to be set.

Effect of ATP depletion on kinetics of Na/Ca exchange-mediated Ca influx in Na-loaded heart cells.

The kinetics of Ca influx by Na/Ca exchange into adult rat heart cells loaded with Na and depleted of ATP were investigated, to further elucidate how ATP regulates exchanger properties in the intact heart cell. We found an eight-fold reduction in Vmax for Ca uptake by ATP depletion, with no significant change in the K(m) for extracellular Ca or the K1 for inhibition by extracellular Na. Autoradiography of ATP depleted cells after45Ca uptake showed no gross heterogeneity of Ca uptake, as would occur if a small fraction of cells still retained some ATP. We conclude that the reduction in Vmax is a property of all of the ATP depleted cells. These kinetics are consistent with regulation of the putative calmodulin binding site on the exchanger. They are also consistent with sequestration of the exchanger at the intracellular face of the sarcolemma by intracellular Na dependent inactivation, a property of the exchanger observed in membrane patches to be relieved by ATP. However, no effect of heptalysine was observed on exchange activity in cells with ATP, nor was the restoration of exchange activity to ATP depleted cells by ATP resynthesis blocked by heptalysine. This suggests that in intact cells the activation of exchange activity by ATP is not caused by phosphatidylserine translocation to the cytosolic leaflet of the sarcolemma. Also, no effect of cytochalasin D was observed on exchanger activity or kinetics, in contrast with observations in Chinese hamster ovary (CHO) cells transfected with bovine cardiac Na/Ca exchanger by ATP, and these are differently expressed depending on the exchanger environment.

Regulation of sodium-calcium exchange in intact myocytes by ATP and calcium.

Regulation of Na-Ca exchange activity by ATP and by intracellular Ca (Cai) has been studied in suspensions of intact Na-loaded adult rat cardiac myocytes using 45Ca uptake and exchange of 22Na. ATP depletion of Na-loaded myocytes results in a strong inhibition of the Na-Ca exchanger, manifested as a strong inhibition of intracellular Na-dependent Ca uptake. Ca uptake by Na-loaded cells in the course of ATP depletion can be very heterogeneous because of the heterogeneity amongst cells of the extent of ATP depletion. This can result in a false measure of the dependence of exchanger activity on cell ATP content. Under conditions intended to maximize the uniformity of cell ATP content amongst cells we found a half maximal rate of Ca uptake with a cell ATP content of 1.96 nmol/mg, about 10% of the normal cell ATP level. The results suggest that ATP depletion after ischemia plus reperfusion is unlikely to limit the rate of Ca uptake by Na-Ca exchange in the whole heart if at least one quarter of the ATP is restored. Ca addition to myocytes loaded with Na in the absence of Ca results in a strong activation of the Na-Ca exchanger at an intracellular site, manifested as a large activation of Na-Na exchange activity. A similar activation of the exchanger is observed in cells with a normal level of intracellular Na, suspended in a medium containing physiological levels of Ca, when the cells are stimulated to beat by application of an electric field. This suggests that regulation of the exchanger by Cai is important physiologically, in the regulation of excitation-contraction coupling. Cells depleted of ATP show not only a strongly inhibited rate of Na-Ca exchange and Na-Na exchange, but also a strongly reduced degree of activation by Cai, even in ATP-depleted cells with no acidosis. This could result from the combined effect of ATP loss and an elevated intracellular Mg concentration on Ca binding affinity at the regulatory site.

Inhibition of sodium/calcium exchange and calcium channels of heart cells by volatile anesthestics.

BACKGROUND: Volatile anesthetics exert profound effects on the heart, probably through their effect on Ca2+ movements during the cardiac cycle. Ca2+ movements across the sarcolemma are thought to involve mainly Ca2+ channels and the Na+/Ca2+ exchanger. We have therefore investigated the action of halothane, isoflurane, and enflurane on Na+/Ca2+ exchange and Ca2+ channel activity to assess the contribution of these pathways to the observed effect of the anesthetics on the myocardium. METHODS: Sarcolemmal ion fluxes were investigated using radioisotope uptake by isolated adult rat heart cells in suspension. Na+/Ca2+ exchange activity was measured from 45Ca2+ uptake by Na(+)-loaded cells. Ca2+ channel activity was measured from verapamil-sensitive trace 54Mn2+ uptake during electric stimulation. RESULTS: Halothane, isoflurane, and enflurane inhibited Na+/Ca2+ exchange completely, with similar potency when concentrations were expressed in millimolar units in aqueous medium but not when expressed as minimum alveolar concentration (MAC). The inhibition by enflurane was particularly strong, > 50%, at 2 MAC. In contrast, the three anesthetics inhibited Ca2+ channels with similar potency when concentrations were expressed as MAC but not when expressed in millimolar units in aqueous medium. Hill plots of pooled data with all three anesthetics showed a slope of -3.87 +/- 0.50 for inhibition of Na+/Ca2+ exchange and -1.73 +/- 0.19 for inhibition of Ca2+ channels. CONCLUSIONS: Halothane, isoflurane, and enflurane inhibit both Na+/Ca2+ exchange and Ca2+ channels at concentrations relevant to anesthesia, although they exhibit differences in potency and number of sites of action. At 1.5 MAC, halothane inhibits Ca2+ channels more than Na+/Ca2+ exchange, whereas enflurane inhibits Na+/Ca2+ exchange more than Ca2+ channels. Isoflurane inhibited both systems equally. The inhibition of Ca2+ influx by these agents is likely to contribute to their negative inotropic effect in the heart. The inhibition of Na+/Ca2+ exchange by enflurane may account for its observed action of delaying relaxation in species lacking sarcoplasmic reticulum.

The effect of hemoglobin and its metabolites on energy metabolism in cultured cerebrovascular smooth-muscle cells.

Cerebral arteries in spasm have been found to contain low levels of adenosine triphosphate (ATP), and it has been postulated that this change in levels results from hypoxia produced by arterial encasement in clotted material. This study was undertaken to determine whether any of four blood-derived agents, ferrous hemoglobin, methemoglobin, hemin, or bilirubin, is capable of reducing energy levels in cerebral artery smooth-muscle cells. Twenty-four-hour exposure of cultured canine basilar artery cells to ferrous hemoglobin and bilirubin led to a significant decline in ATP levels (to 8.9 nmol/mg protein and 2.8 nmol/mg protein, respectively) versus control (16.6 nmol/mg protein); methemoglobin and hemin showed no effect. Bilirubin but not hemoglobin was found to interfere with electron transport and with creatine phosphokinase activity in intact cells; however, bilirubin showed no inhibitory effect on this enzyme in cell-free conditions. The findings indicate that hemoglobin and bilirubin may be responsible for diminished energy levels in cerebral arteries. These observations also suggest that bilirubin may exert its effect on ATP by impairing mitochondrial function.

Bilirubin levels in subarachnoid clot and effects on canine arterial smooth muscle cells.

BACKGROUND AND PURPOSE: Previous studies have suggested that bilirubin is a potential contributor to cerebral vasospasm. The purpose of this investigation was to determine whether bilirubin accrues in subarachnoid clot, whether its vasoconstrictive effect could involve a direct action on arterial smooth muscle cells, and, if so, whether bilirubin affects their Ca2+ uptake. METHODS: Subarachnoid clots were analyzed for bilirubin using high-performance liquid chromatography. The length and 45Ca2+ uptake of vascular smooth muscle cells enzymatically dissociated from canine carotid arteries were measured before and after exposure to bilirubin solution. Additional experiments were conducted on cultured smooth muscle cells from canine basilar artery and on ATP-depleted cardiac myocytes. RESULTS: Mean +/- SE bilirubin concentration in experimental clot was 263 +/- 35.7 mumol/L. Vascular smooth muscle cells exposed to bilirubin showed progressive shortening (P < .01) and an increased uptake of 45Ca2+ (P < .001). Contraction was prevented by Ca(2+)-free media but not by verapamil. Experiments with heart myocytes showed that bilirubin caused an increased uptake of 45Ca2+ but not of [14C]sucrose. CONCLUSIONS: The results indicate that bilirubin accrues in subarachnoid clot, that it exerts a direct constrictive effect on arterial smooth muscle cells, and that this effect is associated with an increased uptake of Ca2+. Studies on heart myocytes suggest that the Ca2+ uptake induced by bilirubin could be due to a selective increase in membrane permeability to Ca2+.

Hemin: levels in experimental subarachnoid hematoma and effects on dissociated vascular smooth-muscle cells.

Although hemin is known to exert toxic effects on a variety of cell types, its possible participation in the genesis of cerebral vasospasm has received little attention. The authors measured the concentration of hemin in experimental subarachnoid clot and studied its effects on the morphology and 45Ca++ uptake of vascular smooth-muscle cells dissociated from canine carotid artery. Craniectomies were performed in five dogs under general anesthesia, and 3 to 5 ml of autologous whole blood was deposited in the supraclinoid subarachnoid compartment. The concentration of hemin recovered by Folch extraction from clotted material removed 7 days after surgery was 390 +/- 247 microM (mean +/- standard error of the mean). Mean vascular smooth-muscle cell length after 40 minutes of exposure to 50 microM hemin was 37.3 +/- 1.2 microns (control 51.6 +/- 1.6 microns) (p < 0.01). The mean percent permeation of 45Ca++, measured by a dual label technique, of cells exposed to hemin was 200.9% +/- 23% (control 102.9% +/- 4.3%) (p < 0.01). These findings indicate that hemin accrues in subarachnoid hematoma, that it exerts a constrictive effect on vascular smooth-muscle cells, and that this effect is associated with an increased uptake of Ca++. This study demonstrates that hemin should be included in the list of potential agents that participate in the development of cerebral vasospasm.

ATP dependence of calcium uptake by the Na-Ca exchanger of adult heart cells.

The ATP dependence of the Na-Ca exchanger was investigated in isolated adult rat heart cells to evaluate the extent to which ATP depletion after a period of ischemia plus reperfusion in whole hearts could limit calcium uptake by Na-Ca exchange. A standard state for measurement of Na-Ca exchange activity that could be used with cells depleted of ATP to different degrees was defined. This was a state of zero sarcolemmal gradient for sodium, potassium, and pH and was achieved by incubation of the cells for 5 minutes with EDTA, EGTA, ouabain, and nigericin. Heterogeneity of cell ATP levels was minimized by using a protocol of total ATP depletion by incubation under conditions similar to ischemia, followed by reoxygenation to give partial restoration of ATP levels. No ATP was regenerated when cells were reoxygenated in the presence of rotenone, and such cells showed a very low rate of calcium uptake. Without rotenone, cells showed an almost complete restoration of Na-Ca exchange activity, in spite of a restoration of ATP levels to only one third of control values. Thus, the dependence of calcium uptake on ATP was highly nonlinear under these conditions. The calculated Km for ATP was no more than 10% of normal ATP levels. We conclude that ATP depletion after ischemia plus reperfusion is unlikely to limit the rate of calcium uptake through Na-Ca exchange in the whole heart if at least one quarter of the ATP is restored. In addition, we measured the apparent ATP dependence of calcium uptake by Na-Ca exchange in cells under conditions in which we previously had concluded that cell ATP distributions were very heterogeneous: when cells undergo contracture during incubation with oligomycin and without glucose. A linear relation between calcium uptake rate and ATP was observed at all ATP levels. This can be understood if cells in contracture that are incubated with oligomycin cannot take up calcium because of low ATP, whereas rod-shaped cells are able to retain a full uptake capability. This result further supports our conclusion that the ATP level declines catastrophically to near zero in these oligomycin-incubated cells just before contracture.

Control of the Na-Ca exchanger in isolated heart cells. I. Induction of Na-Na exchange in sodium-loaded cells by intracellular calcium.

Isolated adult rat heart cells in suspension were loaded with sodium by incubation with ouabain in the absence of calcium for 30 minutes. Addition of low levels of calcium induced accelerated rates of sodium influx and efflux, as measured with 22Na. The magnitude of calcium-induced 22Na efflux was 50-fold greater than the net rate of calcium uptake and required extracellular sodium, but not extracellular calcium, once some calcium was taken up. Calcium did not induce 86Rb efflux. The accelerated rate of 22Na efflux was prevented by verapamil, but verapamil was ineffective when added after calcium. Addition of EGTA after calcium reversed the effect of calcium, but only after incubation. Dichlorobenzamil, unlike verapamil, both prevented and reversed the induction of sodium fluxes by calcium. We conclude 1) that intracellular calcium induces Na-Na exchange through the Na-Ca exchanger in sodium-loaded cells exposed to calcium; and 2) that Na-Na exchange can be activated by calcium that enters the cell through calcium channels. We propose that this Na-Na exchange reflects the intrinsic activity of the Na-Ca exchanger.

Control of the Na-Ca exchanger in isolated heart cells. II. Beat-dependent activation in normal cells by intracellular calcium.

Electrical stimulation of isolated adult rat heart cells in suspension at 4 Hz resulted in a fourfold increase in the rate of sodium influx and efflux across the sarcolemma, with no change in total cell sodium, as measured with 22Na. The magnitude of stimulation-dependent sodium fluxes under these conditions averaged 17 nmol/min/mg protein. The increased rate of efflux was inhibited by tetrodotoxin, verapamil, or dichlorobenzamil and required extracellular calcium. The inhibition by tetrodotoxin was overcome by Bay K 8644. The basal rate of 22Na efflux in cells at rest was inhibited only slightly by dichlorobenzamil. The stimulation-induced efflux was not inhibited by ouabain, but in the presence of ouabain, stimulation increased the rate of accumulation of total sodium by 4 nmol/min/mg. This increase was inhibited by tetrodotoxin or verapamil. A calcium-dependent increase in rate of 22Na influx and efflux could also be induced by KCl addition. This was inhibited by verapamil and dichlorobenzamil but not by tetrodotoxin and was reversed by EGTA, but only after a delay. We conclude the following. 1) The Na-Ca exchanger in cells at rest is no more than 10% activated. 2) The exchanger becomes activated directly or indirectly by calcium that enters the cell through calcium channels during excitation. 3) In this preparation the major part of excitation-induced sodium fluxes are mediated by the Na-Ca exchanger, with only a relatively small direct participation of sodium channels. These channels participate indirectly by promoting calcium channel activation. 4) If all the calcium-dependent sodium fluxes were Na-Ca exchange, then calcium flux through the exchanger per beat would be about sevenfold larger than that through the calcium channels. An undetermined part of the calcium-dependent sodium fluxes, however, could be a direct Na-Na exchange through the activated Na-Ca exchanger.

The use of myocytes as a model for developing successful heart preservation solutions.

The development of a successful method to preserve the heart for relatively long periods (24-48 hr) requires demonstrating successful orthotopic transplantation and long-term survival after preservation. There are, however, multiple variables that may affect the quality of heart preservation, and it is nearly impossible to systematically study all the variables in this complicated model. One model that may be useful to study how preservation parameters affect heart cell preservation is the isolated myocyte preparation. In this study myocytes were isolated from the rabbit heart and the effects of up to 24 hr cold storage on viability measured to determine if this would be a suitable preservation model. Myocytes were stored in various preservation solutions including; EuroCollins (EC), two cardioplegic solutions (Stanford [ST] and Bretschneider solution [HTK]) and the University of Wisconsin solution (UW) with or without the addition of polyethylene glycol. The viability of myocytes was judged by measuring the effects of preservation and rewarming after preservation on cellular morphology (percent rod-shaped cells), ATP concentration, and LDH release. Myocytes preserved in the cardioplegic solutions were least well preserved after 12 and 24 hr storage, as judged by the loss of rod-shaped morphology and lower ATP concentration. Preservation in EC resulted in a decrease in the percent rod-shaped cells after 12 hr and 24 hr storage that was greater than obtained in the UW solutions. The best preservation of myocyte morphology and highest content of ATP was obtained in myocytes stored in the UW solutions, especially those containing PEG. The myocyte model of heart preservation shows a loss of cell integrity that is related to the preservation solution (HTK greater than ST greater than EC greater than UW-PEG) and these results are similar to what has been shown in the past with other models of heart preservation. Thus the myocyte model appears to be a useful method to test how many preservation solutions and preservation variables affect heart cell metabolism. In the future, results from these types of studies may find use in developing improved heart preservation solutions for testing in the orthotopic transplant model.