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)     

Protein and diacylglycerol phosphorylation in the stimulus-secretion coupling of rat mast cells.

The pattern of endogenous protein phosphorylation during stimulation of rat peritoneal mast cells by two types of agonists has been compared. Compound 48/80, substance P and histone, which do not require the presence of external Ca2+ to trigger histamine release, induced a similar profile of phosphorylation comprising an increased phosphorylation of a 35,000 molecular weight (MW) protein and dephosphorylation of a 15,000 MW protein. The same profile was seen when the cells were stimulated with phorbol-12-myristate-13-acetate. The phorbol ester also induced histamine release, although less than that caused by the other secretagogues. The pattern of phosphorylation shared by both the phorbol ester and the basic secretagogues represented only part of that observed when the cells were stimulated in a Ca2+-free medium with anti-IgE. Under those conditions, two additional proteins of 68,000 and 56,000 MW became phosphorylated. The phosphorylation of these two proteins increased when anti-IgE was applied in the presence of Ca2+. In contrast, the extent of phosphorylation of the 35,000 MW protein was diminished. Both the basic secretagogues and anti-IgE, but not the phorbol ester, also enhanced the production of phosphatidic acid, indicating that diacylglycerol was generated. This process was independent of the presence of external Ca2+. It is suggested that protein kinase C activation is responsible for the phosphorylation observed with the basic secretagogues but not entirely with IgE-directed ligands.     

Capacitative Ca2+ influx and activation of the neutrophil respiratory burst. Different regulation of plasma membrane- and granule-localized NADPH-oxidase

The neutrophil NADPH-oxidase may be activated in the plasma membrane, resulting in release of oxygen metabolites extracellularly, or in the granule or phagosomal membranes, giving intracellular production of oxidants. An increase in [Ca2+]i mediated through binding of fMLF to its receptor is part of a signaling cascade that activates the plasma membrane-localized oxidase. In contrast, a rise in [Ca2+]i induced by a Ca2+ ionophore results in activation of the intracellular pool of oxidase. We mimicked fMLF-induced emptying of intracellular Ca2+ stores with thapsigargin. This induced a pronounced intracellular oxidase activity but no extracellular release of oxidants. The thapsigargin-induced effect was dependent on capacitative Ca2+ influx, because the effect was inhibited dose-dependently by EGTA and the Ca2+ channel blocker La3+. At La3+ concentrations between 200 and 400 microM, thapsigargin also induced a massive extracellular production of superoxide anion. No other channel blockers tested induced a similar effect. We conclude that elevation in [Ca2+]i by capacitative Ca2+ influx induces NADPH-oxidase activation at an intracellular site. Further, activation of the plasma membrane-localized NADPH-oxidase is regulated by a more complex Ca2+ signaling, involving capacitative Ca2+ influx and possibly the specific action of La3+-sensitive Ca2+ channels.     

Dual actions of phorbol esters to decrease calcium and potassium conductances of mouse neurons

Phorbol esters, which substitute for diacylglycerol to activate protein kinase C, were applied to mouse dorsal root ganglion and cerebral hemisphere neurons in cell culture. The phorbol esters, phorbol 12,13-dibutyrate and 12-O-tetradecanoyl-phorbol-13-acetate, prolonged calcium-dependent action potential duration at resting membrane potential and at more negative membrane potentials but decreased action potential duration following membrane depolarization to less than -45 mV. When calcium and potassium currents were recorded using the single electrode voltage-clamp technique, the phorbol esters were shown to reduce both voltage-dependent calcium and potassium currents. These studies have demonstrated directly that phorbol esters, presumably by activating protein kinase C, can modify more than one membrane conductance in individual neurons.     

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.     

Evidence that TRH stimulates secretion of TSH by two calcium-mediated mechanisms

Thyrotropin-released hormone (TRH) stimulation of thyrotropin (TSH) release from mouse thyrotropic tumor (TtT) cells is dependent on Ca2+. We demonstrate that TRH action in TtT cells does not require extracellular Ca2+ but that Ca2+ influx induced by TRH can augment TSH secretion. TRH caused a 46% increase in 45Ca2+ uptake by TtT cells in medium with 100 micro M Ca2+. The increment in 45Ca2+ uptake caused by TRH was dependent on the concentration of Ca2+ in the medium. In contrast to the effect of 50 mM K+, which also causes Ca2+ influx, TRH caused 45Ca2+ efflux and TSH release from TtT cells even when the concentration of Ca2+ in the medium was lowered below 100 micro M. TRH stimulated TSH release during perifusion in medium in which the free Ca2+ concentration was lowered to approximately 0.02 micro M, and reintroduction of Ca2+ into the medium simultaneously with TRH markedly increased TSH release. We suggest that TRH may affect Ca2+ metabolism in TtT cells by both extracellular Ca2+-independent and -dependent mechanisms and that this dual mechanism of action serves to augment further TSH secretion induced by TRH.     

Cholecystokinin-stimulated insulin secretion and protein kinase C in rat pancreatic islets

In isolated rat pancreatic islets, the possible involvement of protein kinase C in cholecystokinin-8-stimulated insulin secretion was investigated. In islets exposed for 24 hours to the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (500 nmol l-1), a procedure known to down-regulate islet protein kinase C-activity, the insulinotropic effect of cholecystokinin-8 (10(-7) mol l-1) was partially reduced (by 34 +/- 8%, P less than 0.001). In contrast the insulinotropic response to acute exposure to 12-O-tetradecanoyl phorbol 13-acetate (10(-6) mol l-1) was totally abolished (P less than 0.001), whereas the insulin response to glucose (8.3 mmol l-1) was not affected. In normal islets, the protein kinase C-inhibitor, staurosporine, inhibited 12-O-tetradecanoyl phorbol 13-acetate- and glucose-stimulated insulin secretion (P less than 0.01), but was without effect on cholecystokinin-8-stimulated insulin release. Furthermore, in normal islets, cholecystokinin-8 had no effect on insulin release at a low glucose level (3.3 mmol l-1). However, at this low glucose level, cholecystokinin-8 clearly potentiated insulin release induced by acute exposure to 12-O-tetradecanoyl phorbol 13-acetate (10(-8) -10(-6) mol l-1, P less than 0.001). This potentiating effect was abolished by the removal of extracellular Ca2+. It is concluded that the insulinotropic effect of cholecystokinin-8 in rat islets is partially mediated by the protein kinase C pathway. Furthermore, the lack of effect of cholecystokinin-8 on insulin secretion at a low glucose level might be explained by an insufficient activation of protein kinase C under these conditions.     

The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate,enhances the evoked quanta release of acetylcholine at the frog neuromuscular junction

The possible role of protein kinase C in the regulation of quantal transmitter release was studied at the frog neuromuscular junction by using the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a compound known to mimic the effects of the physiological activator of the enzyme, endogenous diacylglycerol. The main effect of the phorbol ester was to increase the quantal content,m, of the endplate potential. The initial values ofm were adjusted over a wide range by changing the Ca²⁺ concentration of the extracellular medium, and the TPA-induced fractional increase inm was significantly greater at junctions with a lower initial quantal content. On the other hand, the absolute increases inm induced by the phorbol ester were positively correlated with the square root of the initial quantal content. The possible physiological significance of this correlation is discussed in view of the well known relationship between extracellular Ca²⁺ concentration and the quantal content of the end plate potential.     

The ionic mechanism of the excitatory action of glutamate upon the membranes of motoneurones of the frog

Simultaneous intra- and extracellular recordings with K+, Na+, Ca2+, and Cl- sensitive microelectrodes were performed in motoneurones of the spinal cord of the frog during depolarizations mediated by glutamate (GLUT) and by experimentally increased extracellular K+. Depolarization resulting from increased K+ activity (alpha K+) in the bathing solution evoked a decrease of intracellular Na+ activity (alpha Na+i); a transient increase of alpha Na+i accompanied by a decrease of alpha Na+e was observed during the depolarization induced by GLUT. Both modes of depolarization led to an increase of alpha Cl-i and a concomitant decrease of alpha Cl-e. An experimental increase of alpha K+e led to a threshold dependent increase of alpha Ca2+i by at least one order of magnitude and to an equally threshold dependent strong decrease of alpha Ca2+e. The threshold of these changes of alpha Ca2+ was at a membrane potential of -25 mV. During a depolarization of half the amplitude induced by GLUT a comparable increase of alpha Ca2+i and a smaller decrease of alpha Ca2+e were observed. The GLUT mediated changes of alpha Ca2+ were not threshold dependent and occurred synchronously with the onset of depolarization. A transient decrease of alpha K+i and a parallel strong increase of alpha K+e occurred during the GLUT induced depolarization. Depolarization evoked by an experimental increase of alpha K+e led to an increase of alpha K+i. The observed changes in the ionic composition of the intra- and extracellular fluids indicate that GLUT evokes an increase in membrane permeability to Na+ and Ca2+ and a subsequent influx of these ions into motoneurones, while the inward shift of Cl- and the outward shift of K+ are presumably passive. A voltage dependent Ca2+ influx is triggered at -25 mV membrane potential.     

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.     

Mitochondrial Ca2+ flux is a critical determinant of the Ca2+ dependence of mast cell degranulation

An increase in intracellular Ca2+ ([Ca2+]i) is necessary for mast cell exocytosis, but there is controversy over the requirement for Ca2+ in the extracellular medium. Here, we demonstrate that mitochondrial function is a critical determinant of Ca2+ dependence. In the presence of extracellular Ca2+, mitochondrial metabolic inhibitors, including rotenone, antimycin A, and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), significantly reduced degranulation induced by immunoglobulin E (IgE) antigen or by thapsigargin, as measured by beta-hexosaminidase release. In the absence of extracellular Ca2+; however, antimycin A and FCCP, but not rotenone, enhanced, rather than reduced, degranulation to a maximum of 76% of that observed in the presence of extracellular Ca2+. This enhancement of extracellular, Ca2+-independent degranulation was concomitant with a rapid collapse of the mitochondrial transmembrane potential. Mitochondrial depolarization did not enhance degranulation induced by thapsigargin, irrespective of the presence or absence of extracellular Ca2+. IgE antigen was more effective than thapsigargin as an inducer of [Ca2+]i release, and mitochondrial depolarization augmented IgE-mediated but not thapsigargin-induced Ca2+ store release and mitochondrial Ca2+ ([Ca2+]m) release. Finally, atractyloside and bongkrekic acid [an agonist and an antagonist, respectively, of the mitochondrial permeability transition pore (mPTP)], respectively, augmented and reduced IgE-mediated Ca2+ store release, [Ca2+]m release, and/or degranulation, whereas they had no effects on thapsigargin-induced Ca2+ store release. These data suggest that the mPTP is involved in the regulation of Ca2+ signaling, thereby affecting the mode of mast cell degranulation. This finding may shed light on a new role for mitochondria in the regulation of mast cell activation.     

Ca2+ influx in human T lymphocytes is induced independently of inositol phosphate production by mobilization of intracellular Ca2+ stores. A study with the Ca2+ endoplasmic reticulum-ATPase inhibitor thapsigargin.

Thapsigargin (TG), a sesquiterpene lactone and non-phorbol 12-myristate 13-acetate tumor promoter, stimulates a rapid increase in intracellular free Ca2+ [( Ca2+]i) in human T lymphocytes clone P28. The [Ca2+]i response to TG is sustained in the presence of 1 mM extracellular Ca2+, while it becomes transient in Ca2(+)-free medium suggesting that TG activates both the release of Ca2+ from intracellular stores and the entry of Ca2+ from extracellular spaces. TG-induced Ca2+ influx is completely abolished after cell depolarization caused by increased extracellular concentrations of K+. The rise in [Ca2+]i stimulated by TG occurs in the absence of detectable production of inositol phosphates. Moreover, TG does not alter the early biochemical events of T cell activation triggered through the CD2 or the CD3 T cell antigens. Indeed, both inositol phosphate production and intracellular pH increase induced by specific monoclonal antibodies (mAb) remain unchanged after TG treatment. These data suggest that in human T lymphocytes TG releases Ca2+ from an intracellular pool by a mechanism which is independent of the phospholipase C metabolic pathway. Preincubation with TG of T cell clone P28 empties both the CD2 and the CD3-sensitive intracellular Ca2+ pool(s). Conversely, prestimulation of T cell clone P28 by CD3 or CD2-specific mAb inhibits the Ca2(+)-mobilizing effect of TG. Thus it appears that TG and CD2- or CD3-specific mAb mobilize Ca2+ from common Ca2+ pool(s). Taken together, these results demonstrate that Ca2+ influx in human T cells may be linked to mobilization of intracellular Ca2+ pools and by a mechanism independent of phosphoinositide hydrolysis. They further indicate that the release of intracellular Ca2+ pool(s) may play a major role in the opening of cell membrane Ca2+ channels observed during the CD2- or CD3-induced stimulation of human T lymphocytes.     

Further characterization of [3H]gamma-aminobutyric acid release from isolated neuronal growth cones: role of intracellular Ca2+ stores

We have recently shown that growth cones isolated from neonatal rat forebrain possess uptake and release mechanisms for the neurotransmitter gamma-aminobutyric acid. About half of the K+-induced release of [3H]gamma-aminobutyric acid from isolated growth cones is dependent on extracellular Ca2+. The remaining component of the [3H]gamma-aminobutyric acid release is unaffected by removal of extracellular Ca2+ and is resistant to blockade by the voltage-sensitive Ca2+-channel blocker methoxyverapamil. In the present series of experiments we have used caffeine to assess the possible role of intracellular stores of Ca2+ in supporting that component of the K+-induced release of [3H]gamma-aminobutyric acid from isolated growth cones that is independent of extracellular Ca2+. We have chosen caffeine because of its well established effect of releasing Ca2+ from smooth endoplasmic reticulum in muscle. We found that caffeine can release [3H]gamma-aminobutyric acid from isolated growth cones. This effect persists in Ca2+-free medium, in the presence of methoxyverapamil and in the absence of Na+. Furthermore, isobutylmethylxanthine could not substitute for caffeine suggesting that the caffeine effect is not due to phosphodiesterase inhibition and the subsequent rise in intracellular cyclic nucleotides. A combination of the mitochondrial poisons, Antimycin A and sodium azide had no effect on the release of [3H]gamma-aminobutyric acid induced either by caffeine or by high K+. We conclude that caffeine causes the release of Ca2+ from a non-mitochondrial store within the growth cone and that this Ca2+ store supports that component of the K+-induced release of [3H]gamma-aminobutyric acid that is independent of extracellular Ca2+.     

Macelignan Inhibits Histamine Release and Inflammatory Mediator Production in Activated Rat Basophilic Leukemia Mast Cells

Type I allergy is characterized by the release of granule-associated mediators, lipid-derived substances, cytokines, and chemokines by activated mast cells. To evaluate the anti-allergic effects of macelignan isolated from Myristica fragrans Houtt., we determined its ability to inhibit calcium (Ca(2+)) influx, degranulation, and inflammatory mediator production in RBL-2?H3 cells stimulated with A23187 and phorbol 12-myristate 13-acetate. Macelignan inhibited Ca(2+) influx and the secretion of β-hexosaminidase, histamine, prostaglandin E(2), and leukotriene C(4); decreased mRNA levels of cyclooxygenase-2, 5-lipoxygenase, interleukin-4 (IL-4), IL-13, and tumor necrosis factor-α; and attenuated phosphorylation of Akt and the mitogen-activated protein kinases extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase. These results indicate the potential of macelignan as a type I allergy treatment.     

Extracellular accumulation of glutamate in the hippocampus induced by ischemia is not calcium dependent--in vitro and in vivo evidence

Calcium dependency of ischemia-induced increase in extracellular glutamate in the hippocampus was studied in vitro and in vivo. Perfusion of a low pO2 medium without glucose (in vitro ischemia) induced an increase in extracellular glutamate in rat hippocampal slices. This increase did not depend on Ca2+, which is in contrast with the observation that about 40% of membrane depolarization (50 mM KCl)-evoked release was Ca2+-dependent. In vivo cerebral ischemia of 5 min duration in gerbils also caused Ca2+-independent increase in extracellular glutamate in the hippocampus. The data suggest that the increase in extracellular glutamate induced by ischemia is not due to the enhanced release of neurotransmitter glutamate.     

Role of calcium in neurotransmitter release evoked by alpha-latrotoxin or hypertonic sucrose

At the synapse, neurotransmitter release is triggered physiologically by Ca(2+) influx through voltage-gated Ca(2+) channels. Non-physiologically, release can be evoked by a potent neurotoxin, alpha-latrotoxin, and by hypertonic sucrose. Controversy has arisen on whether release evoked by alpha-latrotoxin and hypertonic sucrose requires extracellular Ca(2+) or Ca(2+) from intracellular stores. Using synaptosomes, we have studied the Ca(2+) dependence of alpha-latrotoxin and sucrose action in different neurotransmitter systems. In agreement with previous data, no requirement for extracellular Ca(2+) in sucrose-induced secretion of norepinephrine, dopamine, glutamate or GABA was detected. Unexpectedly, we observed large differences between these neurotransmitters in the Ca(2+) dependence of alpha-latrotoxin-stimulated release: norepinephrine release required Ca(2+), dopamine release was only partially Ca(2+) dependent, and glutamate and GABA release did not require Ca(2+). To test if Ca(2+) derived from intracellular Ca(2+) stores participates in neurotransmitter release triggered by alpha-latrotoxin or hypertonic sucrose, we employed thapsigargin, a Ca(2+)-ATPase inhibitor that empties Ca(2+) stores. Thapsigargin did not induce neurotransmitter release, nor did it inhibit subsequent release stimulated by KCl depolarization, hypertonic sucrose or alpha-latrotoxin. However, intracellular Ca(2+) performs an important regulatory function, since thapsigargin increased the size of the readily releasable pool as measured by stimulation with hypertonic sucrose. This effect required extracellular Ca(2+) and protein kinase C, suggesting that depletion of internal Ca(2+) stores leads to store-operated Ca(2+) entry. The resulting Ca(2+) influx does not trigger release by itself, but activates protein kinase C that increases the readily releasable pool of neurotransmitters.Our data show that internal and external Ca(2+) is not acutely involved in hypertonic sucrose-evoked neurotransmitter release, while alpha-latrotoxin-triggered release requires external Ca(2+) for a subset of neurotransmitters. Although internal Ca(2+) is not essential for release, it modulates its extent, implying that the emptying of intracellular stores by activation of presynaptic receptors plays an important regulatory role in neurotransmitter release.     

RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels

The molecular organization of presynaptic active zones is important for the neurotransmitter release that is triggered by depolarization-induced Ca2+ influx. Here, we demonstrate a previously unknown interaction between two components of the presynaptic active zone, RIM1 and voltage-dependent Ca2+ channels (VDCCs), that controls neurotransmitter release in mammalian neurons. RIM1 associated with VDCC beta-subunits via its C terminus to markedly suppress voltage-dependent inactivation among different neuronal VDCCs. Consistently, in pheochromocytoma neuroendocrine PC12 cells, acetylcholine release was significantly potentiated by the full-length and C-terminal RIM1 constructs, but membrane docking of vesicles was enhanced only by the full-length RIM1. The beta construct beta-AID dominant negative, which disrupts the RIM1-beta association, accelerated the inactivation of native VDCC currents, suppressed vesicle docking and acetylcholine release in PC12 cells, and inhibited glutamate release in cultured cerebellar neurons. Thus, RIM1 association with beta in the presynaptic active zone supports release via two distinct mechanisms: sustaining Ca2+ influx through inhibition of channel inactivation, and anchoring neurotransmitter-containing vesicles in the vicinity of VDCCs.     

Calcium-dependent potentials in mammalian red nucleus neurons in vitro

Intracellular recordings were made from red nucleus (RN) neurons in guinea-pig slice preparations. The slow afterhyperpolarization (AHP) following an action potential was reversibly abolished by Co2+ or Mn2+. Its amplitude was dependent on the extracellular K+ concentration. When tetraethylammonium was added to the perfusing solution, a tetrodotoxin-resistant regenerative depolarization was evoked which was blocked by Co2+ or Mn2+. There results suggest that the slow AHP is produced by an increase in Ca2+-dependent K+ conductance and that RN neurons have a voltage-dependent Ca2+ conductance.     

Source of calcium for contractions induced by depolarization and muscarinic receptor stimulation in rabbit urinary bladder

Omission of calcium or the inclusion of lanthanum in the bathing medium resulted in an almost complete inhibition of contractile responses induced by either K+ depolarization or carbachol in strips of rabbit urinary bladder. D-600 inhibited K+-induced contractions significantly more than carbachol-induced responses. The influx of 45Ca into cells was stimulated both by K+ depolarization and carbachol. Over a 2-min period the increase in 45Ca influx induced by high K+ and carbachol was 98 and 65%, respectively. Both lanthanum and D-600 blocked 45Ca influx stimulated by either K+ depolarization or carbachol. The inhibition of 45Ca influx by these calcium-channel blocking agents, particularly by D-600, was dependent on the length of exposure. Application of carbachol during 45Ca efflux in pre-loaded muscle strips had no effect on the rate of 45Ca efflux. These results indicate that the contractile responses of the urinary bladder to depolarization and to carbachol are highly dependent on an extracellular source of calcium.