Free Ringer Research Articles

Intracellular mediators of Na+-K+ pump activity in guinea pig pancreatic acinar cells

The involvement of Ca2+ and cyclic nucleotides in neurohormonal regulation of Na+-K+-ATPase (Na+-K+ pump) activity in guinea pig pancreatic acinar cells was investigated. Changes in Na+-K+ pump activity elicited by secretagogues were assessed by [3H]ouabain binding and by ouabain-sensitive 86Rb+ uptake. Carbachol (CCh) and cholecystokinin octapeptide (CCK-8) each stimulated both ouabain-sensitive 86Rb+ uptake and equilibrium binding of [3H]ouabain by approximately 60%. Secretin increased both indicators of Na+-K+ pump activity by approximately 40% as did forskolin, 8-bromo- and dibutyryl cAMP, theophylline, and isobutylmethylxanthine. Incubation of acinar cells in Ca2+-free HEPES-buffered Ringer (HR) with 0.5 mM EGTA reduced the stimulatory effects of CCh and CCK-8 by up to 90% but caused only a small reduction in the effects of secretin, forskolin, and cAMP analogues. In addition, CCh, CCK-8, secretin, and forskolin each stimulated ouabain-insensitive 86Rb+ uptake by acinar cells. The increase elicited by CCh and CCK-8 was greatly reduced in the absence of extracellular Ca2+, while that caused by the latter two agents was not substantially altered. The effects of secretagogues on free Ca2+ levels in pancreatic acinar cells also were investigated with quin-2, a fluorescent Ca2+ chelator. Basal intracellular Ca2+ concentration ([Ca2+]i) was 161 nM in resting cells and increased to 713 and 803 nM within 15 s after addition of 100 microM CCh or 10 nM CCK-8, respectively. Forskolin, secretin, and cAMP analogues had no effect on [Ca2+]i, nor did they either reduce or potentiate the rise in [Ca2+]i evoked by CCh.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+-sensitive, spontaneously fluctuating, cation channels in the apical membrane of the adult frog skin epithelium

The fluctuations in transepithelial current through the abdominal skin of bullfrogs (Rana catesbeiana) were analysed while the transepithelial voltage was clamped to zero. A Lorentzian component in the power spectrum was recorded when the skin was bathed with Ca2+ free NaCl Ringer's on both sides. After replacement of all mucosal Na+ by choline the Lorentzian component disappeared. The application of mucosa positive potentials enhanced the plateau of the relaxation noise component while it was depressed by mucosa negative potentials. These observations showed that the current associated with the relaxation noise, was carried by Na+ moving in the inward direction. Divalent cations added to the mucosal solution in micromolar concentrations depressed the relaxation noise immediately, which is indicative for an apical localization of the fluctuating channels. The relaxation noise depended strongly on the pH of the mucosal medium: alkalinization enhanced the relaxation noise while acidification depressed the fluctuations. Micromolar concentrations of the diuretic amiloride, which is known to block the Na+ entry into the cellular compartment, enhanced the Na+-dependent relaxation noise while at higher concentrations an inhibitory effect was observed. From these observations it was concluded that the relaxation noise is caused by inward Na+ movement through fluctuating channels which are localized in the apical membrane. These channels seem to constitute a pathway in parallel with the amiloride-blockable channels. Ionic substitution of Na+ by other monovalent cations showed that these channels are also permeable for K+, Rb+, NH4+, Cs+ and Tl+, but not for Li+. Divalent cations in micromolar concentrations completely occlude these fluctuating channels. Therefore, this pathway will be blocked for monovalent cations when normal Ca2+ containing Ringer's are used as mucosal bathing medium.

Effects of mammalian brain extracts and chlormadinone acetate on neuronal Na +, K +-ATPase and electrogenic Na +, K +-pump activity in vitro

Acid-acetone extracts of brain (from beef and guinea pig) and chlormadinone acetate (CMA) were compared with ouabain for their ability to inhibit the electrogenic Na +,K +-pump and the Na +,K +-ATPase of neuronal tissues. The membrane potential of neurones in the paravertebral sympathetic ganglion of the bullfrog was recorded in K +-free Ringer's solution by means of the sucrose gap technique. The potassium activated hyperpolarization (K H +), induced by the re-introduction of potassium, was used as an index of electrogenic Na +,K +-pumping. The K H + was blocked by 1 μM ouabain. Na +,K +-ATPase activity was measured in microsomal membrane preparations of frog and beef brain using a continuous spectrophotometric assay. Although ouabain consistently inhibited beef brain Na +,K +-ATPase (IC 50 = 2.2 μM), acid-acetone extracts prepared from guinea pig and beef brain produced only partial inhibition. Neither of the extracts significantly reduced the K H + of the frog ganglion. CMA inhibited Na +,K +-ATPase prepared from bullfrog brain and spinal cord with slightly greater potency (IC 50 = 4.5 μM) than did ouabain (IC 50 = 10 μM). In contrast, electrogenic Na +,K +-pumping (i.e. the K H +) in the frog ganglion was not affected by this steroid. It is concluded that although both the extracts and CMA inhibited Na +,K +-ATPase, neither can be considered ouabain-like due to their failure to affect the electrogenic Na +,K +-pump in situ.

Three components of active membrane current in the C-neurons of rabbit cervical nodose ganglion under voltage clamp

In the C-neurons of rabbit nodose ganglion there is a persistent slow outward current at Vm levels positive to −80 mV. This current was detectable in Na+-free Ringer and disappeared in Ca2+-free medium. Therefore it may be the Ca2+-activated K+ current. This K+ current shows a unique time and voltage dependency, suggesting that it may have a regulatory role on the excitability of C-neurons. Two other types of current also observed in C-neurons were IQ- and IA-like currents. In A-neurons, however, a Ca2+-activated K+ current was not observed at all.

Forskolin mimics the hydrosmotic action of vasopressin in the urinary bladder of toads Bufo marinus.

Net water flow JW was measured across the urinary bladder of toads Bufo marinus and averaged over periods of 1 min by means of a volumetric, automatic technique. The diterpene forskolin, an activator of adenylate cyclase bypassing the hormonal receptor subunit, induced a rapid, reversible, dose-dependent increase in osmotic water permeability, Pf, very similar to that induced by vasopressin. At 1.1 microM, forskolin induced a half-maximal response. At 5 microM forskolin caused a near maximal response and Pf increased from 1.66 +/- 0.15 to 66.6 +/- 2.99 microns s-1. In bladders pre-exposed to 5 microM-forskolin, further significant increases in Pf were obtained by their subsequent exposure to vasopressin, cyclic AMP, theophylline or serosal hypertonicity. The similarity of the forskolin and vasopressin actions was further demonstrated by the finding that substances causing enhancement (quercetin) or inhibition (trifluoperazine, vanadate, silver, cobalt, manganese and Ca2+-free Ringer solution) of the vasopressin response, induced parallel changes in the forskolin response. Three agents, however, induced dissimilar effects on vasopressin and forskolin: high K+ potentiated vasopressin but inhibited forskolin; methohexital and diamide inhibited vasopressin but had no effect on forskolin. The forskolin-induced hydrosmotic response can be viewed as a new criterion for ascertaining the messenger role of cycle AMP in the the hydrosmotic effect of vasopressin.

Effect of aminooxyacetic acid on γ-aminobutyric acid release from frog retina

The Ca 2+-dependence of the release of [ 3H]γ-aminobutyric acid (GABA) from the pre-loaded, superfused frog retina was investigated in the presence and absence of the GABA-transaminase inhibitor, aminooxyacetic acid (AOAA). In the latter case, an ion-exchange column Chromatographic technique was used to separate [ 3H]GABA from tritiated metabolites released with it into the superfusate. In the presence of aminooxyacetic acid, a 2 min 30 mM K + pulse released similar amounts of [ 3H]GABA in normal and Ca 2+-free Ringer solutions. However, when aminooxyacetic acid was absent, the K +-evoked release of [ 3h]gaba was substantially reduced under Ca 2+-free conditions. It is concluded that the presence of aminooxyacetic acid can reduce the Ca 2+-dependence of K +-evoked release of GABA from the frog retina. This is in accordance with its effect on cerebral cortex of the rat, reported previously.

Reversal of the static component of spindle potential by imposed depolarizing current in the frog muscle spindle

The static component of the spindle potential provoked during stretch of isolated muscle spindles of the frog was reversed during the application of depolarizing currents ranging from 0.2 to 5 nA in normal Ringer solution and also in Na +-free Ringer solution. In the same range of current intensities, spontaneous rhythmic hyperpolarizations due to [Ca 2+] i-activated G K, an attenuation of membrane impedance, and an anomalous decrease in amplitude of the afferent spikes were observed. All 4 phenomena were abolished by K + channel blockers (10 mM CsCl, 1–2 mM 4-aminopyridine (4-AP), or 20 mM tetraethylammonium chloride (TEA)), Ca 2+ channel blockers (5–10 mM CoCl 2, MnCl 2, 1–2mM CdCl 2 or 0.5 mM verapamil) or 0.1 mM quinine. The amplitude of the static component of the spindle potential was markedly increased at threshold concentration of the K + channel blockers (5 mM CsCl, 0.1–0.5 mM 4-AP or 5–10 mM TEA), but the component disappeared at that of the Ca 2+ channel blockers. The rhythmic hyperpolarizations are associated with the spindle potential, except for its dynamic component, which often triggers a hyperpolarizing deflection. We suggest that both the static component of the spindle potential and rhythmic hyperpolarizations are due to G K(Ca) in the intracapsular axon, either along the terminal or at the branching nodes, or both; and that the receptor potential contributes to, but is not the same as, the spindle potential.

Relationship between coupled Na+?K+?Cl? transport and water absorption across the seawater eel intestine

Simultaneous measurements of net ion and water fluxes were made in the stripped intestine of the seawater eel, and the relationship between Na+, K+, Cl− and water transport were examined in the presence of mucosal KCl and serosal NaCl Ringer (standard condition). When Cl− was removed from both sides of the intestine, net K+ flux from mucosa to serosa was reduced, accompanied by complete blockage of water absorption. Since it has been shown that net Cl− and water fluxes depend on K+ transport under the standard condition (Ando 1983), the interdependence of K+ and Cl− transport suggests the existence of a coupled KCl transport system, while the parallelism between the net Cl− and water fluxes suggests that water absorption is linked to the coupled KCl transport. The coupled KCl and water transport were inhibited by treatment with ouabain or with Na+-free Ringer solutions, suggesting the existence of a Na+-dependent KCl transport system and linkage of water absorption to the coupled Na+−K+−Cl− transport. Since ouabain blocked the active Na+−K+−Cl− transport almost completely, the permeability coefficients for K+ and Na+ through the paracellular shunt pathway were estimated as PK=0.076 and PNa=0.058 cm/h, and PCl was calculated as 0.005 cm/h. Although Na+-independent K+ and Cltt- fluxes were observed again in the present study, these fluxes were not inhibited by CN−, ouabain or diuretics, and evoked even after blocking the Na+−K+−Cl− transport completely with ouabain. These results indicate that the Na+-independent K+ and Cl− fluxes are distinct from the active Na+−K+−Cl− transport and are not themselves active.

Na+-H+ exchange in gastric glands as measured with a cytoplasmic-trapped, fluorescent pH indicator.

We have used the pH-sensitive, fluorescent, cytoplasmic-trapped dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to identify Na+-H+ exchange in gastric glands isolated from rabbit stomachs by high-pressure perfusion and collagenase digestion. The fluorescence of BCECF-loaded glands was calibrated in terms of cytosolic pH (pHc) by permeabilizing the cell membranes and titrating the extracellular solution to different pH values. In one set of experiments in Cl--free solutions, glands were treated with 0.1 mM ouabain for 45 min to increase cellular cytosolic molar sodium ion concentration [( Na+]c) to high levels. Subsequent suspension of these cells in a Na+-free Ringer's solution (to generate [Na+]c greater than [Na+]o) caused cells to acidify rapidly (t1/2 approximately equal to 60 sec) from pHc approximately equal to 7.15 to pHc approximately equal to 6.55. Subsequent addition of 100 mM Na+ or Li+, but not K+, caused cells rapidly to increase pHc (t1/2 approximately equal to 30 sec) toward the control value. These changes of pHc were blocked when ouabain-treated glands had been preequilibrated for 10 min with 1 mM amiloride, and this block was overcome by adding 10 microM monensin (an ionophore that artificially exchanges Na+ for H+). In another set of experiments in Cl--containing Ringer's solution, glands were acid-loaded by treatment with 30 mM NH4Cl for 4 min, followed by washing the NH4Cl from the solutions. Under these conditions, pHc decreased from 7.02 to approximately equal to 6.5; subsequent alkalinization of cells back to control pHc was stimulated by Na+ (t1/2 approximately equal to 60 sec), but not K+, and was inhibited by 1 mM amiloride. This amiloride block also was overcome by further addition of 10 microM monensin. We conclude that gastric glands contain a Na+-H+ exchanger that appears independent of Cl-, not activated by K+, and blocked by 1 mM amiloride. This exchanger is likely localized to the serosal membrane of gland cells. Na+-H+ exchange may play an important role in regulation of pHc in oxyntic and chief cells exposed to high luminal acidity, where back diffusion of H+ into cells may occur at rapid rates.

Influence of calcium and cyclic nucleotides on beta-adrenergic sweat secretion in equine sweat glands

The effects of Ca2+, the cyclic nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP), and other parameters of sweat secretion from single equine sweat glands were examined in vitro. Extracellular Ca2+, the Ca2+ ionophore A23187, and the Ca2+ channel antagonist verapamil were all without effect on sweat secretion. Prolonged rinsing of the glands in Ca2+-free Ringer solution with 5 mM ethylenediaminetetraacetic acid decreased the secretion to 30% of the control sweat rate in response to the beta-adrenergic agonist isoproterenol; the sweat response was restored upon adding Ca2+ to the Ringer. cAMP but not cGMP analogues were as effective in stimulating sweat rates as isoproterenol, which elicited maximal secretory rates in vitro. cAMP stimulation was not inhibited by the beta-adrenergic antagonist propranolol. Because the equine sweat gland is predominantly stimulated via the beta-adrenergic receptor, we conclude that cAMP is a principal intracellular messenger in coupling this type of stimulus to the fluid secretion response in this tissue.

Effect of external sodium on intracellular chloride activity in the surface cells of frog gastric mucosa. Microelectrode studies.

The intracellular chloride activity and its dependence on ionic substitutions in the bathing media was studied in individual surface cells of resting gastric mucosa using conventional and Cl- selective microelectrodes. When the tissue was perfused with control NaCl-Ringer the cell membrane p.d.'s, cell-lumen (psi cm) and cell-serosa (psi cs) were -40.9 +/- 0.6 mV and -66.8 +/- 0.5 mV (n = 175) respectively and the p.d. measured by the Cl- selective microelectrodes across the serosal membrane (psi csCl-) averaged -32.4 +/- 0.7 mV (n = 138). From these values an intracellular Cl- activity (acCl-) of 15.3 mmol/l can be estimated. The data indicate that chloride ion is distributed close to equilibrium at the luminal membrane while it is accumulated by an energy requiring step at the serosal membrane. Reduction (2 mmol/l) or absence of chloride from the luminal bath did not result in any detectable change of acCl-; on the other hand, after removal of Cl- from the serosal bath the intracellular Cl- activity fell to 7.1 mmol/l. When the tissue was exposed to serosal Na+-free Ringer (Na+ replaced by choline or TMA), although the acCl- remained unaffected, a marked reduction of the electrochemical gradient for Cl- at the serosal membrane was observed. These data indicate that: chloride is accumulated in the surface cells against its electrochemical potential difference at the serosal membrane; the luminal membrane has a negligible conductance to Cl-, while the serosal membrane represents a conductive pathway to chloride; the uphill entry of chloride at the serosal membrane seems to be, at least partially, Na+-dependent.

The time course of GABA action on the crayfish stretch receptor: Evidence for a saturable GABA uptake

The conductance increase induced by bath application of GABA has been measured in voltage-clamped stretch-receptor neurones of crayfish. A rapid conductance increase was obtained only at GABA concentrations above 3.3 × 10 −4 M . The response to lower GABA concentrations (between 10 −4 and 10 −6 M) developed slowly over 30–60 min. Repetitive application of intermediate GABA concentrations induced postsynaptic conductance changes which were progressively enhanced in their onset and magnitude. In the presence of nipecotic acid or in Na +-free Ringer solutions, the response to all GABA concentrations was rapid and constant for each concentration. The time course of inhibitory postsynaptic currents was unaffected by nipecotic acid. These results suggest the presence of a saturable GABA uptake system which limits the access of bath-applied GABA to postsynaptic receptors. This system has little if any effect on the termination of response to synaptically released GABA.

H+ Disposal by Rabbit Gastric Mucosal Surface Cells

Exposure of isolated gastric mucosal surface cells to NH4+ results in acidification of cells as determined by a fluorescent dye technique using acridine orange. The resulting intracellular pH gradient is maintained when cells are suspended in either buffered HCO3- -free Ringer's or choline chloride solution. Cells suspended in a Na+-containing but K+-free solution exhibit dissipation of the proton gradient. When Na+ is added to cells suspended in Na+, K+-free solution, the gradient rapidly dissipates with a half-maximal response occurring at 56 mM Na+. The effect of Na+ is amiloride sensitive with half-maximal inhibition occurring at 38 microM at a Na+ concentration of 50 mM. The K+ does not cause dissipation of the gradient and neither ouabain nor valinomycin have an effect. Yet, K+ has a modulating influence on Na+/H+ exchange by the isolated surface cells. The addition of K+ to acid-loaded cells resuspended in Na+-free solution decreases the ability of subsequent Na+ addition to evoke gradient dissipation. The data suggest that Na+/H+ exchange appears to be at least one mechanism whereby gastric mucosal surface cells could protect themselves against diffusing acid. This ion exchange mechanism is amiloride sensitive and appears to be unrelated to Na+, K+ adenosine triphosphatase activity, but is affected by the external K+ concentration.

Mode of inhibition of active chloride transport in the frog cornea by furosemide.

The mechanism of inhibition of active Cl- secretion by 1 mM furosemide and 0.1 mM bumetanide was characterized in the isolated frog corneal epithelium. Transepithelial and transmembrane cell electrical parameters as well as transmembrane Cl- electrochemical potential difference were measured with conventional glass microelectrodes and Cl- selective microelectrodes. Furosemide caused the potential difference across the apical membrane to hyperpolarize by 20 mV while the transepithelial potential difference declined by 13 mV. The apical-to-basolateral membrane resistance ratio increased 3-4 times after furosemide or bumetanide addition. Preincubation with furosemide prevented a 30-mV depolarization of the apical membrane potential difference normally observed when Cl- was removed from the tear side bathing solution. In control conditions, intracellular Cl- activity was above equilibrium. Bumetanide further increased the Cl- electrochemical gradient between the cell compartment and the bathing solutions even though intracellular Cl- activity fell from 18 to 12 mM. In contrast, perfusion with Cl- -free Ringer in the stromal side bathing solution decreased the Cl- electrochemical gradient across the apical membrane to zero, indicating an equilibrium distribution. Adenosine, which selectively increases Cl- permeability of the apical membrane, also decreased the Cl- electrochemical gradient across the apical membrane. These results suggest that the diuretics inhibit active Cl- transport primarily by decreasing the Cl- permeability of the apical membrane.

Effects of Ruthenium ions on the sensory terminal discharges of the frog muscle spindle

The presence of a mixed Na + Ca 2+ spike along the sensory terminal of the frog muscle spindle was verified. When the terminal was perfused with Ringer's solution containing 0.1–0.5 mM ruthenium red (RuR), the amplitude and duration of the spike were increased, occurring as a prolonged or a long-lasting depolarization of up to 20–30 s duration following individual afferent spikes evoked spontaneously or antidromically by electrical stimulation. In an isotonic TEA solution, the amplitude and duration of the afferent spikes were increased; however, no prolonged depolarization occurred. Adding 0.2 mM RuR to the TEA solution produced the prolonged and long-lasting depolarization. All responses disappeared in the presence of 3 μM TTX or Na +-free Ringer's solution. An impedance decrease along the terminal was observed during the prolonged or long-lasting depolarization. The prolonged depolarization was blocked by the addition of Ca 2+-blockers; the afferent spikes remained. In preparations preincubated with 0.1 mM RuR, increasing CaCl 2 in Ringer's solution from 0.2 mM, resulted in shortening of the duration of individual spikes with prolonged depolarization and in increase in the maximum rate of rise (MMR) of the spikes. Preincubation with higher concentrations of RuR produced higher sensitivities in the modifications of the duration and MRR to the change in [Ca 2+] o. The responses were retained by adding RuR or RuCl 3 to Ca 2+-free Ringer's solution containing 0.1–5 mM EGTA, although all responses disappeared in Ca 2+-free EGTA Ringer's solution. It is concluded that the RuR-induced prolonged response is produced by an influx of Na +.

The spindle potential in the frog muscle spindle does not require external Na +

Spindle potential recorded from the sensory nerve terminal of isolated frog muscle spindles disappeared within 20–30 min after the spindle receptor was perfused with Na +-free (Li, Tris or choline) Ringer's solution, whereas the amplitude of spindle potential was not attenuated for periods up to 60 min when the spindles were perfused in a Na +-free Ringer's solution containing both 10 mM TEA and 0.1 mM 4-aminopyridine after being washed with a normal Ringer's solution containing both the K +-channel blockers. It is concluded that the time-dependent decrease in the amplitude of spindle potential during the application of Na +-free solution is not ascribable to a decrease in the inward current carried by Na +, but is due to an increase in an outward current carried by K +.

Comparison of GABA analogues at the crayfish stretch receptor neurone

The conductance increase induced by GABA and structurally related compounds has been measured in voltage clamped stretch receptor neurones of crayfish. GABA induced only at 10 −3 M a rapid conductance increase. The response to lower concentrations between 10 −6 and 10 −4 M developed slowly (20–60 min). The postsynaptic conductance increase induced by repetitive application of the same GABA concentration was progressively enhanced in the speed and magnitude. In the presence of nipecotic acid or in Na +-free Ringer solutions, the response to all GABA concentrations was instantaneous and constant for each concentration. Muscimol between 10 −6 and 10 −3 M caused instantaneous dose-dependent conductance increases. These results suggest the presence of a saturable GABA uptake system limiting the access of bath applied GABA, but not of muscimol, to postsynaptic receptor sites.

Raised intracellular free calcium within the lens causes opacification and cellular uncoupling in the frog.

Ion-sensitive micro-electrodes were used to measure the levels of intracellular free Ca2+ within the intact amphibian lens. The free [Ca2+] was found to constitute 0.4% of the total lens calcium. The pCa measured at the anterior lens surface was found to 6.59, while that at the posterior was 5.70. An 8-fold anterior/posterior Ca2+ gradient thus exists along the optical axis. The intracellular free Ca2+ could be manipulated by incubating the lens in high-Ca2+ or cA2+-free EGTA Ringer solutions. Raising the intracellular free Ca2+ to 0.22 mM caused lens opacification and cellular uncoupling; the coupling ratio was reduced from 1 in control to 0.41 in high Ca2+.

Pardaxin Produces Sodium Influx in Theteleost Gill-Like Opercular Epithelia

ABSTRACT Transport by the gill-like opercular epithelium of the teleost, Fundulus heteroclitus, was affected by pardaxin, a protein that is toxic to fish, isolated from the Red Sea flatfish Pardachirus marmoratus. Administration of pardaxin to the mucosal (seawater) side of the isolated short-circuited opercular epithelium, caused a transient stimulation of the active transport of ions (Isc), followed by an inhibition. The Isc stimulation was abolished by ouabain or/and in Na+-free Ringer but not in Cl−-or HCO3−-Ringer. When applied to the serosal (blood) side, pardaxin did not affect the Isc. Pardaxin produced a net transient Na+ current from the mucosal side to the serosal side of 2·2μequivcm−2h−1. It is concluded that this Na+ influx caused the Isc stimulation. The influx is suggested to be the mechanism of pardaxin’s toxicity in fish.

Aequorin-calcium transients in frog twitch muscle fibres.

Intracellular Ca2+ transients, evoked either by action potentials or depolarizing clamp pulses, were studied in frog sartorius muscle fibres injected with aequorin. The time course of the Ca2+ transients became shorter as the temperature was increased. The half rise time and decay time constants showed straight lines between 3 and 30 degrees C in Arrhenius plots, with a Q10 of 2.5 and 2.3 respectively. The potential dependence of the Ca2+ transient was examined under voltage clamp. The peak light amplitude reached a plateau at around +50 mV, suggesting that Ca2+ release continues beyond the potential level at which contraction was saturated. During a prolonged depolarization, the Ca2+ transient gradually declined. The time course of decline became faster when long depolarizing pulses were repeated, or when the temperature was increased. The Q10 for half duration of the Ca2+ transient evoked by prolonged depolarization was 2.2. A Ca2+ transient could be evoked in Ca2+-free Ringer solution containing EGTA. Formamide, which is known to abolish excitation-contraction coupling, also abolished the Ca2+ transient. During maintained depolarization, the time integral of the Ca2+ transient was larger for larger depolarizations, suggesting that the total amount of Ca2+ released was greater for the more intense depolarization. The decline of the Ca2+ transient during maintained depolarization is probably due to inactivation of excitation-contraction coupling rather than the depletion of intracellular Ca2+ stores. These findings support the view that in frog skeletal muscle fibres the increase in intracellular Ca2+, caused by membrane depolarization, is produced by the release of Ca2+ from intracellular stores and that any influx of Ca2+ from the external medium does not contribute appreciably to the aequorin-Ca2+ transient.