A possible coupling of postjunctional ATP release and transmitters' receptor stimulation in smooth muscles.

The nature of ATP release from mainly smooth muscles of guinea-pig was evaluated with KCl and agonists for different kinds of receptors. In ileal longitudinal muscles, amounts of net ATP release by ACh and bethanechol (1-10 microM) were much larger (about 10 fold) than that by other drugs, e.g., histamine, 5-hydroxytryptamine, prostaglandin-F2 alpha, substance P and bradykinin, including KC1, although differences between contractions of the tissue evoked by test drugs were approximately 1.5 times at most. The ATP release, as well as the contraction, evoked by ACh or bethanechol was markedly reduced by atropine (0.3 microM), thus, indicating primarily postjunctional release of ATP. The remarkable ATP release from vas deferens by norepinephrine (NE), but not by substance P, was abolished almost completely by prazosin (0.3 microM). Increases in intracellular Ca2+ and subsequent contraction in the ileal tissue were produced by ATP and these responses were fully antagonized by nifedipine (0.1 microM). These findings provide evidence that the drugs-stimulated ATP release from smooth muscles does not result from contractility of muscles, but is substantially elicited only by stimulation of neurotransmitter (NE or ACh) receptors, suggesting the existence of the receptor-stimulus-postjunctional ATP release coupling. The released ATP may contribute, in part, to the muscle contractility via increase of Ca2(+)-influx, presumably, in a manner related to the voltage-gated Ca2(+)-channels.     

Characterization of ATP receptor which mediates norepinephrine release in PC12 cells.

PC12 cells, a rat pheochromocytoma cell line, has been reported to release norepinephrine in response to extracellular ATP in the presence of extracellular Ca2+. The potency order of ATP analogues was adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than adenosine 5'-O-(1-thiotriphosphate) = 2-methylthioadenosine 5'-triphosphate (MeSATP) greater than 2'- and 3'-O-(4-benzoyl-benzoyl)ATP (BzATP) greater than ADP greater than 5-adenylylimidodiphosphate. Adenosine 5'-O-(2-thiodiphosphate), beta, gamma-methyleneadenosine 5'-triphosphate, AMP and adenosine were inactive. The ATP action in the absence of extracellular Ca2+, suggests a small but appreciable contribution of intracellular Ca2+ mobilization, for norepinephrine release. However, for some ATP derivatives, like BzATP, almost no contribution of the phospholipase C-Ca2+ pathway is suggested, based on their low activity in inositol phosphates production. To identify the ATP-receptor protein, PC12 cell membranes were photoaffinity-labeled with [32P]BzATP. SDS-PAGE analysis showed that a 53-kDa protein labeling was inhibited by ATP and its derivatives, as well as by P2-antagonists, suramin and reactive blue 2, which inhibit the nucleotide-induced norepinephrine release. The inhibitory activity of the nucleotides was, in parallel with their potency, to induce norepinephrine release. Despite their inability to release norepinephrine, GTP and GTP gamma S inhibited the BzATP labeling, suggesting the participation of a putative G protein in the ATP-receptor-mediated actions. We suggest that the 53-kDa protein on the PC12 cell surface is an ATP receptor, which mediates the norepinephrine release, depending, mainly, on extracellular Ca2+ gating.     

Intracellular adenosine formation and release by freshly-isolated vascular endothelial cells from rat skeletal muscle: effects of hypoxia and/or acidosis

Previous studies suggested indirectly that vascular endothelial cells (VECs) might be able to release intracellularly-formed adenosine. We isolated VECs from the rat soleus muscle using collagenase digestion and magnetic-activated cell sorting (MACS). The VEC preparation had >90% purity based on cell morphology, fluorescence immunostaining, and RT-PCR of endothelial markers. The kinetic properties of endothelial cytosolic 5′-nucleotidase suggested it was the AMP-preferring N-I isoform: its catalytic activity was 4 times higher than ecto-5′nucleotidase. Adenosine kinase had 50 times greater catalytic activity than adenosine deaminase, suggesting that adenosine removal in VECs is mainly through incorporation into adenine nucleotides. The maximal activities of cytosolic 5′-nucleotidase and adenosine kinase were similar. Adenosine and ATP accumulated in the medium surrounding VECs in primary culture. Hypoxia doubled the adenosine, but ATP was unchanged; AOPCP did not alter medium adenosine, suggesting that hypoxic VECs had released intracellularly-formed adenosine. Acidosis increased medium ATP, but extracellular conversion of ATP to AMP was inhibited, and adenosine remained unchanged. Acidosis in the buffer-perfused rat gracilis muscle elevated AMP and adenosine in the venous effluent, but AOPCP abolished the increase in adenosine, suggesting that adenosine is formed extracellularly by non-endothelial tissues during acidosis in vivo. Hypoxia plus acidosis increased medium ATP by a similar amount to acidosis alone and adenosine 6-fold; AOPCP returned the medium adenosine to the level seen with hypoxia alone. These data suggest that VECs release intracellularly formed adenosine in hypoxia, ATP during acidosis, and both under simulated ischaemic conditions, with further extracellular conversion of ATP to adenosine.     

Adenosine triphosphate induces a low [Ca2+]i sensitivity of phosphorylation and an unusual form of receptor desensitization in smooth muscle.

The contractile sensitivity of smooth muscle to changes in myoplasmic [Ca2+] is dependent on the form of stimulation. Both myosin phosphorylation and force are less sensitive to increases in [Ca2+]i derived from Ca2+ entry through L-type Ca2+ channels than to increases in [Ca2+] induced by agents which release internal Ca2+ stores. We hypothesized that activation of receptor-operated channels should produce a [Ca2+]i sensitivity similar to that induced by opening L channels. Aequorin-estimated myoplasmic [Ca2+] and myosin light chain phosphorylation were measured in swine carotid media tissues stimulated with ATP, an activator of the only known receptor-operated cation channel in smooth muscle. ATP, via activation of a P2x purinergic receptor, induced large, transient increases in [Ca2+]i, yet only small transient elevations in phosphorylation or force. Rapid desensitization to ATP was partially, but not completely, caused by hydrolysis of ATP into adenosine since 1) alpha-beta-methylene ATP (a poorly hydrolyzable analog of ATP) produced larger, yet still transient increases in [Ca2+]i, phosphorylation, and force; 2) BW A1433U, a P1 (adenosine) receptor antagonist, enhanced ATP-induced contractions; and 3) ATP, but not alpha-beta-methylene ATP increased bath [adenosine]. The [Ca2+]i sensitivity of phosphorylation during P2x receptor activation was similar to that observed with KCl-depolarization-induced opening of L channels, supporting the hypothesis that transplasmalemmal Ca2+ influx produces less phosphorylation and force than mobilization of intracellular Ca2+ stores. Cumulative additions of higher alpha-beta-methylene ATP concentrations induced repeated transient contractions, indicative of an unusual form of receptor desensitization which could be explained if the affinity of the P2x receptor for ATP, but not the receptor number were rapidly reduced.     

Diadenosine Tetraphosphate Is Co-Released with ATP and Catecholamines from Bovine Adrenal Medulla

Secretion of adenosine(5')tetraphospho(5')adenosine (Ap4A) and ATP from perfused bovine adrenal glands stimulated with acetylcholine or elevated potassium levels was measured and compared with that of catecholamines. We have found a close correlation between the release of Ap4A and catecholamines elicited with all the secretagogues used in the presence of either Ca2+ or Ba2+, suggesting co-release of both constituents from the chromaffin granules. By contrast, ATP secretion, as measured with luciferase, showed a significantly different time course regardless of the secretagogue used. ATP secretion consistently decreased after 1-2 min of stimulation at a time when Ap4A and catecholamine secretions were still increasing. Measures of degradation of injected [3H]ATP to the gland during stimulation showed little difference in the level of uptake or decomposition of ATP throughout the pulse. However, a reexamination of ATP secretion by monitoring its products of degradation (AMP, adenosine, and inosine) by HPLC techniques showed that Ap4A, ATP, and catecholamines are indeed secreted in parallel from the perfused adrenal gland.     

Further Characterization of Dopamine Release by Permeabilized PC 12 Cells

Rat pheochromocytoma cells (PC12) permeabilized with staphylococcal alpha-toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+-ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+-dependent [3H]-dopamine release from digitonin-permeabilized PC12 cells. Permeabilization with alpha-toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+-ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+-induced release. The release was unaltered by thiol-, hydroxyl-, or calmodulin-interfering substances. Thus Mg2+-ATP, calmodulin, or proteins containing -SH or -OH groups are not necessary for exocytosis in permeabilized PC12 cells.     

Adenosine linking reduced O2 to arteriolar NO release in intestine is not formed from extracellular ATP

We have previously reported that adenosine formed in response to reduced arteriolar and/or tissue PO(2) preserves endothelial nitric oxide (NO) synthesis during sympathetic vasoconstriction in the rat intestine. To more precisely identify the site and mechanism of adenosine formation under these conditions, we tested the hypothesis that ATP released in response to reduced O(2) levels serves as a source of adenosine. Direct application of ATP to the wall of first-order arterioles elicited dose-dependent dilations of 15-33% above resting diameter that were reduced by 71-80% by the 5'-ectonucleotidase inhibitor alpha,beta-methyleneadenosine 5'-diphosphate (AOPCP, 4.5 x 10(-5) M) and completely abolished by N(G)-monomethyl-L-arginine (L-NMMA, 10(-4) M). Under control conditions, sympathetic nerve stimulation at 3 and 8 Hz induced arteriolar constrictions of 11 +/- 1 and 19 +/- 1 microm, respectively. These responses were enhanced by 58-69% in the presence of L-NMMA or when local PO(2) was maintained at resting levels. However, in the presence of AOPCP, the enhancing effects of L-NMMA and the high O(2) superfusate on sympathetic constriction were preserved. These results suggest that, although exogenously applied ATP can stimulate arteriolar NO release in the intestine largely through its sequential extracellular hydrolysis to adenosine, this process does not contribute to adenosine formation and sustained NO release during sympathetic constriction in this vascular bed.     

Exogenous ATP enhances calcium influx in intact thymocytes

Recent observations have indicated that exogenous adenosine triphosphate (ATP) may influence lymphocyte functions such as proliferation and cytoxicity. Here we report a novel activity of extracellular ATP--it specifically increases Ca2+ uptake in murine lymphocytes. ATP added to thymocytes increases the rate of [45Ca2+] uptake by up to 20-fold. The increased rate is seen with ATP concentrations as low as 500 microM and is half-maximal at approximately 2 mM ATP. The magnitude of stimulation by ATP is dependent on Mg2+ concentration, and ATP-Mg2+ complex is probably the true activator. Of the high-energy phosphate-containing compounds tested, including deoxy-ATP, only GTP showed a modest stimulation of calcium uptake. ADP, AMP, cyclic AMP, and adenosine did not significantly increase calcium uptake. Cellular integrity as indicated by trypan blue exclusion and ethidium bromide/acridine orange staining was unaffected by ATP. Ca2+ influx is the major mode of action of ATP in raising intrathymocyte Ca2+ levels, because neither the Ca2+ efflux nor the [45Ca2+]-Ca2+ exchange was significantly altered in the presence of ATP. Verapamil, a Ca2+ channel blocking agent, could not prevent the ATP effect, suggesting that ATP may be acting by a mechanism other than the voltage-dependent Ca2+ channel. An analysis of intracellular and extracellular ATP levels by chemiluminescence assay indicated no significant ATP entry into intact lymphocytes. Also, ATP added to the medium containing thymocytes was destroyed (approximately 50% by 20 min). The nonhydrolyzable ATP analogs, AMPPCP and AMPPNP, were unable to stimulate a significant amount of Ca2+ uptake, suggesting the involvement of a cell surface phosphotransferase activity. This was supported by the demonstration of a threefold to fivefold increase in the labeling of protein and phospholipid fractions obtained from intact thymocytes exposed to [gamma 32P]ATP for 30 min. Ca2+ is believed to play an important role in a variety of lymphocyte functions, including mitogenesis and natural killer cell activity. The data herein thus provide a potential mechanism for the action of exogenous ATP on these lymphocyte functions.     

Preferential Release of ATP and Its Extracellular Catabolism as a Source of Adenosine upon High- but Not Low-Frequency Stimulation of Rat Hippocampal Slices

Abstract: The release of adenosine and ATP evoked by electrical field stimulation in rat hippocampal slices was investigated with the following two patterns of stimulation: (1) a brief, high-frequency burst stimulation (trains of stimuli at 100 Hz for 50 ms applied every 2 s for 1 min), to mimic a long-term potentiation (LTP) stimulation paradigm, and (2) a more prolonged (3 min) and low-frequency (5 Hz) train stimulation, to mimic a long-term depression (LTD) stimulation paradigm. The release of ATP was greater at a brief, high-frequency burst stimulation, whereas the release of [³H]adenosine was slightly greater at a more prolonged and low-frequency stimulation. To investigate the source of extracellular adenosine, the following two pharmacological tools were used; α,β-methylene ADP (AOPCP), an inhibitor of ecto-5′-nucleotidase, to assess the contribution of the catabolism of released adenine nucleotides as a source of extracellular adenosine, and S-(4-nitrobenzyl)-6-thioinosine (NBTI), an inhibitor of adenosine transporters, to assess the contribution of the release of adenosine, as such, as a source of extracellular adenosine. At low-frequency stimulation, NBTI inhibited by nearly 50% the evoked outflow of [³H]adenosine, whereas AOPCP inhibited [³H]adenosine outflow only marginally. In contrast, at high-frequency stimulation, AOPCP inhibited by 30% the evoked release of [³H]adenosine, whereas NBTI produced a 40% inhibition of [³H]adenosine outflow. At both frequencies, the kinetics of evoked [³H]adenosine outflow was affected in different manners by AOPCP and NBTI; NBTI mainly depressed the rate of evoked [³H]adenosine outflow, whereas AOPCP mainly inhibited the later phase of evoked [³H]adenosine accumulation. These results show that there is a simultaneous, but quantitatively different, release of ATP and adenosine from rat hippocampal slices stimulated at frequencies that can induce plasticity phenomena such as LTP (100 Hz) or LTD (5 Hz). The source of extracellular adenosine is also different according to the frequency of stimulation; i.e., at a brief, high-frequency stimulation there is a greater contribution of released adenine nucleotides for the formation of extracellular adenosine than at a low frequency with a more prolonged stimulation.     

Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 microM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 microM reserpine, 30 microM NH4+, or 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules with digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 microM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-beta-hydroxylase. The studies demonstrate that 20 microM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.     

Reserpine attenuates interstitial adenosine-mediated activation of ecto-5'-nucleotidase in rat hearts in vivo

We examined whether reserpine-induced norepinephrine (NE) depletion attenuated the products of adenosine in rat heart. A flexibly mounted microdialysis technique was used to measure the concentration of interstitial adenosine and to assess the activity of ecto-5'-nucleotidase in rat hearts in situ. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and perfused with Tyrode solution containing adenosine 5'-monophosphate (AMP) at rate of 1.0 microliter/min. The baseline level of dialysate adenosine was 0.51 +/- 0.09 microM. The introduction of AMP (100 microM) through the probe increased markedly the dialysate adenosine to 8.95 +/- 0.86 microM, and this increase was inhibited by ecto-5'-nucleotidase inhibitor, alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP, 100 microM), to 0.66 +/- 0.38 microM. Thus, the level of dialysate adenosine is a measure of the ecto-5'-nucleotidase activity in the tissue in situ. AMP concentration for the half-maximal effect of adenosine release (EC(50)) was 107.3 microM. The maximum attainable concentration of dialysate adenosine (E(max)) by AMP was 21.1 microM. However, the EC(50) and E(max) values with reserpinized animals were 106.9 and 7.1 microM, respectively. Electrical stimulation of the left stellate ganglion increased significantly dialysate adenosine concentration, from the control level of 8.66 +/- 0.96 microM to 12.38 +/- 1.11 microM. After stimulation, dialysate adenosine returned to near the prestimulation level. When corresponding experiments were performed with reserpinized animals, the effect of electrical stimulation was abolished. Tyramine (endogenous catecholamine trigger) increased the adenosine concentration in a concentration-dependent manner. However, the elevation of adenosine concentration with reserpinized animals was not observed. These results suggest that reserpine attenuates NE-induced adenosine via stimulation of alpha(1)-adrenoceptor and protein kinase C mediated activation of ecto-5'-nucleotidase in rat heart.     

Effects of clentiazem (TA-3090) and nifedipine on basal circulating catecholamine levels and on stimulation-evoked adrenal catecholamine secretion in anesthetized dogs.

The effects of TA-3090 (clentiazem) and nifedipine on basal sympathoadrenal activity and on the adrenal medullary response during splanchnic nerve stimulation were studied in dogs anesthetized with sodium pentobarbital. Plasma concentrations of epinephrine and norepinephrine were measured in aortic and adrenal venous blood before and after acute administration of the drugs, as well as during left splanchnic nerve stimulation before and after administration of drugs. Following intravenous injections, TA-3090 (30, 100, and 300 micrograms/kg) did not affect basal circulating catecholamine levels, whereas nifedipine (10, 30, and 100 micrograms/kg) markedly increased aortic epinephrine and norepinephrine concentrations in a dose-dependent manner in correlation with progressive decreases in mean arterial pressure. The changes in aortic epinephrine and norepinephrine concentrations were inversely related to those in mean arterial pressure (r = 0.603, p < 0.01; r = 0.536, p < 0.01; respectively). In response to direct splanchnic nerve stimulation (2 Hz, 2 ms, 1 min, 12 V), adrenal venous epinephrine and norepinephrine concentrations significantly increased, with a high degree of reproducibility. The catecholamine responses to splanchnic nerve stimulation were not affected by either TA-3090 or nifedipine at any dose tested. The present results suggest that the increases in circulating catecholamine levels following nifedipine administration are due to baroreflex activation secondary to the drug-induced hypotension. The study indicates that both TA-3090 and nifedipine did not significantly affect L-type Ca2+ channels related to catecholamine release in the adrenal medulla under the present experimental conditions.     

Adenosine A2A and beta-adrenergic calcium transient and contractile responses in rat ventricular myocytes

The adenosine A2A receptor (A2AR) enhances cardiac contractility, and the adenosine A1R receptor (A1R) is antiadrenergic by reducing the adrenergic beta1 receptor (beta1R)-elicited increase in contractility. In this study we compared the A2AR-, A1R-, and beta1R-elicited actions on isolated rat ventricular myocytes in terms of Ca transient and contractile responses involving PKA and PKC. Stimulation of A2AR with 2 microM (approximately EC50) CGS-21680 (CGS) produced a 17-28% increase in the Ca transient ratio (CTR) and maximum velocities (Vmax) of transient ratio increase (+MVT) and recovery (-MVT) but no change in the time-to-50% recovery (TTR). CGS increased myocyte sarcomere shortening (MSS) and the maximum velocities of shortening (+MVS) and relaxation (-MVS) by 31-34% with no change in time-to-50% relengthening (TTL). beta1R stimulation using 2 nM (approximately EC50) isoproterenol (Iso) increased CTR, +MVT, and -MVT by 67-162% and decreased TTR by 43%. Iso increased MSS, +MVS, and -MVS by 153-174% and decreased TTL by 31%. The A2AR and beta1R Ca transient and contractile responses were not additive. The PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphorothioate triethylamonium salt prevented both the CGS- and Iso-elicited contractile responses. The PKC inhibitors chelerythrine and KIE1-1 peptide (PKCepsilon specific) prevented the antiadrenergic action of A1R but did not influence A2AR-mediated increases in contractile variables. The findings suggest that cardiac A2AR utilize cAMP/PKA like beta1R, but the Ca transient and contractile responses are less in magnitude and not equally affected. Although PKC is important in the A1R antiadrenergic action, it does not seem to play a role in A2AR-elicited Ca transient and contractile events.     

ATP-induced Secretion in PC12 Cells and Photoaffinity Labeling of Receptors

Abstract— Secretion of catecholamines by rat PC12 cells is strongly stimulated by extracellular ATP via a P₂-type pur-inergic receptor. ATP-induced norepinephrine release was inhibited 80% when extracellular Ca²⁺ was absent. Only four nucleotides, ATP, ATPγS, benzoylbenzoyl ATP (BzATP), and 2-methylthio-ATP, gave substantial stimulation of norepinephrine release from PC12 cells. ATP-induced secretion was inhibited by Mg²⁺, and this inhibition was overcome by the addition of excess ATP suggesting that ATP^4-was the active ligand. ATP-induced secretion of catecholamine release was enhanced by treatment of cells with pertussis toxin or 12- O -tetradecanoylphorbol 13-acetate. The stimulatory effects of 12- O -tetradecanoyl-phorbol 13-acetate and pertussis toxin on norepinephrine release were additive. After brief exposure of intact cells to the photoaffinity analog, [α-³²P]BzATP, two major proteins of 44 and 50 kDa and a minor protein of 97 kDa were labeled. An excess of ATP-γS and BzATP but not GTP blocked labeling of the proteins by [³²P]BzATP. Labeling of the 50-kDa protein was more sensitive to competition by 2-methylthio-ATP than the other labeled proteins, suggesting that the 50-kDa protein represents the P₂ receptor responsible for ATP-stimulated secretion in these cells.     

Adenosine induces ATP release via an inositol 1,4,5-trisphosphate signaling pathway in MDCK cells

ATP is released into extracellular space as an autocrine/paracrine molecule by mechanical stress and pharmacological-receptor activation. Released ATP is partly metabolized by ectoenzymes to adenosine. In the present study, we found that adenosine causes ATP release in Madin-Darby canine kidney cells. This release was completely inhibited by CPT (an A1 receptor antagonist), U-73122 (a phospholipase C inhibitor), 2-APB (an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptor blocker), thapsigargin (a Ca2+-ATPase inhibitor), and BAPTA/AM (an intracellular Ca2+ chelator), but not by DMPX (an A2 receptor antagonist). However, forskolin, epinephrine, and isoproterenol, inducers of cAMP accumulation, failed to release ATP. Adenosine increased intracellular Ca2+ concentrations that were strongly blocked by CPT, U-73122, 2-APB, and thapsigargin. Moreover, adenosine enhanced accumulations of Ins(1,4,5)P3 that were significantly reduced by U-73122 and CPT. These data suggest that adenosine induces the release of ATP by activating an Ins(1,4,5)P3 sensitive-Ca2+ pathway through the stimulation of A1 receptors.     

The Adenosine Analogue N6-L-Phenylisopropyladenosine Inhibits Catecholamine Secretion from Bovine Adrenal Medulla Cells by Inhibiting Calcium Influx

We reported earlier that adenine nucleotides and adenosine inhibit acetylcholine-induced catecholamine secretion from bovine adrenal medulla chromaffin cells. In this article, we used an adenosine analogue, N6-L-phenylisopropyladenosine (PIA), to study the mechanism underlying inhibition of catecholamine secretion by adenosine. PIA inhibits secretion induced by a nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium, or by elevated external K+. The half-maximal effect on 1,1-dimethyl-4-phenylpiperazinium-induced secretion occurred at approximately 5 x 10(-5) M. The inhibition is immediate and reversible. Fura-2 measurements of cytosolic free Ca2+ indicate that PIA inhibits Ca2+ elevation caused by stimulation; measurements of 45Ca2+ influx show that PIA inhibits uptake of Ca2+. PIA does not inhibit calcium-evoked secretion from digitonin-permeabilized cells, nor does PIA cause any significant change in the dependence of catecholamine secretion on calcium concentration. These data suggest that inhibition by PIA occurs at the level of the voltage-sensitive calcium channel.     

Characterization of the calcium signaling system in the submandibular cell line SMG-C6

Establishment of salivary cell lines retaining normal morphological and physiological characteristics is important in the investigation of salivary cell function. A submandibular gland cell line, SMG-C6, has recently been established. In the present study, we characterized the phosphoinositide (PI)-Ca2+ signaling system in this cell line. Inositol 1,4,5-trisphosphate(1,4,5-IP3) formation, as well as Ca2+ storage, release, and influx in response to muscarinic, alpha1-adrenergic, P2Y-nucleotide, and cytokine receptor agonists were determined. Ca2+ release from intracellular stores was strongly stimulated by acetylcholine (ACh) and ATP, but not by norepinephrine (NA), epidermal growth factor (EGF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFalpha). Consistently, 1, 4,5-IP3 formation was dramatically stimulated by ACh and ATP. ACh-stimulated cytosolic free Ca2+ concentration [Ca2+]i increase was inhibited by ryanodine, suggesting that the Ca2+-induced Ca2+ release mechanism is involved in the ACh-elicited Ca2+ release process. Furthermore, ACh and ATP partially discharged the IP3-sensitive Ca2+ store, and a subsequent exposure to thapsigargin (TG) induced further [Ca2+]i increase. However, exposure to TG depleted the store and a subsequent stimulation with ACh or ATP did not induce further [Ca2+]i increase, suggesting that ACh and ATP discharge the same storage site sensitive to TG. As in freshly isolated submandibular acinar cells, exposure to ionomycin and monensin following ACh or TG induced further [Ca2+]i increase, suggesting that IP3-insensitive stores exist in SMG-C6 cells. Ca2+ influx was activated by ACh, ATP, or TG, and was significantly inhibited by La3+, suggesting the involvement of store-operated Ca2+ entry (SOCE) pathway. These results indicate that in SMG-C6 cells: (i) Ca2+ release is triggered by muscarinic and P2Y-nucleotide receptor agonists through formation of IP3; (ii) both the IP3-sensitive and -insensitive Ca2+ stores are present; and (iii) Ca2+ influx is mediated by the store-operated Ca2+ entry pathway. We conclude that Ca2+ regulation in SMG-C6 cells is similar to that in freshly isolated SMG acinar cells; therefore, this cell line represents an excellent SMG cell model in terms of intracellular Ca2+ signaling.     

Effects of Hypoxia on the Catecholamine Release, Ca2+ Uptake, and Cytosolic Free Ca2+ Concentration in Cultured Bovine Adrenal Chromaffin Cells

The purpose of the present study is to clarify the effects of hypoxia on catecholamine release and its mechanism of action. For this purpose, using cultured bovine adrenal chromaffin cells, we examined the effects of hypoxia on high (55 mM) K(+)-induced increases in catecholamine release, in cytosolic free Ca2+ concentration ([Ca2+]i), and in 45Ca2+ uptake. Experiments were carried out in media preequilibrated with a gas mixture of either 21% O2/79% N2 (control) or 100% N2 (hypoxia). High K(+)-induced catecholamine release was inhibited by hypoxia to approximately 40% of the control value, but on reoxygenation the release returned to control levels. Hypoxia had little effect on ATP concentrations in the cells. In the hypoxic medium, [Ca2+]i (measured using fura-2) gradually increased and reached a plateau of approximately 1.0 microM at 30 min, whereas the level was constant in the control medium (approximately 200 nM). High K(+)-induced increases in [Ca2+]i were inhibited by hypoxia to approximately 30% of the control value. In the cells permeabilized by digitonin, catecholamine release induced by Ca2+ was unaffected by hypoxia. Hypoxia had little effect on basal 45Ca2+ uptake into the cells, but high K(+)-induced 45Ca2+ uptake was inhibited by hypoxia. These results suggest that hypoxia inhibits high K(+)-induced catecholamine release and that this inhibition is mainly the result of the inhibition of high K(+)-induced increases in [Ca2+]i subsequent to the inhibition of Ca2+ influx through voltage-dependent Ca2+ channels.     

The relationship between constitutive ATP release and its extracellular metabolism in isolated rat kidney glomeruli.

ATP and adenosine are important extracellular regulators of glomerular functions. In this study, ATP release from glomeruli suspension and its extracellular metabolism were investigated. Basal extraglomerular ATP concentration (1nM) increased several fold during inhibition of ecto-ATPase activity, reflecting the basal ATP release rate. Mechanical perturbation increased the amounts of ATP released from glomeruli. ATP added to glomeruli was almost completely degraded within 20 minutes. In that time, AMP was the main product of extracellular ATP metabolism. Significant accumulation of AMP was observed after 5 min (194 +/-16 microM) and 20 min (271 +/-11 microM), whereas at the same time concentration of adenosine was only 10 muM. A competitive inhibitor of ecto-5-nucleotidase alpha-beta-methylene-ADP (AOPCP), decreased extraglomerular ATP and adenosine concentration by 80% and 50%, respectively. Similarly, AMP (100 microM) also markedly reduced extraglomerular ATP accumulation, whereas IMP, its deamination product, was not effective. P1, P5-diadenosine pentaphosphate (Ap5A) - an inhibitor of ecto-adenylate kinase prevented significantly the disappearance of ATP from extraglomerular media caused by AMP. These findings demonstrate that the decrease in extracellular ATP concentration observed after addition of AOPCP or AMP is caused by the presence of ecto-adenylate kinase activity in the glomeruli. The enzyme catalyses reversible reaction 2ADP<->ATP+AMP, and a rise in the AMP concentration can lead to fall in ATP level. The present study provides evidence the extraglomerular accumulation of ATP reflects both release of ATP from glomeruli cells and its metabolism by ecto-enzymes. Our data suggest that AMP, produced from ATP in the Bowman's capsular space, might plays a dual role as a substrate for ecto-adenylate kinase and ecto-nucleotidase reactions being responsible for the regulation of intracapsular ATP and adenosine concentration. We conclude that AMP degrading and converting ecto-enzymes effectively determine the balance between ATP and adenosine concentration and thus the activation of P2 and/or adenosine receptors.     

Adenosine and catecholamine agonists speed the flagellar beat of mammalian sperm by a non-receptor-mediated mechanism

Activation of rapid motility apparently is one of the first steps of sperm capacitation and can be studied in vitro. Previously we found that 2-chloro-2'-deoxyadenosine or the catecholamine isoproterenol activates mouse sperm motility in vitro via a pathway mediated by cAMP that requires extracellular Ca2+, the atypical sperm adenylyl cyclase, and sperm-specific protein kinase A. We now show that several other adenosine analogs and catecholamines accelerate the flagellar beat of mouse and human sperm. Unexpectedly, the potent adenosine receptor agonist CGS21680 does not accelerate the beat, and the adenosine receptor antagonist DPCPX does not diminish the accelerating action of 2-chloro-2'-deoxyadenosine. The pharmacological profile for activation by catecholamines is also unusual. Both agonists and antagonists of beta-adrenergic receptors elevate the beat frequency. Moreover, both l-(-) and d-+ isomers of epinephrine, norepinephrine, and isoproterenol produce similar acceleration of the beat. In contrast, inhibitors of equilibrative nucleoside transporters effectively slow the onset of the accelerating action of adenosine analogs. Replacement of external Na+ with Li+ also diminishes the accumulation of cAMP and slows the resultant accelerating action of 2-chloro-2'-deoxyadenosine, suggesting the involvement of a Na+-dependent concentrative nucleoside transporter. Our results show that adenosine and catecholamine agonists act in a novel signaling pathway that does not involve G protein-coupled cell-surface receptors that link to conventional adenylyl cyclases. Instead, adenosine and analogs may be transported into sperm via equilibrative and concentrative nucleoside transporters to act on unknown intracellular targets.