Modulation Of Calcium Influx Research Articles

Transmural Gradients of Repolarization and Excitation–Contraction Coupling in Mouse Ventricle

See related article, pages 1306–1313 The effects of changes in action potential waveform on excitation–contraction coupling in mammalian ventricle were first identified almost 40 years ago.1 Many important details concerning the relationship between action potential duration and tension development have been elucidated.2 Recent work has demonstrated significant effects on the intracellular Ca transient and excitation–contraction coupling of early repolarization of the action potential, which is strongly regulated by a Ca2+-independent transient outward K+ current denoted I to.3–6 The molecular physiology and some aspects of the pharmacology of this current are now quite well understood, based, in part, on a series of comprehensive articles from the Nerbonne et al,7,8 Strauss and Campbell,9 and others.6 Interestingly, a somewhat similar transient outward K+ current is expressed in neurons10 and in selected regions near the intraventricular septum of mammalian hearts.11–13 One of the most striking features of the transient outward K+ current in mammalian ventricle is the difference in its transmural expression, with significantly higher expression levels in epi- than in endocardium14 (see also references 8, 9, 11). The basis for this transmural heterogeneity, and also for the higher levels of expression in the right ventricle compared with the left, continues to be a topic of intense investigation. Significant new information and plausible working hypotheses for the way in which this heterogeneity …

Taurine modulates calcium influx through L-type voltage-gated calcium channels in isolated cochlear outer hair cells in guinea pigs

Taurine has been proposed to play a role in calcium modulation. To explore the effect of taurine on intracellular calcium homeostasis of isolated cochlear outer hair cells and on the gentamycin-induced inhibition of calcium influx evoked by high K + depolarization, we employed fluo-3 imaging of intracellular calcium ([Ca 2+] i) via confocal laser scanning microscopy to measure real-time changes of [Ca 2+] i. We found that the sole application of taurine (5, 10, 20 mM) induced a transient [Ca 2+] i increase in a concentration-dependent manner, which was inhibited either by the application of an L-type calcium-channel blocker nifedipine or a calcium-free medium. Pre-incubation with 1 mM gentamicin induced inhibition of [C a 2+] i elevation evoked by high K +. Short-term (10 min) exposure with a high level of taurine (20 mM) prevented this inhibition. These results indicated that taurine at a high concentration was able to promote calcium influx through L-type calcium channels in isolated outer hair cells and antagonize gentamycin-induced inhibition of calcium elevation evoked by high K + by its calcium homeostatic effect.

Sensing the Right Path

During nervous system development, axonal growth cones find their way to their targets with the help of various chemoattractant and chemorepellant cues. Several neurite guidance signals modulate calcium influx into the growth cone, but the underlying mechanisms and the specific channels involved have been unclear (see Gomez). Now two groups, Li et al. and Wang and Poo, provide evidence implicating transient receptor potential canonical (TRPC) channels--a family of channels known for mediating sensory responses--in the response to the chemoattractants brain-derived neurotrophic factor (BDNF) and netrin-1. Wang and Poo performed perforated whole-cell patch clamp analysis of cultured Xenopus spinal neurons and found that netrin-1 elicited membrane depolarization that was inhibited by an antibody to its receptor, DDC. Netrin-induced current present during pharmacological blockade of most voltage-dependent ion channels was inhibited by SKF-96365, which blocks TRP channels, as was a similar current produced by BDNF. Further, growth cone turning in response to netrin-1 or BDNF was abolished by SKF-96365. Injection of morpholino oligonucleotides (MOs) that knocked down xTRPC1 expression inhibited the putative TRP currents and abolished growth cone turning in response to a netrin-1 gradient. Moreover, MO or SKF-96365 abolished the netrin-induced increase in growth cone calcium concentration. Li et al. investigated TRPC channels in BDNF-mediated guidance of cultured cerebellar granule cells, which was blocked by antibodies to the TrkB receptor, incubation with a membrane-permeable calcium chelator, or pharmacological depletion of intracellular calcium stores. Although pharmacological blockade of several voltage-sensitive sodium and calcium channels had no effect on growth cone turning elicited by BDNF, turning was abolished by SKF-96365, as well as by pharmacological inhibition of phospholipase C (PLC), phosphatidylinositol 3-kinase (PI3K), or blockade of the inositol 1,4,5-trisphosphate receptor (IP 3 R). In contrast, a PLC activator elicited turning, an effect that depended on extracellular calcium and was abolished by SKF-96365. BDNF increased growth cone calcium concentration, an effect that depended on both calcium influx and release from internal stores and that was reduced by SKF-96365, pharmacological inhibition of PLC, or IP 3 R blockade. BDNF-induced turning was abolished by siRNA directed against TRPC3 or by overexpression of dominant-negative mutant forms of TRPC3 or TRPC6. Thus, activation of TRPC channels appears to play a key role in calcium-dependent neurite pathfinding in response to both netrin-1 and BDNF. Y. Li, Y.-C. Jia, K. Cui, N. Li, Z.-Y. Zheng, Y.-Z. Wang, X.-B. Yuan, Essential role of TRPC channels in the guidance of nerve growth cones by brain-derived neurotrophic factor. Nature 434 , 894-898 (2005). [PubMed] G. X. Wang, M.-M. Poo, Requirement of TRPC channels in netrin-1-induced chemotropic turning of nerve growth cones. Nature 434 , 898-904 (2005). [PubMed] T. Gomez, Channels for pathfinding. Nature 434 , 835-838 (2005). [PubMed]

Mechanism of Adrenomedullin-induced Prolactin Release from Human Prolactin-releasing Adenoma Cells

The mechanism of adrenomedullin-induced prolactin release was investigated in prolactin-secreting human pituitary adenoma cells by intracellular calcium measurement and static incubation study. Adrenomedullin stimulated prolactin release in a concentration-dependent manner. The stimulation was dependent on extracellular sodium and voltage-gated calcium channels. PKA inhibitor attenuated adrenomedullin-induced prolactin release. The mechanism of adrenomedullin action was studied by fura 2-based intracellular calcium measurement. Adrenomedullin increased intracellular calcium concentration in these cells. The increase was dependent on extracellular sodium and voltage-gated calcium channels. PKA inhibitor attenuated the calcium response. These data indicate that adrenomedullin stimulates prolactin release by modulating calcium influx through voltage-gated calcium channels dependently on extracellular sodium. Mechanisms involving sodium-influx mediated depolarization may play a role in the stimulatory action.

Propofol: Therapeutic Indications and Side-Effects

Propofol (2, 6-diisopropylphenol) is a potent intravenous hypnotic agent which is widely used for the induction and maintenance of anesthesia and for sedation in the intensive care unit. Propofol is an oil at room temperature and insoluble in aqueous solution. Present formulations consists of 1% or 2% (w/v) propofol, 10% soybean oil, 2.25% glycerol, and 1.2% egg phosphatide. Disodium edetate (EDTA) or metabisulfite is added to retard bacterial and fungal growth. Propofol is a global central nervous system depressant. It directly activates GABA(A) receptors. In addition, propofol inhibits the NMDA receptor and modulates calcium influx through slow calcium ion channels. Propofol has a rapid onset of action with a dose-related hypnotic effect. Recovery is rapid even after prolonged use. Propofol decreases cerebral oxygen consumption, reduces intracranial pressure and has potent anti-convulsant properties. It is a potent antioxidant, has anti-inflammatory properties and is a bronchodilator. As a consequence of these properties propofol is being increasingly used in the management of traumatic head injury, status epilepticus, delirium tremens, status asthmaticus and in critically ill septic patients. Propofol has a remarkable safety profile. Dose dependent hypotension is the commonest complication; particularly in volume depleted patients. Hypertriglyceridemia and pancreatitis are uncommon complications. Allergic complications, which may include bronchospasm, have been reported with the formulation containing metabisulfite. In addition, this formulation has been demonstrated to result in the generation of oxygen free radicals. High dose propofol infusions have been associated with the "propofol syndrome"; this is a potentially fatal complication characterized by severe metabolic acidosis and circulatory collapse. This is a rare complication first reported in pediatric patients and believed to be due to decreased transmembrane electrical potential and alteration of electron transport across the inner mitochondrial membrane.

Interaction of TRPC2 and TRPC6 in erythropoietin modulation of calcium influx.

Erythropoietin (Epo) modulates calcium influx through voltage-independent calcium-permeable channel(s). Here, we characterized the expression of transient receptor potential channels (TRPCs) in primary erythroid cells and examined their regulation. Erythroblasts were isolated from the spleens of phenylhydrazine-treated mice, and Epo stimulation resulted in a significant and dose-dependent increase in [Ca](i). Among the classical TRPC channels, expression of three N-terminal splice variants of TRPC2 (clones 14, 17, and alpha) and of TRPC6 were demonstrated in these erythroblasts by both reverse transcriptase-PCR and Western blotting. Confocal microscopy confirmed localization to the plasma membrane. To determine the function of individual TRPC channels in erythropoietin modulation of calcium influx, digital video imaging was used to measure calcium influx through these TRPCs in a Chinese hamster ovary (CHO) cell model. Single CHO-S cells, expressing transfected Epo-R, were identified by detection of green fluorescent protein. Cells that express transfected TRPCs were identified by detection of blue fluorescent protein. [Ca](i) was monitored with Fura Red. Epo stimulation of CHO-S cells transfected with single TRPC2 isoforms (clone 14, 17, or alpha) and Epo-R resulted in a significant increase in [Ca](i). This was not observed in cells transfected with Epo-R and TRPC6. In addition, coexpression of TRPC6 with TRPC2 and Epo-R inhibited the increase in [Ca](i) observed after Epo stimulation. Immunoprecipitation experiments demonstrated that TRPC2 associates with TRPC6, indicating that these TRPCs can form multimeric channels. These data demonstrate that specific TRPCs are expressed in primary erythroid cells and that two of these channels, TRPC2 and TRPC6, can interact to modulate calcium influx stimulated by erythropoietin.

A lipoxygenase product, hepoxilin A 3, enhances nerve growth factor-dependent neurite regeneration post-axotomy in rat superior cervical ganglion neurons in vitro

Hepoxilins are 12-lipoxygenase metabolites of arachidonic acid found in the CNS. They can modulate neuronal signaling but their functions are not known. We examined the effects of hepoxilin A 3 on neurite outgrowth post-axotomy in an in vitro model of spinal cord transection using superior cervical ganglion neurons. In the absence of nerve growth factor, hepoxilin A 3 did not support neuronal survival, or regeneration post-axotomy but did significantly enhance neurite regeneration in the presence of nerve growth factor. As early as 1 h post-injury hepoxilin A 3-treated cultures (+nerve growth factor) had significantly more neurites than controls (nerve growth factor alone). Average hourly rates of outgrowth in hepoxilin A 3-treated cultures were significantly higher than in controls for at least 12 h post-injury, suggesting that the effect of hepoxilin A 3 is maintained in vitro for several hours post-injury. In uninjured neurons hepoxilin A 3 caused a rapid but transient increase in intracellular calcium in the somata; by 2 min post-addition, calcium levels decreased to a new stable plateau significantly higher than pre treatment levels. In injured neurons, hepoxilin A 3 addition immediately post-transection caused a rapid transient increase in intracellular calcium in cell bodies; however, peak calcium levels were significantly lower than in uninjured neurons and the new baseline lower than in uninjured cells. In uninjured cells hepoxilin A 3 addition in zero calcium produced the same pattern, a transient elevation and subsequent decline to a new stable baseline significantly above rest but in injured cells levels fell rapidly to pretreatment values. Taken overall, these findings demonstrate a novel role for hepoxilins as a potentiator of neurite regeneration. They also provide the first evidence that this lipoxygenase metabolite can alter intracellular calcium in neurons by causing release of calcium from intracellular stores and modulating calcium influx mechanisms.

Erythropoietin Modulates Calcium Influx through TRPC2

Mammalian isoforms of calcium-permeable Drosophila transient receptor potential channels (TRPC) are involved in the sustained phase of calcium entry in nonexcitable cells. Erythropoietin (Epo) stimulates a rise in intracellular calcium ([Ca](i)) via activation of voltage-independent calcium channel(s) in erythroid cells. Here, involvement of murine orthologs of classical TRPC in the Epo-modulated increase in [Ca](i) was examined. RT-PCR of TRPC 1-6 revealed high expression of only TRPC2 in Epo-dependent cell lines HCD-57 and Ba/F3 Epo-R, in which Epo stimulates a rise in [Ca](i). Using RT-PCR, Western blotting, and immunolocalization, expression of the longest isoform of mTRPC2, clone 14, was demonstrated in HCD-57 cells, Ba/F3 Epo-R cells, and primary murine erythroblasts. To determine whether erythropoietin is capable of modulating calcium influx through TRPC2, CHO cells were cotransfected with Epo-R subcloned into pTracer-CMV and either murine TRPC2 clone 14 or TRPC6, a negative control, into pQBI50. Successful transfection of Epo-R was verified in single cells by detection of green fluorescent protein from pTracer-CMV using digital video imaging, and successful transfection of TRPC was confirmed by detection of blue fluorescent protein fused through a flexible linker to TRPC. [Ca](i) changes were simultaneously monitored in cells loaded with Rhod-2 or Fura Red. Epo stimulation of CHO cells cotransfected with Epo-R and TRPC2 resulted in a rise in [Ca](i) above base line (372 +/- 71%), which was significantly greater (p < or = 0.0007) than that seen in cells transfected with TRPC6 or empty pQBI50 vector. This rise in [Ca](i) required Epo and extracellular calcium. These results identify a calcium-permeable channel, TRPC2, in erythroid cells and demonstrate modulation of calcium influx through this channel by erythropoietin.

Endoplasmic reticulum calcium release is modulated by actin polymerization.

Intracellular calcium ions regulate the structure and functions of cytoskeletal proteins. On the other hand, recent studies have shown that the cytoskeleton, and actin filaments in particular, can modulate calcium influx through plasma membrane ligand- and voltage-gated channels. We now report that calcium release from inositol trisphosphate (IP3) and ryanodine-sensitive endoplasmic reticulum (ER) stores is modulated by polymerization and depolymerization of actin filaments in cultured hippocampal neurons. Depolymerization of actin filaments with cytochalasin D attenuates calcium release induced by carbamylcholine (CCh; a muscarinic agonist for IP3 pathway), caffeine (a ryanodine receptor agonist) and thapsigargin (an inhibitor of the ER calcium- ATPase) in both the presence and absence of extracellular calcium. Conversely, the actin polymerizing agent jasplakinolide potentiates calcium release induced by CCh, caffeine and thapsigargin. Cytochalasin D attenuated, while jasplakinolide augmented, thapsigargin-induced JNK activation and neuronal cell death. Our data show that the actin cytoskeleton regulates ER calcium release, suggesting roles for actin in the various physiological and pathological processes that involve calcium release.

A nitric oxide/cyclic GMP-dependent protein kinase pathway alters transmitter release and inhibition by somatostatin at a site downstream of calcium entry.

We have examined the somatostatin-mediated modulation of acetylcholine release from intact chick embryo choroid tissue and compared these data with those obtained using acutely dissociated neuronal cell bodies from the chick ciliary ganglion. Acetylcholine release, evoked in a calcium-dependent manner by a high potassium (55 mM KCI) stimulation in both preparations, was inhibited almost completely by 100 nM somatostatin. Measurement of intracellular calcium in these neurons revealed that somatostatin blocked the large calcium transient that was observed in control neurons following KCI exposure. The modulatory effect of somatostatin on transmitter release was significantly attenuated by pre-treatment with pharmacologic agents that selectively block cyclic GMP (cGMP)-dependent protein kinase (PKG) or nitric oxide (NO) synthase. It is interesting that this prevention of somatostatin-mediated acetylcholine release inhibition occurred without reversal of the somatostatin-mediated block of the KCl-evoked calcium transient. Furthermore, a NO donor or cGMP analogue could block KCI-evoked acetylcholine release, but only cGMP could reduce the KCI-evoked calcium transient. Although cGMP could reduce the KCI-evoked calcium transient, a cGMP analogue was shown to reduce calcium ionophore-evoked transmitter release. Thus, somatostatin reduces acetylcholine release by modulating calcium influx, but the NO-PKG pathway can inhibit acetylcholine release, and alter somatostatin-mediated inhibition, by affecting transmitter release at some point after calcium entry.

Fluoxetine Action on Murine T-Lymphocyte Proliferation: Participation of PKC Activation and Calcium Mobilisation

The present study was undertaken to analyse the effect of fluoxetine upon murine T-lymphocyte proliferation. We found that fluoxetine exerted a dual effect, which depended on the degree of lymphocyte activation: at mitogenic concentration (2 μg/mL) of concavalin A (Con A), we observed an inhibitory effect on cellular proliferation, whereas, on submitogenic Con A concentration (1 μg/mL), fluoxetine stimulated the cellular response. Given these facts, we studied PKC activation and calcium mobilisation in both stimulatory and inhibitory effects of fluoxetine on T-cell proliferation. We observed that fluoxetine increased PKC translocation obtained with 1 μg/mL Con A concentration, whereas PKC was degraded when 2 μg/mL was used. This mechanism is thought to be mediated by calcium mobilisation. According to our results, fluoxetine seemed to modulate calcium influx, which, in turn, would influence PKC translocation, modulating the immune response.

Adenosine A 2A receptors facilitate 45Ca 2+ uptake through class A calcium channels in rat hippocampal CA3 but not CA1 synaptosomes

In the hippocampus, the neuromodulatory role of adenosine depends on a balance between inhibitory A 1 responses and facilitatory A 2A responses. Since the presynaptic effects of hippocampal inhibitory A 1 adenosine receptors are mostly mediated by inhibition of Ca 2+ channels, we now investigated whether presynaptic facilitatory A 2A adenosine receptors would modulate calcium influx in the hippocampus. The mixed A 1/A 2 agonist, 2-chloroadenosine (CADO; 1 μM) inhibited veratridine (20 μM)-evoked 45Ca 2+ influx into hippocampal synaptosomes of the CA1 or CA3 areas by 24.2±4.5% and 17.2±5.8%, respectively. In the presence of the A, antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 100 nM), the inhibitory effect of CADO (1 μM) on 45Ca 2+ influx was prevented in CA1 synaptosomes, but was converted into a facilitatory effect (14.2±6.7%) in CA3 synaptosomes. The A 2A agonist, CGS 21680 (3–30 nM) facilitated 45Ca 2+ influx in CA3 synaptosomes, with a maximum increase of 22.9±3.9% at 10 nM, and was virtually devoid of effect in CA1 synaptosomes. This facilitatory effect of CGS 21680 (10 nM) in CA3 synaptosomes was prevented by the A 2A antagonist 8-(3-chlorostyryl)caffeine (CSC; 200 nM), but not by the A 1 antagonist, DPCPX (20 or 100 nM). The facilitatory effect of CGS 21680 on 45Ca 2+ uptake by CA3 synaptosomes was prevented by the class A calcium channel blocker, ω-agatoxin-IVA (200 nM). These results indicate that presynaptic adenosine A 2A receptors facilitate calcium influx in the CA3 but not the CA1 area of the rat hippocampus through activation of class A calcium channels.

Selective Inhibition of Mitogen-induced Transactivation of the HIV Long Terminal Repeat by Carboxyamidotriazole: CALCIUM INFLUX BLOCKADE REPRESSES HIV-1 TRANSCRIPTIONAL ACTIVATION

Carboxyamidotriazole (CAI) is a calcium influx inhibitor that has both antiproliferative and antimetastatic activities. Pretreatment of human T-cells with micromolar concentrations of CAI causes a near complete inhibition of calcium-regulated mitogen-induced transcription from the human immunodeficiency virus (HIV) long terminal repeat (LTR). This inhibition is selective since other mitogen-activated gene regulatory elements, such as the 12-O-tetradecanoylphorbol-13-acetate response element, are not influenced by the drug. HIV LTR transcription inhibition is maximal at 1.0 microM CAI, requires a pretreatment interval of at least 8 h for optimum inhibition, and shows no acute interference with the growth properties of the cells. Moreover, the inhibition is rapidly reversible upon removal of the drug from the medium. Studies to identify enhancer elements within the HIV LTR that are functionally sensitive to low-dose long-term pretreatment with CAI indicate that the NF-kappaB-binding sites are among the major targets of drug action. In vitro DNA binding studies with nuclear extracts prepared from mitogen-induced T-cells stimulated in the presence of CAI indicate that the drug differentially influences the calcium-regulated downstream signal transduction pathways necessary for specific NF-kappaB DNA binding activity at the two kappaB sites within the HIV LTR. Studies with ionomycin and thapsigargin show that repression is specific for selected modes of inducible calcium entry and indicate that, in T-cells, a major mechanism of CAI action is to modulate calcium influx at a level that is proximal to the regulated release of calcium from intracellular stores. Measurement of calcium influx in CAI-treated cells reveals a dramatic and reversible inhibition of mitogen-induced calcium influx. These results indicate that CAI can be an important and effective pharmacological tool for analysis of the calcium-dependent modulation of HIV LTR transcription.

Inhibition of phosphatases and increased Ca2+ channel activity by inositol hexakisphosphate.

Inositol hexakisphosphate (InsP6), the dominant inositol phosphate in insulin-secreting pancreatic beta cells, inhibited the serine-threonine protein phosphatases type 1, type 2A, and type 3 in a concentration-dependent manner. The activity of voltage-gated L-type calcium channels is increased in cells treated with inhibitors of serine-threonine protein phosphatases. Thus, the increased calcium channel activity obtained in the presence of InsP6 might result from the inhibition of phosphatase activity. Glucose elicited a transient increase in InsP6 concentration, which indicates that this inositol polyphosphate may modulate calcium influx over the plasma membrane and serve as a signal in the pancreatic beta cell stimulus-secretion coupling.

Action potential duration modulates calcium influx, Na(+)-Ca2+ exchange, and intracellular calcium release in rat ventricular myocytes.

The experimental work summarized in this paper and described in more detail in our previous publication demonstrates a very important functional role for Na(+)-Ca2+ exchange in intracellular Ca2+ homeostasis in ventricular myocytes from rat hearts. Ca2+ homeostasis in mammalian cardiac myocytes can be considered to be the result of four interactive processes: (i) Ca2+ influx through L-type Ca2+ channels, (ii) Ca2+ release from the SR and its subsequent re-uptake, (iii) intracellular Ca/+ buffering, and (iv) Ca2+ extrusion across the sarcolemma. Our results demonstrate a number of interesting features of these processes. (1) When the action potential voltage-clamp technique is used to identify the size and time-course of Ca2+ fluxes during the action potential, both the peak current and the associated influx of Ca2+ are relatively large as was previously demonstrated by Isenberg and his colleagues. (2) Nevertheless, this source of Ca2+ is unable, by itself, to produce a significant twitch, which is consistent with previous data from rat ventricle. (3) This Ca2+ influx, however, does represent the trigger for SR Ca2+ release. (4) The Na(+)-Ca2+ exchanger on the SR is able, on average, to extrude all the Ca2+ which enters through L-type Ca2+ channels, although it provides relatively little Ca2+, i.e., during the course of the normal action potential there is no significant reverse Na(+)-Ca2+ exchange activity, at least under our experimental conditions. Our results also suggest that although the L-type Ca2+ current cannot by itself trigger and control contraction its amplitude, frequency, and time-course can alter the rate and the extent of Ca2+ release from the SR. Recently, detailed mathematical formulations and a direct demonstration of some of these phenomena have been published. Stern and Stern and Lakatta predicted more than three years ago that the concentration and the time-course of change in concentration of Ca2+ very near the release sites of the SR may be critical determinants of the overall release process. Within the past year Wier and his colleagues and also Lederer et al. have combined electrophysiological measurements with recordings of localized intracellular Ca2+ (made using a confocal microscope) and have shown that rapid, and relatively large, but very localized changes in intracellular Ca2+ due to Ca2+ influx through L-type Ca2+ channels are responsible for triggering, and to some extent, controlling the release of Ca2+ from the SR. However, it has also been shown that this release depends importantly on the loading or priming state of the SR. Perhaps not surprisingly, the massive release of Ca2+ from the SR can, itself, alter the pattern of subsequent SR release events (cf. Ref. 46) and the time-course of Ca2+ influx through the L-type Ca2+ channels. Thus, although our relatively crude measurements have clearly demonstrated the relationship between L-type Ca2+ channel activity and Na+-Ca2+ exchanger function during a normal cardiac action potential in rat ventricle, they fall far short of any delineation of the functional roles of either of these processes in overall Ca2+ homeostasis. This additional information can, in principle, be obtained from studies in which cellular microanatomy can be visualized dynamically in conjunction with localized changes in intracellular Ca2+ as well as Ca2+ of L-type Ca2+ channels, SR release, and cell shortening.

Developmental changes and second messenger modulation of the electrical properties of cells of the cockroach corpora allata

The duration of evoked action potentials in the cells of the corpora allata of recently emerged adult mated female cockroaches was dramatically increased following replacement of calcium ions by barium ions in the bathing saline. The magnitude of this increase was age-dependent. Furthermore, following exposure of corpora allata to barium, spontaneous action potentials (approx. 10–40 mV in amplitude) and small depolarizing potentials (approx. 2–5 mV in amplitude) were observed that were never seen in the absence of barium. This suggests that barium increases the excitability of the corpora allata. 8-bromo-cyclic AMP and the phorbol ester, phorbol 12,13-dibutyrate (PDBu), both increased the duration of barium action potentials in corpora allata from day-1 and day-6 females. However, 8-bromo-cyclic GMP, and the nonspecific protein kinase inhibitor H7 both reduced the barium action potential in day-1 corpora allata but had no significant effect in day-6 corpora allata. Furthemore, the inactive phorbol ester 4a-phorbol 12,13-didecanoate (PDD) did not significantly affect the barium action potential duration in day-1 or day-6 corpora allata, suggesting that PDBu is acting through phospholipid/calcium-dependent protein kinase C and not nonspecifically. 8-Br-cAMP and protein kinase C appear to modulate the electrophysiological properties of the cell membranes of the corpora allata, which suggests that they may modulate calcium influx. In addition, cAMP, protein kinase C and calcium have previously been implicated in the regulation of juvenile hormone biosynthesis. In view of the effects of cAMP and protein kinase C on both membrane properties and juvenile hormone biosynthesis, the cell membrane may be a site for integration of the calcium, cAMP and protein kinase C second messenger systems involved in the regulation of juvenile hormone biosynthesis. Furthermore, because juvenile hormone biosynthesis undergoes cyclical changes in adult mated female cockroaches and there are developmental changes in the electrical properties of the corpora allata, such changes may be important in the regulation of juvenile hormone biosynthesis, possibly by altering calcium influx.

Facilitatory and inhibitory transmitters modulate calcium influx during action potentials in Aplysia sensory neurons

Serotonin (5-HT) produces presynaptic facilitation and FMRFamide produces presynaptic inhibition in Aplysia sensory neurons. These effects may involve the modulation of Ca 2+ influx into sensory neuron terminals during action potentials. Here, we have used the Ca 2+ indicator dye fura-2 to monitor directly the effects of 5-HT and FMRFamide on internal Ca 2+ concentration ([Ca 2+] i). 5-HT caused a 50% increase in the transient rise in [Ca 2+] i in response to action potentials, whereas FMRFamide decreased the [Ca 2+] i transient by 40%. Neither transmitter altered the resting [Ca 2+] i, the time course of recovery of the [Ca 2+] i transient, or the [Ca 2+] i transients produced by intracellular injection of CaCl 2 or inositol 1,4,5-trisphosphate. We conclude that the effects of the transmitters on the action potential-induced [Ca 2+] i transient are due to changes in Ca 2+ influx and not in intracellular Ca 2+ homeostasis.

Structural analysis of the calcium channel by photoaffinity labelling and limited proteolysis

The L-type calcium channel of rabbit skeletal muscle triads, purified from digitonin extracts, was photolabelled with the dihydropyridine (+)[ 3H]PN 200-110. This photolabelled form was then subjected to limited proteolysis with papain and staphylococcus V-8 protease and analyzed by polyacrylamide gel electrophoresis. In the absence of proteolysis, the photolabelled channel was represented by a single protein with an apparent molecular weight of 160 kDa in the presence or absence of reducing agents. Following proteolysis, numerous photoadducts were observed with smaller molecular weights. The V-8 protease digestion pattern indicated that photoinsertion occurred in at least two distinct domains of 33 and 28 kDa. Papain digests were more extensive, generating smaller fragments of 28 and ca. 10 kDa. The results suggest that at least two distinct regions of the calcium channel interface at or near the dihydropyridine binding site, and that the binding site for these calcium antagonists resides within the channel proper, thereby modulating calcium influx.

Dopamine Inhibits Prolactin Secretion Stimulated by the Calcium Channel Agonist Bay-K-8644 through a Pertussis Toxin-Sensitive G Protein in Anterior Pituitary Cells*

In primary culture of anterior pituitary cells, BAY-K-8644, a calcium channel agonist, stimulated PRL secretion by 83% with EC50 of 18 nM. This effect was blocked by nifedipine, a calcium channel antagonist. The stimulations of PRL secretion induced by potassium (50 mM) and BAY-K-8644 were additive. Dopamine inhibited basal as well as BAY-K-8644-stimulated PRL secretion by 64% and 75%, respectively, and with respective EC50 values of 4.5 and 0.6 nM. In the presence of 50 mM K+, dopamine only partially blocks the dose-dependent stimulation of PRL secretion induced by the calcium channel agonist. The inhibitory dopamine effect was blocked by (+)butaclamol, a specific dopamine receptor antagonist. The dopamine response was also blocked by 1-sulpiride, a specific dopamine D2 receptor antagonist, and mimicked by RU 24926, a specific dopamine D2 receptor agonist, suggesting that the dopamine effect on BAY-K-8644-stimulated PRL secretion was mediated through a D2 dopamine receptor. Although unknown, the mechanism by which dopamine inhibited the BAY-K-8644-stimulated PRL secretion involves a GTP binding protein sensitive to Bordetella pertussis toxin. In fact, the dopamine inhibition of PRL secretion induced by the calcium channel agonist was blocked by the pretreatment of cells with the toxin. These results suggest that dopamine D2 receptors in lactotroph cells modulate calcium influx through a GTP binding protein.

Calcium pools involved in histamine release from rat mast cells.

Basic secretagogues, antigen, concanavalin A, the ionophore A23187 and, to a lesser extent, anti-rat IgE produce a significant release of histamine from rat peritoneal mast cells in the absence of extracellular calcium. This release is due to the mobilization of intracellular reservoirs of calcium. The release is abolished by prolonged exposure to chelating agents, but is potentiated by brief exposure to these substances. It is suggested that the latter treatment removes calcium from a superficial, regulatory site and thus facilitates the mobilization of more internal pools of the ion. By analogy with smooth muscle, these regulatory sites may also modulate calcium influx into the cell. On the basis of these and other results, the possible calcium pools important in histamine secretion are discussed.