Cellular mechanisms underlying carbachol-induced oscillations of calcium-dependent membrane current in smooth muscle cells from mouse anococcygeus

1. At a holding potential of -40 mV, carbachol (50 microM) produced a complex pattern of inward currents in single smooth muscle cells freshly isolated from the mouse anococcygeus. Membrane currents were monitored by the whole-cell configuration of the patch-clamp technique. Previous work has identified the first, transient component as a calcium-activated chloride current (ICl(Ca)) and the second sustained component as a store depletion-operated non-selective cation current (I(DOC)). The object of the present study was to examine the cellular mechanisms underlying the third component, a series of inward current oscillations (I(oscil)) superimposed on I(DOC). 2. Carbachol-induced I(oscil) (amplitude 97 +/- 11 pA; frequency 0.26 +/- 0.02 Hz) was inhibited by the chloride channel blocker anthracene-9-carboxylic acid (A-9-C; 1 mM), and by inclusion of 1 mM EGTA in the patch-pipette filling solution. 3. In calcium-free extracellular medium (plus 1 mM EGTA), carbachol produced an initial burst of oscillatory current which lasted 94 s before decaying to zero; I(oscil) could be restored by re-admission of calcium. The frequency, but not the amplitude, of I(oscil) increased with increasing concentrations of extracellular calcium (0.5-10 mM). 4. Inclusion of the inositol triphosphate (IP3) receptor antagonist heparin (5 mg ml(-1) in the patch-pipette filling solution, or pretreatment of cells with the sarcoplasmic reticulum (SR) calcium ATPase inhibitor cyclopiazonic acid (CPA; 10 microM), prevented the activation of I(oscil) by carbachol. Caffeine (10 mM) activated both ICl(Ca) and I(DOC) and prevented the induction of I(oscil) by carbachol. Caffeine and CPA also abolished I(oscil) in the presence of carbachol, as did both a low (3 microM) and a high (30 microM) concentration of ryanodine. 5. Carbachol-induced I(oscil) was abolished by the general calcium entry blocker SKF 96365 (10 MM) and by Cd2+ (100 microM), but was unaffected by La3+ (400 microM). As found previously, I(DOC) was also blocked by SKF 96365 and Cd2+, but not La3+; the inhibition of I(DOC) preceded the abolition of I(oscil) by 27 s with SKF 96365 and by 30 s with Cd2+. Nifedipine (1 microM) produced a partial inhibition of the carbachol-induced I(oscil) frequency at holding potentials of -20 mV and -60 mV and, in addition, reduced I(DOC) at -60 mV by 18%. 6. It is concluded that carbachol-induced inward current oscillations in mouse anococcygeus cells are due to a calcium-activated chloride current, and reflect oscillatory changes in cytoplasmic calcium ion concentration. These calcium oscillations are derived primarily from the SR stores, but entry of calcium into the cell is necessary for store replenishment and maintenance of the oscillations. Capacitative calcium entry (via I(DOC) appears to be important not only for sustained contraction of this tissue, but also as a route for re-filling of the SR and, therefore, represents an important target for the development of novel and selective drugs.     

Calcium released from intracellular stores inhibits GABAA-mediated currents in ganglion cells of the turtle retina

We studied spiking neurons isolated from turtle retina by the whole cell version of the patch clamp. The studied cells had perikaryal diameters > 15 microns and fired multiple spikes in response to depolarizing current steps, indicating they were ganglion cells. In symmetrical [Cl-], currents elicited by puffs of 100 microM gamma-aminobutyric acid (GABA) were inward at a holding potential of -80 mV. All of the GABA-evoked current was blocked by SR95331 (20 microM), indicating that it was mediated by a GABAA receptor. The GABA-evoked currents were unaltered by eliciting a transmembrane calcium current either just before or during the response to GABA. On the other hand caffeine (10 mM), which induces Ca2+ release from intracellular stores, inhibited the GABA-evoked current on average by 30%. The caffeine effect was blocked by introducing the calcium buffer bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) into the cell but was unaffected by replacing [Ca2+]o with equimolar cobalt. Thapsigargin (10 microM), an inhibitor of intracellular calcium pumps, and ryanodine (20 microM), which depletes intracellular calcium stores, both markedly reduced a caffeine-induced inhibition of the GABA-evoked current. Another activator of intracellular calcium release, inositol trisphosphate (IP3; 50 microM), also progressively reduced the GABA-induced current when introduced into the cell. Dibutyryl adenosine 3'5'-cyclic monophosphate (cAMP; 0.5 mM), a membrane-permeable analogue of cAMP, did not reduce GABA-evoked currents, suggesting that cAMP-dependent kinases are not involved in suppressing GABAA currents, whereas calmidazolium (30 microM) and cyclosporin A (20 microM), which inhibit Ca/calmodulin-dependent phosphatases, did reduce the caffeine-induced inhibition of the GABA-evoked current. Alkaline phosphatase (150 micrograms/ml) and calcineurin (300 micrograms/ml) had a similar action to caffeine or IP3. Antibodies directed against the ryanodine receptor or the IP3 receptor reacted with the great majority of neurons in the ganglion cell layer. We found that these two antibodies colocalized in large ganglion cells. In summary, intracellular calcium plays a role in reducing the currents elicited by GABA, acting through GABAA receptors. The modulatory action of calcium on GABA responses appears to work through one or more Ca-dependent phosphatases.     

Depletion of ryanodine-sensitive Ca2+ store activates Ca2+ entry in rat submandibular gland acinar cells

The existence of ryanodine-sensitive Ca2+ stores and their role in the Ca2+ entry mechanism were examined in the rat submandibular gland acinar cells, using the microfluorimetry of intracellular Ca2+ concentration ([Ca2+]i). In the presence of thapsigargin, a Ca(2+)-ATPase inhibitor of inositol (1, 4, 5) triphosphate (InsP3)-sensitive Ca2+ stores, caffeine caused an increase in [Ca2+]i, which was inhibited by treatment with ryanodine (a ligand to the Ca(2+)-induced Ca2+ release channels). In the cells treated with ryanodine, 1 mM Ca2+ addition to a Ca(2+)-free solution caused a marked increase in [Ca2+]i, which was eliminated by application of Ni2+ or SK & F 96365, suggesting a Ca2+ entry triggered by ryanodine. The maximal change in the net increase in [Ca2+]i caused by the ryanodine-coupled Ca2+ entry, was 104.0 +/- 16.0 nM, which intense was caused by 10 microM ryanodine. Emptying the InsP3-sensitive stores by treatment with thapsigargin also caused Ca2+ entry, which maximally changed [Ca2+]i by 349.6 +/- 15.1 nM. Ten mumol/liter ryanodine was confirmed to cause a release of 45Ca2+ from the parotidic microsomal fraction enriched in endopalsmic reticulum. We propose that ryanodine-sensitive Ca2+ stores are present in rat submandibular gland acinar cells. We further propose that release of Ca2+ from the ryanodine-sensitive stores, which means eventually depletion of the ryanodine-sensitive Ca2+ stores, can activate the Ca2+ entry. The ability for Ca2+ entry coupled with the ryanodine-sensitive Ca2+ stores seems to be about 30% of the ability for Ca2+ entry coupled with the thapsigargin-sensitive Ca2+ stores.     

Relative maxillary retrusion as a natural consequence of aging: combining skeletal and soft-tissue changes into an integrated model of midfacial aging

Depletion of intracellular calcium stores by agonist stimulation is coupled to calcium influx across the plasma membrane, a process termed capacitative calcium entry. Capacitative calcium entry was examined in cultured guinea pig enteric glial cells exposed to endothelin 3. Endothelin 3 (10 nM) caused mobilization of intracellular calcium stores followed by influx of extracellular calcium. This capacitative calcium influx was inhibited by Ni2+ (89 +/- 2%) and by La3+ (78 +/- 2%) but was not affected by L-, N-, or P-type calcium channel blockers. Chelerythrine, a specific antagonist of protein kinase C, dose-dependently inhibited capacitative calcium entry. The nitric oxide synthase inhibitor NG-nitro-L-arginine decreased calcium influx in a dose-dependent manner. The combination of chelerythrine and NG-nitro-L-arginine produced synergistic inhibitory effects. Capacitative calcium entry occurs in enteric glial cells via lanthanum-inhibitable channels through a process regulated by protein kinase C and nitric oxide.     

Stereotactic microsurgical craniotomy for the treatment of third ventricular colloid cysts

The process of myoblast fusion during skeletal myogenesis is calcium regulated. Both dihydropyridine receptor and ryanodine receptor are already present on muscle precursors, at the prefusional stage, before they are required for excitation-contraction coupling. Previous pharmacological studies have shown the need for a special pool of Ca2+ associated with the membrane for the fusion process to occur. We hypothesized that this pool of Ca2+ is mobilized via a machinery similar to that involved in excitation-contraction coupling. The process of fusion in rat L6 muscle precursors was either totally or partially abolished in the presence of the L-type calcium channel inhibitors SR33557 and nifedipine (half inhibition towards 2 microM), respectively. The inhibition was reversible and dose-dependent. Drugs able to deplete internal calcium stores (caffeine, ryanodine, and thapsigargin) were also tested on the fusion. Both caffeine and thapsigargin drastically inhibited fusion whereas ryanodine had no effect. This suggests that fusion may be controlled by internal pools of Ca2+ but that its regulation may be insensitive to ryanodine. We presumed that an early form of the ryanodine receptor may exist, with different pharmacological properties than the adult forms. Indeed, Western blot analysis of pre- and postfusional L6 cells demonstrated the presence, at the prefusional stage, of a transient form of the ryanodine receptor protein with an apparent molecular weight slightly different from those of the classical skeletal and cardiac forms. Taken together, these results support the hypothesis that the fusion process is driven by a mechanism involving both the dihydropyridine receptor (alpha1 subunit of the L-type Ca2+ channel) and the internal stores of Ca2+. The machinery underlying this mechanism might consist of slightly different forms of the classic molecules that in adult muscle ensure excitation-contraction coupling. It remains to be seen, however, whether the mobilization of the internal pool of Ca2+ is triggered by the type of mechanism already described in skeletal muscle.     

The phospholipase C inhibitor U73122 increases cytosolic calcium in MDCK cells by activating calcium influx and releasing stored calcium

The effects of the phospholipase C (PLC) inhibitor U73122 on intracellular calcium levels ([Ca2+]i) were studied in MDCK cells. U73122 elevated [Ca2+]i dose-dependently. Ca2+ influx contributed to 75% of 20 microM U73122-induced Ca2+ signals. U73122 pretreatment abolished the [Ca2+]i transients evoked by ATP and bradykinin, suggesting that U73122 inhibited PLC. The Ca2+ signals among individual cells varied considerably. The internal Ca2+ source for the U73122 response was the endoplasmic reticulum (ER) since the response was abolished by thapsigargin. The depletion of the ER Ca2+ store triggered a La3+-sensitive capacitative Ca2+ entry. Independently of the internal release and capacitative Ca2 entry, U73122 directly evoked Ca2+ influx through a La3+-insensitive pathway. The U73122 response was augmented by pretreatment of carbonylcyanide m-chlorophynylhydrozone (CCCP), but not by Na+ removal, implicating that mitochondria contributed significantly in buffering the Ca2+ signal, and that efflux via Na+/Ca2+ exchange was insignificant.     

Use of topiramate, a new anti-epileptic as a mood stabilizer

2-Hydroxycarbazole was shown to induce Ca2+ release from skeletal muscle and cardiac muscle sarcoplasmic reticulum at concentrations between 100-500 microM. This release was blocked by both 1 mM tetracaine and 30 microM ruthenium red which inhibit the ryanodine receptor or by pre-treatment with 10 mM caffeine which depletes the ryanodine receptor-containing Ca2+ stores. This, in addition to the fact that 2-hydroxycarbazole has little effect on Ca2+ ATPase activity, indicates that it activates Ca2+ release through the ryanodine receptor. The apparent EC50 value for release from both skeletal muscle and cardiac muscle sarcoplasmic reticulum was approximately 200 microM and maximal release occurred at 400-500 microM, making it approximately 20 times more potent than caffeine. The dose-dependency in the extent of Ca2+ release induced by 2-hydroxycarbazole was also apparently highly cooperative for both preparations. That 2-hydroxycarbazole was able to mobilize Ca2+ from non-muscle cell microsomes and in intact TM4 cells (which contain ryanodine receptors), makes this compound a more potent and commercially available alternative to caffeine in studying the role of this intracellular Ca2+ channel in a variety of systems.     

Blood blisterlike aneurysms of the internal carotid artery

1. The effects of ryanodine, procaine, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on noradrenaline (NA)- and caffeine-induced contractions of human vas deferens were investigated. 2. In the presence of nifedipine (1 microM), NA ( 100 microM) evoked biphasic contractions. Caffeine (20 mM) evoked repeatable tonic contractions. 3. Ryanodine (30 microM) inhibited the initial but not the secondary component of NA contractions. Procaine (1 and 10 mM) inhibited both components. Contractions induced by caffeine were unaffected by ryanodine or procaine. 4. The calmodulin antagonist W-7 (100 microM) reduced, in a reversible manner, both components of NA-induced response. Caffeine-induced contractions were also reduced in most preparations (8 of 11). In all preparations, contractions induced by caffeine were markedly inhibited after the washout of W-7. Higher doses of W-7 (300 microM) induced an increase in basal tension. 5. These results indicate that NA contracts the longitudinal muscle of human vas deferens by a ryanodine-sensitive calcium-induced calcium release (CICR) mechanism and, in addition, a ryanodine-insensitive pathway: both are sensitive to procaine. In contrast, contraction induced by caffeine is mediated by a pathway that is atypically insensitive to either ryanodine or procaine. The sensitivity of NA- and caffeine-induced contraction to W-7 suggests a role for calcium and its interaction with calmodulin in the response to both agents. The paradoxical action of W-7 is discussed.     

Hydrogen peroxide induces intracellular calcium overload by activation of a non-selective cation channel in an insulin-secreting cell line

Fura-2 fluorescence was used to investigate the effects of H2O2 on [Ca2+]i in the insulin-secreting cell line CRI-G1. H2O2 (1-10 mM) caused a biphasic increase in free [Ca2+]i, an initial rise observed within 3 min and a second, much larger rise following a 30-min exposure. Extracellular calcium removal blocked the late, but not the initial, rise in [Ca2+]i. Thapsigargin did not affect either response to H2O2, but activated capacitive calcium entry, an action abolished by 10 microM La3+. Simultaneous recordings of membrane potential and [Ca2+]i demonstrated the same biphasic [Ca2+]i response to H2O2 and showed that the late increase in [Ca2+]i coincided temporally with cell membrane potential collapse. Buffering Ca2+i to low nanomolar levels prevented both phases of increased [Ca2+]i and the H2O2-induced depolarization. The H2O2-induced late rise in [Ca2+]i was prevented by extracellular application of 100 microM La3+. La3+ (100 microM) inhibited the H2O2-induced cation current and NAD-activated cation (NSNAD) channel activity in these cells. H2O2 increased the NAD/NADH ratio in intact CRI-G1 cells, consistent with increased cellular [NAD]. These data suggest that H2O2 increases [NAD], which, coupled with increased [Ca2+]i, activates NSNAD channels, causing unregulated Ca2+ entry and consequent cell death.     

Role of the cytoskeleton in calcium signaling in NIH 3T3 cells. An intact cytoskeleton is required for agonist-induced [Ca2+]i signaling, but not for capacitative calcium entry

Treatment of NIH 3T3 cells with cytochalasin D (10 microM, 1 h at 37 degrees C) disrupted the actin cytoskeleton and changed the cells from a planar, extended morphology, to a rounded shape. Calcium mobilization by ATP or by platelet-derived growth factor was abolished, while the ability of thapsigargin (2 microM) to empty calcium stores and activate calcium influx was unaffected. Similar experiments with nocodazole to depolymerize the tubulin network yielded identical results. Platelet-derived growth factor induced an increase in inositol phosphates, and this increase was undiminished in the presence of cytochalasin D. Therefore, the blockade of agonist responses by this drug does not result from decreased phospholipase C. Injection of inositol 1,4,5-trisphosphate (IP3) released calcium to the same extent in control and cytochalasin D-treated cells. Confocal microscopic studies revealed a significant rearrangement of the endoplasmic reticulum after cytochalasin D treatment. Thus, disruption of the cytoskeleton blocks agonist-elicited [Ca2+]i mobilization, but this effect does not result from a lower calcium storage capacity, impaired function of the IP3 receptor, or diminished phospholipase C activity. We suggest that cytoskeletal disruption alters the spatial relationship between phospholipase C and IP3 receptors, impairing phospholipase C-dependent calcium signaling. Capacitative calcium entry was not altered under these conditions, indicating that the coupling between depletion of intracellular calcium stores and calcium entry does not depend on a precise structural relationship between intracellular stores and plasma membrane calcium channels.     

Role of intracellular Ca2+ in the K channel opener action of CGRP in the guinea-pig ureter

1. The aim of this study was to assess the role of sarcoplasmic reticulum (SR) calcium (Ca2+) in the smooth muscle relaxant and hyperpolarizing actions of calcitonin gene-related peptide (CGRP) in the guinea-pig ureter. 2. CGRP (0.1 microM) rapidly and transiently reduced myogenic phasic contractions (twitches) produced by electrical field stimulation (EFS). Approximately 70% of the response to CGRP was antagonized by glibenclamide (1 microM). 3. Cyclopiazonic acid (CPA, 10 microM), ryanodine (100 microM) and thapsigargin (1 microM) reduced only the glibenclamide-sensitive component of the response to CGRP (0.1 microM) but did not modify the mechano-inhibitory effect of cromakalim (3 microM). A low concentration of CPA (1 microM), assumed to produce a limited impairment of Ca2+ uptake from the stores, prolonged the duration of the inhibitory response to CGRP. Pre-exposure to caffeine (5 mM) inhibited the suppression of twitches by CGRP or cromakalim. 4. When the frequency of EFS was increased, the suppression of twitches by CGRP was reduced. Under these conditions, CPA (1 microM) again prolonged the duration of the inhibitory response to CGRP. 5. CGRP (0.1 microM) and cromakalim (3 microM) markedly depressed the phasic component of contractions to 80 mM KCl. CPA (10 microM) antagonized the inhibitory effect of CGRP but not that of cromakalim. Inhibition of the tonic contraction to 80 mM KCl by CGRP was insensitive to CPA. 6. In sucrose gap experiments, a 5 min exposure to CGRP (0.1 microM) or cromakalim (3 microM) produced a sustained membrane hyperpolarization. Caffeine (5 mM) produced a glibenclamide-sensitive transient hyperpolarization followed by a sustained depolarization. When tested in a Ca(2+)-free medium the hyperpolarization produced by CGRP, cromakalim or caffeine was reduced. In normal Krebs, pre-exposure to CPA (10 microM, 60 min) only abolished the hyperpolarization induced by CGRP. In contrast, 5 min after a caffeine challenge (5 mM) the hyperpolarizations induced by CGRP or cromakalim were reduced. The CGRP-induced hyperpolarization was insensitive to apamin (0.1 microM) or charybdotoxin (0.1 microM). 7. We conclude that the K channel-opening action of CGRP in the guinea-pig ureter requires the mobilization of intracellular Ca2+ from a caffeine- and CPA-sensitive store, leading to transient activation of glibenclamide-sensitive K channels. The K channel-opening action of caffeine appears to involve Ca2+ mobilization from a store which is insensitive to depletion by CPA.     

Calmodulin regulation of calcium stores in phototransduction of Drosophila

Phototransduction in Drosophila occurs through the ubiquitous phosphoinositide-mediated signal transduction system. Major unresolved questions in this pathway are the identity and role of the internal calcium stores in light excitation and the mechanism underlying regulation of Ca2+ release from internal stores. Treatment of Drosophila photoreceptors with ryanodine and caffeine disrupted the current induced by light, whereas subsequent application of calcium-calmodulin (Ca-CaM) rescued the inactivated photoresponse. In calcium-deprived wild-type Drosophila and in calmodulin-deficient transgenic flies, the current induced by light was disrupted by a specific inhibitor of Ca-CaM. Furthermore, inhibition of Ca-CaM revealed light-induced release of calcium from intracellular stores. It appears that functional ryanodine-sensitive stores are essential for the photoresponse. Moreover, calcium release from these stores appears to be a component of Drosophila phototransduction, and Ca-CaM regulates this process.     

Intracellular calcium stores modulate miniature GABA-mediated synaptic currents in neonatal rat hippocampal neurons

The whole-cell configuration of the patch clamp technique was used to record miniature gamma-aminobutyric acidA (GABAA) receptor-mediated currents (in tetrodotoxin, 1 microM and kynurenic acid 1 mM) from CA3 pyramidal cells in thin hippocampal slices obtained from postnatal (P) day (P6-9) old rats. Switching from a Ca2+-containing to a nominally Ca2+-free medium (in which Ca2+ was substituted with Mg2+, in the presence or in the absence of 100 microM EGTA) did not change significantly the frequency or amplitude of miniature events. Superfusion of thapsigargin induced a concentration-dependent increase in frequency but not in amplitude of tetrodotoxin-resistant currents that lasted for the entire period of drug application. Mean frequency ratio (thapsigargin 10 microM over control) was 1.8+/-0.5, (n = 9). In nominally Ca2+-free solutions thapsigargin was ineffective. When bath applied, caffeine (10 mM), reversibly reduced the amplitude of miniature postsynaptic currents whereas, if applied by brief pressure pulses, it produced an increase in frequency but not in amplitude of spontaneous GABAergic currents. Superfusion of caffeine (10 mM) reversibly reduced the amplitude of the current induced by GABA (100 microM) indicating a clear postsynaptic effect on GABAA receptor. Superfusion of ryanodine (30 microM), in the majority of the cells (n = 7) did not significantly modify the amplitude or frequency of miniature events. In two of nine cells it induced a transient increase in frequency of miniature postsynaptic currents. These results indicate that in neonatal hippocampal neurons, mobilization of calcium from caffeine-ryanodine-sensitive stores facilitates GABA release.     

Alpha 1-adrenoceptors and calcium sources in adrenergic neurogenic contractions of rat vas deferens

1. The involvement of alpha 1-adrenoceptor subtypes in adrenergic neurogenic contractions of different type was studied in epididymal and prostatic portions of the rat vas deferens. 2. The adrenergic component of neurogenic contractions was isolated by suramin (300 microM). Twitch-like and tonic contractions were elicited by appropriate pulse patterns of electrical field stimulation, and contractions relying on intracellular calcium mobilization and calcium entry were isolated by means of nifedipine (10 microM) and ryanodine (20 microM), respectively. Increasing concentrations of 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)- amino)-propyl)benzeneacetonitrile (HV 723), prazosin and 5-methylurapidil progressively, monophasically and with potency decreasing in that order reduced and finally abolished all types of contraction, with one exception: concentration-effect curves of 5-methylurapidil in epididymal segments in the presence of ryanodine levelled off at about 75% inhibition. In the presence of both nifedipine (10 microM) and ryanodine (20 microM), contractions were abolished. 3. Contractions elicited by exogenous noradrenaline were also studied in the presence of either nifedipine 10 microM (prostatic segments) or ryanodine 20 microM (epididymal segments). Increasing concentrations of tamsulosin, WB 4101, benoxathian, HV 723, prazosin, 5-methylurapidil and urapidil progressively, monophasically and with potency decreasing in that order reduced and eventually abolished both kinds of contraction, with two exceptions: in epididymal segments in the presence of ryanodine, the concentration-effect curve of 5-methylurapidil was biphasic and the curve of urapidil levelled off at only partial inhibition. 4. In slices prepared from the prostatic end and preincubated with [3H]-noradrenaline, WB 4101, HV 723, prazosin and 5-methylurapidil, at the highest concentrations tested against neurogenic contractions, increased only slightly the overflow of tritium elicited by trains of 50 pulses at 5 Hz. 5. It is concluded that two alpha l-adrenoceptor subtypes mediate adrenergic neurogenic contractions of rat vas deferens. The main one, pharmacologically alpha 1A, activates both calcium mobilization and entry. In addition there is a second receptor, not previously detected in the vas deferens and not corresponding to any named alpha l subtype, characterized by high and similar affinity for tamsulosin, WB 4101, benoxathian,HV 723 and prazosin and very low affinity for 5-methylurapidil and urapidil, and linked exclusively to calcium entry. Both subtypes and their respective transduction pathways also contribute to contractions elicited by exogenous noradrenaline. An alpha 1B-adrenoceptor-mediated contraction was not found under any experimental conditions.     

Sphingosine-induced inhibition of capacitative calcium influx in CFPAC-1 cells

Sphingosine (10 microM) induced mobilization of intracellular Ca2+ stores in the pancreatic duct adenocarcinoma cell line CFPAC-1. The effect was specific for sphingosine, since the sphingosine analog C2-ceramide had no effect. Sphingosine did not cause Ca2+ entry from extracellular medium, as also shown by following Mn2+ quenching of Fura-2 fluorescence. Furthermore, sphingosine, similarly to the mitochondrial inhibitors rotenone and oligomycin, strongly inhibited the rate of Mn2+ entry triggered by both thapsigargin- and agonist-induced depletion of intracellular stores. The uptake of rhodamine 123, a lipophilic cation which estimates mitochondrial energy level, was reduced by sphingosine to an extent similar to that observed in the presence of mitochondrial inhibitors. It is suggested that impairment of mitochondrial function might be responsible for inhibition of capacitative Ca2+ entry caused by sphingosine.     

Caffeine stimulates amyloid beta-peptide release from beta-amyloid precursor protein-transfected HEK293 cells

Extracellular amyloid beta-peptide (A beta) deposition is a pathological feature of Alzheimer's disease and the aging brain. Intracellular A beta accumulation is observed in the human muscle disease, inclusion body myositis. A beta has been reported to be toxic to neurons through disruption of normal calcium homeostasis. The pathogenic role of A beta in inclusion body myositis is not as clear. Elevation of intracellular calcium following application of calcium ionophore increases the generation of A beta from its precursor protein (betaAPP). A receptor-based mechanism for the increase in A beta production has not been reported to our knowledge. Here, we use caffeine to stimulate ryanodine receptor (RYR)-regulated intracellular calcium release channels and show that internal calcium stores also participate in the genesis of A beta. In cultured HEK293 cells transfected with betaAPP cDNA, caffeine (5-10 mM) significantly increased the release of A beta fourfold compared with control. These actions of caffeine were saturable, modulated by ryanodine, and inhibited by the RYR antagonists ruthenium red and procaine. The calcium reuptake inhibitors thapsigargin and cyclopiazonic acid potentiated caffeine-stimulated A beta release. NH4Cl and monensin, agents that alter acidic gradients in intracellular vesicles, abolished both the caffeine and ionophore effects. Immunocytochemical studies showed some correspondence between the distribution patterns of RYR and cellular betaAPP immunoreactivities. The relevance of these findings to Alzheimer's disease and inclusion body myositis is discussed.     

Oscillations of receptor-operated cationic current and internal calcium in single guinea-pig ileal smooth muscle cells

In single cells isolated from guinea-pig ileal smooth muscle, held under voltage clamp at -40 mV or -50 mV by patch pipette in the whole-cell recording mode, carbachol (CCh) evoked an oscillatory inward cationic current. The frequency of current oscillations increased with increasing CCh concentration. CCh-evoked current oscillations were followed very closely by oscillations in intracellular free Ca2+ estimated from the Indo-1 signal, and were abolished by inclusion of EGTA in the pipette solution. Ryanodine and heparin, but not nifedipine, blocked the generation of current oscillations. CCh-evoked current oscillations were abolished upon withdrawal of extracellular calcium and restored upon its reintroduction. Inclusion of GTP[gamma S] in the pipette solution caused the generation of an oscillatory inward current, which was blocked by ryanodine. The present results are consistent with the hypothesis that CCh-evoked cationic current is gated by activation of a G protein and is steeply dependent on [Ca2+]i, fluctuations in the release of Ca2+ from stores during carbachol's action produce oscillations in [Ca2+]i which cause similar oscillations in the cationic current.     

Calcium channel subtypes responsible for voltage-gated intracellular calcium elevations in embryonic rat motoneurons

The central role of electrical activity and Ca2+ influx in motoneuron development raises important questions about the regulation of Ca2+ signalling induced by voltage-dependent Ca2+ influx. In the purified embryonic rat motoneuron preparation, we recorded barium currents through voltage-activated Ca2+ channels using the whole-cell configuration of the patch-clamp technique. We found that motoneurons express at least four types of high-voltage-activated Ca2+ channels, based on their kinetics, voltage-dependences and pharmacological properties. Of the sustained Ca2+ current activated at 0 mV from a holding potential of -100 mV, approximately 45% was omega-conotoxin-GVIA (1 microM) sensitive, 25% was omega-agatoxin-IVA (30 nM) sensitive and 20% was nitrendipine (250 nM) sensitive. The residual current, after applying these three antagonists, was an inactivating current that differs from classical T-type Ca2+ currents. Based on this pharmacology, changes in intracellular free Ca2+ concentrations were then monitored by Fura 2 digital imaging microspectrofluorimetry. Upon K+ depolarization, the intracellular Ca2+ transient induced by the activation of each type of Ca2+ channel appeared to be quantitatively proportional to their Ca2+ influx. The existence of a calcium-induced calcium release mechanism through activation of caffeine-, ryanodine-sensitive intracellular stores was then investigated. High doses of caffeine and low doses of ryanodine failed to increase intracellular free calcium concentrations and low concentrations of caffeine and high concentrations of ryanodine did not affect K+-induced intracellular free calcium concentration transients indicating both the absence of Ca2+-gated Ca2+-release channels and of a Ca2+-induced Ca2+ release mechanism. Together, these data provide evidence that embryonic motoneurons express multiple Ca2+ channels that function as important regulators of intracellular Ca2+ signalling and may be involved in their development.     

Three-dimensional modeling of renal glomerular capillary networks

Using whole cell patch clamp recordings on unfertilized eggs of the ascidian Ciona intestinalis, we are able to detect ryanodine receptors within the oocytes. Our approach is based on measurements of the voltage-activated inward calcium currents. Two types of Ca2+ currents have been described on the oocyte membrane of Ciona: a low threshold slowly activating current, and a high threshold faster one. We show here that caffeine induces a decrease in the intensity of the Ca2+ currents, when applied either externally or internally from the mouth of a patch pipette. Caffeine application mimics fertilization which transiently decreases the high threshold Ca2+ current density during density during the first meiotic cycle. Ryanodine (> 1 nM) has an effect similar to caffeine. This partial decrease in Ca2+ current density elicited by caffeine or ryanodine is prevented by intracellular application of the calcium chelator BAPTA, then imputable to calcium release. In summary, the depolarization-induced Ca2+ current intensity allows monitoring of an intracellular calcium store which is sensitive to low concentrations of ryanodine in Ciona oocytes. Further identification of a ryanodine receptor was obtained by immunological staining with antibodies against mammalian skeletal muscle ryanodine receptor. Ryanodine receptors were asymmetrically localized in the cortex of Ciona eggs. We discuss the methodological relevance of our patch-clamp approach, in connection with the possible biological role of such a ryanodine receptor in the early stages of development.     

Rat oligodendrocytes express muscarinic receptors coupled to phosphoinositide hydrolysis and adenylyl cyclase

Muscarinic receptors expressed by rat oligodendrocyte primary cultures were examined by measuring changes in second messengers following exposure to carbachol, an acetylcholine analog, and by polymerase chain reaction. Inositol phosphate levels were measured in [3H]myo-inositol-labelled young oligodendrocyte cultures following stimulation with carbachol. Atropine, a specific muscarinic antagonist, prevented the carbachol-induced accumulation of inositol phosphates. The formation of inositol trisphosphate was concentration- and time-dependent, with the peak at 100 microM carbachol and 10 min. Carbachol increased intracellular calcium levels, which were dependent both on the mobilization of intracellular stores and influx of extracellular calcium. In initial experiments with more selective antagonists, the mobilization of intracellular calcium was preferentially inhibited by pirenzepine, a selective M1 antagonist, but not methoctramine, a selective M2 antagonist, suggesting M1 muscarinic receptor involvement. A role for protein kinase C in the regulation of carbachol-stimulated inositol phosphate formation and intracellular calcium mobilization was demonstrated, as acute pretreatment with phorbol-12,13-myristate acetate abolished the formation of both second messengers. Pretreatment with 100 microM carbachol abolished the 40% increase in the cyclic AMP accumulation stimulated by isoproterenol, a specific beta-adrenergic agonist. In turn, the inhibition was alleviated by pretreatment with atropine, suggesting muscarinic receptor involvement. Polymerase chain reaction carried out with specific m1 and m2 muscarinic receptor oligonucleotide primers, confirmed that these cells express, at least, the two muscarinic receptor subtypes. Without excluding the expression of other subtypes, these results suggest that developing oligodendrocytes express m1 (M1) and m2 (M2) muscarinic receptors capable of mediating phosphoinositide hydrolysis, mobilization of intracellular calcium and the attenuation of beta-adrenergic stimulation of cyclic AMP formation.