Involvement of protein kinase C and intracellular Ca in goldfish brain somatostatin-28 inhibitory action on growth hormone release in goldfish

Goldfish brain somatostatin-28 (gbSS-28) is present in brain and pituitary tissues of goldfish. We assessed whether gbSS-28 targets Ca²⁺ and/or protein kinase C (PKC)-dependent signaling cascades in inhibiting growth hormone (GH) release. gbSS-28 decreased basal GH release from primary cultures of dispersed goldfish pituitary cells and intracellular free calcium levels ([Ca²⁺]_i) in goldfish somatotropes. gbSS-28 partially reduced [Ca²⁺]_i and GH responses induced by two endogeneous gonadotropin-releasing hormones (GnRHs), salmon (s)GnRH and chicken (c)GnRH-II. Furthermore, gbSS-28 reduced GH increases and abolished [Ca²⁺]_i elevations elicited by two PKC activators, tetradecanoyl 4β-phorbol-13-acetate and dioctanyl glycerol. The PKC inhibitors Gö6976 and Bis II abolished [Ca²⁺]_i responses to PKC activators, but only attenuated GnRH-induced increases in [Ca²⁺]_i and did not alter basal [Ca²⁺]_i. In cells pretreated with Bis II, gbSS-28 further reduced basal [Ca²⁺]_i. Our results suggest that gbSS-28 inhibits GnRH-induced GH release in part by attenuating PKC-mediated GnRH [Ca²⁺]_i signals. gbSS-28 reduces basal GH release also via reduction in [Ca²⁺]_i but PKC is not involved in this regard.     

Diabetes-induced activation of protein kinase C inhibits store-operated Ca<Superscript>2+</Superscript> uptake in rat retinal microvascular smooth muscle

Aims/Hypothesis To assess the effects of diabetes-induced activation of protein kinase C (PKC) on voltage-dependent and voltage-independent Ca²⁺ influx pathways in retinal microvascular smooth muscle cells.Methods Cytosolic Ca²⁺ was estimated in freshly isolated rat retinal arterioles from streptozotocin-induced diabetic and non-diabetic rats using fura-2 microfluorimetry. Voltage-dependent Ca²⁺ influx was tested by measuring rises in [Ca²⁺]_i with KCl (100 mmol/l) and store-operated Ca²⁺ influx was assessed by depleting [Ca²⁺]_i stores with Ca²⁺ free medium containing 5 µmol/l cyclopiazonic acid over 10 min and subsequently measuring the rate of rise in Ca²⁺ on adding 2 mmol/l or 10 mmol/l Ca²⁺solution.Results Ca²⁺ entry through voltage-dependent L-type Ca²⁺ channels was unaffected by diabetes. In contrast, store-operated Ca²⁺ influx was attenuated. In microvessels from non-diabetic rats 20 mmol/l D-mannitol had no effect on store-operated Ca²⁺ influx. Diabetic rats injected daily with insulin had store-operated Ca²⁺ influx rates similar to non-diabetic control rats. The reduced Ca²⁺ entry in diabetic microvessels was reversed by 2-h exposure to 100 nmol/l staurosporine, a non-specific PKC antagonist and was mimicked in microvessels from non-diabetic rats by 10-min exposure to the PKC activator phorbol myristate acetate (100 nmol/l). The specific PKC antagonist LY379196 (100 nmol/l) also reversed the poor Ca²⁺ influx although its action was less efficacious than staurosporine.Conclusion/interpretation These results show that store-operated Ca²⁺ influx is inhibited in retinal arterioles from rats having sustained increased blood glucose and that PKC seems to play a role in mediating this effect.Abbreviations DAG Diacylglycerol - PKC protein kinase C - [Ca²⁺]_i intracellular calcium concentration - STZ streptozotocin - SPP staurosporine - SR sarcoplasmic reticulum - MVSM microvascular smooth muscle - CPA cyclopiazonic acid - PMA phorbol myristate acetate - VDCC voltage-dependent Ca²⁺ channels     

Aging differentially modifies agonist-evoked mouse detrusor contraction and calcium signals

Although aging-induced changes in urinary bladder neurotransmission have been studied in some detail, information regarding alterations in detrusor muscle is scanty and addresses only partial aspects of the myogenic response of detrusor. Rodent bladder aging shows several features similar to those reported in humans. The aim of this study was to characterize in aged mouse the alterations of detrusor muscle contraction and the putative underlying changes in Ca²⁺ signals. We studied in vitro the myogenic contraction induced by agonists in detrusor strips from adult (3 months old) or aged (23–25 months old) mice. In addition, we determined the agonist-induced [Ca²⁺]_i signals by epifluorescence microscopy in fura-2 loaded isolated detrusor cells. Aging impaired the contractile response of bladder strips to cholinergic stimulation with bethanechol and to chemical depolarization with KCl-containing solutions. On the contrary, the response to purinergic stimulation (ATP) was enhanced. Aging also diminished the transient Ca²⁺ signal evoked by bethanechol and the Ca²⁺ influx induced by KCl in bladder strips. Treatments aimed to release calcium from intracellular stores (caffeine and a low level of ionomycin in Ca²⁺-free medium) showed that aging reduces the size of agonist-releasable stores. Similar to contraction, the mobilization of Ca²⁺ by ATP was increased in aged cells. Therefore, the differential effects of aging on detrusor contraction are associated to alterations of [Ca²⁺]_i signals: the cholinergic inhibition is due to inhibition of voltage-operated Ca²⁺ influx and reduction of the size of intracellular Ca²⁺ stores, while the age-induced ATP response is accompanied by an enhanced Ca²⁺ mobilization.     

Calcium sensitivity, force frequency relationship and cardiac troponin I: Critical role of PKA and PKC phosphorylation sites

Transgenic models with pseudo phosphorylation mutants of troponin I, PKA sites at Ser 22 and 23 (cTnIDD_22,23 mice) or PKC sites at Ser 42 and 44 (cTnIAD_22,23DD_42,44) displayed differential force-frequency relationships and afterload relaxation delay in vivo. We hypothesized that cTnI PKA and PKC phosphomimics impact cardiac muscle rate-related developed twitch force and relaxation kinetics in opposite directions. cTnIDD_22,23 transgenic mice produce a force frequency relationship (FFR) equivalent to control NTG albeit at lower peak [Ca²⁺]_i, while cTnIAD_22,23DD_42,44 TG mice had a flat FFR with normal peak systolic [Ca²⁺]_i, thus suggestive of diminished responsiveness to [Ca²⁺]_i at higher frequencies. Force-[Ca²⁺]_i hysteresis analysis revealed that cTnIDD_22,23 mice have a combined enhanced myofilament calcium peak response with an enhanced slope of force development and decline per unit of [Ca²⁺]_i, whereas cTnIAD_22,23DD_42,44 transgenic mice showed the opposite. The computational ECME model predicts that the TG lines may be distinct from each other due to different rate constants for association/dissociation of Ca²⁺ at the regulatory site of cTnC. Our data indicate that cTnI phosphorylation at PKA sites plays a critical role in the FFR by increasing relative myofilament responsiveness, and results in a distinctive transition between activation and relaxation, as displayed by force-[Ca²⁺]_i hysteresis loops. These findings may have important implications for understanding the specific contribution of cTnI to β-adrenergic inotropy and lusitropy and to adverse contractile effects of PKC activation, which is relevant during heart failure development.     

Mechanical stretch increases intracellular calcium concentration in cultured ventricular cells from neonatal rats

Summary We investigated the effects of mechanical stretch on intracellular calcium concentration ([Ca²⁺]_i) of cultured neonatal rat ventricular cells using micro-fluorometry with fura-2. Myocytes were cultured on laminin-coated silicon rubber and stretched by pulling the rubber with a manipulator. Myocytes were either mildly stretched (to less than 115% of control length), moderately so (to 115%–125% of control length), or extensively (to over 125% of the control length). “Quick stretches” (accomplished within 10s) of moderate to extensive intensities produced a large transient increase of [Ca²⁺]_i in the early phase of stretch (30s-2 min), followed by a small but sustained increase during the late phase of stretch (5–10 min). The initial transient increase in [Ca²⁺]_i after the “quick stretch” was preserved in the presence of gallopamil (10⁻⁷M) or ryanodine (10⁻⁵ M), but was absent in Ca²⁺-free medium or in the presence of gadolinium (10⁻⁷M). The late or steady state [Ca²⁺]_i increase was observed in the presence of gadolinium, gallopamil, or ryanodine but was abolished in Ca²⁺-free medium. A steady-state increase in [Ca²⁺]_i was also evoked by “slow stretch” in which cells were slowly pulled to the final length within 1–2min. As the presence of external Ca²⁺ was indispensable, increased trans-sarcolemmal Ca²⁺ influx appears to be involved in both initial and steady-state increases in [Ca²⁺]_i. The initial increase in [Ca²⁺]_i after the “quick stretch” can be attributed to the activation of gadolinium-sensitive, stretch-activated channels.     

Sarcoplasmic Reticulum Function in Murine Ventricular Myocytes Overexpressing SR CaATPase

To examine the effects of the overexpression of sarcoplasmic reticulum (SR) CaATPase on function of the SR and Ca²⁺homeostasis, we measured [Ca²⁺]_itransients (fluo-3), and L-type Ca²⁺currents (I_Ca,L), Na/Ca exchanger currents (I_Na/Ca), and SR Ca²⁺content with voltage clamp in ventricular myocytes isolated from wild type (WT) mice and transgenic (SRTG) mice. The amplitude of [Ca²⁺]_itransients was insignificantly increased in SRTG myocytes, while the diastolic [Ca²⁺]_itended to be lower. The initial and terminal declines of [Ca²⁺]_itransients were significantly accelerated in SRTG myocytes, implying a functional upregulation of the SR CaATPase. We examined the functional contribution of only the SR CaATPase to the initial and the terminal phase of the decline of [Ca²⁺]_i, by abruptly inhibiting Na/Ca exchange with a rapid switcher device. The rate of [Ca²⁺] decline mediated by the SR CaATPase was increased by 40% in SRTG compared with WT myocytes. The function of the L-type Ca²⁺channel was unchanged in SRTG myocytes, while INa/Ca density was slightly (10%) decreased. Measured SR Ca²⁺content was significantly increased by 29% in SRTG myocytes. Thus, overexpression of SR CaATPase markedly accelerates the decline of [Ca²⁺]_itransients, and induces an increase in SR Ca²⁺content, with some downregulation of the Na/Ca exchanger.     

Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca²⁺ concentration inside pancreatic acinar cells ([Ca²⁺]_i), due to excessive release of Ca²⁺ stored inside the cells followed by Ca²⁺ entry from the interstitial fluid. The sustained [Ca²⁺]_i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca²⁺ release-activated Ca²⁺ channels (CRAC) would prevent sustained elevation of [Ca²⁺]_i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca²⁺ ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca²⁺. Ca²⁺ entry then occurred upon admission of Ca²⁺ to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca²⁺ entry in a concentration-dependent manner within the range of 1 to 50 μM (IC₅₀ = 3.4 μM), but had little or no effect on the physiological Ca²⁺ spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca²⁺]_i, which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca²⁺]_i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.     

Intracellular calcium ion response to glucose in beta-cells of calbindin-D28k nullmutant mice and in betaHC13 cells overexpressing calbindin-D28k

This article describes studies on the glucose-induced responses of intracellular Ca²⁺ concentration ([Ca²⁺]_i), insulin release, and redistribution of calbindin-D_28k, a calcium-binding regulatory protein, in β-cells of pancreatic islets of calbindin-D_28k knockout (KO) and wild-type mice (C57BL6) as well as in βHC-13 control cells and βHC-13 CaBP40 cells (β-cell line overexpressing calbindin-D_28k). Upon increasing the glucose concentration from 2.8 to 30 mM, islets of KO mice showed a significantly greater increase in [Ca²⁺]_i (mean increase in [Ca²⁺]_i, i.e., Δ[Ca²⁺], was 296 nM) compared with wild-type mice (Δ[Ca²⁺]_i=97 nM). βHC-13 CaBP40 cells showed little change in [Ca²⁺]_i upon elevation of glucose from 5.5 to 32.7 mM, whereas βHC-13 control cells exhibited significant increases in [Ca²⁺]_i (Δ[Ca²⁺]_i=510 nM). Similarly, upon addition of 30 mM glucose, the rate of insulin release increased from 25.2 (basal rate) to 145.2 pg/mL/min in βHC-13 control cells, whereas in βHC-13 CaBP40 cells the rate of insulin release was only 27.5 pg/mL/min in high glucose. Thus, levels of calbindin-D_28k in β-cells affect both [Ca²⁺]_i and insulin secretion in response to glucose. The three-dimensional reconstruct of confocal immunofluorescent images showed that glucose caused redistribution of calbindin-D_28k resulting in co-localization in the region of L-type voltage-dependent calcium channels (VDCC). This colocalization may be an important regulatory function concerning Ca²⁺ influx via L-type VDCC and exocytosis of insulin granules.     

Intracellular Ca- and PKC-dependent upregulation of T-type Ca channels in LPC-stimulated cardiomyocytes

Lysophosphatidylcholine (LPC) accumulation in intracellular and/or interstitial space in cardiomyocytes may underlie as a mechanism for tachycardia and various arrhythmias during cardiac ischemia, which is usually accompanied by elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The present study was therefore designed to investigate possible mechanisms responsible for [Ca²⁺]_i elevation by LPC focusing on T-type Ca²⁺ channel current (I_Ca.T). LPC as well as phorbol 12-myristate 13-acetate (PMA) significantly accelerated the beating rates of neonatal rat cardiomyocytes. Augmentation of I_Ca.T by LPC was dependent on the intracellular Ca²⁺ concentration: an increase of I_Ca.T was significantly larger in high [Ca²⁺]_i condition (pCa = 7) than those in low [Ca²⁺]_i condition (pCa = 11). In heterologous expression system by use of human cardiac Ca_V3.1 and Ca_V3.2 channels expressed in HEK293 cells, LPC augmented Ca_V3.2 channel current (I_Cav3.2) in a concentration-dependent manner but not Ca_V3.1 channel current (I_Cav3.1). Augmentation of I_Cav3.2 by LPC was highly [Ca²⁺]_i dependent: I_Cav3.2 was unchanged when pCa was 11 but was markedly increased when [Ca²⁺]_i was higher than 10⁻¹⁰ M (pCa ≤ 10) by LPC application (10-50 μM). A specific inhibitor of protein kinase Cα (Ro-32-0432) attenuated the increase of I_Cav3.2 by LPC. LPC stimulates I_Ca.T in a [Ca²⁺]_i-dependent manner via PKCα activation, which may play a role in triggering arrhythmias in pathophysiological conditions of the heart.     

Experimental diabetes enhances Ca2+ mobilization and glutamate exocytosis in cerebral synaptosomes from mice

The present study was conducted to investigate the effects of the diabetic condition on the Ca²⁺ mobilization and glutamate release in cerebral nerve terminals (synaptosomes). Diabetes was induced in male mice by intraperitoneal injection of streptozotocin. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release in synaptosomes were determined using fura-2 and enzyme-linked fluorometric assay, respectively. Diabetes significantly enhanced the ability of the depolarizing agents K⁺ and 4-aminopyridine (4-AP) to increase [Ca²⁺]_i. In addition, diabetes significantly enhanced K⁺- and 4-AP-evoked Ca²⁺-dependent glutamate release. The pretreatment of synaptosomes with a combination of ω-agatoxin IVA (a P-type Ca²⁺ channel blocker) and ω-conotoxin GVIA (an N-type Ca²⁺ channel blocker) inhibited K⁺- or 4-AP-induced increases in [Ca²⁺]_i and Ca²⁺-dependent glutamate release in synaptosomes from the control and diabetic mice to a similar extent, respectively. These results indicate that diabetes enhances a K⁺- or 4-AP-evoked Ca²⁺-dependent glutamate release by increasing [Ca²⁺]_i via stimulation of Ca²⁺ entry through both P- and N-type Ca²⁺ channels.     

Insulin-secreting INS-1E cells express functional TRPV1 channels

We have studied whether functional TRPV1 channels exist in the INS-1E cells, a cell type used as a model for β-cells, and in primary β-cells from rat and human. The effects of the TRPV1 agonists capsaicin and AM404 on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the INS-1E cells were studied by fura-2 based microfluorometry. Capsaicin increased [Ca²⁺]_i in a concentration-dependent manner, and the [Ca²⁺]_i increase was dependent on extracellular Ca²⁺. AM404 also increased [Ca²⁺]_i in the INS-1E cells. Capsazepine, a specific antagonist of TRPV1, completely blocked the capsaicin- and AM404-induced [Ca²⁺]_i increases. Capsaicin did not increase [Ca²⁺]_i in the primary β-cells from rat and human. Whole cell patch clamp configuration was used to record currents across the plasma membrane in the INS-1E cells. Capsaicin elicited inward currents that were inhibited by capsazepine. Western blot analysis detected TRPV1 proteins in the INS-1E cells and the human islets. Immunohistochemistry was used to study the expression of TRPV1, but no TRPV1 protein immunoreactivity was detected in the human islet cells and the human insulinoma cells. We conclude that the INS-1E cells, but not the primary β-cells, express functional TRPV1 channels.     

Intracellular calcium activates TRPM2 and its alternative spliced isoforms

Melastatin-related transient receptor potential channel 2 (TRPM2) is a Ca²⁺-permeable, nonselective cation channel that is involved in oxidative stress-induced cell death and inflammation processes. Although TRPM2 can be activated by ADP-ribose (ADPR) in vitro, it was unknown how TRPM2 is gated in vivo. Moreover, several alternative spliced isoforms of TRPM2 identified recently are insensitive to ADPR, and their gating mechanisms remain unclear. Here, we report that intracellular Ca²⁺ ([Ca²⁺]_i) can activate TRPM2 as well as its spliced isoforms. We demonstrate that TRPM2 mutants with disrupted ADPR-binding sites can be activated readily by [Ca²⁺]_i, indicating that [Ca²⁺]_i gating of TRPM2 is independent of ADPR. The mechanism by which [Ca²⁺]_i activates TRPM2 is via a calmodulin (CaM)-binding domain in the N terminus of TRPM2. Whereas Ca²⁺-mediated TRPM2 activation is independent of ADPR and ADPR-binding sites, both [Ca²⁺]_i and the CaM-binding motif are required for ADPR-mediated TRPM2 gating. Importantly, we demonstrate that intracellular Ca²⁺ release activates both recombinant and endogenous TRPM2 in intact cells. Moreover, receptor activation-induced Ca²⁺ release is capable of activating TRPM2. These results indicate that [Ca²⁺]_i is a key activator of TRPM2 and the only known activator of the spliced isoforms of TRPM2. Our findings suggest that [Ca²⁺]_i-mediated activation of TRPM2 and its alternative spliced isoforms may represent a major gating mechanism in vivo, therefore conferring important physiological and pathological functions of TRPM2 and its spliced isoforms in response to elevation of [Ca²⁺]_i.     

Effect of stimulation and veratrine on total cellular calcium in rat and guinea-pig ventricular myocytes

Summary In rat cardiac myocytes, calcium efflux by Na⁺/Ca²⁺-exchange is expected only during ventricular systole following initial action potential repolarization. In contrast, in guinea-pigs, calcium influx via Na⁺/Ca²⁺-exchange is expected only during the initial portion of the action potential. Thus electrical stimulation is expected to result in reduced intracellular calcium ([Ca²⁺]_i) in rat and an increase in guinea pig. We tested this hypothesis by measuring total cellular calcium ([Ca]_tot) using⁴⁵Ca following stimulation of isolated rat and guinea-pig ventricular myocytes. Many studies have also emphasized that the rate and the direction of Na⁺/Ca²⁺-exchange across the sarcolemma are in part dependent on the magnitude of the transsarcolemmal sodium gradient. Thus, increasing intracellular sodium ([Na⁺]_i) is expected to result in an increased [Ca²⁺]_i. This hypothesis was also tested by measuring [Ca]_tot following veratrine administration. Enzymatically isolated rat and guinea-pig ventricular myocytes were divided into two groups; non-stimulated and stimulated (1 Hz). The concentration-dependent effects of veratrine (1,10,100 g/ml) on [Ca]_tot were determined in both these groups. In the absence of veratrine, non-stimulated rat myocytes had a significantly higher [Ca]_tot than did stimulated ones. Non-stimulated guinea-pig myocytes had a significantly lower [Ca]_tot when compared with stimulated ones Veratrine increased [Ca]_tot in both species in a concentration-dependent fashion. In addition, following veratrine the difference between [Ca]_tot in non-stimulated and stimulated rat myocytes was no longer significant. These results support those of others who have demonstrated that stimulation is associated with a gain of cellular calcium in both rabbit and guinea-pig ventricle and a calcium loss in rat ventricle. In addition, the increase in [Ca]_tot following veratrine was similar to the results of others that have shown an increase in [Ca²⁺]_i following exposure to veratrum alkaloids. Thus, we have demonstrated, using a technique limited to determination of [Ca]_tot, that this parameter may reflect changes produced by alteration of Na⁺/Ca²⁺-exchange.     

Migration of primary cultured rabbit gastric epithelial cells requires intact protein kinase C and Ca2+/calmodulin activity

Superficial gastric mucosal injury is rapidly repaired by epithelial cell migration. This study aims to characterize the intracellular signal transduction pathways underlying the repair process. Primary monolayer cultures of rabbit gastric epithelial cells were wounded. The measured spontaneous cell migration speed at the edge of the wound was 457 ± 89 m/24 hr. Epidermal growth factor stimulated and genistein (receptor tyrosine protein kinase inhibitor) inhibited cell migration significantly. Down-regulation of protein Kinase C (PKC) with long-term phorbol 12-myristate 13-acsetate or inhibition with calphostin-C significantly inhibited cell migration. Blocking of Ca²⁺ channels with verapamil and endogenous Ca²⁺ release with TMB-8 or inhibition of the Ca²⁺/calmodulin complex with calmidazolium likewise significantly inhibited migration speed and also abolished the rise of [Ca²⁺]_i, which was measured in migrating cells. Modulation of the cAMP-PKA pathway or prostaglandin synthesis had no influence on cell migration. Gastric epithelial cell migration implies activation of receptor tyrosine kinase. It is associated with increased [Ca²⁺]_i and requires an intact Ca²⁺/calmodulin complex. Intact PKC activity also is needed.     

Crosstalk between membrane potential and cytosolic Ca2+ concentration in beta cells from Sur1 −/− mice

Aims/hypothesis Islets or beta cells from Sur1^–/– mice were used to determine whether changes in plasma membrane potential (V_m) remain coupled to changes in cytosolic Ca²⁺ ([Ca²⁺]_i) in the absence of K_ATP channels and thus provide a triggering signal for insulin secretion. The study also sought to elucidate whether [Ca²⁺]_i influences oscillations in V_m in sur1^–/– beta cells.Methods Plasma membrane potential and ion currents were measured with microelectrodes and the patch–clamp technique. [Ca²⁺]_i was monitored with the fluorescent dye fura-2. Insulin secretion from isolated islets was determined by static incubations.Results Membrane depolarisation of Sur1^–/– islets by arginine or increased extracellular K⁺, elevated [Ca²⁺]_i and augmented insulin secretion. Oligomycin completely abolished glucose-stimulated insulin release from Sur1^–/– islets. Oscillations in V_m were influenced by [Ca²⁺]_i as follows: (1) elevation of extracellular Ca²⁺ lengthened phases of membrane hyperpolarisation; (2) simulating a burst of action potentials induced a Ca²⁺-dependent outward current that was augmented by increased Ca²⁺ influx through L-type Ca²⁺ channels; (3) Ca²⁺ depletion of intracellular stores by cyclopiazonic acid increased the burst frequency in Sur1^–/– islets, elevating [Ca²⁺]_i and insulin secretion; (4) store depletion activated a Ca²⁺ influx that was not inhibitable by the L-type Ca²⁺ channel blocker D600.Conclusions/interpretation Although V_m is largely uncoupled from glucose metabolism in the absence of K_ATP channels, increased electrical activity leads to elevations of [Ca²⁺]_i that are sufficient to stimulate insulin secretion. In Sur1^–/– beta cells, [Ca²⁺]_i exerts feedback mechanisms on V_m by activating a hyperpolarising outward current and by depolarising V_m via store-operated ion channels.     

Hyperpolarization induced by K+ channel openers inhibits Ca2+ influx and Ca2+ release in coronary artery

The vasodilating mechanisms of the K⁺ channel openers—cromakalim, pinacidil, nicorandil, KRN2391, and Ki4032—were examined by measurement of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) using the fura-2 method in canine or porcine coronary arterial smooth muscle. The five K⁺ channel openers all produced a reduction of [Ca²⁺]_i in 5 and 30 mM KCl physiological salt solution (PSS), the effects of which were antagonized by tetrabutylammonium (TBA) or glibenclamide, but failed to affect [Ca²⁺]_i in 45 and 90 mM MCl-PSS. Cromakalim and Ki4032 only partially inhibited the 30 mM KCl-induced contractures, whereas pinacidil, nicorandil, and KRN2391 nearly abolished contractions produced by high KCl-PSS. The increased [Ca²⁺]_i and force produced by a thromboxane A₂ analogue, U46619, were inhibited by K⁺ channel openers and verapamil. In the absence of extracellular Ca²⁺, U46619 induced a transient increase in [Ca²⁺]_i with a contraction, which is effectively inhibited by cromakalim and Ki4032. Their inhibitory effects were blocked by TBA and counteracted by 20 mM KCl-induced depolarization. Cromakalim and Ki4032 did not affect caffeine-induced Ca²⁺ release. Cromakalim reduced U46619-induced IP₃ production and TBA blocked this inhibitory effect. Thus, cromakalim and Ki4032 are more specific K⁺ channel openers than pinacidil, nicorandil, and KRN2391. The vasodilation related with a reduction of [Ca²⁺]_i produced by K⁺ channel openers is due to the hyperpolarization of the plasma membrane resulting in not only the closure of voltage-dependent Ca²⁺ channels but also inhibition of the production of IP₃ and Ca²⁺ release from intracellular stores related to stimulation of the thromboxane A₂ receptor.     

Influence of Infrasound Exposure on the Whole L-type Calcium Currents in Rat Ventricular Myocytes

This study was designed to examine the effect of infrasound exposure (5 Hz at 130 dB) on whole-cell L-type Ca²⁺ currents (WLCC) in rat ventricular myocytes and the underlying mechanism(s) involved. Thirty-two adult Sprague-Dawley rats were randomly assigned to infrasound exposure and control groups. [Ca²⁺]_i, WLCC, mRNA expression of the a_1c subunit of L-type Ca²⁺ channels (LCC), and SERCA2 protein were examined on day 1, 7, and 14 after initiation of infrasound exposure. Fluo-3/AM fluorescence and the laser scanning confocal microscope techniques were used to measure [Ca²⁺]_i in freshly isolated ventricular myocytes. The Ca²⁺ fluorescence intensity (FI), denoting [Ca²⁺]_i in cardiomyocytes, was significantly elevated in a time-dependent manner in the exposure groups. There was a significant increase in WLCC in the 1-day group and a further significant increase in the 7- and 14-day groups. LCC mRNA expression measured by RT-PCR revealed a significant rise in the 1-day group and a significant additional rise in the 7- and 14-day groups compared with control group. SERCA2 expression was significantly upregulated in the 1-day group followed by an overt decrease in the 7- and 14-day groups. Prolonged exposure of infrasound altered WLCC in rat cardiomyocytes by shifting the steady-state inactivation curves to the right (more depolarized direction) without altering the slope and biophysical properties of I _Ca,L. Taken together, our data suggest that changes in [Ca²⁺]_I levels as well as expression of LCC and SERCA2 may contribute to the infrasound exposure-elicited cardiac response. Zhaohui Pei and Zhiqiang Zhuang contributed equally to this work.     

Epinephrine-induced Ca2+ influx in vascular endothelial cells is mediated by CNGA2 channels

Epinephrine, through its action on β-adrenoceptors, may induce endothelium-dependent vascular dilation, and this action is partly mediated by a cytosolic Ca²⁺ ([Ca²⁺]_i) change in endothelial cells. In the present study, we explored the molecular identity of the channels that mediate epinephrine-induced endothelial Ca²⁺ influx and subsequent vascular relaxation. Patch clamp recorded an epinephrine- and cAMP-activated cation current in the primary cultured bovine aortic endothelial cells (BAECs) and H5V endothelial cells. L-cis-diltiazem and LY-83583, two selective inhibitors for cyclic nucleotide-gated channels, diminished this cation current. Furthermore, this cation current was greatly reduced by a CNGA2-specific siRNA in H5V cells. With the use of fluorescent Ca²⁺ dye, it was found that epinephrine and isoprenaline, a β-adrenoceptor agonist, induced endothelial Ca²⁺ influx in the presence of bradykinin. This Ca²⁺ influx was inhibited by L-cis-diltiazem and LY-83583, and by a β₂-adrenoceptor antagonist ICI-118551. CNGA2-specific siRNA also diminished this Ca²⁺ influx in H5V cells. Furthermore, L-cis-diltiazem and LY-83583 inhibited the endothelial Ca²⁺ influx in isolated mouse aortic strips. L-cis-diltiazem also markedly reduced the endothelium-dependent vascular dilation to isoprenaline in isolated mouse aortic segments. In summary, CNG channels, CNGA2 in particular, mediate β-adrenoceptor agonist-induced endothelial Ca²⁺ influx and subsequent vascular dilation.     

Intracellular calcium ion responses to somatostatin in cells from human somatotroph adenomas

OBJECTIVE Various GH secretory responses to long-acting somatostatin (SRIH) analogues have been observed during the treatment of acromegalic patients. The effects of SRIH on intracellular Ca²⁺ homeostasis in human somatotroph adenoma cells has not been examined in detail, and the underlying mechanisms therefore remain to be determined. Using isolated cells from human somatotroph adenomas, we have investigated the SRIH-induced intracellular Ca²⁺ responses at a single-cell level with computerized real time intracellular calcium ion (Ca²⁺_i) imaging. PATIENTS Adenoma specimens were obtained from 4 male and 11 female acromegalic patients (mean age 56, range 26–72 years) undergoing transsphenoidal hypophysectomy. METHODS The identity of the biopsy material obtained was confirmed by immunocytochemistry for hGH and in situ hybridization histochemistry using a ³⁵S end-labelled hGH oligodeoxynucleotide probe and probes complementary to proopiomelanocortin and prolactin. Genomic DNA coding for somatostatin receptor (SSTR2) from each adenoma was PCR amplified and sequenced. Cells cultured from these adenoma were subject to computerized real time intracellular Ca²⁺_i imaging at a single cell level. RESULTS In cells from 11 of the 15 adenomas, SRIH produced a reversible, dose-independent reduction in [Ca²⁺]_i from the mean of 167±11 to 43±3 nM within 51±1.8s, and blocked the growth hormone releasing hormone (GRH)-induced increase in [Ca²⁺]_i as expected. In the same adenomas, withdrawal of SRIH after a 30 second exposure produced a small but significant increase in resting [Ca²⁺]_i. Pretreatment with pertussis toxin abolished the SRIH-induced inhibition of [Ca²⁺]_i and prevented the SRIH-induced inhibition of the effect of GRH on [Ca²⁺]_i. One of the remaining 4 adenomas was completely unresponsive to SRIH despite responding vigorously to other ligands and immunostaining strongly for GH. Surprisingly, cells from 3 adenomas showed a paradoxical increase in [Ca²⁺]_i in response to SRIH in some or, in one case, all of the cells examined. In all adenomas the sequence of SSTR2 corresponded to wild-type. CONCLUSIONS In the majority of cells derived from human somatotrophic adenomas, SRIH caused a reduction in baseline [Ca²⁺]_i and inhibition of GRH-induced [Ca²⁺]_i increase, as observed in somatotrophs of other species. In addition, SRIH was found either to induce a paradoxical increase in [Ca²⁺]_i or to have no effect on [Ca²⁺]_i in a small proportion of somatotroph adenomas examined. This finding corroborates the clinical observation that the response to SRIH analogues varies markedly between somatotroph adenoma patients. There was no evidence of SSTR2 mutations in any of the adenomas examined.