Microtubule disruption by colchicine reversibly enhances calcium signaling in intact rat cardiac myocytes

Using the whole-cell patch-clamp configuration in rat ventricular myocytes, we recently reported that microtubule disruption increases calcium current (I(Ca)) and [Ca(2+)](i) transient and accelerates their kinetics by adenylyl cyclase activation. In the present report, we further analyzed the effects of microtubule disruption by 1 micromol/L colchicine on Ca(2+) signaling in cardiac myocytes with intact sarcolemma. In quiescent intact cells, it is possible to investigate ryanodine receptor (RyR) activity by analyzing the characteristics of spontaneous Ca(2+) sparks. Colchicine treatment decreased Ca(2+) spark amplitude (F/F(0): 1.78+/-0.01, n=983, versus 1.64+/-0.01, n=1660, recorded in control versus colchicine-treated cells; P<0.0001) without modifying the sarcoplasmic reticulum Ca(2+) load and enhanced their time to peak (in ms: 6.85+/-0.09, n=1185, versus 7.33+/-0.13, n=1647; P<0.0001). Microtubule disruption also induced the appearance of Ca(2+) sparks in doublets. These alterations may reflect RyR phosphorylation. To further investigate Ca(2+) signaling in cardiac myocytes with intact sarcolemma, we analyzed [Ca(2+)](i) transient evoked by field stimulation. Cells were loaded with the fluorescence Ca(2+) indicator, Fluo-3 cell permeant, and stimulated at 1 HZ: [Ca(2+)](i) transient amplitude was greater and its decay was accelerated in colchicine-treated, field-stimulated myocytes. This effect is reversible. When colchicine-treated myocytes were placed in a colchicine-free solution for 30 minutes, tubulin was repolymerized into microtubules, as shown by immunofluorescence, and the increase in [Ca(2+)](i) transient was reversed. In summary, we demonstrate that microtubule disruption by colchicine reversibly modulates Ca(2+) signaling in cardiac cells with intact sarcolemma.     

Mechanism of D(2) agonist-induced inhibition of GH secretion from human GH-secreting adenoma cells

The mechanism of dopamine D(2) agonist-induced inhibition of GH secretion from GH-secreting adenoma cells was investigated by measurement of intracellular calcium concentration ([Ca(2+)] (i)) and static incubation experiment. Bromocriptine decreased [Ca(2+)](i) in a concentration-dependent manner through D(2) receptor. The inhibition was abolished by pertussis toxin pretreatment. Bromocriptine did not decrease [Ca (2+)](i) after nitrendipine had decreased it. 8Br-cAMP increased [Ca(2+)](i) but application of bromocriptine decreased it, suggesting that bromocriptine-induced inhibition of [Ca(2+)](i) is not dependent on bromocriptine-induced inhibition of adenylyl cyclase. Static incubation experiment revealed that bromocriptine inhibited GH secretion in a concentration-dependent manner. The inhibition was through D(2) receptor and was abolished by pertussis toxin pretreatment. 8Br-cAMP increased GH secretion. Bromocriptine decreased GH secretion even after 8Br-cAMP pretreatment. However, the GH release from cells incubated with bromocriptine alone was significantly less than that from cells incubated with bromocriptine after 8Br-cAMP pretreatment, suggesting a modulatory action of cAMP system in bromocriptine response.     

Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells

The pancreatic acinar cell produces powerful digestive enzymes packaged in zymogen granules in the apical pole. Ca(2+) signals elicited by acetylcholine or cholecystokinin (CCK) initiate enzyme secretion by exocytosis through the apical membrane. Intracellular enzyme activation is normally kept to a minimum, but in the often-fatal human disease acute pancreatitis, autodigestion occurs. How the enzymes become inappropriately activated is unknown. We monitored the cytosolic Ca(2+) concentration ([Ca(2+)](i)), intracellular trypsin activation, and its localization in isolated living cells with specific fluorescent probes and studied intracellular vacuole formation by electron microscopy as well as quantitative image analysis (light microscopy). A physiological CCK level (10 pM) eliciting regular Ca(2+) spiking did not evoke intracellular trypsin activation or vacuole formation. However, stimulation with 10 nM CCK, evoking a sustained rise in [Ca(2+)](i), induced pronounced trypsin activation and extensive vacuole formation, both localized in the apical pole. Both processes were abolished by preventing abnormal [Ca(2+)](i) elevation, either by preincubation with the specific Ca(2+) chelator 1, 2-bis(O-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid (BAPTA) or by removal of external Ca(2+). CCK hyperstimulation evokes intracellular trypsin activation and vacuole formation in the apical granular pole. Both of these processes are mediated by an abnormal sustained rise in [Ca(2+)](i).     

Microtubule disruption modulates Ca(2+) signaling in rat cardiac myocytes

Microtubules have been shown to alter contraction in cardiac myocytes through changes in cellular stiffness. However, an effect on excitation-contraction coupling has not been examined. Here we analyze the effects of microtubule disruption by 1 micromol/L colchicine on calcium currents (I(Ca)) and [Ca(2+)](i) transients in rat ventricular myocytes. I(Ca) was studied using the whole-cell patch-clamp technique. Colchicine treatment increased I(Ca) density (peak values, -4.6+/-0.4 and -9.1+/-1.3 pA/pF in 11 control and 12 colchicine-treated myocytes, respectively; P<0.05). I(Ca) inactivation was well fitted by a biexponential function. The slow component of inactivation was unchanged, whereas the fast component was accelerated after colchicine treatment (at -10 mV, 11.8+/-1.0 versus 6.7+/-1.0 ms in control versus colchicine-treated cells; P<0.005). [Ca(2+)](i) transients were analyzed by fluo-3 epifluorescence simultaneously with I(Ca). Peak [Ca(2+)](i) transients were significantly increased in cardiac myocytes treated with colchicine. The values of F/F(0) at 0 mV were 1.1+/-0.02 in 9 control cells and 1.4+/-0.1 in 11 colchicine-treated cells (P<0.05). beta-Adrenergic stimulation with 1 micromol/L isoproterenol increased both I(Ca) and [Ca(2+)](i) transient in control cells. However, no significant change was induced by isoproterenol on colchicine-treated cells. Colchicine and isoproterenol effects were similar and not additive. Inhibition of adenylyl cyclase by 200 micromol/L 2'-deoxyadenosine 3'-monophosphate blunted the colchicine effect. We suggest that beta-adrenergic stimulation and microtubule disruption share a common pathway to enhance I(Ca) and [Ca(2+)](i) transient.     

Activation of 5-HT(7) receptor in rat glomerulosa cells is associated with an increase in adenylyl cyclase activity and calcium influx through T-type calcium channels

Serotonin (5-HT) stimulates aldosterone secretion from the rat adrenal gland through 5-HT(7) receptors. The aim of the present study was to investigate the transduction mechanisms associated with activation of 5-HT(7) receptors in rat glomerulosa cells. The stimulatory effect of 5-HT on aldosterone secretion and cAMP formation was significantly reduced by the 5-HT(7) receptor antagonist LY 215840. Pretreatment of cells with the adenylyl cyclase inhibitor SQ 22536 or the PKA inhibitor H-89 markedly attenuated the effect of 5-HT on aldosterone secretion. Conversely, type 2 and 4 phosphodiesterase inhibitors potentiated the 5-HT-induced stimulation of aldosterone secretion. Administration of 5-HT in the vicinity of cultured glomerulosa cells induced a slowly developing and robust increase in cytosolic calcium concentration ([Ca(2+)](i)). The effect of 5-HT on [Ca(2+)](i) was suppressed by mibefradil, a T-type calcium channel blocker. Patch-clamp studies confirmed that 5-HT activated a T-type calcium current. Mibefradil also induced a dose-dependent inhibition of 5-HT-induced aldosterone secretion. The sequence of events associated with activation of 5-HT(7) receptors was investigated. The PKA inhibitor H-89 markedly attenuated both the [Ca(2+)](i) response and the activation of T-type calcium current induced by 5-HT. In contrast, reduction of the calcium concentration in the incubation medium did not affect 5-HT- induced cAMP formation. Preincubation of glomerulosa cells with cholera toxin abolished the stimulatory effect of 5-HT on aldosterone secretion, but pertussis toxin had no effect. Taken together, these data demonstrate that, in rat glomerulosa cells, activation of native 5-HT(7) receptors stimulates cAMP formation through a G(salpha) protein, which in turn provokes calcium influx through T-type calcium channels. Both the adenylyl cyclase/PKA pathway and the calcium influx are involved in 5-HT-induced aldosterone secretion.     

A novel mechanism for the melatonin inhibition of testosterone secretion by rat Leydig cells: reduction of GnRH-induced increase in cytosolic Ca2+

The site of inhibition, by melatonin, of GnRH-dependent testosterone secretion was investigated in adult rat Leydig cells cultured in vitro. The various effects downstream of the binding of GnRH to its own receptor were isolated and mimicked by specific drugs. Testosterone secretion was then evaluated after 3 h stimulation with GnRH, thapsigargin (1 microM), phorbol-12-myristate-13-acetate (100 nM), arachidonic acid (20 microM), and ionomycin (1 microM) in the presence or absence of melatonin (215 nM). The effect of melatonin on the GnRH-induced changes in cytoplasmic calcium concentration ([Ca(2+)](i)) was also studied, using Fura-2 as fluorescent Ca(2+) indicator. Melatonin attenuated the increase in [Ca(2+)](i) and inhibited the testosterone secretion induced by GnRH, but not that induced by ionomycin. Both ionomycin and thapsigargin potentiated GnRH-induced testosterone secretion; however, ionomycin, but not thapsigargin, partially prevented the inhibitory effect of melatonin on cells stimulated with GnRH. The effect of melatonin was probably dependent on the binding of melatonin to its Gi-protein-coupled receptor, as the inhibitory effect on GnRH-induced secretion was supressed in cells pretreated with pertussis toxin in a concentration of 180 ng/ml for 20 h. Assay of 17-hydroxy-progesterone showed that, irrespective of the treatment, cells cultured with melatonin secreted greater amounts than controls. We conclude that melatonin reduces GnRH-induced testosterone secretion by 1) decreasing [Ca(2+)](i), through impairment of the GnRH-dependent release of Ca(2+) from intracellular stores and 2) blocking 17-20 desmolase enzymatic activity, an effect that occurs irrespective of changes in [Ca(2+)](i).     

Modulation of Ca(2+) signaling by microtubule disruption in rat ventricular myocytes and its dependence on the ruptured patch-clamp configuration

In the absence of hypertrophic proliferation of microtubules, microtubule disruption by colchicine does not modulate contraction of adult cardiac myocytes. However, Gomez et al (Circ Res. 2000;86:30-36) recently reported that disruption of microtubules by colchicine in ruptured patch-clamped myocytes increased I(Ca,L) density and [Ca(2+)](i) transient amplitude and depressed the response of these parameters to the beta-adrenoceptor agonist isoproterenol. These effects were ascribed to stimulation of adenylyl cyclase by increased intracellular free tubulin. In the present study, we show that in intact rat ventricular myocytes, 2 to 4 hours of exposure to 10 micromol/L colchicine had no effect on shortening or [Ca(2+)](i) transient amplitude or on the amplitude of I(Ca,L) in perforated patch-clamped cells, under basal conditions and after stimulation with 1 micromol/L isoproterenol. However, in ruptured patch-clamped myocytes, basal I(Ca,L) was 2-fold higher after treatment with colchicine compared with vehicle and, in contrast to vehicle-treated cells, I(Ca,L) did not increase in response to isoproterenol. Cell width decreased during ruptured patch-clamp experiments in colchicine-treated but not vehicle-treated myocytes. We conclude that in cells with intact sarcolemma, colchicine does not modulate Ca(2+) signaling or the response to beta stimulation. However, the combination of microtubule disruption by colchicine and the ruptured patch configuration activates I(Ca,L) and attenuates the response to beta stimulation. We propose that these effects may be due to loss of free tubulin by intracellular dialysis or to increased sensitivity to mechanical stimulation as a result of microtubule disruption. These findings have important implications for cardiomyopathies associated with decreased free tubulin or a diminished microtubular network. The full text of this article is available at http://www.circresaha.org.     

Reactive oxygen intermediates induce regulated secretion of von Willebrand factor from cultured human vascular endothelial cells

Exocytosis from Weibel-Palade bodies, the secretory granules of vascular endothelial cells, causes the rapid release of von Willebrand factor (vWF), an adhesive glycoprotein involved in primary hemostasis, and cell surface expression of P-selectin, a membrane protein involved in neutrophil binding. Thus, exocytosis may represent a link between hemostasis and inflammation. We investigated the effect of reactive oxygen intermediates (ROIs) on vWF secretion. Incubation of cultured endothelial cells with xanthine oxidase (XO), which generates superoxide anions (O2-), induces a potent, rapid secretory response. However, vWF release was not observed in response to H2O2. Extracellular, subendothelial vWF deposits typically seen after exocytosis from Weibel-Palade bodies were observed after exposure to XO. XO caused a rapid, sustained increase in intracellular free calcium concentration ([Ca2+]i). vWF secretion was markedly inhibited by BAPTA- AM, a cell-permeant calcium chelator. Removal of extracellular calcium did not inhibit vWF release, although the sustained phase of the [Ca2+]i increase was suppressed. These results suggest that XO-induced vWF release is mediated by the initial increase in [Ca2+]i which is caused by calcium mobilization from intracellular stores rather than by calcium influx. Exocytosis from Weibel-Palade bodies may contribute to the pathogenic effect of ROIs in atherosclerosis and inflammation.     

Evidence of diminished glucose stimulation and endoplasmic reticulum function in nonoscillatory pancreatic islets

Pulsatility is a fundamental feature of pancreatic islets and a hallmark of hormone secretion. Isolated pancreatic islets endogenously generate rhythms in secretion, metabolic activity, and intracellular calcium ([Ca(2+)](i)) that are important to normal physiological function. Few studies have directly compared oscillatory and nonoscillatory islets to identify possible differences in function. We investigated the hypothesis that the loss of these oscillations is a leading indicator of islet dysfunction by comparing oscillatory and nonoscillatory mouse islets for multiple parameters of function. Nonoscillatory islets displayed elevated basal [Ca(2+)](i) and diminished [Ca(2+)](i) response and insulin secretory response to 3-28 mm glucose stimulation compared with oscillatory islets, suggesting diminished glucose sensitivity. We investigated several possible mechanisms to explain these differences. No differences were observed in mitochondrial membrane potential, estimated ATP-sensitive potassium channel and L-type calcium channel activity, or cell death rates. Nonoscillatory islets, however, showed a reduced response to the sarco(endo)plasmic reticulum calcium ATPase inhibitor thapsigargin, suggesting a disruption in calcium homeostasis in the endoplasmic reticulum (ER) compared with oscillatory islets. The diminished ER calcium homeostasis among nonoscillatory islets was also consistent with the higher cytosolic calcium levels observed in 3 mm glucose. Inducing mild damage with low-dose proinflammatory cytokines reduced islet oscillatory capacity and produced similar effects on glucose-stimulated [Ca(2+)](i), basal [Ca(2+)](i), and thapsigargin response observed among untreated nonoscillatory islets. Our data suggest the loss of oscillatory capacity may be an early indicator of diminished islet glucose sensitivity and ER dysfunction, suggesting targets to improve islet assessment.     

A Galphas protein-coupled membrane receptor,distinct from the classical oestrogen receptor,transduces rapid effects of oestradiol on [Ca2+]i in female rat distal colon

We examined the hypothesis whether rapid non-genomic effects of oestradiol (E2) on [Ca(2+)](i) are mediated via a membrane-located oestrogen receptor (ER) and further elucidated the signalling pathways involved in rapid non-genomic effects of E2 on [Ca(2+)](i) in distal colonic crypts. Basal [Ca(2+)](i) was significantly increased, within minutes, in response to physiological concentrations of E2. Oestradiol linked to bovine serum albumin (E2-BSA), which renders the E2 membrane impermeable, rapidly increased [Ca(2+)](i) suggesting mediation by a membrane surface receptor. A classical ER is not involved however, as no inhibition of either the E2 or E2-BSA [Ca(2+)](i) response was seen in the presence of the classical ER antagonist ICI 182,780. Treatment with the Galphas inhibitor cholera toxin abolished both E2 and E2-BSA induced Ca(2+) increases. In contrast, treatment with pertussis toxin, an inhibitor of Galphai and Galphao, had no inhibitory effect. Following subsequent additions of E2 and E2-BSA, no further increases in [Ca(2+)](i) were observed, indicating receptor desensitisation. The E2-induced increase in [Ca(2+)](i) was completely abolished by the PKCdelta-specific inhibitor rottlerin, whereas Go6976, an inhibitor of Ca(2+)-sensitive PKC isoforms, was without inhibitory effect. The phospholipase A2 antagonist, quinacrine, and the COX1 inhibitor, indomethacin, abolished the E2-induced increase in [Ca(2+)](i). MAP kinase activation is not involved in rapid stimulatory effects of E2 on [Ca(2+)](i) as the specific inhibitor PD98059 did not inhibit the E2 response. These results demonstrate that rapid E2-induced stimulation of [Ca(2+)](i), in femal rat distal colonic crypts, occurs via a CTx-sensitive Galphas-coupled membrane receptor distinct from the classical ER. PKCdelta and fatty acids are involved in the E2 signalling pathway. In contrast, PKCalpha and MAP kinase are not required.     

Disorganization of cytoplasmic Ca<Superscript>2+</Superscript> oscillations and pulsatile insulin secretion in islets from <Emphasis Type="Italic">ob</Emphasis>/<Emphasis Type="Italic">ob </Emphasis>mice

AIMS/HYPOTHESIS: In normal mouse islets, glucose induces synchronous cytoplasmic [Ca(2+)](i) oscillations in beta cells and pulses of insulin secretion. We investigated whether this fine regulation of islet function is preserved in hyperglycaemic and hyperinsulinaemic ob/ obmice. METHODS: Intact islets from ob/ ob mice and their lean littermates were used after overnight culture for measurement of [Ca(2+)](i) and insulin secretion. RESULTS: We observed three types of [Ca(2+)](i) responses during stimulation by 9 to 12 mmol/l of glucose: sustained increase, rapid oscillations and slow (or mixed) oscillations. They occurred in 8, 18 and 74% of lean islets and 9, 0 and 91% of ob/ ob islets, respectively. Subtle desynchronisation of [Ca(2+)](i) oscillations between regions occurred in 11% of lean islets. In ob/ ob islets, desynchronisation was frequent (66-82% depending on conditions) and prominent: oscillations were out of phase in different regions because of distinct periods and shapes. Only small ob/ ob islets were well synchronised, but sizes of synchronised lean and desynchronised ob/ ob islets were markedly overlapped. The occurrence of desynchronisation in clusters of 5 to 50 islet cells from ob/ obmice and not from lean mice further indicates that islet hypertrophy is not the only causal factor. In both types of islets, synchronous [Ca(2+)](i) oscillations were accompanied by oscillations of insulin secretion. In poorly synchronised ob/ ob islets, secretion was irregular but followed the pattern of the global [Ca(2+)](i) changes. CONCLUSIONS/INTERPRETATION: The regularity of glucose-induced [Ca(2+)](i) oscillations is disrupted in islets from ob/ ob mice and this desynchronisation perturbs the pulsatility of insulin secretion. A similar mechanism could contribute to the irregularity of insulin oscillations in Type II (non-insulin-dependent) diabetes mellitus.     

Aggregation Fails to Increase Cytosolic [Ca(2+)] in Aequorin-loaded Human Platelets

Studies were performed to determine whether formation of platelet aggregates itself, could cause an increase in cytosolic [Ca(2+)]([Ca(2+)](i)) which is independent of that resulting from the addition of agonists which induce aggregation. An increase in [Ca(2+)](i) did not coincide with aggregate formation when this response was dissociated from the addition of ADP or thrombin by delay either in initiating stirring or, for ADP, in adding fibrinogen. No increase in [Ca(2+)](i) occurred when aggregation was induced by addition of 1,2-dioctanoylglycerol or of ristocetin, or for chymotrypsin-treated platelets by addition of fibrinogen. The results demonstrate clearly that aggregate formation does not cause an increase in [Ca(2+)](i), and therefore exclude this possibility as an explanation for the discrepancies observed when [Ca(2+)](i) is measured, using aequorin and Fura2 as probes and as an underlying mechanism to account for contact-induced responses.     

Inhibitory control of growth hormone secretion by somatostatin in rat pituitary GC cells: sst(2) but not sst(1) receptors are coupled to inhibition of single-cell intracellular free calcium concentrations

Rat pituitary tumor cells (GC cells) exhibit spontaneous oscillations of intracellular free calcium concentration ([Ca(2+)](i)) that allow continuous release of growth hormone (GH). Of the somatostatin (SRIH) receptor subtypes (sst receptors) mediating SRIH action, sst(1) and sst(2) receptors are highly expressed by GC cell membranes. In the present study, the effects of sst(1) or sst(2) receptor activation on single-cell [Ca(2+)](i) were investigated in GC cells by confocal fluorescence microscopy. In addition, the effects of sst(1) or sst(2) receptor activation on GH secretion were also studied. Our results demonstrate that SRIH decreases [Ca(2+)](i) baseline and almost completely blocks Ca(2+) transients through activation of sst(2) but not of sst(1) receptors. In contrast, SRIH effectively inhibits GH secretion through activation of both sst(1) and sst(2) receptors. Blocking Ca(2+) transients is less efficient than SRIH to inhibit GH release. The cyclic octapeptide, CYN-154806, antagonizes sst(2) receptors at [Ca(2+)](i) since it abolishes the sst(2) receptor-mediated inhibition of [Ca(2+)](i) without affecting single-cell Ca(2+) signals. On the other hand, CYN-154806 alone potently inhibits GH secretion through the involvement of pertussis toxin-sensitive G proteins. In conclusion, the present results demonstrate that SRIH inhibition of GH release in GC cells involves mechanisms either dependent or independent on SRIH modulation of [Ca(2+)](i). The implications of CYN-154806 inhibition of GH secretion are discussed.     

Growth hormone signaling in pancreatic beta-cells--calcium handling regulated by growth hormone

Deficiency in insulin secretion is a fundamental part in the pathogenesis of all forms of diabetes, determined by impaired secretory function and inadequate beta-cell mass. Growth hormone (GH) is a multifunctional hormone, involved in cellular metabolism, mitogenesis and differentiation. In pancreatic islets, GH is involved in maintaining beta-cell mass, stimulating islet hormone production and insulin secretion, and, therefore, plays a role in maintaining normal insulin sensitivity and glucose homeostasis. The intracellular events that convey the GH signal into various cellular responses remain incompletely understood. In this review, we discuss GH signaling in the beta-cells, with emphasis on Ca(2+) handling and insulin secretion regulated by human GH (hGH). hGH-stimulated rise in [Ca(2+)](i) is dependent on extracellular Ca(2+) and is mediated by Ca(2+)-induced Ca(2+) release (CICR) in the beta-cell. This process is triggered by hGH-stimulated activation of the non-receptor tyrosine kinases JAK2 and c-Src, which causes tyrosine phosphorylation of RyRs, resulting in sensitization of CICR. The rise in [Ca(2+)](i) elicited by hGH is associated with an enhanced insulin secretion, effects that are mediated mainly through the prolactin receptor. These mechanisms indicate that a rise in [Ca(2+)](i) elicited by activation of PRLR is JAK2-dependent and is associated with enhanced insulin secretion. In contrast, GH receptor-mediated increase in [Ca(2+)](i) is JAK2-independent and is dissociated from insulin secretion.     

GABA(B) receptors in anterior pituitary cells. Mechanism of action coupled to endocrine effects

The activation of pituitary GABA(B) receptors by the specific agonist baclofen inhibits pituitary hormone secretion in vitro. Here we studied the mechanism of action of GABA(B) receptors in rat adenohypophysis. Anterior pituitary cells were obtained by trypsinization and were either plated for hormonal studies and cAMP determination or incubated in FURA 2AM for calcium measurements. Baclofen (BACL: 1 x 10(-5) M) significantly inhibited basal and thyrotropic releasing hormone (TRH)-stimulated (1 x 10(-7) M) PRL secretion in anterior pituitary cells from proestrous rats. In the presence of pertussis toxin (PTX: 150 ng/ml, 20 h), which leads to the uncoupling of the G(i/o)-protein from the receptor, both effects of BACL were abolished while the effect of dopamine (DA: 1 x 10(-8) M), used as an inhibitory control, was reduced from 70 to 25%. PTX also reversed BACL-induced inhibition of gonadotropin-releasing hormone (GnRH)-elicited luteinizing hormone (LH) secretion in anterior pituitary cells from 15-day-old female rats. In addition, though working in a pituitary mixed cell population, in which only some cell types possess GABA(B) receptors, BACL (1 x 10(-5) M) attenuated the forskolin-induced (0.5 microM) increase in cAMP. This effect was prevented by co-incubation with the antagonist 2 hydroxysaclofen and by preincubation with PTX. BACL (5 x 10(-5) M) and DA (5 x 10(-7) M) inhibited basal intracellular calcium concentrations ([Ca(2+)](i)) in pituitary cells and the effect of the latter was significantly stronger. The effect of BACL on [Ca(2+)](i) was abolished after preincubation with PTX. In the presence of the potassium channel blocking agents barium (200 microM and 1 mM) and tetraethylammonium (10 mM), BACL was still able to inhibit [Ca(2+)](i). Blockade of voltage-sensitive calcium channels (VSCC) with either verapamil (5 x 10(-6) M) or nifedipine (1 x 10(-6) M) completely abolished the effect of BACL on [Ca(2+)](i). In the presence of 12.5 mM potassium concentration baclofen significantly inhibited [Ca(2+)](i). In conclusion, our results describe the negative coupling of adenohypophyseal GABA(B) receptors to VSCC through PTX-sensitive G-proteins. These characteristics suggest a resemblance of these receptors to the typical presynaptic GABA(B) sites described in the central nervous system.     

Transient receptor potential channel TRPM8 agonists stimulate calcium influx and neurotensin secretion in neuroendocrine tumor cells

TRPM8 is a member of the melastatin-type transient receptor potential ion channel family. Activation by cold or by agonists (menthol, icilin) induces a transient rise in intracellular free calcium concentration ([Ca(2+)](i)). Our previous study demonstrated that Ca(2+)-permeable cation channels play a role in IGF-1-induced secretion of chromogranin A in human neuroendocrine tumor (NET) cell line BON [Mergler et al.: Neuroendocrinology 2006;82:87-102]. Here, we extend our earlier study by investigating the expression of TRPM8 and characterizing its impact on [Ca(2+)](i) and the secretion of neurotensin (NT). We identified TRPM8 expression in NET BON cells by RT-PCR, Western blotting and immunofluorescence staining. Icilin increased [Ca(2+)](i) in TRPM8-transfected human embryonic kidney cells (HEK293) but not in mock-transfected cells. Icilin and menthol induced Ca(2+) transients in BON cells as well as in primary NET cell cultures of two different pancreatic NETs as detected by single cell fluorescence imaging. Icilin increased non-selective cation channel currents in BON cells as detected by patch-clamp recordings. This activation was associated with increased NT secretion. Taken together, this study demonstrates for the first time the expression TRPM8 in NET cells and its role in regulating [Ca(2+)](i) and NT secretion. The regulation of NT secretion in NETs by TRPM8 may have a potential clinical implication in diagnosis or therapy.     

Stimulatory effect of ghrelin on isolated porcine somatotropes

Research on the mechanism for growth hormone secretagogue (GHS) induction of growth hormone secretion led to the discovery of the GHS receptor (GHS-R) and later to ghrelin, an endogenous ligand for GHS-R. The ability of ghrelin to induce an increase in the intracellular Ca(2+) concentration - [Ca(2+)](i) - in somatotropes was examined in dispersed porcine pituitary cells using a calcium imaging system. Somatotropes were functionally identified by application of human growth hormone releasing hormone. Ghrelin increased the [Ca(2+)](i) in a dose-dependent manner in 98% of the cells that responded to human growth hormone releasing hormone. In the presence of (D-Lys(3))-GHRP-6, a specific receptor antagonist of GHS-R, the increase in [Ca(2+)](i) evoked by ghrelin was decreased. Pretreatment of cultures with somatostatin or neuropeptide Y reduced the ghrelin-induced increase of [Ca(2+)](i). The stimulatory effect of ghrelin on somatotropes was greatly attenuated in low-calcium saline and blocked by nifedipine, an L-type calcium channel blocker, suggesting involvement of calcium channels. In a zero Na(+) solution, the stimulatory effect of ghrelin on somatotropes was decreased, suggesting that besides calcium channels, sodium channels are also involved in ghrelin-induced calcium transients. Either SQ-22536, an adenylyl cyclase inhibitor, or U73122, a phospholipase C inhibitor, decreased the stimulatory effects of ghrelin on [Ca(2+)](i) transiently, indicating the involvement of adenylyl cyclase-cyclic adenosine monophosphate and phospholipase C inositol 1,4,5-trisphosphate pathways. The nonpeptidyl GHS, L-692,585 (L-585), induced changes in [Ca(2+)](i) similar to those observed with ghrelin. Application of L-585 after ghrelin did not have additive effects on [Ca(2+)](i). Preapplication of L-585 blocked the stimulatory effect of ghrelin on somatotropes. Simultaneous application of ghrelin and L-585 did not cause an additive increase in [Ca(2+)](i). Our results suggest that the actions of ghrelin and synthetic GHS closely parallel each other, in a manner that is consistent with an increase of hormone secretion.     

Extracellular ATP stimulates estradiol secretion in rat Sertoli cells in vitro: modulation by external sodium

In this study, we examined the effects of extracellular ATP (ATPe) on [Ca(2+)](i), [Na(+)](i), plasma membrane potential changes and estradiol secretion in rat Sertoli cells. ATPe caused a rapid rise of [Ca(2+)](i) with an initial spike followed by a long lasting plateau. The first rapid spike was dependent on the release of Ca(2+) from internal stores as it also occurred in Ca(2+)-free medium while the long lasting plateau phase was dependent on Ca(2+) influx from the external medium. ATPe stimulated a rapid plasma membrane depolarization that was dependent on an influx of Na(+) from the external medium as demonstrated by plasma membrane potential monitoring in Na(+)-free medium and by [Na(+)](i) measurement with the Na(+)-sensitive fluorescent dye SBFI. ATPe stimulated estradiol secretion in a dose dependent manner and was fully dependent on the presence of Na(+) in the external medium while the presence of Ca(2+) was not necessary. Among the different nucleotides tested, only ATP, ATP-5'-[gamma-thio]triphosphate, UTP, alpha,beta-methylene-ATP were effective in stimulating estradiol secretion. These results demonstrate that rat Sertoli cells possess P2-purinergic receptors belonging to the P2X and P2Y subfamily which activation induces [Ca(2+)](i) and [Na(+)](i) rise and Na(+)-dependent plasma membrane depolarization leading to estradiol secretion.     

Shear stress-induced von Willebrand factor binding to platelet glycoprotein Ib initiates calcium influx associated with aggregation.

Platelets subjected to elevated levels of fluid shear stress in the absence of exogenous agonists will aggregate. Shear stress-induced aggregation requires von Willebrand factor (vWF) multimers, extracellular calcium (Ca2+), adenosine diphosphate (ADP), and platelet membrane glycoprotein (GP)Ib and GPIIb-IIIa. The sequence of interaction of vWF multimers with platelet surface receptors and the effect of these interactions on platelet activation have not been determined. To elucidate the mechanism of shear stress-induced platelet aggregation, suspensions of washed platelets were subjected to different levels of uniform shear stress (15 to 120 dyne/cm2) in an optically modified cone and plate viscometer. Cytoplasmic ionized calcium ([Ca2+]i) and aggregation of platelets were monitored simultaneously during the application of shear stress; [Ca2+]i was measured using indo-1 loaded platelets and aggregation was measured as changes in light transmission. Basal [Ca2+]i was approximately 60 to 100 nmol/L. An increase of [Ca2+]i (up to greater than 1,000 nmol/L) was accompanied by synchronous aggregation, and both responses were dependent on the shear force and the presence of vWF multimers. EGTA chelation of extracellular Ca2+ completely inhibited vWF-mediated [Ca2+]i and aggregation responses to shear stress. Aurin tricarboxylic acid, which blocks the GPIb recognition site on the vWF monomer, and 6D1, a monoclonal antibody to GPIb, also completely inhibited platelet responses to shear stress. The tetrapeptide RGDS and the monoclonal antibody 10E5, which inhibit vWF binding to GPIIb-IIIa, partially inhibited shear stress-induced [Ca2+]i and aggregation responses. The combination of creatine phosphate/creatine phosphokinase, which converts ADP to adenosine triphosphate and blocks the effect of ADP released from stimulated platelets, inhibited shear stress-induced platelet aggregation without affecting the increase of [Ca2+]i. Neither the [Ca2+]i nor aggregation response to shear stress was inhibited by blocking platelet cyclooxygenase metabolism with acetylsalicylic acid. These results indicate that GPIb and extracellular Ca2+ are absolutely required for vWF-mediated [Ca2+]i and aggregation responses to imposed shear stress, and that the interaction of vWF multimers with GPIIb-IIIa potentiates these responses. Shear stress-induced elevation of platelet [Ca2+]i, but not aggregation, is independent of the effects of release ADP, and both responses occur independently of platelet cyclooxygenase metabolism. These results suggest that shear stress induces the binding of vWF multimers to platelet GPIb and this vWF-GPIb interaction causes an increase of [Ca2+]i and platelet aggregation, both of which are potentiated by vWF binding to the platelet GPIIb-IIIa complex.     

Negative-feedback loop attenuates hydrostatic lung edema via a cGMP-dependent regulation of transient receptor potential vanilloid 4

Although the formation of hydrostatic lung edema is generally attributed to imbalanced Starling forces, recent data show that lung endothelial cells respond to increased vascular pressure and may thus regulate vascular permeability and edema formation. In combining real-time optical imaging of the endothelial Ca(2+) concentration ([Ca(2+)](i)) and NO production with filtration coefficient (K(f)) measurements in the isolated perfused lung, we identified a series of endothelial responses that constitute a negative-feedback loop to protect the microvascular barrier. Elevation of lung microvascular pressure was shown to increase endothelial [Ca(2+)](i) via activation of transient receptor potential vanilloid 4 (TRPV4) channels. The endothelial [Ca(2+)](i) transient increased K(f) via activation of myosin light-chain kinase and simultaneously stimulated NO synthesis. In TRPV4 deficient mice, pressure-induced increases in endothelial [Ca(2+)](i), NO synthesis, and lung wet/dry weight ratio were largely blocked. Endothelial NO formation limited the permeability increase by a cGMP-dependent attenuation of the pressure-induced [Ca(2+)](i) response. Inactivation of TRPV4 channels by cGMP was confirmed by whole-cell patch-clamp of pulmonary microvascular endothelial cells and intravital imaging of endothelial [Ca(2+)](i). Hence, pressure-induced endothelial Ca(2+) influx via TRPV4 channels increases lung vascular permeability yet concomitantly activates an NO-mediated negative-feedback loop that protects the vascular barrier by a cGMP-dependent attenuation of the endothelial [Ca(2+)](i) response. The identification of this novel regulatory pathway gives rise to new treatment strategies, as demonstrated in vivo in rats with acute myocardial infarction in which inhibition of cGMP degradation by the phosphodiesterase 5 inhibitor sildenafil reduced hydrostatic lung edema.