The Ventral Photoreceptor Cells of Limulus : III. A voltage-clamp study

In the dark, the ventral photoreceptor of Limulus exhibits time-variant currents under voltage-clamp conditions; that is, if the membrane potential of the cell is clamped to a depolarized value there is an initial large outward current which slowly declines to a steady level. The current-voltage relation of the cell in the dark is nonlinear. The only ion tested which has any effect on the current-voltage relation is potassium; high potassium shifts the reversal potential towards zero and introduces a negative slope-conductance region. When the cell is illuminated under voltage-clamp conditions, an additional current, the light-induced current, flows across the cell membrane. The time course of this current mimics the time course of the light response (receptor potential) in the unclamped cell; namely, an initial transient phase is followed by a steady-state phase. The amplitude of the peak transient current can be as large as 60 times the amplitude of the steady-state current, while in the unclamped cell the amplitude of the peak transient voltage never exceeds 4 times the amplitude of the steady-state voltage. The current-voltage relations of the additional light-induced current obtained for different instants of time are also nonlinear, but differ from the current-voltage relations of the dark current. The ions tested which have the greatest effect on the light-induced current are sodium and calcium; low sodium decreases the current, while low calcium increases the current. The data strongly support the hypothesis that two systems of electric current exist in the membrane. Thus the total ionic current which flows in the membrane is accounted for as the sum of a dark current and a light-induced current.     

Effect of intracellular cyclic AMP injection on calcium current in identifiedHelix pomatia neurons

The effect of intracellular injection of cyclic AMP (cAMP) and extracellular application of theophylline on the inward calcium current was investigated in neurons RPa3 and LPa3 ofHelix pomatia. Iontophoretic injection of cyclic AMP (current 10–35 nA, duration about 1 min) led to a decrease in amplitude of the calcium current to a new stationary level, which depended on the injection current. After the end of injection the calcium current was restored to its initial level. Current-voltage characteristic curves of the calcium current were not shifted along the voltage axis by cAMP injection, indicating that the reduction in this current was connected with a change in maximal calcium conductance. An increase in the frequency of depolarizing shifts from 0.1 to 0.5 Hz caused a decrease in the calcium current but did not affect the time course of the decrease in calcium current in response to injection of cAMP or the time course of its recovery after the end of injection. Theophylline an inhibitor of cyclic nucleotide phosphodiesterase, in a concentration of 1 mM in the external solution, lowered the amplitude of the calcium current by 50–75% of its initial value. In 40% of neurons, abolition of the action of theophylline by rinsing was incomplete, but in the rest the effect of theophylline was irreversible. It is postulated on the basis of the results that cytoplasmic compounds take part in regulation of the calcium current of molluscan neurons. The possible physiological role of this process is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 3, pp. 290–297, May–June, 1982.     

Ionic currents in dispersed chemoreceptor cells of the mammalian carotid body

Ionic currents of enzymatically dispersed type I and type II cells of the carotid body have been studied using the whole cell variant of the patch-clamp technique. Type II cells only have a tiny, slowly activating outward potassium current. By contrast, in every type I chemoreceptor cell studied we found (a) sodium, (b) calcium, and (c) potassium currents. (a) The sodium current has a fast activation time course and an activation threshold at approximately -40 mV. At all voltages inactivation follows a single exponential time course. The time constant of inactivation is 0.67 ms at 0 mV. Half steady state inactivation occurs at a membrane potential of approximately -50 mV. (b) The calcium current is almost totally abolished when most of the external calcium is replaced by magnesium. The activation threshold of this current is at approximately -40 mV and at 0 mV it reaches a peak amplitude in 6-8 ms. The calcium current inactivates very slowly and only decreases to 27% of the maximal value at the end of 300-ms pulses to 40 mV. The calcium current was about two times larger when barium ions were used as charge carriers instead of calcium ions. Barium ions also shifted 15-20 mV toward negative voltages the conductance vs. voltage curve. Deactivation kinetics of the calcium current follows a biphasic time course well fitted by the sum of two exponentials. At -80 mV the slow component has a time constant of 1.3 +/- 0.4 ms whereas the fast component, with an amplitude about 20 times larger than the slow component, has a time constant of 0.16 +/- 0.03 ms. These results suggest that type I cells have predominantly fast deactivating calcium channels. The slow component of the tails may represent the activity of a small population of slowly deactivating calcium channels, although other possibilities are considered. (c) Potassium current seems to be mainly due to the activity of voltage-dependent potassium channels, but a small percentage of calcium-activated channels may also exist. This current activates slowly, reaches a peak amplitude in 5-10 ms, and thereafter slowly inactivates. Inactivation is almost complete in 250-300 ms. The potassium current is reversibly blocked by tetraethylammonium. Under current-clamp conditions type I cells can spontaneously fire large action potentials. These results indicate that type I cells are excitable and have a variety of ionic conductances. We suggest a possible participation of these conductances in chemoreception.     

Ethanol effects on voltage-dependent membrane conductances: comparative sensitivity of channel populations inAplysia neurons

The study of ethanol (EtOH) action is interesting because of its clinical relevance and for the insights it provides into structure-function relationships of excitable membranes. This paper describes the concentration dependencies of various parameters of four currents in Aplysia cells. ICa is the most sensitive of the currents studied. There was a significant reduction of ICa at concentrations of 50 mM EtOH. At low concentrations, the reduction of amplitude was the primary effect of ethanol, with the kinetics and voltage dependency of activation not affected. INa and IA were also affected, but at EtOH levels higher than those which altered ICa. The primary effect of EtOH on INa was a reduction in its amplitude, although the time to peak current flow was increased by EtOH. The effects of EtOH on IA were cell specific and, for the purposes of this paper, we examined the giant metacerebral cell (MCC). In MCC, the primary effect of EtOH on IA was an increase in the time course of inactivation. The time to peak IA was also increased by high concentrations of EtOH, but its amplitude was unaffected even at high concentrations. The delayed rectifier current, IK, was the most EtOH resistant of the currents examined. High EtOH concentrations augmented the amplitude of IK, although even at 600 mM concentrations, the percentage change was only 30%. Our results indicate that the calcium channel is very susceptible to the influence of ethanol and is a serious candidate to be the primary target of EtOH action in the nervous system. The differential sensitivity of voltage-dependent currents and individual components of a given current suggests further experiments to probe the relationship between membrane structure and channel function in excitable membranes.     

Activation of NMDA receptors linked to modulation of voltage-gated ion channels and functional implications

Catfish (Ictaluruspunctatus) cone horizontal cells containN-methyl-D-aspartate (NMDA) receptors, thefunction of which has yet to be determined. In the present study, wehave examined the effect of NMDA receptor activation on voltage-gatedion channel activity. NMDA receptor activation produced a long-termdownregulation of voltage-gated sodium and calcium currents but had noeffect on the delayed rectifying potassium current. NMDA'seffect was eliminated in the presence of AP-7. To determine whetherNMDA receptor activation had functional implications, isolated catfish cone horizontal cells were current clamped to mimic the cell's physiological response. When horizontal cells were depolarized, theyelicited a single depolarizing overshoot and maintained a depolarizedsteady state membrane potential. NMDA reduced the amplitude of thedepolarizing overshoot and increased the depolarized steady-statemembrane potential. Both effects of NMDA were eliminated in thepresence of AP-7. These results support the hypothesis that activationof NMDA receptors in catfish horizontal cells may affect the type ofvisual information conveyed through the distal retina.

     

Calcium transients associated with the T type calcium current in myotubes

Immature skeletal muscle cells, both in vivo and in vitro, express a high density of T type calcium current and a relatively low density of the dihydropyridine receptor, the protein thought to function as the Islow calcium channel and as the voltage sensor for excitation- contraction coupling. Although the role of the voltage sensor in eliciting elevations of myoplasmic, free calcium (calcium transients) has been examined, the role of the T type current has not. In this study we examined calcium transients associated with the T type current in cultured myotubes from normal and dysgenic mice, using the whole cell configuration of the patch clamp technique in conjunction with the calcium indicator dye Fluo-3. In both normal and dysgenic myotubes, the T type current was activated by weak depolarizations and was maximal for test pulses to approximately -20 mV. In normal myotubes that displayed T type calcium current, the calcium transient followed the amplitude and the integral of the current at low membrane potentials (- 40 to -20 mV) but not at high potentials, where the calcium transient is caused by SR calcium release. The amplitude of the calcium transient for a pulse to -20 mV measured at 15 ms after depolarization represented, on average, 4.26 +/- 0.68% (n = 19) of the maximum amplitude of the calcium transient elicited by strong, 15-ms test depolarizations. In dysgenic myotubes, the calcium transient followed the integral of the calcium current at all test potentials, in cells expressing only T type current as well as in cells possessing both T type current and the L type current Idys. Moreover, the calcium transient also followed the amplitude and time course of current in dysgenic myotubes expressing the cardiac, DHP-sensitive calcium channel. Thus, in those cases where the transient appears to be a consequence of calcium entry, it has the same time course as the integral of the calcium current. Inactivation of the T type calcium current with 1-s prepulses, or block of the current by the addition of amiloride (0.3-1.0 mM) caused a reduction in the calcium transient which was similar in normal and dysgenic myotubes. To allow calculation of expected changes of intracellular calcium in response to influx, myotubes were converted to a roughly spherical shape (myoballs) by adding 0.5 microM colchicine to culture dishes of normal cells. Calcium currents and calcium transients recorded from myoballs were similar to those in normal myotubes.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the effects of divalent cations and ethylene glycol-bis-(beta- aminoethyl ether) N,N,N'',N''-tetraacetate on action potential duration in frog heart

Resting and action potentials were recorded from superfused strips of frog ventricle. Reducing the bathing calcium concentration ([Ca2+]0) with or without ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) prolongs the action potential (AP). The change in the duration of the AP extends over many minutes, but is rapidly reversed by restoring calcium ions. Other changes (e.g., in resting potential and overshoot) are, however, only more slowly reversed. Reducing [Ca2+]0 with 0.2, 2, or 5 mM EGTA produces progressively greater prolongation of AP; maximum values were well in excess of 1 min. This prolongation can be reversed by other divalent cations in EGTA (Mg2+, Sr2+) or Ca-free (Mn2+) solutions, or by acetylcholine. Barium ions increase AP duration in keeping with their known effect on potassium conductance. D600, which blocks the slow inward current in cardiac muscle, is without effect on the action potentials recorded in EGTA solutions, or on the time course and extent of the recovery to normal duration upon restoring calcium ions. It is concluded that divalent cations exert an influence on membrane potassium conductance extracellularly in frog heart. The cell membrane does not become excessively "leaky" in EGTA solutions.     

The effects of catecholamines on tension reactivation in cardiac muscle

The effects of adrenaline and the beta-agonist isoprenaline on the time course of tension reactivation were studied in several cardiac tissues. The aim of the study was to assess whether experimental evidence can be found for a role of the sarcoplasmic reticulum in the reactivation of tension. It was assumed that calcium recycles between different parts of the reticulum, and that this recycling may affect tension repriming. Isoprenaline was assumed to enhance such recycling by increasing the uptake of calcium, following its release during a preceding contraction. Isoprenaline (in the range of 40 nM to 4 microM) was found to enhance tension repriming in adult guinea pig atria. However, in adult rat atria, isoprenaline often gave a complex effect, with a smaller degree of repriming at short intervals, and enhanced repriming at longer intervals. This was thought to reflect the balance between the enhancing effect of the drug on calcium recycling and an augmented release from the sarcoplasmic reticulum (SR). In striking contrast, there was no effect of isoprenaline on tension repriming in neonatal guinea pig atria and a retardation in neonatal rat atria. This was interpreted as reflecting the lack of a sarcoplasmic network in the neonatal tissue. The effects of isoprenaline on tension repriming in the frog atrium (which also has a sparse sarcoplasmic reticulum network) were also found to be complex; low concentrations (40 nM) enhanced the process, and high concentrations (0.4 microM) retarded it. Intermediate levels often produced a 'crossover' effect: more reactivation at short intervals, and less at long intervals. The interpretation of these results was that there are two processes which interact to determine the amount of tension produced at short intervals after each contraction: the basal reactivation process and some augmenting mechanism superimposed on it. This mechanism is probably related to other behavioural features of cardiac muscle, such as rate-dependent increases in membrane calcium currents. It is relevant mainly in those cases where tension repriming depends on membrane calcium currents. Further experiments (in the frog atrium) with elevated calcium and with the alpha-adrenergic agonist phenylephrine (both of which slowed down the reactivation process) also support this idea. These agents elevate internal calcium levels, and presumably saturate the augmenting mechanism (by producing maximal tension responses).(ABSTRACT TRUNCATED AT 400 WORDS)     

Adenosine inhibits calcium channel currents via A1 receptors on salamander retinal ganglion cells in a mini-slice preparation

The effects of adenosine on high-voltage-activated calcium channel currents in tiger salamander retinal ganglion cells were investigated in a mini-slice preparation. Adenosine produced a concentration-dependent decrease in the amplitude of calcium channel current with a maximum inhibition of 26%. The effects of adenosine on calcium channel current were both time- and voltage-dependent. In cells dialyzed with GTP-gamma-s, adenosine caused a sustained and irreversible inhibition of calcium channel current, suggesting involvement of a GTP-binding protein. The inhibitory effect of adenosine on calcium channel current was blocked by the A1 antagonist 8-cyclopentyltheophylline (DPCPX, 1-10 microm), but not by the A2 antagonist 3-7-dimethyl-1-propargylxanthine (DMPX, 10 microm), and was mimicked by the A1 agonist N6-cyclohexyladenosine (CHA, 1 microm) but not by the A2 agonist 5'-(N-cyclopropyl) carbox-amidoadenosine (CPCA, 1 microm). Adenosine's inhibition of calcium channel current was not affected by the L-type calcium channel blocker nifedipine (5 microm). However, adenosine's inhibition of calcium channel current was reduced to approximately 10% after application of omega-conotoxin GVIA (1 microm), suggesting that adenosine inhibits N-type calcium channels. These results show that adenosine acts on an A1 adenosine receptor subtype via a G protein-coupled pathway to inhibit the component of calcium channel current carried in N-type calcium channels.     

Omega-conotoxin blockade of calcium currents in cultured neonatal rat cardiomyocytes: different action on EGTA-modified calcium channels

Calcium currents from neonatal rat ventricular heart muscle cells grown in primary culture were examined using the "whole-cell" voltage clamp technique. An inward current characterized by large amplitude and slow inactivation decay was induced when the extracellular Ca2+ concentration was reduced by EGTA. This current was suppressed by extracellular Na+ removal, or by calcium antagonists, and increased by epinephrine and BAY K 8644. These findings suggest that this current is carried by sodium ions through Ca channels. Both Ca and Na currents through calcium channels were irreversibly blocked by omega-conotoxin. Complete blockade developed 10-15 minutes after the toxin introduction in the extracellular solution. Blockade of Na currents through calcium channels was characterized by a transient increase of current amplitude without any changes in its kinetics and voltage-dependent properties. Structural differences between calcium channels in rat and guinea-pig and frog cardiomyocytes were suggested.     

Presynaptic facilitation at the crayfish neuromuscular junction. Role of calcium-activated potassium conductance

Membrane potential was recorded intracellularly near presynaptic terminals of the excitor axon of the crayfish opener neuromuscular junction (NMJ), while transmitter release was recorded postsynaptically. This study focused on the effects of a presynaptic calcium-activated potassium conductance, gK(Ca), on the transmitter release evoked by single and paired depolarizing current pulses. Blocking gK(Ca) by adding tetraethylammonium ion (TEA; 5-20 mM) to a solution containing tetrodotoxin and aminopyridines caused the relation between presynaptic potential and transmitter release to steepen and shift to less depolarized potentials. When two depolarizing current pulses were applied at 20-ms intervals with gK(Ca) not blocked, the presynaptic voltage change to the second (test) pulse was inversely related to the amplitude of the first (conditioning) pulse. This effect of the conditioning prepulse on the response to the test pulse was eliminated by 20 mM TEA and by solutions containing 0 mM Ca2+/1 mM EGTA, suggesting that the reduction in the amplitude of the test pulse was due to activation of gK(Ca) by calcium remaining from the conditioning pulse. In the absence of TEA, facilitation of transmitter release evoked by a test pulse increased as the conditioning pulse grew from -40 to -20 mV, but then decreased with further increase in the conditioning depolarization. A similar nonmonotonic relationship between facilitation and the amplitude of the conditioning depolarization was reported in previous studies using extracellular recording, and interpreted as supporting an additional voltage-dependent step in the activation of transmitter release. We suggest that this result was due instead to activation of a gK(Ca) by the conditioning depolarization, since facilitation of transmitter release increased monotonically with the amplitude of the conditioning depolarization, and the early time course of the decay of facilitation was prolonged when gK(Ca) was blocked. The different time courses for decay of the presynaptic potential (20 ms) and facilitation (greater than 50 ms) suggest either that residual free calcium does not account for facilitation at the crayfish NMJ or that the transmitter release mechanism has a markedly higher affinity or stoichiometry for internal free calcium than does gK(Ca). Finally, our data suggest that the calcium channels responsible for transmitter release at the crayfish NMJ are not of the L, N, or T type.     

Effect of protein tyrosine kinase inhibitors on the current through the Ca(V)3.1 channel

In the present study, we have investigated the effects of protein tyrosine kinase (PTK) inhibitors on the Ca(V)3.1 calcium channel stably transfected in HEK293 cells using the whole-cell configuration of the patch-clamp technique. We have tested two different tyrosine kinase inhibitors, genistein and tyrphostin AG213, and their inactive analogs, genistin and tyrphostin AG9. Bath application of genistein, but not genistin, decreased the T-type calcium current amplitude in a concentration-dependent manner with an IC(50) of 24.7+/-2.0 microM. This effect of genistein was accompanied by deceleration of channel activation and acceleration of channel inactivation. Intracellular application of neither genistein nor genistin had a significant effect on the calcium current. Extracellular application of 50 microM tyrphostin AG213 and its inactive analogue, tyrphostin AG9, did not affect the current through the Ca(V)3.1 channel. The effect of genistein on the channel was also not affected by the presence of catalytically active PTK, p60(c-src) inside the cell. We have concluded that genistein directly inhibited the channel. This mechanism does not involve a PTK-dependent pathway. The alteration of the channel kinetics by genistein suggests an interaction with the voltage sensor of the channel together with the channel pore occlusion.     

Stretch of active muscle during the declining phase of the calcium transient produces biphasic changes in calcium binding to the activating sites

In voltage-clamped barnacle single muscle fibers, muscle shortening during the declining phase of the calcium transient increases myoplasmic calcium. This extra calcium is probably released from the activating sites by a change in affinity when cross-bridges break (Gordon, A. M., and E. B. Ridgway, 1987. J. Gen. Physiol. 90:321-340). Stretching the muscle at similar times causes a more complex response, a rapid increase in intracellular calcium followed by a transient decrease. The amplitudes of both phases increase with the rate and amplitude of stretch. The rapid increase, however, appears only when the muscle is stretched more than approximately 0.4%. This is above the length change that produces the breakpoint in the force record during a ramp stretch. This positive phase in response to large stretches is similar to that seen on equivalent shortening at the same point in the contraction. For stretches at different times during the calcium transient, the peak amplitude of the positive phase has a time course that is delayed relative to the calcium transient, while the peak decrease during the negative phase has an earlier time course that is more similar to the calcium transient. The amplitudes of both phases increase with increasing strength of stimulation and consequent force. When the initial muscle the active force. A large decrease in length (which drops the active force to zero) decreases the extra calcium seen on a subsequent restretch. After such a shortening step, the extra calcium on stretch recovers (50 ms half time) toward the control level with the same time course as the redeveloped force. Conversely, stretching an active fiber decreases the extra calcium on a subsequent shortening step that is imposed shortly afterward. Enhanced calcium binding due to increased length alone cannot explain our data. We hypothesize that the calcium affinity of the activating sites increases with cross-bridge attachment and further with cross-bridge strain. This accounts for the biphasic response to stretch as follows: cross-bridges detached by stretch first decrease calcium affinity, then upon reattachment increase calcium affinity due to the strained configuration brought on by the stretch. The experiments suggest that cross-bridge attachment and strain can modify calcium binding to the activating sites in intact muscle.     

Measurement of calcium transients and slow calcium current in myotubes

The purpose of this study was to characterize excitation-contraction (e- c) coupling in myotubes for comparison with e-c coupling of adult skeletal muscle. The whole cell configuration of the patch clamp technique was used in conjunction with the calcium indicator dye Fluo-3 to study the calcium transients and slow calcium currents elicited by voltage clamp pulses in cultured myotubes obtained from neonatal mice. Cells were held at -80 mV and stimulated with 15-20 ms test depolarizations preceded and followed by voltage steps designed to isolate the slow calcium current. The slow calcium current had a threshold for activation of about 0 mV; the peak amplitude of the current reached a maximum at 30 to 40 mV a and then declined for still stronger depolarizations. The calcium transient had a threshold of about -10 mV, and its amplitude increased as a sigmoidal function of test potential and did not decrease again even for test depolarizations sufficiently strong (> or = 50 mV) that the amplitude of the slow calcium current became very small. Thus, the slow calcium current in myotubes appears to have a negligible role in the process of depolarization-induced release of intracellular calcium and this process in myotubes is essentially like that in adult skeletal muscle. After repolarization, however, the decay of the calcium transient in myotubes was very slow (hundreds of ms) compared to adult muscle, particularly after strong depolarizations that triggered larger calcium transients. Moreover, when cells were repolarized after strong depolarizations, the transient typically continued to increase slowly for up to several tens of ms before the onset of decay. This continued increase after repolarization was abolished by the addition of 5 mM BAPTA to the patch pipette although the rapid depolarization-induced release was not, suggesting that the slow increase might be a regenerative response triggered by the depolarization-induced release of calcium. The addition of either 0.5 mM Cd2+ + 0.1 mM La3+ or the dihydropyridine (+)-PN 200-110 (1 microM) reduced the amplitude of the calcium transient by mechanisms that appeared to be unrelated to the block of current that these agents produce. In the majority of cells, the decay of the transient was accelerated by the addition of the heavy metals or the dihydropyridine, consistent with the idea that the removal system becomes saturated for large calcium releases and becomes more efficient when the size of the release is reduced.     

Guanosine-5''-O-(3-thiotriphosphate) modifies kinetics of voltage-dependent calcium current in chick sensory neurons.

Internal perfusion with the G-protein activator guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S) mimics the effect of noradrenaline and dopamine on the voltage-dependent calcium current in chick dorsal root ganglion (DRG) cells. With 100 microM GTP-gamma S in the pipette, the current at +10 mV was depressed by approximately 50%, with a 10-fold increase of its time to peak. The activation time course of the control calcium current could be approximated with a single exponential curve, whereas with GTP-gamma S the activation time course was double exponential, with time constants tau 1 and tau 2. 2 mM Mg-ATP in the pipette prevented the GTP-gamma S-induced current decrease in 70% of the cells, but the time course of the current was always double exponential. From -50 mV, the current at +10 mV was best fitted with tau 1 = 1.7 +/- 0.5 and tau 2 = 25.6 +/- 5.5 in seven cells. Both time constants decreased with increasing depolarizations. In the first 2 min of recording, the current changed with time. However, both tau 1 and tau 2 were constant, whereas the relative contribution of the slow component increased from 10 to 70%. In addition, the effect was independent of the holding potential in the range from -100 to -30 mV. These results suggest that the activation of a G-protein causes a fraction of the high-threshold calcium channels to switch to a new closed state, with slower opening kinetics.     

Calcium current in embryonic Xenopus muscle cells in culture

We have investigated the appearance of calcium current in Xenopus muscle cells in 1- to 6-day-old cultures. Whole cell currents were recorded using a patch-clamp amplifier with sodium and potassium replaced with tetraethylammonium and cesium, respectively, and BaCl2 used in place of CaCl2. When the muscle membrane was depolarized above -30 mV, a slow inward current was activated, the current reached a peak amplitude near 0 mV, and an outward current became apparent above +10 mV. This slow current was enhanced by adding barium or Bay K 8644 to the extracellular recording solution and was blocked by the addition of cobalt, cadmium, or the dihydropyridines nifedipine or (+)PN 200-110. Taken together these results indicate the presence of an inward calcium current mediated through L-type channels. Thirty-one percent of the cells examined on the first day in culture showed no discernible slow inward current; however, as the age of the culture increased, all cells showed slow inward current and there was an increase in the amplitude of the current. A small proportion of the muscle cells (5 out of 34) also showed a fast activating and inactivating inward current. This current, which activated at more hyperpolarized potentials (-40 mV) was only present when 5 mM ATP was included in the internal recording solution. It also appeared to be mediated through a calcium channel but not a dihydropyridine, sensitive channel.     

Control of spontaneous synchronous Ca<Superscript>2+</Superscript> oscillations in hippocampal neurons by GABAergic neurons containing kainate receptors without desensitization

Previously we have shown that in culture of rat hippocampal neurons, the calcium responses of individual cells (changes of cytoplasmic free Ca²⁺ concentration in response to agonists of glutamate kainate receptors) differed in shape and amplitude (Kononov A.V., Bal’ N.V., Zinchenko V.P. 2011. Biochemistry (Moscow) Suppl. Series A: Membrane and Cell Biology. 5 (2), 162–170). In the majority of neurons, the amplitudes of calcium response were regularly distributed, although there were a small number of cells that generated the desensitization-free signals of far greater amplitudes. In these cells, the desensitization inhibitors did not increase the amplitude of calcium response. We identified these neurons and revealed their function. The agonists of kainate receptors inhibited the synchronized spontaneous Ca²⁺ oscillations, decreased the baseline calcium level in the majority of neurons, and considerably elevated it in some of them. After washout of the agonists, the oscillations were restored in all neurons only after a certain time lag determined by the period needed for calcium concentration to decrease to subbasal level in specific neurons with high calcium signal amplitude. This observation indicates the command role of these neurons in synchronizing the activity of the entire population. To identify the subtype of KA receptors in these neurons, we used especially selective agonists and showed that KA receptors of the neurons characterized with desensitization-free calcium signals of unusually great amplitude contained GluR5/GLUK1 subunits. These receptors are known to be located mostly in the presynaptic membrane, where they promote exocytosis of neurotransmitters due to elevation of the Ca²⁺ conductivity. Having marked the positions of these neurons, we fixed the preparation and stained the cells with fluorescently labeled antibodies raised against glutamate decarboxylase, an enzyme which is selectively expressed in GABAergic neurons. The experiments demonstrated that antibodies were localized only in the neurons, where the kainate receptor agonist evoked desensitization-free calcium responses of especially large amplitude. Thus, GABAergic neurons control the synchronous activity of a large number of neurons via glutamate-evoked activation of specific presynaptic kainate receptors with GluR5/GLUK1 subunits leading to desensitization-free calcium signals of especially large amplitude.     

The reactivation of demembranated human spermatozoa lacking outer dynein arms is independent of pH

The effect of pH, Mg-ATP, and free calcium on activity of the inner dynein arm was investigated using demembranated human spermatozoa lacking the outer dynein arms (LODA). The results were compared with those obtained for demembranated-reactivated normal spermatozoa to evaluate the functional properties of the inner and outer dynein arms in axonemal motility. The reactivation of Triton X-100–demembranated LODA spermatozoa was analysed at various pHs and concentrations of Mg-ATP and calcium using video recordings. The percentage of reactivated LODA spermatozoa as a function of Mg-ATP concentration was not dependent on pH, whereas reactivation of normal human spermatozoa is pH dependent. This suggests that there may be a pH-dependent regulatory mechanism associated with the outer dynein arms. A delay in the principal bend propagation of normal and LODA reactivated cells was found at pH 7.1. This disappeared at pH 7.8 in normal but not in LODA populations. This suggests a role for outer dynein arms in the initiation of the propagation of flagellar bends at alkaline pH. The level of LODA and normal sperm reactivation both depended on the calcium concentration in the medium. At lower free calcium concentrations, the reactivation level and beat frequency of reactivated cells were higher. Our results suggest a functional difference between outer and inner dynein arms of human spermatozoa based on a differential pH sensitivity. Moreover, calcium seems to exert its regulatory action elsewhere than on the outer dynein arms. Mol. Reprod. Dev. 49:416–425, 1998. © 1998 Wiley-Liss, Inc.     

Effects of calcium and chelating agents on the ability of various agonists to increase cyclic GMP in pancreatic acinar cells.

In dispersed acinar cells from guinea pig pancreas we found that chelating extracellular calcium with EDTA did not alter cellular cyclic GMP but caused a 50% reduction in the increase in cyclic GMP caused by the synthetic C-terminal octapeptide of porcine cholecystokinin (cholecystokinin octapeptide). This effect was maximal within 2 min and preincubating the cells with EDTA for as long as 30 min caused no further reduction in the action of cholecystokinin octapeptide. In acinar cells preincubated without calcium, adding calcium caused a time dependent increase in the action of cholecystokinin octapeptide and this increase was maximal after 10 min of incubation. An effect of extracellular calcium on the action of cholecystokinin octapeptide could be detected with 0.5 mM calcium and was maximal with 2.0 mM calcium. Magnesium alone or with calcium did not alter the action of cholecystokinin octapeptide. Extracellular calcium did not alter the time course or the configuration of the dose vs. response curve for the action of cholecystokinin octapeptide on cellular cyclic GMP. Low concentrations of EGTA (0.1 mM) decreased the effect of cholecystokinin octapeptide on cellular cyclic GMP to the same extent as did EDTA or preincubating acinar cells without calcium. Increasing EGTA above 0.1 mM caused progressive augmentation of the action of cholecystokinin octapeptide on cellular cyclic GMP and this augmentation did not require extracellular calcium or magnesium. Results similar to those obtained with cholecystokinin octapeptide were also obtained with bombesin, carbamylcholine, litorin and eledoisin. In contrast, the action of sodium nitroprusside on cyclic GMP in pancreatic acinar cells was not altered by adding EDTA or EGTA. These results indicate that the ability of extracellular calcium to influence the action of cholecystokinin octapeptide and other agents on cyclic GMP results from changes in cellular calcium and not from effects of extracellular calcium per se. The action of low concentrations of EGTA on the increase in cyclic GMP caused by various agents reflects the ability of EGTA to chelate extracellular calcium. The actions of high concentrations of EGTA were independent of extracellular calcium or magnesium and appear to reflect a direct action of EGTA on pancreatic acinar cells.     

Effects of calcium and chelating agents on the ability of various agonists to increase cyclic GMP in pancreatic acinar cells

In dispersed acinar cells from guinea pig pancreas we found that chelating extracellular calcium with EDTA did not alter cellular cyclic GMP but caused a 50% reduction in the increase in cyclic GMP caused by the synthetic C-terminal octapeptide of porcine cholecystokinin (cholecystokinin octapeptide). This effect was maximal within 2 min and preincubating the cells with EDTA for as long as 30 min caused no further reduction in the action of cholecystokinin octapeptide. In acinar cells preincubated without calcium, adding calcium caused a time dependent increase in the action of cholecystokinin octapeptide and this increase was maximal after 10 min of incubation. An effect of extracellular calcium on the action of cholecystokinin octapeptide could be detected with 0.5 mM calcium and was maximal with 2.0 mM calcium. Magnesium alone or with calcium did not alter the action of cholecystokinin octapeptide. Extracellular calcium did not alter the time course or the configuration of the dose vs. response curve for the action of cholecystokinin octapeptide on cellular cyclic GMP. Low concentrations of EGTA (0.1 mM) decreased the effect of cholecystokinin octapeptide on cellular cyclic GMP to the same extent as did EDTA or preincubating acinar cells without calcium. Increasing EGTA above 0.1 mM caused progressive augmentation of the action of cholecystokinin octapeptide on cellular cyclic GMP and this augmentation did not require extracellular calcium or magnesium. Results similar to those obtained with cholecystokinin octapeptide were also obtained with bombesin, carbamylcholine, litorin and eledoisin. In contrast, the action of sodium nitroprusside on cyclic GMP in pancreatic acinar cells was not altered by adding EDTA or EGTA.These results indicate that the ability of extracellular calcium to influence the action of cholecystokinin octapeptide and other agents on cyclic GMP results from changes in cellular calcium and not from effects of extracellular calcium per se. The action of low concentrations of EGTA on the increase in cyclic GMP caused by various agents reflects the ability of EGTA to chelate extracellular calcium. The actions of high concentrations of EGTA were independent of extracellular calcium or magnesium and appear to reflect a direct action of EGTA on pancreatic acinar cells.