Increased chloride conductance as the proximate cause of hydrogen ion concentration effects in Aplysia neurons

A fall in extracellular pH increased membrane conductance of the giant cell in the abdominal ganglion of Aplysia californica. Chloride conductance was trebled whereas potassium conductance was increased by 50%. Half the giant cells were hyperpolarized (2–8 mv) and half were depolarized (3–10 mv) by lowering the pH. The hyperpolarizing response always became a depolarizing response in half-chloride solutions. When internal chloride was increased electrophoretically, the hyperpolarization was either decreased or changed to depolarization. The depolarizing response was reduced or became a hyperpolarizing response after soaking the cell in 10.0 mM chloride, artificial seawater solution for 1 hr. Depolarization was unaffected when either external sodium, calcium, or magnesium was omitted. A glass micropipette having an organic liquid chloride ion exchanger in its tip was used to measure intracellular chloride activity in 14 giant cells; 7 had values of 27.7 ± 1.8 mM (SEM) and 7 others 40.7 ± 1.5 mM. Three of the first group were hyperpolarized when pH was lowered and three of the second group were depolarized. In all six cells, these changes of membrane potential were in the direction of the chloride equilibrium potential. Intracellular potassium activity was measured by means of a potassium ion exchanger microelectrode.     

The Dual Effect of Lithium Ions on Sodium Efflux in Skeletal Muscle

Sartorius muscle cells from the frog were stored in a K-free Ringer solution at 3°C until their average sodium contents rose to around 23 mM/kg fiber (about 40 mM/liter fiber water). Such muscles, when placed in Ringer''s solution containing 60 mM LiCl and 50 mM NaCl at 20°C, extruded 9.8 mM/kg of sodium and gained an equivalent quantity of lithium in a 2 hr period. The presence of 10^-5 M strophanthidin in the 60 mM LiCl/50 mM NaCl Ringer solution prevented the net extrusion of sodium from the muscles. Lithium ions were found to enter muscles with a lowered internal sodium concentration at a rate about half that for entry into sodium-enriched muscles. When sodium-enriched muscles labeled with radioactive sodium ions were transferred from Ringer''s solution to a sodium-free lithium-substituted Ringer solution, an increase in the rate of tracer sodium output was observed. When the lithium-substituted Ringer solution contained 10^-5 M strophanthidin, a large decrease in the rate of tracer sodium output was observed upon transferring labeled sodium-enriched muscles from Ringer''s solution to the sodium-free medium. It is concluded that lithium ions have a direct stimulating action on the sodium pump in skeletal muscle cells and that a significantly large external sodium-dependent component of sodium efflux is present in muscles with an elevated sodium content. In the sodium-rich muscles, about 23% of the total sodium efflux was due to strophanthidin-insensitive Na-for-Na interchange, about 67% being due to strophanthidin-sensitive sodium pumping.     

An active electrical response in fibroblasts

L cells have a resting potential of about -16 mv (internal negative) at 37°C in Dulbecco''s modified Eagle''s medium containing 10% fetal calf serum and a potassium concentration of 5.4 mM. Membrane resistivity is about 20,000 Ωcm² when the surface filopodia described by others are taken into account. Mechanical and electrical stimuli can evoke an active response from mouse L cells, cells of the 3T3 line, and normal fibroblasts which we have termed hyperpolarizing activation or the H.A. response. This consists of a prolonged (3–5 sec) increase in the membrane permeability by a factor of 2–10 with a parallel increase in membrane potential to about -50 mv. The reversal potential for the H.A. response is -80 mv. The resting cells are depolarized to about -12 mv when the external medium contains 27 mM potassium, and the potential reached at the peak of the H.A. response is about -30 mv. The reversal potential for the H.A. response is about -40 mv in 27 mM external potassium. This effect of potassium ions on the reversal potential of the H.A. response leads us to conclude that the response represents an increase in membrane permeability, predominantly to potassium, by at least a factor of five. This increase must be greater than 20-fold if previous measurements of the ratio of potassium permeability to chloride permeability in L cells are valid for the preparation used in the present study.     

Choline permeability in cardiac muscle cells of the cat

Permeability of the cardiac cell membrane to choline ions was estimated by measuring radioactive choline influx and efflux in cat ventricular muscle. Maximum values for choline influx in 3.5 and 137 mM choline were respectively 0.56 and 9 pmoles/cm²·sec. In 3.5 mM choline the intracellular choline concentration was raised more than five times above the extracellular concentration after 2 hr of incubation. In 137 mM choline, choline influx corresponded to the combined loss of intracellular Na and K ions. Paper chromatography of muscle extracts indicated that choline was not metabolized to any important degree. The accumulation of intracellular choline rules out the existence of an efficient active pumping mechanism. By measuring simultaneously choline and sucrose exchange, choline efflux was analyzed in an extracellular phase, followed by two intracellular phases: a rapid and a slow one. Efflux corresponding to the rapid phase was estimated at 16–45 pmoles/cm²·sec in 137 mM choline and at 1.3–3.5 pmoles/cm²·sec in 3.5 mM choline; efflux in 3.5 mM choline was proportional to the intracellular choline concentration. The absolute figures for unidirectional efflux were much larger than the net influx values. The data are compared to Na and Li exchange in heart cells. Possible mechanisms for explaining the choline behavior in heart muscle are discussed.     

The interaction between caffeine and calcium in the desensitization of muscle postjunctional membrane receptors

The interaction between caffeine and calcium on the rate of desensitization of muscle postjunctional membrane (PJM) receptors during the sustained application of 0.27 mM carbamylcholine (CARB) has been studied in vitro on the sartorius muscle of the frog. The rate of PJM repolarization with CARB added to the solution bathing the muscle or the recovery of the effective transmembrane resistance (EMR) during the microperfusion of CARB directly onto the end-plate region of individual fibers was used as an index of the rate of desensitization. Caffeine (1.5 mM) increased the rate of PJM repolarization with bulk application of CARB in a 1.8 or 10 mM calcium Ringer solution but had no effect on PJM repolarization in a calcium-deficient, 4 mM magnesium Ringer solution. For EMR measurements the preparation was rendered mechanically quiescent by repeated challenges with isotonic KCl during an exposure of several hours to a calcium-free, 4 mM magnesium-1 mM EGTA Ringer solution. In these fibers, the microperfusion of 0.27 mM CARB together with 1.8 mM calcium plus 1.5 mM caffeine significantly increased the rate of EMR recovery above that observed in the absence of caffeine. Raising the calcium concentration to 10 mM had a similar effect; however, no additional increase was noted by the inclusion of 1.5 mM caffeine. It is suggested that the major role of caffeine in PJM desensitization is to increase the calcium permeability of the surface membrane. The transmembrane movement of calcium and the consequent intracellular accumulation of calcium is seen as a critical factor in controlling the rate of PJM desensitization.     

Studies of cardiac muscle with a high permeability to calcium produced by treatment with ethylenediaminetetraacetic acid

Thin strips of frog ventricle were isolated and bathed for 15 min in a solution containing 140 mM KCl, 5 mM Na₂ATP, 3 mM EDTA, and 10 mM Tris buffer at pH 7.0. The muscle was then exposed to contracture solutions containing 140 mM KCl, 5 mM Na₂ATP, 1 mM MgCl₂, 10 mM Tris, 3 mM EGTA, and CaCl₂ in amounts to produce concentrations of free calcium from 10^-4.8 M to 10^-9 M. The muscles developed some tension at approximately 10^-8 M, and maximum tension was achieved in 10^-5 M Ca⁺⁺. They relaxed in Ca⁺⁺ concentrations less than 10^-8 M. The development of tension by the EDTA-treated muscles was normalized by comparison with twitch tension at a stimulation rate of 9 per min before exposure to EDTA. In 10^-5 M Ca⁺⁺ tension was always several times the twitch tension and was greater than the contracture tension of a frog ventricular strip in KCl low Na-Ringer. Tension equal to half-maximum was produced at approximately 10^-6.2 M Ca⁺⁺. Intracellular recording of membrane potential indicated that after EDTA treatment the resting potential of cells in Ringer solution with 10^-5 M Ca or less was between 5 and 20 mv. Contracture solutions did not produce tension without prior treatment with EDTA. The high permeability of the membrane produced by EDTA was reversed and the normal resting and action potentials restored in 1 mM Ca-Ringer. Similar studies of EDTA-treated rabbit right ventricular papillary muscle produced a similar tension vs. Ca⁺⁺ concentration relation, and the high permeability state reversed with exposure to normal Krebs solution.     

MAINTENANCE OF IMAGINAL DISCS OF DROSOPHILA MELANOGASTER IN CHEMICALLY DEFINED MEDIA

A phosphate-buffered saline and a chemically defined synthetic medium for in vitro maintenance of imaginal discs of Drosophila melanogaster were developed. The composition of the chemically defined medium was varied in order to optimize the incorporation of tritiated uridine into RNA and tritiated amino acids into acid-insoluble protein. The optimal ranges obtained were: pH, 6.75–7.35; osmolarity, 285–345 milliosmoles/liter; sodium concentration, 40–60 mM/liter; potassium concentration, 40–60 mM/liter; magnesium concentration, 0.5–3.5 mM/liter; calcium concentration, 0.3–1.5 mM/liter; and inorganic phosphate concentration, 1.5–4.0 mM/liter. The phosphate-buffered saline is superior to a commonly used insect Ringer solution in maintaining total RNA and acid-insoluble protein synthesis in culture. The chemically defined synthetic medium permits linear total RNA and acid-insoluble protein synthesis for more than 48 hr, DNA synthesis for several hours, normal differentiation to occur after 74 hr in vitro, and trypsinization of imaginal discs into single cell suspensions without developmental damage.     

Ca 2+ -specific removal of Z lines from rabbit skeletal muscle

Removal of rabbit psoas strips immediately after death and incubation in a saline solution containing 1 mM Ca²⁺ and 5 nM Mg²⁺ for 9 hr at 37°C and pH 7.1 causes complete Z-line removal but has no ultrastructurally detectable effect on other parts of the myofibril. Z lines remain ultrastructurally intact if 1 mM 1,2-bis-(2-dicarboxymethylaminoethoxy)-ethane (EGTA) is substituted for 1 mM Ca²⁺ and the other conditions remain unchanged. Z lines are broadened and amorphous but are still present after incubation for 9 hr at 37°C if 1 mM ethylenediaminetetraacetate (EDTA) is substituted for 1 mM Ca²⁺ and 5 mM Mg²⁺ in the saline solution. A protein fraction that causes Z-line removal from myofibrils in the presence of Ca²⁺ at pH 7.0 can be isolated by extraction of ground muscle with 4 mM EDTA at pH 7.0–7.6 followed by isoelectric precipitation and fractionation between 0 and 40% ammonium sulfate saturation. Z-line removal by this protein fraction requires Ca²⁺ levels higher than 0.1 mM, but Z lines are removed without causing any other ultrastructurally detectable degradation of the myofibril. This is the first report of a protein endogenous to muscle that is able to catalyze degradation of the myofibril. The very low level of unbound Ca²⁺ in muscle cells in vivo may regulate activity of this protein fraction, or alternatively, this protein fraction may be localized in lysosomes.     

Comparison of tetrodotoxin and procaine in internally perfused squid giant axons

Squid giant axons were internally perfused with tetrodotoxin and procaine, and excitability and electrical properties were studied by means of current-clamp and sucrose-gap voltage-clamp methods. Internally perfused tetrodotoxin was virtually without effect on the resting potential, the action potential, the early transient membrane ionic current, and the late steady-state membrane ionic current even at very high concentrations (1,000–10,000 nM) for a long period of time (up to 36 min). Externally applied tetrodotoxin at a concentration of 100 nM blocked the action potential and the early transient current in 2–3 min. Internally perfused procaine at concentrations of 1–10 mM reversibly depressed or blocked the action potential with an accompanying hyperpolarization of 2–4 mv, and inhibited both the early transient and late steady-state currents to the same extent. The time to peak early transient current was increased. The present results and the insolubility of tetrodotoxin in lipids have led to the conclusion that the gate controlling the flow of sodium ions through channels is located on the outer surface of the nerve membrane.     

Cation regulation in the smooth muscle of frog stomach

Cation composition of frog smooth muscle cells was investigated. Fresh stomach muscle rings resembled skeletal muscle, but marked Na gain and K loss followed immersion. Mean Na (49.8–79.7 mM/kg tissue) and K (61.8–80.1 mM/kg tissue) varied between batches, but were stable for long periods in vitro. Exchange of 6–30 mM Na/kg tissue with ²²Na was extremely slow and distinct. Extracellular water was estimated from sucrose-¹⁴C uptake. Calculated exchangeable intracellular Na was 9 mM/kg cell water, and varied little. Thus steady-state transmembrane cation gradients appeared to be steep. K-free solution had only slight effects. Ouabain (10^-4 M) caused marked Na gain and reciprocal K loss; at 30°C, Na and K varied linearly with time over a wide range of contents, indicating constant net fluxes. Net fluxes decreased with temperature decrease. ²²Na exchange in ouabain-treated tissue at 20–30°C was rapid and difficult to analyze. The best minimum estimates of unidirectional Na fluxes at 30°C were 10–12 times the constant net flux; constant pump efflux may explain these findings. The rapidity of Na exchange may not reflect very high permeability, but it does require a high rate of transport work.     

NOTES ON ULTRASTRUCTURE AND SOME PROPERTIES OF TRANSPORT WITHIN THE LIVING MITOTIC SPINDLE

The sites of lead phosphate precipitation in mouse bladder smooth muscle incubated with adenosine triphosphate and lead nitrate were studied by electron microscopy. The media constituents and incubating conditions were independently varied so that we could determine optimal conditions for histochemical demonstration of ATPase activity in agranular endoplasmic reticulum. Specimens of glutaraldehyde-fixed bladder muscle, frozen, cut into 10–40-µ sections, and incubated for 1 hr at 25°C in medium containing 0.025 M ATP, 0.0025 M lead nitrate, 0.05 M magnesium chloride, and 0.09 M sodium acetate buffer at pH 6.2, exhibited microcrystalline deposits in agranular endoplasmic reticulum and pinocytotic vesicles. Lead salt deposition was also noted in terminal cisternae of sarcoplasmic reticulum in skeletal muscle similarly treated, suggesting that the organelle systems in the two types of muscle cells subserve a common function.     

Sodium fluxes in internally dialyzed squid axons

The effects which alterations in the concentrations of internal sodium and high energy phosphate compounds had on the sodium influx and efflux of internally dialyzed squid axons were examined. Nine naturally occurring high energy phosphate compounds were ineffective in supporting significant sodium extrusion. These compounds were: AcP, PEP, G-3-P, ADP, AMP, GTP, CTP, PA, and UTP.¹ the compound d-ATP supported 25–50% of the normal sodium extrusion, while ATP supported 80–100%. The relation between internal ATP and sodium efflux was nonlinear, rising most steeply in the range 1 to 10 µM and more gradually in the range 10 to 10,000 µM. There was no evidence of saturation of efflux even at internal ATP concentrations of 10,000 µM. The relation between internal sodium and sodium efflux was linear in the range 2 to 240 mM. The presence of external strophanthidin (10 µM) changed the sodium efflux to about 8–12 pmoles/cm² sec regardless of the initial level of efflux; this changed level was not altered by subsequent dialysis with large concentrations of ATP. Sodium influx was reduced about 50 % by removal of either ATP or Na and about 70 % by removing both ATP and Na from inside the axon.     

Effects of Sodium Azide on Sodium Fluxes in Frog Striated Muscle

Unidirectional Na fluxes from frog''s striated muscle were measured in the presence of 0 to 5 mM sodium azide. With azide concentrations of 2 and 5 mM the Na efflux was markedly stimulated; the Na efflux with 5 mM azide was about 300 per cent greater than normal. A similar increase was present when all but the 5.0 mM sodium added with azide was replaced by choline. 10^-5 M strophanthidin abolished the azide effect on Na²⁴ efflux. Concentrations of azide of 1.0 mM or less had no effect on Na efflux. The Na influx, on the other hand, was only increased by 41 per cent in the presence of 5 mM NaN₃. From these findings it is concluded that the active transport of Na is stimulated by the higher concentrations of azide. The hypothesis is advanced that the active transport of Na is controlled by the transmembrane potential and that the stimulation of Na efflux is produced as a consequence of the membrane depolarization caused by the azide.     

Further studies of the effect of calcium on the time course of action of carbamylcholine at the neuromuscular junction

The rate at which the postjunctional membrane of muscle fibers becomes desensitized to the action of carbamylcholine is increased after the muscle has been soaked in solutions containing increased concentrations of calcium. Some further aspects of this effect of calcium were investigated by measuring changes in the input resistance of single fibers of the frog sartorius during local perfusion of the neuromuscular junction with 2.73 x 10^-3 M carbamylcholine in isolated muscles immersed in 165 mM potassium acetate. It was found that (a) sudden changes in the local concentration of calcium brought about by perfusing fibers with carbamylcholine solutions containing 20 mM calcium, 40 mM oxalate, or 40 mM EDTA were followed within 20 sec by marked changes in the rate of desensitization; (b) prior to 13 sec after the introduction of carbamylcholine, however, no effect on the input resistance could be detected even though the muscle had been presoaked in 10 mM calcium; (c) the ability of high concentrations of calcium to bring about rapid desensitization disappears when a lower concentration of carbamylcholine (0.137 x 10^-3 M) is applied to the muscle fiber. These findings suggest that calcium present in the extracellular fluid can act directly on the postjunctional membrane to promote the desensitization process and that an increased permeability of the membrane to calcium brought about by the presence of carbamylcholine is a factor which contributes to this action.     

Nature of the Schwann cell electrical potential. Effects of the external ionic concentrations and a cardiac glycoside

The effects on the Schwann cell electrical potential of external ionic concentrations and of K-strophanthoside were investigated. Increasing (K)_o depolarized the cell. The potential is related to the logarithm of (K)_o in a quasi-linear fashion. The linear portion of the curve has a slope of 45 mv/ten-fold change in (K)_o. Diminutions of (Na)_o and (Cl)_o produced only small variations in the potential. Calcium and magnesium can be replaced by 44 mM calcium without altering the potential. Increase of (Ca)_o to 88 mM produced about 10 mv hyperpolarization. The cell was hyperpolarized by 11 mv and 4 mv within 1 min after applying K-strophanthoside at concentrations of 10^-3 and 10^-5 M, respectively. No variations of cellular potassium, sodium, or chloride were observed 3 min after applying the glycoside. The hyperpolarization caused by 10^-3 M K-strophanthoside was not observed when (K)_o was diminished to 1 or 0.1 mM or was increased to 30 mM. At a (K)_o of 30 mM, 10^-2 M strophanthoside was required to produce the hyperpolarizing effect. In high calcium, the cell was further hyperpolarized by the glycoside. The initial hyperpolarization caused by the glycoside was followed by a gradual depolarization and a decrease of the cellular potassium concentration. The results indicate that the Schwann cell potential of about -40 mv is due to ionic diffusion, mainly of potassium, and to a cardiac glycoside-sensitive ion transport process.     

Effects of caffeine on crayfish muscle fibers. I. Activation of contraction and induction of Ca spike electrogenesis

Contractions are evoked in single muscle fibers of crayfish by intracellular as well as extracellular applications of caffeine. Responses to external applications in concentrations above 2 mM could be induced indefinitely. With concentrations above 5 mM the caffeine-induced responses were highly repeatable. Tensions were transient even when the caffeine remained in the bath. There was no change in resting potential, but during the contraction the effective resistance decreased about 10%. A number of factors (change in pH, Ca, K, and Cl) modified the responses. The time course of the tension was greatly prolonged when the transverse tubular system (TTS) was s swollen and was again shortened when the TTS was caused to shrink. An increased permeability to Ca induced by caffeine was evidenced by the transformation of the normally graded electrical responses to Ca spikes, which are insensitive to tetrodotoxin. The overshoot is a function of both external Ca and caffeine. A 10-fold change in Ca changed the overshoot by 19 mv in the presence of 10 mM caffeine and by 29 mv in 80 mM caffeine. The role of the increased permeability to Ca for caffeine-induced contractions will be analyzed in the accompanying paper.     

Some factors influencing sodium extrusion by internally dialyzed squid axons

Squid giant axons were internally dialyzed by a technique previously described. In an axon exposed to cyanide seawater for 1 hr and dialyzed with an ATP-free medium, the Na efflux had a mean value of 1.3 pmole/cm²sec when [Na]_i was 88 mM, in quantitative agreement with flux ratio calculations for a purely passive Na movement. When ATP at a concentration of 5–10 mM was supplied to the axoplasm by dialysis, Na efflux rose almost 30-fold, while if phosphoarginine, 10 mM, was supplied instead of ATP, the Na efflux rose only about 15-fold. The substitution of Li for Na in the seawater outside did not affect the Na efflux from an axon supplied with ATP, while a change to K-free Na seawater reduced the Na efflux to about one-half. When special means were used to free an axon of virtually all ADP, the response of the Na efflux to dialysis with phosphoarginine (PA) at 10 mM was very small (an increment of ca. 3 pmole/cm²sec) and it can be concluded that more than 96% of the Na efflux from an axon is fueled by ATP rather than PA. Measurements of [ATP] in the fluid flowing out of the dialysis tube when the [ATP] supplied was 5 mM made it possible to have a continuous measurement of ATP consumption by the axon. This averaged 43 pmole/cm²sec. The ATP content of axons was also measured and averaged 4.4 mM. Estimates were made of the activities of the following enzymes in axoplasm: ATPase, adenylate kinase, and arginine phosphokinase. Values are scaled to 13°C.     

Cytochemistry of phosphatases of the sarcoplasmic reticulum. II. In situ localization of the Mg-dependent enzyme

The distribution of the Mg-dependent ATPase associated with a microsomal fraction of rabbit psoas muscle was studied histochemically and its localization in relation to the vesicles of the fraction and to the structure of intact fixed muscle was determined. Although enzyme activity was retained after fixation in hydroxyadipaldehyde and in glyoxal, it was lost after fixation in glutaraldehyde or after 4 hr fixation in formaldehyde. Activity was optimally demonstrated when incubations were conducted at 17°C, in media containing 125 mM Trismaleate buffer, pH 7.5, 5 mM ATP, 4 mM MgCl₂, and 1 mM Pb(NO₃)₂. After such incubations, activity was present throughout the sarcoplasmic reticulum, but was absent from the T system. Activation by Na or K could not be demonstrated histochemically. However, the other biochemical properties of the enzyme in the isolated vesicles and in intact muscle were similar with respect to Mg dependence, substrate specificity, inhibition by Ca, N-ethyl maleimide, p-hydroxymercuribenzoate, and lack of inhibition by ouabain.     

Effects of External Potassium and Strophanthidin on Sodium Fluxes in Frog Striated Muscle

Unidirectional Na fluxes in isolated fibers from the frog''s semitendinosus muscle were measured in the presence of strophanthidin and increased external potassium ion concentrations. Strophanthidin at a concentration of 10^-5 M inhibited about 80 per cent of the resting Na efflux without having any detectable effect on the resting Na influx. From this it is concluded that the major portion of the resting Na efflux is caused by active transport processes. External potassium concentrations from 2.5 to 7.5 mM had little effect on resting Na efflux. Above 7.5 mM and up to 15 mM external K, the Na efflux was markedly stimulated; with 15 mM K the Na influx was 250 to 300 per cent greater than normal. On the other hand, Na influx was unchanged with 15 mM K. The stimulated Na efflux with the higher concentrations was not appreciably reduced when choline or Li was substituted for external Na, but was completely inhibited by 10^-5 M strophanthidin. From these findings it is concluded that the active transport of Na is stimulated by the higher concentrations of K. It is postulated that this effect on the Na "pump" is produced as a result of the depolarization of the muscle membranes and is related to the increased metabolism and heat production found under conditions of high external K.     

The ionic requirements for the initiation of action potentials in insect muscle fibers

Electrical properties of locust leg muscle fibers were studied by means of intracellular electrodes. In most fibers, a depolarizing current pulse initiated a local response. A delayed decrease in membrane resistance appeared with more than about 10 mv depolarization. In some fibers a regenerative response also was found. Membrane constants were measured, applying the short cable model. The value of the space constant λ was 1.6 mm and the calculated value of R_m was about 1750 ohm cm². Action potentials could be elicited when the bathing fluid contained more than 2–5 mM Ba or Sr. Similar responses were seen with 2 mM Ca in the presence of tetraethylammonium (TEA). The overshoot of these action potentials increased with increasing [Ca⁺⁺]_o, [Sr⁺⁺]_o, or [Ba⁺⁺]_o, the increment for a 10-fold increase being about 29 mv for Ca and Sr and between 40 and 50 mv for Ba. These action potentials were inhibited by Mn ions but were not affected by tetrodotoxin or procaine. In solutions containing Ba or Sr, action potentials generated were suppressed by addition of Ca. The removal of Na ions did not change the configuration of the action potential. The results suggest that an increase in permeability to Ca, Ba, or Sr ions makes a major contribution to the initiation of action potentials in this tissue.