Kir4.1 channels mediate a depolarization of hippocampal astrocytes under hyperammonemic conditions in situ.

Increased ammonium (NH(4) (+) ) concentration in the brain is the prime candidate responsible for hepatic encephalopathy (HE), a serious neurological disorder caused by liver failure and characterized by disturbed glutamatergic neurotransmission and impaired glial function. We investigated the mechanisms of NH(4) (+) -induced depolarization of astrocytes in mouse hippocampal slices using whole-cell patch-clamp and potassium-selective microelectrodes. At postnatal days (P) 18-21, perfusion with 5 mM NH(4) (+) evoked a transient increase in the extracellular potassium concentration ([K(+) ](o) ) by about 1 mM. Astrocytes depolarized by on average 8 mV and then slowly repolarized to a plateau depolarization of 6 mV, which was maintained during NH(4) (+) perfusion. In voltage-clamped astrocytes, NH(4) (+) induced an inward current and a reduction in membrane resistance. Amplitudes of [K(+) ](o) transients and astrocyte depolarization/inward currents increased from P3-4 to P18-21. Perfusion with 100 µM Ba(2+) did not alter [K(+) ](o) transients but strongly reduced both astrocyte depolarization and inward currents. NH(4) (+) -induced depolarization and inward currents were also virtually absent in slices from Kir4.1 -/- mice, while [K(+) ](o) transients were unaltered. Blocking Na(+) /K(+) -ATPase with ouabain caused an immediate and complex increase in [K(+) ](o) . Taken together, our results are in agreement with the hypothesis that reduced uptake of K(+) by the Na(+) , K(+) -ATPase in the presence of NH(4) (+) disturbs the extracellular K(+) homeostasis. Furthermore, astrocytes depolarize in response to the increase in [K(+) ](o) and by influx of NH(4) (+) through Kir4.1 channels. The depolarization reduces the astrocytes' capacity for channel-mediated flux of K(+) and for uptake of glutamate and might hereby contribute to the pathology of HE.

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

The hyperpolarisation-activated cation current (I(h)) has been described in many vertebrate and invertebrate species and cell types. In neurons, I(h) is involved in rhythmogenesis, membrane potential stabilisation and many other functions. In this work, we investigate the distribution and functional properties of I(h) in identified leech neurons of intact segmental ganglia. We found I(h) 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. I(h) 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 p(Na)/p(K) 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.

Swelling-activated chloride channels in leech Retzius neurons.

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.

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.

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.

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.