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.     

Effects of purinoceptor agonists on cytosolic Ca2+ concentration in swine tracheal smooth muscle cells in culture.

1. The effects of various purinoceptor agonists on intracellular Ca2+ concentration ([Ca2+]i) in swine tracheal smooth muscle cells in primary culture were examined to investigate the subtype of purinoceptors in these cells. 2. ATP (1 microM to 1 mM) concentration-dependently increased [Ca2+]i which was measured by monitoring the fluorescence signal of fura2. 3. alpha, beta-Me ATP at concentrations higher than 10 microM increased [ca2+]i in the presence of extracellular Ca2+. Responses to the drug were 12 +/- 5 and 61 +/- 4% of responses to ATP (100 microM) at 100 microM and 1 mM, respectively (n = 7). The response to 100 microM ATP was inhibited by 62% in the presence of 1 mM alpha, beta-Me ATP (n = 8), though the drug at concentrations lower than that did not affect the response to ATP. 4. ATP increased [Ca2+]i in the absence of extracellular Ca2+. The response to ATP in this condition was 40% of that in the presence of extracellular Ca2+ (n = 8). 5. Neither cibacron blue 3GA (10 microM) (n = 8) nor suramin (10 and 100 microM) (n = 10) affected the response to ATP (1 microM to 100 microM). 6. The rank order of potency in the absence of extracellular Ca2+ was UTP > ATP > adenosine 5'-o-(3-thiotriphosphate) > > ADP = alpha, beta-methylene adenosine 5'-triphosphate > 2-(methylthio)-adenosine 5'-(tetrahydrogen triphosphate). 7. UTP (1 microM to 100 microM) concentration-dependently increased inositol 1,4,5-triphosphate (IP3) production. 8. These results suggest that the increase in [Ca2+]i induced by purinoceptor agonists is mediated mainly via a nucleotide receptor in swine tracheal smooth muscle cells in primary culture.     

Purinergic receptor regulation of signal transduction in NCB-20 cells.

In the present paper, P1 and P2 purinergic receptors and their control of signal transduction pathways were investigated in NCB-20 cells. ATP elicited an increase in [Ca2+]i. The purinergic receptor subtype involved was identified by comparing the actions of a range of nucleotides. UTP was the most potent agonist in elevating [Ca2+]i, with an EC50 value of 6.2 +/- 0.5 microM. UTP, ATP (EC50, 17.3 +/- 1.5 microM), adenosine-5'-O-(3-thio)triphosphate (23 +/- 3 microM), and ITP (55 +/- 4 microM) exerted similar maximal effects. Other nucleotides tested, including beta, gamma-methylene-ATP and 2-methylthio-ATP, which are considered prototypic agonists for P2x and P2y receptors, respectively, were ineffective; in general, modifications in the ribose-triphosphate chain and substitution on the 2-position of the purines reduced the efficacy of nucleotides. This pharmacological characterization indicated that a putative P2u receptor mediates the [Ca2+]i elevation elicited by nucleotides in NCB-20 cells. The increase in [Ca2+]i originates from intracellular Ca2+ stores; blockade of Ca2+ entry does not affect the rise in [Ca2+]i. In contrast, pretreatment with the Ca(2+)-ATPase inhibitor thapsigargin or with bradykinin, a hormone that releases Ca2+ from inositol trisphosphate-sensitive stores, does preclude the increase in [Ca2+]i induced by ATP. ATP and UTP also transiently inhibit cAMP accumulation in the intact cell, presumably via a Ca(2+)-mediated mechanism. The finding of a P2u receptor in NCB-20 cells adds to a growing perception that P2 receptors are widely distributed. Besides the P2u receptor, NCB-20 cells express adenosine A2 receptors, coupled to stimulation of cAMP accumulation. The presence of both P1 and P2 purinergic receptors permits a sequential modulation of distinct second messenger levels associated with a common stimulus, ATP.     

P2-purinoceptor-mediated formation of inositol phosphates and intracellular Ca2+ transients in human coronary artery smooth muscle cells.

1. The effects of extracellular adenosine 5''-triphosphate (ATP) on smooth muscles are mediated by a variety of purinoceptors. In this study we addressed the identity of the purinoceptors on smooth muscle cells (SMC) cultured from human large coronary arteries. Purinoceptor-mediated increases in [Ca2+]i were measured in single fura-2 loaded cells by applying a digital imaging technique, and the formation of inositol phosphate compounds was quantified after separation on an anion exchange column. 2. Stimulation of the human coronary artery SMC (HCASMC) with extracellular ATP at concentrations of 0.1-100 microM induced a transient increase in [Ca2+]i from a resting level of 49 +/- 21 nM to a maximum of 436 +/- 19 nM. The effect was dose-dependent with an EC50 value for ATP of 2.2 microM. 3. The rise in [Ca2+]i was independent of the presence of external Ca2+, but was abolished after depletion of intracellular stores by incubation with 100 nM thapsigargin. 4. [Ca2+]i was measured upon stimulation of the cells with 0.1-100 microM of the more specific P2-purinoceptor agonists alpha, beta-methyleneadenosine 5''-triphosphate (alpha,beta-MeATP), 2-methylthioadenosine 5''-triphosphate (2MeSATP) and uridine 5''-triphosphate (UTP). alpha, beta-MeATP was without effect, whereas 2MeSATP and UTP induced release of Ca2+ from internal stores with 2MeSATP being the most potent agonist (EC50 = 0.17 microM), and UTP having a potency similar to ATP. The P1 purinoceptor agonist adenosine (100 microM) did not induce any changes in [Ca2+]i. 5. Stimulation with a submaximal concentration of UTP (10 microM) abolished a subsequent ATP-induced increase in [Ca2+]i, whereas an increase was induced by ATP after stimulation with 10 microM 2MeSATP. 6. The phospholipase C (PLC) inhibitor U73122 (5 microM) abolished the purinoceptor-activated rise in [Ca2+]i, whereas pretreatment with the Gi protein inhibitor pertussis toxin (PTX, 500 ng ml-1) was without effect on ATP-evoked [Ca2+]i increases. 7. Receptor activation with UTP and ATP resulted in formation of inositol phosphates with peak levels of inositol 1, 4, 5-trisphosphate (Ins(1, 4, 5)P3) observed 5-20 s after stimulation. 8. These findings show, that cultured HCASMC express G protein-coupled purinoceptors, which upon stimulation activate PLC to induce enhanced Ins(1, 4, 5)P3 production causing release of Ca2+ from internal stores. Since a release of Ca2+ was induced by 2MeSATP as well as by UTP, the data indicate that P2y- as well as P2U-purinoceptors are expressed by the HCASMC.     

Rise in cytosolic Ca2+ concentration induced by P2-purinoceptor activation in isolated myocytes from the rat gastrointestinal tract.

1. The changes in the free cytosolic Ca2+ concentration ([Ca2+]i) in response to agonists of P2-purinoceptors were studied in myocytes isolated from the longitudinal muscle layer of different regions of the rat gastrointestinal tract (stomach, jejunum, ileum, caecum and colon). [Ca2+]i was estimated by emission from the fluorescent dye, indo-1. 2. ATP and the P2Y-purinoceptor agonist, 2-methylthio-ATP (2-MeSATP), transiently increased [Ca2+]i in single myocytes from all segments of the gastrointestinal tract, whereas alpha,beta-methylene-ATP, a P2x-purinoceptor agonist, had no effect. 3. The rise in [Ca2+]i induced by ATP and 2-MeSATP was maintained in Ca(2+)-free solution but was abolished by depletion of the intracellular store with thapsigargin (1 microM). 4. Single myocytes from stomach, caecum and colon also responded to UTP by a transient increase in [Ca2+]i. 5. Individual myocytes responded to ATP, 2-McSATP and UTP in a nearly all-or-nothing manner. The increasing of agonist concentration enhanced the number of responding cells but did not increase the amplitude of the [Ca2+]i rise. 6. These results suggest that myocytes from the longitudinal layer of gastrointestinal muscle do not possess functional P2x-purinoceptors and that agonists of P2Y and P2U-purinoceptors induced a rise in [Ca2+]i, probably via an all-or-nothing mobilization of Ca2+ from intracellular stores.     

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.     

Histamine-stimulated increases in intracellular calcium in the smooth muscle cell line, DDT1MF-2

Suspensions of undifferentiated cultured vas deferens smooth muscle cells (DDT1MF-2) were loaded with the calcium-sensitive fluorescent dye fura-2. Exposure to histamine elicited a rapid and maintained increase in intracellular free calcium ([Ca2+] i) with an EC50 of 1.3 +/- 0.7 x 10(-5) M. The initial rise is a consequence of calcium release from intracellular stores, whereas the maintained or plateau phase, which is dependent upon the presence of extracellular calcium, is associated with calcium influx. Experiments in nominally Ca(2+)-free buffer attenuated the initial rise in [Ca2+]i (i.e. peak height) and virtually abolished the plateau phase. Re-addition of 2 mM Ca2+ (during experiments performed in nominally Ca(2+)-free buffer) resulted in a return of the plateau phase. Pretreatment with the H1-antagonist mepyramine (100 nM; Kd = 1.0 +/- 0.4 nM, N = 3) completely blocks the response to histamine, whereas tiotidine (2 microM; H2-antagonist) had no effect. In conclusion, the present data would suggest that functional H1-receptors found in hamster vas deferens smooth muscle cells are typical of the "classical" H1-receptor in both its control of intracellular Ca2+ and sensitivity to antagonism by mepyramine.     

ATP对大鼠三叉神经节小直径神经元胞内钙浓度的调制作用及其机制

目的探讨神经递质ATP通过何种途径引起大鼠三叉神经节(trigeminal ganglion,TG)小直径神经元胞内钙离子浓度升高。方法在急性分离的TG神经元上,应用钙离子成像技术检测胞内游离Ca2+浓度([Ca2+]i)的变化。结果在大鼠TG小直径神经元中,ATP(100μmol·L-1),thap-sigargin(1μmol·L-1,内质网钙泵抑制剂)和咖啡因(20mmol·L-1,内质网钙离子通道开放剂)在正常细胞外液和去除细胞外Ca2+的情况下,均能够引起细胞[Ca2+]i升高。在细胞外无Ca2+条件下,thapsigargin能够可逆地抑制ATP引起细胞内[Ca2+]i升高(n=8,P<0.01),而咖啡因对ATP引起的细胞内[Ca2+]i升高无影响(n=6,P>0.05)。然而在正常外液中,thapsigargin不能完全抑制ATP引起的细胞内[Ca2+]i升高,不过ATP引起的细胞内[Ca2+]i升高的幅度明显地低于thapsigargin处理前(n=7,P<0.05)。结论在大鼠TG小直径神经元中,存在有IP3敏感钙库和Ryanod-ine敏感钙库。ATP可通过激动P2Y受体引起IP3敏感钙库的Ca2+释放,也可通过激动P2X受体引起细胞外钙内流。     

Two subtypes of G protein-coupled nucleotide receptors, P2Y(1) and P2Y(2) are involved in calcium signalling in glioma C6 cells

1. In glioma C6 cells, the stimulation of P2Y receptors by ADP, ATP and UTP initiated an increase in the intracellular Ca2+ concentration, in a process that involved the release of Ca2+ from InsP(3)-sensitive store and the capacitative, extracellular Ca2+ entry. The presence of external Ca2+ was not necessary to elevate Ca(2+). 2. The rank order of potencies of nucleotide analogues in stimulating [Ca2+](i) was: 2MeSADP > ADP > 2MeSATP = 2ClATP > ATP > UTP. alpha,beta-Methylene ATP, adenosine and AMP were ineffective. 3. ADP and UTP effects were additive, while actions of ATP and UTP were not additive on [Ca2+](i) increase. Similarly, cross-desensitization between ATP and UTP but not between ADP and UTP occurred. 4. Suramin, a non-specific nucleotide receptors inhibitor, antagonized ATP-, UTP- and ADP-evoked Ca2+ responses. PPADS, a selective antagonist of the P2Y(1) receptor-generated InsP(3) accumulation, decreased ADP-initiated Ca2+ response with no effect on ATP and UTP. 5. Pertussis toxin (PTX) reduced ADP- and ATP-induced Ca2+ increases. Short-term treatment with TPA, inhibited both ATP and ADP stimulatory effects on [Ca2+](i). 6. ADP inhibited isoproterenol-induced cyclic AMP accumulation. PTX blocked this effect, but PPADS did not. 7. RT - PCR analysis revealed the molecular identity of P2Y receptors expressed by glioma C6 cells to be both P2Y(1) and P2Y(2). 8. It is concluded that both P2Y(1) and P2Y(2) receptors co-exist in glioma C6 cells. ADP acts as agonist of the first, and ATP and UTP of the second one. Both receptors are linked to phospholipase C (PLC).     

The effect of L-type calcium channel modulators on the mobilization of intracellular calcium stores in guinea-pig intestinal smooth muscle.

1. The action of Ca2+ channel modulators has been examined on the intracellular Ca2+ signal in the longitudinal smooth muscle cells of the guinea-pig intestine after exposure to histamine and to agents known to affect intracellular Ca2+ stores. Isometric contraction has been measured simultaneously with front-surface fluorometry of fura 2-loaded preparations. 2. Histamine (10 microM) evoked a phasic and tonic increase in [Ca2+]i and contraction which were both sensitive to the Ca2+ channel blockers, nimodipine and D600. 3. Caffeine (10 mM) evoked in rapid increase in [Ca2+]i which was sustained as long as the preparation was exposed to the drug, whereas the contractile response was only phasic. In the presence of nimodipine 1 microM, the phasic contraction was absent although the fura 2-Ca2+ signal amounted to 32% of the control. 4. Ryanodine (10 microM) evoked a slow increase in [Ca2+]i and a contraction, both of which were reversed after exposure to nimodipine (1 microM) or D600 (10 microM). In the presence of diazoxide (500 microM), a hyperpolarizing agent, the ryanodine-evoked increase in [Ca2+]i and in muscle tone were inhibited. 5. Thapsigargin (1 microM) also produced an increase in [Ca2+]i and a contraction both of which were blocked by nimodipine (1 microM). 6. In Ca2+-free solution, histamine 10 microM evoked non-reproducible phasic Ca2+ signal and contraction. This response was recovered after refilling in Ca2+ containing solution. The recovery was blocked by nimodipine, D600 or diazoxide and was facilitated by the Ca2+ channel activator, Bay K 8644. When the refilling medium was supplemented with thapsigargin, the recovered response was significantly reduced, but Bay K 8644 still had some action. 7. The present results show that blockage of L-type Ca2+ channels inhibited changes in [Ca2+]i evoked by histamine, caffeine and ryanodine which are generally attributed to Ca2+ mobilization from intracellular stores. They also show that when the tissue was exposed to nimodipine, D600 and diazoxide during the procedure of refilling after depletion of intracellular stores, the action of histamine on [Ca2+]i and contraction was blocked. Bay K 8644 had an opposite effect even when the Ca2+ pumping activity of the sarcoplasmic reticulum was reduced by thapsigargin. This indicates that refilling of intracellular Ca2+ stores depleted by histamine in guinea-pig intestine mainly occurred through L-type Ca2+ channels.     

Actions of ADP,but not ATP,on cytosolic free Ca2+ in single rat hepatocytes mimicked by 2-methylthioATP.

1. Aequorin-injected, single rat hepatocytes generate series of repetitive transients in cytosolic free calcium concentration ([Ca2+]i) when stimulated with agonists acting through the phosphoinositide signalling pathway, including ADP and ATP. We have previously described differences in the [Ca2+]i responses of aequorin-injected hepatocytes to ADP and ATP. 2. The effects of the phosphorothioate analogue of ATP, 2-methylthioATP (2-meSATP), have been examined on single rat hepatocytes. This analogue is belived to be the most potent agonist at the P2Y1 subclass of purinoceptor. 3. The [Ca2+]i transients induced by 2-meSATP were indistinguishable from those induced by ADP, and in contrast to those induced by ATP. 4. At hig concentrations, 2-meSATP and ADP both induced transients at high frequency. In contrast, hepatocytes responded to high concentrations of ATP with an initial rapid rise in [Ca2+]i, followed by a slowly decaying fall. 5. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both 2-meSATP- and ADP-induced [Ca2+]i transients; the peak height and frequency of transients were enhanced. ATP-induced transients, however, underwent either an increase in duration or conversion into a sustained rise in [Ca2+]i. 6. ATP-induced transients were specifically potentiated by the co-addition of alpha, beta-methyleneATP, whereas 2-meSATP- and ADP-induced transients were unaffected by this treatment. 7. We conclude that 2-meSATP acts at the same receptor as ADP on rat hepatocytes, and that this is distinct from teh receptor(s) mediating the effects of ATP.     

The ether lipid ET-18-OCH3 increases cytosolic Ca2+ concentrations in Madin Darby canine kidney cells.

The effect of the ether lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3) on the intracellular free Ca2+ concentration ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was studied using fura-2 as the Ca2+ probe. In Ca2+ medium, ET-18-OCH3 induced a significant rise in [Ca2+]i at concentrations between 10-100 microM with a concentration-dependent delay of 45-175 s. The [Ca2+]i signal was composed of a gradual rise and a sustained plateau. In Ca2+-free medium, ET-18-OCH3 (10-100 microM) induced a Ca2+ release from internal Ca2+ stores with a concentration-dependent delay of 45-175 s. This discharge of internal Ca2+ triggered capacitative Ca2+ entry in a concentration-dependent manner. This capacitative Ca2+ entry was not inhibited by econazole (25 microM), 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365; 50 microM), nifedipine (10 microM), verapamil (10 microM), diltiazem (10 microM) and cadmium (0.5 microM). Methyl 2-(phenylthio)ethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-dicarboxylat e (PCA-4248), a platelet-activating factor (PAF) receptor antagonist, inhibited 25 microM ET-18-OCH3-induced [Ca2+]i rise in a concentration-dependent manner between 1-20 microM, with 20 microM exerting a complete block. The [Ca2+]i rise induced by ET-18-OCH3 (25 microM) was not altered when the production of inositol 1,4,5-trisphosphate (IP3) was suppressed by the phospholipase C inhibitor U73122 (2 microM), but was partly inhibited by the phospholipase D inhibitor propranolol (0.1 mM) or the phospholipase A2 inhibitor aristolochic acid (20-40 microM). In Ca2+-free medium, pretreatment with 25 microM ET-18-OCH3 completely depleted the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin-sensitive Ca2+ store. In contrast, pretreatment with thapsigargin abolished 0.1 mM ATP-induced [Ca2+]i rise without altering the ET-18-OCH3-induced [Ca2+]i rise. This suggests that ET-18-OCH3 depleted thapsigargin-sensitive Ca2+ stores and also released Ca2+ from thapsigargin-insensitive stores. The thapsigargin-insensitive stores involve mitochondria because the mitochondria uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) induced a release of mitochondrial Ca2+ which was abolished by pretreatment with 25 microM ET-18-OCH3. ET-18-OCH3 (25 microM) induced a significant Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength confirming that ET-18-OCH3 induced capacitative Ca2+ entry. La3+ (0.1 mM) or Gd3+ (50 microM) abolished the ET-18-OCH3-induced Mn2+ quench and [Ca2+]i rise. Our data imply that ET-18-OCH3 induced a [Ca2+]i rise in MDCK cells by activating PAF receptors leading to an internal Ca2+ release followed by capacitative Ca2+ entry. Phospholipase D and phospholipase A2, but not phospholipase C, might be involved in mediating the capacitative Ca2+ entry. La3+ abolished the ET-18-OCH3-induced [Ca2+]i rise presumably by inhibiting PAF receptors.     

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)     

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.     

A novel effect of bifemelane, a nootropic drug, on intracellular Ca2+ levels in rat cerebral astrocytes

We investigated the effects of bifemelane, a nootropic drug, on the intracellular calcium concentration ([Ca2+]i) in rat cerebral astrocytes using a Ca2+ imaging device. At concentrations of 10 - 30 microM, bifemelane induced a slow onset and small increase in the [Ca2+]i, while at higher concentrations (100 - 300 microM), it induced a rapid transient increase in the [Ca2+]i during administration and a second large increase was seen during drug washout. The first peak was observed in Ca2+-free medium, but its onset was significantly delayed, and no second peak was seen. Neither of these effects was seen in cells treated with thapsigargin, a specific inhibitor of endoplasmic reticulum Ca2+-ATPase, in Ca2+-free medium. When thapsigargin-treated astrocytes were returned to normal medium containing Ca2+ (1.8 mM), the [Ca2+]i increased significantly, and this effect was reversely inhibited by bifemelane. We conclude that bifemelane causes the first peak by stimulating release from intracellular Ca2+ stores and the second by capacitive entry through store-operated Ca2+ channels. Although the detail mechanisms of action of the drug are still unknown, bifemelane will be provided as a pharmacological tool for basic studies on astrocytes.     

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.     

Purinoceptor-mediated calcium mobilization and cellular proliferation in cultured bovine corneal endothelial cells

In the present study, we investigated the effect of adenosine triphosphate (ATP) on cytosolic free calcium mobilization and mitogenic activity in cultured bovine corneal endothelial cells (BCEC). The [Ca2+]i was determined using a Ca2+ sensitive indicator, Fura-2/AM, and cell proliferation was evaluated by counting the cell number. ATP, its metabolites and analogs caused transient increase in [Ca2+]i in a concentration-dependent manner (10(-7) M-10(-3) M) and the potency of agonists was ordered as follows: 2-methylthio-ATP > uridine triphosphate > ATP > adenosine diphosphate. Adenosine monophosphate and adenosine did not affect [Ca2+]i. ATP (10(-4) M) also promoted the accumulation of inositol trisphosphate (IP3). The ATP-induced transient [Ca2+]i increase and IP3 accumulation were attenuated by pretreatment with a phospholipase C inhibitor, U-73122 (5 microM), for 30 min. ATP (10(-5) M) significantly enhanced the proliferation of BCEC. ATP-induced [Ca2+]i increase and cell proliferation were inhibited by a purinoceptor antagonist, suramin (10(-4) M). Thus, the present study indicates that BCEC contain P2 purinoceptors that regulate their proliferation.     

Characterization of the P2Y-purinoceptor involved in the ATP-induced rise in cytosolic Ca2+ concentration in rat ileal myocytes.

1. The P2-purinoceptor subtype and the intracellular signalling mechanism(s) involved in the rise in the free cytosolic Ca2+ concentration ([Ca2+]i) induced by ATP and analogues were analyzed in myocytes isolated from the longitudinal muscle layer of rat ileum by means of molecular and physiological techniques. 2. The P2-purinoceptor expressed by ileal smooth muscle cells shared 100% amino acid identity with the rat P2Y1-receptor. 3. Short applications of the purinoceptor agonists induced a transient rise in [Ca2+]i in an all-or-nothing manner. The rank order of potency of the analogues of ATP and ADP, determined by measuring the percentage of responding cells was 2-methylthioATP = 2-chloro-ATP > ADP > ATP, with concentrations giving [Ca2+]i response in 50% of cells ranging between 3 nM and 0.6 microM. The concentration-response curves to ADP and ATP were shifted to the right by 10 microM pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). 4. Although the rise in [Ca2+]i induced by stimulation of the ileal P2v-purinoceptor was inhibited by heparin (5 mg ml-1), we were not able to detect stimulation of phospholipase C under conditions (37 degrees C) where muscarinic cholinoceptor activation markedly increased inositol phosphate (InsP) accumulation. However, the carbachol (CCh)-induced increase in InsP accumulation was suppressed when the agonist was applied at 20 degrees C while a CCh-induced [Ca2+]i rise similar to that obtained in response to the P2-purinoceptor agonist was still observed. 5. Our results indicate that the rat ileal myocytes express a PPADS-sensitive P2-purinoceptor similar to the P2Y1-receptor subtype. Although there is no detectable increase in InsP production, stimulation of these receptors leads to a rise in [Ca2+]i by activation of the inositol 1,4,5-trisphosphate receptor-channel of the intracellular Ca2+ store, indicating that they couple to phospholipase C.     

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.