Substitution Of External Na Research Articles

Functional analysis of plasma membrane Ca2+ATPase 3 and its primary aldosteronism-associated mutations.

Primary aldosteronism (PA) is the most common form of secondary hypertension and can lead to higher rates of cardiovascular complications and death than essential hypertension. Aldosterone-producing adenomas (APAs) are the most frequent cause of PA. Most APAs have mutations in plasma membrane ion-transport proteins with 0.6%-9% carrying a mutation in the Plasma Membrane Ca2+ ATPase 3 (PMCA3). We have developed a Xenopus oocyte expression system to measure the function of PMCA3 wildtype (PMCA3WT) and an APA associated deletion mutant (PMCA3L425_V426del.) with minimal endogenous contamination. We measured ATPase activity at 37 °C in plasma membrane preparations from oocytes expressing PMCA3. Ca2+-dependent ATPase was observed in preparations from oocytes expressing PMCA3WT, with a [Ca2+] dependence well described with a rectangular hyperbola (K0.5 = 25.6 ± 5.1 μM) (triplicates from three independent membrane preparations). In contrast, membrane preparations from PMCA3L425_V426del. expressing oocytes lacked Ca2+-dependent ATPase activity, indicating loss of enzymatic activity in the mutant. We used two-electrode voltage clamp electrophysiology to evaluate the presence of PMCA3-variant mediated currents. At resting intracellular [Ca2+], PMCA3WT-injected oocytes had currents indistinguishable from those in uninjected oocytes, at all voltages. When bathed by extracellular 125 mM Na+, PMCA3L425_V426del.-injected oocytes presented inward currents at negative membrane potentials and outward currents at positive ones, consistent with induction of an aberrant channel-like current. The currents were outwardly directed upon substitution of external Na+ with N-methyl D-glucamine+ and were insensitive to changes in extracellular [Ca2+]. These results indicate that PMCA3L425_V426del. causes hyperaldosteronism due to the concomitant loss of active Ca2+ transport and induction of a depolarizing Na+-mediated current. Current experiments are evaluating the ATPase and physiological characteristics of other PA-associated PMCA3 mutations. Funded by NSF-MCB 2003251.

Involvement of both sodium influx and potassium efflux in ciguatoxin-induced nodal swelling of frog myelinated axons

Ciguatoxins, mainly produced by benthic dinoflagellate Gambierdiscus species, are responsible for a complex human poisoning known as ciguatera. Previous pharmacological studies revealed that these toxins activate voltage-gated Na+ channels. In frog nodes of Ranvier, ciguatoxins induce spontaneous and repetitive action potentials (APs) and increase axonal volume that may explain alterations of nerve functioning in intoxicated humans. The present study aimed determining the ionic mechanisms involved in Pacific ciguatoxin-1B (P-CTX-1B)-induced membrane hyperexcitability and subsequent volume increase in frog nodes of Ranvier, using electrophysiology and confocal microscopy. The results reveal that P-CTX-1B action is not dependent on external Cl− ions since it was not affected by substituting Cl− by methylsulfate ions. In contrast, substitution of external Na+ by Li+ ions suppressed spontaneous APs and prevented nodal swelling. This suggests that P-CTX-1B-modified Na+ channels are not selective to Li+ ions and/or are blocked by these ions, and that Na+ influx through Na+ channels opened during spontaneous APs is required for axonal swelling. The fact that the K+ channel blocker tetraethylammonium modified, but did not suppress, spontaneous APs and greatly reduced nodal swelling induced by P-CTX-1B indicates that K+ efflux might also be involved. This is supported by the fact that P-CTX-1B, when tested in the presence of both tetraethylammonium and the K+ ionophore valinomycin, produced the characteristic nodal swelling. It is concluded that, during the action of P-CTX-1B, water movements responsible for axonal swelling depend on both Na+ influx and K+ efflux. These results pave the way for further studies regarding ciguatera treatment.

Characterizing the conductance underlying depolarization-induced slow current in cerebellar Purkinje cells

Brief strong depolarization of cerebellar Purkinje cells produces a slow inward cation current [depolarization-induced slow current (DISC)]. Previous work has shown that DISC is triggered by voltage-sensitive Ca influx in the Purkinje cell and is attenuated by blockers of vesicular loading and fusion. Here, we have sought to characterize the ion channel(s) underlying the DISC conductance. While the brief depolarizing steps that triggered DISC were associated with a large Ca transient, the onset of DISC current corresponded only with the Ca transient decay phase. Furthermore, substitution of external Na with the impermeant cation N-methyl-d-glucamine produced a complete and reversible block of DISC, suggesting that the DISC conductance was not Ca permeant. Transient receptor potential cation channel, subfamily M, members 4 (TRPM4) and 5 (TRPM5) are nonselective cation channels that are opened by Ca transients but do not flux Ca. They are expressed in Purkinje cells of the posterior cerebellum, where DISC is large, and, in these cells, DISC is strongly attenuated by nonselective blockers of TRPM4/5. However, measurement of DISC currents in Purkinje cells derived from TRPM4 null, TRPM5 null, and double null mice as well as wild-type mice with TRPM4 short hairpin RNA knockdown showed a partial attenuation with 35-46% of current remaining. Thus, while the DISC conductance is Ca triggered, Na permeant, and Ca impermeant, suggesting a role for TRPM4 and TRPM5, these ion channels are not absolutely required for DISC.

The effect of external sodium concentration on sodium–calcium exchange in frog olfactory receptor cells

During the response of vertebrate olfactory receptor cells to stimulation, Ca(2+) enters the cilia via cyclic nucleotide-gated channels and is extruded by Na(+)-Ca(2+) exchange. The rise in Ca(2+) concentration opens a Ca(2+)-activated Cl(-) conductance which carries most of the inward receptor current. The dependence of Ca(2+) extrusion upon external Na(+) concentration was studied by using the falling phase of the Ca(2+)-activated Cl(-) current following a brief exposure to the phosphodiesterase inhibitor IBMX to monitor indirectly the decay in intraciliary Ca(2+) concentration. External Na(+) concentration was reduced by partial substitution with guanidinium, an ion which permeates the cyclic nucleotide-gated channel but does not support Na(+)-Ca(2+) exchange. The time constant describing the decay in current following IBMX stimulation was surprisingly little affected by substitution of external Na(+), being substantially retarded only when its concentration was reduced to a third or less of its normal value in Ringer solution. When the cilia were returned to Ringer solution after a period in reduced-Na(+) solution, the time constant for the final decay of current was similar to that seen when returning immediately to IBMX-free Ringer solution. This observation suggests that Ca(2+) extrusion via Na(+)-Ca(2+) exchange dominates the falling phase of the response to IBMX, which can therefore be used to assess exchanger activity. Rate constants derived from the time constants for current decay at different external Na(+) concentrations could be fitted by the Hill equation with a K(d) of 54 +/- 4 mm and Hill coefficient of 3.7 +/- 0.4. The cooperativity of the dependence upon external Na(+) concentration indicates that at least three Na(+) ions enter for each exchanger cycle, while the high affinity for external Na(+) contrasts with the photoreceptor and cardiac exchangers. The functional importance of this observation is that the relative insensitivity of the Na(+)-Ca(2+) exchanger to external Na(+) concentration allows normal response termination even following partial dilution or concentration of the olfactory mucus.

Ionic conductances in sustentacular cells of the mouse olfactory epithelium

The electrical properties of sustentacular cells (SCs) in the olfactory epithelium (OE) were investigated in tissue slices taken from neonatal mice (P0-P4). Conventional whole-cell recordings were obtained from SCs and also from olfactory receptor neurones (ORNs) in situ. SCs had a larger apparent cell capacitance (C(cell)) (18.6 +/- 0.5 pF) than ORNs (4.4 +/- 0.4 pF) and a lower apparent membrane resistance (R(m)) (160 +/- 11 MOmega versus 664 +/- 195 MOmega, respectively). When corrected for a seal resistance of 1 GOmega, these mean R(m) values were increased to 190 MOmega and 2 GOmega in SCs and ORNs, respectively. SCs generated a TTX (1 microm)-resistant voltage-activated Na(+) current (I(Na)) that had a peak density at -38 mV of -44 pA pF(-1) and supported action potential firing. Peak current density of I(Na) in neurones was 510 +/- 96 pA pF(-1). The outward K(+) current in SCs was composed (> 70%) of a TEA (2 mm)-sensitive component that was mediated by the opening of large-conductance (237 +/- 10 pS; BK) channels. The resting leak conductance (g(L)) of SCs was permeable to monovalent cations and anions and was largely inhibited by substitution of external Na(+) with NMDG and by internal F(-) with gluconate. g(L) deactivated up to 50% at potentials negative of -70 mV and was inhibited by 18beta-glycyrrhetinic acid (20 mum). SCs were identified using fluorescent dyes (Lucifer Yellow and Alexa Fluor 488) in the whole-cell patch pipette-filling solution. Our findings indicate that SCs in the OE of neonates are electrically excitable and are distinguishable from neurones by a having a resting g(L).

Action potential afterdepolarization mediated by a Ca 2+-activated cation conductance in myenteric AH neurons

We investigated the nature of afterdepolarizing potentials in AH neurons from the guinea-pig duodenum using whole-cell patch-clamp recordings in intact myenteric ganglia. Afterdepolarizing potentials were minimally activated following action-potential firing under normal conditions, but after application of charybdotoxin (40 nM) or tetraethyl ammonium (TEA; 10–20 mM) to the bathing solution, prominent afterdepolarizing potentials followed action potentials. The whole-cell current underlying afterdepolarizing potentials ( I ADP) in the presence of TEA (10–20 mM) reversed at −38 mV and was not voltage-dependent. Reduction of NaCl in the bathing (Krebs) solution to 58 mM shifted the reversal potential of the I ADP to −58 mV, suggesting that the current underlying the afterdepolarizing potential was carried by a mixture of cations. The relative contributions of Na + and K + to this current were estimated to be about 1:5. Substitution of external Na + with N-methyl D-glucamine blocked the current while replacement of internal Cl − with gluconate did not block the I ADP. The I ADP was also inhibited when CsCl-filled patch pipettes were used. The I ADP was blocked or substantially decreased in amplitude in the presence of N-type Ca 2+ channel antagonists, ω-conotoxin GVIA and ω-conotoxin MVIIC, respectively, and was eliminated by external Cd 2+, indicating that it was dependent on Ca 2+ entry. The I ADP was also inhibited by ryanodine (10–20 μM), indicating that Ca 2+-induced Ca 2+ release was involved in its activation. Niflumic acid consistently inhibited the I ADP with an IC 50 of 63 μM. Using antibodies against the pore-forming subunits of L-, N- and P/Q-type voltage-gated Ca 2+ channels, we have demonstrated that myenteric AH neurons express N- and P/Q, but not L-type voltage-gated Ca 2+ channels. We conclude that the ADP in myenteric AH neurons, in the presence of an L-type Ca 2+-channel blocker, is generated by the opening of Ca 2+-activated non-selective cation channels following action potential-mediated Ca 2+ entry mainly through N-type Ca 2+ channels. Ca 2+ release from ryanodine-sensitive stores triggered by Ca 2+ entry contributes significantly to the activation of this current.

Developmental changes in hyperpolarization-activated currents I(h) and I(K(IR)) in isolated rat intracardiac neurons.

The hyperpolarization-activated nonselective cation current, I(h), was investigated in neonatal and adult rat intracardiac neurons. I(h) was observed in all neurons studied and displayed slow time-dependent rectification. I(h) was isolated by blockade with external Cs(+) (2 mM) and was inhibited irreversibly by the bradycardic agent, ZD 7288. Current density of I(h) was approximately twofold greater in neurons from neonatal (-4.1 pA/pF at -130 mV) as compared with adult (-2.3 pA/pF) rats; however, the reversal potential and activation parameters were unchanged. The reversal potential and amplitude of I(h) was sensitive to changes in external Na(+) and K(+) concentrations. An inwardly rectifying K(+) current, I(K(IR)), was also present in intracardiac neurons from adult but not neonatal rats and was blocked by extracellular Ba(2+). I(K(IR)) was present in approximately one-third of the adult intracardiac neurons studied, with a current density of -0.6 pA/pF at -130 mV. I(K(IR)) displayed rapid activation kinetics and no time-dependent rectification consistent with the rapidly activating, inward K(+) rectifier described in other mammalian autonomic neurons. I(K(IR)) was sensitive to changes in external K(+), whereby raising the external K(+) concentration from 3 to 15 mM shifted the reversal potential by approximately +36 mV. Substitution of external Na(+) had no effect on the reversal potential or amplitude of I(K(IR)). I(K(IR)) density increases as a function of postnatal development in a population of rat intracardiac neurons, which together with a concomitant decrease in I(h) may contribute to changes in the modulation of neuronal excitability in adult versus neonatal rat intracardiac ganglia.

Sodium effects on 4-aminopyridine-sensitive transient outward current in canine ventricular cells.

Tetrodotoxin (TTX) or substitution of external Na+ reduces the 4-aminopyridine-sensitive transient outward current (Ito1) in rat ventricular myocytes. We investigated the outcome of reducing external sodium on the kinetics, gating, and selectivity of Ito1 with a dual-patch electrode technique to record whole cell currents and transmembrane potentials independently of the voltage clamp in canine midmyocardial cells. Steps from -80 to 0 mV produced overlapping inward sodium and outward potassium currents, accompanied by a loss of voltage control associated with activation of INa. Substitution of external Na+ or application of TTX abolished INa, restored voltage control, and reduced Ito1. Inactivation of INa with a 10-ms prestep to -45 mV decreased Ito1 to the same extent as external Na+ substitution. The kinetics, gating, and selectivity of Ito1 recorded after inactivation of INa were unaffected by drastic reductions in external Na+. Our findings suggest that a larger Ito1 in the presence of normal external Na+ is due to 1) transient loss of voltage control and concomitant changes in activation of Ito1 and/or 2) facilitation of an outward current by intracellular Na+. We conclude that reduction of external sodium has no direct effect on the kinetics or gating of Ito1, non does Na+ contribute to current flow through Ito1 channels in canine midmyocardial cells.

Properties and functions of Na(+)-activated K+ channels in the soma of rat motoneurones.

1. Properties and functions of Na(+)-activated K+ (KNa) channels in the soma of motoneurones were studied in spinal cord slices of newborn rat. KNa channels had a conductance of 44.8 pS in 5.6 mM external K+ (Ko+)/106 mM internal K+ (Ki+) solutions and 139.2 pS in 155 mM Ko+/85 mM Ki+ solutions. KNa channels were voltage independent and needed a relatively high [Na+]i to become active (EC50 = 39.9 mM). Li+ could not substitute for Na+ in activation of KNa channels. The channels were predominantly found in the vicinity of cell processes, in the regions of most probable accumulation of cytoplasmic Na+. 2. In current-clamp experiments, the shape of the single action potential (AP) recorded in Ca(2+)-free Ringer solution was not changed after substitution of external Na+ with Li+. However, 0.4-0.8 s trains of APs were followed by a slow (1-2s) after-hyperpolarization (sAHP), which reversibly disappeared when external Na+ was replaced by Li+. Na(+)-activated sAHP persisted after addition of ouabain and its amplitude was even increased in K(+)-free Ringer solution. sAHP disappeared when the membrane potential was equal to the K+ equilibrium potential. This indicated that sAHP resulted from activation of a Na(+)-dependent K+ conductance, rather than from activation of the electrogenic Na(+)-K+ pump. 3. In conclusion, KNa channels can play an important role in excitability of motoneurones. KNa channels do not make a contribution to the single AP, but they can be activated by a local accumulation of internal Na+ during trains of APs. A Na(+)-activated K+ conductance can reduce membrane excitability and contribute to regulation of AP firing in motoneurones.

Contribution of Na+/Ca2+ exchange to action potential of human atrial myocytes.

The Ca2+ dye indo 1 was used to record internal Ca2+ (Cai) transients in order to investigate the role of the Na+/Ca2+ exchange current (INa/Ca) in whole cell patch-clamped human atrial myocytes After the activation of the L-type Ca2+ current by test pulses (20 ms) at +20 mV, a tail current (I(tail)) was activated at a holding potential of -80 mV with a density of -1.29 +/- 0.06 pA/pF. The time course of I(tail) followed that of Cai transients I(tail) was suppressed by dialyzing cells with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, applying 5 mM caffeine, or substituting external Na+ with Li+, indicating that this current was mainly generated by INa/Ca. Two types of action potential were recorded: type A, which is characterized by a narrow early plateau followed by a late low plateau phase, and type B, which is characterized by a small initial peak followed by a prolonged high plateau phase. Type B action potentials were found in larger cells than type A action potentials (membrane capacitance 81.8 +/- 4.5 and 122.4 +/- 7.0 pF in types A and B, respectively, P < 0.001). Substitution of external Na+ with Li+ shortened the late plateau of the type A action potential and the prolonged plateau of the type B action potential. Suppression of Cai transients by caffeine shortens the late part of both types of action potentials, whereas its lengthening effect on the initial phase of action potentials can result from several different mechanisms. The beat-to-beat dependent relationship between Cai transients and action potentials could be mediated by Ina/Ca- Delayed afterdepolarizations were present in a significant proportion of atrial myocytes in our experimental conditions. They were reversibly suppressed by Li+ substitution for Na+, suggesting that they are generated by INa/Ca. We conclude that INa/Ca plays a major role in the development of action potentials and delayed afterdepolarizations in isolated human atrial myocytes.

Photolysis of caged Ca2+ facilitates and inactivates but does not directly excite light-sensitive channels in Drosophila photoreceptors

Substantial evidence implicates the phosphoinositide cascade in invertebrate phototransduction, but the final pathway of excitation remains obscure. In order to test the hypothesis that Ca2+ is the excitatory messenger rapid concentration jumps of cytosolic Ca2+ were achieved in dissociated Drosophila photoreceptors by flash photolysis of the caged Ca2+ compounds DM-nitrophen and nitr-5. Both compounds were introduced via patch pipettes used to record whole-cell currents. Calibrations using INDO-1 and Mag-INDO-1 indicated that photolysis of DM-nitrophen (5 mM loaded with 4 mM Ca2+), raised Ca, to ca. 20-50 microM, and nitr-5 (same loading) to ca. 1-2 microM. In mutants lacking light responses (ora, lacking rhodopsin; norpA, lacking phospholipase C; trp, which is inactivated by conditioning lights), the only current evoked by photolysis of DM-nitrophen was a small inward current with no detectable latency. This current did not reverse at +80 mV and was blocked by substitution of external Na+ for Li+, suggesting it represents activation of an electrogenic Na+/Ca2+ exchanger. A similar current was also the only current elicited by caged Ca2+ during the 5 msec latent period in wild type (WT) photoreceptors. To investigate possible modulatory effects of caged Ca2+ on the light-activated conductance, cells were first stimulated with a saturating light stimulus, itself incapable of releasing significant Ca2+, and then the photolytic flash was discharged during the response. During the rising phase of the response, photolysis of DM-nitrophen (but not nitr-5) induced a pronounced facilitation in WT photoreceptors. When photolysed during the plateau phase both DM-nitrophen and nitr-5 induced a rapid inactivation of the light-induced current. By contrast, in trp photoreceptors, which lack one class of Ca2+ permeable light-sensitive channel, photolysis of DM-nitrophen induced a significant facilitation during the falling phase of the response, but during the rising phase photolysis significantly depressed the overall response. In conclusion, caged Ca2+ failed to activate any channels in Drosophila photoreceptors but profoundly affected the light-dependent channels once they have been activated.

Trans-ACPD, a metabotropic receptor agonist, produces calcium mobilization and an inward current in cultured cerebellar Purkinje neurons.

1. 1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), a racemic mixture of 1-aminocyclopentane-1S,3R-dicarboxylic acid and 1-aminocyclopentane-1R,3S-dicarboxylic acid, a selective agonist of the metabotropic glutamate receptor, was applied to mouse Purkinje neurons (PNs) in culture. Measurements of free intracellular Ca2+ were made using fura-2 microfluorimetric imaging and of membrane current using perforated-patch voltage-clamp recording in separate experiments. 2. Brief pulses of t-ACPD (< or = 100 microM, 1-5 s) consistently produced a large (200-600 nM) increase in dendritic Ca2+ that was sometimes followed by a somatic increase. The dendrites typically returned to basal Ca2+ levels within 10-30 s. 3. Ca2+ increases produced by t-ACPD were measured in Ca(2+)-free external saline [0.5 mM ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)], suggesting that they result from intracellular mobilization rather than influx. In addition, Ca2+ increases were not attenuated by a mixture of DL-AP5 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) [antagonists of N-methyl-D-aspartate (NMDA) and AMPA/kainate receptors, respectively], but were almost entirely eliminated by L-AP3 (100 microM), a putative metabotropic receptor antagonist or by preincubation of the cultures in pertussis toxin. 4. Brief pulses of t-ACPD (10 microM) produced a small inward current that was associated with an increase in membrane conductance. This current was reversibly blocked by L-AP3 but not by treatments that attenuate some voltage-gated K+ currents. Thus this current is unlikely to underlie the depolarization that is produced by metabotropic agonists in hippocampal pyramidal cells by K(+)-channel closure. 5. The t-ACPD induced inward current was attenuated by substitution of external Na+ with Li+ or choline, or by application of the membrane-permeable Ca2+ chelator, bis-(2-aminophenoxy)-N,N,N',N'- tetraacetic acid (BAPTA)/AM. One mechanism that could mediate this current is electrogenic Nao/Cai exchange, triggered by Ca2+ mobilization.

A patch clamp study of the nicotinic acetylcholine receptor of bovine adrenomedullary chromaffin cells in culture.

1. Acetylcholine-induced currents recorded from bovine adrenal medullary chromaffin cells maintained in culture were studied during pressure or ionophoretic applications of ACh, using the 'whole-cell' and 'outside-out' configurations of the patch clamp technique. In standard salines, ACh evoked whole-cell currents of -38 pA to -1 nA at -60 mV, which had a reversal potential (EACh) of -7.1 +/- 0.6 mV. The ACh current-voltage relationship was characteristically linear at negative holding potentials and biphasic at positive holding potentials, displaying a region of almost zero slope conductance between 0 and +40 mV followed by a region of positive slope conductance at more positive potentials. 2. Relative permeation to cations was examined. Substitution of external Na+ by sucrose resulted in a -42 mV shift of EACh for a 10-fold reduction in [Na+]o. Using isotonic substitutions, the permeability ratios (relative to Na+) for monovalent cations were determined to be 1.32 +/- 0.02 for Cs+ (n = 11), 1.03 +/- 0.02 for Li+ (n = 8) and 0.18 +/- 0.02 for Tris+ (n = 7). Elevated external Ca2+ salines were found to shift EACh to more positive potentials, especially in the presence of low external Na+. 3. The nicotinic agonists nicotine, tetramethylammonium and lobeline evoked inward currents in bovine chromaffin cells. In contrast, decamethonium and the muscarinic agonist, methacholine, had no effect. 4. The nicotinic antagonists mecamylamine, trimetaphan, (+)-tubocurarine and hexamethonium caused dose-dependent reductions in the amplitude of ACh-evoked inward currents. The estimated IC50's were 0.25, 0.33, 0.63 and 2.2 microM respectively, for cells voltage clamped at -60 mV. High concentrations (> 2 microM) of the muscarinic antagonist, atropine, also produced a dose-dependent reduction in the amplitude of ACh-induced currents. 5. Inhibition by trimetaphan was voltage independent. With the other drugs the antagonism was voltage sensitive, increasing with membrane hyperpolarization. The voltage sensitivity was most marked for hexamethonium. Neither hexamethonium nor mecamylamine were found to depress ACh-evoked outward currents at concentrations which severely depressed inward currents. In addition to its antagonist actions, (+)-tubocurarine activated unitary currents in these cells and on isolated membrane patches. 6. The results indicate that nicotinic ion channels of bovine chromaffin cells have a similar ionic selectivity to monovalent cations, but that Ca2+ ions permeate the channels to a greater degree than at the motor endplate. The ACh current-voltage relationship resembles that described for other types of 'neuronal' nicotinic receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Light-induced calcium release and re-uptake in toad rods

Laser micromass analysis was used to investigate the effect of light on the Ca content of rod outer segments in the isolated retina and eyecup of the toad, Bufo marinus. Isolated retinas were incubated in Ringer for which most of the Ca (normally 97% 40Ca) was replaced with 44Ca, so that release of internal Ca (as 40Ca) could be distinguished from the uptake of 44Ca from the external medium. Continuous illumination produced a decline in outer segment 40Ca content. In bright light, the decrease in 40Ca was sigmoidal, beginning with a delay of at least 4 sec, reaching a maximal rate of 1-2 x 10(8) Ca/rod/sec after 8-16 sec, and declining to zero as the light-releasable pool of 40Ca was exhausted within 1-2 min. Decreases of similar magnitude and time course to those observed in isolated retina were also seen in an eyecup preparation. The rate of light-dependent release was reduced 20- to 60-fold by substitution of external Na with choline or Li. The rate of 44Ca uptake from the external medium into rods was little affected during continuous illumination. Uptake, however, was markedly increased in darkness following light exposure. This Ca re-uptake occurred at a rate approximately 2-fold greater in eyecups than in isolated retinas. We interpret our results to show that light causes an increase in the permeability or transport of Ca across the disk membrane and that the Ca is extruded from the rod via Na/Ca (or Na/Ca-K) exchange across the plasma membrane. Cessation of illumination stimulates the resequestration of Ca back into the disks, and this process is somehow enhanced by the presence of the pigment epithelium.

Unique properties of non-N-methyl-D-aspartate excitatory responses in cultured purkinje neurons.

Cerebellar Purkinje neurons respond to glutamate and to the agonists quisqualate (QA) and kainate (KA) with prolonged, multiphasic, voltage-dependent depolarizations. In contrast, N-methyl-D-aspartate (NMDA) at equivalent doses is not effective as an agonist for Purkinje neurons. The responses to QA and KA are reduced by extracellular Cd2+ (30 microM), by increased Mg2+ or Ca2+ (12 mM), and by the glutamate antagonist kynurenic acid (1 mM) but not by the NMDA-selective antagonist 2-amino-5-phosphonovalerate (100 microM). The short pressure application of 1 microM QA (less than or equal to 0.5 s) produces a response often exceeding 1 min in duration, which consists of several phases: rapid initial depolarization, followed by a long plateau, repolarization, and a subsequent small hyperpolarization. A similar response is evoked by glutamate and KA at higher doses (30-50 microM). The initial and plateau depolarizations are dependent on Na+, being reduced by substitution of external Na+ with sucrose or choline, but are not affected by the Na+ channel blocker tetrodotoxin. Rectification, observed at hyperpolarized potentials below -60 mV set by current clamp, is attributed in part to an intrinsic voltage sensitivity of the agonist-activated response. Both the duration and the magnitude of the excitatory responses were found to be voltage-dependent. Single-channel recordings of a Ca2+-sensitive K+ channel, activated selectively during the excitatory response, suggest that intracellular Ca2+ increases during the plateau phase. Certain properties of the excitatory responses in the Purkinje neuron resemble those associated with NMDA-receptor activation in other regions of the central nervous system, including voltage-sensitive rectification, blockade by divalent cations, and the induction of increased intracellular Ca2+ during the excitatory response. These unique properties may enable the Purkinje neuron to express both rapid and long-term effects of glutamatergic transmission with non-NMDA receptors alone.

PH regulation in the vertebrate central nervous system: microelectrode studies in the brain stem of the lamprey.

Studies of intracellular pH (pHi) in nervous tissue are summarized and recent investigation of intracellular and extracellular pH (pHo) in the isolated brain stem of the lamprey is reviewed. In the lamprey, pHi regulation was studied in single reticulospinal neurons using double-barrel ion-selective microelectrodes (ISMs). In nominally HCO3(-)-free HEPES-buffered media, acute acid loading was followed by a spontaneous recovery of pHi requiring 10-20 min and was associated with a prolonged rise in intracellular Na+. The recovery of pHi was blocked by 1-2 mM amiloride. Amiloride also caused a small rise in pHo. Substitution of external Na+ caused a slow intracellular acidification and extracellular alkalinization. Return of external Na+ reversed these effects. Transition from HEPES to HCO3(-)-buffered media increased the rate of acid extrusion during recovery of pHi. Recovery in HCO3(-)-buffered media was inhibited by 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid and was slowed after exposure to Cl(-)-free media. Following inhibition of acid extrusion by amiloride, transition to HCO3- media restored pHi recovery. These data indicate that lamprey neurons recover from acute acid loads by both Na+-H+ exchange and an independent HCO3(-)-dependent mechanism. Evidence for HCO3(-)-dependent acid extrusion in other vertebrate cells and the protocols of pHi studies using ISMs are discussed.

Regulation of intracellular sodium and pH by the electrogenic H+ pump in frog skin

We have investigated the role of the electrogenic hydrogen ion pump in the regulation of intracellular sodium ion activity (aiNa) and intracellular pH (pHi) in frog skin epithelial cells using double-barreled ion sensitive microelectrodes. When Rana esculenta skin is mounted in an Ussing chamber and bathed in 1 mM Na2SO4 buffered to pH 7.34 with imidazole on the apical side and in normal Ringer on the serosal side, the apical addition of the carbonic anhydrase inhibitor ethoxzolamide (10(-4)M) blocks net H+ ion excretion and Na absorption, producing a depolarization of 25-30 mV of the apical membrane potential (psi mc). We demonstrate that these changes are accompanied by a fall in aiNa from 6.2 +/- 0.5 mmol/l to 3.4 +/- 0.6 mmol/l and an increase in pHi from 7.20 +/- 0.03 to 7.38 +/- 0.08 (n = 12 skins). Voltage clamping psi mc to its control value in the presence of ethoxzolamide restored aiNa but the pHi remained alkaline. Furthermore, the fall in aiNa produced by ethoxzolamide could be mimicked by voltage clamping psi mc towards the value of the Nernst potential for Na at the apical membrane. These results indicate that the maintenance of the cellular Na+ transport pool is dependent on a favourable electrical driving force and counter-current generated by an electrogenic H+ pump at the apical membrane. Addition of amiloride (10(-5) mol/l) or substitution of external Na+ by Mg2+ or K+ caused a hyperpolarization of psi mc and a fall in aiNa.2+ e

Intracellular pH regulation in the sensory neurone of the stretch receptor of the crayfish (Astacus fluviatilis).

The ionic mechanisms of intracellular pH (pHi) regulation were studied in the slowly adapting sensory cell of the crayfish stretch receptor by using pH-, Na+- and Cl(-)-sensitive liquid ion exchanger electrodes. Under control conditions a mean pHi of 7.23 +/- 0.12 (S.D.) at a mean membrane potential of 68.3 +/- 4.1 mV S.D. was found in sixteen cells. Thus pHi is about 1 pH unit more alkaline than predicted from passive distribution, implying the presence of an acid extrusion mechanism. In order to acidify the cytoplasm, the cell was either acid-loaded by NH4Cl or exposed to CO2 and CO2/HCO3- solutions. During CO2 exposures pHi was regulated only if calculated amounts of HCO3- were added to keep external pH (pHo) constant. The pHo per se was found to be an important determinant of pHi and its regulation. Substitution of external Na+ by choline inhibited pHi recovery almost completely. As soon as Na+ was readmitted H+ extrusion occurred immediately at a rate similar to that of the control. The internal Na+ activity (aiNa) ranged between 6 and 13 mM with a mean of approximately 9.1 +/- 2.5 mM (S.D.; n = 8). The effects of various solutions on aiNa and the temporal relationship between aiNa and pHi in NH4Cl acid-loaded cells were investigated. The amount of aiNa increased during cell internal acidification and recovered in parallel with pHi recovery in NH4Cl acid-loaded cells. Experiments with 10(-4) M-ouabain and K+-free conditions suggest that neither the Na+-K+ pump nor external K+ are directly involved in pHi regulation. The internal chloride activity (aiCl), which was lower than predicted from a passive distribution, fell during exposure to HCO3-/CO2. Regulation of pHi was inhibited if the cell was completely depleted of Cl- by prolonged exposures to Cl(-)-free solution (isethionate and/or gluconate substituted). The pHi-regulating system of the sensory cell requires Na+ and Cl- which probably operate in a combined mechanism such as Na+ -H+-Cl(-)-HCO3- or an equivalent.

Effects of substance P on neurones of the inferior mesenteric ganglia of the guinea-pig.

1. The membrane effects of substance P on neurones of isolated inferior mesenteric ganglia and the underlying ionic mechanisms were investigated by means of intracellular recording techniques.2. When applied to the neurones by superfusion, substance P (0.5 mum) caused a membrane depolarization; in a few neurones, the depolarization was preceded by a small hyperpolarization. Substance P effects were not altered in a low Ca(2+)/high Mg(2+) solution or in a solution containing d-tubocurarine and atropine.3. When the membrane potential was clamped manually at the resting level between -50 and -60 mV, substance P caused, in about an equal number of neurones, a slight to moderate decrease and also increase of membrane resistance; a brief increase occurred prior to the decrease of membrane resistance.4. In neurones with high resting membrane potential (> -70 mV), substance P elicited a large depolarization accompanied by a marked increase in membrane resistance; the latter was probably due to anomalous rectification.5. Conditioning hyperpolarization of the membrane close to the level of E(K) increased and decreased substance P-induced depolarization in eleven and two neurones, respectively.6. Substitution of external Na(+) with an equimolar amount of either sucrose or Tris buffer markedly attenuated the depolarizing effect of substance P.7. The substance P-induced depolarization was diminished in a high K(+) (10 mm) solution, and it could be augmented when membrane was hyperpolarized to E(K). On the other hand, the effect of substance P was not appreciably affected in a low Cl(-) solution.8. It is concluded that substance P depolarizes the sympathetic neurones by increasing and decreasing membrane permeability to Na(+) and K(+), respectively, and that the concomitant membrane resistance change depends on interaction of G(Na) activation and G(K) inactivation.9. The possibility that substance P is the transmitter mediating the non-cholinergic slow excitatory potential elicited by repetitive preganglionic stimulation in the neurones of the inferior mesenteric ganglia is suggested.

Catecholamine activation of electrogenous Na + −K + -pump in identified neurones of lymnaea stagnalis

1. Using the voltage-clamp technique catecholamine-induced hyperpolarization has been shown to be accompanied by outward currents. 2. The currents do not reverse even at membrane potential shift to −150 mV. 3. The catacholamine-induced currents are inhibited by strophanthidine, lowering the temperature and substitution of external Na + by Li +. 4. The similarity between potential dependences of catecholamine-induced currents and the strophanthidine-sensitive part of membrane potential has been found. 5. The results strongly suggest catecholamines stimulate Na + −K +-pump in the neurones studied.