Lead inhibits meso-2,3-dimercaptosuccinic acid induced calcium transients in cultured rhesus monkey kidney cells.

Previously we have shown that meso-2,3-dimercaptosuccinic acid (DMSA, 15-500 microM) elicits concentration-dependent increases in intracellular calcium levels ([Ca2+]i) in untreated rhesus monkey kidney cells (LLC-MK2) (Pokorski et al., 1997, unpublished results). Little is known about the restorative effects of the chelating agent 2,3-dimercaptosuccinic acid on intracellular calcium homeostasis in the presence of lead. Lead interacts at numerous sites in Ca2+ homeostasis and may mimic Ca2+ to interfere with Ca2+-mediated intracellular signaling. To examine the effects of lead on [Ca2+]i and DMSA-induced calcium transients, LLC-MK2 were plated on 35 mm coverslip dishes (10(4) cells/dish) and pre-treated with non-cytotoxic concentrations of lead (0-100 microM) for 24 h. Cells were washed, loaded with the calcium-sensitive probe Fura-2/AM, rinsed again, and examined in loading buffer in the absence of any additional lead. Intracellular calcium was measured using a dual-wavelength calcium imaging system. Basal [Ca2+]i levels did not change between Pb-exposed (0-50 microM, 24 h) and non-lead exposed cells. In cells treated with > or = 10 microM lead for 24 h, the ability of DMSA to elicit a calcium response was blocked. These results provide evidence that pre-exposure to lead blocks the entry of extracellular calcium into LLC-MK2 cells when stimulated by specific calcium mobilizing agents.     

The role of caffeine-sensitive calcium stores in the regulation of the intracellular free calcium concentration in rat sympathetic neurons in vitro

Intracellular Ca2+ stores were studied in sympathetic neurons grown in primary culture from the superior cervical ganglion of the rat. The [Ca2+]i was measured in single cells using the fluorescent Ca2+ indicator fura-2 and a sensitive microfluorimeter. Superfusion of the cells with 10 mM caffeine elicited a rapid and transient increase in [Ca2+]i in the absence of extracellular Ca2+, indicating the presence of a caffeine-sensitive intracellular Ca2+ storage site. After depletion of the store by mobilization of Ca2+ with caffeine, it could be refilled by elevating [Ca2+]i, allowing multiple caffeine-induced [Ca2+]i transients to be elicited from a single neuron. Ryanodine (1 microM), an alkaloid that promotes Ca2+ release from the sarcoplasmic reticulum, was an effective inhibitor of the caffeine-induced [Ca2+]i transients in sympathetic neurons. Exposure to ryanodine in the presence of caffeine was required to produce a subsequent inhibition of the caffeine-induced response, suggesting a "use-dependent" inhibition that may result from depletion of the Ca2+ stores. In contrast, dantrolene Na (10 microM), an agent known to interfere with Ca2+ release from the sarcoplasmic reticulum, also blocked the caffeine-induced [Ca2+]i transients, but in a time-dependent rather than a use-dependent manner. Electrophysiological measurements using the whole cell version of the patch-clamp technique were made simultaneously with [Ca2+]i microfluorimetric recordings. The magnitude of the [Ca2+]i transients elicited by step depolarizations closely paralleled the magnitude of Ca2+ influx via voltage-sensitive Ca2+ channels, regardless of whether the magnitude of the Ca2+ current was modified by varying the test pulse duration or potential. The relationship between the magnitude of Ca2+ influx and the resulting increase in [Ca2+]i saturated at large Ca2+ influxes resulting from long depolarizations, consistent with the activation of a large capacity, low affinity [Ca2+]i buffering mechanism. Caffeine (10 mM) and ryanodine (10 microM), applied singly or together, produced a small and variable decrease in the [Ca2+]i transient resulting from cell depolarization using the whole-cell patch-clamp technique. We conclude that mammalian sympathetic neurons possess intracellular Ca2+ stores with pharmacological characteristics that closely resemble those found in muscle but that these are relatively small and produce little amplification of [Ca2+]i transients resulting from Ca2+ influx through voltage-sensitive Ca2+ channels.     

The effects of ATP and alpha,beta-methylene-ATP on cytosolic Ca2+ level and force in rat isolated aorta.

1. The effects of a non-selective P2-receptor agonist ATP and a selective P2x-receptor agonist alpha,beta-methylene-ATP on intracellular free Ca2+ level ([Ca2+]i) and force were examined in rat isolated aorta without endothelium. 2. Both ATP (1-1000 microM) and alpha,beta-methylene-ATP (0.1-100 microM) induced transient increase followed by small sustained increase in [Ca2+]i in a concentration-dependent manner. Compared with the force induced by a high concentration of KCl, the force induced by alpha,beta-methylene-ATP was smaller and that induced by ATP was much smaller at a given [Ca2+]i. 3. An L-type Ca2+ channel blocker, verapamil (10 microM), completely inhibited the high K(+)-stimulated [Ca2+]i and force. Verapamil partially inhibited the transient and sustained increases in [Ca2+]i induced by 10 microM alpha,beta-methylene-ATP and the sustained increase but not the transient increase induced by 1 mM ATP. 4. In the absence of extracellular Ca2+ (with 0.5 mM EGTA) 1 mM ATP caused transient increase in [Ca2+]i while 10 microM alpha,beta-methylene-ATP was ineffective 5. ATP, but not alpha,beta-methylene-ATP, increased the tissue adenosine 3':5'-cyclic monophosphate (cyclic AMP) level. 6. These data suggest that ATP and alpha,beta-methylene-ATP increase [Ca2+]i by an activation of both L-type and non-L-type Ca2+ channels. In addition, ATP, but not alpha,beta-methylene-ATP, increases [Ca2+]i by a release of Ca2+ from an intracellular Ca2+ store. Possible reasons are discussed as to why the increase in [Ca2+]i due to ATP and alpha,beta-methylene-ATP resulted in only a small contraction.     

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.     

Dissociation of P2 purinoceptor-mediated increase in intracellular Ca2+ level from myosin light chain phosphorylation and contraction in rat aorta.

1. The effects of P2 agonists, adenosine-5'-triphosphate (ATP), alpha, beta-methylene-adenosine-5'-triphosphate (alpha, beta-me-ATP) and adenosine 5-O-(3-thiotriphosphate) (ATP gamma S), on the intracellular free Ca2+ level ([Ca2+]i), myosin light chain (MLC) phosphorylation and force of contraction were examined in vascular smooth muscle of rat aorta. 2. ATP (0.1 microM-1 mM), alpha, beta-me-ATP (0.1-100 microM) and ATP gamma S (1-100 microM) induced transient increases followed by sustained increase in [Ca2+]i. The effects of these agonists were concentration-dependent. Compared with the effects of a high concentration of KCl (17.5-72.4 mM), the contractions induced by these P2 purinoceptor agonists were smaller at a given [Ca2+]i. 3. In the absence of extracellular Ca2+ (with 0.5 mM EGTA), ATP gamma S (10 microM) induced large transient increase in [Ca2+]i with only small contraction in Ca(2+)-free solution. In contrast, alpha, beta-me-ATP (10 microM) induced only a very small increase in [Ca2+]i and contraction. 4. ATP (1 mM), alpha, beta-me-ATP (10 microM) and ATP gamma S (10 microM), added during stimulation with 0.1 microM noradrenaline, induced additional and transient increases in [Ca2+]i which were also not associated with contraction. 5. High K+ (72.4 mM) increased MLC phosphorylation with a similar time course to that of the increase in [Ca2+]i (peak phosphorylation was 56% when [Ca2+]i increased to 100%). In contrast, the time course of the increase in MLC phosphorylation due to ATP (1 mM) did not coincide with that of the large increases in [Ca2+]i; MLC phosphorylation increased to only 31% when [Ca2+]i increased to 163%. The MLC phosphorylation due to alpha, beta-me-ATP (10 microM) and ATP gamma S (10 microM), measured at peak [Ca2+]i, were only 19% and 14%, respectively, irrespective of a large increase in [Ca2+]i (138% and 188%, respectively). 6. The absence of a clear relationship between P2-purinoceptor-mediated increase in [Ca2+]i (either by Ca2+ influx or Ca2+ release) and MLC phosphorylation or force generation appears to imply that elevation in [Ca2+]i does not contribute to these responses.     

Characteristics of nucleotide receptors that cause elevation of cytoplasmic calcium in immortalized rat brain endothelial cells (RBE4) and in primary cultures.

1. A dual-wavelength microfluorimetric method using Fura-2 as calcium indicator was applied to cells from an immortalized cell line of rat brain microvascular endothelial cells (RBE4), and to primary cultured rat brain endothelial cells. 2. In RBE4 cells, a brief (20 s) pulse of extracellular ATP (100 microM) induced a transient increase in the cytoplasmic calcium level ([Ca2+]i). Control responses to 100 microM ATP consisted of a ratio increase of 0.64 +/- 0.03 (mean +/- s.e., n = 51). Responses were seen at a concentration of 2.5 microM and were maximal at 100-1000 microM. When extracellular calcium was chelated with EGTA, the transient increase in [Ca2+]i was not affected. The results are consistent with Ca2+ mobilization from intracellular stores. 3. The purinoceptor involved belongs to the P2 subtype, since the agonist potency order among the adenine nucleotides was ATP > ADP > AMP. Moreover, the increase in [Ca2+]i evoked by ATP was partially inhibited by the P2 antagonist, suramin but was not affected by 8-phenyltheophylline, a P1-purinoceptor antagonist. The strong desensitization observed with repeated applications of ATP is also typical of a P2 receptor. 4. 2-Methylthio-ATP (2meS-ATP 100 microM), a P2Y agonist, elevated [Ca2+]i in only 17% of the cells tested; however, 2meS-ATP was found to antagonize the effect of ATP in all cells tested. The increase in [Ca2+]i evoked by ATP was inhibited by 500 s application of the P2Y purinoceptor antagonist, Reactive Blue 2 at 10 microM, while 60 s application of 100 microM was ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid actions of calcitriol and its side chain analogues CB1093 and GS1500 on intracellular calcium levels in skeletal muscle cells: a comparative study.

1. The ability of synthetic analogues of the secosteroid hormone 1alpha,25-dihydroxy-vitamin-D3 [calcitriol, CT; 1,25(OH)2D3] to exert non-genomic (rapid) effects on target cells has been scarcely studied. To evaluate the pharmacological potential of the CT side-chain analogues CB1093 and GS1500, we compared their fast effects on intracellular calcium concentration ([Ca2+]i) in chick skeletal muscle cells with those elicited by the natural hormone. 2. Both analogues, similarly to CT, specifically induced rapid (30-60 s) and sustained rises in [Ca2+]i levels. CB1093 and GS1500 were more potent than the natural hormone at concentrations as low as 10(-13) M (4.5 fold stimulation) and 10(-12) M (2.5 fold), respectively, whereas higher concentrations (10(-9)- 10(-8) M) of CT were more effective than the analogues in elevating [Ca2+]i. Cyclic AMP was markedly increased by both analogues pointing for a role of this messenger in the fast actions of the synthetic compounds. 3. In Ca2+ free medium CT and analogues elicited a transient elevation in [Ca2+]i. The PLC inhibitors U73122 (2 microM) and neomycin (0.5 mM), as well as depletion of intracellular stores with thapsigargin (1 microM), completely prevented CB1093/GS1500-dependent changes in [Ca2+]i suggesting that, similarly to CT, these analogues mobilized Ca2+ from an IP3/thapsigargin-sensitive store. 4. The voltage-dependent calcium channel (VDCC) blocker nifedipine (2 microM) reduced by 50-60% the influx phase of the [Ca2+]i response to CB1093 and GS1500, indicating that VDCC contributed partially to Ca2+ entry. The Ca2+ readdition protocol suggested that analogue-dependent activation of a SOC entry pathway accounted, to the same extent as for CT, for the remaining non-VDCC mediated Ca2+ influx.     

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.     

P2X2 characteristics of the ATP receptor coupled to [Ca2+]i increases in cultured Purkinje neurons from neonatal rat cerebellum.

P2X receptors present in cerebellar Purkinje cells have been studied by recording ATP-elicited [Ca2+]i signals from immuno-identified (calbindin+) cells in culture using fura-2 microfluorescence. The [Ca2+]i increases evoked by ATP were mimicked by 2MeSATP but not by alpha, beta-meATP and other purinoceptor agonists. The selective P2X1 antagonist diinosine pentaphosphate failed to inhibit ATP-elicited [Ca2+]i transients, but suramin and PPADS rapidly and reversibly blocked the [Ca2+]i responses to ATP and 2MeSATP. The IC50 values for suramin and PPADS inhibition were 48.7 +/- 4.4 and 5.9 +/- 0.3 microM, respectively. Both antagonists blocked completely the signal elicited by ATP, revealing that there was not a separate antagonist-insensitive P2X receptor population in Purkinje cells. The effect of ATP was potentiated by Zn2+ and H+ ions. A one unit acidification from pH 7.4 to 6.4 enhanced by 172% the [Ca2+]i transient elicited by an intermediate concentration of ATP. Conversely, alkalinization of the medium to pH 8.4 reduced the ATP response by 88%. This combination of pharmacological and modulatory properties indicates that endogenous P2X receptors present in Purkinje neurons are formed by P2X2 subunits, rather than the more abundantly expressed P2X4 purinoceptor subunits.     

Extracellular ATP increases [Ca2+]i in primarily cultured pig coronary smooth muscle cells via a P2Y purinoceptor subtype

In primarily cultured pig coronary smooth muscle cells, extracellular adenosine triphosphate (ATP; 10(-9) to 10(-3) M) dose-dependently increases intracellular calcium ([Ca2+]i). The [Ca2+]i transients measured by fura-2 fluorescence consist of peak and plateau phases with [Ca2+]i values of 191.84 +/- 5.67 nM (n = 10) and 91.67 +/- 1.89 nM, respectively. In Ca(2+)-free solution, the peak phases persisted, but there was a loss of the plateau response, indicating an initial ATP-stimulated intracellular Ca2+ release and a subsequent transarcolemmal Ca2+ entry. Various agonists have been used to characterize the P2 purinoceptor subtype involved in the ATP-induced Ca2+ transients. The rank order of potency was uridine triphosphate (UTP) > ATP > 2-meSATP > beta,gamma-meATP = alpha,beta-meATP = adenosine = 0. To examine the refilling of ATP-sensitive stores, four repetitive 60-s ATP responses were produced throughout with a 5-min recovery period in between. Now the ATP peaks gradually declined in Ca(2+)-free solution, indicating the emptying of the stores. If, however, Ca2+ entry was allowed in the "refilling period" (i.e., between the ATP pulses), the Ca2+ peaks could be maintained or restored, respectively. The data suggest that the ATP-dependent [Ca2+]i transients may be mediated via a UTP > ATP-activated P2Y purinoceptor subtype, mediating both an intracellular Ca2+ release and a transarcolemmal Ca2+ influx. The refilling of Ca2+ stores may occur through the unstimulated membrane after agonist stimulation. A putative pathway may be a "capacitative" Ca2+ entry induced on depletion of intracellular Ca2+ stores.     

Inositol 1,4,5-trisphosphate generation and calcium mobilisation via activation of an atypical P2 receptor in the neuronal cell line, N1E-115.

1. Alterations in the levels of intracellular calcium ([Ca2+]i) and D-myo-inositol-1,4,5-trisphosphate (InsP3) were measured in the murine neuroblastoma cell line clone, N1E-115, by use of the calcium-sensitive dye, fura-2 and a radioreceptor assay, respectively. 2. Exposure of the cells to ATP (100 microM) elicited rapid and transient increases in [Ca2+]i and InsP3, with both responses reaching a maximum between 10-20 s after agonist addition. 3. Investigation of concentration-response data by use of various analogues of ATP suggests the presence of an extracellular receptor which fails to fit into the current classification of purinoceptors. 4. Cross-desensitization experiments suggest that the same receptor can also be activated by the structurally different pyrimidine base, UTP. 5. Application of the tumour-promoting agent, beta-phorbol-12,13 dibutyrate (PDBu) caused a reduction in the increases in both [Ca2+]i and InsP3, suggesting a role for protein kinase C in feedback inhibition of purinoceptor responses in this cell line. 6. In summary, we present the first evidence for the existence of an atypical purinoceptor on a cell line of CNS origin. This receptor is linked to stimulation of phosphoinositide turnover and subsequent mobilisation of intracellular calcium.     

Dual action of palmitoyl trifluoromethyl ketone (PACOCF3) on Ca2+ signaling: activation of extracellular Ca2+ influx and alteration of ATP- and bradykinin-induced Ca2+ responses in Madin Darby canine kidney cells

The effect of the phospholipase A2 inhibitor palmitoyl trifluoromethyl ketone (PACOCF3) on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was examined using fura-2 as the fluorescent Ca2+ indicator. At a concentration of 20 microM, PACOCF3 did not change basal cytosolic free calcium concentrations ([Ca2+]i), but at concentrations of 50-250 microM PACOCF3 induced an increase in [Ca2+]i by activating extracellular Ca2+ entry which was partly suppressed by 50 microM La3+. The effect of PACOCF3 was abolished by removal of extracellular Ca2+. PACOCF3 (10 microM) enhanced both the peak value and the area under the curve of the [Ca2+]i increase induced by 10 microM ATP and 1 microM bradykinin by potentiating extracellular Ca2+ influx without affecting internal Ca2+ release. Several other phospholipase A2 inhibitors had no effect on basal [Ca2+]i or agonist-induced [Ca2+]i increases. Collectively, the results suggest that PACOCF3 alters Ca2+ signaling in renal tubular cells in a manner independent of phospholipase A2 inhibition.     

Increases in intracellular calcium via activation of an endogenous P2-purinoceptor in cultured CHO-K1 cells.

1. Increases in intracellular calcium ([Ca2+]i) were measured in chinese hamster cultured ovary cells (clone, CHO-K1), by use of the fluorescent, calcium-sensitive dye, fura-2. 2. Addition of both ATP and UTP elicited rapid increases in [Ca2+]i due to mobilization from intracellular stores and calcium entry across the plasma membrane. 3. Omission of calcium from the extracellular medium and pre-incubation with the inorganic calcium channel blocker, nickel (Ni2+) prevented the calcium entry components of the responses. 4. Investigation of the concentration-response relationships of various analogues of ATP suggests the presence of a purinoceptor which cannot be characterized as P2X or P2Y. In addition, there appears to be a sub-population of P2Y-purinoceptors which do not cross-react with the 'nucleotide' receptor population. 5. Cross-desensitization and additivity experiments suggest that both ATP and UTP activate the same receptor. 6. Pre-incubation with the tumour-promoting agent, beta-phorbol-12,13 dibutyrate (PDBu), caused a reduction in the increases in [Ca2+]i, suggesting a role for protein kinase C in feedback inhibition of purinoceptor responses in this cell line. 7. In summary, we present evidence for the existence of an endogenous P2U-purinoceptor (or 'nucleotide receptor') which is linked to increases in [Ca2+]i in CHO-K1 cells.     

Dual effects of mastoparan on intracellular free Ca2+ concentrations in human astrocytoma cells.

1. The effect of mastoparan, a wasp venom toxin, on intracellular free Ca2+ concentration ([Ca2+]i) was examined in human astrocytoma cells. Mastoparan inhibited [Ca2+]i induced by carbachol (100 microM) in a concentration-dependent manner in the absence of extracellular Ca2+, consistent with our previous results showing that mastoparan inhibits phosphoinositide hydrolysis in human astrocytoma cells. 2. In contrast, mastoparan itself increased [Ca2+]i and augmented carbachol-induced increase in the [Ca2+]i in the presence of extracellular Ca2+, suggesting that mastoparan elicited Ca2+ influx from the extracellular medium. The increase appeared to be maximum at extracellular Ca2+ concentrations of 0.1-0.2 mM. The higher concentrations of extracellular Ca2+ depressed the influx. 3. Pertussis toxin did not affect mastoparan-induced inhibition of [Ca2+]i in the absence of extracellular Ca2+, consistent with the previous results that pertussis toxin did not affect mastoparan-induced inhibition of phosphoinositide hydrolysis. 4. Pertussis toxin augmented mastoparan-induced increase in [Ca2+]i in the presence of extracellular Ca2+, suggesting that pertussis toxin substrate(s) seems to be inhibitory for Ca2+ influx induced by mastoparan. 5. Verapamil, nifedipine and diltiazem (each 10 microM), L-type Ca2+ antagonists, did not affect mastoparan-induced Ca2+ influx. However, verapamil (10 microM) slightly inhibited the increase in [Ca2+]i induced by carbachol in the presence of mastoparan. 6. The results obtained in the present study indicate that mastoparan has two opposite effects on [Ca2+]i in human astrocytoma cells and possibly has at least two sites of action.     

Effect of riluzole on Ca2+ movement and cytotoxicity in Madin-Darby canine kidney cells

Riluzole is a drug used in the treatment of amyotrophic lateral sclerosis; however, its in vitro action is unclear. In this study, the effect of riluzole on intracellular Ca2+ concentration ([Ca2+]i) in Madin-Darby canine kidney (MDCK) cells was investigated using the Ca2+ -sensitive fluorescent dye, fura-2. Riluzole (100-500 microM) caused a rapid and sustained increase of [Ca2+]i in a concentration-dependent manner (EC50 = 150 microM). Some 40 and 50% of this [Ca2+]i increase was prevented by the removal of extracellular Ca2+ and the addition of La3+, respectively, but was unchanged by dihydropyridines, verapamil and diltiazem. In Ca2+ -free medium, thapsigargin - an inhibitor of the endoplasmic reticulum (ER) Caz+ -ATPase--caused a monophasic [Ca2+]i increase, after which the increasing effect of riluzole on [Ca2+]i was attenuated by 70%; in addition, pre-treatment with riluzole abolished thapsigargin-induced [Ca2+]i increases. U73122, an inhibitor of phospholipase C (PLC), abolished ATP (but not riluzole)-induced [Ca2+]i increases. At concentrations of 250 and 500 microM, riluzole killed 40 and 95% cells, respectively. The cytotoxic effect of riluzole (250 microM) was unaltered by pre-chelating cytosolic Ca2+ with BAPTA. Collectively, in MDCK cells, riluzole rapidly increased [Ca2+]i by stimulating extracellular Ca2+ influx via an La3+ -sensitive pathway and intracellular Ca2+ release from the ER via, as yet, unidentified mechanisms. Furthermore, riluzole caused Ca2+ -unrelated cytotoxicity in a concentration-dependent manner.     

Differential effect of diltiazem and TA-3090 on calcium homeostasis of neutrophils.

The effects of diltiazem and TA-3090, an 8-chloro analog of diltiazem, on cellular responses and calcium homeostasis of human neutrophils were investigated. TA-3090, at 10 to 20 microM, enhanced lysozyme release and superoxide generation induced in neutrophils by n-formyl-methionyl-leucyl-phenylalanine (FMLP). Higher concentrations of TA-3090 inhibited responses at IC50s between 70 and 85 microM. Diltiazem by comparison inhibited responses at an IC50 of about 200 microM. The two drugs had little or no effect on early signaling events: inositol 1,4,5-trisphosphate formation triggered by FMLP was not affected. Moreover, 500 microM TA-3090 or diltiazem did not significantly affect FMLP-triggered Ca2+ transients. (Cytoplasmic free Ca2+ levels ([Ca2+]i) were monitored in fura-2-loaded neutrophils.) Diltiazem alone caused a limited influx of extracellular Ca2+ which increased basal [Ca2+]i by twofold. Internal Ca2+ stores were not released. TA-3090, in contrast, induced a biphasic rise in [Ca2+]i--an initial mobilization of intracellular Ca2+ stores was followed after 10-15 min by a persistent influx of extracellular Ca2+ which increased [Ca2+]i to 1.3 +/- 0.7 (SD) microM. Complementary studies with semipermeabilized neutrophils showed that TA-3090 but not diltiazem directly released Ca2+ from intracellular stores. In TA-3090-treated cells, lactate dehydrogenase release was correlated with delayed influx of extracellular Ca2+. The chelation of extracellular Ca2+ by EGTA prevented LDH release. Present results show that TA-3090 and diltiazem initially blocked cell signaling at steps subsequent to phospholipase C activity. With TA-3090-treated cells, elevated [Ca2+]i ensuing from prolonged incubations likely activated inappropriate reactions leading to cell lysis and death.     

Effect of extracellular ATP on contraction,cytosolic calcium activity,membrane voltage and ion currents of rat mesangial cells in primary culture.

1. The effects of extracellular ATP on contraction, membrane voltage (Vm), ion currents and intracellular calcium activity [Ca2+]i were studied in rat mesangial cells (MC) in primary culture. 2. Addition of extracellular ATP (10(-5) and 10(-4) M) to MC led to a cell contraction which was independent of extracellular calcium. 3. Membrane voltage (Vm) and ion currents were measured with the nystatin patch clamp technique. ATP induced a concentration-dependent transient depolarization of Vm (ED50: 2 x 10(-6) M). During the transient depolarization ion currents were monitored simultaneously and showed an increase of the inward- and outward current. 4. In a buffer with a reduced extracellular chloride concentration (from 145 to 30 mM) ATP induced a depolarization augmented to -4 +/- 4 mV. 5. ATP-gamma-S and 2-methylthio-ATP depolarized Vm to the same extent as ATP, whereas alpha,beta-methylene-ATP (all 10(-5) M) had no effect on Vm. 6. The Ca2+ ionophore, A23187, depolarized Vm transiently from -51 +/- 2 to -28 +/- 4 mV and caused an increase of the inward current. 7. The intracellular calcium activity [Ca2+]i was measured with the fura-2 technique. ATP stimulated a concentration-dependent increase of [Ca2+]i (ED50: 5 x 10(-6) M). The increase of [Ca2+]i was biphasic with an initial peak followed by a sustained plateau. 8. The [Ca2+]i peak was still present in an extracellular Ca(2+)-free buffer, whereas the plateau was abolished. Verapamil (10(-4) M) did not inhibit the [Ca2+]i increase induced by ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Co-existence of P2Y-and PPADS-insensitive P2U-purinoceptors in endothelial cells from adrenal medulla.

1. We have studied the effects of purinoceptor stimulation on Ca2+ signals in bovine adrenomedullary endothelial cells. [Ca2+]i was determined with the fluorescent probe fura-2 both in population samples and in single, isolated, endothelial cells in primary culture and after subculturing. 2. In endothelial cells, maintained in culture for more than one passage, several purinoceptor agonists elicited clear [Ca2+]i transient peaks that remained in the absence of extracellular Ca2+. Adenosine 5''-triphosphate (ATP) and uridine 5''-triphosphate (UTP) were equipotently active, with EC50 values of 8.5 +/- 0.9 microM and 6.9 +/- 1.5 microM, respectively, whereas 2-methylthioadenosine 5''-triphosphate (2MeSATP), adenosine 5''-(alpha, beta-methylene)triphosphate (alpha, beta-MeATP) and adenosine(5'')tetraphospho(5'')adenosine (Ap4A) were basically inactive. Adenosine 5''-O-(2-thiodiphosphate) (ADP beta S) was a weak agonist. The apparent potency order was UTP = ATP > ADP beta S >> 2MeSATP > alpha, beta-MeATP. 3. Cross-desensitization experiments revealed that UTP or ATP, added sequentially at concentrations of maximal effect, could completely abolish the [Ca2+]i response to the second agonist. ADP beta S exerted only a partial desensitization of the response to maximal ATP, in accordance with its lower potency in raising [Ca2+]i. 4. The effect on [Ca2+]i of 100 microM ATP in subcultured cells was reduced by only 25% with 100 microM suramin pretreatment and was negligibly affected by exposure to 10 microM pyridoxalphosphate-6-azophenyl-2'', 4''-disulphonic acid (PPADS). The concentration-effect curve for ATP was not significantly affected by PPADS, but was displaced to the right by a factor of 6.5 by 100 microM suramin. 5. In primary cultures, clear [Ca2+]i responses were elicited by 2MeSATP. Suramin totally and selectively blocked 2MeSATP responses, whereas UTP-evoked [Ca2+]i transients were mainly unaffected by suramin or PPADS. Over 80% of cells tested showed responses to both 2MeSATP and UTP. The [Ca2+]i response to UTP was not desensitized in the presence of 2MeSATP. 6. ATP and UTP stimulated the release of preloaded [3H]-arachidonic acid ([3H]-AA), both in the presence and in the absence of extracellular Ca2+, by approximately 135% with respect to basal levels. Suramin and PPADS enhanced, rather than inhibited, the [3H]-AA releasing effect of ATP by 2.5 times. Suramin also potentiated the effect of the calcium ionophore A23187. 7. These results indicate that endothelial cells from adrenomedullary capillaries co-express both P2Y- and P2U-purinoceptors. P2Y-purinoceptors are lost in culture with the first passage of the cells. The P2U-purinoceptor subtype present in these cells is insensitive to PPADS and thus similar to that found in aortic endothelial cells.     

粉防己碱对培养乳牛基底动脉平滑肌细胞内游离钙浓度的影响

目的：研究粉防己碱对培养乳牛基底动脉平滑肌细胞游离钙浓度（[Ca^2 ]i)的影响。方法：利用AR－CM－MIC阳离子测定系统,采用Fura 2-AM为指示剂,测量单个细胞内[Ca^2 ]i。结果：粉防己碱10－100μmol/L对培养乳牛基底动脉平滑肌细胞静息[Ca^2 ]i无明显影响。在细胞外钙为1．3mmol/L,粉防己碱可浓度依赖性地抑制KC1引起[Ca^2 ]i的升高。咖啡因10mmol/L可诱导一次[Ca^2 ]i瞬间快速升高,随后自发回复到静息水平,粉防己碱10和30μmol/L对咖啡因诱导的[Ca^2 ]i瞬间升高没有作用,但高浓度（100μmol/L）粉防己碱抑制了[Ca^2 ]i瞬间升高。在细胞外钙为1.3mmol/L,苯肾上腺素10μmol/L可引起双相[Ca^2 ]i变化,包括快速升高相和持续升高相。在细胞外钙为零,苯肾上腺素仅引起[Ca^2 ]i的快速升高相。粉防己碱可浓度依赖性地抑制苯肾上腺素引起[Ca^2 ]i快速升高相。结论：在培养乳牛基底动脉平滑肌细胞,粉防己碱可能通过影响电压依赖性和苯肾上腺素受体介导的钙通道而抑制钙内流。高浓度粉防己碱也可能影响肌浆网钙释放或钙摄取。