Relaxation in ferret ventricular myocytes: unusual interplay among calcium transport systems.

Transport systems responsible for removing Ca2+ from the myoplasm during relaxation in isolated ferret ventricular myocytes were studied using caffeine-induced contractures. Internal calcium concentration ([Ca2+]i) was measured with the fluorescent calcium indicator indo-1, and the results were compared with our recent detailed characterizations in rabbit and rat myocytes. Relaxation and [Ca2+]i decline during a twitch in ferret myocytes were fast and similar to that in rat myocytes (i.e. half-time, t 1/2 approximately 100-160 ms). During a caffeine-induced contracture (SR Ca2+ accumulation prevented), relaxation was still relatively fast (t 1/2 = 0.57 s) and similar to relaxation in rabbit supported mainly by a strong Na(+)-Ca2+ exchange. When both the SR Ca2+ uptake and Na(+)-Ca2+ exchange are blocked (by caffeine and 0 Na+, 0 Ca2+ solution) relaxation in the ferret myocyte is remarkably fast (approximately 5-fold) compared with rabbit and rat myocytes. The decline of the Cai2+ transient was also fast under these conditions. These values were similar to those in rat under conditions where relaxation is due primarily to Na(+)-Ca2+ exchange. Additional inhibition of either the sarcolemmal Ca(2+)-ATPase or mitochondrial Ca2+ uptake caused only modest slowing of the relaxation of caffeine-induced contracture in 0 Na+, 0 Ca2+ (t 1/2 increased to approximately 3 s). In rabbit myocytes the relaxation t 1/2 is slowed to 20-30 s by these procedures. Even when the systems responsible for slow relaxation in rabbit ventricular myocytes are inhibited (i.e. sarcolemmal Ca(2+)-ATPase and mitochondrial Ca2+ uptake) along with the SR Ca(2+)-ATPase and Na(+)-Ca2+ exchange, relaxation and [Ca2+]i decline in ferret myocytes remain rapid compared with rabbit myocytes. Ca2+ taken up by mitochondria in rabbit myocytes during a caffeine contracture in 0 Na+, 0 Ca2+ solution gradually returns to the SR after caffeine removal, but this component appears to be much smaller in ferret myocytes under the same conditions. We tested for possible residual Ca2+ transport by each of the four systems which suffice to explain Ca2+ removal from the cytoplasm in rabbit (SR Ca(2+)-ATPase, Na(+)-Ca2+ exchange, sarcolemmal Ca(2+)-ATPase and mitochondrial Ca2+ uptake). We conclude that there is an additional calcium transport system at work in ferret myocytes. For this additional system, our results are most compatible with a trans-sarcolemmal Ca2+ transport, but neither a cation exchanger nor a Ca(2+)-ATPase with characteristics like that in other cardiac cells. This additional system appears able to transport Ca2+ nearly as fast as the Na(+)-Ca2+ exchange in rat ventricular myocytes.     

Processes that remove calcium from the cytoplasm during excitation-contraction coupling in intact rat heart cells.

1. The processes that remove Ca2+ rapidly from the cytoplasm were studied in isolated rat ventricular myocytes subjected to whole-cell voltage clamp and internal perfusion with the Ca2+ indicator, indo-1. Na(+)-Ca2+ exchange was eliminated in most experiments by removing Na+ both internally and externally. 2. When the Ca(2+)-pumping ATPase of the sarcoplasmic reticulum (SR) was inhibited with cyclopiazonic acid and ryanodine interfered with the release of Ca2+ from the SR, [Ca2+]i transients rose slowly and declined extremely slowly. We concluded that transport of Ca2+ by mitochondria and the surface membrane Ca(2+)-pumping ATPase would be negligible over the time course of a single [Ca2+]i transient. 3. The influence of cytoplasmic Ca2+ ligands was characterized by internal perfusion with high concentrations of diffusible Ca2+ ligands (indo-1) or by superfusion with the membrane-permeant Ca2+ ligand, BAPTA AM. As the concentration of indo-1 in the cell increased from < 0.1 mM to at least 0.5 mM, the time constant of the decline of [Ca2+]i increased from about 0.15 s to nearly 3 s. 4. Calcium bound to endogenous Ca2+ ligands during depolarizing clamp pulses was characterized quantitatively as the difference between the total Ca2+ entering the cell via L-type Ca2+ channels and [Ca2+]i, in experiments in which SR function had been abolished. As total calcium increased during the entry of Ca2+, total calcium was found to agree reasonably well with that predicted by assuming that Ca2+ could bind to endogenous intracellular Ca2+ ligands and to indo-1. 5. The results indicate that, in the absence of Na+, the major factors determining the removal of cytoplasmic free Ca2+ are the Ca(2+)-pumping ATPase of the SR and the binding of Ca2+ to endogenous and exogenous Ca2+ ligands. 6. Several hypothetical 'Ca2+ removal functions' were fitted to the declining phase of [Ca2+]i transients. The best fit was one in which the flux of Ca2+ through the SR Ca(2+)-pumping ATPase was described by a Michaelis-Menten-type equation. The decline of the [Ca2+]i transient was thus described by a linear, first-order differential equation having terms giving the rate of Ca2+ transport by the SR Ca(2+)-pumping ATPase (Vmax and KM), the rates of complexation of Ca2+ with the various Ca2+ ligands (L), and a leak of Ca2+ into the cytoplasm from the SR (FSR,leak).(ABSTRACT TRUNCATED AT 400 WORDS)     

Na+-Ca2+ exchange induces low Na+contracture in frog skeletal muscle fibers after partial inhibition of sarcoplasmic reticulum Ca2+-ATPase

Contractile responses due to reduction in external sodium concentration ([Na+]o) were investigated in twitch skeletal muscle fibers of frog semitendinosus. Experiments were conducted after partial inhibition of sarcoplasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). In the absence of CPA, Na+ withdrawal failed to produce any change in resting tension. In the presence of CPA (2-10 microM), [Na+]o reduction induced a transient contracture without a significant change in the resting membrane potential. The amplitude of the contracture displayed a step dependence on [Na+]o, was increased by K(+)-free medium and was prevented in Ca(2+)-free medium. This contracture was inhibited by various blockers of the Na(+)-Ca2+ exchange but was little affected by inhibitors of sarcolemmal Ca(2+)-ATPase or mitochondria. When sarcoplasmic reticulum function was impaired, low-Na+ solutions caused no contracture. These results provide evidence that skeletal muscle fibers possess a functional Na(+)-Ca2+ exchange which can mediate sufficient Ca2+ entry to activate contraction by triggering Ca2+ release from sarcoplasmic reticulum when the sodium electrochemical gradient is reduced, and sarcoplasmic reticulum Ca(2+)-ATPase is partially inhibited. This indicates that when the sarcoplasmic reticulum Ca(2+)-ATPase is working (no CPA), Ca2+ fluxes produced by the exchanger are buffered by the sarcoplasmic reticulum. Thus the Na(+)-Ca2+ exchange may be one of the factors determining sarcoplasmic reticulum Ca2+ content and thence the magnitude of the release of Ca2+ from the sarcoplasmic reticulum.     

Measurement of calcium entry and exit in quiescent rat ventricular myocytes

The aim of this work was to obtain the first quantitative measurements of Ca2+ influx and efflux in quiescent cardiac cells. The relationship between free and total Ca2+ was obtained during a caffeine application. This buffering curve was then used to calculate changes of total Ca2+ from measurements of free cytosolic [Ca2+] ([Ca2+]i) made with Indo-1. The rate of Ca2+ removal from the cytoplasm was calculated by differentiating total Ca2+ with respect to time. The dependence of d(total Ca2+)/dt on [Ca2+]i was hyperbolic. Inhibition of either Na+-Ca2+ exchange (by addition of 10 mmol l(-1) NiCl2 or removal of external Na+) or the sarcolemmal Ca2+-activated adenosine triphosphatase (Ca2+-ATPase) (with carboxyeosin) decreased the calculated efflux. In both cases, the main effect was on the apparent maximum rate (Vmax) with little effect on the Michaelis-Menten constant (Km). These results suggest that the Na+-Ca2+ exchange and Ca2+-ATPase have very similar affinities for [Ca2+]i and that their fractional contributions do not change over the systolic range of [Ca2+]i. Ca2+ influx was quantified in two ways. The first method was to extrapolate the curve relating Ca2+ efflux to [Ca2+]i to zero [Ca2+]i. This gave a value of 4.49+/-0.54 micromol l(-1) s(-1) which was reduced to zero by either removal of external Ca2+ or addition of Ni2+. In other experiments external Ca2+ was removed and the maximum rate of fall of total Ca2+ calculated as 2.53+/-0.93 micromol l(-1) s(-1). This approach can be used to provide a quantitative analysis of the control of resting [Ca2+]i.     

Calcium-dependent modulation of the plateau phase of action potential in isolated ventricular cells of rabbit heart

[Ca2+]i-dependent modulation of the action potential has been studied in Fura-2 dialysed ventricular myocytes of the rabbit using the whole-cell current-clamp method. Fifteen consecutive action potentials (AP1-AP15) and [Ca2+]i transients were elicited at a frequency of 0.2 Hz. A single, brief application of caffeine (during AP9) first enhanced and thereafter attenuated the [Ca2+]i transients accompanying AP9 and AP10-AP12, respectively. This approach provided direct comparison between time courses of action potentials: during the initial steady state (e.g. AP8) and when Ca2+ release from the sarcoplasmic reticulum was increased by caffeine (AP9) or decreased by depletion (AP10). The increase in [Ca2+]i facilitated repolarization and decreased action potential duration. However, action potentials at reduced Ca2+ release (AP10) had longer duration than during steady state. The caffeine-induced changes in L-type Ca2+ current (ICa,L), during voltage-clamp conditions partially explained the effects of caffeine on action potentials. When ICa,L was blocked by 500 micromol L-1 Cd2+, enhanced [Ca2+]i transients revealed an extra current component which was outward at +10 mV and inward at the resting membrane potential (most probably the transient inward current). In the presence of Cd2+, however, AP8 and AP10 had identical time courses, suggesting that ICa,L alone was responsible for the lengthening of AP10. Alterations in the transmembrane Na+ gradient resulted in changes of the steady state action potential durations (AP8) consistently with the expected modulation of the Na+-Ca2+ exchange current. However, the contribution of this current to the [Ca2+]i-dependent behaviour of action potential plateau could not be demonstrated.     

Caffeine stimulates the reverse mode of Na/Ca2+ exchanger in ferret ventricular muscle.

This study investigated the effect of caffeine on the sarcolemmal mechanisms involved in intracellular calcium control. Ferret cardiac preparations were treated with ryanodine and thapsigargin in order to eliminate the sarcoplasmic reticulum (SR) function. This treatment abolished caffeine contracture irreversibly in normal solution. The perfusion with K-free medium that blocked the Na+--K+ pump resulted in a recovery of slow relaxing caffeine contractures similar to Na-free contractures. The amplitude of caffeine contractures was dependent on the bathing [caffeine]o and [Ca2+]o. Divalent cations Ni2+ and Cd2+, which have an inhibitory effect on the Na+/Ca2+ exchanger, produced dose-dependent inhibition of caffeine responses with apparent Ki of 780 +/- 19 and 132 +/- 5 microM, respectively. Caffeine also caused dose-dependent inhibition of Na-free contractures (Ki=4.62 +/- 1.5 mM), and the reduction or removal of [Na+]o exerted an inhibitory effect on caffeine contractures (Ki=73.5 +/- 17.12 mM). These experiments indicate that the increase in resting tension following exposure to caffeine was mediated by Na+/Ca2+ exchanger, which represents an additional element of complexity in caffeine action on cardiac muscle.     

Transport of magnesium by two isoforms of the Na+-Ca2+ exchanger expressed in CCL39 fibroblasts

Cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were measured with fluorescent indicators in CCL39 cells, a cell line established from Chinese hamster lung fibroblasts, transfected with complementary deoxyribonucleic acid (cDNA) of the Na+-Ca2+ exchanger isolated either from canine heart (NCX1) or from rat brain (NCX3). Raising extracellular [Mg2+] to 10 mM increased Mg2+ influx and the resultant change in [Mg2+]i (delta[Mg2+]i) was monitored with furaptra under Ca2+-free conditions. In control (vector-transfected) cells, delta[Mg2+]i at 45 min was similar with or without extracellular Na+ (130 mM or 0 mM) and when [Na+]i was raised by 1 mM ouabain treatment. delta[Mg2+]i in NCX1-transfected cells was attenuated significantly in the presence of 130 mM Na+, but became comparable to (or slightly larger than) that in control cells on either removal of extracellular Na+ or treatment with 1 mM ouabain. Cells expressing NCX3 showed an intermediate dependence of delta[Mg2+]i on Na+, probably reflecting a lower degree of expression of the exchanger protein. Extracellular Na+-dependent changes in [Ca2+]i (measured with fura-2 in the presence of extracellular Ca2+ and 10 microM ionomycin, a Ca2+ ionophore) were minimal in control cells, marked in the NCX1-transfected cells and intermediate in the NCX3-transfected cells. These results suggest that the Na+-Ca2+ exchanger (either NCX1 or NCX3) can transport Mg2+ and may play a role in the extrusion of magnesium from cells.     

Intracellular [Ca(2+)] transients in Ca(2+) wave in single rat ventricular myocyte

Ca(2+) release from the sarcoplasmic reticulum (SR) in heart muscle grades depending on Ca(2+) influx in the physiological twitch; Ca(2+( wave results from regenerative Ca(2+) release from the SR. To examine if the Ca(2+) release from the SR in the Ca(2+) wave takes a duration similar to the physiological one, a transient rise of intracellular [Ca(2+)] ([Ca(2+)](i) transient) was recorded during both a propagating Ca(2+) wave and an electrically evoked twitch with single rat ventricular myocytes, using a laser scanning confocal microscope. Care was taken to record the fluo-3 fluorescence from a segmental region with little lateral movement, especially during a propagating Ca(2+) wave. During a typical Ca(2+) wave, the time-to-peak (TP) and the half-width (HD) of the averaged [Ca(2+)](i) transient were 161 and 253 ms respectively, but they were 76 and 145 ms during an electrically evoked twitch. The difference in the duration between the two types of [Ca(2+)](i) transients could not be accounted for by modification of duration of [Ca(2+)](i) transient by possible asynchronous Ca(2+) release from the SR during a Ca(2+) wave, suggesting that the regenerative Ca2+) release from the SR in the Ca2+) wave occurs more slowly than the physiological one in rat ventricular myocytes.     

Decrease in sodium-calcium exchange and calcium currents in diabetic rat ventricular myocytes.

This study was designed in order to gain insight into possible changes in the inward sodium-calcium exchange current (INa-Ca) and the L-type calcium current (ICa), in ventricular myocytes isolated from streptozotocin-induced diabetic rats. Recordings were made using the nystatin-perforated patch technique which minimizes interference with the normal intracellular Ca2+ buffering mechanisms. The averaged INa-Ca current density elicited by Ca2+ current was smaller in diabetic than in normal myocytes at all potentials tested. INa-Ca activated by rapid application of caffeine was significantly reduced and the decay phase was prolonged. The density of ICa was also significantly reduced by diabetes in the range of test potentials between -10 and +50 mV. In addition, the fast time constant of ICa inactivation, which represents mainly the sarcoplasmic reticulum (SR) Ca2+ release-induced inactivation, was significantly higher in diabetic than in normal myocytes. The decrease in ICa, which is the main source of trigger Ca2+ for SR Ca2+ release, may explain the significantly lowered peak systolic [Ca2+]i previously shown in diabetic myocytes. As activation of ICa is essential for subsequent stimulation of INa-Ca, reduced ICa may contribute to decreasing activation of the Na+-Ca2+ exchanger.     

Relationship between global cytosolic Ca2+ concentration and Ca2+-activated K+ current in rabbit cerebral arterial myocyte.

In smooth muscle cells, the sarcoplasmic reticulum (SR) has been identified as the primary storage site for intracellular Ca2+. The peripheral SR is in close proximity with plasma membrane to make a narrow subsarcolemmal space. In this study, we investigated the regulation of subsarcolemmal [Ca2+] ([Ca2+]sl) and global cytosolic [Ca2+] ([Ca2+]c) of rabbit arterial smooth muscle using whole cell patch clamp technique and microspectrofluorimetry. The Ca2+-activated K+ current (IK(Ca)) and the ratio of fura-2 fluorescence (R340/380) were considered to reflect the [Ca2+]sl and [Ca2+]c, respectively. At a holding potential of 0 mV, extracellular application of 10 mM caffeine, a well known Ca2+-releasing agent, induced transient increase of IK(Ca) and R340/380 (IK(Ca)-transient and R340/380-transient, respectively). The increase and decay of IK(Ca) transient was faster than R340/380-transient. By repetitive application of caffeine, when the refilling state of SR was supposed to be lower than the control condition, IK(Ca)-transient and R340/380 transient were suppressed to different levels; e.g. the second application 20 sec after the first could induce smaller IK(Ca) transient than R340/380-transient. Dissociation of IK(Ca)-transient and R340/380-transient was removed by sufficient (>3 min) washout of caffeine. Recovery from the dissociation was also dependent upon the membrane potential; faster recovery was observed at negative (-40 mV) holding potential than at depolarized (0 mV) condition. Dissociation of IK(Ca) from [Ca2+]c was also partially prevented by perfusion with Na+-free (replaced by NMDG+) extracellular solution. These results suggest that, 1) there is prominent spatial inhomogeneity of [Ca2+] in cerebral arterial myocyte, 2) [Ca2+]Sl is preferentially affected by the interference from nearby plasmalemmal Ca2+ regulation mechanism which is partly dependent upon extracellular Na+.     

KB-R7943 reveals possible involvement of Na(+)-Ca2+ exchanger in elevation of intracellular Ca2+ in rat carotid arterial myocytes.

A Na(+)/Ca(2+) exchanger (NCX) is one of the major regulators of intracellular Ca(2+) concentration ([Ca(2+)](i)) in cardiac muscle cells. Although vascular smooth muscle myocytes also express NCX proteins, their functional role has not been clear, mainly due to the lack of specific inhibitors of NCX and relatively low levels of expression of NCX. In the present study, we have examined the involvement of NCX in the Na(+) deficient (0 Na(+)) elevation of [Ca(2+)](i) in rat carotid arterial myocytes using KB-R7943, an inhibitor of NCX. Perfusion with a Na(+)-free bathing solution, prepared by replacement of Na(+) with N-methyl-D-glucamine, induced an elevation of [Ca(2+)](i), which was effectively inhibited by KB-R7943 (IC(50)=3.5 microM). This inhibition was reversed by washout of KB-R7943. In contrast, D600, a blocker of voltage dependent L-type Ca(2+) channels (VDCC), did not affect the 0 Na(+)-induced elevation of [Ca(2+)](i). Treatment of myocytes with ryanodine abolished the elevation of [Ca(2+)](i) caused by caffeine but not that caused by 0 Na(+). Application of Cd(2+), which is known to block NCX as well as VDCC, also significantly inhibited the 0 Na(+) induced elevation. These results suggest that KB-R7943 inhibits the extracellular Na(+) dependent ([Na(+)](o)) change in [Ca(2+)](i) in rat carotid arterial myocytes, which is presumably activated by the reverse mode of NCX.     

Effects of gender on intracellular [Ca2+] in rat cardiac myocytes

The present study investigated the effects of gender on intracellular [Ca2+] ([Ca2+]i) in freshly isolated rat cardiac myocytes. Changes in [Ca2+]i in response to varied extracellular [Ca2+], different stimulus frequencies and addition of caffeine and isoprenaline were monitored using fura-2 in both male and female cardiac myocytes. Increasing extracellular [Ca2+] and stimulus frequency resulted in significant increases in peak [Ca2+] and the amplitude of the Ca2+ transient in both male and female cardiac myocytes. However, as extracellular [Ca2+] was raised, peak [Ca2+] and the amplitude of the Ca2+ transient increased significantly more in male than female cardiac myocytes. In addition a significant difference between male and female cells at each stimulus frequency was apparent. The time course of decay of the Ca2+ transient was significantly slower in female cardiac myocytes when compared with male cardiac myocytes, along with significantly slowed times to peak shortening and 50% relaxation, and a reduced extent of shortening. There was no significant difference in the amplitude of caffeine-induced [Ca2+]i responses between male and female cells, however, [Ca2+]i increased more readily in male cells than in female cells when isoprenaline was added. The data demonstrate that, under a variety of conditions, intracellular [Ca2+] rises to higher levels in cardiac myocytes from male as compared to female rats.     

Mechanism of activation of the tonic component of contraction in myocytes of guinea pig heart

AIM: Contractions of myocytes of guinea pig heart consist of a phasic component relaxing independently on the voltage and a tonic component relaxing upon repolarization. We found previously that Ca(2+) activating the tonic component is released from the sarcoplasmic reticulum. In this study, we analysed the mechanism of activation and maintenance of this release. METHODS: Experiments were performed at 37 degrees C in ventricular myocytes of guinea pig heart. Voltage-clamped myocytes were stimulated by the pulses of the duration of 300 ms to 15-45 s from the holding potential of -40 to +5 mV. [Ca(2+)](i) was monitored as fluorescence of Indo-1 and contractions were recorded with the TV edge-tracking system. RESULTS: Myocytes responded to the short and long pulses with phasic contraction or Ca(2+) transient followed by the sustained contraction or increase in [Ca(2+)](i). Repolarization brought about relaxation. 10 mmol L(-1) Ni(2+) blocking Na(+)/Ca(2+) exchange superfused during the tonic component increased its amplitude. Superfusion of Ca(2+)-free solution during sustained contraction brought about relaxation both in normal cells and in cells superfused with Ni(2+) despite preserved sarcoplasmic reticulum Ca(2+) content assessed with caffeine spritz. Relaxing effect of Ca(2+)-free solution was not affected by carboxyeosin, a blocker of sarcolemmal Ca(2+)-ATPase. Tonic component of contraction and of Ca(2+) transient was inhibited by 200 micromol L(-1) ryanodine, a blocker of Ca(2+) release channels of the sarcoplasmic reticulum and by 20 micromol L(-1) nifedipine, a blocker of L-type I(Ca). CONCLUSION: Tonic component of contraction results from Ca(2+) release via the sarcoplasmic reticulum Ca(2+) channels activated by sustained, nifedipine-sensitive and Ni(2+)-insensitive Ca(2+) influx. Alternatively, the SR Ca(2+) release is activated by voltage, the dihydropyridine receptors acting like voltage sensors.     

The actions of altered osmolarity on guinea-pig detrusor smooth muscle contractility and intracellular calcium

The study measured the effects in vitro of changing extracellular osmolarity on the contractility of detrusor smooth muscle strips. The data were interpreted in the context of separate measurements from isolated cells of alterations to the intracellular [Ca2+], [Ca2+]i. Increased osmolarity (300-700 mosmol l-1) reduced phasic contractions but increased resting tension regardless of whether sucrose, LiCl or NaCl were used as osmolytes. [Ca2+]i was decreased slightly only when NaCl increased osmolarity, otherwise it was unchanged. The contractile effects may be explained by tissue shrinkage and reduction of detrusor excitability. Lowered osmolarity (300-64 mosmol l-1) decreased phasic contractions but increased resting tension and [Ca2+]i. The raised resting tension was due solely to low osmolarity and was independent of changes to [Na], [Cl] or ionic strength. The rise of [Ca2+]i was due partly to Ca2+ influx through Na(+)-Ca2+ exchange but a fraction was independent of extracellular Ca, unaffected by Gd3+, and persisted in the presence of caffeine. By contrast, reduction of phasic tension was due mainly to the reduced ionic strength, not osmolarity. The results do not support the presence of functional stretch-activated channels and suggest only a minor role for Na(+)-Ca2+ exchange under these conditions. However, they do suggest an intracellular source of Ca2+, which is independent of the sarcoplasmic reticulum.     

External calcium sensitivity of low sodium contractures in the control and hypertrophied right ventricle of the ferret.

The existence of possible differences of calcium (Ca2+) fluxes through the sarcolemmal sodium-calcium (Na+/Ca2+) exchanger during hypertrophy has been tested by comparing the characteristics of the contracture--as an indicator of the intracellular Ca2+ concentration--induced by partial or total withdrawal of external sodium (Na+), in the absence of external potassium, in the right ventricular trabeculae of adult ferret hearts. Pressure-overload was induced by pulmonary artery clipping and led to an increase of the right ventricular weight of 60%. At an external Ca2+ concentration ([Ca2+]o) of 3 mM, the dependence of the contractures on extracellular sodium concentration ([Na+]o), the rate of tension development, the time course of spontaneous relaxation and the time course for the repriming of the contracture were unchanged by hypertrophy. However, the relationship between [Ca2+]o and contracture amplitude at various [Na+]o showed that the apparent affinity of the contracture for [Ca2+]o was decreased in hypertrophied preparations. Thus, in 0 mM [Na+]o, half-maximal contracture was induced at a [Ca2+]o of 0.012 +/- 0.016 mM and 0.171 +/- 0.021 mM in control (n = 11) and hypertrophy (n = 12) respectively (P less than 0.001). Although these data may be indicative of a decreased Ca2+ influx through the Na+/Ca2+ exchanger, it cannot be excluded that intracellular buffering mechanism may also be involved in this differential response to [Na+]o withdrawal.     

Difference in propagation of Ca2+ release in atrial and ventricular myocytes

Intracellular [Ca2+] ([Ca2+]i) was imaged in atrial and ventricular rat myocytes by means of a high-speed Nipkow confocal microscope. Atrial myocytes with an absent t-tubule system on 8-di- ANEPPS staining showed an initial rise in Ca2+ at the periphery of the cell, which propagated to the interior of the cell. Ventricular myocytes showed a uniform rise in [Ca2+]i after electrical stimulation, consistent with a prominent t-tubular network. In atrial myocytes, there was a much shorter time between the peak of the [Ca2+]i transient and the peak contraction as compared to ventricular myocytes. A regional release of Ca2+ induced by an exposure of one end of the myocyte to caffeine with a rapid solution switcher resulted in a uniform propagation of Ca2+ down the length of the cell in atrial myocytes, but we found no propagation in ventricular myocytes. A staining with rhodamine 123 indicated a much greater density of mitochondria in ventricular myocytes than in atrial myocytes. Thus the atrial myocytes display a lack of "local control" of Ca2+ release, with propagation after the Ca2+ release at the periphery induced by stimulation or at one end of the cell induced by exposure to caffeine. Ventricular myocytes showed the presence of local control, as indicated by an absence of the propagation of a local caffeine-induced Ca2+ transient. We suggest that this finding, as well as a reduced delay between the peak of the [Ca2+]i transient and the peak shortening in atrial myocytes, could be due in part to reduced Ca2+ buffering provided by mitochondria in atrial myocytes as opposed to ventricular myocytes.     

Calcium homeostasis in dissociated embryonic neurons: a flow cytometric analysis.

1. Ca2+ homeostasis in freshly dissociated neurons from embryonic rat hypothalamus, cortex, and brain stem was investigated with flow cytometry. Cells were dissociated from embryonic brain by enzymatic and mechanical means and were incubated with the acetoxymethylester derivative of the Ca(2+)-sensitive dye indo-1. Neurons hydrolyzed and retained the dye as determined by the intensity of fluorescence emission, whereas similarly treated cultured astrocytes gave very low-level fluorescence. 2. The fluorescence of the indo-1 dye was measured at two wavelengths (405 and 485 nm) for each cell. Data were collected only from those cells (presumptive neurons) with high levels of fluorescence. Methods were developed to calibrate the level of intracellular free calcium ([Ca2+]i) as the ratio of fluorescence at 410 and 485 nm. The level of intracellular free Ca2+ was then calculated for each neuron. 3. A wide distribution of resting [Ca2+]i was found, with a median of approximately 90 nM. After addition of ionomycin to cells in Ca(2+)-free medium, there was a transient increase in [Ca2+]i, suggesting that all embryonic neurons had internal Ca2+ stores. The presence of active calcium extrusion mechanisms was demonstrated with the use of ionomycin in Ca(2+)-containing medium and with metabolic inhibitors. Furthermore, incubation in sodium-free medium resulted in a transient increase in [Ca2+]i and a reduced ability to eliminate elevated [Ca2+]i from the cytoplasm, suggesting that calcium homeostasis was dependent on the activity of the Na(+)-Ca2+ exchange mechanism. 4. Depolarization with K+ or veratrine increased [Ca2+]i in approximately 20% of the cells. This increase was blocked by eliminating extracellular free Ca2+ or adding Co2+, nifedipine, or verapamil, suggesting mediation by voltage-sensitive calcium channels. 5. Neurons were sorted on the basis of high [Ca2+]i and placed into dissociated culture. After 24 h, neurons in culture retained indo-1 fluorescence, suggesting that populations of neurons can be collected on the basis of their levels of [Ca2+]i. 6. These results demonstrate that flow cytometric analysis allows the characterization of a variety of Ca(2+)-regulatory mechanisms in populations of freshly dissociated embryonic neurons. Although only a proportion of embryonic day 17 neurons exhibit voltage-sensitive calcium channels, all neurons have developed the ability to sequester and extrude Ca2+.     

Is there a calcium paradox in isolated bovine aortic endothelial cells?

When bovine aortic endothelial (BAE) cells are superfused with a solution containing low calcium (approximately 10 nM) and subsequently exposed to a solution containing normal Ca2+ (2 mM) a large transient increase in intracellular calcium is seen. This elevation in [Ca2+]i is similar to that described as the calcium paradox in cardiac cells. If the cells are exposed to the agonist, ATP, during the period in low-Ca2+ solution the paradoxical rise in [Ca2+]i is increased. Removal of external Na+ from the low-Ca2+ solution reduces the rise in [Ca2+]i on returning to 2mM-Ca2+ solution. These data are consistent with the presence of a calcium paradox in these cells and with the hypothesis that the underlying mechanism involves the loading of the cell with Na+ during the period in low Ca2+. This process may occur as a result of the altered selectivity of the ATP-activated Ca2+ influx mechanism.     

Contribution of Na+/Ca2+ exchanger to glucose-induced [Ca2+]i increase in rat pancreatic islets

The present study was carried out to elucidate the role of the reverse mode of the Na+/Ca2+ exchanger in an increase in intracellular Ca2+ concentration ([Ca2+]i) induced by a stimulatory concentration of glucose in rat pancreatic islets. The effects of KB-R7943, a selective inhibitor of reverse Na+/Ca2+ exchanger, on Na+o removal-induced [Ca2+]i changes were examined by a microfluorimetric method using fura-2 in perifused preparations of isolated rat pancreatic islets. Na+o removal induced a rapid increase in [Ca2+]i under 100 or 5 mM K+ conditions, respectively. The increases in [Ca2+]i induced by Na+o removal were inhibited by KB-R7943. The net amount of the [Ca2+]i increases during Na+o removal (Delta[Ca2+]i), obtained by subtracting the KB-R7943-independent Delta[Ca2+]i in the presence of KB-R7943 from Delta[Ca2+]i in the absence of KB-R7943, was significantly increased when extracellular K+ was raised. Increasing the external glucose concentration from 3 to 20 mM caused a biphasic increase in [Ca2+]i, which exhibited a transient increase (first phase) followed by a sustained increase (second phase) in [Ca2+]i. KB-R7943 (10 microM) partially inhibited the second phase of the [Ca2+]i increase rather than the first phase. These results suggest that the increase in [Ca2+]i induced by Na+o removal may be enhanced when plasma membrane is depolarized, and consequently, Ca2+ influx through the reverse Na+/Ca2+ exchanger may partially contribute to the glucose-induced [Ca2+]i dynamics in rat pancreatic islet cells.     

Na(+)-Ca(2+) exchanger controls the gain of the Ca(2+) amplifier in the dendrites of amacrine cells

We have previously shown that disabling forward-mode Na(+)-Ca(2+) exchange in amacrine cells greatly prolongs the depolarization-induced release of transmitter. To investigate the mechanism for this, we imaged [Ca(2+)](i) in segments of dendrites during depolarization. Removal of [Na(+)](o) produced no immediate effect on resting [Ca(2+)](i) but did prolong [Ca(2+)](i) transients induced by brief depolarization in both voltage-clamped and unclamped cells. In some cells, depolarization gave rise to stable patterns of higher and lower [Ca(2+)] over micrometer-length scales that collapsed once [Na(+)](o) was restored. Prolongation of [Ca(2+)](i) transients by removal of [Na(+)](o) is not due to reverse mode operation of Na(+)-Ca(2+) exchange but is instead a consequence of Ca(2+) release from endoplasmic reticulum (ER) stores over which Na(+)-Ca(2+) exchange normally exercises control. Even in normal [Na(+)](o), hotspots for [Ca(2+)] could be seen following depolarization, that are attributable to local Ca(2+)-induced Ca(2+) release. Hotspots were seen to be labile, probably reflecting the state of local stores or their Ca(2+) release channels. When ER stores were emptied of Ca(2+) by thapsigargin, [Ca(2+)] transients in dendrites were greatly reduced and unaffected by the removal of [Na(+)](o) implying that even when Na(+)-Ca(2+) exchange is working normally, the majority of the [Ca(2+)](i) increase by depolarization is due to internal release rather than influx across the plasma membrane. Na(+)-Ca(2+) exchange has an important role in controlling [Ca(2+)] dynamics in amacrine cell dendrites chiefly by moderating the positive feedback of the Ca(2+) amplifier.