Calcium-free External Solution Research Articles

Properties of Calcium-Activated Chloride Currents in Rat Purkinje Cerebellum Neurons

The presence of calcium-activated chloride current was shown using on freshly isolated rat Purkinje cerebellum neiurones and the pacth-clamp method in the whole-cell configuration. Chloride currents appeared in sodium-free external solution and reversibly disappeared in chloride-free or calcium-free external solution. Replacing of K+ ions (120 mM) to Cs+ ions in micropipette (120 mM) show the chloride currents even with 140 mM Na+ in external solution. This current was blocked to 80–100% by nifluminic acid (25–100 ΜM). It was found out that well known blockers of potassium channels tetraethylammonium (TEA) and 4-aminopyridine (4-AP) also effectively blocked chloride channels. The IC50 values for TEA and 4-AP were 130 ΜM, and 110 ΜM respectively. The action of TEA was reversible, while 4-AP at concentration 100 ΜM and above irreversibly blocked chloride channels.

GluN2A Subunit-Containing NMDA Receptors Are the Preferential Neuronal Targets of Homocysteine.

Homocysteine (HCY) is an endogenous redox active amino acid, best known as contributor to various neurodegenerative disorders. Although it is known that HCY can activate NMDA receptors (NMDARs), the mechanisms of its action on receptors composed of different NMDA receptor subunits remains almost unknown. In this study, using imaging and patch clamp technique in cultured cortical neurons and heterologous expression in HEK293T cells we tested the agonist activity of HCY on NMDARs composed of GluN1 and GluN2A subunits (GluN1/2A receptors) and GluN1 and GluN2B subunits (GluN1/2B receptors). We demonstrate that the time courses of Ca2+ transients and membrane currents activated by HCY and NMDA in cortical neurons are drastically different. Application of HCY to cortical neurons induced responses, which in contrast to currents induced by NMDA (both in the presence of glycine) considerably decreased to steady state of small amplitude. In contrast to NMDA, HCY-activated currents at steady state were resistant to the selective GluN2B subunit inhibitor ifenprodil. In calcium-free external solution the decrease of NMDA evoked currents was abolished, suggesting the Ca2+-dependent NMDAR desensitization. Under these conditions HCY evoked currents still declined almost to the baseline suggesting Ca2+-independent desensitization. In HEK293T cells HCY activated NMDARs of GluN1/2A and GluN1/2B subunit compositions with EC50s of 9.7 ± 1.8 and 61.8 ± 8.9 μM, respectively. Recombinant GluN1/2A receptors, however, did not desensitize by HCY, whereas GluN1/2B receptors were almost fully desensitized by HCY. Thus, HCY is a high affinity agonist of NMDARs preferring the GluN1/2A subunit composition. Our data suggest that HCY induced native NMDAR currents in neurons are mainly mediated by the “synaptic type” GluN1/2A NMDARs. This implies that in hyperhomocysteinemia, a disorder with enlarged level of HCY in plasma, HCY may persistently contribute to post-synaptic responses mediated by GluN2A-containing NMDA receptors. On the other hand, HCY toxicity may be limited by desensitization typical for HCY-induced activation of GluN2B-containing extrasynaptic receptors. Our findings, therefore, provide an evidence for the physiological relevance of endogenous HCY, which may represent an effective endogenous modulator of the central excitatory neurotransmission.

Localised calcium release events in cells from the muscle of guinea-pig gastric fundus.

After enzymatic dispersion of the muscle of the guinea-pig gastric fundus, single elongated cells were observed which differed from archetypal smooth muscle cells due to their knurled, tuberose or otherwise irregular surface morphology. These, but not archetypal smooth muscle cells, consistently displayed spontaneous localized (i.e. non-propagating) intracellular calcium ([Ca(2+)](i)) release events. Such calcium events were novel in their magnitude and kinetic profiles. They included short transient events, plateau events and events which coalesced spatially or temporally (compound events). Quantitative analysis of the events with an automatic detection programme showed that their spatio-temporal characteristics (full width and full duration at half-maximum amplitude) were approximately exponentially distributed. Their amplitude distribution suggested the presence of two release modes. Carbachol application caused an initial cell-wide calcium transient followed by an increase in localized calcium release events. Pharmacological analysis suggested that localized calcium release was largely dependent on external calcium entry acting on both inositol trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) to release stored calcium. Nominally calcium-free external solution immediately and reversibly abolished all localized calcium release without blocking the initial transient calcium release response to carbachol. This was inhibited by 2-APB (100 microm), ryanodine (10 or 50 microm) or U-73122 (1 microm). 2-APB (100 microm), xestospongin C (XeC, 10 microm) or U-73122 (1 microm) blocked both spontaneous localized calcium release and localized release stimulated by 10 microm carbachol. Ryanodine (50 microm) also inhibited spontaneous release, but enhanced localized release in response to carbachol. This study represents the first characterization of localized calcium release events in cells from the gastric fundus.

Bile acids induce calcium signals in mouse pancreatic acinar cells: implications for bile-induced pancreatic pathology.

The effect of the natural bile acid, taurolithocholic acid 3-sulfate (TLC-S), on calcium signalling in pancreatic acinar cells has been investigated. TLC-S induced global calcium oscillations and extended calcium transients as well as calcium signals localised to the secretory granule (apical) region of acinar cells. These calcium signals could still be triggered by TLC-S in a calcium-free external solution. TLC-S-induced calcium signals were not inhibited by atropine, but were abolished by caffeine or by depletion of calcium stores, due to prolonged application of ACh. Global calcium signals, produced by TLC-S application, displayed vectorial apical-to-basal polarity. The signals originated in the apical part and were then propagated to the basal region. Other natural bile acids, taurocholate (TC) and taurodeoxycholate (TDC), were also able to produce local and global calcium oscillations (but at higher concentrations than TLC-S). Bile, which can enter pancreas by reflux, has been implicated in the pathology of acute pancreatitis. The calcium releasing properties of bile acids suggest that calcium toxicity could be an important contributing factor in the bile acid-induced cellular damage.

Norepinephrine-induced calcium signaling pathways in afferent arterioles of genetically hypertensive rats.

This study provides new information about the relative importance of calcium mobilization and entry in the renal vascular response to adrenoceptor activation in afferent arterioles isolated from 7- to 8-wk-old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Intracellular free calcium concentration ([Ca(2+)](i)) was measured in microdissected arterioles utilizing ratiometric photometry of fura 2 fluorescence. There was no significant strain difference in baseline [Ca(2+)](i). Norepinephrine (NE; 10(-6) and 10(-7) M) elicited immediate, sustained increases in [Ca(2+)](i). The general temporal pattern of response to 10(-6) M NE consisted of an initial peak and a maintained plateau phase. The response to NE was partially blocked by nifedipine (10(-6) M) or 8-(N,N-diethylamino) octyl-3,4,5-trimetoxybenzoate (TMB-8; 10(-5) M). A calcium-free external solution abolished the sustained [Ca(2+)](i) plateau response to NE, with less influence on the peak response. In the absence of calcium entry, TMB-8 (10(-5) M) completely blocked the calcium response to NE in WKY but not SHR, suggesting strain differences in mobilization. A higher concentration of TMB-8 (10(-4) M), however, blocked all discernible mobilization in both strains. We conclude that there are differences in Ca(2+) handling in renal resistance vessels between young WKY and SHR with respect to mobilization stimulated by alpha-adrenoceptors. Afferent arterioles of young SHR appear to have a larger inositol-1,4,5-trisphosphate-sensitive pool or release from a site less accessible to TMB-8.

An examination of the secretion-like coupling model for the activation of the Ca2+ release-activated Ca2+ current I(CRAC) in RBL-1 cells.

One popular model for the activation of store-operated Ca2+ influx is the secretion-like coupling mechanism, in which peripheral endoplasmic reticulum moves to the plasma membrane upon store depletion thereby enabling inositol 1,4,5-trisphosphate (InsP3) receptors on the stores to bind to, and thus activate, store-operated Ca2+ channels. This movement is regulated by the underlying cytoskeleton. We have examined the validity of this mechanism for the activation of I(CRAC), the most widely distributed and best characterised store-operated Ca2+ current, in a model system, the RBL-1 rat basophilic cell line. Stabilisation of the peripheral cytoskeleton, disassembly of actin microfilaments and disaggregation of microtubules all consistently failed to alter the rate or extent of activation of I(CRAC). Rhodamine-phalloidin labelling was used wherever possible, and revealed that the cytoskeleton had been significantly modified by drug treatment. Interference with the cytoskeleton also failed to affect the intracellular calcium signal that occurred when external calcium was re-admitted to cells in which the calcium stores had been previously depleted by exposure to thapsigargin/ionomycin in calcium-free external solution. Application of positive pressure through the patch pipette separated the plasma membrane from underlying structures (cell ballooning). However, I(CRAC) was unaffected irrespective of whether cell ballooning occurred before or after depletion of stores. Pre-treatment with the membrane-permeable InsP3 receptor antagonist 2-APB blocked the activation of I(CRAC). However, intracellular dialysis with 2-APB failed to prevent I(CRAC) from activating, even at higher concentrations than those used extracellularly to achieve full block. Local application of 2-APB, once I(CRAC) had been activated, resulted in a rapid loss of the current at a rate similar to that seen with the rapid channel blocker La3+. Studies with the more conventional InsP3 receptor antagonist heparin revealed that occupation of the intracellular InsP3-sensitive receptors was not necessary for the activation or maintenance of I(CRAC). Similarly, the InsP3 receptor inhibitor caffeine failed to alter the rate or extent of activation of I(CRAC). Exposure to Li+, which reduces InsP3 levels by interfering with inositol monophosphatase, also failed to alter I(CRAC). Caffeine and Li+ did not affect the size of the intracellular Ca2+ signal that arose when external Ca2+ was re-admitted to cells which had been pre-exposed to thapsigargin/ionomycin in Ca2+-free external solution. Our findings demonstrate that the cytoskeleton does not seem to regulate calcium influx and that functional InsP3 receptors are not required for activation of I(CRAC). If the secretion-like coupling model indeed accounts for the activation of I(CRAC) in RBL-1 cells, then it needs to be revised significantly. Possible modifications to the model are discussed.

Role of the Na+/K+-ATPase in regulating the membrane potential in rat peritoneal mast cells.

1. The aim of this study was to investigate the effect of the Na+/K+-ATPase on the membrane potential of peritoneal mast cells isolated from male Sprague-Dawley SPF-rats. 2. Experiments were performed at 22-26 degrees C in the tight-seal whole-cell configuration of the patch-clamp technique by use of Sylgard-coated patch pipettes (3-6 M[omega]). High-resolution membrane currents were recorded with an EPC-9 patch-clamp amplifier controlled by the 'E9SCREEN' software. In addition, a charting programme on another computer synchronously recorded at low resolution (2 Hz) membrane potential and holding current (low-pass filtered at 500 Hz). 3. Na+/K+-ATPase activity was measured as the ouabain-sensitive change in the zero-current potential. The zero-current potential in rat peritoneal mast cells measured 2 min after obtaining whole-cell configuration amounted to 1.7 +/- 2.5 mV (n = 21). Ouabain (5 mM), a Na+/K+-ATPase-inhibitor, had only a very minor effect upon the membrane potential under resting conditions (n = 3). 4. When mast cells were superfused with nominal calcium-free external solution, the cells hyperpolarized (delta mV: 20.2 +/- 3.8 mV (n = 5)). In addition, when the mast cells were preincubated in nominal calcium-free external solution for 12 +/- 1.6 min before whole-cell configuration, the membrane potential amounted to -53.7 +/- 9.8 mV (n = 8). A subsequent superfusion with ouabain (5 mM) depolarized the membrane potential (ouabain-sensitive hyperpolarization (delta mV): 23.0 +/- 8.4 mV (n = 8)). 5. A high intracellular concentration of Na+ ([Na+]i) (26.6 mM) also resulted in hyperpolarization (delta mV: 20.2 +/- 9.1 mV (n = 7)), but only when ATP was present. A subsequent superfusion with ouabain (5 mM) repolarized these cells to -1.2 +/- 14 mV (ouabain-sensitive hyperpolarization (delta mV): 19.7 +/- 7.7 mV (n = 7)). 6. The size of the [Na+]i-dependent hyperpolarization was dose-dependent. Low [Na+]i (1 mM) had no effect on membrane potential and these cells were unaffected by superfusion with calcium-free external solution. 7. These data thus directly confirm that the stimulant effect of calcium-free external solutions on the ouabain-sensitive changes in the zero-current potential, and hence the Na+/K+-ATPase, is mediated through [Na+]i and that the activity of the Na+/K+-ATPase can have an important influence on the resting membrane potential in rat peritoneal mast cells.

Extracellular Ca2+-dependent and independent calcium transient in fetal myotubes

Spatio-temporal changes in the intracellular calcium concentration [Ca2+]i of dissociated mice myotubes from 14-day and 18-day-old fetuses were studied using digital imaging analysis of the Ca2+ indicator fura-2. Myotubes from 18-day-old fetuses displayed a transient [Ca2+]i increase upon electrical stimulation either in nominally calcium-free external solution or in Krebs solution containing 100 microM lanthanum. Thus, at this developmental stage, membrane depolarization appears to increase [Ca2+]i by stimulating Ca2+ release from the sarcoplasmic reticulum independently of extracellular Ca2+ influx. Similarly, myotubes from 14-day-old fetuses also showed a calcium transient upon electrical stimulation in Krebs solution. However, in 46% of these myotubes the calcium transient was abolished when Ca2+ entry through calcium channels was suppressed.

Membrane responses to norepinephrine in cultured brown fat cells.

We used the "perforated-patch" technique (Horn, R., and A. Marty, 1988. Journal of General Physiology. 92:145-159) to examine the effects of adrenergic agonists on the membrane potentials and membrane currents in isolated cultured brown fat cells from neonatal rats. In contrast to our previous results using traditional whole-cell patch clamp, 1-23-d cultured brown fat cells clamped with the perforated patch consistently showed vigorous membrane responses to both alpha- and beta-adrenergic agonists, suggesting that cytoplasmic components essential for the thermogenic response are lost in whole-cell experiments. The membrane responses to adrenergic stimulation varied from cell to cell but were consistent for a given cell. Responses to bath-applied norepinephrine in voltage-clamped cells had three possible components: (a) a fast transient inward current, (b) a slower outward current carried by K+ that often oscillated in amplitude, and (c) a sustained inward current largely by Na+. The fast inward and outward currents were activated by alpha-adrenergic agonists while the slow inward current was mediated by beta-adrenergic agonists. Oscillating outward currents were the most frequently seen response to norepinephrine stimulation. Activation of this current, termed IK,NE, was independent of voltage and seemed to be carried by Ca2(+)-activated K channels since the current oscillated in amplitude at constant membrane potential and gradually decreased when the cells were bathed with calcium-free external solution. IK,NE had a novel pharmacology in that it could be blocked by 4-aminopyridine, tetraethylammonium, apamin, and charybdotoxin. Both IK,NE and the voltage-gated K channels also present in brown fat (Lucero, M. T., and P. A. Pappone, 1989a. Journal of General Physiology. 93:451-472) may play a role in maintaining cellular homeostasis in the face of the high metabolic activity involved in thermogenesis.

Kinetics of the sodium current through EDTA-modified calcium channels of the molluscan neuron somatic membrane

The kinetics of the slow current carried by sodium ions through potential-dependent calcium channels after addition of EDTA to calcium-free external solution was investigated in experiments by the intracellular dialysis method on isolatedHelix pomatia neurons. The activation kinetics of this current was similar to that of the calcium current and could be described by the use of the square of the activation variable m in Hodgkin-Huxley equations. The decay (inactivation) kinetics of the induced sodium current during prolonged depolarization is biexponential in character. It is suggested that decay of the sodium currents takes place as a result of two independent processes: potential-dependent inactivation with a time constant τh∼1 sec, taking place as far as a certain steady-state level h∞, and a decrease in current connected with Na+ accumulation inside the cell during passage of the current and a consequent change in the sodium electrochemical potential (τc∼10 sec). It is concluded that modification of the calcium channels, so that they acquire the ability to conduct sodium, has no significant effect on the gating mechanisms responsible for opening and closing of the channels.

Selectivity of edta-modified calcium channels to monovalent cations

The characteristics of slow inward sodium currents arising in response to membrane depolarization were studied in experiments on isolated dialyzed neurons of the snailHelix pomatia when the calcium-chelating agent EDTA was added to the calcium-free external solution. Values of the relative permeability of the corresponding ionic channels, determined from the shift of the equilibrium potential, were: PNa+:PLi+: $$P_{NH_2 NH_3 } + :P_{OHNH_3 } $$ +=1.00:0.80:0.55:0.21. The ratio between these values for "fast" sodium channels was 1.00:1.04:0.44:0.19. The induced sodium current was blocked by D-600 and nifedipine, which block calcium channels, more effectively than the calcium current of the same membrane (the corresponding dissociation constants were 10−5 and 0.8·10−5 mole/liter for the induced sodium current compared with 2.6·10−5 and 2.3·10−5 mole/liter for the calcium current). It is postulated on the basis of these data that the calcium channels have a principal selective filter similar to that of sodium channels, but also an additional binding site for bivalent cations, which prevents entry of monovalent cations into the channel. The addition of calcium-chelating agents to the calcium-free external solution liberates this site and thereby modifies the calcium channel into a sodium channel.

Prolonged action potentials in Aplysia neurones injected with egta

1. 1. Aplysia neurones injected with EGTA generate prolonged action potentials during repetitive activity. 2. 2. The prolonged action potentials were abolished in calcium-free external solution and following the injection of a calcium-EGTA buffer containing 5·25 × 10 −7 M free calcium. 3. 3. The ionic mechanism may be similar to that of action potentials recorded from cardiac muscle.