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The Na/Ca exchanger has been examined with respect to its molecular biology, its cellular function, and its role in excitation-contraction coupling. The Na/Ca exchanger plays a central part in excitation-contraction coupling, setting the level of sarcoplasmic reticular calcium and contributing to the triggering of sarcoplasmic reticular calcium release. Functional biophysical studies with isolated single cells and caged calcium provide evidence that the Na/Ca exchanger works as a two step sequential transporter. In the heart there are about 250 exchangers.mu-2, operating at a turnover rate of up to about 2500.s-1, with the exchanger carrying -2.56 charges under normal conditions. The Na/Ca exchanger has been recently cloned from diverse mammalian species and several tissues and is largely conserved. It is clear, however, that the function of the Na/Ca exchanger is different in the different tissues. Thus work is in progress in several laboratories, including ours, to determine how the Na/Ca exchanger achieves its tissue specific function. Several modulatory motifs have been seen in studies of the exchanger that may explain some of the tissue specific differences. Interestingly the modulation of the Na/Ca exchanger (for example, by protons, sodium, calcium, ATP, calmodulin) seems to arise from interactions with the intracellular loop.

Altered Na+/Ca2+ exchanger (NCX) protein expression or activity is thought to contribute to various aspects of cardiac pathology. In guinea pig ventricular myocytes, NCX-mediated Ca2+ entry is almost entirely responsible for Ca2+ overload during hypoxia-reoxygenation. Because Ca2+ overload is a common initiator of apoptosis, the purpose of this study was to test the hypotheses that NCX activity is critically involved in initiating apoptosis after hypoxia-reoxygenation and that hypoxia-reoxygenation-induced apoptosis can be modulated by changes in NCX protein expression or activity. An NCX antisense oligonucleotide was used to reduce NCX protein expression in cultured adult guinea pig ventricular myocytes. Caspase-3 activation and cytochrome c release were used as markers of apoptosis. Hypoxia-reoxygenation-induced apoptosis was significantly decreased in antisense-treated myocytes compared with untreated control or nonsense-treated myocytes. Pretreatment of cultured myocytes for 24 h with either endothelin-1 or phenylephrine was found to increase both NCX protein expression and evoked NCX activity as well as enhance hypoxia-reoxygenation-induced apoptosis. Control experiments demonstrated that endothelin-1 and phenylephrine did not induce apoptosis on their own nor did they enhance the apoptotic response in a model of Ca2+-dependent, NCX-independent apoptosis. Additional control experiments demonstrated that the NCX antisense oligonucleotide did not alter the apoptotic response of myocytes to either H2O2 or isoproterenol. Taken together, these data suggest that the NCX has a critical and specific role in the initiation of apoptosis after hypoxia-reoxygenation in guinea pig myocytes and that hypoxia-reoxygenation-induced apoptosis is quite sensitive to changes in NCX activity.

Considerable evidence supports a Ca2+dysregulation hypothesis of brain aging and Alzheimer's disease. However, it is still not known whether (1) intracellular [Ca2+]iis altered in aged brain neurons during synaptically activated neuronal activity; (2) altered [Ca2+]iis directly correlated with impaired neuronal plasticity; or (3) the previously observed age-related increase in L-type voltage-sensitive Ca2+channel (L-VSCC) density in hippocampal neurons is sufficient to impair synaptic plasticity. Here, we used confocal microscopy to image [Ca2+]iin single CA1 neurons in hippocampal slices of young-adult and aged rats during repetitive synaptic activation. Simultaneously, we recorded intracellular EPSP frequency facilitation (FF), a form of short-term synaptic plasticity that is impaired with aging and inversely correlated with cognitive function. Resting [Ca2+]idid not differ clearly with age. Greater elevation of somatic [Ca2+]iand greater depression of FF developed in aged neurons during 20 sec trains of 7 Hz synaptic activation, but only if the activation triggered repetitive action potentials for several seconds. Elevated [Ca2+]iand FF also were negatively correlated in individual aged neurons. In addition, the selective L-VSCC agonist Bay K8644 increased the afterhyperpolarization and mimicked the depressive effects of aging on FF in young-adult neurons. Thus, during physiologically relevant firing patterns in aging neurons, postsynaptic Ca2+elevation is closely associated with altered neuronal plasticity. Moreover, selectively increasing postsynaptic L-VSCC activity, as occurs in aging, negatively regulated a form of short-term plasticity that enhances synaptic throughput. Together, the results elucidate novel processes that may contribute to impaired cognitive function in aging.

This study investigated the role of the Na+/Ca2+exchanger (NCX) in regulating cytosolic intracellular Ca2+concentration ([Ca2+]i) during anoxia/reoxygenation in guinea pig ventricular myocytes. The hypothesis that the NCX is the predominant mechanism mediating [Ca2+]ioverload in this model was tested through inhibition of NCX expression by an antisense oligonucleotide. Immunocytochemistry revealed that this antisense oligonucleotide, directed at the area around the start site of the guinea pig NCX1, specifically reduced NCX expression in cultured adult myocytes by 90 ± 4%. Antisense treatment inhibited evoked NCX activity by 94 ± 3% and decreased the rise in [Ca2+]iduring anoxia/reoxygenation by 95 ± 3%. These data suggest that NCX is the predominant mechanism mediating Ca2+overload during anoxia/reoxygenation in guinea-pig ventricular myocytes.

Adriamycin (ADM) is a broad-spectrum antineoplastic antibiotic used to treat cancer patients. However, the usefulness of this drug is presently limited by the development of a dose-dependent cardiotoxicity. A current hypothesis for the ADM-induced cardiotoxicity is the production of reactive oxygen radicals by the drug. We utilized the fluorescent indicator 2',7'-dichlorodihydrofluorescein diacetate (DCFH/DA), in which fluorescence appears if reactive oxygen species (ROS) are present, to investigate the ability of ADM to generate reactive oxygen species and the potential protective effect of antioxidants in a cultured cardiomyocyte model. All three of the antioxidants (alpha-phenyl-tert-butyl nitrone (PBN), trolox, and 5-aminosalicylic acid (5-ASA)) tested in our ADM-treated myocytes provided protection against the oxidative stress induced by the drug. These findings suggest that antioxidants modulate ADM-induced oxidative stress, and they are discussed in terms of a possible therapeutic strategy in the prevention of cardiotoxicity resulting from ADM administration.

We have constructed a modular instrument to measure intracellular [Ca2+] ([Ca2+]i) in single isolated cells while simultaneously imposing step changes in [Ca2+]i using "caged Ca2+". By combining the outputs of a xenon arc lamp with a frequency-tripled (Nd:YAG) laser, the instrument can operate with low maintained illumination to measure [Ca2+]i using a ratiometric Ca(2+)-sensitive fluorophore and also activate the release of Ca2+ from a caged-Ca2+ compound with a high energy pulse of ultraviolet light. This instrument is simple to assemble, introduces little electrical noise, provides a wide range of illumination power, produces only moderate photobleaching of the Ca2+ indicator and can be readily adapted to diverse cellular preparations. We demonstrate the use of this system to measure step changes in [Ca2+]i in adult rat ventricular myocytes and a human embryonic kidney cell line (293 cells) in culture.

Currents through time-dependent K+ channels (also referred to as IK or the delayed rectifier) were studied with the whole cell patch-clamp technique in isolated guinea pig ventricular myocytes. IK measurements were restricted to the examination of deactivation tail currents. Substitution of various monovalent cations for external K+ produced shifts of the reversal potential of IK. These shifts were used to calculate permeability ratios relative to K+. The permeability sequence for the IK channels was K+ = Rb+ greater than NH4+ = Cs+ greater than Na+. Time-dependent outward currents were also examined when the myocytes were dialyzed with Cs+ instead of K+. A sizeable time-dependent outward current, quite similar to that seen with K+ dialysis, was demonstrated. This current was primarily carried by intracellular Cs+, as the reversal potential of the current shifted 46 mV per 10-fold change of external Cs+ concentration. The significance of Cs+ permeation through IK channels is discussed with respect to the common use of Cs+ in isolating other currents.

Experimental data do not support the idea that excitation-contraction coupling in heart muscle can be explained by a simple calcium-induced calcium release mechanism alone or a simple voltage-dependent calcium release mechanism alone. Our data on excitation-contraction coupling combined with the results of others suggest the need to either develop more complex and more sophisticated single-mechanism models, or to establish dual-control models.

3-Nitropropionic acid (3NP), an irreversible inhibitor of succinate dehydrogenase, induces both rapid necrotic and slow apoptotic death in rat hippocampal neurons. Low levels of extracellular glutamate (10 microM) shift the 3NP-induced cell death mechanism to necrosis, while NMDA receptor blockade results in predominantly apoptotic death. In this study, we examined the 3NP-induced alterations in free cytosolic and mitochondrial calcium levels, ATP levels, mitochondrial membrane potential, and calpain and caspase activity, under conditions resulting in the activation of apoptotic and necrotic pathways. In the presence of 10 microM glutamate, 3NP administration resulted in a massive elevation in [Ca(2+)](c) and [Ca(2+)](m), decreased ATP, rapid mitochondrial membrane depolarization, and a rapid activation of calpain but not caspase activity. In the presence of the NMDA receptor antagonist MK-801, 3NP did not induce a significant elevation of [Ca(2+)](c) within the 24h time period examined, nor increase [Ca(2+)](m) within 1h. ATP was maintained at control levels during the first hour of treatment, but declined 64% by 16h. Calpain and caspase activity were first evident at 24h following 3NP administration. 3NP treatment alone resulted in a more rapid decline in ATP, more rapid calpain activation (within 8h), and elevated [Ca(2+)](m) as compared to the results obtained with added MK-801. Together, the results demonstrate that 3NP-induced necrotic neuron death is associated with a massive calcium influx through NMDA receptors, resulting in mitochondrial depolarization and calpain activation; while 3NP-induced apoptotic neuron death is not associated with significant elevations in [Ca(2+)](c), nor with early changes in [Ca(2+)](m), mitochondrial membrane potential, ATP levels, or calpain activity.

The aim of this study was to quantify the contribution of the Na+/H+exchanger (NHE) and the Na+channel to the rise in cytosolic Na+concentration ([Na+]) that is seen in anoxic guinea pig ventricular myocytes. [Na+] was measured with the use of microfluorometry and was found to rise to 44 mM after prolonged anoxia. This rise was partially sensitive to either TTX or HOE-642, selective inhibitors of the Na+channel and NHE1, respectively. [Na+] did not significantly rise when both drugs were present, suggesting that other routes of Na+entry were insignificant. However, the relative contributions of the NHE and the Na+channel were found to be remarkably sensitive to ionic conditions expected to occur during ischemia. The Na+channel was the dominant Na+source during acidic anoxia. However, the NHE was the dominant Na+source during both hyperkalemic anoxia and simulated ischemia (hyperkalemia, low pH, and anoxia). The data suggest that the NHE may prove to be the best pharmacological target to reduce Na+entry during true ischemia and that inhibition of Na+influx could contribute strongly to the cardioprotective effects of NHE inhibitors.

Previous work has shown that the genes encoding each alpha1-adrenoceptor subtype are coexpressed throughout the peripheral vascular system. We have evaluated subtype-selective antibodies as tools to determine the extent of protein expression in arteries. The alpha1A-, alpha1B-, and alpha1D-adrenoceptors were detected in the medial layer of the aorta, caudal, femoral, iliac, renal, superior mesenteric, and mesenteric resistance arteries. In Rat1 fibroblasts expressing each subtype, immunoreactivity was noted both on the cell surface and in a perinuclear orientation. Intense alpha1B-adrenoceptor immunostaining was similarly localized in cultured femoral and renal vascular smooth muscle cells. Although the cellular localization appeared to be the same, immunoreactivity obtained with alpha1A- and alpha1D-adrenoceptors was much less intense than that with the alpha1B-adrenoceptor. The alpha1A-adrenoceptor selective agonist A-61603 was 22-fold more potent in activating renal artery contraction when compared with the femoral artery. The expression of each alpha1-adrenoceptor was significantly decreased by in vivo application of antisense oligonucleotides targeted against each subtype. Inhibition of the expression of only one, the alpha1A in renal and the alpha1D in femoral arteries, reduced the contractile response to naphazoline. The results show: 1) subtype-selective antibodies can be used in tissues and cell culture to localize the alpha1-adrenoceptor subtypes, 2) in addition to expression on the cell surface, the alpha1-adrenoceptors are expressed intracellularly, and 3) despite expression of all adrenoceptors, a single subtype mediates the contractile response in the femoral and renal arteries.

The aim of this study was to investigate the role of mitochondrial ionic homeostasis in promoting reoxygenation-induced hypercontracture in cardiac muscle. Mitochondrial membrane potential and intramitochondrial Ca2+concentration ([Ca2+]) were measured using confocal imaging in guinea pig ventricular myocytes exposed to anoxia and reoxygenation. Anoxia produced a variable, but often profound, mitochondrial depolarization. Some cells mounted a recovery of their mitochondrial membrane potential during reoxygenation; the depolarization was sustained in other cells. Recovery of the mitochondrial membrane potential seemed essential to avoid reoxygenation-induced hypercontracture. Reoxygenation also caused a sizable elevation in intramitochondrial [Ca2+], the amplitude of which was correlated with the likelihood of a cell undergoing hypercontracture. A sustained Ca2+load analogous to that seen during reoxygenation was imposed on cardiac mitochondria through permeabilization of the plasma membrane. Elevation of intracellular [Ca2+] to 800 nM caused a substantial mitochondrial depolarization. We propose that the conditions seen in guinea pig ventricular myocytes during reoxygenation are well suited to produce Ca2+-dependent mitochondrial depolarization, which may play a significant role in promoting irreversible cell injury.

The contribution of the alpha-1B adrenergic receptor (AR) to vascular smooth muscle contraction has been assessed using a combination of immunological, molecular biological and pharmacological approaches. A subtype-selective antibody detected alpha-1B immunoreactivity in the medial layer of the aorta, caudal, femoral, iliac, mesenteric resistance, renal and superior mesenteric arteries. Receptor protection assays and antisense oligonucleotides were used to assess the contribution of the alpha-1B AR to contraction. The alpha-1B AR was implicated in mediating the phenylephrine-induced contraction of the mesenteric resistance artery. The alpha-1D AR was implicated in mediating the contraction of the aorta, femoral, iliac and superior mesenteric arteries. Similarly, the alpha-1A AR was implicated in mediating contraction of the caudal and renal arteries. In vivo application of antisense oligonucleotides targeted to the translational start site of the alpha-1B AR had no effect on the phenylephrine-induced contraction of the femoral or renal arteries. In contrast, antisense oligonucleotides directed against the alpha-1D AR significantly inhibited the phenylephrine response in the femoral artery but had no effect on the renal artery. Application of alpha-1A AR antisense oligonucleotides inhibited the contraction of the renal artery without effect on the femoral artery. These data show that (1) alpha-1B AR immunoreactivity is widely distributed in the same peripheral arteries in which previous studies detected its mRNA, and (2) despite this distribution, receptor protection and antisense oligonucleotide studies indicate that the alpha-1B AR mediates the contraction of only the mesenteric resistance artery.

The aim of this study was to evaluate whether the magnitude and time course of the intracellular acidification observed in anoxic cardiac myocytes was sufficient to protect against reoxygenation-induced hypercontracture. Cytosolic [Ca2+], [Na+], and pH were measured using fluorescent indicators in myocytes that were first subjected to both anoxia and glucose deprivation and then oxygen and glucose restoration 15-30 min after the onset of rigor. The cytosol underwent a profound acidification early in anoxia (pH 7.21 to 6.84) that reached a plateau at the time of rigor contracture. In contrast, [Na+] rose throughout anoxia. Cytosolic [Ca2+] underwent little rise during anoxia, but reoxygenation induced a large spike in [Ca2+]. Reoxygenation also induced a significant secondary acidification of the cytosol that was apparently induced by the spike in [Ca2+]. The characteristics of this secondary acidification were deemed sufficient to provide partial protection against the hypercontracture associated with the reoxygenation-induced [Ca2+] transient.

L-type Ca2+ channels were studied in guinea pig ventricular myocytes by examining how photoinactivation of nifedipine affected the Ca2+ current (ICa) and the Ca2+ channel gating current (Ig). ICa, blocked by nifedipine, reappeared in qualitatively different phases (immediate and delayed) following photoinactivation of nifedipine. Immediate recovery was attributed to unblock of closed Ca2+ channels, while delayed recovery was attributed to unblock of inactivated channels. In contrast to the ICa results, photoinactivation of nifedipine produced only delayed recovery of Ig. Analysis of these results suggests the following conclusions. First, the actions of inhibitory dihydropyridines can be attributed to binding to either the inactivated or the closed conformation, but only binding to the inactivated state is associated with reduction of Ig. Second, the action of inhibitory dihydropyridines on closed channels is to retard their movement through a final, voltage-independent transition to the open state. This effect seems to be the converse of a major action of stimulatory dihydropyridines and thus is the principal mechanistic difference between stimulatory and inhibitory dihydropyridines.

Effects of (-)-BAY K 8644 on Ca2+ channel function were studied in guinea pig ventricular myocytes. It was found that the compound has both voltage-dependent stimulatory and inhibitory effects on the Ca2+ current (ICa), in agreement with prior studies. The basis for these effects was studied by evaluating the effects of (-)-BAY K 8644 on the Ca2+ channel gating current. It was found that the voltage-dependent inhibitory effects of the drug on ICa could be well explained by similar reductions in the amount of gating charge moved. However, the stimulatory effect of (-)-BAY K 8644 on ICa could not be simply correlated with changes in the amount of gating charge moved. Although the drug produced a shift of the charge-voltage relationship to more negative potentials, the drug actually reduced the total amount of movable gating charge. Thus it could be demonstrated that there are membrane potentials where (-)-BAY K 8644 reduced the Ca2+ channel gating current while enhancing ICa. In addition, the drug was found to slow the decay of the gating current during repolarization. It seems likely that (-)-BAY K 8644 has a dual effect on Ca2+ channels: affecting both the voltage dependence of gating charge and the relationship between open probability and charge movement.

1. L-type Ca2+ currents and Ca2+ channel gating currents were studied in isolated guinea-pig ventricular heart cells using the whole-cell patch-clamp technique, in order to investigate the mechanism of Ca(2+)-dependent inactivation. The effect of altering the intracellular Ca2+ concentration ([Ca2+]i) on these currents was studied through photorelease of intracellular Ca2+ ions using the photolabile Ca2+ chelators DM-nitrophen and nitr-5. 2. We found that step increases in [Ca2+]i produced by photorelease could either increase or decrease the L-type Ca2+ current. Specifically, Ca2+ photorelease from DM-nitrophen almost exclusively caused inactivation of the Ca2+ current. In contrast, Ca2+ photorelease from nitr-5 had a biphasic effect: a small, rapid inactivation of the Ca2+ current was followed by a slow potentiation. These two Ca(2+)-dependent processes seemed to differ in their Ca2+ dependence, as small Ca2+ photoreleases elicited potentiation without a preceding inactivation, whereas larger photoreleases elicited both inactivation and potentiation. 3. The mechanism of the Ca(2+)-dependent inactivation of Ca2+ channels was explored by comparing the effects of voltage and photoreleased Ca2+ on the Ca2+ current and the Ca2+ channel gating current. Voltage was found to reduce both the Ca2+ current and the gating current proportionally. However, Ca2+ photorelease from intracellular DM-nitrophen inactivated the Ca2+ current without having any effect on the gating current. 4. The dephosphorylation hypothesis for Ca(2+)-dependent inactivation was tested by applying isoprenaline to the cells before eliciting a maximal rise of [Ca2+]i (maximal flash intensity, zero external [Na+]i). Isoprenaline could completely prevent Ca(2+)-dependent inactivation under these conditions, even when [Ca2+]i rose so high as to cause an irreversible contracture of the cell. 5. We concluded from these experiments that voltage and Ca2+ ions inactivate the L-type Ca2+ channel through separate, independent mechanisms. In addition, we found that Ca(2+)-dependent inactivation does not result in the immobilization of gating charge, and apparently closes the Ca2+ permeation pathway through a mechanism that does not involve the voltage-sensing region of the channel. Furthermore, we found that Ca(2+)-dependent inactivation is entirely sensitive to beta-adrenergic stimulation. These facts suggest that either Ca(2+)-dependent inactivation results from Ca(2+)-dependent dephosphorylation of the Ca2+ channel, or that Ca(2+)-dependent inactivation is modulated by protein kinase A.

Nonlinear capacitative current (charge movement) was compared to the Ca current (ICa) in single guinea pig ventricular myocytes. It was concluded that the charge movement seen with depolarizing test steps from -50 mV is dominated by L-type Ca channel gating current, because of the following observations. (a) Ca channel inactivation and the immobilization of the gating current had similar voltage and time dependencies. The degree of channel inactivation was directly proportional to the amount of charge immobilization, unlike what has been reported for Na channels. (b) The degree of Ca channel activation was closely correlated with the amount of charge moved at all test potentials between -40 and +60 mV. (c) D600 was found to reduce the gating current in a voltage- and use-dependent manner. D600 was also found to induce "extra" charge movement at negative potentials. (d) Nitrendipine reduced the gating current in a voltage-dependent manner (KD = 200 nM at -40 mV). However, nitrendipine did not increase charge movement at negative test potentials. Although contamination of the Ca channel gating current from other sources cannot be fully excluded, it was not evident in the data and would appear to be small. However, it was noted that the amount of Ca channel gating charge was quite large compared with the magnitude of the Ca current. Indeed, the gating current was found to be a significant contaminant (19 +/- 7%) of the Ca tail currents in these cells. In addition, it was found that Ca channel rundown did not diminish the gating current. These results suggest that Ca channels can be "inactivated" by means that do not affect the voltage sensor.

The cellular localization of the alpha1D-adrenergic receptor (alpha1D-AR) is controversial. Studies in heterologous cell systems have shown that this receptor is expressed in intracellular compartments. Other studies show that dimerization with other ARs promotes the cell surface expression of the alpha1D-AR. To assess the cellular localization in vascular smooth muscle cells, we developed an adenoviral vector for the efficient expression of a GFP labeled alpha1D-AR. We also measured cellular localization with immunocytochemistry. Intracellular calcium levels, measurement of reactive oxygen species and contraction of the rat aorta were used as measures of functional activity.The adenovirally expressed alpha1D-AR was expressed in intracellular compartments in human aortic smooth muscle cells. The intracellular localization of the alpha1D-AR was also demonstrated with immunocytochemistry using an alpha1D-AR specific antibody. RT-PCR analysis detected mRNA transcripts corresponding to the alpha1A-alpha1B- and alpha1D-ARs in these aortic smooth muscle cells. Therefore, the presence of the other alpha1-ARs, and the potential for dimerization with these receptors, does not alter the intracellular expression of the alpha1D-AR. Despite the predominant intracellular localization in vascular smooth muscle cells, the alpha1D-AR remained signaling competent and mediated the phenylephrine-induced increases in intracellular calcium. The alpha1D-AR also was coupled to the generation of reactive oxygen species in smooth muscle cells. There is evidence from heterologous systems that the alpha1D-AR heterodimerizes with the beta2-AR and that desensitization of the beta2-AR results in alpha1D-AR desensitization. In the rat aorta, desensitization of the beta2-AR had no effect on contractile responses mediated by the alpha1D-AR.Our results suggest that the dimerization of the alpha1D-AR with other ARs does not alter the cellular expression or functional response characteristics of the alpha1D-AR.

Etude de la pompe Na + -Ca ++ dans les cellules excitables, notamment les cellules du muscle cardiaque, et de son influence sur les concentrations de calcium intracellulaire et la contraction. Suite aux publications recentes d'autres auteurs, discussion sur la possible existence d'un espace intracellulaire proche de la membrane ou la diffusion des ions Na + et Ca ++ serait freinee

Na-loading single frog atrial cells produce changes in membrane currents that are similar to the creep currents originally observed in Na-loaded cardiac Purkinje fibers. Exposure to the Na ionophore, monensin, was used to induce creep currents in isolated atrial cells. The sensitivity of myocardial creep currents to three compounds that have been shown to be inhibitors of Na-Ca exchange flux activity in isolated sarcolemmal vesicles was assessed. Dodecylamine, quinacrine, and the amiloride analog, 3',4'-dichlorobenzamil block creep currents at concentrations well below those required to block Na-dependent Ca uptake in sarcolemmal vesicles. The estimated Ki's for inhibition of myocardial creep currents were 3 microM for dodecylamin, 10 micron for quinacrine, and 4 microM for 3',4'-dichlorobenzamil. The sensitivity of creep currents to these compounds is consistent with the hypothesis that creep currents may represent the electrogenic activity of a Na-Ca exchange carrier. In an additional series of experiments, the relative specificity of these compounds was tested by examining their effects on myocardial membrane channels. Both dodecylamine and 3',4'-dichlorobenzamil were found to inhibit myocardial Ca and K currents over the same range of concentrations in which block of exchange activity occurs. These results seriously question the use of these exchange carrier inhibitors as selective experimental probes for defining the role of Na-Ca exchange in various physiological processes.

Single Na+ channel currents have been examined in isolated guinea pig ventricular myocytes using the patch-clamp technique. The effects of lidocaine, extracellular calcium [(Ca)o], and tetrodotoxin on patch Na+ channel availability were assessed using ensemble averages of Na+-channel openings during depolarizing test potential steps from 7 to 10 different patch-holding potentials in each cell-attached patch. In six control patches, the potential for 50% channel availability (Vh) was -15 mV (relative to an average resting membrane potential of -80 mV). Exposure of patches to either lidocaine or elevated (Ca)o produced the expected shifts in Vh [average -22 mV for lidocaine and +10 mV for 6 mM (Ca)o]. Exposure of patches to tetrodotoxin (0.5 microM or 1.0 microM) produced a dose-dependent hyperpolarizing shift of Vh (average -10 and -17 mV) compared with control patches. The hyperpolarizing shift by tetrodotoxin was observed with pulses applied at frequencies of 1.0 or 0.067 Hz. In agreement with earlier maximal upstroke velocity studies in the same preparation, we conclude that block of ventricular Na+ channels by tetrodotoxin exhibits genuine steady-state voltage dependence.

Emission spectra of the photolabile Ca2+ chelators DM-nitrophen, nitr-5, and diazo-2 were studied alone, and in the presence of indo-1, to investigate potential interactions that would make the simultaneous manipulation and ratiometric measurement of the intracellular Ca2+ concentration difficult. Neither diazo-2 nor its photoproduct were found to be significantly fluorescent, and consequently concentrations of diazo-2 up to 20 times that of indo-1 did not distort the emission spectra of indo-1. DM-nitrophen was scarcely fluorescent, but its fluorescence did increase upon photolysis. In contrast to diazo-2 and DM-nitrophen, nitr-5 itself was found to be quite fluorescent, and this fluorescence was significantly increased upon photolysis. Thus, combined use of nitr-5 and indo-1 poses the most difficulty. The emission spectra of all the investigated compounds were used to define experimental conditions and calibration procedures that make possible simultaneous measurement and manipulation of the intracellular Ca2+ concentration.

Ventricular muscle of rat heart has two classes of receptors which are responsible for the positive inotropic effect of ouabain. Low affinity receptors are apparently related to Na+, K+-ATPase. To determine if high affinity receptors are also sarcolemmal Na+, K+-ATPase of muscle cells, their characteristics were examined. Binding of [3H]ouabain to the high affinity binding site required ATP in the presence of Mg2+ and Na+, was stimulated by Na+ in the presence of Mg2+ and ATP, and was inhibited by K+. Digoxin, digitoxin and cassaine all inhibited [3H]ouabain binding to the high affinity site. Cassaine was about an order of magnitude less potent than the glycosides. These results indicate similarities in high affinity ouabain binding sites in ventricular muscle of rat heart and Na+, K+-ATPase obtained from other sources. Destruction of sympathetic nerve terminals with 6-hydroxydopamine failed to affect the high affinity ouabain binding sites indicating that high affinity sites do not represent the Na+, K+-ATPase in sympathetic nerve terminals. Labeling of Na+, K+-ATPase from [gamma-32P]ATP indicates that high affinity ouabain binding sites account for 25% of the total enzyme molecules present in ventricular muscle of rat heart.

The effects of Bay K 8644 on the high-threshold calcium channel was investigated by means of the whole-cell patch-clamp technique in single guinea pig ventricular myocytes. The goal of the experiments was to characterize the inhibitory effects of Bay K 8644 on the calcium channel, and identify the factors that influence the inhibition. Bay K 8644 was found to have strong calcium channel antagonist properties, which were both dose- and voltage-dependent. Channel block by Bay K 8644 had both a tonic and a use-dependent component. The stimulatory effect of the drug was found to have little obvious dependence on the holding potential. The accumulation of use-dependent block during trains of pulses was facilitated by faster rates of stimulation, longer pulse durations, and more positive holding potentials. Application of the drug induced the appearance of a second, slow component of calcium channel recovery. Both the time-constant and relative proportion of the slow component of recovery were found to be voltage-dependent. Bay K 8644 was also found to cause a hyperpolarizing shift of the inactivation curve for the calcium current, suggesting that it has strong interactions with the inactivated state of the calcium channel. Thus, Bay K 8644 has, along with its stimulatory effects, inhibitory effects that strongly resemble those of typical calcium channel antagonists.

1. Non-linear capacitative current (charge movement) was studied in isolated guinea-pig and rat ventricular myocytes. Linear capacitance was subtracted using standard procedures. Most of the experiments were done with guinea-pig myocytes, while rat myocytes were used for comparison. 2. When a myocyte was held at -100 mV, depolarizing clamp steps produced a rapid outward current transient, which was followed by an inward current transient upon repolarization. This current was identified as the movement of charged particles in the cell membrane, rather than ionic movement across the membrane, for the following reasons: (1) the current saturated at membrane potentials positive to +20 mV; (2) the current was capacitative in nature, having no reversal potential; (3) in general, the charge moved during depolarization (Qon) approximated the charge moved during repolarization (Qoff). 3. Qoff was significantly less than Qon for a depolarization from -100 mV to 0 mV. However, the Qoff/Qon ratio approached unity if the cell was instead repolarized to -140 mV. This was interpreted as being due to the immobilization of a fraction of the charge during the depolarization, which recovered rapidly enough to be measured at -140 mV, but recovered too slowly at -100 mV. 4. Charge movement in these cells had a sigmoidal dependence on the membrane potential, which could be empirically described by the two-state Boltzmann equation Q = Qmax/(1 + exp[-(V-V*)/kappa]), where Q is the charge movement at potential V, Qmax is the maximum charge, V* is the membrane potential at Q = Qmax/2, and kappa is a slope factor. Qmax was 11.7 nC/microF, V* was -18 mV and kappa was 16 mV in guinea-pig myocytes held at -100 mV, while the values in rat myocytes were 10.9 nC/microF, -32 mV and 13 mV. 5. The charge movement could be partially immobilized by a prior depolarization. This effect developed over a broad voltage range, from -120 to +20 mV. The fraction of charge that could be immobilized by a 10 s pre-pulse to +20 mV was 59%. 6. The time course of decay of both Qon and Qoff could basically be described as a single-exponential process. The time constant was largest at -40 mV and decreased at both more positive and negative test potentials. A second, slower Qoff time constant, possibly representing remobilization of immobilized charge, could be seen under some conditions. 7. The temperature dependence of charge movement was studied between 11 and 35 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Currents through Ca2+ channels of single guinea-pig ventricular myocytes were studied using patch electrodes for whole-cell recording. Currents through Na+ and K+ channels were suppressed by the application of drugs or the substitution of impermeant ions. 2. Inactivation of the Ca2+ current (ICa) was investigated using a two-pulse protocol. The amount of inactivation left behind by a pre-pulse appeared to be related to current magnitude, as others have reported. The dependence of inactivation on the pre-pulse potential was partially U-shaped, as the amount of inactivation peaked at 0 mV and then declined with more positive pre-pulses. 3. Non-specific current carried by monovalent ions through Ca2+ channels (Ins) was induced by lowering the extracellular Ca2+ concentration with EGTA. Ins peaked in an inward direction at -20 mV, reversed direction at +22 mV, and became a large outward current at more positive potentials. 4. Ins inactivated with a slow time course. The inactivation was not due to accumulation or depletion phenomena. Studies using two-pulse protocols showed that the amount of inactivation left by a pre-pulse was directly related to the pre-pulse potential. 5. The addition of micromolar amounts of free Ca2+ to the external solution induced outward rectification of Ins. Inward currents were small or absent, while larger outward currents could still be seen at very positive potentials. Ca2+-channel inactivation still occurred under these conditions, even in the absence of any significant ionic movement. 6. The time courses of Ins inactivation and recovery were studied. The half-time of Ins inactivation decreased with larger depolarizations. Recovery of Ins was very slow, but could be accounted for by changes in the surface charge of the membrane. 7. It is concluded that Ins inactivation is due solely to a voltage-dependent inactivation process which is intrinsic to myocardial Ca2+ channels. Voltage-dependent inactivation appears to account for a significant proportion of total Ca2+-channel inactivation at negative potentials, and appears to account for almost all of the inactivation at very positive potentials, even in the presence of millimolar concentration of external Ca2+.

The direct actions of phencyclidine (PCP) on mammalian sarcolemma were examined by determination of the drug's effects on the action potentials of isolated guinea-pig ventricular cells, and on the underlying ionic currents. PCP (10(-6) to 10(-4) M) did not alter the resting membrane potential but produced a dose-dependent prolongation of the duration of the action potential, and a reduction of the rate of depolarization of phase 0 (Vmax) of the action potential. Voltage clamp experiments revealed that PCP blocks both myocardial Ca++ channels and myocardial time-dependent K+ channels. The K+ channel blockade was shown to exhibit an apparent voltage-dependence. The effects of PCP on these ionic channels could explain previous reports of it prolonging myocardial action potentials and conflicting reports of positive and negative inotropism.

In many cells including cardiac myocytes, cytoplasmic Ca is importantly controlled by the plasmalemmal Na-Ca exchanger (3, 8). The tissue diversity and differences in cellular environment raise the question whether the same exchanger is found in all tissues. Recent experiments using rod cells have demonstrated that at least two forms of Na-dependent Ca transport exist. We have examined this issue in various rat and human tissues using the cloned human cardiac Na-Ca exchanger cDNA. Northern blot analysis in these two species show that the major transcript of the Na-Ca exchanger is 7.2 kilobases in heart, brain, kidney, liver, pancreas, skeletal muscle, placenta, and lung. Furthermore, ribonuclease protection analysis in rats shows conservation of the 348-base pair segment tested in heart, brain, kidney, skeletal muscle, and liver. Additionally, Southern blot analysis suggests that a single gene encodes this Na-Ca exchanger. Finally, we show that the clone used to generate our probes encodes a completely functional Na-Ca exchanger. With the use of COS cells and 293 cells transfected with the cloned human cardiac Na-Ca exchanger, we tested the Ca transport properties of the Na-Ca exchanger, the voltage dependence of the Na-Ca exchanger, as well as the Na dependence of the transport function of the Na-Ca exchanger. We conclude that the cardiac form of the Na-Ca exchanger is completely functional when the cDNA is expressed in mammalian cell lines, and, furthermore, this "cardiac" form of the Na-Ca exchanger is naturally expressed in all human and rat tissues tested (but at varying levels).