Wolf-R. Schlue

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.

CAFFEINE MEDIATES CATION INFLUX AND INTRACELLULAR CA2+ RELEASE IN LEECH P NEURONES

We investigated the effect of caffeine on the intracellular free Ca2+ concentration ([Ca2+]i) of leech P neurones by using the fluorescent indicator Fura-2. Caffeine induced a [Ca2+]i increase that was strongly reduced, but not abolished, in Ca(2+)-free solution. The effect of caffeine on [Ca2+]i was dose-dependent: while 5 mM caffeine evoked a persistent [Ca2+]i increase that could be elicited repetitively, 10 mM caffeine or more induced a transient [Ca2+]i increase that was strongly reduced upon subsequent applications at the same concentration. Surprisingly, the cells remained fully responsive to a moderately increased caffeine concentration. The caffeine-induced [Ca2+]i increase was not blocked by millimolar concentrations of La3+, Mg2+, Cd2+, Zn2+, Co2+, Ni2+, or Mn2+. While La3+ and Mg2+ had no effect on the caffeine response, the other cations caused irreversible changes in the Fura-2 fluorescence. The inhibitors of intracellular Ca2+ pumps-thapsigargin, cyclopiazonic acid (CPA), and 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ)--had no effect on the caffeine-induced [Ca2+]i increase at normal extracellular Ca2+ concentration, but they reduced it in Ca(2+)-free solution. Ryanodine had no effect on the caffeine-induced [Ca2+]i increase at normal extracellular Ca2+ concentration, and also in Ca(2+)-free solution it seemed to be largely ineffective. Caffeine evoked complete fluctuations of the membrane potential. The effect in Ca2+ free and in Na(+)-free solution suggests that the depolarizing response components were mainly due to Na+ influx, while Ca2+ reduced the Na+ influx and/or activated mechanisms which re- or hyperpolarize the cells. It is concluded that leech P neurones possess caffeine-sensitive intracellular Ca2+ stores, as well as caffeine-sensitive ion channels, in the plasma membrane that are activated by a voltage-independent mechanism. The plasma membrane channels are permeable to various divalent cations including Ca2+, and possibly also to Na+.

Effects of glutamatergic agonists and antagonists on membrane potential and intracellular Na+ activity of leech glial and nerve cells.

The membrane potential of neuropile glial cells and Retzius neurones in the central nervous system of the leech Hirudo medicinalis was measured using electrolyte-filled single-barreled microelectrodes. Intracellular Na+ activity (aNai) was recorded with Na(+)-sensitive double-barreled microelectrodes. Bath-application of kainate, quisqualate and L-glutamate elicited concentration-dependent membrane depolarizations in both cell types as demonstrated by dose-response curves. The competitive quinoxalinedione antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to the non-NMDA glutamate receptor inhibited the membrane depolarizations in neuropile glial cells completely, but in Retzius neurones only partially. These results confirm that leech neuropile glial cells have a kainate- and quisqualate-preferring non-NMDA glutamate receptor similar to that in the Retzius neurones. The initial decrease in aNai in neuropile glial cells in kainate- or quisqualate-containing solutions and the afterhyperpolarization in these glial cells and the Retzius neurones following the removal of both glutamate antagonists, were blocked in the presence of the cardiac glycoside ouabain (10(-4) M). In saline solutions containing 42.5 mM Li+ instead of Na+ the afterhyperpolarizations were blocked in neuropile glial cells and Retzius neurones. We conclude that the initial aNai changes and the afterhyperpolarization could be due to the stimulation of the electrogenic Na+/K+ pump in the glial and neuronal membranes.

ATP-inhibited and Ca2+-dependent K+ channels in the soma membrane of cultured leech Retzius neurons

The properties of one ATP-inhibited and one Ca2+-dependent K+ channel were investigated by the patch-clamp technique in the soma membrane of leech Retzius neurons in primary culture. Both channels rectify at negative potentials. The ATP-inhibited K+ channel with a mean conductance of 112 pS is reversibly blocked by ATP (Ki = 100 μm), TEA (Ki=0.8 mm) and 10 mm Ba2+ and irreversibly blocked by 10 nm glibenclamide and 10 μm tolbutamide. It is Ca2+ and voltage independent. Its open state probability (Po) decreases significantly when the pH at the cytoplasmic face of inside-out patches is altered from physiological to acid pH values. The Ca2+-dependent K+ channel with a mean conductance of 114 pS shows a bell-shaped Ca2+ dependence of Po with a maximum at pCa 7–8 at the cytoplasmic face of the membrane. The Po is voltage independent at the physiologically relevant V range. Ba2+ (10 mm) reduces the single channel amplitude by around 25% (ATP, TEA, glibenclamide, tolbutamide, and Ba2+ were applied to the cytoplasmic face of the membrane).We conclude that the ATP-dependent K+ channel may play a role in maintaining the membrane potential constant—independently from the energy state of the cell. The Ca2+-dependent K+ channel may play a role in generating the resting membrane potential of leech Retzius neurons as it shows maximum activity at the physiological intracellular Ca2+ concentration.

Chloride-dependent pH regulation in connective glial cells of the leech nervous system

We used double-barreled pH-sensitive microelectrodes to study the mechanisms by which the intracellular pH is regulated in the connective glial cells of the medicinal leech. The experiments indicate that a Cl(-)- and HCO3(-)-dependent mechanism mediates some recovery from intracellular alkalosis even in the absence of external Na+. This suggests the presence of Na(+)-independent Cl-/HCO3- exchange in the connective glial membrane. At alkaline pHi, this exchange most likely operates in the direction of net acid loading (i.e. HCO3- efflux).

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

The intracellular pH, pHi, 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 pHi of these neurons is due to amiloride-sensitive Na-H exchange and hence requires extracellular Na, Nao. We have measured a decrease of pHi following the removal of Nao. 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-dimethylammonium (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 pHi from acidification upon readdition of Nao was fast, only when Li had replaced Na was the pHi recovery considerably delayed. In conclusion, in all experiments using different Na substitutes the removal of Nao caused a substantial intracellular acidification presumably due to inhibition of Na-H exchange. These changes in pHi might be relevant for results obtained by experiments in which Na-free solutions are used.

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.