A. Ya. Tsyndrenko

Diadenosine polyphosphates selectively potentiate N-type Ca2+ channels in rat central neurons

The action of diadenosine polyphosphates on Ca2+ channels was studied in two preparations: isolated hippocampal neurons and synaptosomes, both from the rat brain. High-voltage-activated Ca2+ channels were recorded in freshly isolated CA3 neurons using a whole-cell patch-clamp technique. Current-voltage relationships were measured in the control and after incubation in 5 microM diadenosine pentaphosphate. In the majority of tested pyramidal neurons, the latter procedure led to a reversible increase in the high-voltage-activated current through Ca2+ channels when measured at the holding potential of -100 mV but not at -40 mV. In experiments on synaptosomes from the whole brain, diadenosine pentaphosphate taken at a concentration of 100 microM increased the intrasynaptosomal calcium level measured by means of spectrofluorimetry for 26 +/- 1.8 nM (by 24 +/- 2%). Nifedipine failed to block this effect both in synaptosomes and hippocampal neurons. Potentiation of the current through Ca2+ channels in hippocampal neurons as well as the increase in intrasynaptosomal Ca2+ were irreversibly blocked by 5 microM omega-conotoxin, but not by 200 nM omega-Agatoxin-IVA. These data indicate that diadenosine polyphosphates enhance the activity of N-type Ca2+ channels in many central neurons of the rat brain.

R56865 AND FLUNARIZINE AS Na+-CHANNEL BLOCKERS IN ISOLATED PURKINJE NEURONS OF RAT CEREBELLUM

Dose-related blocking effects of R56865, flunarizine and nimodipine on voltage-activated Na+ currents recorded in the whole-cell voltage clamp mode were studied in acutely isolated Purkinje neurons of rat cerebellum. The dose-dependences of blocking action were obtained for all drugs at a holding potential of -110 mV and rare stimulation. At stimulation frequencies 5 and 15 Hz the block produced by R56865 was increased showing a shift of dose-dependence to lower concentrations of antagonist. This shift was less pronounced for flunarizine, practically absent for nimodipine, and increased for all drugs with an increase in the amplitude of stimulating voltage pulse. With the change in holding potential to -80 mV the block produced by R56865 and flunarizine increased showing a dose-dependence shift to lower concentrations of antagonists. All the drugs tested induced parallel shifts of the steady-state voltage-dependence of inactivation of Na+ channels to more negative membrane potentials. R56865, and to a lesser extent flunarizine, slowed down the recovery of Na+ channels from steady-state inactivation increasing the relative number of channels which showed slow recovery. In the absence of Na+ current inactivation (treatment by intracellular pronase) R56865 at a concentration of 1 microM blocked modified channels preferentially in the open state, while the block produced by flunarizine showed no dependence on voltage pulse protocol. R56865 was shown to decrease the cell leakage while other drugs produced little or no effect. It is concluded that R56865 and flunarizine block Na+ currents predominantly by interacting with inactivated Na+ channels. The higher ability of R56865 to block open channels and to increase slow inactivation underlies its higher frequency-dependence. These characteristics suggest the use of R56865 and flunarizine in the treatment of cerebral ischemia.

Calcium-dependent potassium conductance studied on internally dialysed nerve cells.

Abstract The Ca-dependent tetraethylammonium-resistant potassium conductance was studied in the membrane of internally dialysed isolated snail neurones. In these conditions the tetraethylammoniumresistant noninactivating outward potassium current decreased when the transmembrane influx of Ca 2+ was suppressed either by blocking the calcium channels with external Cd 2+ or internal F − or by substituting Mg 2+ for Ca 2+ in the external solution. Elevation of intracellular Ca 2+ concentration by dialysing the cell with solution containing Ca-EGTA buffer resulted in an increase of potassium outward current. The effect could be measured with Ca 2+ concentrations as low as 10 −7 M. Much higher concentrations of Sr 2+ or Ba 2+ inside the cell did not potentiate the potassium current. The corresponding ionic channels are shown to be less selective for potassium ions as compared to the Ca-insensitive voltage-dependent channels. A conclusion is made that the changes in Ca 2+ concentration near the inner surface of the membrane are the key factor which makes these channels ready for activation.

R56865 as Ca2+-channel blocker in Purkinje neurons of rat: Comparison with flunarizine and nimodipine

The blocking action of recently synthesized benzothiazolamine derivative R56865 was compared with that of dihydropyridine (nimodipine) and diphenylalkylamine (flunarizine) on low-voltage-activated and non-inactivating high-voltage-activated Ca2+ currents. The experiments were carried out on freshly isolated Purkinje neurons of rat cerebellum using patch-clamp technique in the whole-cell configuration. Among the substances tested R56865 was found to be the most effective blocker of the Ca2+ current. In the sequence R56865, flunarizine and nimodipine, apparent Kd values for low-voltage-activated current are 0.1, 0.9 and 3.5 microM, and for high-voltage-activated current 3.1, 9.5 and 38 microM, respectively. The current-voltage relationships for both types of currents displayed little or no shift under either flunarizine or R56865 but showed a 10-mV shift in the positive direction under the action of nimodipine. The steady-state inactivation curves for low-voltage-activated calcium currents were shifted under the action of R56865, flunarizine and nimodipine (in concentrations which blocked 50-60% of the current) to more negative membrane potentials for 20, 10 and 6 mV, respectively. In contrast to R56865, flunarizine blocked both types of Ca2+ channel in a use-dependent manner. It is concluded that the order of potency of Ca2+ antagonist for both types of channels studied is R56865 > flunarizine > nimodipine. Strong shift of steady-state inactivation relationship by R56865 can further facilitate its blocking action in in vivo conditions.

Separation of sodium and calcium channels in the surface membrane of molluscan nerve cells

By intracellular dialysis of isolated neurons of the mollusksHelix pomatia andLimnaea stagnalis and by a voltage clamp technique the characteristics of transmembrane ionic currents were studied during controlled changes in the ionic composition of the extracellular and intracellular medium. By replacing the intracellular potassium ions by Tris ions, functional blocking of the outward potassium currents was achieved and the inward current distinguished in a pure form. Replacement of Ringers solution in the extracellular medium with sodium-free or calcium-free solution enabled the inward current to be separated into two additive components, one carried by sodium ions, the other by calcium ions. Sodium and calcium inward currents were found to have different kinetics and different potential-dependence: τmNa=1±0.5 msec, τmCa=3±1 msec, τhNa=8±2 msec, τhCa=115±10 msec (Vm=0), GNa=0.5 (Vm=−21±2 mV), GCa=0.5 (Vm=−8±2 mV). Both currents remained unchanged by tetrodotoxin, but the calcium current was specifically blocked by cadmium ions (2·10−3 M), verapamil, and D=600, and also by fluorine ions if injected intracellularly. All these results are regarded as evidence that the soma membrane of the neurons tested possesses separate systems of sodium and calcium ion-conducting channels. Quantitative differences are observed in the relative importance of the systems of sodium and calcium channels in different species of mollusks.

A highly potent and selective N-methyl-D-aspartate receptor antagonist from the venom of the Agelenopsis aperta spider

Agatoxin-489, extracted from the venom of the Agelenopsis aperta spider, was studied on acutely isolated perfused hippocampal neurons of rat using the concentration clamp technique. Agatoxin-489 proved to be a selective N-methyl-D-aspartate antagonist; responses to applications of N-methyl-D-aspartate or L-aspartate were blocked by concentrations of agatoxin-489 ranging between 0.1 nM and 1 microM, while responses to kainate were not affected by agatoxin-489 at concentrations up to 10 microM. The actions of agatoxin-489 against responses to N-methyl-D-aspartate or L-aspartate were use- and voltage-dependent, being less pronounced with an increase in the holding potential from -100 to -30 mV. The action of agatoxin-489 could be completely or partially reversed only after washout in the presence of an N-methyl-D-aspartate agonist. The washout was more effective at positive membrane potentials ranging from 0 to +20 mV. These results imply that the spider toxin agatoxin-489, like dizocilpine, is a potent and selective N-methyl-D-aspartate antagonist which preferentially interacts with activated N-methyl-D-aspartate receptors and/or open N-methyl-D-aspartate-activated ionic channels.

Argiopine, argiopinines, and pseudoargiopinines as glutamate receptor blockers in hippocampal neurons

A homologous set of low-molecular weight compounds selectively blocking ionic currents were purified from venom from the spiderArgiope lobata with a selective blocking action on ionic currents activated by applying glutamate and its agonist kainic acid (KA) to the membrane of neurons isolated from the rat hippocampus. Three groups of these compounds — argiopine, argiopinines, and pseudoargiopinines, produced voltage-dependent glutamate- and KA-activated ionic currents at concentrations of 10−6-10−4 M, interacting primarily with agonist-activated ionic channels without affecting Kd values of the agonist. The blocking action could be partially reversed by argiopine application but only slightly when argiopinines and pseudoargiopinines were used. Kinetics of toxin effects on Ka-activated ionic currents showed at least two exponential components with different time constants. Simple and reversed rate constants of interaction between toxins and ionic channels were estimated from the plot of the kinetics of ionic current blockade and recovery against toxin concentration. Argiopine, argiopinines, and pseudoargiopinines lend themselves to further research into glutamate receptors of the mammalian CNS employing electrophysiological and biochemical techniques.

Investigation of the TEA-resistant outward current in the somatic membrane of perfused nerve cells

Outward currents remaining after addition of 20–50 mM of tetraethylammonium (TEA) ions to the extracellular or intracellular solution, were investigated in perfused isolatedHelix neurons. After this addition, the inactivated inward current carried by potassium ions, the potential-dependent and kinetic characteristics of which differ from those of potassium outward currents suppressed by TEA, is preserved in the membrane. A component dependent on the inward calcium current was found in this TEA-resistant outward current; it was abolished by replacement of the extra-cellular calcium ions by magnesium ions, by blocking of the calcium channels by extracellular cadmium ions, and by their destruction by intracellular fluoride ions. Increasing the intracellular concentration of free calcium ions by perfusing the cell with solutions containing calcium-EGTA buffer potentiated the TEA-resistant component of the outward current, whereas removal of these ions with EGTA weakened it. It is concluded that a system of outward current channels whose activation depends on the presence of calcium ions near the inner surface of the membrane is present in the somatic membrane. It is suggested that to keep these channels capable of being activated, calcium ions must bind with the structures forming their internal opening.

Action of calcium ions on channels of inward and outward currents in the molluscan neuron membrane

Changes in the characteristics of activity of sodium, calcium, and potassium channels in the surface membrane during variation of the calcium ion concentration in the extracellular and intracellular medium were investigated by the voltage clamp method during intracellular dialysis of isolated neurons of the mollusksLimnea stagnalis andHelix pomatia. Besides their direct role in passage of the current through the membrane, calcium ions were shown to have two actions, differing in their mechanism, on the functional properties of this membrane. The first was caused by the electrostatic action of calcium ions on the outer surface of the membrane and was manifested as a shift of the potential-dependent characteristics of the ion transport channels along the potential axis; the second is determined by closer interaction of calcium ions with the specific structures of the channels. During the action of calcium-chelating agents EGTA and EDTA on the inner side of the membrane the conductivity of the potassium channels is substantially reduced. With an increase in the intracellular free calcium concentration the conductivity is partially restored. The action of EGTA and EDTA on the outer side of the membrane causes a substantial decrease in the ion selectivity of the calcium channels and changes the kinetics of the portal mechanism. These changes are easily abolished by rinsing off the chelating agents or by returning calcium ions to the external medium. A specific blocking action of an increase in the intracellular free calcium concentration on conductivity of the calcium channels was found.

Action of intracellular calcium on the inward calcium current

Calcium is one of the mos t importar~ e lements f r o m the physiological point of view. Its p resence in the ex t race l lu la r medium is an almost essent ia l condition for normal cel l function. The in t race l lu la r f r ee calc ium concentra t ion, however, is ex t r eme ly low [6, 10]. This fact ma y be of cons iderable physiological impor tance . F o r mos t enzymes functioning inside the cell, ca lc ium ions a re antagonistic inhibitors, whereas magnesium ions a re natural ac t iva tors (this is t rue for m o r e than 9 out of 10 of the enzymes l i s ted by Dixon and Webb [1]). The ra t io between the concentra t ions of f r ee ca lc ium and magnes ium in the in t raee l lu la r medium may the re fo re be a fac to r de termining the metabol ic act ivi ty of the cel l [5], i .e . , the ve loc i ty of enzyme react ions may be modif ied by t rans ien t changes in the f r ee in t race l lu la r ca lc ium concentra t ion.