Multiple subtypes of voltage-gated calcium channel mediate transmitter release from parasympathetic neurons in the mouse bladder

Multiple subtypes of voltage-gated calcium channels are coupled to transmitter release from central neurons; however, only N-type channels have been shown to play a role in autonomic neurons. The aim of the present study was to investigate potential roles for other channel subtypes in transmitter release from parasympathetic neurons in the mouse bladder using calcium channel toxins alone and in combination. Transmitter release was measured indirectly by recording the contraction of bladder dome strips in response to electrical stimulation of the neurons by single pulses or trains of 20 pulses at 1-50 Hz. omega-Conotoxin-GVIA (GVIA) and omega-conotoxin-MVIIC (MVIIC) inhibited contractions in a concentration-dependent manner, with IC50 values of approximately 30 and 200 nM, respectively, at low stimulation frequencies. omega-Agatoxin-IVA (agatoxin) alone did not have any significant effect up to 300 nM. Cumulative addition of the toxins demonstrated that 300 nM agatoxin had a significant effect after N-type channels were blocked with 100 nM GVIA. MVIIC (3 microM) reduced the contraction amplitude further. Testing the toxins on the cholinergic or purinergic component of the contraction separately showed that acetylcholine release depends primarily on N-type channels and, to a lesser extent, on P- and Q-type channels, whereas ATP release involves predominantly P- and Q-type channels. In conclusion, parasympathetic neurons in the mouse bladder, like central neurons, use multiple calcium channel subtypes. Furthermore, the release of the two main transmitters in these neurons has differing dependencies on the calcium channel subtypes.     

Antagonists-resistant calcium currents in rat embryo motoneurons

Ca2+ channels diversity of cultured rat embryo motoneurons was investigated with whole-cell current recordings. In 5-20 mM Ba2+, the whole-cell currents were separated in low- (LVA) and high-voltage-activated (HVA) current. The LVA current was evident since the first day in culture, while the HVA component was small and increased with time. Recordings after 4 days revealed approximately 20% L-, approximately 45% N- and approximately 35% P- and R-type currents. P-type currents were revealed only in 40% of motoneurons, in which 20-200 nM omega-Aga-IVA caused 20% irreversible block of total current. The remaining 60% of cells were insensitive even to higher doses of the toxin (500 nM in 5 mM Ba2+), suggesting weak expression and heterogeneous distribution of P-type channels compensated by high densities of HVA Ca2+ channels resistant to all the antagonists (R-type). A significant residual current could also be resolved after prolonged applications of 5 microM omega-CTx-MVIIC, which allowed separation of N- and P-type currents by the distinct onset of toxin block. The antagonists-resistant current reveals biophysical characteristics typical of HVA channels, but distinct from the alphaE channel. The current activates around -20 mV in 20 mM Ba2+; inactivates slowly and independently of Ca2+; is blocked by low [Cd2+] and high [Ni2+]; and is larger with Ba2+ than Ca2+. The uncovered R-type calcium current can account for part of the presynaptic Ca2+ current controlling neurotransmitter release at the mammalian neuromuscular junction whose activity is resistant to DHP-and omega-CTx-GVIA, and displays anomalous sensitivity to omega-Aga-IVA and omega-CTx-MVIIC.     

Calcium channel subtypes in lamprey sensory and motor neurons

Pharmacologically distinct calcium channels have been characterized in dissociated cutaneous sensory neurons and motoneurons of the larval lamprey spinal cord. To enable cell identification, sensory dorsal cells and motoneurons were selectively labeled with fluorescein-coupled dextran amine in the intact spinal cord in vitro before dissociation. Calcium channels present in sensory dorsal cells, motoneurons, and other spinal cord neurons were characterized with the use of whole cell voltage-clamp recordings and specific calcium channel agonist and antagonists. The results show that a transient low-voltage-activated (LVA) calcium current was present in a proportion of sensory dorsal cells but not in motoneurons, whereas high-voltage-activated (HVA) calcium currents were seen in all neurons recorded. The different components of HVA current were dissected pharmacologically and similar results were obtained for both dorsal cells and motoneurons. The N-type calcium channel antagonist omega-conotoxin-GVIA (omega-CgTx) blocked >70% of the HVA current. A large part of the omega-CgTx block was reversed after washout of the toxin. The L-type calcium channel antagonist nimodipine blocked approximately 15% of the total HVA current. The dihydropyridine agonist (+/-)-BayK 8644 markedly increased the amplitude of the calcium channel current. The BayK-potentiated current was not affected by omega-CgTx, indicating that the reversibility of the omega-CgTx effect is not due to a blockade of L-type channels. Simultaneous application of omega-CgTx and nimodipine left approximately 15% of the HVA calcium channel current, a small part of which was blocked by the P/Q-type channel antagonist omega-agatoxin-IVA. In the presence of the three antagonists, the persistent residual current (approximately 10%) was completely blocked by cadmium. Our results provide evidence for the existence of HVA calcium channels of the N, L, and P/Q types and other HVA calcium channels in lamprey sensory neurons and motoneurons. In addition, certain types of neurons express LVA calcium channels.     

Single tottering mutations responsible for the neuropathic phenotype of the P-type calcium channel

Recent genetic and molecular biological analyses have revealed many forms of inherited channelopathies. Homozygous ataxic mice, tottering (tg) and leaner (tgla) mice, have mutations in the P/Q-type Ca2+ channel alpha1A subunit gene. Although their clinical phenotypes, histological changes, and locations of gene mutations are known, it remains unclear what phenotypes the mutant Ca2+ channels manifest, or whether the altered channel properties are the primary consequence of the mutations. To address these questions, we have characterized the electrophysiological properties of Ca2+ channels in cerebellar Purkinje cells, where the P-type is the dominant Ca2+ channel, dissociated from the normal, tg, and tgla mice, and compared them with the properties of the wild-type and mutant alpha1A channels recombinantly expressed with the alpha2 and beta subunits in baby hamster kidney cells. The most striking feature of Ca2+ channel currents of mutant Purkinje cells was a marked reduction in current density, being reduced to approximately 60 and approximately 40% of control in tg and tgla mice, respectively, without changes of cell size. The Ca2+ channel currents in the tg Purkinje cells showed a relative increase in non-inactivating component in voltage-dependent inactivation. Besides the same change, those of the tgla mice showed a more distinct change in voltage dependence of activation and inactivation, being shifted in the depolarizing direction by approximately 10 mV, with a broader voltage dependence of inactivation. In the recombinant expression system, the tg channel with a missense mutation (P601L) and one form of the two possible tgla aberrant splicing products, tgla (short) channel, showed a significant reduction in current density, while the other form of the tgla channels, tgla (long), had a current density comparable to the normal control. On the other hand, the shift in voltage dependence of activation and inactivation was observed only for the tgla (long) channel. Comparison of properties of the native and recombinant mutant channels suggests that single tottering mutations are directly responsible for the neuropathic phenotypes of reduction in current density and deviations in gating behavior, which lead to neuronal death and cerebellar atrophy.     

Nutrient-induced intracellular calcium movement in rat pancreatic B cell

Several insulin secretagogues increase 45Ca efflux from islets prelabeled with the tracer in the presence of glucose, an effect attributable to stimulation of 40Ca entry into islet cells. When the islets are prelabeled in the absence instead of presence of glucose, the 45Ca becomes more readily releasable, and the secretory response to nutrient or ionic secretagogues is decreased. Such a decrease is more marked in response to glucose than other secretagogues because the prior nutrient deprivation also impairs the metabolism of glucose in islet cells. In the islets prelabeled in the absence of glucose, both glyceraldehyde and 2-ketoisocaprote dramatically stimulated 45Ca efflux. This effect persists in the absence of extracellular Ca2+ or presence of the calmodulin-antagonist trifluoperazine. It may correspond to a nutrient-induced intracellular Ca2+ movement, the existence of which would be unmasked in islets prelabeled in the absence of glucose. This so-far-undetected Ca2+ movement may play a role in the secretory response to nutrient secretagogues.     

Cloning and functional expression of a voltage-gated calcium channel alpha1 subunit from jellyfish

Voltage-gated Ca2+ channels in vertebrates comprise at least seven molecular subtypes, each of which produces a current with distinct kinetics and pharmacology. Although several invertebrate Ca2+ channel alpha1 subunits have also been cloned, their functional characteristics remain unclear, as heterologous expression of a full-length invertebrate channel has not previously been reported. We have cloned a cDNA encoding the alpha1 subunit of a voltage-gated Ca2+ channel from the scyphozoan jellyfish Cyanea capillata, one of the earliest existing organisms to possess neural and muscle tissue. The deduced amino acid sequence of this subunit, named CyCaalpha1, is more similar to vertebrate L-type channels (alpha1S, alpha1C, and alpha1D) than to non-L-type channels (alpha1A, alpha1B, and alpha1E) or low voltage-activated channels (alpha1G). Expression of CyCaalpha1 in Xenopus oocytes produces a high voltage-activated Ca2+ current that, unlike vertebrate L-type currents, is only weakly sensitive to 1,4-dihydropyridine or phenylalkylamine Ca2+ channel blockers and is not potentiated by the agonist S(-)-BayK 8644. In addition, the channel is less permeable to Ba2+ than to Ca2+ and is more permeable to Sr2+. CyCaalpha1 thus represents an ancestral L-type alpha1 subunit with significant functional differences from mammalian L-type channels.     

Advancing age alters intracellular calcium buffering in rat adrenergic nerves

There is a marked increase with advancing age of stimulation-evoked neurotransmitter release from vascular adrenergic nerves in the rat, an effect correlated with increased levels of plasma norepinephrine. This increase in norepinephrine release could not be accounted for by an alteration in neuronal and extraneuronal uptake of norepinephrine or a decline in feedback inhibition of release by prejunctional alpha2-adrenergic receptors. Measurement of intracellular calcium in fura-2-labeled superior cervical ganglion cells revealed elevated K+-evoked calcium transients in old compared to young neurons. Blockade of mitochondrial calcium uptake with dinitrophenol resulted in increased calcium transients in old neurons only. Furthermore, following blockade of mitochondrial calcium uptake the rate of return of calcium to resting levels was reduced to a greater degree in old cells as compared to young cells. The effects of dinitrophenol in old cells were attenuated when extracellular calcium was reduced. These findings suggest that older cells are more dependent on mitochondrial calcium buffering, perhaps due to changes in ATP dependent calcium uptake. Increased calcium transients as a result of altered intracellular calcium buffering offer a reasonable explanation for our previous observation of increased stimulation evoked norepinephrine release.     

Calcium channel blocker decreases pentagastrin-stimulated alkaline-tide: a role for extracellular calcium in gastric acid secretion

The value of magnetic resonance imaging (MRI) in the diagnosis and staging of endometrial carcinoma was studied in 43 cases of clinically suspected endometrial carcinoma and 7 normal women. All of the 43 cases showed abnormal endometria measured by MRI, among which were pathologically proven 40 cases of endometrial carcinoma, 2 cases of endometrial polyps and 1 case of adenomyosis. This suggested that MRI showed a high susceptibility in the diagnosis of endometrial carcinoma, and a lack of specificity. MRI could predict myometrial invasion, its accuracy being verified by surgico-pathological findings in 11 of 13 cases. MRI staging was correct in 10 of the 13 primarily operated cases, and only half of the clinical staging was in conformity with surgical staging. The results suggest that MRI is useful in the staging of endometrial carcinoma and therefore is of value in the choice of treatment planning, although it is not yet an ideal diagnostic aid to detect small metastatic pelvic lesions.     

The thrombin receptor elevates intracellular calcium in adult rat ventricular myocytes

While there is evidence that thrombin receptor activation leads to contractile dysfunction and induces arrhythmias in ischemic/reperfused cardiac tissue, thrombin is variably reported to modulate intracellular calcium in cardiomyocytes. The present study demonstrates that thrombin receptor activation leads to a rise in intracellular calcium in adult ventricular myocytes and serves to reconcile previous discrepant findings. The thrombin receptor-derived agonist peptide (SFLLRN, a portion of the tethered ligand created by thrombin's proteolytic actions) increases cytosolic calcium and twitch amplitude in cardiomyocytes isolated from adult ventricles. The truncated control peptide FLLRN has no effect, establishing that the response to SFLLRN results from a specific agonist peptide-receptor interaction. However, the response to SFLLRN occurs only at high agonist peptide concentrations and thrombin itself is inactive. This result is not compatible with an action of SFLLRN at a distinct protease-activated receptor (PAR-2; which is activated by SFLLRN, but not by thrombin), since SLIGRL (a ligand which is selective for PAR-2, but not the thrombin receptor) has no effect. Rather, the enzyme-based cell isolation procedure may partially cleave the thrombin receptor and influence cell responses, since concentrations of SFLLRN which are sub-threshold in enzymatically disaggregated myocytes significantly increase the force of isometric contraction of intact rat papillary muscles. These studies provide the first evidence that thrombin receptor activation leads to a change in intracellular calcium and a positive inotropic response in adult ventricular myocardium.     

The effect of calcium channel blockers and calcium on methotrexate accumulation in rat hepatocytes

The effects of diltiazem (DIL), verapamil (VRP) and Ca2+ on the accumulation of methotrexate (MTX) were investigated in isolated rat hepatocytes. At the physiological 2 mM Ca2+, the calcium-channel blockers DIL (100 microM) and VRP (50 microM) significantly reduced the hepatocellular accumulation of MTX. By increasing the Ca2+ concentration to 7 mM control MTX levels (at 2 mM Ca2+) were restored with VRP, and resulted in MTX levels above the controls for DIL. Ca2+ at 7 mM significantly enhanced MTX accumulation in the hepatocyte suspensions after 60 min. The concentration time curves for MTX indicated that for the first 10 min influx was the dominating process. Dixon plot analysis of this uptake phase revealed Ki values of 140 microM for DIL and 75 microM for VRP. The data suggested that DIL was a non-competitive, and VRP a competitive inhibitor of MTX influx. Hence, the inhibitory effect on MTX accumulation mediated by DIL and VRP could be due to different mechanisms.     

Developmental changes in intracellular calcium regulation in rat cerebral cortex during hypoxia

During the first weeks of life, injury to the central nervous system caused by brief periods of oxygen deprivation greatly increases. To investigate possible causes for this change, the effects of hypoxia or application of the excitatory neurotransmitter glutamate on intracellular calcium ([Ca2+]i) and ATP were studied in rat cerebrocortical brain slices. [Ca2+]i was measured fluorometrically with the indicator Fura-2. Hypoxia (95% N2/5% CO2) or 100 microM sodium cyanide produced gradual elevations in [Ca2+]i and ATP depletion in slices from rats < 2 weeks old, but rapid changes in older rats. After 20 min, [Ca2+]i in adult slices exposed to cyanide was 1,980 +/- 310 nM; in day 1-14 animals, it was 796 +/- 181 nM (p < 0.05). Combination of cyanide and a glycolytic inhibitor (iodoacetate) rapidly elevated [Ca2+]i and depleted ATP in all age groups. Energy utilization during anoxia, assessed by measuring ATP fall in cyanide/iodoacetate-treated brain slices, increased with age. Elevations in [Ca2+]i caused by application of 500 microM glutamate increased 240% from days 1-2 to day 28, but ATP loss caused by glutamate did not change with age. The N-methyl-D-aspartate antagonist MK-801 delayed calcium entry during the initial 5-7 min of hypoxia or cyanide in rats < 2 weeks old. We conclude that anaerobic ATP production, conservation of energy by reduced ATP consumption, and reduced sensitivity to glutamate contribute to delaying elevation in [Ca2+]i in neonatal rat brain during hypoxia.     

Conditioned adrenocortical steroid elevations in the rat

An illness-induced taste aversion paradigm was used to condition an elevation in plasma corticosterone level. Rats were injected with cyclophosphamide 30 min after consuming a novel saccharin drinking solution. Plasma corticosterone levels were measured before conditioning to determine unconditioned steroid levels and 3 and 6 days after training when conditioned and nonconditioned animals were provided with the saccharin solution or plain water, or were left deprived. The pairing of saccharin and cyclophosphamide was effective in inducing a passive avoidance response. There were no differences between the steroid levels of conditioned and nonconditioned animals supplied with plain water or those that remained deprived, although deprivation increased corticosterone levels. Nonconditioned rats presented with saccharin had steroid levels that did not differ from control values. Conditioned animals presented with saccharin showed an elevation in steroid level which was significantly greater than that observed in any other group. Comparable results were obtained when LiCl was used as the unconditioned stimulus.     

Antibodies to synthetic peptides of the alpha1A subunit of the voltage-gated calcium channel in Lambert-Eaton myasthenic syndrome

STUDY OBJECTIVE: This study aimed to evaluate the effects of nasal continuous positive airway pressure (nCPAP) therapy on sleep and daytime symptoms of bed partners and patients with obstructive sleep apnea (OSA). DESIGN: A cross-sectional questionnaire survey. SETTING: The sleep laboratory of a university teaching hospital. PATIENTS: Ninety-one consecutive OSA patients within 2 to 12 months of being prescribed nCPAP. RESULTS: Eighty-five replies (93% of sample population) were received. Twelve patients (14% of replies) had discontinued nCPAP therapy; two patients had not yet been supplied with an nCPAP device. Seventy-one patients continued nightly nCPAP therapy. Bed partners of these patients (n = 55) answered a separate questionnaire assessing improvements in their own sleep quality, daytime alertness, mood and quality of life (questions 1 to 4), and evaluated the same parameters for the patients (questions 5 to 8). Possible scores ranged from -1 (worse) to +3 (marked improvement). Questions 1 to 4 yielded median scores of 2, 1, 1, and 2, respectively, and scores of 3, 3, 2, and 3 for questions 5 to 8. A ninth question addressing perceived changes in the quality of their relationship resulted in a median score of 2. Mean (SD) Epworth sleepiness scores improved from 14.3 (5.8) to 5.2 (4.3) in patients receiving therapy (p < 0.005). CONCLUSIONS: These data indicate that bed partners of OSA patients treated with nCPAP experience important improvements in symptoms and personal relationships. The findings are of practical clinical use when counseling patients with OSA and their partners on the likely impact of nCPAP therapy on their quality of life.     

The type 1 angiotensin-II receptor mediates intracellular calcium mobilization in rat luteal cells

The intracellular cytosolic calcium concentration ([Ca]i) was determined in cultured rat luteal cells using the calcium-chelating dye fura-2 and microspectrofluorimetry. Angiotensin-II (Ang-II) induced a dose-dependent transient increase in [Ca]i (ED50, 9.0 +/- 6.5 nM). After the initial peak in [Ca]i, cytosolic calcium returned to a secondary elevated basal level that was dependent upon the presence of extracellular calcium. Pretreatment of rat luteal cells with Ang-II (100 nM) desensitized a subsequent response to a higher concentration (1 microM), but did not desensitize a prostaglandin F2 alpha (PGF2 alpha)-induced calcium flux. Although the peak increases in [Ca]i induced by Ang-II (1 microM) and PGF2 alpha (10 microM) were not significantly different, the plateau phase stimulated by PGF2 alpha was significantly higher (P < 0.05) than that stimulated by Ang-II (1 microM). Pretreatment of luteal cells with the type 2 Ang-II receptor antagonist PD 123319 (10 microM) did not inhibit calcium mobilization; however, Ang-II (1 microM)-induced calcium mobilization was dose dependently blocked by the type 1 Ang-II receptor antagonist Losartan (DuP 753). The ID50 for Losartan was 5.2 +/- 1.8 nM. Pretreatment of the luteal cells with the endoplasmic reticulum calcium ATPase inhibitor thapsigargin (1 microM) also blocked Ang-II-induced calcium mobilization. These data demonstrate the presence of the type 1 Ang-II receptor in rat luteal cells, through which Ang-II dose dependently mobilizes calcium from an intracellular source, probably the endoplasmic reticulum.     

Calcium currents and calcium signaling in rod bipolar cells of rat retinal slices

Combined electrophysiological and imaging techniques were used to study calcium currents (ICa) and their sites of origin at rod bipolar cells in rat retinal slices. We report here for the first time the successful whole-cell patch-clamp recording from presynaptic boutons that were compared with somatic recordings. TTX-resistant inward currents were elicited in response to depolarization. The kinetic and pharmacological properties of ICa were very similar for recordings obtained from the soma and the presynaptic terminals. ICa activated maximally between -30 and -20 mV was enhanced by Bay K 8644 and was blocked by isradipine and nifedipine. Peak amplitude and time to peak were -31.3 +/- 1.2 pA and 3.2 +/- 0.2 msec with somatic recordings (n = 54), whereas the corresponding values were -31.6 +/- 6.1 pA and 3.2 +/- 0.7 msec in recordings obtained directly from terminals (n = 6). ICa showed little inactivation during sustained depolarizations. No T-type ICa was observed with depolarizations from -90 mV. Concomitant with Ca2+ entry, depolarization induced the appearance of transient outward currents that resembled IPSCs and were blocked by GABA and glycine receptor antagonists, suggesting that they arise from activation of amacrine feedback synapses. Upon depolarization, intracellular Ca2+ ([Ca2+]i) rises were restricted to the presynaptic terminals with no somatic or axonal changes and were linearly dependent on pulse duration when using a low-affinity Ca2+ indicator. In cone bipolar cells, ICa inactivated markedly, and [Ca2+]i rises occurred in the axon, as well as in the presynaptic terminals.     

Expression of Shaker-type voltage-gated potassium channel genes in the guinea-pig

PROBLEM: Inhibin A concentrations in serum may reflect the ovarian granulosa cell compartment. To characterize the correlation between ovarian function after gonadotoxic chemotherapy for Hodgkin's or non-Hodgkin's lymphoma in young women, the immunoreactive inhibin A concentrations in the sera of these patients was measured before, during, and after the gonadotoxic chemotherapy. METHOD OF STUDY: A prospective clinical protocol was undertaken in 20 cycling women with lymphoma, aged 15-40 years. A monthly injection of depot D-TRP6-GnRH-a (Decapeptyl CR, Ferring) was administered from before starting the chemotherapy until its conclusion, up to a maximum of six monthly injections. Most of the patients were treated with the mustargen-oncovin-procarbazine-prednisone (MOPP)/actinomycin D-bleomycin-vincristine (ABV) chemotherapy combination; 13 with and 7 without radiotherapy. A hormonal profile [follicle-stimulating hormone (FSH), luteinizing hormone (LH), 17-beta-estradiol (E2), testosterone (T), progesterone (P4), insulin-like growth factor (IGF)-1, IGF-BP3, and prolactin (PRL)] was taken before starting the gonadotropin-releasing hormone agonist (GnRH-a)/chemotherapy co-treatment and monthly thereafter until resuming spontaneous ovulation and menstrual cyclicity. This group of prospectively treated lymphoma patients was compared with a control group of 22 regularly cycling women who had been treated with chemotherapy (mostly MOPP/ABV) with or without radiotherapy for Hodgkin's or non-Hodgkin's lymphoma. Inhibin A immunoactivity developed by Nigel Groome was measured by an enzyme-linked immunoadsorbent assay (ELISA) commercial kit (Serotec). RESULTS: Whereas all but one (40 years of age) of the surviving patients in the GnRH-a/chemotherapy co-treatment group resumed spontaneous ovulation and menses within 6 months, only one half of the patients in the "control" group (chemotherapy without GnRH-a co-treatment) resumed ovarian function and regular cyclic activity (P < 0.05). The remaining 50% experienced premature ovarian failure (POF). Temporarily increased FSH concentrations were experienced by approximately one third of the patients resuming cyclic ovarian function, suggesting a reversible ovarian damage in a larger proportion of women than those experiencing POF. The inhibin A immunoactive concentrations decreased during the GnRH-a/chemotherapy co-treatment but increased to normal levels in patients who resumed regular ovarian cyclicity, and/or spontaneously conceived, as compared to low levels in menopausal women and those who had developed POF. CONCLUSIONS: If these preliminary data are consistent in a larger group of patients, inhibin A concentration may serve as a prognostic factor for predicting the resumption of ovarian function, in addition to the levels of FSH, LH, and E2.     

Properties of the voltage-gated calcium channels mediating dopamine and acetylcholine release from the isolated rat retina

We examined the properties of voltage-gated calcium channels mediating endogenous dopamine (DA) and acetylcholine (ACh) release in the isolated rat retina. Application of 30 mM KCl elicited the release of DA and ACh, and these releases were abolished in Ca(2+)-free medium. The high K(+)-evoked DA release was largely blocked by both of omega-agatoxin IVA and omega-conotoxin MVIIC, P- and Q-type calcium channel antagonists, and partly blocked by isradipine, and L-type calcium channel antagonist, and omega-conotoxin GVIA, an N-type calcium channel antagonist. omega-Agatoxin IVA at a small dose, sufficient to block P-type channels alone, was however without effect. On the other hand, the high K(+)-evoked ACh release was partly blocked by omega-agatoxin IVA and omega-conotoxin MVIIC, but was resistant to isradipine and omega-conotoxin GVIA. Flunarizine, a non-selective T-type calcium channel antagonist, did not inhibit the release of DA and ACh. Cd2+ markedly blocked the release of both DA and ACh, Co2+ and Ni2+ slightly blocked the release of DA, and the release of ACh was not blocked by these two divalent cations. These results suggest that the high K(+)-evoked release of retinal DA is largely mediated by omega-agatoxin IVA and omega-conotoxin MVIIC sensitive calcium channels (probably Q-type channels), while the release of retinal ACh is largely mediated by as yet uncharacterized Cd2+ sensitive calcium channels. The properties of voltage-gated calcium channels involved in the release of ACh in the rat retina differ from those of DA.     

Calcium channel blockers in heart failure

Patients with heart failure are particularly susceptible to the negative effects of calcium channel blockers because the failing heart demonstrates a defect in the delivery of calcium to the contractile proteins, and an attenuation of the normal sympathetic reflexes. Currently these drugs have no place in the treatment of heart failure caused by systolic dysfunction of the left ventricle. Calcium channel blockers should probably not be described for patients with coronary artery disease and left ventricular dysfunction. When the patient needs additional treatment for angina and beta-blockers or nitrates have not given satisfactory results, it may be appropriate to prescribe amlodipine or felodipine.     

Selective ligand-induced intracellular calcium changes in a population of rat isolated gastric endocrine cells

BACKGROUND & AIMS: Peripheral regulation of acid secretion depends mainly on stimulation or inhibition of the three major gastric endocrine cells (enterochromaffin-like, gastrin, and somatostatin). The aim of this paper was to define physiological responses of enterochromaffin-like, gastrin, and somatostatin cells in a mixed endocrine cell population by measuring ligand-selective changes of intracellular calcium ([Ca2+]i) in individual cells. METHODS: Endocrine cells were enriched from a rat gastric cell suspension by elutriation, a density-gradient fractionation, and a 48-hour short-term culture. [Ca2+]i responses of individual cells to various ligands such as gastrin/carboxy-terminal cholecystokinin octapeptide and selective cholecystokinin antagonists, carbachol, and gastrin-releasing peptide were monitored using video imaging in a perfusion chamber. Characteristic [Ca2+]i changes distinguished the three cell types, confirmed by immunostaining. RESULTS: All enterochromaffin-like cells respond to cholecystokinin-B receptor stimulation, but only a few respond to carbachol. Gastrin cells respond to both gastrin-releasing peptide and carbachol but not to cholecystokinin-receptor agonists. Somatostatin cells have both stimulatory cholecystokinin-A and cholecystokinin-B receptors and inhibitory muscarinic receptors. All cells have inhibitory somatostatin receptors. CONCLUSIONS: Calcium-signaling responses of gastric endocrine cells are distinctive. This allows individual cell types in a mixed population to be characterized and permits an analysis of the hormones and transmitters that act directly on a specific cell type.     

Rapid effects of cytochalasin-D on contraction and intracellular calcium in single rat ventricular myocytes

Contraction and intracellular calcium ([Ca2+]i) transients were recorded using a video edge detector and fluorescence spectrophotometry, respectively, in rat ventricular myocytes at 22-24 degreesC stimulated at a frequency of 1 Hz. Application of the F-actin disrupter cytochalasin-D (Cyt-D) caused a large reduction in the amplitude of contraction and a small increase in the [Ca2+]i transient. These responses began within a few seconds of application and were complete after 2 min of exposure. Phase-plane relationships of contraction and [Ca2+]i were consistent with cytochalasin-D causing a decrease in myofilament responsiveness to Ca2+.