Differential effects of divalent cations on spontaneous and evoked glycine release from spinal interneurons

The effects of Ca2+, Sr2+, and Ba2+ on spontaneous and evoked glycinergic inhibitory postsynaptic currents (mIPSCs and eIPSCs) were studied using the "synaptic bouton" preparation of rat spinal neurons and conventional whole cell recording under voltage-clamp conditions. In response to application of Ca2+-free solution, the frequency of mIPSC initially rapidly decreased to 40 approximately 50% of control followed by a gradual further decline in mIPSC frequency to approximately 30% of control. Once mIPSC frequency had significantly decreased in Ca2+-free solution, application of Ca2+, Sr2+, or Ba2+ increased mIPSC frequency. The rank order of effect in restoring mIPSCs was Ba2+>Ca2+>Sr2+. Moreover, the application of excess external [K+]o solution (30 mM) containing Sr2+ or Ba2+ after 2 h in Ca2+-free solution also increased mIPSC frequency in the order Sr2+>or==Ba2+>Ca2+. The mean mIPSC amplitude was not affected at all. In contrast, eIPSCs produced by focal stimulation of single boutons were completely abolished in Ca2+-free solution or when Ca2+ was replaced by Sr2+ or Ba2+ (2 mM each). However, eIPSCs were restored in increased concentrations of Sr2+ or Ba2+ (5 mM each). The results show that these divalent cations affect mIPSC and eIPSCs differently and indicate that the mechanisms underlying transmitter release that generates eIPSCs and mIPSC in presynaptic nerve terminals are different. The different mechanisms might be explained by the different sensitivity of synaptotagmin isoforms to Ca2+, Sr2+, and Ba2+.     

The effects of strontium and barium ions at synapses in sympathetic ganglia.

1. A study has been made of the effects of Sr2+ and Ba2+ ions at synapses in isolated superior cervical ganglia of guinea-pigs. Intracellular recordings of membrane potential were made from ganglion cells in the presence of different concentrations of Ca2+, Sr2+ and Ba2+ ions. 2. The addition of Sr2+ (2-5 mM) caused little change in resting membrane potential; in contrast, Ba2+ (1-6 mM) always depolarized the cells and prolonged the duration of action potentials. 3. The resting frequency of spontaneous miniature excitatory post-synaptic potentials (min. e.p.s.p.s) was briefly accelerated by the addition of either Sr2+ or Ba2+, but subsequently returned to about control levels. 4. Following replacement of Ca2+ by Sr2+, e.p.s.p.s could always be evoked during repetitive stimulation of preganglionic axons at a fixed latency after the nerve impulses ('phasic' transmitter release). Replacement of Ca2+ by Ba2+ produced many asynchronous e.p.s.p.s during trains of impulses ('residual' transmitter release). 5. By analysis of the interaction between Sr2+ and Ca2+, Sr2+ was shown to have a partial agonist action on 'phasic' transmitter release. The same analysis applied to Ba2+ failed to demonstrate either a partial agonist or antagonist action. 6. Both Sr2+ and Ba2+ prolonged e.p.s.p.s. Changes in Sr2+ could mainly be attributed to its effect on cell input resistance; Ba2+ may also prolong the time course of transmitter release. 7. The increased frequency of min. e.p.s.p.s which occurs during repetitive stimulation was potentiated by both Sr2+ and Ba2+, Ba2+ being about twice as potent as Sr2+. This activation of 'residual' transmitter release is independent of the action of these ions on 'phasic' release. 8. It is concluded that the reported maintenance by Ba2+ of acetyl-choline output from perfused ganglia results from the asynchronous release of large numbers of quanta during trains of impulses.     

The effect of alpha-latrotoxin on the neurosecretory PC12 cell line: studies on toxin binding and stimulation of transmitter release

alpha-Latrotoxin of black widow spider venom was found to bind with high affinity (KA = 1.8 X 10(9)M-1) to specific sites present in discrete number (approximately 6300/cell, approximately 12/micron2) at the surface membrane of PC12 cells. This binding correlated with (and therefore, probably caused) the secretory response produced by the toxin. Binding was enhanced (approximately 2-fold) in the presence of mM concentrations of various divalent cations (Ca2+, Mn2+ and Co2+) while Ba2+ and Sr2+ had a smaller effect and Mg2+ was inactive. Hypertonicity, concanavalin A and trypsin pretreatment of the cells blocked the binding interaction. The alpha-latrotoxin-induced stimulation of 3H-dopamine release was massive and occurred very rapidly when cells were exposed to the toxin in a Ca2+-containing Krebs-Ringer medium, whereas it occurred at a much slower rate in a Ca2+-free, Mg2+-containing Ringer. Introduction of Ca2+ into the latter medium resulted in a shift of the release rate from slow to fast. In contrast, in divalent cation-free medium the response was abolished. The toxin-induced secretory response was unaffected by Na+ and Ca2+ channel blockers (tetrodotoxin and D600) as well as by calmodulin inhibitors (calmidazolium and trifluoperazine). The effects of Ca2+ and Mg2+ were found to be concentration-dependent, with half maximal responses occurring at approximately 0.3 and 1.5 mM for the two divalent cations, respectively. Other divalent cations could substitute for Ca2+ and Mg2+, the relative efficacy being Sr2+ greater than Ca2+ greater than Ba2+ much greater than Mn2+ greater than Mg2+ greater than Co2+. Moreover, the response occurring at suboptimal concentration of Ca2+ (0.4 mM) was potentiated by the concomitant addition of either Mg2+, Mn2+ or Co2+. The effect(s) of divalent cations in supporting the alpha-latrotoxin-induced release response seem(s) to occur primarily at step(s) beyond toxin binding because (a) the stimulatory effects of the various cations on release were not matched by parallel effects on binding, and (b) Ca2+ maintained its ability to stimulate fast release even when toxin binding had occurred in a Ca2+-free medium. Delays in the release responses were observed when cells were exposed to alpha LTx in Na+-free, glucosamine or methylamine-based media, or depolarized with high K+ (in the presence of D600) before toxin treatment. Moreover, in these two conditions the ability of Mg2+ to support the alpha LTx response was considerably decreased.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electrical excitability in the rat clonal pituitary cell and its relation to hormone secretion

Membrane electrical properties of the clonal anterior pituitary cell (GH3), were studied using intracellular recording techniques. The resting potential in the GH3 cell was -48.0 +/- 1.1 mV (mean +/- S.E., n=39) in normal saline. The input resistance estimated from the linear portion of the current-voltage relationship was 453 +/- 30 Momega (n=21). The maximum rate of rise of the action potential was 7.0 +/- 1.1 V/sec (n=12) at room temperature (23-25 degrees C). The action potential had both Na and Ca components. The Ca component was abolished by addition of 4 mM Co2+. Sr2+ could substitute for Ca2+ in supporting spike initiation. As the concentration of Sr2+ was increased, the maximum rate of rise of the action potential increased. After replacement of Ca2+ with isomolar Ba2+ the membrane potential shifted to -6.1 +/- 1.1 mV (n=11). In the Ba solution, prolonged action potentials were evoked by a depolarizing current pulse after maintaining the membrane potential more negative than -50 mV. The release of both prolactin and growth hormone was enhanced by increasing the external K+ concentration to 50 mM in the presence of Ca2+. Sr2+ could substitute for Ca2+. Ba2+ enhanced the release of both hormones, even if the K+ concentration was unaltered. The facilitatory effects of high K+ and Ba2+ were markedly suppressed by addition of 2-4 mM Co2+. These results may suggest that the potential-dependent increase in the membrane permeability to Ca2+, responsible for initiation of the Ca spike, plays a significant role in stimulation of hormone secretion in GH3 cells.     

Effects of divalent cations on the inhibitory potentials in the duodenal smooth muscle cells of the guinea-pig

The effects of divalent cations and some Ca2+ antagonists on the non-adrenergic inhibitory potential (i.p.) in the duodenal smooth muscle cells of the guinea-pig were investigated intracellularly. The membrane potential was a function of the external Ca2+ (0.25-7.5 mM) and Mg2+ (1.2-24 mM) concentrations. The latency and the time to peak of the i.p. were prolonged by low Ca2+ and excess Mg2+. The amplitude and the rate of hyperpolarization of the i.p. were reduced by low Ca2+ and excess Mg2+. The effects of Mn2+ (5-10 mM) on the i.p. were similar to those by excess Mg2+ except for the small depolarization. The i.p. evoked in the Ca2+-free solution was considerably restored by the addition of Ba2+ (1.25 mM) and Sr2+ (2 mM). The actions of Ba2+ and Sr2+ on the i.p. were inhibited by verapamil (10(-4)g/ml). Verapamil (2 X 10(-5)-2 X 10(-4)g/ml) and gentamicin (10(-3)g/ml) reduced the amplitude and the rate of hyperpolarization of the i.p. The latency and the time to peak of the i.p. were prolonged by verapamil but not by gentamicin. In nifedipine (10(-4)g/ml), the two-peaked i.p. was evoked by a single stimulus. This potential was similar to that evoked by a paired pulse in normal solution. The results obtained suggest that the released of the non-adrenergic inhibitory substance requires Ca2+ which moves into the non-adrenergic inhibitory neurons through the Ca2+ conductance pathway.     

Roles of Ca2+ influx through ATP-activated channels in catecholamine release from pheochromocytoma PC12 cells.

1. Extracellular ATP evokes catecholamine release concomitant with depolarization in pheochromocytoma PC12 cells. Roles of Ca2+ influx through ATP-activated channels during the catecholamine release were investigated. 2. Norepinephrine or dopamine release induced by > or = 100-microM concentrations of ATP was insensitive to 300 microM Cd2+, whereas the release induced by increasing extracellular KCl (50-150 mM) was completely blocked by this concentration of Cd2+. 3. ATP (100 microM) increased the intracellular free Ca2+ concentration measured with fura-2. The increase was not affected by 300 microM Cd2+ or 100 microM nicardipine, suggesting that Ca2+ influx through ATP-activated channels but not through voltage-gated Ca2+ channels contributes to the ATP-evoked catecholamine release. 4. Inward currents permeating through voltage-gated Ca2+ channels were measured using the whole-cell voltage clamp. In the presence of 10 microM ATP, a concentration that induces an ATP-activated channel-mediated current equivalent to that induced by 100 microM ATP during the depolarization in "non-voltage clamped" cells, the Ca2+ current activated by a voltage step to +10 mV was reduced. The reduction in the Ca2+ channel-mediated current was not observed when the extracellular Ca2+ was replaced with Ba2+. 5. The ATP (100 microM)-evoked dopamine release was inhibited by 300 microM Cd2+ when measured with extracellular Ba2+ instead of Ca2+. This effect of Ba2+ may not be related to K+ channel-blocking activity, because the ATP-evoked dopamine release obtained with 5 mM tetraethylammonium (TEA) was not inhibited by Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ enhances U-type inactivation of N-type (CaV2.2) calcium current in rat sympathetic neurons

Ca2+ -dependent inactivation (CDI) has recently been shown in heterologously expressed N-type calcium channels (CaV2.2), but CDI has been inconsistently observed in native N-current. We examined the effect of Ca2+ on N-channel inactivation in rat sympathetic neurons to determine the role of CDI on mammalian N-channels. N-current inactivated with fast (tau approximately 150 ms) and slow (tau approximately 3 s) components in Ba2+. Ca2+ differentially affected these components by accelerating the slow component (slow inactivation) and enhancing the amplitude of the fast component (fast inactivation). Lowering intracellular BAPTA concentration from 20 to 0.1 mM accelerated slow inactivation, but only in Ca2+ as expected from CDI. However, low BAPTA accelerated fast inactivation in either Ca2+ or Ba2+, which was unexpected. Fast inactivation was abolished with monovalent cations as the charge carrier, but slow inactivation was similar to that in Ba2+. Increased Ca2+, but not Ba2+, concentration (5-30 mM) enhanced the amplitude of fast inactivation and accelerated slow inactivation. However, the enhancement of fast inactivation was independent of Ca2+ influx, which indicates the relevant site is exposed to the extracellular solution and is inconsistent with CDI. Fast inactivation showed U-shaped voltage dependence in both Ba2+ and Ca2+, which appears to result from preferential inactivation from intermediate closed states (U-type inactivation). Taken together, the data support a role for extracellular divalent cations in modulating U-type inactivation. CDI appears to play a role in N-channel inactivation, but on a slower (sec) time scale.     

Elementary currents through Ca2+ channels in Guinea pig myocytes

Elementary Ca2+ and Ba2+ currents were recorded from cell-attached membrane patches of ventricular myocytes from adult guinea pig hearts using the improved patch-clamp technique (Hamill et al. 1981). High concentrations of Ba2+ or Ca2+ (50 or 90 mM) were used in the pipettes to increase the signal-to-noise ratio. All data were derived from elementary current analyses in patches containing only one channel. 1) In response to voltage steps, channel openings occurred singly or in bursts of closely spaced unitary current pulses separated by wider shut intervals. During depolarizations of small amplitude from the resting potential, channel openings occurred almost randomly, whereas during larger depolarizations the events were grouped preferentially at the beginning. 2) Channel openings became more probable with increased depolarization; simultaneously, unitary current amplitudes declined in an ohmic manner. Elementary current amplitudes were slightly larger, when 50 mM Ba2+ replaced 50 mM Ca2+ in the pipettes (slope conductances 9 and 10 pS, respectively), but more than doubled, when Ba2+ was increased to 90 mM (slope conductance 18 pS). Clear outward currents through Ca2+ channels were not observed under these conditions. 3) Peak amplitudes of reconstructed mean currents doubled when 50 mM Ba2+ replaced 50 mM Ca2+ and were larger still when 90 mM Ba2+ was used in the pipettes. The current-voltage relations of the reconstructed mean currents showed a positive shift along the voltage axis as Ba2+ was increased or substituted equimolarly by Ca2+. correspondingly, the open state probability-voltage relations (activation curves) showed a parallel shift as Ba2+ was increased, which was less pronounced when Ba2+ was replaced equimolarly by Ca2+. 4) Determination of Ca2+ channel inactivation using 90 mM Ba2+ in the pipettes indicated an overlap with channel activation in a limited voltage range, resulting in a steady-state "window" current. Inactivation can occur without divalent cation influx. 5) Formation of an inside-out patch resulted in a fast rundown of elementary Ca2+ channel currents. 6) Channel openings were often grouped in bursts. The lifetimes of the open state, the bursts, and the closed states were estimated for Ba2+ and Ca2+ as permeating ions. At least two exponentials were needed to fit the histogram of the lifetimes of all closed states. The lifetimes of the individual openings and bursts were mono-exponentially distributed. The kinetics of the Ca+ channel depended on the voltage and the permeating ion. During +30 mV depolarizations, no significant effect on the permeating ion on channel gating could be detected.(ABSTRACT TRUNCATED AT 400 WORDS)     

Divalent cations and the action potential of leech Retzius cells

Summary The effects of Sr, Ba, Mn, La and Co on the action potential of the leech Retzius cell were examined using intracellular recording techniques. A previous paper showed that these cells could fire Ca-dependent action potentials in Na-free solution provided TEA was present (Kleinhaus and Prichard, 1975). Under the same conditions Sr 1.5-20 mM was capable of substituting as a current carrier. Ba 2–25 mM added to normal Ringer prolonged the duration and increased the amplitude of the action potential of the Retzius cell, and supported action potentials without requiring TEA in Na-free solutions. The overshoots of the Sr- and Ba-dependent action potentials varied with a slope of 40 mM and 75 mV, respectively per 10-fold change in divalent cation concentration. Mn and La selectively blocked that portion of the action potential resulting from an inward movement of Ca, Sr or Ba without affecting the Na-dependent depolarization. The actions of Ca 1 mM on Sr-dependent action potentials were compatible with reversible competitive antagonism. In conclusion the findings: 1. support the proposition that outward K current must be blocked in order for divalent cations to dominate the Retzius cell's behavior during excitation. 2. characterize the divalent cation conductance channel as pharmacologically distinct from the Na conductance channel in the Retzius cell and similar to those described in several other excitable membranes. 3. suggest that the current carrying divalent cations probably flow through the same channel.     

Mechanism of action of magnesium on acetylcholine-evoked secretory responses in isolated rat pancreas.

This study investigates the effects of magnesium (Mg2+) on acetylcholine (ACh)-evoked secretory responses and calcium (Ca2+) mobilization in the isolated rat pancreas. ACh induced marked dose-dependent increases in total protein output and amylase release from superfused pancreatic segments in zero, normal (1 x 1 mM) and elevated (10 mM) extracellular Mg2+. Elevated Mg2+ attenuated the ACh-evoked secretory responses compared to zero and normal Mg2+. In the absence of extracellular Ca2+, but presence of 1 mM-EGTA (ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid), ACh elicited a small transient release of protein from pancreatic segments compared to a larger and more sustained secretion in the absence of both Ca2+ and Mg2+. Incubation of pancreatic segments with 45Ca2+ resulted in time-dependent uptake with maximum influx of 45Ca2+ occurring after 20 min of incubation period. ACh stimulated markedly the 45Ca2+ uptake compared to control tissues. In elevated extracellular Mg2+ the ACh-induced 45Ca2+ influx was significantly (P less than 0.001) reduced compared to zero and normal Mg2+. ACh also evoked dose-dependent increases in cytosolic free Ca2+ concentrations ([Ca2+]i) in pancreatic acinar cells loaded with the fluorescent dye Fura-2 AM. In elevated Mg2+ the ACh-induced cytosolic [Ca2+]i was significantly (P less than 0.001) reduced compared to zero and normal Mg2+. These results indicate that Mg2+ can influence ACh-evoked secretory responses possibly by controlling both Ca2+ influx and release in pancreatic acinar cells.     

Effects of the calcium ionophore A23187 on pancreatic acinar cell membrane potentials and amylase release.

1. The effects of the Ca2+-ionophore A23187 and the non-metabolizable cholinergic agonist bethanechol on acinar cell membrane potentials and amylase release from the superfused mouse pancreas were studied. 2. In the presence of extracellular Ca2+ (2.56 mM), A23187 (10(-5)M) and bethanechol (3 X 10(-5)M) caused an equal increase in the release of amylase. Both stimulants depolarized theacinar cells, A23187 by 6-0 mV and bethanechol by 12-3 mV. 3. When Ca2+ and Mg2+ were removed from the superfusate, the ability of A23187 to increase the rate of amylase release was virtually abolished, while the effect of bethanechol remained unaltered. Similarly, in the absence of these divalent cations, A23187 did not cause depolarization of the acinar cells, while depolarization in response to bethanechol was largely normal. Consequently it is unlikely that cholinergic agonists initiate secretion by activating a Ca2+-ionophore-like mechanism in the cell membrane. 4. When the concentration of Ca2+ in the medium was raised to 10 mM was the only extracellular divalent cation present, the depolarization in response to A23187 was increased to 11-8 mV. When Mg2+ in a concentration of 10 mM was the only extracellular divalent cation, the depolarization was only 2-1 mV. 5. The Ca2+ dependent, A23187-induced depolarization was abolished in the absence of Na+ (Tris substitution). Addition of Na+ to the superfusate caused an immediate depolarization. 6. It is concluded that the Ca2+ dependent depolarization of pancreatic acinar cells induced by A23187 is not directly due to an increased divalent cation conductance. Our findings are consistent with the view that the depolarization is due to an increased influx of Na+ resulting from a Ca2+ mediated increase in Na+ permeability.     

Gap junction hemichannel-mediated release of glutathione from cultured rat astrocytes

Release of glutathione (GSH) from astrocytes is essential for the supply of neurons with the GSH precursor cysteine. In order to test whether gap junction hemichannels could contribute to GSH release from astrocytes, we incubated astrocyte-rich primary cultures from neonatal rat brain in the absence of divalent cations, a condition that is known to increase the opening probability of hemichannels. During incubation in divalent cation free incubation solution (DCFS) the cells remained viable and released about 50% of the initial cellular GSH within 15 min. This extracellular GSH accumulation in DCFS was lowered by the presence of Ca2+ in a concentration dependent manner with a half-maximal inhibition at a Ca2+ concentration of 107+/-46 microM. Extracellular GSH accumulation in DCFS was also blocked by the divalent cations Mg2+, Ba2+ and Sr2+ as well as by the known gap junction inhibitors carbenoxolone (CBX), flufenamic acid (FFA) and lanthanum chloride. In contrast, the P2X7 receptor blocker brilliant blue G (BBG) did not affect GSH release in divalent cation free solution. This pharmacological profile strongly suggests that astrocytes are able to release GSH via open hemichannels. This release of GSH may have severe consequences for the antioxidative defense and for the GSH homeostasis in pathological brain.     

Divalent cation dependent inactivation of the high-voltage-activated Ca-channel current in chick sensory neurons

We have used the whole-cell clamp technique to investigate inactivation of the -conotoxin sensitive high-voltage-activated Ca-channel current (HVA current [2]) carried either by Ca, Ba or Sr (2.5 mM) in chick sensory neurons. At a low internal EGTA concentration (0.1 mM), Ca-channel currents clearly inactivated irrespective of the species of divalent cation carrying the current. During 150 ms pulses, current inactivated to 0.57, 0.67 and 0.75 of the peak current in Ca, Ba and Sr solution, respectively. Time constants of inactivation (26±10 ms and 280±50 ms, mean±S.D., in Ba) were largely independent of the membrane potential. Double-pulse experiments showed that the amount of inactivation left by a pre-pulse was proportional to the amplitude of the current evoked by the pre-pulse. No inactivation was induced by an outward current elicited by a strong depolarization to ü60 mV. With an internal EGTA concentration of 20 mM, the amount of inactivation was significantly smaller. In conclusion, the inactivation of the HVA Ca-channel currents during current flow depends mostly on the entry of divalent cations irrespective of their species.     

Effects of divalent cations on biphasic potassium contractures and on contractile inactivation in low calcium solutions in frog single twitch muscle fibers

When the concentration of external Ca2+ was reduced for 30 sec in a single twitch muscle fiber of a frog, the peak tension of the initial component of biphasic 80 mM K+ contractures was potentiated, whereas that of the secondary component was markedly inhibited, despite the fact that in the early stage it was potentiated usually in case of contractures in 60 and 40 nM K+ but rarely in 80 mM K+. These changes were not observed, however, in the presence of 0.5-1 nM Mn2+, i.e., Mn2+ having been substituted for Ca2+. The foregoing result and the authors' previously reported data indicated the following. First, the concentrations of divalent cations having an equal effect in bringing about the peak tensions of both components are 3 mM Mg2+, 0.7 mM Mn2+, 0.5 mM Ni2+, and 1.8 mM Ca2+. Secondly, this sequence constitutes their increasing order of effectiveness on the time course of the secondary component. Meanwhile, a similar order was found to exist in another experiment concerning the effectiveness in inhibiting the inactivation of K contractures facilitated by lowering the concentration of external Ca2+. All these findings indicated that the divalent cations act on the activation processes of both components in a stabilizing manner, differing from the way in which they act on the inactivation process of the secondary component. The mechanisms in which the peak tensions of the initial and secondary components are inhibited in a low Ca2+ solution and the divalent cations act on both components are discussed. Finally, another experiment made in the absence and presence of Ca2+ revealed that the effect of high concentrations of Mn2+ in the initial component is different from that on the secondary component.     

Inhibitory effects of cations on the Ca2+ response of water fibers in the frog tongue.

The effects of various cations on water fibers in the frog (Rana catesbeiana) tongue were investigated. The following results were obtained. 1. Water fibers responded well to Ca2+ and Sr2+. 2. SO2-(4) did not inhibit the Ca2+ response. Cl- at high concentrations had an inhibitory effect on the response to high Ca2+ stimulation. However, when the Ca2+ concentration was low, Cl- did not induce any inhibition. Therefore, low Ca2+ stimulation (0.1 mM CaCl2) was used to examine the inhibitory effect of cation. 3. MgCl2 and MgSO4 strongly inhibited the response to low Ca2+ stimulation. The inhibition by Mg salts was not caused by anion but by Mg2+. 4. The Ca2+ response was inhibited by various cations. The order of the inhibitory effects of cations was as follows: La3+ greater than Mn2+ = Mg2+ greater than or equal to Ni2+ greater than Co2+ greater than monovalent cations (Na+, K+, NH4+ and choline+). 5. The Sr2+ response, as well as the Ca2+ response, was inhibited by Na+ and Mg2+.     

Effects of guanidine on transmitter release and neuronal excitability.

1. Guanidine hydrochloride (CH5N3-HCl) was applied to frog neuromuscular junctions blocked by reduced external Ca2+, or increased external Mg2+ concentration, or by both. Guanidine produced a dose-dependent increase in the average number of quanta released by presynaptic action potentials, the threshold dose being 0-1-0-2 mM. No post-synaptic effects were observed. 2. Guanidine also increased the excitability of the motor nerve fibres, as evidenced by multiple firing to single electrical stimuli and finally by spontaneous action potentials. These effects were studied in greater detail in giant axons (Müller axons) in the spinal cord of lamprey. Exposure to guanidine produced in these axons a progressive increase in excitability, manifested by repetitive firing to a single electrical stimulus, spontaneous membrane potential oscillations and spontaneous bursts of action potentials. Guanidine had no effect on the resting potential. 3. The effect of guanidine on the excitability of Müller axons was mimicked in every detail simply by reducing the divalent cation concentration of the bathing solution. 4. Guanidine also produced dose-dependent increases in the duration of action potentials in Müller axons. This effect always preceded in time the appearance of the excitability effects and was not mimicked by reducing the divalent cation concentration. It is suggested that the broadening of the action potential is separate from the excitability effects and may reflect a decrease of delayed rectification. 5. Guanidine (0-3 mM) increased the frequency of miniature end-plate potentials (min. e.p.p.) in solutions containing 2-11 mM-K+ in such a way as to shift the relationship between min. e.p.p. frequency and extracellular K+ toward lower values of K+. This effect was interpreted to mean that guanidine produced a depolarization of the nerve terminal which summed with the depolarization produced by a given concentration of K+. The calculated depolarization produced by 0-3 mM guanidine was 5-7 mV. 6. The effects of guanidine on evoked transmitter release, excitability, and min. e.p.p. frequency are consistent with a hypothesis which states that guanidine binds at or near fixed negative changes on the outside of nerve membrane and reduces the screening effect of divalent cations.     

Catecholamine release from bovine chromaffin cells: the role of sodium-calcium exchange in ouabain-evoked release

Spontaneous catecholamine (CA) release from bovine chromaffin cells maintained in primary tissue culture has been measured after pre-loading the cells with [3H]noradrenaline. Ouabain inhibited 86Rb+ uptake and increased 3H release in a concentration-dependent manner during a 60 min incubation period. Low external Na+ (5 mM: Li+ substitution) also increased 3H release. Whereas the 3H-releasing action of ouabain was maintained, the Li(+)-evoked release decreased with time. The effects of both ouabain and low Na+ solution on 3H release were completely inhibited by removal of Ca2+ from the external medium even though in Ca2(+)-free solution ouabain further inhibited 86Rb+ uptake into the cells. Readmission of Ca2+ to Na(+)-loaded cells (10-4 M-ouabain in Ca2(+)-free-1 mM-EGTA solution for 60 min) markedly increased the release of 3H. In the additional presence of diphenylhydantoin (DPH, 10-4 M) 3H release was significantly less on Ca2+ readmission. The 3H release from Na(+)-loaded cells was proportional to the concentration of Ca2+ readmitted. The 3H release was further increased from Na(+)-loaded cells in response to Ca2+ readmission when [Na+]o was lowered from 149 to 5 mM (Li+, choline+, Tris+ or sucrose substitution) though Li+ was less effective than the other Na+ substitutes. Potassium removal from the external medium significantly inhibited the 3H release evoked by Ca2+ readmission to Na(+)-loaded cells, even when [Ca2+]o was greater than normal (7.5 mM) or if Ca2+ was readmitted in low [Na+]o solution. Rb+, Cs+ or Li+ could substitute for K+ with the order of potency: Rb+ greater than or equal to K+ greater than Cs+ greater than Li+. A slight increase of external K+ (10.8 mM) potentiated the 3H release from Na(+)-loaded cells on Ca2+ readmission, but a higher concentration of K+ (149.4 mM) had the opposite action. The data is consistent with the hypothesis that ouabain-evoked CA release from bovine chromaffin cells is, in part, a consequence of an internal Na(+)-dependent Ca2+ influx. The evidence also suggests that there is Na(+)-Ca2+ competition at the external arm of the exchanger together with a monovalent cation activation site.     

Interaction Between Store-Operated Non-Selective Cation Channels and the Na+-Ca2+ Exchanger During Secretion in the Rat Colon

The properties of capacitative Ca(2+) influx were studied using the whole-cell patch-clamp technique in crypts isolated from rat distal colon. Store-operated cation influx was evoked by increasing the intracellular buffering capacity for Ca(2+) in the pipette solution; contamination by Cl(-) currents was reduced by the use of NMDG gluconate as the main electrolyte in the pipette solution. The permeability of the non-selective cation conductance stimulated by store depletion had the following sequence for monovalent cations: Cs(+) > Na(+) > or = Li(+). The store-operated conductance is permeable to Na(+) and Ca(2+), but in contrast to Na(+), Ca(2+) also exerts a (feedback) inhibition on its own influx. Other divalent cations shared this inhibitory action with the sequence: Ca(2+) > or = Mg(2+) > or = Ba(2+) > or = Sr(2+). Fura-2 experiments revealed that replacement of extracellular Na(+) by NMDG(+) induced an increase in the intracellular Ca(2+) concentration, which was suppressed by the Na(+)-Ca(2+) exchange inhibitor, dichlorobenzamil, indicating the presence of a Na(+)-Ca(2+) exchanger within the colonic crypt cells. In Ussing chamber experiments dichlorobenzamil induced an increase in short-circuit current (I(sc)) in the majority of tissues tested indicating that this exchanger acts as a Ca(2+)-extruding transporter under physiological conditions. When Ca(2+)-dependent anion secretion was stimulated by the acetylcholine analogue carbachol, dichlorobenzamil no longer evoked an increase in I(sc), indicating that after stimulation of the store-operated cation conductance the Na(+)-Ca(2+) exchanger is turned off. Therefore, it is concluded that the influx of Na(+) across the non-selective store-operated cation conductance serves to reduce the driving force for Ca(2+) extrusion via the Na(+)-Ca(2+) exchanger and thereby maintains the increase in the intracellular Ca(2+) concentration during induction of secretion. Experimental Physiology (2001) 86.4, 461-468.     

Influence of divalent cations on potassium contracture duration in frog muscle fibres

Single fibres or small fibre bundles were dissected from twitch muscles of frogs and washed in low Cl-solutions. Contractures were provoked by 122.5 mM K⁺. At room temperature (17–20° C) the contracture duration was about 1.5 s in the absence of divalent cations and about 3 s in the presence of 2 mM Ca²⁺. Contractures were prolonged when Ca²⁺ was replaced by Ni²⁺ showing that inward Ca current was not the factor responsible for the contracture prolongation. K contracture duration was prolonged in the cold (3° C) by a factor of about 4 in the presence of non-permeating divalent cations (Ni, Co), when 0.1 mM La³⁺ was applied together with 2 mM Ca²⁺, and in the virtual absence of divalent cations. The contractures were prolonged in the cold by a factor of 6 or more in the presence of permeant divalent cations (Ca, Sr, Ba, Mg, and Mn at 8 mM). Diffusion of divalent cations in the transverse tubules of the muscle fibres was shown to have a Q₁₀ similar to that in free solution. It was concluded that inward current of divalent cations may shorten contracture duration by causing ionic depletion of the transverse tubules.     

A study of the mechanism of quantal transmitter release at a chemical synapse

1. The nerve-muscle preparation of the cutaneous pectoris of the frog has been used to study quantal transmitter release.2. When the osmotic pressure of the external solution is raised 1.5-2 fold, the frequency of miniature end-plate potentials (m.e.p.p.s) rises by 1.5-2 orders of magnitude. This effect is independent of the presence of Ca(2+) ions and of the nature of the substances by which the osmotic pressure has been increased.3. In Ca(2+) free hypertonic solution the nerve impulse still invades the nerve terminals but does not alter the frequency of the m.e.p.p.s.4. The arrival of the impulse in the terminals causes an immediate increase in the rate of quantal release, provided divalent cations are present whose passage through the axon membrane is facilitated by excitation (Ca(2+), Sr(2+), Ba(2+)).5. Divalent cations which penetrate only slightly (Mg(2+), Be(2+)) lower the frequency of m.e.p.p.s and suppress the end-plate potential (e.p.p.) evoked by an impulse, in the presence of Ca(2+) ions. Be(2+) is a more effective inhibitor than Mg(2+).6. In Ca(2+) free solutions, adding Mg(2+) causes an increase in the frequency of m.e.p.p.s evoked by depolarization of the nerve endings or by treatment with ethanol.7. The trivalent cation La(3+) is more effective than divalent cations are in increasing the frequency of m.e.p.p.s. The tetravalent cation Th(4+) also raises the m.e.p.p. frequency.8. The observations summarized in paragraphs 2-7 indicate that the frequency of m.e.p.p.s at a constant temperature depends only on the concentration of uni-, di- and trivalent cations inside the nerve ending. It is suggested that the internal cation concentration influences the adhesion between synaptic vesicles and the membrane of the nerve ending.9. For a model experiment, artificial phospholipid membranes have been used to study the effect of uni-, di-, tri- and tetravalent cations on the adhesion process. At pH 7-7.4, the time required for adhesion to take place decreases with increasing cation concentration in the bath. Ca(2+) ions are 100-1000 times more effective than K(+) ions; La(3+) and Th(4+) ions are still more effective. The ;adhesion time' decreases when the pH is lowered; it increases greatly with lowering of temperature.10. The hypothesis is put forward that the mutual adhesion of artificial vesicles made of phospholipid membranes, and the adhesion between synaptic vesicles and the membrane of the nerve ending arise by a common mechanism. In both cases, the important factor is the influence of cations on the electric double layer at the membrane surface.