Caffeine-induced depolarization in amphibian skeletal muscle fibres: role of Na+/Ca2+ exchange and K+ release

Caffeine (4 mM ) produces a depolarization of about 10 mV in frog muscle fibres (Leptodactylus ocellatus). The aim of this work was to study the mechanisms of this effect. An approximately threefold rise in membrane resistance [Cl⁻-free (SO₄^2–) medium] substantially increased, and both Na⁺-free medium and Ni²⁺ (5 mM ) reduced, the caffeine-induced depolarization. In voltage-clamped (–60 mV) short fibres from lumbricalis muscle of the toad (Buffo arenarum), caffeine generated an inward current of 4.13 ± 0.48 μA cm^–2. This caffeine-induced current was reduced by 60% in Na⁺-free medium, 44% in the presence of 5 mM amiloride and 48% by 5 mM Ni²⁺, suggesting that the activation of the Na⁺–Ca²⁺ exchanger in its forward mode may play a role in the observed electrical effects of the drug. Caffeine also produced a marked release of K⁺. Net K⁺ efflux increased from 3.5 ± 0.2 (control) to 22.1 ± 2.3 pmol s^–1 cm^–2 (caffeine). It is shown that in the presence of the drug, [K⁺] in the lumen of the T tubules may well increase to levels which could produce, in part, both the observed depolarization and the caffeine-induced current under voltage clamp conditions. The caffeine-induced K⁺ efflux was not reduced by 5 mM Ni²⁺. At a holding potential of 30 mV the caffeine-induced current was reversed (outward) and roughly halved by 5 mM Ni²⁺. The Ni²⁺-sensitive fraction of the caffeine-induced current, assumed to represent the Na⁺–Ca²⁺ exchanger current, had an estimated reversal potential close to 12 mV ([Na⁺]_o=115 mM ; [Ca²⁺]_o=1 mM ). In conclusion, the depolarizing effect of caffeine described here would be produced by two mechanisms: (a) an inward current generated by the activation of the Na⁺–Ca²⁺ exchanger in its forward mode, and (b) the rise of the external [K⁺] in restricted spaces like the T tubules.     

Influence of extracellular Na+ removal on cytosolic Ca2+ concentration in smooth muscle cells of rabbit cerebral artery.

There are some controversies over the contribution of Na+/Ca2+ exchanger (NCX) to the regulation of cytosolic Ca2+ concentration ([Ca2+]c) in smooth muscle. To prove the functional role of Na+/Ca2+ exchanger, we examined whether the removal of extracelluar Na+ could affect [Ca2+]c of rabbit cerebral arterial smooth muscle. The fluorescence ratio of fura-2 (R(340/380)) was measured in the single myocyte of rabbit middle cerebral artery and Na+ was substituted with the same concentration of NMDG+ or Li+. In 21 out of 230 cells tested, Na+ removal increased R(340,380) (deltaR(340/380)) by 115 +/- 16.5% of the deltaR(340/380) induced by 10 mM caffeine in the same cell. The Na+ removal-induced deltaR(340/380) was blocked by a selective inhibitor of cardiac type NCX exchanger (KB-R7943, (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea, 10 microM). In those cells where the Na+ removal by itself did not increase R(340/380), the caffeine-induced deltaR(340/380) was increased by Na+-removal (130 +/- 9.8% of control response, n=30). Under the whole-cell patch clamp condition, short application of caffeine induced transient increase of outward current (I(K,Ca)-transient) which reflect the change of subsarcolemmal [Ca2+]. The application of KB-R7943 increased the amplitude of I(K,Ca)-transient (n=4). These results suggest the functional existence of NCX in rabbit cerebral artery smooth muscle.     

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.     

Modulation of intracellular Na+ and Ca2+ induced by the cardiac Na+-Ca2+ exchanger in guinea pig ventricular myocytes

The Na+-Ca2+ exchanger current was measured in single guinea pig ventricular myocytes, using the whole-cell voltage-clamp technique, and intracellular free calcium concentration ([Ca2+](i)) was monitored simultaneously with the fluorescent probe Indo-1 applied intracellularly through a perfused patch pipette. In external solutions, which have levels of Ca2+ (approximately 66 microM Ca2+) thought low enough to inhibit exchanger turnover, the removal of external Na+ (by replacement with Li+) induced both an outward shift of the holding current and an increase in [Ca2+](i), even though the recording pipette contained 30 mM bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), sufficient to completely block phasic contractions. The effects of Na+ removal were blocked either by the extracellular application of 2 mM Ni2+ or by chelating extracellular Ca2+ with 1 mM EGTA. In the presence of 10 microM Ryanodine, the effects of external Na+ substitution with Li(+) on both membrane current and [Ca2+](i) were attenuated markedly in amplitude and at a much slower time course. Reversal potentials were estimated by using ramp pulses and by defining exchange currents as the Ni2+-sensitive components. The experimental values of the reversal potential and [Ca2+](i) were used to calculate cytosolic Na+ ([Na+](i)) by assuming an exchanger stoichiometry of 3Na+ : 1Ca2+. These calculations suggested that in the nominal absence of external Ca2+ ( approximately 66 microM under our experimental conditions), the exchanger operates at -40 mV as though approximately 40 mM Na+ had accumulated in the vicinity of the intracellular binding sites. We conclude that under the conditions of low extracellular Ca2+ and high intracellular Ca2+ buffering, the Na+-Ca2+ exchanger can still generate sufficient Ca2+ influx on the removal of external Na+ to markedly increase cytosolic free Ca2+.     

Properties of the inwardly rectifying K+ conductance in the toad retinal pigment epithelium.

An inwardly rectifying K+ current was analysed in isolated toad retinal pigment epithelial (RPE) cells using the perforated-patch clamp technique. The zero-current potential (Vo) of RPE cells averaged -71 mV when the extracellular K+ concentration ([K+]o) was 2 mM. Increasing [K+]o from 0.5 to 5 mM shifted V0 by +43 mV, indicating a relative K+ conductance (TK) of 0.74. At [K+]o greater than 5 mM, TK decreased to 0.53. Currents were larger in response to hyperpolarizing voltage pulses than depolarizing pulses, indicating an inwardly rectifying conductance. Currents were time independent except in response to voltage pulses to potentials positive to 0 mV, where the outward current decayed with an exponential time course. Both the inwardly rectifying current and the transient outward current were eliminated by the addition of 0.5 mM Ba2+, 5 mM Cs+ or 2 mM Rb+ to the extracellular solution. The current blocked by these ions reversed near the K+ equilibrium potential (EK) over a wide range of [K+]o, indicating a highly selective K+ channel. The current-voltage relationship of the isolated K+ current exhibited mild inward rectification at voltages negative to -20 mV and a negative slope conductance at voltages positive to -20 mV. The Cs(+)- and Ba(2+)-induced blocks of the K+ current were concentration dependent but voltage independent. The apparent dissociation constants were 0.8 mM for Cs+ and 40 microM for Ba2+. The K+ conductance decreased when extracellular Na+ was removed. Increasing [K+]o decreased the K+ chord conductance (gK) at negative membrane potentials. In the physiological voltage range, increasing [K+]o from 2 to 5 mM caused gK to decrease by approximately 25%. We conclude that the inwardly rectifying K+ conductance represents the resting K+ conductance of the toad RPE apical membrane. The unusual properties of this conductance may enhance the ability of the RPE to buffer [K+]o changes that take place in the subretinal space at the transition between dark and light.     

Contribution of Na+/Ca2+ exchanger to the regulation of myogenic tone in isolated rat small arteries.

The contribution of the Na+/Ca2+ exchanger to the myogenic vascular tone was examined in rat isolated skeletal muscle small arteries (ASK) with pronounced myogenic tone and mesenteric small arteries (AMS) with little myogenic tone. Myogenic tone was assessed by the vascular inner diameter at transmural pressures of 40 and 100 mmHg. To depress the Na+/Ca2+ exchanger, the extracellular Na+ concentration ([Na+]o) was lowered from 143 to 1.2 mM by substituting choline-Cl for NaCl. The ASK developed significant myogenic tone and constricted further in low [Na+]o. Nifedipine (1 microM) reduced both myogenic tone and low [Na+]o-induced contraction. Because the membrane potential of ASK was not changed by low [Na+]o (-35 +/- 2 mV at 143 mM [Na+]o, -37 +/- 3 mV at 1.2 mM [Na+]o), depolarization-induced Ca2+ influx was not a cause of the low [Na+]o-induced contraction. The AMS did not develop significant myogenic tone. Although low [Na+]o also constricted AMS, the magnitude of constriction was significantly weaker than that in ASK (17 +/- 4 vs. 47 +/- 6%, P < 0.01, at 58 mM Na+). With Bay K 8644, AMS developed myogenic tone, and low [Na+]o-induced constriction was significantly increased. In conclusion, Na+/Ca2+ exchanger may play an important role in regulating myogenic tone, likely via mediating Ca2+-extrusion.     

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.     

Whole-cell K+ currents in isolated rabbit corneal epithelial cells.

Membrane currents were recorded from enzymatically isolated cells from basal layers of rabbit corneal epithelium by the whole-cell clamp technique. Pipettes contained 140.4 mM KCl and extracellular K+ concentration was varied. The membrane currents on step voltage changes were rectangular currents with some fluctuations. The fluctuations disappeared near the zero-current potential. The reversal potential in normal Tyrode's solution with 5.4 mM K+ was -57.8 +/- 6.2 mV (mean +/- S.D., n = 10). Increasing [K+]o from 5.4 to 140.4 mM shifted the reversal potentials in the positive direction with a slope of 41.0 mV/decade. Concomitant depolarization of the resting potential was observed on increasing [K+]o. The whole-cell currents were blocked by Cs+ or Ba2+. These suggest that the major current component in the corneal epithelial cells in K+.     

Characterization of the relationship between Na+–Ca2+ exchange rate and cytosolic calcium in trout cardiac myocytes

The whole-cell patch-clamp technique combined with rapid caffeine (CAF) applications was used to measure Na+-Ca2+ exchange (NCX) currents (I(NCX)). The rate of Ca2+ extrusion and the amount of Ca2+ extruded from the cell upon a rapid CAF exposure were obtained from I(NCX) and its time integral, respectively. This gave a maximal NCX rate (V(NCX)) of 151 amol pF(-1) s(-1) or 2.3 mM s(-1) and a half-maximal V(NCX) (K0.5) at a total cellular [Ca2+] ([Ca2+]tot) of 15.4 amol pF(-1). Using the same approach for the tail current induced by repolarization to -80 mV gave a K0.5 of 7.0 amol pF(-1) corresponding to 108 microM total or 2-4 microM free Ca2+. The relationship between [Ca2+]tot and V(NCX) was linear in the physiological range. Inhibition of the SR function with cyclopiazonic acid plus ryanodine reduced the slope significantly from 23.2+/-1.4 to 17.6+/-1.6 s(-1), while ryanodine alone had no effect. The relationship between [Ca2+]tot and V(NCX) was steeper at more negative membrane potentials, and with identical SR Ca2+ loads the maximal VNCX at -10 mV was reduced to 39.7+/-2.7% of the value at -90 mV. Long depolarizations caused SR Ca2+ loading through reverse-mode NCX. Between -30 and +10 mV reverse mode V(NCX)=Vm.0.047 amol pF(-1) s(-1) mV(-1)+2.51 amol pF(-1) s(-1), giving a reversal potential of -54 mV. In conclusion, the relationship between V(NCX) and [Ca2+]tot shows that the NCX is capable of removing a total Ca2+ transient of 60 microM at physiological heart rates, while reverse-mode NCX reloads the sarcoplasmic reticulum (SR) during depolarization. Furthermore, small alterations in the action potential configuration are predicted to change significantly the relative importance of the NCX in the regulation of cytosolic [Ca2+] and SR Ca2+ loading.     

Caffeine-induced current in embryonic heart cells: time course and voltage dependence

Abrupt exposure of 90- to 130-micron diameter chick embryonic myocardial cell aggregates to 10 mM caffeine has been shown to induce a transient inward current. In the present study, we recorded a similar current in small cell clusters (less than 10 cells) in which access of caffeine to each of the cells was rapid. The resulting inward current consisted of a single peak, which decayed exponentially (predominant time constant 335 +/- 130 ms at -40 mV) and had a peak amplitude of up to 15.5 microA/cm2. The caffeine-induced current persisted when the slow inward current was abolished by a 30-s pretreatment with 2 microM D 600 and could be observed at potentials where the fast sodium channels were fully inactivated. The current-voltage relation of the caffeine response was linear between -110 and -40 mV, giving an extrapolated voltage intercept of +12 mV. However, the inward current did not diminish or reverse with further depolarization. A substantial inward current occurred at potentials up to +60 mV, which is more positive than the reversal potential of the tetrodotoxin-sensitive inward current. We conclude that the caffeine-induced current is mediated in part by electrogenic Na+-Ca2+ exchange.     

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+.     

Sodium-activated potassium current in guinea pig gastric myocytes

This study was designed to identify and characterize Na+-activated K+ current (I(K(Na))) in guinea pig gastric myocytes under whole-cell patch clamp. After whole-cell configuration was established under 110 mM intracellular Na+ concentration ([Na+]i) at holding potential of -60 mV, a large inward current was produced by external 60 mM K+([K+]degrees). This inward current was not affected by removal of external Ca2+. K+ channel blockers had little effects on the current (p>0.05). Only TEA (5 mM) inhibited steady-state current to 68+/-2.7% of the control (p<0.05). In the presence of K+ channel blocker cocktail (mixture of Ba2+, glibenclamide, 4-AP, apamin, quinidine and TEA), a large inward current was activated. However, the amplitude of the steady-state current produced under [K+]degrees (140 mM) was significantly smaller when Na+ in pipette solution was replaced with K+- and Li+ in the presence of K+ channel blocker cocktail than under 110 mM [Na+]i. In the presence of K+ channel blocker cocktail under low Cl- pipette solution, this current was still activated and seemed K+-selective, since reversal potentials (E(rev)) of various concentrations of [K+]degrees-induced current in current/voltage (I/V) relationship were nearly identical to expected values. R-56865 (10-20 microM), a blocker of I(K(Na)), completely and reversibly inhibited this current. The characteristics of the current coincide with those of I(K(Na)) of other cells. Our results indicate the presence of I(K(Na)) in guinea pig gastric myocytes.     

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.     

Inhibition of Ca2+ mobilization by caffeine in a cultured vascular smooth muscle cell line (A7r5).

The effects of caffeine on the resting level and agonist-induced changes in intracellular calcium ([Ca2+]i) have been studied in the vascular smooth muscle cell line A7r5. Caffeine (1-30 mM) lowers the resting [Ca2+]i by reducing the entry of Ca2+ and inhibits completely the mobilization of Ca2+ by arginine vasopressin. Application of forskolin, to elevate cAMP, does not affect the resting level of Ca2+i but does abolish the agonist-induced rise. These data add to the complexity of caffeine-induced changes in [Ca2+]i and point to a possible interaction between cAMP and other second messenger systems mobilizing Ca2+i in this cell type.     

Rhythmic hyperpolarizations and depolarization of sympathetic ganglion cells induced by caffeine.

Superfusion of the isolated sympathetic ganglion of the bullfrog with a caffeine-containing (1-6 mM) solution caused in many cells an initial slow hyperpolarization which was followed by a subliminal depolarization interruped by rhythmic hyperpolarizations. A hyperpolarization, similar to one of the rhythmic hyperpolarizations, could be triggered by an action potential in the presence of caffeine. The action potential itself was not markedly affected by caffeine except for its afterhyperpolarization which was prolonged. All these caffeine-induced hyperpolarizations were associated with a marked reduction of the membrane resistance, their amplitude was increased in a K+-free solution and decreased in a high-K+ solution, and their polarity was reversed at the same level at which the afterhyperpolarization was also inverted. This reversal level was not altered by omission of Na+ or C1- from the external medium. These hyperpolarizations were reversibly abolished by depletion of external Ca2+ or replacement of external Ca2+ by Mg2+. Excess of external Ca2+ caused a shortening of the interval between rhythmic hyperpolarizations. Furthermore, iontophoretic injection of EDTA into the cytoplasm markedly depressed the initial caffeine hyperpolarizatin and abolished both the rhythmic and evoked caffeine hyperpolarizations. The caffeine-induced depolarization was not affected by omission of external Cl-. It was decreased in a Na+-free medium, but completely eliminated by omission of both Na+ and Ca2+ from the external medium. Tetrodotoxin did not impair the production of the initial and the rhythmic hyperpolarizations. A strong depolarizing pulse could evoke a typical hyperpolarizing response in the presence of this compound. Dibutyryl cyclic AMP, d-tubocurarine, atropine, and phenoxybenzamine were without effect on the caffeine-induced hyperpolarizations and depolarization. It was concluded that each caffeine-induced hyperpolarization is the result of an increased K+ permeability, which is probably caused by a rise in the internal Ca2+ concentration. It was also concluded that the caffeine-induced depolarization is due to an increased membrane permeability to Ca2+ and Na+.     

Ba2+ and K+ alteration of K+ conductance in spontaneously active vascular muscle.

The spontaneous spiking of vascular muscle in hepatic portal vein was converted from a burst pattern to single spiking by K+ concentrations greater than 20 mM. A pronounced decrease in contraction amplitude was always associated with the conversion of bursts of spikes to single spikes. The single spikes were accompanied by contractions that were only 1-5% of the magnitude of the contractions associated with spike bursts. Ba2+ (1 mM) increased the number of spikes per burst in myovascular cells at low(less than 10 mM) K+ concentrations. Even though Ba2+ caused depolarization, which itself tended to cause conversion to single spiking, the number of spikes per burst and contraction amplitude were greater in Ba2+ solutions. The slope of the Em-log [K+]o relationship depended on the time allowed for changes in [K+], and a maximum slope of 36 mV/decade and an extrapolated [K+]i of 160 mM were the highest slope and lowest [K+]i values recorded. Ba2+ slightly decreased (to 28 mV/decade) the slope of the curve but did not alter the intercept at [K+] = 160 mM. Ba2+ caused an increase in input resistance (rin) and depolarization independent of CL- concentration, and thus appeared to decrease K+ conductance. K+ concentration increases tended to increase K+ conductance (decrease rin) with depolarization. Spontaneously active vascular muscle thus appears to have a K+ conductance that can be altered and to show different spike patterns and altered conduction, resulting in a marked change in contractions. The spike pattern appears to depend at least partially on the combination of K+ conductance and Em.     

Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms.

The roles of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase and Na(+)-Ca2+ exchange in Ca2+ removal from cytosol were compared in isolated rabbit and rat ventricular myocytes during caffeine contractures and electrically stimulated twitches. Cell shortening and intracellular calcium concentration ([Ca2+]i) were measured in indo-1-loaded cells. Na(+)-Ca2+ exchange was inhibited by replacement of external Na+ by Li+. To avoid net changes in cell or SR Ca2+ load during a twitch in 0 Na+ solution, intracellular Na+ (Na+i) was depleted using a long pre-perfusion with 0 Na+, 0 Ca2+ solution. SR Ca2+ accumulation was inhibited by caffeine or thapsigargin (TG). Relaxation of steady-state twitches was 2-fold faster in rat than in rabbit (before and after Na+i depletion). In contrast, caffeine contractures (where SR Ca2+ accumulation is inhibited), relaxed faster in rabbit cells. Removal of external Na+ increased the half-time for relaxation of caffeine contractures 15- and 5-fold in rabbit and rat myocytes respectively (and increased contracture amplitude in rabbit cells only). The time course of relaxation in 0 Na+, 0 Ca2+ solution was similar in the two species. Inhibition of the Na(+)-Ca2+ exchange during a twitch increased the [Ca2+]i transient amplitude (delta[Ca2+]i) by 50% and the time constant of [Ca2+]i decline (tau) by 45% in rabbit myocytes. A smaller increase in tau (20%) and no change in delta[Ca2+]i were observed in rat cells in 0 Na+ solution. [Ca2+]i transients remained more rapid in rat cells. Inhibition of the SR Ca(2+)-ATPase during a twitch enhanced delta[Ca2+]i by 25% in both species. The increase in tau after TG exposure was greater in rat (9-fold) than in rabbit myocytes (2-fold), which caused [Ca2+]i decline to be 70% slower in rat compared with rabbit cells. The time course of [Ca2+]i decline during twitch in TG-treated cells was similar to that during caffeine application in control cells. Combined inhibition of these Ca2+ transport systems markedly slowed the time course of [Ca2+]i decline, so that tau was virtually the same in both species and comparable to that during caffeine application in 0 Na+, 0 Ca2+ solution. Thus, the combined participation of slow Ca2+ transport mechanisms (mitochondrial Ca2+ uptake and sarcolemmal Ca(2+)-ATPase) is similar in these species. We conclude that during the decline of the [Ca2+]i transient, the Na(+)-Ca2+ exchange is about 2- to 3-fold faster in rabbit than in rat, whereas the SR Ca(2+)-ATPase is 2- to 3-fold faster in the rat.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcium release induced by high K+ and caffeine in cultured skeletal muscle cells of embryonic chicken

To further understand the function of excitation-contraction coupling in skeletal muscle cells developing in vitro, Ca2+ transients elicited by high-K+ depolarization in the presence and absence of extracellular Ca2+ were compared with Ca2+ release induced by caffeine in cultured skeletal muscle cells isolated from 9-day-old chicken embryos (E9). Almost all myoblasts and myotubes cultured for 1 (E9I1) to 8 (E9I8) days responded to 80 mM [K+]O with an elevation of [Ca2+]i. Although all myotubes cultured for more than 4 days exhibited Ca2+ release independent of extracellular Ca2+, only about 50% of E9I1 and E9I2 cells maintained their response to Ca(2+)-free high-[K+]O solution. Strikingly, a considerable proportion of cells of short-term culture were insensitive to 10 mM caffeine. Moreover, 46.8% of the caffeine-insensitive E9I1 and E9I2 cells, 29 out of 62, was still responsive to 80 mM [K+]O in the absence of extracellular Ca2+. Western blot and immunocytochemistry showed that ryanodine receptor (RyRs) expression increases with culture. The Ca2+ release from caffeine-insensitive cells induced by Ca(2+)-free high-[K+]O solution could be blocked by 100-200 microM ryanodine, which suggests the involvement of RyRs. Evidence is presented to show that a low resting [Ca2+]i may be one factor responsible for the caffeine insensitivity of RyRs in cells of short-term culture.     

Influences of pressure-injected cyclic AMP on the membrane current and characteristics of an identified neuron of Aplysia kurodai

The ionic mechanism of the effect of intracellularly injected adenosine 3',5'-cyclic monophosphate (cAMP) on the membrane of identified neuron L5 of Aplysia kurodai was investigated with conventional voltage-clamp and ion-substitution techniques. The intracellular elevation of cAMP caused an inward current (IcAMP), which was not accompanied by a significant change in membrane conductance at potentials more hyperpolarized than -60 mV. The current increased over the voltage range (-50 to -30 mV) associated with a conductance decrease and decreased at potentials more hyperpolarized than -60 mV. Elevated intracellular cAMP was found to enhance a region of negative slope resistance in steady-state I-V relations. Duration of the IcAMP was greatly prolonged by bath-applied isobutylmethylxanthine (50 microM), but imidazole (10 mM) had an opposite effect on the IcAMP. Tolbutamide (5 mM), a protein kinase inhibitor, reduced the IcAMP. The current was not affected by the presence of bath-applied TTX (50 microM), ouabain (50 microM), or triaminopyrimidine (5 mM). Reduction of [Na+]0 reversibly decreased the IcAMP. Li+ could largely substitute for Na+. Alterations of [K+]0, and bath application of 4-AP (5 mM) and TEA (30 mM) did not affect the IcAMP. In the presence of Na+, Cl-, and divalent cations such as Ca2+ and Ba2+ inhibited the IcAMP. These results suggest that fast elevation of intracellular cAMP induces a TTX-resistant slow Na+ inward current, and the current might be due to activation of cAMP-dependent protein kinase.     

Ionic currents in the uterine smooth muscle.

1. Short segments of isolated longitudinal myometrium from the pregnant rate uterus have been studied in a double sucrose-gap voltage-clamp arrangement. The clamped segment averaged 65 mum times 240 mum times 100 mum, has an average total capacitance of 0-14 muF, and may contain 50-200 individual myometrial cells. 2. A significant resistance exists in series with the membrane, and limits theprecision of the quantitative information. However, it is argued that some qualitative and some comparative information is useful. 3. In Krebs-bicarbonate solution, depolarizing steps produced initial transient inward currents followed by delayed outward currents. 4. When [Na+]o was reduced by 50%, the equilibrium potential Ea shifted by an average of -17-6 mV, the maximum inward current was reduced to 0-5, the time to peak of the early current was delayed by 1-1 msec, and the maximum chord conductances for the early(Ga) and late (GK) currents remained unchanged as compared with those in normal [Na+]o. 5. When [Ca2+] was reduced to 25% of normal, Ea shifted by an average of -20-3 mV, the maximum inward current was reduced to 0-5, the time to peak was delayed 3-1 msec, and Ga was significantly reduced, while GK was unaffected. 6. The early current, and its tail when repolarization was imposed, reversed direction from inward to outward when [Na+]o was reduced from 143 mM to zero, with [Ca2+]o remaining constant at 1-9 mM. 7. From the observations in 4, 5 and 6, it was concluded that Na+ is the main charge carrier for the early current, and that Ca2+ is important in regulating Ga. 8. The late current is outwards when [K+]o equals 5-9 mM, but inwards in some voltage range when [K+]o was elevated to 120 or 148 mM. K+ is the main charge carrier for the late current. 9. The equilibrium potential for the late current, EK, is about 15 mV more negative than the natural resting potential. 10. Prolonged holding of the preparations at voltages that differ significantly from the natural resting potential tends to shift EK in a way consistent with passive changes in [K+]i by the holding current. 11. The steady-state inactivation of the early current, h, is unusual. Inward current is macimum around the resting potential, and declines with both hyperpolarizing and depolarizing changes. Half-inactivation occurred with about 9 mV depolarization and 15 mV hyperpolarization. 12. The instantaneous current-voltage relations of both early and late currents are linear. The chord conductances Ga and GKare similar in form to those in other tissues.