Injection Of Ca2 Research Articles

Spatiotemporal distribution of Ca2+ following axotomy and throughout the recovery process of cultured Aplysia neurons.

This study investigates the alterations in the spatiotemporal distribution pattern of the free intracellular Ca2+ concentration ([Ca2+]i) during axotomy and throughout the recovery process of cultured Aplysia neurons, and correlates these alterations with changes in the neurons input resistance and trans-membrane potential. For the experiments, the axons were transected while imaging the changes in [Ca2+]i with fura-2, and monitoring the neurons' resting potential and input resistance (Ri) with an intracellular microelectrode inserted into the cell body. The alterations in the spatiotemporal distribution pattern of [Ca2+]i were essentially the same in the proximal and the distal segments, and occurred in two distinct steps: concomitantly with the rupturing of the axolemma, as evidenced by membrane depolarization and a decrease in the input resistance, [Ca2+]i increased from resting levels of 0.05-0.1 microM to 1-1.5 microM along the entire axon. This is followed by a slower process in which a [Ca2+]i front propagates at a rate of 11-16 microns/s from the point of transection towards the intact ends, elevating [Ca2+]i to 3-18 microM. Following the resealing of the cut end 0.5-2 min post-axotomy, [Ca2+]i recovers in a typical pattern of a retreating front, travelling from the intact ends towards the cut regions. The [Ca2+]i recovers to the control level 7-10 min post-axotomy. In Ca(2+)-free artificial sea water (2.5 mM EGTA) axotomy does not lead to increased [Ca2+]i and a membrane seal is not formed over the cut end. Upon reperfusion with normal artificial sea water, [Ca2+]i is elevated at the tip of the cut axon and a membrane seal is formed. This experiment, together with the observations that injections of Ca2+, Mg2+ and Na+ into intact axons do not induce the release of Ca2+ from intracellular stores, indicates that Ca2+ influx through voltage gated Ca2+ channels and through the cut end are the primary sources of [Ca2+]i following axotomy. However, examination of the spatiotemporal distribution pattern of [Ca2+]i following axotomy and during the recovery process indicates that diffusion is not the dominating process in shaping the [Ca2+]i gradients. Other Ca2+ regulatory mechanisms seem to be very effective in limiting these gradients, thus enabling the neuron to survive the injury.

The behavioral syndrome induced by adreno corticotropic hormone in rats is prevented by Ca++ channel blockade.

The most typical signs (stretchings, yawnings, penile erections, excessive grooming) of the behavioral syndrome induced in rats by the intracerebroventricular administration of ACTH- (1-24) (4 micrograms/rat) were dose-dependently antagonized by the intraperitoneal injection of the selective Ca++ channel inhibitor, nicardipine (dose range: 0.1-1 mg/kg). These data suggest that the influx of Ca++ into target neurons is a step of key importance for the occurrence of ACTH-induced behavioral signs, and that Ca++ may play the role of second intracellular messenger for the behavioral effects of melanocortins.

Effects of Ca2+ and vitamin E on posttranslational regulation of acetylcholine receptor expression in the somata of identified molluscan neurons

The effects of Ca2+ and vitamin E (α-tocopherol) on acetylcholine (Ach)-induced Cl− currents in LP11 and RBc4 neurons of the snail Helix pomatia have been studied. Injection of Ca2+ into the cells and application of vitamin E (10−5 mole/liter) induced the appearance of potentiation of Ach-induced currents in membrane parts more remote from the axon than the Ach-sensitive regions in the control. The Hill coefficient (n) for such Ach receptors was equal to 0.8, unlike 1.8 for Ach receptors active in the control. Arachidonic acid (10−5 mole/liter) and phorbol ester TPA (10−6 mole/liter) inhibited Ach responses, while oleoylacetyglycerol (10−6 mole/liter) produced no effect. Calmidazolium (10−6 mole/liter) decreased the effects of Ca2+ and vitamin E on Ach responses, while nordihydroquiaretic acid (5 · 10−6 mole/liter) enhanced the modulating effect of vitamin E and weakened that of arachidonic acid. It is suggested that the expression of Ach receptors activated by Ca2+ and vitamin E is mediated through posttranslational mechanisms, since cycloheximide and actinomycin D, inhibitors of protein synthesis, did not influence the effects of C2+ and vitamin E. The mechanisms responsible for the stimulating effects of Ca2+ and vitamin E are discussed.

InsP3 and Ins(1,3,4,5)P4 act in synergy to stimulate influx of extracellular Ca2+ in Xenopus oocytes.

To investigate the role of D-myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] in the regulation of Ca2+ influx, we injected inositol phosphates into Xenopus oocytes and measured Ca(2+)-gated Cl- current to assay intracellular free Ca2+ concentration ([Ca2+]i). To assess Ca2+ influx, we removed extracellular Ca2+ or added the inorganic Ca2+ channel blocker Mn2+ to the extracellular bath and measured the resulting change in Cl- current. Ins(1,3,4,5)P4 did not cause Ca2+ influx when injected alone or when preceded by an injection of Ca2+. In contrast, Ins(1,3,4,5)P4 stimulated Ca2+ influx when injected after the poorly metabolized inositol trisphosphate (InsP3) analogues D-myo-inositol 1,4,5-trisphosphorothioate [Ins(1,4,5)P3S3] or D-myo-inositol 2,4,5-trisphosphate [Ins(2,4,5)P3]. These results indicate that Ins(1,3,4,5)P4 is not sufficient to stimulate Ca2+ influx but acts in synergy with InsP3s to cause Ca2+ influx. We also studied the effect of Ca2+ influx on the immediate metabolism of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in single oocytes. Ca2+ influx shunted the metabolism of Ins(1,4,5)P3 toward the formation of Ins(1,3,4,5)P4 and away from D-myo-inositol 1,4-bisphosphate [Ins(1,4)P2]. These results suggest that there is a positive feedback regulatory mechanism in which Ca2+ influx stimulates Ins(1,3,4,5)P4 production and Ins(1,3,4,5)P4 stimulates further Ca2+ influx.

Regulation of a major cloned voltage-gated K + channel from human T lymphocytes

When expressed into Xenopus oocytes, HLK 3 K + channel (Kvl-3) induced a slowly inactivating voltage-dependent K + current. We have studied the modulation of this K + current by co-expressing a cloned 5-HT 2 receptor together with HLK 3K + channel protein. Application of 5-HT caused a long-lasting inhibition of the voltage-gated K + current. This inhibitory modulation was mimicked by intracellular injection of inositol triphosphate or Ca 2+, as well as by incubation with phorbol esters or diacylglycerol analogs. Oocytes pretreatment with staurosporine and EGTA fully prevented 5-HT inhibitory action. Elevation of cAMP and cGMP levels into oocytes did not produce any detectable effect on the current recorded in the absence or the presence of 5-HT. These data suggest that the second messengers generated by phospholipase C activation may be important modulators of HLK 3 K + channels in the immune and the central nervous systems.

Calcium is necessary for light excitation in barnacle photoreceptors.

Illumination of barnacle (Balanus amphitrite) photoreceptors is known to increase the membrane permeability to sodium and Ca2+ ions resulting in a depolarizing receptor potential. In this report, we show that lanthanum (La3+), a known inhibitor of Ca-binding proteins, reversibly eliminates the receptor potential of barnacle photoreceptors when applied to the extracellular space. Similar reversible elimination of the light response was obtained by removing extracellular Ca2+ by application of the calcium chelating agent EGTA. Iontophoretic injection of Ca2+, but not K+ into the cells protected both the transient and the steady-state phases of the receptor potential from elimination by EGTA while only the transient phase was protected in the presence of La3+. The EGTA experiments suggest that internal Ca2+ is necessary for light excitation of barnacle photoreceptors while the La3+ experiments suggest that La(3+)-sensitive inward current is necessary to maintain excitation during prolonged light.

Heat production in chemically skinned smooth muscle of guinea‐pig taenia coli.

1. The rate of heat production of chemically skinned guinea-pig taenia coli smooth muscle at 25 degrees C was measured using microcalorimetric techniques. 2. Muscle strips were mounted isometrically and incubated in solutions containing MgATP (3.2 mM) and phosphocreatine (PCr, 12 mM), pH 6.9. Activation was obtained by the injection of Ca2+ into the sample compartment of the calorimeter. 3. The heat production rate of the resting preparation (pCa 9) was 0.40 +/- 0.03 mW g-1 wet weight (n = 23). During maximal activation (pCa 4.8) the heat rate increased to 1.12 +/- 0.07 mW g-1 (mean +/- S.E.M., n = 15). With stepwise increase in [Ca2+] from pCa 9 to 4.8 the energetic cost of force maintenance tended to increase at higher [Ca2+]. 4. After activation by Ca2+, the heat production rate reached its maximum while force was still increasing. 5. Changing ionic strength from 90 to 150 mM had no effect on either basal or activated heat rate. Oligomycin, amphotericin B and the adenylate kinase inhibitor Ap5A had no effect on the basal heat rate. 6. Exchanging ATP in the incubation medium for inosine triphosphate (ITP) reduced the force and heat production after injection of Ca2+. The basal heat production was not lowered when ATP was exchanged for ITP. 7. The observed enthalpy change for PCr splitting at 25 degrees C (pH 6.9, ionic strength 90 mM) was -28 +/- 3 kJ mol-1 (mean +/- S.E.M., n = 9). After correction for the phosphate equilibrium, buffer reactions, and Mg2+ binding to PCr and HPO42-, the net enthalpy change is calculated to be -39 +/- 3 kJ mol-1. 8. Heat production in the skinned smooth muscle consists of one basal component present in relaxed muscle, and one component associated with contraction. The nature of the basal heat production is unclear but does not seem to involve turnover of phosphate on the myosin light chains. The increase in the energetic tension cost with increasing activation by Ca2+ has implications for the understanding of the contractile mechanism in smooth muscle.

Pentylenetetrazole suppresses the potassium current in Euhadra neurons which is coupled with Ca 2+/calmodulin-dependent protein phosphorylation

The Ca 2+/calmodulin-dependent protein phosphorylation-related mechanism underlying pentylenetetrazole (PTZ)-induced reduction of delayed potassium current ( I KD) was examined in identified Euhadra neurons. PTZ gradually reduced peak I KD in a dose-dependent manner, as well as an inhibition of Ca 2+/calmodulin-dependent protein phosphorylation. Similar effects were observed by a general protein kinase inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpoperazine, whose saturating dose occluded the action of PTZ on the I KD. Intracellular injection of Ca 2+/calmodulin-dependent protein kinase II transiently restored the PTZ-suppressed I KD nearly to the pre-PTZ level, whereas either CaCl 2 or calmodulin, injected in the same way, had little effect. However, this restoration was not detectable in the presence of N-(6-aminohexyl)-5-chloronaphthalenesulfonamide, a calmodulin inhibitor, in the perfusate. These results suggest that PTZ suppresses the potassium current coupled with Ca 2+/calmodulin-dependent protein phosphorylation.

Muscarinic suppression of the M-current is mediated by a rise in internal Ca 2+ concentration

The role of intracellular Ca 2+ in the muscarinic suppression of M-current was examined. Intracellular injection of Ca 2+ buffer into cells in the intact ganglion reduced the response to muscarinic agonist. In similar experiments on isolated cells, Ca 2+ buffer was introduced into the cytoplasm using a perfused recording pipette. Ca 2+ buffer (20 mM) with the free Ca 2+ concentration set to normal resting levels produced a reversible reduction of the muscarinic response. In a second line of investigation, it was found that pharmacological procedures designed to deplete internal stores of Ca 2+ produced a decrease in the muscarinic response. These results, taken together with previous work, support the hypothesis that the muscarinic suppression of M-current is mediated by the release of Ca 2+ from intracellular stores.

Effects of intra-axonal injection of Ca 2+ buffers on evoked release and on facilitation in the crayfish neuromuscular junction

Ca 2+ buffers were injected into the excitatory axon of the crayfish opener muscle. The magnitude and time course of evoked release and of facilitation were measured. EGTA (on-rate about 10 6 M −1s −1 had no effect on evoked release but reduced facilitation. BAPTA and nitr-5, buffers with similar K d's but faster on-rates, reduced both evoked release and facilitation. However, these buffers had no effect on the time course of evoked release. These results show that fast Ca 2+ buffers reduce the Ca 2+ transient associated with evoked release and also the level of residual Ca 2+ involved in facilitation. However, Ca 2+ buffering is not the mechanism which controls the time course of release.

Muscarinic enhancement of the voltage‐dependent calcium current in an identified snail neuron.

1. In the F1 neuron of the snail Helix aspersa bathed in a Ba2+ and 4-aminopyridine-containing saline, carbamylcholine (CCh) enhanced the inward current carried by Ba2+ through the voltage-dependent Ca2+ channels. 2. This effect of CCh on the F1 neuron was not affected by the nicotinic antagonists (+)-tubocurarine and hexamethonium, but it was mimicked by oxotremorine and blocked by both atropine and pirenzepine. 3. The intracellular injection of GTP gamma S (guanosine 5'-O-(3- thiotriphosphate] into the F1 neuron caused both a decrease in Ca2+ current and a blockade of the CCh-induced enhancement of the Ca2+ current. 4. Neither cyclic AMP, cyclic GMP nor arachidonic acid mimicked the effect of CCh on the Ca2+ current in the F1 neuron. In contrast, the intracellular injection of EGTA blocked the CCh-induced enhancement of the Ca2+ current thus suggesting that cytosolic Ca2+ is involved in the CCh-induced response. 5. We then investigated the possible role of inositol 1,4,5-trisphosphate (InsP3) and Ca(2+)-dependent protein kinases in the CCh-induced enhancement of the Ca2+ current. The intracellular injection of InsP3 in the F1 neuron elicited no consistent change in the Ca2+ current. Diacylglycerol analogues (OAG and DOG) decreased the Ca2+ current amplitude, i.e. an effect opposite to that produced by CCh. This effect of the diacylglycerol analogues resulted from the activation of protein kinase C (PKC) since it was blocked by staurosporine. In addition, staurosporine did not affect the CCh-induced increase in Ca2+ current. 6. The intracellular injection of either Ca(2+)-calmodulin-dependent protein kinase II (Ca(2+)-CaM-PK) or a peptide inhibitor of this enzyme into the F1 neuron affected neither the Ca2+ current nor its enhancement by CCh. 7. We conclude that the CCh-induced enhancement of the Ca2+ current in the snail F1 neuron involves the activation via muscarinic receptors of an intracellular transduction mechanism in which cytosolic Ca2+ plays a key role. However, InsP3, protein kinase C and Ca(2+)-CaM-PK do not appear to be directly involved in this CCh-induced response.

Caffeine inhibits inositol trisphosphate‐mediated liberation of intracellular calcium in Xenopus oocytes.

1. Voltage-clamp recording of Ca(2+)-activated chloride currents in Xenopus oocytes was used to study the effects of caffeine on the liberation of intracellular Ca2+ induced by photo-release of inositol 1,4,5-trisphosphate (InsP3) from caged InsP3. Bath application of caffeine, at concentrations between 0.1 and 10 mM, reduced or abolished the current evoked by photo-release of InsP3 and by microinjection of InsP3. 2. Caffeine did not appreciably reduce currents evoked by injection of Ca2+ into oocytes, whereas measurements using the Ca2+ indicator Rhod-2 showed that it instead inhibited the liberation of Ca2+ by InsP3. 3. Caffeine increased the threshold amount of InsP3 required to evoke a current response and proportionally reduced the currents evoked by suprathreshold levels of InsP3. 4. Theophylline and 3-isobutyl-1-methylxanthine (IBMX) were much less potent than caffeine, and few changes were seen in the InsP3 responses following application of forskolin or intracellular injection of cyclic AMP. Thus, inhibition of InsP3 responses by caffeine does not arise through inhibition of phosphodiesterase enzymes. 5. Even at high (10 mM) concentrations, caffeine did not itself elicit any clear Ca(2+)-activated current. It is therefore unlikely that inhibition of the InsP3 responses arise because caffeine itself liberates Ca2+ from intracellular stores. 6. The site of action of caffeine is intracellular, because injections of caffeine into the oocyte strongly inhibited responses to InsP3, whereas local extracellular applications of similar amounts were almost without effect.

Effects of inositol 1,4,5-trisphosphate injections into cat spinal motoneurons

Inositol 1,4,5-trisphosphate (IP3) was injected iontophoretically into cat spinal motoneurons in pentobarbital-anaesthetized cats and nonanaesthetized, decerebrate cats. Injections of IP3 induced a long-lasting, reproducible hyperpolarization without consistent change in input resistance. The peak amplitude of post-spike afterhyperpolarization (AHP) was significantly increased by IP3 when the membrane potential was adjusted to the control level. Intracellular injections of Ca2+ chelators, which depressed the Ca2(+)-activated AHP, prevented the IP3-induced long-lasting hyperpolarization, suggesting that IP3 acts by a Ca2(+)-dependent mechanism. Intracellular injections of myo-inositol did not consistently induce hyperpolarizations. Also intracellular injections of Li+, which blocks IP3 catabolism, did not prevent the IP3-evoked hyperpolarization. These data suggest that IP3 itself, rather than its breakdown product myo-inositol, is mainly responsible for the hyperpolarizing effect. Possible mechanisms for the IP3-induced hyperpolarization are discussed.

Concentrations of essential elements after repeated administrations of tin and selenium

To study effects of simultaneous administration of tin (Sn) and selenium (Se) on concentrations of several essential elements, mice were injected with either SnCl2 (ip) or Na2SeO3 (sc), alone or both compounds at a daily dose of 5 mumol/kg each for 12 consecutive days. Mice were sacrificed at 20 h after the last injection and concentrations of Sn, Se, Na, Ca, Zn, P, Fe, K, and Mg in the liver, kidney, spleen, pancreas, testis, seminal vesicle, lung, femoral muscle, and femoral bone were determined. In the control mice, Sn and Se concentrations were the highest in bone (0.69 micrograms Sn and 6.93 micrograms Se/g dry wt). Administered Sn was found to accumulate in all organs except the testis. Among the essential elements determined, Na was the most affected in terms of concentration in the organs and Mg was the least affected element in these organs. Among the organs tested, each elemental concentration in the pancreas was most affected. Simultaneous injections of Sn and Se appeared to keep the correlation coefficients between elements similar to those found in the control mice.

Short-and long-term desensitization of serotonergic response in Xenopus oocytes injected with brain RNA: roles for inositol 1,4,5-trisphosphate and protein kinase C

In Xenopus oocytes injected with rat brain RNA, serotonin (5HT) and acetylcholine (ACh) evoke membrane responses through a common biochemical cascade that includes activation of phospholipase C, production of inositol 1,4,5-trisphosphate (Ins1,4,5-P3), release of Ca2+ from intracellular stores, and opening of Ca-dependent Cl- channels. The response is a Cl- current composed of a transient component (5HT1 or ACh1) and a slow, long-lasting component (5HT2 or ACh2). Here we show that only the fast, but not the slow, component of the response is subject to desensitization that follows a previous application of the transmitter. The recovery of 5HT1 from desensitization is biphasic, suggesting the existence of two types of desensitization: short-term desensitization (STD), which lasts for less than 0.5 h; and long-term desensitization (LTD) lasting for up to 4 h. The desensitization between 5HT and ACh is heterologous and long-lasting. We searched for (a) the molecular target and (b) the cause of desensitization. (a) Pre-exposure to 5HT does not reduce the response evoked by intracellular injection of Ca2+ and by Ca2+ influx. Cl- current evoked by intracellular injection of Ins1,4,5-P3 was reduced shortly after application of 5HT, but fully recovered 30 min later. Thus, the Cl- channel is not a target for desensitization. Neither Ins1,4,5-P3 receptor nor the Ca2+ store is a target of LTD but they may be the targets of STD. (b) Ca2+ injection did not inhibit the 5HT response, suggesting that Ca2+ is not a sole cause of STD or LTD.(ABSTRACT TRUNCATED AT 250 WORDS)

Age constraints on the geological evolution of the Narryer Gneiss Complex, Western Australia

Zircon U‐Th‐Pb and mineral K‐Ar and 40Ar/39Ar isotopic studies indicate that the maximum deposition age of the Mt Narryer quartzite (which contains detrital zircons up to 4200 Ma old) is 3280 Ma, or by association with other sequences possibly 3100 Ma. This postdates a major episode of high‐grade metamorphism, granite emplacement and deformation at 3300 Ma, which affected adjacent gneiss terranes and which previously had been considered to have affected metasediments and basement gneisses alike. Prograde metamorphism of the Narryer metasediments to amphibolite facies evidently took place during a younger event culminating ca 2700 Ma, prior to injection of granite sheets ca 2650 Ma in age, by which time the present tectonic framework had been assembled.

Effects of cyclic nucleotides and intracellular Ca on voltage-activated ciliary beating in Paramecium

1. Coupling mechanisms between ciliary beating and the membrane potential in Paramecium were investigated under voltage clamp applying intracellular pressure injection of cAMP, cGMP and Ca-EGTA buffer. Ciliary responses following step changes in membrane potential were recorded by high-speed video on magnetic tape. 2. Injections of cAMP and cGMP up to millimolar concentrations caused no detectable changes in the frequency voltage relationship. A minor effect was that the ciliary reorientation towards the anterior cell end (reversal) tended to be inhibited with depolarization up to 10 mV. 3. Injection of Ca2+ into the cell clamped at the resting potential caused a transient anteriad ciliary reorientation and a simultaneous increase in the beating frequency. 4. Injection of EGTA (to buffer Ca2+ below 10−8M) was ineffective in relation to frequency for several minutes. After this time, hyperpolarization- and depolarization activated frequency responses of EGTA-injected cells were increasingly inhibited. The ciliary reorientation following depolarization was not affected by EGTA. 5. A posterior contraction of the cell diameter was noticed upon membrane hyperpolarization. The contraction coincided in time with the increase in beating frequency. 6. The results support the view that the voltage-dependent augmentation of the ciliary beating rate is not directly mediated by an intracellular increase in either cAMP or cGMP. 7. The role of Ca2+ as intracellular messenger in the ciliary and somatic compartments is discussed.

Activation of a K + conductance by bradykinin and by inositol-1,4,5-trisphosphate in rat glioma cells: involvement of intracellular and extracellular Ca 2+

Extracellular application of bradykinin and injection of inositol-1,4,5-trisphosphate (Ins-P 3) induced a hyperpolarization in polyploid rat glioma cells. Ins-1,4,5-P 3 and Ins-2,4,5-P 3 were effective but not Ins-4,5-P 2, Ins-1,3,4,5-P 4 and Ins-1,3,4,5,6-P 5. The reversal potential of the hyperpolarizing response induced by bradykinin or by Ins-P 3 increased to a comparable degree with increasing the extracellular K + concentration. Certain blockers of K + channels, for example charybdotoxin (5–50 nM), Ba 2+ (5–20 mM), 4-aminopyridine (5–10 mM) and quinidine (0.1–0.5 mM) reversibly suppressed the membrane potential response to bradykinin or to Ins-P 3; however, apamin (1 μM) and d-tubocurarine (0.5 mM) had no effect. Intracellular injection of EGTA made the glioma cells unresponsive to bradykinin. Superfision of the cells with Ca 2+-free medium gradually and reversibly abolished the response to bradykinin, but only slightly reduced the effect of Ins-P 3. The Ca 2+ channel blockers Co 2+ (1–5 mM), Mn 2+ (2–6 mM) and nifedipine (1–20 μM), but not desmethoxyverapamil (100 μM) inhibited the hyperpolarizing effect of bradykinin. The hyperpolarization induced by Ins-P 3, however, was not influenced by Mn 2+ (1–5 mM) or by Co 2+ (7 mM). Injection of Ca 2+ into the glioma cells induced a hyperpolarization susceptible to Ba 2+ and quinidine. Treatment of glioma cells with an activator or with inhibitors of protein kinase C or with pertussis toxin did not affect the response to bradykinin. Incubation of the cells with the Ca 2+ ionophore A23187 (0.1–1 μM) made the cells unresponsive to bradykinin and, somewhat less, to Ins-P 3. At these concentrations the Ca 2+ ionophore primarily depletes intracellular Ca 2+ stores. In summary, bradykinin, via B 2-receptors (blocked by [Thi 5,8, d-Phe 7]-bradykinin) activates a K + conductance in glioma cells following a rise of cytosolic Ca 2+ activity most likely due to Ins-P 3-mediated release of Ca 2+ from internal stores. Entry of extracellular Ca 2+ appears also to be involved in this process.

Paroxysmal afterpotentials and role of calcium-dependent potassium conductivity in neuronal activity of strychninized neocortex

Reactions of cortical suprasylvian gyrus neurons were investigated intracellularly after supracortical strychnine application in immobilized and anaesthetized cats. It was shown that paroxysmal depolarizing shifts of membrane potential could be accompanied by de- and hyperpolarizing afterpotentials. When passing from epileptiform to normal physiological activity, short afterhyperpolarizations, 300–500 ms in duration, were converted into inhibitory postsynaptic potentials which were also accompanied by a decrease in membrane potential. When the frequency of paroxysmal discharge was less than 1 s, prolonged (1–2 s) afterhyperpolarizations were observed; at a higher frequency their summation led to tonic hyperpolarization of the membrane. The ictal discharges were accompanied by postictal hyperpolarizations of up to 30 s duration. The intracellular injection of EGTA blocking Ca 2+-dependent potassium conductivity, eliminated prolonged after- and postictal hyperpolarizations and produced depolarizing afterpotentials and a gradual depolarization of cell membranes. Our results indicate that the development of short hyperpolarizing afterpotentials could be determined by the inhibitory synaptic effects. The activation of Ca 2+-dependent potassium conductivity caused by the development of prolonged afterhyperpolarizations and postictal polarizations, as well as maintained tonic hyperpolarization of cell membranes. Obviously, the depolarizing afterpotentials are of a non-synaptic origin and can be induced by inward calcium current.

Organic calcium channel antagonists provoke acetylcholine receptor autodesensitization on train stimulation of motor nerve

The effects of nicardipine and other organic Ca 2+-channel antagonists on the responses induced by indirect train stimulation (3 s, 50–100 Hz) were studied in mouse phrenic nerve diaphragm preparations. Nicardipine at 1–10μM, which alone did not affect single or tetanic contractions or the amplitude of evoked endplate potentials and spontaneous miniature endplate potentials, caused tetanic contraction to fade completely in the presence of 0.3 μM neostigmine or 50 μM diisopropylfluorophosphate. In combination with these anticholinesterases, nicardipine caused a severe run-down and shortening of endplate potentials in 1–2 s. This effect on endplate potentials was dependent on stimulus frequency and on extracellular Ca 2+. The effect was accelerated by intracellular injection of Ca 2+, but retarded by injection of EGTA. The amplitudes of miniature endplate potentials and the evoked endplate depolarization were also depressed during repetitive stimulation. On termination of repetitive stimulation, all postsynaptic responses, including evoked endplate potentials, miniature endplate potentials and single twitches, recovered to pre-train level in 3–10s. These results suggest that the postsynaptic nicotinic receptors had lost the functional activity during repetitive stimulation. The time-courses of the aforementioned changes initiated by repetitive stimulation were similar to the fast phase of desensitization induced by acetylcholine. The irreversible action of α-bungarotoxin on acetylcholine receptor was attenuated in the presence of nicardipine and neostigmine if repetitive stimulation was applied. The same effects were observed with other organic Ca 2+-channel antagonists (diltiazem, verapamil and nifedipine) as well as agonist (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate, BAY K8644), but not with Mn 2+, theophylline or caffeine. It is inferred that organic Ca 2+-channel antagonists interact directly with acetylcholine receptor ion channel, enhance its autodesensitization liability and thus cause extinction of endplate potentials on repetitive stimulation.