Promoting Calcium Influx Research Articles

The hyperpolarizing impact of glycine on endothelial cells may be anti-atherogenic

Studies to date indicate that endothelial cells express glycine-activated chloride channels, which promote hyperpolarization of the endothelial plasma membrane. If such channels are expressed by endothelial cells lining conduit arteries, glycine is likely to have anti-atherogenic activity. This reflects the fact that endothelial hyperpolarization promotes calcium influx, activating the endothelial isoform of nitric oxide synthase, while also down-regulating the activity of the membrane-bound NADPH oxidase, chief endothelial source of superoxide. Since macrophages express glycine-activated chloride channels that suppress production of oxidants and cytokines, glycine may also oppose atherogenesis by influencing intimal macrophage function. In rats, supplemental glycine exerts anti-inflammatory and anti-angiogenic effects attributed to chloride channel activation. Administration of large daily doses of glycine would appear to be practical and safe, and has already been shown to inhibit protein glycation in human diabetics.

Molecular mechanism of tamoxifen‐induced calcium influx in human platelets

Tamoxifen, a non steroidal estrogen antagonist, is the most widely used drug for treatment and prevention of breast cancer. However, tamoxifen can cause thrombosis by an unknown mechanism. Using fura-2 to measure intracellular calcium in freshly isolated human platelets, we found that tamoxifen and its major metabolite 4-hydroxy tamoxifen at 5–10 uM promote calcium influx into platelets. Tamoxifen also potentiates action of platelet agonists (thrombin, vasopressin, ADP) to raise calcium. Tamoxifen effect was not attenuated by estrogen receptor antagonist ICI 182780 or COX inhibitor aspirin, but was blocked by the PLC inhibitor U-73122. Tamoxifen-induced calcium elevation was blocked by the lipid-soluble antioxidant vitamin E, but was unaffected by water-soluble vitamin C and quercetin. Tamoxifen effect was also blocked by the NADPH inhibitor apocynin and by wortmannin, an inhibitor of PI3 kinase, an upstream effector of NADPH oxidase. High molecular weight derivatives of 4-hydroxy tamoxifen conjugated to poly-methacrylic acid also promoted calcium elevation in platelets. An anti-estrogenic drug raloxifene, which is structurally related to tamoxifen, does not affect platelet calcium entry. Therefore tamoxifen may affect membrane-bound PLC, NADPH oxidase and PI3 kinase, leading to the activation of store-operated calcium channels, calcium entry into platelets, platelet activation and aggregation, which may be an underlying mechanism of tamoxifen-induced thrombosis. (Supported by grants from CHRB of Virginia and Cancer Research and Prevention Foundation to YD and Army Breast Cancer Research Program to RW).

Mechanisms of homocysteine neurotoxicity in neurodegenerative diseases with special reference to dementia

Mild to moderate hyperhomocysteinemia is a risk factor for neurodegenerative diseases. Human studies suggest that homocysteine (Hcy) plays a role in brain damage, cognitive and memory decline. Numerous studies in recent years investigated the role of Hcy as a cause of brain damage. Hcy itself or folate and vitamin B12 deficiency can cause disturbed methylation and/or redox potentials, thus promoting calcium influx, amyloid and tau protein accumulation, apoptosis, and neuronal death. The Hcy effect may also be mediated by activating the N-methyl- d-aspartate receptor subtype. Numerous neurotoxic effects of Hcy can be blocked by folate, glutamate receptor antagonists, or various antioxidants. This review describes the most important mechanisms of Hcy neurotoxicity and pharmacological agents known to reverse Hcy effects.

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.

Nicotinic Signal Transduction Machinery

Nicotinic synapses employ acetylcholine to activate ligand-gated ion channels that are cation-selective in vertebrates. Although the resulting nicotinic cholinergic transmission is famously excitatory at the neuromuscular junction, it plays many additional roles in the CNS. Most prevalent is that of modulation, usually involving calcium and signal transduction. Because of this, it is becoming increasingly important not only to understand the mechanisms that guide nicotinic receptors to appropriate locations but also to identify the postsynaptic machinery making possible the requisite signal transduction. Clearly, the kinds of components tethered in the vicinity of the receptor will assume a major role in determining the consequences of receptor activation. One of the most abundant and interesting nicotinic receptors in this respect is the species comprised of the alpha7 gene product (Broide and Leslie, 1999). These alpha7 homopentameric nicotinic acetylcholine receptors (alpha7 nAChRs) have a high relative permeability to calcium, rivaling that of NMDA receptors. But unlike NMDA receptors, alpha7 nAChRs promote calcium influx without requiring a coincident event such as membrane depolarization. As a result, the receptors are well equipped to regulate calcium-dependent events in neurons, particularly when depolarization might be occluded.

Pituitary cell type-specific electrical activity, calcium signaling and secretion

All secretory anterior pituitary cells exhibit spontaneous and extracellular calcium-dependent electrical activity, but differ with respect to the patterns of firing and associated calcium signaling and hormone secretion. Thus, somatotrophs and lactotrophs fire plateau-bursting action potentials spontaneously and without coupling to calcium release from intracellular stores, which generate calcium signals of sufficient amplitude to keep steady hormone release. In these cells, both spontaneous electrical activity and basal hormone secretion can be further amplified by activation of Gq/11 and Gs-coupled receptors and inhibited by Gi/o/z-coupled receptors. In contrast, gonadotrophs fire single, high-amplitude spikes with limited ability to promote calcium influx and exocytosis, whereas activated Gq/11-coupled receptors in these cells transform single-action potential spiking into the plateau-bursting type of electrical activity and trigger periodic high-amplitude calcium signals and exocytosis of prestored secretory vesicles. Here, we review biochemical and biophysical aspects of spontaneous and receptor-controlled electrical activity, calcium signaling, and hormone secretion in pituitary cells.

GABAB heterodimeric receptors promote Ca2+ influx via store-operated channels in rat cortical neurons and transfected chinese hamster ovary cells

The GABAB receptors are generally considered to be classical Gi-coupled receptors that lack the ability to mobilize intracellular Ca2+ without the aid of promiscuous G proteins. Here, we report the ability of GABAB receptors to promote calcium influx into primary cultures of rat cortical neurons and transfected Chinese hamster ovary cells. Chinese hamster ovary cells were transfected with GABAB1(a) or GABAB1(b) subunits along with GABAB2 subunits. In experiments using the fluorometric imaging plate reader platform, GABA and selective agonists promoted increases in intracellular Ca2+ levels in transfected Chinese hamster ovary cells and cortical neurons with the expected order of potency. These effects were fully antagonized by selective GABAB receptor antagonists. To investigate the intracellular pathways responsible for mediating these effects we employed several pharmacological inhibitors. Pertussis toxin abolished GABAB mediated Ca2+ increases, as did the phospholipase Cβ inhibitor U73122. Inhibitor 2-aminethoxydiphenyl borane acts as an antagonist at inositol 1,4,5-trisphosphate receptors and at store-operated channels. In all cell types, 2-aminethoxydiphenyl borane prevented Ca2+ mobilization. The selective store-operated channel inhibitor 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride prevented increases in intracellular Ca2+ levels as did performing the assays in Ca2+ free buffers. In conclusion, GABAB receptors expressed in Chinese hamster ovary cells and endogenously expressed in rat cortical neurons promote Ca2+ entry into the cell via the activation of store-operated channels, using a mechanism that is dependent on Gi/o heterotrimeric proteins and phospholipase Cβ. These findings suggest that the neuronal effects mediated by GABAB receptors may, in part, rely on the receptor’s ability to promote Ca2+ influx.

Redistribution of STIM Proteins to the Peripheral Structures in Store-Operated Calcium Influx

Stimuli that trigger release of calcium from the endoplasmic reticulum (ER) through inositol trisphosphate (IP 3 ) receptors can deplete this intracellular calcium store and trigger store-operated calcium (SOC) influx from the plasma membrane. Liou et al . designed a siRNA screen against 2304 proteins selected on the basis of the presence of signaling domains in their primary sequence. These were tested to identify proteins that, when depleted, reduced the plateau phase of calcium influx after exposure of cells to histamine (which promotes calcium release through IP 3 receptors) in the presence of thapsigargin (to inhibit calcium reloading of the ER). siRNA for stromal interaction molecules 1 and 2 (STIM1 and STIM2), which have a single transmembrane domain and a single EF-hand calcium-binding motif, suppressed the sustained calcium signal without decreasing the initial transient calcium signal. siRNA for STIM1 or STIM2 reduced the calcium influx observed after extracellular calcium was added after stores were depleted in the absence of extracellular calcium (calcium add-back assay). Overexpression of a yellow fluorescent protein (YFP)-STIM1 resulted in increased opening of the SOC influx channel, based on a Mn 2+ quench assay, in which Mn 2+ entering through the SOC influx channel quenches the Fura-2 signal. YFP-STIM1 was localized to the ER, and store depletion caused redistribution of this signal to punctate structures near the cell periphery even in the absence of extracellular calcium, which suggests that store depletion (rather than subsequent calcium influx through the plasma membrane) may be the cause of the redistribution. Mutation of the EF-hand domain caused redistribution of YFP-STIM1 to the punctate structures in the absence of store depletion and promoted calcium influx in the absence of store depletion, and store depletion did not cause any additional influx. In cells transfected to express a wild-type cyan fluorescent protein (CFP)-STIM1 and the EF-hand YFP-STIM1, store depletion triggered redistribution of the CFP-STIM1 to the YFP-STIM1-positive punctate structures. Thus, it appears that SOC influx may involve the rapid redistribution of ER proteins to a punctate intracellular structure in close proximity to the membrane, which may mediate a direct coupling between proteins in these intracellular structures with the channels in the plasma membrane. J. Liou, M. L. Kim, W. D. Heo, J. T. Jones, J. W. Myers, J. E. Ferrell Jr., T. Meyer, STIM is a Ca 2+ sensor essential for Ca 2+ -store-depletion-triggered Ca 2+ influx. Current Biol. 15 , 1235-1241 (2005). [PubMed]

Calcium and Vitamin D increase mRNA levels for the growth control hIK1 channel in human epidermal keratinocytes but functional channels are not observed.

BackgroundIntermediate-conductance, calcium-activated potassium channels (IKs) modulate proliferation and differentiation in mesodermal cells by enhancing calcium influx, and they contribute to the physiology of fluid movement in certain epithelia. Previous reports suggest that IK channels stimulate proliferative growth in a keratinocyte cell line; however, because these channels indirectly promote calcium influx, a critically unique component of the keratinocyte differentiation program, an alternative hypothesis is that they would be anti-proliferative and pro-differentiating. This study addresses these hypotheses.MethodsReal-time PCR, patch clamp electrophysiology, and proliferation assays were used to determine if human IK1 (hIK1) expression and function are correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes, and skin biopsies grown in explant culture.ResultshIK1 mRNA expression in human keratinocytes and skin was increased in response to anti-proliferative/pro-differentiating stimuli (elevated calcium and Vitamin D). Correspondingly, the hIK1 agonist 1-EBIO inhibited keratinocyte proliferation suggesting that the channel could be anti-proliferative and pro-differentiating. However, this proliferative inhibition by 1-EBIO was not reversed by a panel of hIK1 blockers, calling into question the mechanism of 1-EBIO action. Subsequent patch clamp electrophysiological analysis failed to detect hIK1 channel currents in keratinocytes, even those expressing substantial hIK1 mRNA in response to calcium and Vitamin D induced differentiation. Identical electrophysiological recording conditions were then used to observe robust IK1 currents in fibroblasts which express IK1 mRNA levels comparable to those of keratinocytes. Thus, the absence of observable hIK1 currents in keratinocytes was not a function of the electrophysiological techniques.ConclusionHuman keratinocyte differentiation is stimulated by calcium mobilization and influx, and differentiation stimuli coordinately upregulate mRNA levels of the calcium-activated hIK1 channel. This upregulation is paradoxical in that functional hIK1 channels are not observed in cultured keratinocytes. It appears, therefore, that hIK1 does not contribute to the functional electrophysiology of primary human keratinocytes, nor intact human skin. Further, the results indicate caution is required when interpreting experiments utilizing pharmacological hIK1 modulators in human keratinocytes.

PIPs and pools in insulin secretion

Insulin is secreted from pancreatic β cells in response to glucose metabolism altering intracellular ATP:ADP ratios and promoting calcium influx. Secretion is biphasic with a strong first phase and a sustained second phase. This probably relates to the dynamics of the pools of insulin-containing granules. How this might be regulated by physiological ATP:ADP ratios impinging on phosphatidylinositol kinases and phosphatidylinositol phosphates (PIPs) has recently been addressed. The results suggest an additional layer of control of stimulus – secretion coupling by ATP:ADP – and provide an exciting model for basic research and also new putative targets for therapy.

Hemofiltrate CC chemokine 1[9-74] causes effective internalization of CCR5 and is a potent inhibitor of R5-tropic human immunodeficiency virus type 1 strains in primary T cells and macrophages.

Proteolytic processing of the abundant plasmatic human CC chemokine 1 (HCC-1) generates a truncated form, HCC-1[9-74], which is a potent agonist of CCR1, CCR3, and CCR5; promotes calcium influx and chemotaxis of T lymphoblasts, monocytes, and eosinophils; and inhibits infection by CCR5-tropic human immunodeficiency virus type 1 (HIV-1) isolates. In the present study we demonstrate that HCC-1[9-74] interacts with the second external loop of CCR5 and inhibits replication of CCR5-tropic HIV-1 strains in both primary T cells and monocyte-derived macrophages. Low concentrations of the chemokine, however, frequently enhanced the replication of CCR5-tropic HIV-1 isolates but not the replication of X4-tropic HIV-1 isolates. Only HCC-1[9-74] and HCC-1[10-74], but not other HCC-1 length variants, displayed potent anti-HIV-1 activities. Fluorescence-activated cell sorter analysis revealed that HCC-1[9-74] caused up to 75% down-regulation of CCR5 cell surface expression, whereas RANTES (regulated on activation, normal T-cell expressed and secreted) achieved a reduction of only about 40%. Studies performed with green fluorescent protein-tagged CCR5 confirmed that both HCC-1[9-74] and RANTES, but not full-length HCC-1, mediated specific internalization of the CCR5 HIV-1 entry cofactor. Our results demonstrate that the interaction with HCC-1[9-74] causes effective intracellular sequestration of CCR5, but they also indicate that the effect of HCC-1[9-74] on viral replication is subject to marked cell donor- and HIV-1 isolate-dependent variations.

Amyloid-beta promotes calcium influx and neurodegeneration via stimulation of L voltage-sensitive calcium channels rather than NMDA channels in cultured neurons.

Exposure of cultured neurons and neuronal cells to aggregated amyloid-beta (Abeta) induces multiple neurodegenerative events including accumulation of cytosolic calcium, generation of reactive oxygen species, abnormal levels of phosphorylation of the microtubule-associated protein tau, and apoptosis. Prevention of accumulation of calcium within the cytosol also prevents all other events, suggesting that calcium accumulation is an early and pivotal event in Abeta neurotoxicity. Calcium influx has been suggested to occur via L voltage-sensitive calcium channels or NMDA channels. Calcium influx into differentiated human neuroblastoma cells has been previously attributed to the L voltage-sensitive calcium channel, but the contribution of the NMDA channel was not examined. In the present study, treatment of these cells with MK-801, an antagonist of NMDA channels, failed to attenuate Abeta-induced calcium influx or neurodegeneration, while nimopridine, an antagonist of the L voltage-sensitive calcium channel, blocked Abeta-induced calcium influx. Our findings suggest that NMDA channels do not contribute significantly to Abeta neurotoxicity in these acute cell culture analyses.

Dependence of Pituitary Hormone Secretion on the Pattern of Spontaneus Voltage-gated Calcium Influx

In excitable cells, voltage-gated calcium influx provides an effective mechanism for the activation of exocytosis. In this study, we demonstrate that although rat anterior pituitary lactotrophs, somatotrophs, and gonadotrophs exhibited spontaneous and extracellular calcium-dependent electrical activity, voltage-gated calcium influx triggered secretion only in lactotrophs and somatotrophs. The lack of action potential-driven secretion in gonadotrophs was not due to the proportion of spontaneously firing cells or spike frequency. Gonadotrophs exhibited calcium signals during prolonged depolarization comparable with signals observed in somatotrophs and lactotrophs. The secretory vesicles in all three cell types also had a similar sensitivity to voltage-gated calcium influx. However, the pattern of action potential calcium influx differed among three cell types. Spontaneous activity in gonadotrophs was characterized by high amplitude, sharp spikes that had a limited capacity to promote calcium influx, whereas lactotrophs and somatotrophs fired plateau-bursting action potentials that generated high amplitude calcium signals. Furthermore, a shift in the pattern of firing from sharp spikes to plateau-like spikes in gonadotrophs triggered luteinizing hormone secretion. These results indicate that the cell type-specific action potential secretion coupling in pituitary cells is determined by the capacity of their plasma membrane oscillator to generate threshold calcium signals.

Role of Hyperpolarization Attained by Linoleic Acid in Chick Myoblast Fusion

Our previous report has suggested that hyperpolarization generated by reciprocal activation of calcium-activated potassium (KCa) channels and stretch-activated channels induces calcium influx that triggers myoblast fusion. Here we show that linoleic acid is involved in the process of generating hyperpolarization in cultured chick myoblasts and hence in promotion of the cell fusion. Linoleic acid dramatically hyperpolarized the membrane potential from −14 ± 3 to −58 ± 5 mV within 10 min. This effect was partially blocked by 1 mM tetraethylammonium (TEA) or 30 nM charybdotoxin, a selective KCa channel inhibitor, and completely abolished by 10 mM TEA. Single-channel recordings revealed that linoleic acid activates TEA-resistant potassium channels as well as KCa channels. Furthermore, linoleic acid induced calcium influx from extracellular solution, and this effect was partially blocked by 1 mM TEA and completely prevented at 10 mM, similar to the effect of TEA on linoleic acid-mediated hyperpolarization. Since the valinomycin-mediated hyperpolarization promoted calcium influx, hyperpolarization itself appears capable of inducing calcium influx. In addition, gadolinium prevented the valinomycin-mediated increase in intracellular calcium level under hypotonic conditions, revealing the involvement of stretch-activated channels in calcium influx. Furthermore, linoleic acid stimulated myoblast fusion, and this stimulatory effect could completely be prevented by 10 mM TEA. These results suggest that linoleic acid induces hyperpolarization of membrane potential by activation of potassium channels, which induces calcium influx through stretch-activated channels, and thereby triggers myoblast fusion.

Secreted Dense Granule Adenine Nucleotides Promote Calcium Influx and the Maintenance of Elevated Cytosolic Calcium Levels in Stimulated Human Platelets

Evidence that secreted dense granule adenine nucleotides mediate part of the agonist-induced cytosolic calcium ([Ca2+]i) responses in human platelets was obtained from comparisons of fura-2-loaded platelets from normal subjects and from patients with a form of platelet storage pool deficiency (SPD) in which the secretory dense granules and their contents are virtually absent. SPD platelets had normal initial [Ca2+]i increases induced by thrombin and the endoperoxide analog U46619, but a significantly enhanced decay of elevated [Ca2+]i levels following the initial increases. With thrombin, this enhanced [Ca2+]i decay was associated with decreased Ca2+ influx, as measured by Mn2+ quench of fura-2 fluorescence. Addition of micromolar concentrations of ADP, alone or together with ATP, after stimulation reversed the enhanced [Ca2+]i decay and increased Mn2+ quench in SPD platelets, but had no effect on these responses in normal platelets, while addition of 100-fold higher concentrations of ATP or apyrase before stimulation increased [Ca2+]i decay and decreased Mn2+ quench in normal platelets, but had little effect in SPD platelets. ATP and alpha,beta-methylene ATP, a specific agonist for P2X1 receptors, at micromolar concentrations also increased Mn2+ quench, but to lesser extents than did ADP, in SPD platelets isolated and loaded with fura-2 in the presence of apyrase. Similar effects of ADP and excess ATP were seen in U46619-stimulated platelets, but decreased Ca2+ influx could not be measured directly in SPD platelets, presumably due to the very transient influx response seen with U46619. These results suggest that secreted dense granule ADP and ATP contribute to the maintenance of elevated [Ca2+]i levels, but not to the initial [Ca2+]i increases, in stimulated human platelets, most likely via a nucleotide-specific component of Ca2+ influx which may be mediated by interactions with both P2X1 and P2Y1 purinoceptors.

Prostaglandins induce calcium influx in human spermatozoa.

Progesterone, prostaglandin and follicular fluid are reported to enhance the acrosome reaction through the influx of extracellular calcium into the cytoplasm of human spermatozoa. Prostaglandins are present within the male reproductive tract, and high concentrations of prostaglandins exist in seminal fluid. In order to investigate the mechanisms by which prostaglandins enhance the acrosome reaction through calcium influx, the intracellular calcium response induced by progesterone, prostaglandin E1 (PGE1), prostaglandin E2 (PGE2) and follicular fluid was measured using fura-2. PGE1 and PGE2 promoted calcium influx dose dependently through dihydropyridine insensitive calcium channels. Refractoriness of the elevation of intracellular Ca2+ concentration ([Ca2+]i) to a second stimulus occurred when 60 microg/ml PGE1 was administered 100 s after the prior administration of 60 microg/ml of PGE1, and similarly when 1 microg/ml of progesterone was administered 100 s after the prior administration of 1 microg/ml of progesterone. Refractoriness also occurred when 60 microg/ml PGE1 was administered after the prior addition of 60 microg/ml PGE2, but did not occur between PGE1 and progesterone. Pertussis toxin (PTX) did not modify the changes in [Ca2+]i after the addition of PGE1 or PGE2. In conclusion, PGE1 and PGE2 promoted calcium influx through PTX-insensitive calcium channels which appeared to be recognized by a common receptor different from that of progesterone.

Endothelium-dependent hyperpolarization. Beyond nitric oxide and cyclic GMP.

Blood flow and blood pressure are determined by an integration of reflex, humoral, and local vascular control mechanisms. Knowledge of these mechanisms has mushroomed over the past 15 years, particularly in the area of local endothelium-dependent vasomotor control. This has stemmed from the pioneering report in 1980 by Robert Furchgott1 demonstrating that the endothelium releases a vasodilator substance in response to acetylcholine. This concept has been expanded with knowledge that the endothelium releases a variety of relaxing and contracting factors that regulate the underlying smooth muscle.2 The most widely known endothelium-derived relaxing factor, nitric oxide, is released from endothelial cells in response to shear stress or stimulation of different receptors for a variety of neurohumoral mediators on the endothelial cell surface.3 The increase in endothelial cell calcium initiated by these stimuli increases the activity of a constitutively expressed enzyme, nitric oxide synthase, which converts l-arginine to nitric oxide and citrulline.4 5 6 7 Nitric oxide thus formed diffuses to and inhibits contraction of the underlying vascular smooth muscle.3 The physiological significance of the production of endothelial nitric oxide is suggested by the vasoconstriction observed in most vascular beds8 9 10 11 12 13 and the increase in systemic arterial blood pressure,14 15 16 which occurs on infusion of inhibitors of nitric oxide synthase. This observation further implies that under normal conditions, endothelial cells are locally liberating nitric oxide which effectively inhibits vasoconstriction arising by other mechanisms. Thus, normal vascular homeostasis depends in the periphery on a balance between neurally and humorally mediated vasoconstriction in skeletal muscle, mesentery, and the kidney, and local endothelium-dependent vasodilatation. In the truly vital areas of the heart, brain, and genitalia, vasodilator neural mechanisms reinforce the vasodilator influence of the endothelium.17 All is not known regarding the …

Evidence that Diazoxide Promotes Calcium Influx in Mouse Keratinocyte Cultures by Membrane Hyperpolarization

We have used fura-2/AM to investigate the effect of diazoxide on the cytosolic calcium concentration (Cai) in primary mouse keratinocyte cultures. Treatment of keratinocytes with 100 microM diazoxide induced a transient peak in Cai, followed by a sustained elevation. Depletion of medium calcium by addition of EGTA abolished the diazoxide-induced Cai response, indicating that the agent promoted calcium influx without release of calcium from intracellular stores. The diazoxide-induced rise in Cai was inhibited both by addition of 60 mM KCl to the assay buffer and by preincubation with glibenclamide, a specific K+ channel blocker. In addition, studies with the membrane potential-sensitive fluorescent probe bis-oxonol demonstrated that diazoxide hyperpolarized keratinocyte membranes. These findings suggest that keratinocytes possess K+ channels, and that the previously reported proliferation effects of K+ channel openers such as diazoxide on keratinocytes may result from hyperpolarization-induced elevation of Cai.

Alpha 2-adrenoceptors activate dihydropyridine-sensitive calcium channels via Gi-proteins and protein kinase C in rat portal vein myocytes.

The presence of functional alpha 2-adrenoceptors was investigated in isolated smooth muscle cells from rat portal vein using the nystatin-perforated patch-clamp technique. The free cytoplasmic calcium concentration ([Ca2+]i) was estimated using emission from the dye Fura-2. Activation of alpha 2-adrenoceptors by clonidine (an alpha 2-adrenoceptor agonist) or noradrenaline (a non-selective alpha-adrenoceptor agonist), both in the presence of 0.1 microM prazosin to block alpha 1-adrenoceptors, caused a slow and sustained increase in [Ca2+]i which was inhibited by 0.1 microM rauwolscine (an alpha 2-adrenoceptor antagonist). A similar Ca2+ response was obtained with oxymetazoline (a selective alpha 2A-adrenoceptor agonist) suggesting that the increase in [Ca2+]i resulted from activation of the alpha 2A-adrenoceptor subtype. The increase in [Ca2+]i did not occur in calcium-free solution or in the presence of oxodipine (a voltage-dependent calcium channel blocker), indicating that it depended on a calcium influx. The alpha 2A-adrenoceptor-activated calcium influx was unchanged after complete release of the stored calcium induced by applications of ryanodine and caffeine. In addition, no accumulation of inositol trisphosphate was detected in the presence of 0.1 microM prazosin. Taken together, these results indicate that alpha 2A-adrenoceptor activation does not stimulate phosphoinositide turnover and subsequent calcium release from intracellular stores. Whole-cell patch-clamp experiments showed that alpha 2A-adrenoceptor activation promoted calcium influx through voltage-dependent L-type channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental autoregulation of calcium currents in mammalian central neurones.

The influence of calcium currents expressed at early stages on the subsequent development of calcium currents was studied in embryonic rat hypothalamic neurones in culture. Voltage-activated calcium currents and spontaneous fluctuations of intracellular free calcium concentration ([Ca2+]i) were monitored. Acute application of nickel chloride (0.1 mM) to 6- to 7-day-old cultures strongly reduced calcium currents and [Ca2+]i fluctuations. When cultures were maintained for 6-7 days in the presence of NiCl2 (0.05-0.1 mM), expression of the low voltage-activated current was strongly inhibited; this treatment did not affect high voltage-activated currents. Our results suggest that spontaneous activation of calcium currents early during development promotes calcium influx that regulates expression of calcium current in mature neurones.