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.     

Potency of depolarization-induced transmitter release is determined by divalent cation influx in PC 12 cells.

Evoked release of [3H]dopamine ([3H]DA) from pheochromocytoma cells (PC 12) is dependent on extracellular calcium ([Ca2+]ex), but it can take place if calcium ions (Ca2+) are substituted by other divalent ions such as strontium (Sr2+) and barium (Ba2+). The potency of the divalent cations at supporting release varies with the cell type; in PC 12 cells the order of potency is Ba2+ > Sr2+ > Ca2+. The close correlation between depolarization-evoked Ca2+ entry and depolarization-evoked transmitter release prompted us to examine whether the higher evoked transmitter release in the presence of Sr2+ correlates with an increased evoked Sr2+ influx. Influx studies were conducted on PC12 cells using a radioactive tracer (45Ca2+ or 85Sr2+, < 1 microM) in the presence of either Sr2+ (0.5 mM) or Ca2+ (0.5 mM). Depolarization with K Cl (60 mM) increased evoked 45Ca2+ influx 2-fold when Ca2+ was substituted with Sr2+. Similarly, evoked 85Sr2+ influx increased 1.87-fold by substituting Ca2+ for Sr2+. Thus the amount of evoked cation influx is determined by the type of divalent ion which is accessible in the extracellular medium, independently of the radioactive tracer used. Increased evoked transmitter release in the presence of Sr2+ was associated with increased evoked Sr2+ influx. This suggests that the potency of evoked transmitter release is determined predominantly by the influx of divalent cations. Furthermore, the steps subsequent to cation influx in the release process are equally efficient for both cations.     

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)     

Relaxation of contracted rabbit tracheal and human bronchial smooth muscle by Y-27632 through inhibition of Ca2+ sensitization.

The mechanism of Ca2+ sensitization of contraction has not been elucidated in airway smooth muscle (SM). To determine the role of a small G protein, rhoA p21, and its target protein, rho-associated coiled coil-forming protein kinase (ROCK), in receptor-coupled Ca2+ sensitization of airway SM, we studied the effect of (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexane carboxamide dihydrochloride, monohydrate (Y-27632), a ROCK inhibitor, on isometric contractions in rabbit tracheal and human bronchial SM. Y-27632 completely reversed 1 microM carbachol (CCh)-induced contraction of intact trachea with a concentration producing half-maximum inhibition of effect (IC50) of 1.29 +/- 0.2 microM (n = 5). Although 4beta-phorbol 12,13-dibutyrate (1 microM)-induced Ca2+ sensitization was relatively resistant to Y-27632 in alpha-toxin-permeabilized trachea, CCh (100 microM) plus guanosine triphosphate (GTP) (3 microM)- and guanosine 5'-O-(3'-thiotriphosphate) (10 microM)-induced contractions were relaxed completely by Y-27632 with IC50 of 1.44 +/- 0.3 (n = 6) and 1.15 +/- 0.3 microM (n = 6). Endothelin-1 (1 microM) plus GTP (3 microM)- developed force was also reversed by Y-27632 with IC50 of 4. 10 +/- 1.1 microM (n = 6) in the alpha-toxin-permeabilized bronchus. Both the rabbit and human SM expressed rhoA p21, ROCK I, and its isoform ROCK II. Collectively, rho/ROCK-mediated Ca2+ sensitization plays a central role in the sustained phase of airway SM contraction, and selective inhibition of this pathway may become a new strategy to resolve airflow limitation in asthma.     

Reversal of alpha 1-receptor mediated relaxation in intestinal smooth muscle

The alpha 1-receptor agonist phenylephrine relaxed longitudinal rabbit jejunal muscle contracted in vitro by low concentrations of barium ions (1 mM). When the Ba2+ concentration was increased to 10-15 mM the response to phenylephrine was a contraction, and at Ba2+ concentrations in between the high and low range this response was biphasic--a relaxation followed by a contractile phase. The alpha 2-receptor agonist clonidine did not affect the tone of the Ba2+ contracted preparation. When the muscle preparation was contracted by Sr2+ (1-20 mM) in the presence of Ca2+ (2.5 mM), phenylephrine relaxed it, and no contractile response to phenylephrine was observed. In the absence of extracellular Ca2+, 5 mM Ba2+ caused a contraction. Under these conditions phenylephrine had no effect on the tissue tone. When Ca2+ was added in a low concentration (0.2-2 mM), phenylephrine elicited a gradually increasing contractile response. At 5 mM Ca2+ the contractile response was replaced by the normal relaxation. The contractile response to phenylephrine in the presence of 5 mM Ba2+ and 2.5 mM Ca2+ was partially blocked by low concentrations of verapamil. In higher concentrations verapamil abolished the tissue tonus completely. The contractile response to phenylephrine in the presence of 5 mM Ba2+ and 2.5 mM Ca2+ could be reverted to the normal relaxation by the addition of 20 mM Mg2+. Increasing the K+ concentration from the normal 5.9 to 62.9 mM blocked the phenylephrine-induced relaxation. No contractile response to phenylephrine occurred. It is concluded that Ba2+ could reverse the response of alpha 1 receptor stimulation in rabbit jejunum from a relaxation to a contraction and that this contractile response was dependent on the presence of Ca2+.     

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+.     

Two components of the calcium current in the egg cell membrane of the tunicate.

The Ca current of the egg cell membrane of a certain tunicate, Halocynthia roretzi Drasche, was studied by the voltage-clamp technique. 2. The Ca current in the standard artificial sea water (ASW) was produced at the critical membrane potential of -10 mV after inactivating the Na current by conditioning depolarization, -30 to -15 mV. The Ca current was abolished by replacing Ca in ASW with Mg2+ or Mn2+. The Ca current was not significantly influenced by replacing Na in ASW with choline or Cs. 3. The relation of Ca current to the external Ca concentration was a monotonously increasing function, but was not linear. The current tended to saturate above 50 mM-Ca. In 100 mM-Ca ASW, the maximum peak inward current of Ca ranged from 1 to 7 X 10(-9) A. 4. The kinetics of Ca current was accurately analysed because of the small contribution of K outward current and was found to be relatively slow in comparison with the Na current. The peak time and the half-decay time of the maximum Ca current at about 25 mV were about 25 and 100 msec respectively in 100 mM-Ca ASW at 15 degrees C. 5. Addition of 20 mM-Co2+ to 100 mM-Ca ASW reduced Ca current to one fourth and 1 mM-La3+ to 100 mM-Ca ASW abolished the current. 6. Sr and Ba could substitute for Ca in Ca channels. The selectivity ratios for the 'Ca channels's were Ca (1-00):Sr(1-17):Ba(0-71) at a potential level of +40 mV. The Ca current in the egg cell membrane appeared to be essentially the same as the Ca current in the common excitable membranes, such as the crustacean muscle fibre. 7. The polyvalent cations including Ca ion and monovalent H+ ion showed the stabilizing effect upon both Na and Ca currents, by shifting V-I relations along the voltage axis. From the prediction of a theory of the diffuse double layer, the shift in the V-I relation induced by those cations should be directly related to their binding powers to the membrane. Thus, the sequence of the binding powers was inferred as H+ greater than La3+ greater than Co2+ greater than Mn2+ greater than Ca2+ greater than Sr2+ larger than or equal to Ba2+ greater than Mg2+. 9. In Na-free ASW, such as isotonic Ca ASW, Ca current was composed of two components. The one component was the Ca current described in 1 to 6. The other was also dependent upon the external Ca concentration, but showed the more negative critical membrane potential and the faster kinetics. It was concluded that this component should be the Ca current through Na channels. 10. The selectivity among Ca, Sr and Ba for 'Ca' current through 'Na channels' was significantly different from that of 'Ca' current through 'Ca channels', being Ca greater than Sr larger than or equal to Ba = 0.     

Delayed contractures induced by external cadmium ions in rat soleus muscle fibres

Cd(2+)-induced contractures began with a delay of approximately =4 min after adding 3 mM Cd2+ to external solutions that contained Cl- as the major anion. Tension increased to approximately =20% of peak tetanic tension after 30 min and was maintained after Cd2+ washout. Tension developed more rapidly at higher [Cd2+] (up to 10 mM). There was a lack of correlation between the delay before the contracture and contracture tension: (1) tension was reduced by 2 mM CO2+ or 50 microM nifedipine, although the delay remained at approximately =4 min, and (2) the delay fell to seconds when Cd2+ was added in SO42- solutions, although tension was the same as in Cl- solutions. Since (SO4)2- solutions swell T-tubules, Cd2+ may enter the T-system before inducing contractures. Cd(2+)-induced contractures depended on external [Ca2+] since they were reduced when Ca2+ was omitted from solutions. The contractures did not depend on activation of excitation-contraction coupling, since tension was not altered when the voltage sensor was inactivated by depolarization in 40 mM K+. A small contracture developed with 3 mM Zn2+, but not 3 mM Co2+ or La3+. Both Cd2+ and Zn2+ activated the contractile proteins in skinned fibres. Cd(2+)-induced contractures may depend on external Cd2+ releasing Ca2+ from the sarcoplasmic reticulum (SR), or on Cd2+ entering the fibre, releasing Ca2+ from the SR and/or directly activating the contractile proteins.     

Enhanced Ca2+ sensitization of the bronchial smooth muscle contraction in antigen-induced airway hyperresponsive rats

To investigate the alteration in acetylcholine (ACh)-induced increase in Ca2+ sensitization of bronchial smooth muscle contraction concurrent with the airway hyperresponsiveness (AHR), the ACh-induced increases in cytosolic Ca2+ ([Ca2+]) level and contractile response were simultaneously determined by using Fura-2 loaded bronchial smooth muscle. The left main bronchi were isolated from AHR rats which were sensitized and repeatedly challenged with DNP-Ascaris antigen. The tissue ring preparations were incubated in loading solution containing 10 microM Fura-2AM for 3 hr at room temperature. Then the isometrical contraction and [Ca2+]i (F340/F380) were monitored. Although the ACh (10(-3) M)-induced contractile response in AHR group (322 +/- 60 % of 60 mM K+ induced contraction) was significantly greater than that in control animals (173 +/- 15 %, p<0.05), the ACh (10(-3) M)-induced increase in [Ca2+]i was without significant difference between the two groups (128 +/- 15 and 171 +/- 29% of 60 mM K+ -induced increase in [Ca2+]i, respectively). These findings suggest that an augmentation of ACh-induced Ca2+ sensitization may occur in bronchial smooth muscle of the rats with antigen-induced AHR.     

Effects of different types of K+ channel modulators on the spontaneous myogenic contraction of guinea-pig urinary bladder smooth muscle.

In the present study, effects of different types of K+ channel modulators on the spontaneous rhythmic contractile activity were examined in guinea-pig urinary bladder smooth muscle (UBSM). Guinea-pig UBSM exhibited myogenic rhythmic contraction in the presence of atropine (1 microM), phentolamine (1 microM), propranolol (1 microM), suramin (10 microM) and tetrodotoxin (1 microM). Nisoldipine (100 nM) or diltiazem (10 microM) substantially diminished UBSM contractile activity. Nisoldipine-resistant component of UBSM rhythmic contraction was further inhibited by gadolinium (200 microM). Iberiotoxin (50 nM), a selective blocker of large-conductance, voltage-gated Ca2+-activated K+ (K(Ca)) (BK) channel, dramatically increased both contraction amplitude and frequency whereas NS-1619 (30 microM), which increases BK channel activity, decreased them. Apamin (100 nM), a selective blocker of small-conductance, K(Ca) (SK) channel, increased contraction amplitude but decreased frequency. A blocker of voltage-gated K+ (Kv) channel, 4-aminopyridine (100 microM), significantly increased contraction frequency. E-4031, a blocker of a novel inwardly rectifying K+ channel, i.e. the human ether-a-go-go-related gene (HERG) K+ channel, significantly increased contraction amplitude. Glibenclamide (1-10 microM) (K(ATP) channel blocker) and Ba2+ (10 microM) (conventional K(ir) channel blocker) did not exhibit conspicuous effects on spontaneous contractile activity of UBSM. These findings imply that two types of K(Ca) (BK and SK) channels have prominent roles as negative feedback elements to limit extracellular Ca2+ influx-mediated guinea-pig UBSM contraction by regulating both amplitude and frequency. It was also suggested that both non-K(Ca) type of K+ (Kv and HERG-like K+) channels may contribute to the regulation of UBSM myogenic rhythmic contraction.     

Adenosine affects the release of Ca2+ from the sarcoplasmic reticulum via A2A receptors in ferret skinned cardiac fibres

In this study, it was shown that adenosine potentiates caffeine-induced Ca2+ release. It was then proposed that the enhancement of the caffeine-induced Ca2+ release might occur by a direct effect on the ryanodine Ca2+ release channel or on other Ca2+ regulation mechanisms. Furthermore, A2A receptors may be functional on the ferret cardiac sarcoplasmic reticulum. Using chemically skinned fibres, experiments were conducted on ferret cardiac muscle to find out whether adenosine and the A1 and A2A adenosine receptor agonists (CCPA and CGS 21680) and antagonists (DPCPX and ZM 241385) affected caffeine-induced Ca2+ release and the Ca2+ sensitivity of contractile proteins. Changes in the caffeine-induced contracture brought about by adenosine and by adenosine-receptor agonists and antagonists were recorded in saponin-skinned fibres (50 microg ml(-1)). Tension-pCa relationships were then obtained by exposing Triton X-100-skinned fibres (1% v/v) sequentially to solutions of decreasing pCa. Adenosine (1-100 nm) and the specific A2A receptor agonist CGS 21680 (1-50 nm) produced a concentration-dependant potentiation of the caffeine-induced Ca2+ release from saponin-skinned fibres. The data plotted versus adenosine and CGS 21680 concentrations displayed sigmoid relationships (Hill relationship), with potentiation of Ca2+ release by 22.2 +/- 1.6 (n = 6) and 10.9 +/- 0.4% (n = 6), respectively. In addition, the potentiation of caffeine-induced Ca2+ release by adenosine (50 nm; 15.3 +/- 1.0%; n = 6) and by CGS 21680 (50 nm; 11.2 +/- 0.4%; n = 6) was reduced by the specific A2A receptor antagonist ZM 241385 (50 nm) to 8.0 +/- 1.4 (n = 4) and 5.4 +/- 1.2% (n = 4), respectively. The A1 receptor agonist CCPA (1-50 nm) and antagonist DPCPX (50 nm) had no significant effects on caffeine responses. In Triton X-100-skinned fibres, the maximal Ca(2+)-activated tension of the contractile proteins (41.3 +/- 4.1 mN mm(-2); n = 8), the Hill coefficient (nH = 2.2 +/- 0.1; n = 8) and the pCa50 (6.15 +/- 0.05; n = 8) were not significantly modified by adenosine (100 nm) or by CGS 21680 (50 nm).     

Muscarine affects calcium-currents in rat hippocampal pyramidal cells in vitro

Ca2+-currents were recorded from single CA3 pyramidal cells in hippocampal slice cultures, voltage-clamped through a single Cs+ - or K+-filled microelectrode and perfused with Hanks' balanced salt solution containing 1 microM tetrodotoxin and 10 mM tetraethylammonium. The Ca2+-current was reversibly reduced by bath-perfused muscarine (10-100 microM). This effect was inhibited by pirenzepine (10 microM) or atropine (1 microM). In K+-filled cells, inhibition was preceded by a phase of inward current enhancement; this was considered to be secondary to rapid outward current inhibition induced it by muscarine since it was reduced when outward currents were previously inhibited with Ba2+. In partially clamped or unclamped cells inhibition of Ca2+-current leads to a shortening of the Ca2+-spike plateau.     

Maitotoxin induces insertion of different ion channels into the Xenopus oocyte plasma membrane via Ca2+-stimulated exocytosis

The activation of cation channels in oocytes of Xenopus laevis by the marine poison maitotoxin (MTX) was monitored as membrane current (I(m)), conductance (Gm) and membrane surface area determined by continuous measurements of membrane capacitance (Cm). When MTX (25 pM) was added to the bathing solution there was an abrupt, large increase in inward membrane currents. Current/voltage relationships (I/V curves) were linear and suggested activation of voltage-independent non-selective cation channels (NSCC). MTX-induced Ca(2+)-sensitive currents were mainly carried by Na+ and were suppressed by low (0 mM) or high (40 mM) external Ca2+ concentrations and removal of Na+. Gadolinium (Gd3+, 10-500 microM) also had inhibitory effects, demonstrating the possible involvement of stretch-activated cation channels (SACC). In a high concentration (500 microM), amiloride substantially reduced the MTX-activated current while lower amiloride concentrations (50-100 microM) stimulated the current further. Continuous measurements of Cm revealed that MTX induced exocytotic delivery and functional insertion of new channel proteins into the plasma membrane, indicated by a Ca(2+)-dependent increase in membrane surface area by around 28%. From these data we conclude that MTX activates NSCC that require relatively high concentrations of amiloride to be blocked. Furthermore, MTX possibly stimulates activation of Gd(3+)- and Ca(2+)-sensitive mechanosensitive cation channels. Stimulation of these channels is achieved by exocytotic delivery and functional insertion of new channels into the plasma membrane in a pathway that depends on the presence of extracellular Ca2+.     

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.     

Effects of extracellular calcium and calcium channel blocker on silver-induced contractures in frog skeletal muscle fibers

The mechanism by which Ag+ induces muscle contracture was elucidated by investigating the effect of external Ca2+ concentration and Ca2+ channel blocker on the maximum tension amplitude in single fibers from frog toe skeletal muscle. Five microM Ag+ induced two different types of contracture in the presence of external Ca2+ more than 0.1 mM, i.e., a phasic and a subsequent tonic contracture. The phasic contracture appeared only in fibers with intact T-tubules immersed in a solution with or without Ca2+ after a lag time of 5.7 +/- 0.9 s (N = 5). The maximum amplitude was 58% of the tetanus tension observed in the same fiber immediately before Ag+ exposure. Diltiazem at high-concentration (100 microM) inhibited the Ag+-induced phasic contracture only to a small extent (17%). The contracture was not affected by 1 microM TTX or 1 mM DAP at all. These results indicate that Na+, K+, and Ca2+ channels on the T-tubular membrane would not be attributed to the phasic tension development induced by Ag+. On the contrary, a tonic contracture did not require intact T-tubules. The amplitude and the rate of rise of the contracture depended on external Ca2+ concentrations and were inhibited by a high concentration of diltiazem. Neither 1 microM TTX nor 1 mM DAP affected them. Therefore, the tonic contracture seems to be triggered by Ca2+ which entered the muscle fiber through the surface but not T-tubular membranes.     

Kinetics of the Ca(2+)-activated K+ channel in rat hippocampal neurons

The kinetics of the large-conductance Ca(2+)-activated K+ channel (235 pS in symmetrical 150 mM K+) were examined in the inside-out mode of the patch clamp technique. The open probability of the channel increased when [Ca2+]i, [Sr2+]i, or [Ba2+]i was increased. The [Ca2+]i-response relation was fitted with a Hill coefficient of 2 and half-maximum concentrations of 185, 80, 14.5, and 5.5 microM at -40, -20, +20, and +40 mV, respectively. The channel was blocked by TEA or Ba2+. The open-time histogram showed a single exponential component and the closed-time histogram showed at least two exponential components at various [Ca2+]i. Increasing [Ca2+]i decreased the time constant of the slow component of the closed-time histogram. Cell-attached patch recording revealed activation of the large-conductance Ca(2+)-activated K+ channel (BK channel) during the action potential. The deactivation time course was consistent with the fast after-hyperpolarization. A minimum model of the channel, close(2)-close(1)-open, where the transition from close(2) to close(1) requires the binding of 2 Ca2+, reconstructed quick activation of the channel if [Ca2+]i of 40 microM was assumed.     

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.     

BK channels in human glioma cells

Ion channels in inexcitable cells are involved in proliferation and volume regulation. Glioma cells robustly proliferate and undergo shape and volume changes during invasive migration. We investigated ion channel expression in two human glioma cell lines (D54MG and STTG-1). With low [Ca2+]i, both cell types displayed voltage-dependent currents that activated at positive voltages (more than +50 mV). Current density was sensitive to intracellular cation replacement with the following rank order; K+ > Cs+ approximately = Li+ > Na+. Currents were >80% inhibited by iberiotoxin (33 nM), charybdotoxin (50 nM), quinine (1 mM), tetrandrine (30 microM), and tetraethylammonium ion (TEA; 1 mM). Extracellular phloretin (100 microM), an activator of BK(Ca2+) channels, and elevated intracellular Ca2+ negatively shifted the I-V curve of whole cell currents. With 0, 0.1, and 1 microM [Ca2+]i, the half-maximal voltages, V(0.5), for whole cell current activation were +150, +65, and +12 mV, respectively. Elevating [K+]o potentiated whole cell currents in a fashion proportional to the square-root of [K+]o. Recording from cell-attached patches revealed large conductance channels (150-200 pS) with similar voltage dependence and activation kinetics as whole cell currents. These data indicate that human glioma cells express large-conductance, Ca2+ activated K+ (BK) channels. In amphotericin-perforated patches bradykinin (1 microM) activated TEA-sensitive currents that were abolished by preincubation with bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM). The BK channels described here may influence the responses of glioma cells to stimuli that increase [Ca2+]i.     

The effects of Ca2+ and Sr2+ at different modes of activation in the smooth muscle of the rat portal vein

The dependence of Ca2+ for different modes of activation has been investigated in the rat portal vein in vitro by evaluating the effects of substituting Sr2+ for Ca2+ in the extracellular fluid. For the spontaneous contractile activity as well as for contractures induced by depolarizing (high-K+) solution Sr2+ can to a large extent replace Ca2+. Sr2+ is however a poor Ca2+-substitute for the contractile response induced by alpha-stimulation (noradrenaline). The inhibition of spontaneous contractions by beta-stimulation (isoprenaline) is markedly less pronounced in Sr2+ solutions. Available data in the literature suggest that agonist interaction with alpha-receptors, in contrast to beta-receptors, is Ca2+ independent. Our results thus suggest that there exists a Ca2+ specific step between the alpha-receptor agonist occupancy and membrane excitation.     

Avian shell gland contractility: interaction of PGF2 alpha and arginine vasotocin with Ca2+

The effects of prostaglandin F2 alpha (PGF2 alpha) and arginine vasotocin (AVT) on the isometric contractile activity of avian shell gland longitudinal muscle strips were studied in relation to the role of extracellular Ca2+. PGF2 alpha and AVT stimulated contractile tension in a dose-related manner. This was attenuated when either of the Ca2+ channel blockers verapamil or R33956 was added to the muscle chamber baths. Ca2+-free solution containing 1 mM ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetraacetic acid completely prevented contraction in response to increasing doses of PGF2 alpha or AVT. Washing of the strips with Ca2+-free solution eliminated spontaneous contractile activity, but replenishment of CaCl2 to the medium (0.1-5.0 mM) restored it. Addition of PGF2 alpha to the Ca2+-free medium enhanced contractile tension during Ca2+ replenishment, whereas AVT had no effect on tension generation at low extracellular Ca2+ concentration (0.1-0.5 mM) but increased it at higher extracellular Ca2+ concentration (1.0-5.0 mM). PGF2 alpha stimulation was sensitive to extracellular Na+ concentration, whereas AVT-induced activity was not. Potassium depolarization (20 mM K) potentiated PGF2 alpha-stimulated activity, whereas the response to AVT was unaffected. At 127.5 mM K, AVT-stimulated activity was inhibited. PGF2 alpha-enhanced Ca2+-dependent tension generation was right shifted in a dose-related manner by AVT. These results suggest that extracellular Ca2+ is necessary for the full expression of PGF2 alpha- and AVT-stimulated muscle contraction and suggest that each agonist has a different mechanism of action.