Na-free Saline Research Articles

Lidocaine increases intracellular sodium concentration through voltage-dependent sodium channels in an identified lymnaea neuron.

The local anesthetic lidocaine affects neuronal excitability in the central nervous system; however, the mechanisms of such action remain unclear. The intracellular sodium concentration ([Na]i) and sodium currents (INa) are related to membrane potential and excitability. Using an identifiable respiratory pacemaker neuron from Lymnaea stagnalis, the authors sought to determine whether lidocaine changes [Na]i and membrane potential and whether INa is related to these changes. Intracellular recording and sodium imaging were used simultaneously to measure membrane potentials and [Na]i, respectively. Measurements for [Na]i were made in normal, high-Na, and Na-free salines, with membrane hyperpolarization, and with tetrodotoxin pretreatment trials. Furthermore, changes of INa were measured by whole cell patch clamp configuration. Lidocaine increased [Na]i in a dose-dependent manner concurrent with a depolarization of the membrane potential. In the presence of high-Na saline, [Na]i increased and the membrane potential was depolarized; the addition of lidocaine further increased [Na]i, and the membrane potential was further depolarized. In Na-free saline or in the presence of tetrodotoxin, lidocaine did not change [Na]i. Similarly, hyperpolarization of the membrane by current injections also prevented the lidocaine-induced increase of [Na]i. In the patch clamp configuration, membrane depolarization by lidocaine led to an inward sodium influx. A persistent reduction in membrane potential, resulting from lidocaine, brings the cell within the window current of INa where sodium channel activation occurs. Lidocaine increases intracellular sodium concentration and promotes excitation through voltage-dependent sodium channels by altering membrane potential in the respiratory pacemaker neuron.

Mytilus inhibitory peptide (MIP) induces a Na+-activated K+-current in snail neurons

Two microelectrode voltage-clamp and single-channel recordings were performed on D-cluster neurons of snail right parietal ganglion in order to study the properties of MIP-activated potassium current. It was found that the octapeptide member of the MIP-family, ASHIPRFVa elicits an outward current, which possesses all the properties characteristic for the hexapeptide(s) inward membrane response. The main component of the peptide elicited response is highly [K+]o dependent, however the response was attenuated in Na-free extracellular saline. The peptide elicited response was mimicked by raising the [Na+]i by pressure injection of Na+ into the cell. Single channel recordings indicated that MIP-induced outward K-current is Na-dependent. The probability to find a channel in open state increases with increasing intracellular Na+-concentration. Excised inside-out patches obtained from D-neurons contained I(K(Na)) channels could be activated by exposure of the cytoplasmic face of the patch membrane to 40 mM Na+, and 40 mM Li+, as well. The single channel current amplitude at -60 mV is 15 pA and the single channel conductance is 212 pS between -80 and 0 mV. It was concluded that MIP's activate a novel type of K+-current in the snail neurons. This current is the Na-activated K+-current. The single channel properties of the MIP activated channel is in concert with I(K(Na)) data obtained on different vertebrate and invertebrate preparations.

Opposite effects of molluscan neuropeptides CARP and MIP on LVA Ca-currents in Helix pomatia L. neurons

1. Voltage-clamp recordings from identified neurons of Helix pomatia L. revealed that two Ca-current components may contribute to the inward current in Na-free saline. 2. It was found that the transient component of the inward Ca-current was largely blocked by Ni 2+ but not by Cd 2+. 3. CARP (10 −6 M) selectively suppressed LVA currents. This effect is also reflected by tail current measurements, where in control saline the deactivation could be fitted by two exponentials, while after CARP treatment, by one. 4. MIP (10 −6 M) specifically enhanced the LVA component, or shifted the I-V curve of the composite Ca-current in hyperpolarizing direction. 5. It is concluded that peptides (CARP and MIP) isolated from Mytilus ganglia selectively affect the LVA Ca-channels, and that this effect is supposed to be species-specific.

Intracellular acidosis of identified leech neurones produced by substitution of external sodium

The intracellular pH, pH i, of identified neurones of the central nervous system of the leech Hirudo medicinalis L. was measured with double-barrelled neutral carrier pH-sensitive microelectrodes. The active regulation of pH i of these neurons is due to amiloride-sensitive Na H exchange and hence requires extracellular Na, Na o. We have measured a decrease of pH i following the removal of Na o. The rate of intracellular acidification in Na-free saline was similar to that in the presence of 2 mM amiloride suggesting that the acidification was due to inhibition of the Na H exchange. The rate of intracellular acidification depended on the Na substitute chosen; it was 0.02 ± 0.005 pH units/min (±S.D., n = 17) when Na was replaced by N-methyl- d-glucamine. A similar rate of acidification occurred with tris-hydroxymethyl-aminomethane (Tris) while the rate of acidification was higher with bis-2-hydroxymethyl-dimethyl-ammonium (BDA, 0.033 ± 0.016 pH units/min (±S.D., n = 7) and tetramethylammonium TMA, 0.046 ± 0.017 pH units/min ( n = 3) as Na substitutes. A high, non-linear rate of intracellular acidification was observed, when Li, K or choline were used as Na substitute. The recovery of pH i from acidification upon readdition of Na o was fast, only when Li had replaced Na was the pH i recovery considerably delayed. In conclusion, in all experiments using different Na substitutes the removal of Na o caused a ssubstantial intracellular acidification presumably due to inhibition of Na H exchange. These changes in pH i might be relevant for results obtained by experiments in which Na-free solutions are used.

Comparison of electrical responses of terminals, axons, and somata of a peptidergic neurosecretory system

Spontaneous and evoked electrical activity was recorded intracellularly from somata, axons, and terminal dilatations of an isolated peptidergic neurosecretory system, the X-organ-sinus gland, of the crabs Cardisoma carnifex and Podophthalmus vigil in order to compare their electrical characteristics. Spontaneous impulse activity was present in most penetrations and included irregular and pacemaker-like firing, as well as patterned activity (bursting). Extracellular recording showed that spontaneous impulses and bursting originate in a proximal region of the axon tract. Somata vary from being electrically nonresponsive to having overshooting impulses with a relatively slow rate of rise. Overshooting impulses were consistently recorded from axons and terminals. Regional differences include (1) a longer action potential duration in terminals, (2) ability of axons and terminals but not somata to sustain repetitive firing, (3) presence of depolarizing afterpotentials in axons but of hyperpolarizing afterpotentials in somata and terminals, and (4) occurrence of impulse broadening during repetitive firing in some terminals but not in axons or somata. Somata and terminals sustained reduced and slowed, but regenerative impulses in nominally Na-free saline and showed alterations of waveform in nominally Ca-free salines, while axons showed no regenerative responses in Na-free saline and no change of impulse form in Ca-free saline. Terminal responses in the presence of tetraethylammonium chloride (TEA) (50 mM) or Ba (50 mM) exhibited long depolarized plateaus, while impulses of somata were much less prolonged. Bursts often took the form of impulses superimposed on a depolarized plateau. Bursts could be evoked by single stimuli applied to the axon tract but not by current passed intracellularly. After addition of TTX, axon tract stimulation evoked plateaus without superimposed impulses. Terminals exhibit specialization of their electrical responses by comparison to axons and somata in having long-duration action potentials attributable to participation of Ca, capability of sustained firing, impulse broadening, and channels supporting sustained inward currents, all of which might enhance the admission of Ca for initiation of peptide secretion.

Simultaneous monitoring of electrical and secretory activity in peptidergic neurosecretory terminals of the crab.

Intracellularly recorded responses of peptidergic neurosecretory terminals and somata were correlated with their secretory responsiveness to elevation of the external K concentration ([K+]o). The experiments were performed on in vitro X-organ sinus gland neurosecretory systems from the eyestalk of the crab Cardisoma carnifex. Elevated-K-evoked release was followed in preparations exposed to a pulse-chase radiolabelling regime. The release of [3H]leucine incorporated into neurosecretory peptides could be followed by collecting the separate perfusates of the somata and terminal regions. Elevation of the [K+]o evoked terminal depolarization, an increase in impulse firing frequency and a decrease (50%) in terminal input resistance. Impulse firing ceased (in ca. 2 min) as depolarization reached a sustained maximum level (-17.6 +/- 3.57 mV, n = 9, absolute potential). The terminal depolarization and decreased input resistance were maintained throughout the period of elevated-K treatment. Release from the terminal region of incorporated 3H label paralleled the simultaneously monitored terminal depolarization. Maintained exposure to elevated-K saline was accompanied by sustained high levels of 3H release continuing beyond the loss of regenerative membrane responses. Release declined with a half-time of 47.1 +/- 13.5 min (n = 7). In contrast, terminal release of red pigment concentrating hormone (RPCH) was transitory, reaching peak values and declining to base line within a 10 min period. Removal of external Ca or addition of the Ca antagonists, Cd or Mn, blocked the stimulated 3H release. Addition of Cd or Mn, prior to or during an elevated-K-evoked 3H release produced a reversible suppression of the secretory response. Stimulation in the absence of external Na, under normal Ca conditions, resulted in a normal secretory response. The amplitude and duration of the elevated-K-evoked terminal depolarization was unaffected by nominally Ca- or Na-free saline or addition of Cd. Cd (1mM) and Na-free saline were effective in removing a Ca and Na component, respectively, of spontaneous or evoked terminal action potentials. Somatic responses to direct application of elevated K exhibited membrane depolarization and an accompanying increase in impulse firing. In contrast to recordings from the terminals in elevated K, fast regenerative potentials, electrotonically conducted from the distal axon, persisted in the somatic records. Somatic secretion of RPCH was below detectable limits (less than 0.2 fmol min-1). 3H release was an order of magnitude less than from the terminal region under similar conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Post-stimulus hyperpolarization in retzius nerve cells of leech, Haemopis sanguisuga

Abstract 1. 1. Post-stimulus hyperpolarization (PSH) that occurred in response to direct or indirect (across the electrotonic junction) depolarizing stimulation has been studied in Retzius neurons of horse leech, Haemopis sanguisuga. 2. 2. The amplitude and time course of PSH were dependent on the duration of depolarizing stimulation and passive membrane properties of Retzius cells. The recovery of membrane potential during PSH has an approximately exponential time course and consisted of two phases: the early (fast) one, which has been associated with a decrease in membrane input and transfer resistance and the late (slow) one. 3. 3. Na-free saline (with TRIS as substituing cation) and K-free saline augmented the amplitude of PSH and abolished its late phase. 4. 4. Ouabain completely blocked the late phase and greatly diminished the early one. Verapamil almost completely abolished PSH. 5. 5. Increased external Ca concentrations slightly augmented the amplitude and duration of PSH. 6. 6. Application of strong inward current reversed the PSH. The PSH reversal potential varied with extracellular K concentrations, approx. 40 mV per ten-fold change in external K+. 7. 7. It was concluded that PSH in Retzius nerve cells is mediated by two mechanisms: the prolonged increase in membrane K conductance and increased activity of the electrogenic sodium pump.

Studies of ionic currents in the isolated vestibular hair cell of the chick.

The ionic currents in enzymatically isolated vestibular hair cells of the chick were studied by a whole-cell-clamp variation of the patch voltage clamp, and by single channel recording. At membrane potentials more negative than -80 mV the hair cell showed anomalous rectification, and at potentials more positive than -40 mV large outward K currents were observed in normal saline. The outward K current decreased at large positive potentials, showing an N-shaped I-V relation. The outward K current was carried mostly through the Ca-activated K channel. K currents through the anomalous rectifier channel showed a decay in normal saline. This decay was eliminated reversibly in Na-free saline when the isotonic KCl-EGTA solution was used as the internal medium. However, a fast decay was still observed in Na-free high-K external solution when isotonic CaCl-EGTA was used as the internal medium. An increase in [K]o decreased the decay rate of the inward K current. The single-channel conductance of the anomalous rectifier channel was 50 pS in 160 mM-K saline and 23 pS in 40 mM-K saline. In 100 mM-Ca, -Sr and -Ba salines a large inward current was observed. At positive potentials the inward current carried by Ca and Sr ions showed significant decay; the current became outward at large positive potentials. Since the decay of the inward current was eliminated when 100 microM-quinine was added to the bathing medium, it was probably due to the activation of some Ca-activated K conductance which remained even with isotonic CsCl-EGTA internal medium. The activation kinetics of the Ca channel were studied in 100 mM-Ba solution at low temperatures (9-13 degrees C). From a comparison of the time constants of activation with the time constants of the tail currents, it was concluded that the Ca channel follows Hodgkin-Huxley-type m2 kinetics. A slow component that deviated from m2 kinetics was frequently observed at relatively large positive potentials. The steady-state fluctuations of Ba current showed a power density spectrum reasonably well fitted by a sum of two Lorentzian functions. The spectrum has a low-frequency component which indicates kinetics close to the macroscopic activation process of the Ca channel and a high-frequency component that indicates very fast flickering kinetics operating in the Ca channel.

KCl transport across and insect epithelium: I. Tracer fluxes and the effects of ion substitutions

Models for active Cl transport across epithelia are often assumed to be universal although they are based on detailed studies of a relatively small number of epithelia from vertebrate animals. Epithelial Cl transport is also important in many invertebrates, but little is known regarding its cellular mechanisms.We used short-circuit current, tracer fluxes and ion substitutions to investigate the basic properties of Cl absorption by locust hindgut, an epithelium which is ideally suited for transport studies. Serosal addition of 1 mM adenosine 3':5'-cyclic monophosphate (cAMP), a known stimulant of Cl transport in this tissue, increased short-circuit current (I(sc)) and net reabsorptive (36)C1 flux (J(CI)net) by 1000%. C1 absorption did not exhibit an exchange diffusion component and was highly selective over all anions tested except Br. Several predictions of Na- and HCO3-coupled models for Cl transport were tested: Cl-dependent I(sc) was not affected by sodium removal (<0.05 mM) during the first 75 min. Also, a large stimulation of J(C1)net was elicited by cAMP when recta were bathed for 6 hr in nominally Na-free saline(<0.001 to 0.2 mM) and there was no correlation between C1 transport rate and the presence of micromolar quantities of Na contamination. Increased unidirectional influx of (36)C1 into rectal tissue during cAMP-stimulation was not accompanied by a comparable uptake of (22)Na. J(CI)net was independent of exogenous CO2 and HCO3, but was strongly dependent on the presence of K.These results suggest that the major fraction of C1 transport across this insect epithelium occurs by an unusual K-dependent mechanism that does not directly require Na or HCO3.

Differences in active sodium transport through frog skin with various experimental setups.

Four experimental setups for examining active sodium transport by frog skin were examined and compared. They were: (I) a cannula, the interior of which faces the dermis; (II) a cannula, the interior of which faced the epidermis; (III) an Ussing-type chamber without gaskets; and (IV) an Ussing-type chamber with gaskets. The experiments were out in SO4-Ringer's solution. In control saline, the magnitude of the short circuit current (SCC microA/cm2) was in the order: I greater than III greater than II not equal to IV, while the magnitude of skin resistance (RM, k omega . cm2) and potential difference were: II not equal to IV greater than III greater than I. The responses of the skin to amiloride and cadmium varied greatly according to the various setups employed. Amiloride decreased the SCC and increased the RM. The percent differences from the control SCC and RM showed I not equal to IV greater than II and IV not equal to II greater than I, respectively. On the other hand, cadmium increased the SCC and decreased the RM. The orders of percent differences from the control SCC and RM were IV not equal to II greater than I and II not equal to IV greater than I. The effect of calcium gluconate on the active Na transport was also different depending on the various setups used; the responses to epidermal and dermal Ca-gluconate were quite opposite. The above seemingly curious findings are discussed in terms of the ENa, RNa, and R sigma obtained with Helman's Na-free saline method. From the present investigation, it is clear that careful attention should be given to the interpretation of experimental results in active transport studies when various kinds of setups are employed.

Calcium current inactivation in identified neurones of Helix aspersa.

1. A two-electrode voltage clamp method was used to study Ca inward currents in identified Helix aspersa neurones bathed in 25 mM-Ca, Na-free saline with TEA and 4-AP. 2. Inward currents were blocked by CdCl2. In Cd delayed outward currents appeared at +30 mV. When two identical depolarizations were separated by a gap inward current turned off to the same level during the two pulses up to +20 mV; above this potential the records cross over. 3. The turn-off of inward current at potentials up to +20 mV was not affected by 0.2 mM-quinine, which reduced outward currents at more depolarized potentials. Inward currents declined exponentially over the first 100 msec with a time constant around 60 msec at 0 mV. Double-pulse experiments gave the same time course of turn-off. 4. When Ca inward current was reduced by lowering [Ca]o or by partial block by Cd the rate and extent of turn-off was reduced. 5. The inactivation curve (obtained using a double pulse with gap method) was U-shaped. The curve was not significantly changed by addition of quinine (0.2 mM) or by changing test pulse size. 6. Recovery of inward currents after a depolarizing prepulse was a double-exponential process, with time constants of 120 msec and 9.4 sec at 10--11 degrees C. 7. Our results are discussed in terms of possible Ca-dependent Ca inactivation and in terms of the possibility of development of an outward Ca-dependent K current.

The depolarizing afterpotential of crab muscle fibres. A sodium-dependent process mediated by intracellular calcium.

1. A study was made of the depolarizing afterpotential (d.a.p.) which follows the initial graded electrogenesis of crab muscle fibres. 2. Increasing the strength, duration or amplitude of the stimulating current pulses enhanced both the d.a.p. and the local contractions. 3. Arsenazo III was injected intracellularly and changes in light absorbance by the dye were used to monitor the increase in free sarcoplasmic Ca concentration during excitation-contraction (E-C) coupling. The onset of the absorbance changes occurred during the depolarizing phase of the initial electrogenesis and the maximum value coincided with the peak of the d.a.p. An exponential decay of the absorbance signal occurred during the repolarizing phase of the d.a.p. 4. Ionophoretic injection of EGTA into the sarcoplasm did not affect the initial electrogenesis but did reduce changes in dye absorbance, blocked tension development and abolished the d.a.p. 5. Caffeine (0.1--0.4 mM) markedly enhanced both the d.a.p. and the local contractions, but had no effect on the initial electrogenesis. 6. Replacement of extracellular Na ions with Li, Tris or choline abolished the d.a.p. The initial electrogenesis was enhanced in the choline-containing medium, but was not affected by Li or Tris. The rate of relaxation of the local contractions and the rate of decay of the light absorbance changes were slowed in Na-free saline. 7. Tetrodotoxin (10(-5) g/ml.) had no effect on either the membrane responses or tension development. 8. For initial graded responses of comparable peak amplitude a threefold reduction of [Ca-a1o shortened the d.a.p., but had little effect onits peak amplitude. A fivefold increase in [Ca]o reduced both the amplitude and duration of the d.a.p. 9. Changes in [Mg]o had little effect on the d.a.p., but both Mn (4--10 mM) and La (0.1 mM) blocked the initial electrogenesis and the d.a.p. 10. It is concluded that distinct ionic mechanisms give rise to the initial electrogenesis and the d.a.p. While the former is due to activation of Ca conductance, the d.a.p. is a Na-dependent phenomenon that is tetrodotoxin-insensitive, and mediated by the rise of intracellular Ca concentration during E--C coupling.

Tetrodotoxin sensitivity and Ca Component of action potentials of mouse dorsal root ganglion cells cultured in vitro

In the mouse dorsal root ganglia cultured in vitro, neurons were classified into 3 groups according to the responses of their action potentials to tetrodotoxin (TTX) and removal of Na ions from bathing medium: (1) the neurons whose action potentials were not affected by TTX by TTX (10(-6) - 10 (-5)g/ml) and which generated Ca-dependent regenerative responses under Na-free condition, (2) the neurons whose spike potentials were resistant to TTX but failed to survive in Na-free saline and (3) the neurons whose action potentials were suppressed by TTX(10(-8)g/ml) as well as Na removal. The mean duration of spike and after-hyperpolarization was longest in the first group of the neurons and shortest in the third, probably reflecting the difference in the contribution of Ca currents to action potentials. The unresponsiveness of the neurons to TTX was shown to be due to the insensitivity of Na as well as Ca components of action potentials to the toxin. It was discussed that the occurrence of TTX-resistant action potentials to the toxin. It was discussed that the occurrence of TTX-resistant action might be related to the neuronal development.

The effect of sodium, calcium and metabolic inhibitors on calcium efflux from goldfish heart ventricles.

1. 45Ca efflux and tissue Ca content were examined in goldfish ventricles under conditions known to affect cellular Ca movements. 2. EGTA or Ca-EGTA was added to the washout solutions in sufficient concentration (10 mM) to avoid retardation of the apparent tissue 45Ca efflux by extracellular 45Ca binding or backflux. 3. After a variable initial increase, the cellular Ca content usually stabilizes within 60 min when ventricles are immersed in Li- or K-substituted saline containing 1.8 mM Ca0 (under these conditions the internal Ca2+ concentration is below 10(-5) M). 4. 45Ca efflux is maximally activated by external concentrations of Ca2+ as low as 10(-6) M, in both Na-containing and Na-free saline. 5. 45Ca efflux decreases in Na-free solutions. It is reactivated by Na-saline. The effect of different external Na concentration on 45Ca efflux is comparable at external Ca2+ concentrations between 10(-6) M and 2 x 10(-3) M. 6. Reactivation of Ca efflux after Na0 readmission is inhibited by metabolic poisoning, or in the presence of 10 mM-caffeine. Loading with 45Ca at very low external Ca2+ concentration prevents the inhibition of Ca efflux in Na-free medium. 7. Caffeine (10 mM) produces contractions of about equla size when K-depolarized preparations are immersed in either Na- or Li-saline. At the same time there is a similar increase in 45Ca efflux in absence of Na0 and in its presence. 8. In the virtual absence of Ca2+0 (10(-5) M-Ca, 10(-2) M-EGTA) and Na+0, the residual 45Ca efflux is reversibly inhibited by cyanide (2 mM). 9. The results are roughly compatible with the general concept of ATP-dependent Na-Ca exchange in internal Ca2+ homeostasis. However, this hypothesis should probably be modified to account for the fact that under physiological concentrations Na+0 and Ca2+0 do not compete for activating 45Ca efflux. Metabolic products may be involved in Na0- and Ca0-dependent Ca efflux. It is therefore not excluded that a Na-independent active mechanism co-operates with Na-Ca exchange in Ca extrusion.

The effect of sodium and magnesium and their interaction with quinine and lanthanum on spontaneous activity and related calcium movements of rat ileal smooth muscle

1. Na-free media, with or without atropine, induced tonic contractures and enhanced spontaneous contractions of the rat ileum. 2. Quinine and lanthanum did not significantly inhibit these Na-free contractures, and neither agent, nor Na-free saline itself, caused any significant net gain of tissue calcium. 3. Na-free media enormously stimulated calcium efflux from the ileum whether atropine was present or not, suggesting a high tissue calcium turnover due to stimulation of surface transport sites. Quinine and lanthanum had no significant effect on this calcium efflux. 4. Mg-free media increased tension in the ileum and enhanced spontaneous contractions while high-Mg media inhibited contractility. 5. Mg-free media were without significant effect on calcium efflux from the ileum but they did induce a very small rise in tissue calcium. 6. These results are not consistent with the concept of a simple Na/Ca membrane counter exchange system in the ileum to regulate tissue calcium. On present evidence, a linked counter exchange system of Ca with Mg and Na would seem more likely.

Mephenesin blocks persistent Na + or Ba 2+ currents underlying paroxysmal depolarizations in PTZ- or Ba-treated molluscan neurones

1. Mephenesin blocks the paroxysmal depolarizations (PDS's) induced by PTZ or barium saline on Helix or Aplysia neurones. 2. Under voltage-clamp conditions, the current-voltage relation is mapped with a slow ramp. In PTZ or Ba-treated neurones, the I– V curves have a negative-slope region produced by a persistent inward current. This current disappears with mephenesin. 3. TTX or Na-free saline blocks PDS's and the persistent inward current induced by PTZ, but not those induced by barium. Co 2+ ions (10 mM) have opposite effects. 4. We conclude that a persistent Na current underlies the PDS's induced by PTZ, whereas a persistent Ba current underlies those induced by Ba 2+ and that both Na and Ba currents are blocked by mephenesin.

Action potentials in the rat chromaffin cell and effects of acetylcholine.

1. Electrophysiological properties of the rat chromaffin cell were studied using intracellular recording techniques. 2. The resting potential in the chromaffin cell was -49 +/- 6 mV (mean +/- S.D., n = 14) in standard saline containing 10 mM-Ca whereas that in Na-free saline was -63 +/- 9 mV (n = 17). At rest, the membrane has a substantial Na permeability. 3. Action potentials were evoked by passing current through the recording electrode. In standard saline the major fraction of the action potential disappeared either upon omission of external Na ions from standard saline or addition of 1 muM tetrodotoxin (TTX). We conclude that action potentials in the chromaffin cell are due mainly to an increase in the permeability of the membrane to Na ions. 4. Small but significant regenerative action potentials were observed in Na-free saline, and when Ca in Na-free saline was replaced by Ba, prolonged action potentials occurred. We conclude that action potentials in the chromaffin cell also have a Ca component. 5. Iontophoretic application of acetylcholine (ACh) produced a transient membrane depolarization in standard saline. 6. Spontaneous action potentials were recorded extracellularly by microsuction electrodes. They occurred at a rate of 0-05-0-1/sec in almost all cells. 7. When the perfusion fluid contained 3 x 10(-7) M to 10(-4) M ACh the spike frequency increased up to about 2/sec. This stimulatory effect of ACh was blocked by 10(-7) M atropine but not by 10(-3) M hexamethonium nor by 10(-5) M-d-tubocurarine. 8. The importance of Ca entry during action potentials for catecholamine secretion is discussed

Sodium and calcium components of the action potential in a developing skeletal muscle cell line.

1. Developmental changes in action potential properties were studied in a clonal rat skeletal muscle cell line. 2. Small action potentials were evoked in mononucleate myoblasts. No spike was seen in Na-free saline. A similar spike was evoked in a medium where all NaCl was replaced by LiCl. No spike was evoked when NaCl was replaced by CsCl. 3. Action potentials overshot zero membrane potential in multinucleate myotubes. The action potential was composed of two components, an initial fast spike and a hump on the falling phase or in some cases a distinct second peak. 4. Teh overshoot of the initial fast spike decreased when the external Na concentration was decreased. 5. In saline with 10 mM-Ca the second component often formed a distinct peak following the initial fast spike. A slow regenerative potential was evoked in Na-free media with a depolarizing current pulse. 6. In saline containing BaCl-2 instead of CaCl-2 there was always a second peak, the overshoot of which changed with external Ba concentration. A slow regenerative potential was evoked in Na-free, Ba-saline. The membrane conductance at the peak of the Ba-action potential was larger than in the resting state. 7. In adult rat skeletal muscle, the shape of the action potential was not changed when Ca was replaced by Ba. No action potential was evoked in Na-free Ba-saline or Ba-saline with tetrodotoxin (3 times 10-7 M). 8. The significance of the Ca component in the developing muscle is discussed.

The ionic mechanism of action of GABA and l-glutamate on a crustacean striated muscle (vas deferens of the crayfish)

1. The striated musculature of the vas deferens of the crayfish responds to l-glutamate with a contraction. 2. The action of l-glutamate is reduced or abolished by Na-free saline. It is usually enhanced by picrotoxin (PTX) and is greatly augmented by media of low Cl concentration, even in the absence of external Na. This augmentation is blocked by PTX. 3. GABA either causes contraction or does not; in the latter case it inhibits the contraction caused by l-glutamate. In low Cl media GABA always causes a strong contraction; like its inhibitory action this is blocked by PTX. 4. It is suggested that GABA activates Cl channels and that l-glutamate activates Na and Cl channels. Cl activation, whether caused by GABA or by l-glutamate, is blocked by PTX. 5. Since PTX does not interfere with Na activation, it is likely that the receptors responding with Cl activation are different from those responsible for Na activation and that the receptors responding to GABA can also be activated by l-glutamate.

Slow changes of membrane permeability in giant neurones of Helix pomatia.

1. Membrane currents associated with long-lasting depolarizations were measured on nerve cells of the snail Helix pomatia using the voltage clamp method. 2. During depolarizing pulses of several seconds duration the delayed outward current markedly declined in an exponential fashion with a time constant of 0.5–1.75 sec at 23–26.5°C. The ratio final outward current: peak outward current decreased with increasing depolarization reaching a value of 0.5 at membrane potentials between −28 and −16 mV. The decline of the outward current is tentatively ascribed to a slow inactivation of the permeability of the delayed channel. 3. In saline with veratridine long depolarizing pulses gave rise to a slowly developing, maintained inward current. At the end of the pulses large tails of inward current occurred which slowly declined in an exponential fashion. The slowly developing, maintained inward current and the inward tail current are carried by sodium ions; they disappear in Na-free saline with veratridine. 4. The voltage and time dependence of the veratridine-induced slow sodium permeability was studied by investigating the effect of pulse height, pulse duration and holding potential on the amplitude of the tail currents. The results were compared with the known effects of veratridine on myelinated nerve fibres.