Peter Hochstrate

Voltage-dependent Ca2+ influx into identified leech neurones

We determined the relationships between the intracellular free Ca2+ concentration ([Ca2+]i) and the membrane potential (Em) of six different neurones in the leech central nervous system: Retzius, 50 (Leydig), AP, AE, P, and N neurones. The [Ca2+]i was monitored by using iontophoretically injected fura-2. The membrane depolarization evoked by raising the extracellular K+ concentration ([K+]o) up to 89 mM caused a persistent increase in [Ca2+]i, which was abolished in Ca(2+)-free solution indicating that it was due to Ca2+ influx. The threshold membrane potential that must be reached in the different types of neurones to induce a [Ca2+]i increase ranged between -40 and -25 mV. The different threshold potentials as well as differences in the relationships between [Ca2+]i and EM were partly due to the cell-specific generation of action potentials. In Na(+)-free solution, the action potentials were suppressed and the [Ca2+]i/Em relationships were similar. The K(+)-induced [Ca2+]i increase was inhibited by the polyvalent cations Co2+, Ni2+, Mn2+, Cd2+, and La3+, as well as by the cyclic alcohol menthol. Neither the polyvalent cations nor menthol had a significant effect on the K(+)-induced membrane depolarization. Our results suggest that different leech neurones possess voltage-dependent Ca2+ channels with similar properties.

Ionic mechanism of ouabain-induced swelling of leech Retzius neurons.

By using electrophysiological and microfluorimetric methods, we found that leech Retzius neurons swell after inhibition of the Na+–K+ pump by the cardiac glycoside ouabain. To explore the mechanism of this swelling, we measured the effect of ouabain on [Na+]i, [K+]i, and [Cl−]i, as well as on the membrane potential, by applying triple-barrelled ion-sensitive microelectrodes. As shown previously, ouabain induced a marked [Na+]i increase, a [K+]i decrease, and a membrane depolarization, and it also evoked an increase in [Cl−]i. The analysis of the data revealed a net uptake of NaCl, which quantitatively explained the ouabain-induced cell swelling. In the absence of extracellular Na+ or Cl−, NaCl uptake was excluded, and the cell volume remained unaffected. Likewise, NaCl uptake and, hence, cell swelling did not occur when the Na+–K+ pump was inhibited by omitting bath K+. Also, in K+-free solution, [Na+]i increased and [K+]i dropped, but [Cl−]i slightly decreased, and after an initial, small membrane depolarization, the cells hyperpolarized for a prolonged period. It is concluded that the ouabain-induced NaCl uptake is caused by the depolarization of the plasma membrane, which augments the inwardly directed electrochemical Cl− gradient.

The ouabain-induced [Ca2+]i increase in leech Retzius neurones is mediated by voltage-dependent Ca2+ channels

In leech Retzius neurones the inhibition of the Na+/K+ pump by ouabain causes an increase in the cytosolic free calcium concentration ([Ca2+]i). To elucidate the mechanism of this increase we investigated the changes in [Ca2+]i (measured by Fura-2) and in membrane potential that were induced by inhibiting the Na+/K+ pump in bathing solutions of different ionic composition. The results show that Na+/K+ pump inhibition induced a [Ca2+]i increase only if the cells depolarized sufficiently in the presence of extracellular Ca2+. Specifically, the relationship between [Ca2+]i and the membrane potential upon Na+/K+ pump inhibition closely matched the corresponding relationship upon activation of the voltage-dependent Ca2+ channels by raising the extracellular K+ concentration. It is concluded that the [Ca2+]i increase caused by inhibiting the Na+/K+ pump in leech Retzius neurones is exclusively due to Ca2+ influx through voltage-dependent Ca2+ channels.

L-type Ca2+ channel antagonists block voltage-dependent Ca2+ channels in identified leech neurons

We investigated the effect of L-type Ca2+ channel antagonists on the Ca2+ influx through voltage-gated Ca2+ channels in leech Retzius, Leydig, AP, AE, P, and N neurons. The efficacy of the antagonists was quantified by monitoring their effect on the increase in the intracellular free Ca2+ concentration ([Ca2+]i; measured by Fura-2) that was induced by depolarizing the cell membrane by raising the extracellular K+ concentration. This K+-induced [Ca2+]i increase was blocked by the phenylalkylamines verapamil, gallopamil, and devapamil, the benzothiazepine diltiazem, as well as by the 1,4-dihydropyridine nifedipine. The blocking effect of the three phenylalkylamines was similar, being most pronounced in P and N neurons and smaller in Leydig, Retzius, AP, and AE neurons. Contrastingly, diltiazem and nifedipine were similarly effective in the neurons investigated, whereby their efficacy was like that of the phenylalkylamines in Retzius, Leydig, AP, and AE neurons. Depending on cell type and blocking agent, the concentrations necessary to suppress the K+-induced [Ca2+]i increase by 50% were estimated to vary between 5 and 190 microM. At high concentrations, the phenylalkylamines and diltiazem by themselves caused a marked [Ca2+]i increase in Leydig, P, and N neurons, which is probably due to activation of the caffeine-sensitive ion channels present in the plasma membrane of these cells. Together with previous observations, the results indicate a distant relationship of the voltage-gated Ca2+ channels present in many if not all leech neurons to vertebrate L-type Ca2+ channels.

Modulation of Ca2+ influx in Leech Retzius neurons. I. Effect of extracellular pH

Abstract. We investigated the cytosolic free Ca2+ concentration ([Ca2+]i) of leech Retzius neurons in situ while varying the extracellular and intracellular pH as well as the extracellular ionic strength. Changing these parameters had no significant effect on [Ca2+]i when the membrane potential of the cells was close to its resting value. However, when the cells were depolarized by raising the extracellular K+ concentration or by applying the glutamatergic agonist kainate, extracellular pH and ionic strength markedly affected [Ca2+]i, whereas intracellular pH changes appeared to have virtually no effect. An extracellular acidification decreased [Ca2+]i, while alkalinization or reduction of the ionic strength increased it. Correspondingly, [Ca2+]i also increased when the kainate-induced extracellular acidification was reduced by raising the pH-buffering capacity. At low extracellular pH, the membrane potential to which the cells must be depolarized to evoke a detectable [Ca2+]i increase was shifted to more positive values, and it moved to more negative values at high pH. We conclude that in leech Retzius neurons extracellular pH, but not intracellular pH, affects [Ca2+]i by modulating Ca2+ influx through voltage-dependent Ca2+ channels. The results suggest that this modulation is mediated primarily by shifts in the surface potential at the extracellular side of the plasma membrane.

Swelling-activated chloride channels in leech Retzius neurons.

SUMMARY During periods of high activity neurons are expected to swell due to the uptake of Cl–. To find out whether leech Retzius neurons possess swelling-activated Cl– channels that facilitate Cl– efflux and, hence, volume recovery, we exposed the cells to hypotonic solutions. In hypotonic solutions, the cells slowly swelled but did not undergo a regulatory volume decrease. However, the cell volume increased less than predicted for an ideal osmometer, suggesting the action of a compensatory mechanism. The cell swelling was paralleled by a marked decrease in the input resistance as well as by the activation of a membrane current with a reversal potential close to the Cl– equilibrium potential. This current was substantially diminished by removing bath Cl–, by applying the Cl– channel blocker DIDS, or by treating the cells with the tubulin polymerization inhibitor colchicine. Furthermore, in the presence of colchicine or vinblastine, the cell swelling was substantially increased. It is concluded that leech Retzius neurons possess swelling-activated Cl– channels that require an intact microtubule system for activation. The channels may help to restore cell volume after periods of high neuronal activity.

Functional properties and cell type specific distribution of I(h) channels in leech neurons.

SUMMARY The hyperpolarisation-activated cation current (Ih) has been described in many vertebrate and invertebrate species and cell types. In neurons, Ih is involved in rhythmogenesis, membrane potential stabilisation and many other functions. In this work, we investigate the distribution and functional properties of Ih in identified leech neurons of intact segmental ganglia. We found Ih in the mechanosensory touch (T), pressure (P) and noxious (N) neurons, as well as in Retzius neurons. The current displayed its largest amplitude in P neurons and we investigated its biophysical and pharmacological properties in these cells. Ih was half-maximally activated at –65 mV and fully activated at –100 mV. The current mutually depended on both Na+ and K+ with a permeability ratio pNa/pK of ∼0.21. The reversal potential was approximately –35 mV. The time course of activation could be approximated by a single time constant of ∼370 ms at –60 mV, but required two time constants at –80 mV of ∼80 and ∼560 ms. The current was half-maximally blocked by 0.3 mmol l–1 Cs+ but was insensitive to the bradycardic agent ZD7288. The physiological function of this channel could be a subtle alteration of the firing behaviour of mechanosensory neurons as well as a stabilisation of the resting membrane potential.

Sodium-dependent potassium channels in leech P neurons.

In leech P neurons the inhibition of the Na+-K+ pump by ouabain or omission of bath K+ leaves the membrane potential unaffected for a prolonged period or even induces a marked membrane hyperpolarization, although the concentration gradients for K+ and Na+ are attenuated substantially. As shown previously, this stabilization of the membrane potential is caused by an increase in the K+ conductance of the plasma membrane, which compensates for the reduction of the K+ gradient. The data presented here strongly suggest that the increased K+ conductance is due to Na+-activated K+ (KNa) channels. Specifically, an increase in the cytosolic Na+ concentration ([Na+]i) was paralleled by a membrane hyperpolarization, a decrease in the input resistance (Rin) of the cells, and by the occurrence of an outwardly directed membrane current. The relationship between Rin and [Na+]i followed a simple model in which the Rin decrease was attributed to K+ channels that are activated by the binding of three Na+ ions, with half-maximal activation at [Na+]i between 45 and 70 mM. At maximum channel activation, Rin was reduced by more than 90%, suggesting a significant contribution of the KNa channels to the physiological functioning of the cells, although evidence for such a contribution is still lacking. Injection experiments showed that the KNa channels in leech P neurons are also activated by Li+.

NTP, the photoproduct of nifedipine, activates caffeine-sensitive ion channels in leech neurons.

Leech P neurons possess caffeine-sensitive ion channels in intracellular Ca(2+) stores and in the plasma membrane. The following results indicate that these channels are also activated by 2,6-dimethyl-4-(2-nitrosophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester (NTP), the photoproduct of the L-type Ca(2+) channel-blocker nifedipine: (1) Just like caffeine, NTP evoked Ca(2+) influx and intracellular Ca(2+) release, as well as the influx of various other divalent cations and that of Na(+). (2) In the presence of high NTP or caffeine concentrations the plasma membrane channels close, suggesting desensitization of the channel-activating mechanism. (3) Depending on the concentration, NTP and caffeine induce cross-desensitization or act additively. (4) NTP was effective in the same neurons as caffeine (P, N, Leydig, 101), and it was ineffective in neurons in which caffeine was also ineffective (AP, T, L, 8, AE). (5) In Retzius neurons, NTP and caffeine evoked intracellular Ca(2+) release but no Ca(2+) influx. Despite these parallels, the effects of NTP and caffeine were not identical, which may be due to differences in the mechanisms of channel activation or desensitization and/or to substance-specific side effects. The caffeine-sensitive ion channels were activated by NTP concentrations > or =10 microM, which is almost three orders of magnitude smaller than the threshold concentration of caffeine.

Activation and desensitization of the caffeine-sensitive cation channels and calcium stores have no persistent effect on the electrophysiological properties of leech P neurones.

In leech P neurones caffeine activates unselective ion channels in the plasma membrane and induces intracellular Ca2+ release (Schoppe, J., Hochstrate, P., Schlue, W.-R., 1997. Caffeine mediates cation influx and intracellular Ca2+ release in leech P neurones. Cell Calcium 22, 385-397). These effects are prominent only upon the first caffeine exposure, while subsequent applications are largely ineffective; i.e. both plasma membrane channels and intracellular Ca2+ release mechanism desensitize irreversibly. In order to examine whether this desensitization is paralleled by irreversible changes in the electrophysiological parameters of the cells, we investigated the action of caffeine on changes in membrane potential and the cytosolic free Ca2+ concentration, which were induced by varying the ionic composition of the extracellular fluid or by application of 5-hydroxytryptamine. Neither the resting values nor any of the experimentally induced shifts in membrane potential or cytosolic Ca2+ concentration were affected by caffeine, which suggests strongly that activation and/or desensitization of the caffeine-sensitive ion channels and Ca2+ stores have no long-lasting effect on the relevant electrochemical gradients, membrane conductances, or transport mechanisms.