Zn2+ increases resting cytosolic Ca2+ levels and abolishes capacitative Ca2+ entry induced by ATP in MDCK cells

The effect of Zn2+ on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated by measuring the changes in the fluorescence of the Ca2+-sensitive dye fura-2. Zn2+ significantly increased cytoplasmic free Ca2+ levels ([Ca2+]i) at concentrations of 2-100 microM. The maximum response was obtained at concentrations of 25-100 microM. The [Ca2+]i rise induced by 100 microM Zn2+ consisted of a gradual rise and a plateau phase, and was primarily mediated by La3+-sensitive extracellular Ca2+ influx because the [Ca2+]i rise was abolished by pretreatment with 100 microM La3+ or removal of extracellular Ca2+, and that Zn2+ induced Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength which was prevented by pretreatment with 100 microM La3+. Pretreatment with 100 microM Zn2+ for 220 s did not reduce the [Ca2+]i rise induced by the endoplasmic reticulum (ER) Ca2+ pump inhibitor, thapsigargin, suggesting that Ca2+ release from the ER played a minor role in the Zn2+-induced [Ca2+]i rise. Zn2+ (100 microM) nearly abolished the capacitative Ca2+ entry induced by ATP (100 microM). We also investigated the effect of Zn2+ pretreatment on the [Ca2+]i rise induced by ATP. Zn2+ (100 microM) affected ATP-induced [Ca2+]i rise by abolishing capacitative Ca2+ entry and increasing [Ca2+]i on its own without altering Ca2+ release from intracellular sources. The effect of Zn2+ on [Ca2+]i was dissociated from changes in membrane potential.     

Rise in intracellular calcium concentration elicited by isradipine in Gin-1 cells.

We performed experiments to examine whether isradipine (Isr), a calcium antagonist, would raise the intracellular calcium concentration ([Ca2+]i) in Gin-1 cells and, if so, to elucidate the mechanism of the [Ca2+]i rise. Gin-1 cells, which are human normal gingival fibroblasts were used as the material. The [Ca2+]i was measured with the Ca2+-sensitive fluorescent dye fura-2/AM. Changes in the fluorescence intensity of fura-2 in the cells were recorded with a video-imaging analysis system. Isr concentration-dependently raised the [Ca2+]i. A Ca2+-free saline significantly inhibited the Isr-induced [Ca2+]i rise. Whereas Isr in Ca2+-containing solution weakly raised the [Ca2+]i by pretreatment with thapsigargin, an inhibitor of Ca2+ release from Ca2+ stores, the Ca2+-free saline plus thapsigargin completely depressed the Isr-induced [Ca2+]i rise. The same response was observed in the case of pretreatment with cyclopiazonic acid (1 microM), another inhibitor of Ca2+ release from the Ca2+ stores. Isr raises the [Ca2+]i in Gin-1 cells and that the Isr-induced [Ca2+]i rise is ascribable to both the Ca2+ influx through the plasma membrane and Ca2+ release from the intracellular Ca2+ store.     

Effect of intracellular pH on cytosolic free [Ca2+] in human epidermoid A-431 cells.

This study characterizes the correlation between intracellular pH (pHi) and the cytosolic free Ca2+ concentration ([Ca2+]i) in suspended and adherent human epidermoid A-431 cells. Using the fluorescent dyes 2,7-bis(carboxyethyl)carboxyfluorescein acetoxymethyl ester (BCECF) and fura-2, the resting pHi and [Ca2+]i in suspended cells were 7.23 +/- 0.03 and 209 +/- 30 nM; those in adherent cells were 7.28 +/- 0.02 and 87 +/- 5 nM. Removal of external Ca2+ did not change the resting pHi but reduced the resting [Ca2+]i, indicating the resting level of [Ca2+]i is in part maintained by an influx of Ca2+ from the external medium. When both suspended and adherent cells were acidified or alkalinized, resting [Ca2+]i was altered. An intracellular acidification induced a fall in [Ca2+]i, and a rise in pHi induced a rise in [Ca2+]i. These changes in [Ca2+]i were correlated with an uptake of 45Ca2+ from the external medium, whereas no Ca2+ efflux occurred. The alteration in [Ca2+]i induced by modification of pHi was abolished in the absence of external Ca2+ or by adding 2 mM CoCl2, LaCl3, and attenuated by the addition of 2 mM MnCl2 to the bathing medium. It was insensitive to the voltage-gated Ca2+ channel blockers nifedipine or verapamil (1 mM). CoCl2, LaCl3, and MnCl2 each induced changes in pHi and [Ca2+]i but verapamil and nifedipine did not. Because CoCl2, LaCl3, and MnCl2 are also known to block Na+/Ca2+ exchange, intracellular Na+ ([Na+]i) was measured by flame photometry in acidified or alkalinized cells. In either case no change in [Na+]i was observed. Furthermore, treatment with amiloride (100 microM), a blocker of the Na+/Ca2+ exchanger, did not inhibit the pH-induced changes in [Ca2+]i. 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (100 microM), a Ca2+ chelator, induced a decrease in pHi as well as a reduction of [Ca2+]i, also supporting the direct relation between pHi and [Ca2+]i. 3,4,5-Trimethoxybenzoic acid 8-(diethylamino)ocytl ester HCl (TMB-8) (100 microM), a known blocker of intracellular Ca2+ mobilization, did not change the resting pHi and [Ca2+]i in normal cells or cells acidified or alkalinized. This observation, taken together with data from cells incubated in the absence of external Ca2+, suggests intracellular Ca2+ pools are not involved in changes in [Ca2+]i that result from a modification of pHi. Resting pHi and [Ca2+]i in cells treated with either 8-bromo-dibutyryl cAMP (1 mM) or forskolin (150 microM) are not changed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Meso-2,3-dimercaptosuccinic acid induces calcium transients in cultured rhesus monkey kidney cells

The maintenance of intracellular Ca2+ homeostasis is critical to many cellular functions that rely on the calcium ion as a messenger. While attempting to characterize the effects of lead on intracellular calcium levels ([Ca2+]i) in LLC-MK2 Rhesus Monkey kidney cells, we observed that treatment with the metal chelating drug, meso-2,3-dimer-captosuccinic acid (DMSA) evoked transient increases in [Ca2+]i. Changes in [Ca2+]i were monitored using the Ca2+ indicator dye Fura-2 and a dual wavelength fluorescence imaging system. In the presence of 2 mM extracellular Ca2+, DMSA treatment caused a concentration-dependent (15-500 microM) transient increase in [Ca2+]i returning to baseline levels within 30-60 s. Pharmacologic concentrations of DMSA (30 microM) stimulated a three-fold increase in [Ca2+]i, which was spatiotemporally comparable to Ca2+ transients induced by other calcium agonists. Depletion of inositol trisphosphate (IP3)-sensitive [Ca2+]i stores with the smooth endoplasmic reticulum calcium-ATPase (SERCA) inhibitor thapsigargin did not prevent DMSA-elicited increases in [Ca2+]i, suggesting that Ca2+ mobilized by DMSA was either extracellular or from an non-IP3 releasable Ca2+ pool. Treatment with glutathione, cysteine, or 2-mercaptoethanol caused similar but not identical calcium transients. Adenosine-5'-trisphosphate (ATP) also elicited transient increases in [Ca2+]i similar to those of DMSA. No transient increases in [Ca2+]i were elicited by DMSA or ATP in the absence of extracellular calcium. These data indicate that DMSA and other sulfhydryl compounds trigger an influx of extracellular calcium, suggesting a previously unobserved and unanticipated interaction between DMSA and the Ca2+ messenger system.     

Glutamate-induced increase in intracellular Ca2+ concentration in isolated hippocampal neurones.

A system for real-time quantitative monitoring of intracellular free calcium ion concentration ([Ca2+]i) on a single cell basis was developed by the combination of a fluorescent Ca2+ indicator fura-2, a fluorescence microscope, a video-camera and photometrical devices. It was applied to rat individual hippocampal neurones under culture for detection of L-glutamate-induced alterations in the [Ca2+]i level. L-Glutamate (0.01-100 microM) induced a dose-dependent elevation of the [Ca2+]i. The [Ca2+]i in the rat hippocampal neurone was found to be around 30 nM in the resting state, and was increased up to 500 nM by the application of 100 microM L-glutamate. N-methyl-D-aspartate, kainate and quisqualate in a concentration of 10 microM also increased the [Ca2+]i level in the same single neurone, but their efficacy varied between individual cells. The L-glutamate-induced [Ca2+]i elevation was abolished after removal of extracellular Ca2+ and was much reduced by Mg2+ (3 mM). The increase was, however, still observed in a Na+-free medium. The L-glutamate-induced [Ca2+]i elevation was not affected substantially after treatment with nitrendipine (10 microM) which blocked the increase in [Ca2+]i induced by an isotonic high KCl-medium (50 mM). The present results suggest that the L-glutamate-induced [Ca2+]i elevation in the hippocampal neurone is due to an influx of Ca2+ through both L-glutamate receptor-coupled and voltage-sensitive ionic channels.     

Inhibitory action of dilazep on histamine-stimulated cytosolic Ca2+ increase in cultured human endothelial cells.

Using a fluorescent Ca(2+)-sensitive dye, fura-2, and photometric fluorescence microscopy, we measured changes in cytosolic Ca2+ concentration ([Ca2+]i) in cultured human endothelial cells and studied the effect of dilazep on [Ca2+]i elevation induced by histamine. Histamine (1 microM) caused a rapid transient peak in the average [Ca2+]i of a group of cells (approximately 10(2) cells), followed by a decrease to a sustained elevation. Dilazep as well as diltiazem (1.0 to 100 microM) concentration-dependently inhibited the latter sustained elevation, which was eliminated by removal of extracellular Ca2+, while the initial transient response was not changed by dilazep at concentrations up to 100 microM. The IC50 values of dilazep and diltiazem were 16 and 58 microM, respectively. The patterns of the [Ca2+]i elevation responses to histamine were variable among individual cells. Some single cells showed a transient peak and a sustained elevation as observed in a group of cells. Some single cells caused a repetitive spikelike elevation of [Ca2+]i. Dilazep lowered the sustained elevation to the resting level and in some single cells, changed the sustained elevation to the spikelike elevation. The frequency of the spikelike [Ca2+]i elevation was also decreased by dilazep. Decrease in extracellular [Ca2+] showed the same pattern of inhibitory actions as dilazep did. These results indicate that dilazep inhibits the extracellular Ca2+ influx in endothelial cells.     

Tamoxifen-induced [Ca2+]i rise and apoptosis in corneal epithelial cells

The effect of tamoxifen on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability has not been explored in corneal epithelial cells. This study examined whether tamoxifen altered [Ca2+]i and viability in SIRC corneal epithelial cells. Tamoxifen at concentrations > or = 1 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 6 microM. The Ca2+ signal was reduced substantially by removing extracellular Ca2+. Tamoxifen induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was insensitive to Ca2+ entry inhibitors and protein kinase C modulators. After pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), tamoxifen-induced [Ca2+]i rises were abolished; conversely, tamoxifen pretreatment abolished thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C with U73122 did not change the [Ca2+]i rises. At concentrations of 5-30 microM, tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 15 microM tamoxifen was not reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Apoptosis was induced by 5-30 microM tamoxifen. Tamoxifen (30 microM did not induce production of reactive oxygen species (ROS). Collectively, in SIRC cells, tamoxifen induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via unknown pathways. Tamoxifen-caused cytotoxicity was partly mediated by a Ca2+-independent apoptotic pathway.     

Mechanisms mediating the vasorelaxing action of eugenol, a pungent oil, on rabbit arterial tissue

The inhibitory actions of eugenol on intracellular Ca2+ concentration ([Ca2+]i) and the contractions induced by excess extracellular K+ concentration ([K+]o) in rabbit thoracic aorta were investigated. Application of excess [K+]o solution (30-90 mM) produced contraction and increased the intensity of the Ca2+ fluorescence signal. Pretreatment with eugenol (> or =0.1 mM) reduced both the amplitude of contraction and the intensity of the Ca2+ fluorescence signal, but the contraction was more strongly affected than the [Ca2+]i. Application of eugenol (0.3 mM) to tissue precontracted by 90 mM [K+]o solution (immediately after the removal of the 90 mM [K+]o solution) slowed the decay of the [Ca2+]i signal, but it did not change the rate of relaxation. Carbonyl cyanide m-chlorophenylhydrozone (10 microM), a mitochondrial metabolic inhibitor, produced a reduction in tension despite a slight increase in [Ca2+]i when applied to muscle precontracted by 90 mM [K+]o solution. These results indicate that eugenol relaxes the rabbit thoracic aorta while suppressing the Ca2+-sensitivity and both the uptake and extrusion mechanisms for Ca2+. To judge from the similarities between its actions and those of metabolic inhibitors, eugenol may produce its actions at least partly through metabolic inhibition.     

Mechanisms of AM404-induced [Ca(2+)](i) rise and death in human osteosarcoma cells

The effect of N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca2+ levels ([Ca2+]i) and viability was studied in human MG63 osteosarcoma cells using the fluorescent dyes fura-2 and WST-1, respectively. AM404 at concentrations > or = 5 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 60 microM. The Ca2+ signal was reduced partly by removing extracellular Ca2+. AM404 induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was sensitive to La3+, Ni2+, nifedipine and verapamil. In Ca2+-free medium, after pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), AM404-induced [Ca2+]i rise was abolished; and conversely, AM404 pretreatment totally inhibited thapsigargin-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not change AM404-induced [Ca2+]i rise. At concentrations between 10 and 200 microM, AM404 killed cells in a concentration-dependent manner presumably by inducing apoptotic cell death. The cytotoxic effect of 50 microM AM404 was partly reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Collectively, in MG63 cells, AM404 induced [Ca2+]i rise by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via L-type Ca2+ channels. AM404 caused cytotoxicity which was possibly mediated by apoptosis.     

Effect of carvedilol on Ca2+ movement and cytotoxicity in human MG63 osteosarcoma cells

Carvedilol is a useful cardiovascular drug for treating heart failure, however, the in vitro effect on many cell types is unclear. In human MG63 osteosarcoma cells, the effect of carvedilol on intracellular Ca2+ concentrations ([Ca2+]i) and cytotoxicity was explored by using fura-2 and tetrazolium, respectively. Carvedilol at concentrations greater than 1 microM caused a rapid rise in [Ca2+]i in a concentration-dependent manner (EC50=15 microM). Carvedilol-induced [Ca2+]i rise was reduced by 60% by removal of extracellular Ca2+. Carvedilol-induced Mn2+-associated quench of intracellular fura-2 fluorescence also suggests that carvedilol induced extracellular Ca2+ influx. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of carvedilol on [Ca2+]i was inhibited by 50%. Conversely, pretreatment with carvedilol to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+. U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca2+ mobilizer)-induced, but not carvedilol-induced, [Ca2+]i rise. Pretreatment with phorbol 12-myristate 13-acetate and forskolin to activate protein kinase C and adenylate cyclase, respectively, did not alter carvedilol-induced [Ca2+]i rise. Separately, overnight treatment with 0.1-30 microM carvedilol inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human MG63 osteosarcoma cells, carvedilol increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum and other stores via a phospholipase C-independent manner. Carvedilol may be cytotoxic to osteoblasts.     

Appearance of highly sensitive cells to L-type Ca antagonist in cultured cerebellar granule cells.

We studied intracellular Ca2+ ([Ca2+]i) kinetics within single cells using and image-analyzing system. We prepared a culture dish with a bottom made of quartz (non-fluorescent glass) for reducing the background fluorescence during [Ca2+]i measurement. Furthermore, we changed the concentration of the poly-L-lysine solution which had been used to make cells adhere to the dish bottom from the conventional 5 micrograms/ml to 15 micrograms/ml. This modification improved cell viability, prevented colony formation, and therefore, provided favorable cell samples for detection within single cells. Then, we investigated 25 mM KCl-evoked changes in the [Ca2+]i level of rat cerebellar granule cells. We found that sensitivity to KCl was high in some cells but low in others. In a study using the highly sensitive cells, nicardipine, nimodipine and nifedipine were active even at nanomolar or picomolar levels, although their affinities to the L-type Ca ion channel have been reported to act only at micromolar levels.     

Effect of the antidepressant maprotiline on Ca2+ movement and proliferation in human prostate cancer cells

1. The effect of maprotiline, an antidepressant, on human prostate cells is unclear. In the present study, the effect of maprotiline on [Ca2+]i and growth in PC3 human prostate cancer cells was measured using the fluorescent dyes fura-2 and tetrazolium, respectively. 2. Maprotiline caused a rapid, concentration-dependent increase in [Ca2+]i (EC50 = 200 micromol/L). The maprotiline-induced [Ca2+]i increase was reduced by removal of extracellular Ca2+ or pretreatment with nicardipine. 3. The maprotiline-induced Mn2+ influx-associated fura-2 fluorescence quench directly suggests that maprotiline caused Ca2+ influx. 4. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca2+]i increase, after which the effects of maprotiline of increasing [Ca2+]i were abolished. In addition, pretreatment with maprotiline reduced a major portion of the thapsigargin-induced increase in [Ca2+]i. 5. U73122, an inhibitor of phospholipase C, abolished the ATP (but not maprotiline)-induced increase in [Ca2+]i. 6. Overnight incubation with 1-10 micromol/L maprotiline did not alter cell proliferation, although incubation with 30-50 micromol/L maprotiline decreased cell proliferation. 7, These findings suggest that maprotiline rapidly increases [Ca2+]i in human prostate cancer cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release and that it may modulate cell proliferation in a concentration-dependent manner.     

地塞米松对新生小鼠海马和培养的皮层神经胶质中敏感细胞内钙浓度的影响

目的：研究地塞米松（Ｄｅｘ）对神经元和胶质细胞内钙浓度（Ｃａ＾２＋）ｉ的影响,方法：Ｆｕｒａ２－ＡＭ负载小鼠海马细胞（ＮＭＨＣ）和培养的胶质细胞（ＣＣＮ）,单细胞内（Ｃａ＾２＋）ｉ由ＡＲ－ＣＭ－ＭＩＣ检测系统测定。结果：Ｄｅｘ使多数ＮＭＨＣ（Ｃａ＾２＋）ｉ浓度依赖地迅速升高,９６个ＮＭＨＣ中公１０％出现（Ｃａ＾２＋）ｉ降低,（Ｃａ＾２＋）ｉ,升高被无镁细胞外液阻滞,被氯化镧逆转,但不受氯化锂影响,     

Amiodarone-mediated increase in intracellular free Ca2+ associated with cellular injury to human pulmonary artery endothelial cells

The cardiac antidysrrhythmic drug amiodarone can give rise to potentially fatal pulmonary toxicity in large numbers of patients. The effect of amiodarone on Ca2+ homeostasis and cell injury has been studied using human pulmonary artery endothelial (HPAE) cells in vitro. Amiodarone produced a concentration-dependent increase in intracellular free Ca2+ concentration ( [Ca2+]i) to micromolar levels that are similar to those seen with physiological stimuli that increase [Ca2+]i. Unlike physiological stimuli, the rise in [Ca2+]i produced by amiodarone developed slowly and was maintained over at least 30 min. Omitting Ca2+ from the external medium reversibly prevented the amiodarone-induced rise in [Ca2+]i. Amiodarone treatment increased the apparent first order rate constants for 45Ca2+ influx and efflux in intact HPAE cells. 45Ca2+ accumulation into the endoplasmic reticulum of saponin-permeabilized HPAE cells was decreased by amiodarone treatment. The release of 45Ca2+ from the endoplasmic reticulum stores by the putative intracellular second messengers inositol-1,4,5-trisphosphate, arachidonic acid, and Ca2+ was blocked by amiodarone treatment. The changes in Ca2+ homeostasis coincide with an increase in [3H]deoxyglucose release as a measure of early cell injury by amiodarone. It is concluded that amiodarone can produce an increase in [Ca2+]i by an action on the plasma membrane that allows the influx of external Ca2+. This increase in [Ca2+]i, together with other changes in Ca2+ homeostasis, may be responsible for the early cell injury associated with amiodarone toxicity.     

Adrenergic nerves compensate for a decline in calcium buffering during ageing

1. The ubiquitous involvement of intracellular calcium ([Ca2+]i) in multiple neuronal pathways has led investigators to suggest that dysfunction of calcium homeostasis may be the primary mediator of age-related neuronal degeneration. Recently, it was shown that sympathetic neurones from superior cervical ganglion (SCG) of aged rats demonstrate decreased sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) function and that aged neurones are more dependent upon mitochondria to control K+-evoked [Ca2+]i transients. 2. Therefore, in the present study we investigated age-related changes in ATP-dependent calcium pumps of plasma membrane Ca2+-ATPase (PMCA) and SERCA in acutely dissociated SCG cells from Fischer-344 rats aged 6 and 20 months. To distinguish between PMCA and SERCA pump activity, we applied the Ca2+-ATPase blocker vanadate and measured rates of recovery of K+-evoked [Ca2+]i transients by fura-2 microfluorometry. 3. Young SCG cells showed a biphasic response to vanadate over the vanadate concentration range (0.01-100 microM); however, old SCG cells showed only a single response over the same concentration range. Additionally, old SCG cells showed a greater sensitivity to Ca2+-ATPase blockade by vanadate. 4. The contribution of mitochondrial calcium uptake to regulate [Ca2+]i was also investigated. To measure the impact of mitochondrial calcium uptake, PMCAs and SERCAs were blocked with vanadate (100 microM) and extracellular sodium was replaced with tetraethylammonium (TEA) to block Na+/Ca2+-exchange. Treated SCG cells showed a decline of 50% in rate of recovery of [Ca2+]i in both 6- and 20-month-old cells; however, this effect did not vary with age. 5. These data suggest that there is an age-related decline in function of SERCAs, with an increased reliance on PMCAs to control high K+-evoked [Ca2+]i transients. In addition, there appears to be no age-related change in the capacity of the mitochondria to restore [Ca2+]i transients to basal levels.     

Triphenyltin impairs a protein kinase A (PKA)-dependent increase of cytosolic Na+ and Ca2+ and PKA-independent increase of cytosolic Ca2+ associated with insulin secretion in hamster pancreatic beta-cells

Oral administration of triphenyltin chloride (TPT) (60 mg/kg body weight) inhibits the insulin secretion by decreasing the cytoplasmic Ca2+ concentration ([Ca2+]i) induced by glucose-dependent insulinotropic polypeptide (GIP) in pancreatic beta-cells of the hamster. To test the possibility that the abnormal level of [Ca2+]i induced by TPT administration could be due to a defect in the cAMP-dependent cytoplasmic Na+ concentration ([Na+]i) in the beta-cells, we investigated the effects of TPT administration on the changes of [Na+]i induced by GIP, glucagon-like peptide-1 (GLP-1), or forskolin, an activator of adenylyl cyclase, and on the changes of [Na+]i or [Ca2+]i induced by 6-Bnz-cAMP, an activator of protein kinase A (PKA), and 8-pCPT-2'-O-Me-cAMP, an activator of Epac. The [Na+]i and [Ca2+]i were measured in islet cells loaded with sodium-binding benzofuran isophthalate (SBFI) and fura-2, respectively. In the presence of 135 mM Na+, TPT administration significantly reduced the rise in [Na+]i by 10 nM GLP-1, 10 microM forskolin, and 50 microM 6-Bnz-cAMP, but had not effect in a Na+-free medium. In the presence of 135 mM Na+, TPT administration also reduced the rise in [Ca2+]i by 8-pCPT-2'-O-Me-cAMP plus10 microM H-89, a inhibitor of PKA, and 6-Bnz-cAMP. Moreover, TPT administration significantly reduced the insulin secretion by 2 mM db-cAMP, GLP-1, GIP, and 8-pCPT-2'-O-Me-cAMP with and without H-89, and that by 6-Bnz-cAMP and forskolin. Our study suggested that TPT has inhibitory effects on the cellular Ca2+ response due to a reduced Na+ permeability through PKA-dependent mechanisms in hamster islet cells. Also TPT has the reduction of [Ca2+]i related to Na+-dependent insulin secretion after an activation of Epac.     

Muscarinic receptor regulation of cytoplasmic Ca2+ concentrations in human SK-N-SH neuroblastoma cells: Ca2+ requirements for phospholipase C activation

The relationship between muscarinic receptor-mediated inositol lipid hydrolysis and the generation of Ca2+ signals has been examined in human SK-N-SH neuroblastoma cells. The resting cytoplasmic calcium concentration [( Ca2+]i) as determined by fura-2 fluorescence measurements was 59 +/- 2 nM. Upon the addition of oxotremorine-M, there was a 4-fold increase in [Ca2+]i (293 +/- 18 nM), with half-maximal stimulation obtained at an agonist concentration of 8 microM, a value similar to that previously observed for the enhancement of phosphoinositide hydrolysis. Addition of partial muscarinic agonists for phosphoinositide turnover (bethanechol, oxo-2, and arecoline) elicited correspondingly smaller increases in [Ca2+]i than did oxotremorine-M. Inclusion of EGTA lowered the basal [Ca2+]i within 2 min and markedly reduced (greater than 60%) the magnitude of the agonist-induced rise in [Ca2+]i. Addition of muscarinic agonists to SK-N-SH cells that had been prelabeled with [3H]inositol led to the rapid (5-15 sec) release of inositol mono-, bis-, and triphosphates. When assayed under conditions similar to those employed for the fluorescence measurements, EGTA also inhibited both the basal and oxotremorine-M-stimulated release of inositol phosphates by 45-61%. Conversely, ionomycin both elevated [Ca2+]i and stimulated the release of inositol phosphates. The addition of Ca2+ (10 nM-2 microM) to digitonin-permeabilized cells directly stimulated the release of labeled inositol mono-, bis-, and trisphosphates by 3-4-fold with a half-maximal effect (EC50) observed at 145 nM free Ca2+ (Ca2+f). A further (6-fold) calcium-dependent increase in inositol phosphate release was obtained by inclusion of either guanosine-5-O-(3-thio)-trisphosphate (GTP gamma S) or oxotremorine-M. In the combined presence of agonist and GTP gamma S, a synergistic release of all three inositol phosphates occurred, with half-maximal stimulation observed at 35-40 nM Ca2+f, a value similar to the [Ca2+]i in quiescent cells. These results indicate (i) that the magnitude of the initial rise in [Ca2+]i is directly related to the production of phosphoinositide-derived second messenger molecules and (ii) that the phospholipase C-mediated breakdown of inositol lipids in SK-N-SH cells is particularly sensitive to regulation by physiologically relevant Ca2+ concentrations. It is concluded that, in SK-N-SH cells, either an elevation above or reduction below basal [Ca2+]i can modulate the extent of hydrolysis of inositol lipids and the subsequent generation of calcium signals.     

Mg2+ and caffeine-induced intracellular Ca2+ release in human vascular endothelial cells.

Interaction of ionized magnesium ([Mg2+]o) and caffeine in regulation of intracellular free calcium concentration ([Ca2+]i) in human aortic endothelial cells was studied using fura-2 and digital imaging microscopy. In 1.2 mM [Mg2+]o, basal [Ca2+]i was 73.7 +/- 22.4 nM, with a heterogeneous distribution within the cells. No significant changes of basal [Ca2+]i were found either when cells were treated with 10 mM caffeine or when [Mg2+]o was lowered from 1.2 mM to 0.3 mM. However, a combined superfusion of the cells with 0.3 mM [Mg2+]o and 10 mM caffeine resulted in a significant elevation of [Ca2+]i to 382.8 +/- 57.1 nM, probably by release of Ca2+ from internal stores, which was attenuated by NiCl2 (1 mM). These results suggest that a Ca(2+)-induced Ca2+ release mechanism is involved in regulation of [Ca2+]i in endothelial cells, which may be either regulated or modulated by Mg2+.     

Sensitivity of G-protein involved in endothelin-1-induced Ca2+ influx to pertussis toxin in porcine endothelial cells in situ.

1. We designed a new method to determine quantitatively the intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ, using front-surface fluorometry and fura-2-loaded porcine aortic valvular strips. Using this method, we investigated the characteristics of the G-protein involved in endothelin-1 (ET-1)-induced changes in [Ca2+]i of endothelial cells in situ. 2. Endothelial cells were identified by specific uptake of acetylated-low density lipoprotein labelled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL). Double staining with DiI-Ac-LDL and fura-2 showed that the valvular strip was covered with a monolayer of endothelial cells and that the cellular component which contributed to the fura-2 fluorescence, [Ca2+]i signal, was exclusively endothelial cells. 3. ET-1 (10(-7) M) induced an elevation of [Ca2+]i consisting of two components: the first was a rapid and transient elevation to reach a peak, followed by a second, sustained elevation (the second phase). The first phase was composed of extracellular Ca(2+)-independent and -dependent components, while the second phase was exclusively extracellular Ca(2+)-dependent. The extracellular Ca(2+)-independent component of the first phase was due to the release of Ca2+ from intracellular storage sites. The second phase and part of the first phase of [Ca2+]i elevation were attributed to the influx of extracellular Ca2+. The Ca2+ influx component was completely inhibited by 10(-3) M Ni2+ but was not affected by 10(-5) M diltiazem.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual excitatory and inhibitory effects of opioids on intracellular calcium in neuroblastoma x glioma hybrid NG108-15 cells.

The intracellular free calcium concentration ([Ca2+]i) was measured in single NG108-15 cells using indo-1-based microfluorimetry. In cells differentiated for 6-14 days in serum-free, forskolin (5 microM)-supplemented medium, application of micromolar concentrations of [D-Ala2,D-Leu5]-enkephalin (DADLE) inhibited Ca2+ influx mediated by voltage-gated Ca2+ channels. DADLE, at concentrations ranging from 1 nM to 1 microM, also produced rapid transient increases in [Ca2+]i (EC50 = 10 nM). The [Ca2+]i increases elicited by DADLE did not correlate with the inhibitory effects of the peptide. DADLE-induced [Ca2+]i increases were blocked by naloxone. In single cells, sequential application of selective opioid agonists (30 nM) evoked responses of the rank order DADLE = [D-Pen2,D-Pen5]-enkephalin > (trans)-(+-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl) benzeneacetamide > [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, consistent with activation of a delta-opioid receptor. The response was completely blocked by removal of extracellular Ca2+ or application of 1 microM nitrendipine, indicating that the increase in [Ca2+]i results from Ca2+ influx via dihydropyridine-sensitive, voltage-gated Ca2+ channels. Substitution of N-methyl-D-glucamine for extracellular Na+ or application of 1 microM tetrodotoxin greatly reduced, and in some cases blocked, the DADLE-induced [Ca2+]i increase, consistent with amplification of the response by voltage-gated Na+ channels. The [Ca2+]i increase was mimicked by both dibutyryl-cAMP and phorbol 12,13-dibutyrate. These findings are consistent with a delta-opioid-induced depolarization, possibly mediated by a second messenger, that subsequently recruits voltage-sensitive Ca2+ channels. In contrast to differentiated cells, undifferentiated cells responded to DADLE with a modest [Ca2+]i increase that was not sensitive to nitrendipine. In these cells, activation of the same second messenger system may elevate [Ca2+]i by mobilization from intracellular stores rather than influx. In addition to previously described inhibitory coupling to adenylyl cyclase and Ca2+ channels in NG108-15 cells, these results suggest that a novel, excitatory, effector system may also couple to opioid receptors.