Adenylic dinucleotides produced by CD38 are negative endogenous modulators of platelet aggregation

Diadenosine 5',5'-P1,P2-diphosphate (Ap2A) is one of the adenylic dinucleotides stored in platelet granules. Along with proaggregant ADP, it is released upon platelet activation and is known to stimulate myocyte proliferation. We have previously demonstrated synthesis of Ap2A and of two isomers thereof, called P18 and P24, from their high pressure liquid chromatography retention time, by the ADP-ribosyl cyclase CD38 in mammalian cells. Here we show that Ap2A and its isomers are present in resting human platelets and are released during thrombin-induced platelet activation. The three adenylic dinucleotides were identified by high pressure liquid chromatography through a comparison with the retention times and the absorption spectra of purified standards. Ap2A, P18, and P24 had no direct effect on platelet aggregation, but they inhibited platelet aggregation induced by physiological agonists (thrombin, ADP, and collagen), with mean IC(50) values ranging between 5 and 15 mum. Moreover, the three dinucleotides did not modify the intracellular calcium concentration in resting platelets, whereas they significantly reduced the thrombin-induced intracellular calcium increase. Through binding to the purinergic receptor P2Y(11), exogenously applied Ap2A, P18, and P24 increased the intracellular cAMP concentration and stimulated platelet production of nitric oxide, the most important endogenous antiaggregant. The presence of Ap2A, P18, and P24 in resting platelets and their release during thrombin-induced platelet activation at concentrations equal to or higher than the respective IC(50) value on platelet aggregation suggest a role of these dinucleotides as endogenous negative modulators of aggregation.     

Coexpression of several types of metabotropic nucleotide receptors in single cerebellar astrocytes

We have examined the expression of mRNA for several P2Y nucleotide receptors by northern blot analysis in purified type 1 cerebellar astrocyte cultures. These results suggest that different P2Y subtypes could be responsible for ATP metabotropic calcium responses in single type 1 astrocytes. To identify these subtypes we have studied the pharmacological profile of ATP calcium responses using fura-2 microfluorimetry. All tested astrocytes responded to ATP and UTP stimulations evoking similar calcium transients. Most astrocytes also responded to 2-methylthioATP and ADP challenges. The agonist potency order was 2-methylthioATP > ADP > ATP = UTP. Cross-desensitization experiments carried out with ATP, UTP, and 2-methylthioATP showed that 2-methylthioATP and UTP interact with different receptors, P2Y(1) and P2Y(2) or P2Y(4). In a subpopulation of type 1 astrocytes, ATP prestimulation did not block UTP responses, and UDP elicited clear intracellular Ca(2+) concentration responses at very low concentrations. 2-MethylthioATP and UTP calcium responses exhibited different sensitivity to pertussis toxin and different inhibition patterns in response to P2 antagonists. The P2Y(1)-specific antagonist N:(6)-methyl-2'-deoxyadenosine 3', 5'-bisphosphate (MRS 2179) specifically blocked the 2-methylthio-ATP responses. We can conclude that all single astrocytes coexpressed at least two types of P2Y metabotropic receptors: P2Y(1) and either P2Y(2) or P2Y(4) receptors. Moreover, 30-40% of astrocytes also coexpressed specific pyrimidine receptors of the P2Y(6) subtype, highly selective for UDP coupled to pertussis-toxin insensitive G protein.     

Agonists of the P2Y(AC)-receptor activate MAP kinase by a ras-independent pathway in rat C6 glioma

We have previously shown that an ecto-NPPase modulates the ATP- and ADP-mediated P2Y(AC)-receptor activation in rat C6 glioma. In the present study, 2MeSADP and Ap(3)A induced no detectable PI turnover and were identified as specific agonists of the P2Y(AC)-receptor with EC(50) values of 250 +/- 37 pM and 1 +/- 0.5 microM, respectively. P2Y(AC)-receptor stimulation increased MAP kinase (ERK1/2) activation that returned to the basal level 4 h after stimulation and was correlated with a gradual desensitization of the P2Y(AC)-purinoceptor. The purinoceptor antagonists DIDS and RB2 blocked MAP kinase activation. An IP(3)-independent Ca(2+)-influx was observed after P2Y(AC)-receptor activation. Inhibition of this influx by Ca(2+)-chelation, did not affect MAP kinase activation. Pertussis toxin, toxin B, selective PKC-inhibitors and a specific MEK-inhibitor inhibited the 2MeSADP- and Ap(3)A-induced MAP kinase activation. In addition, transfection with dominant negative RhoA(Asn19) rendered C6 cells insensitive to P2Y(AC)-receptor-mediated MAP kinase activation whereas dominant negative ras was without effect. Immunoprecipitation experiments indicated a significant increase in the phosphorylation of raf-1 after P2Y(AC)-receptor activation. We may conclude that P2Y(AC)-purinoceptor agonists activate MAP kinase through a G(i)-RhoA-PKC-raf-MEK-dependent, but ras- and Ca(2+)-independent cascade.     

Existence of high and low affinity dinucleotides pentaphosphate-induced calcium responses in individual synaptic terminals and lack of correlation with the distribution of P2X1-7 subunits

Individual analysis of synaptic terminals calcium responses, induced by dinucleotides pentaphosphate, Ap(5)A or Gp(5)G, demonstrates the presence of two main groups considering the concentration required for stimulation. The first group corresponds to those responding to Ap(5)A or Gp(5)G at nanomolar concentration, representing 16% and 12%, respectively, and the second one responds to micromolar concentration and represents, respectively, 17% and 14%, of the total functional synaptosomal population in rat midbrain. Dose-response curves in single terminals showed an Ap(5)A EC(50) values of 0.9+/-0.2 nM and 11.8+/-0.9 microM, being the maximal intrasynaptosomal calcium increase of 200+/-0.3 and 125+/-0.2 nM for the high and low affinity responding terminals, respectively. Combination of microfluorimetric and immunocytochemical studies showed lack of correlation between dinucleotides pentaphosphate responses and P2X receptor subunits expression, in spite of the abundance of P2X(2), P2X(3) and P2X(7) at the presynaptic level in rat midbrain synaptosomes. Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a P2X receptors antagonist, showed no effect on low affinity dinucleotides receptors population, and partial inhibition on the high affinity one. On the other hand, diinosine pentaphosphate (Ip(5)I) completely abolished the low affinity dinucleotides responses, and 60% inhibition of the high affinity ones.     

Presence of functional ATP and dinucleotide receptors in glutamatergic synaptic terminals from rat midbrain

Glutamatergic terminals from rat midbrain were characterized by immunolocalization of synaptophysin and the vesicular glutamate transporters, either VGLUT1 or VGLUT2. Terminals containing these markers represent about 31% (VGLUT1) and 16% (VGLUT2) of the total synaptosomal population. VGLUT1-positive glutamatergic terminals responded to ATP or P1,P 5-di(adenosine-5') pentaphosphate (Ap5A) with an increase in the intrasynaptosomal calcium concentration as measured by a microfluorimetric technique in single synaptosomes. Roughly 20% of the VGLUT1-positive terminals responded to ATP, 13% to Ap5A and 11% to both agonists. Finally 56% of the terminals labeled with the anti-VGLUT1 antibody did not show any calcium increase in response to ATP or Ap5A. A similar response distribution was also observed in the VGLUT2-positive terminals. The Ca2+ responses induced by ATP and Ap5A in the glutamatergic terminals could be selectively inhibited by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 80 micro m) and P1,P 5-di(inosine-5') pentaphosphate (Ip5I, 100 nm), respectively. Both ATP and Ap5A, once assayed in the presence of extrasynaptosomal calcium, were able to induce a concentration-dependent glutamate release from synaptosomal populations, EC50 values being 21 micro m and 38 micro m for ATP and Ap5A, respectively. Specific inhibition of glutamate release was obtained with PPADS on the ATP effect and with Ip5I on the dinucleotide response, indicating that separate receptors mediate the secretory effects of both compounds.     

Differential effects of UTP, ATP, and adenosine on ciliary activity of human nasal epithelial cells

The purinergic regulation of ciliary activity was studied using small, continuously superfused explants of human nasal epithelium. The P2Y(2) purinoceptor (P2Y(2)-R) was identified as the major purinoceptor regulating ciliary beat frequency (CBF); UTP (EC(50) = 4.7 microM), ATP, and adenosine-5'-O-(3-thiotriphosphate) elicited similar maximal responses, approximately twofold over baseline. ATP, however, elicited a post-peak sustained plateau in CBF (1.83 +/- 0.1-fold), whereas the post-peak CBF response to UTP declined over 15 min to a low-level plateau (1.36 +/- 0.16-fold). UDP also stimulated ciliary beating, probably via P2Y(6)-R, with a maximal effect approximately one-half that elicited by P2Y(2)-R stimulation. Not indicated were P2Y(1)-R-, P2Y(4)-R-, or P2Y(11)-R-mediated effects. A(2B)-receptor agonists elicited sustained responses in CBF approximately equal to those from UTP/ATP [5'-(N-ethylcarboxamido)adenosine, EC(50) = 0.09 microM; adenosine, EC(50) = 0.7 microM]. Surprisingly, ADP elicited a sustained stimulation in CBF. The ADP effect and the post-peak sustained portion of the ATP response in CBF were inhibited by the A(2)-R antagonist 8-(p-sulfophenyl)theophylline. Hence, ATP affects ciliary activity through P2Y(2)-R and, after an apparent ectohydrolysis to adenosine, through A(2B)AR.     

Y1 receptors for neuropeptide Y are coupled to mobilization of intracellular calcium and inhibition of adenylate cyclase

Two types of binding sites have previously been described for neuropeptide Y (NPY), called Y1 and Y2 receptors. The intracellular events following Y1 receptor activation was studied in the human neuroblastoma cell line SK-N-MC. Both NPY and the specific Y1 receptor ligand, [Leu31,Pro34]-NPY, caused a rapid and transient increase in the concentration of free calcium in the cytoplasm as measured by the fluorescent probe, Fura-2. The effect of both peptides was independent of extracellular calcium as addition of EGTA or manganese neither changed the size nor the shape of the calcium response. The calcium response to NPY was abolished by pretreatment with thapsigargin, which can selectively deplete a calcium store in the endoplasmic reticulum. Y1 receptor stimulation, by both NPY and [Leu31,Pro34]NPY, also inhibited the forskolin-stimulated cAMP production with an EC50 of 3.5 nM. There was a close relation between the receptor binding and the cellular effects as half-maximal displacement of [125I-Tyr36]monoiodoNPY from the receptor was obtained with 2.1 nM NPY. The Y2-specific ligand NPY(16-36)peptide had no effect on either intracellular calcium or cAMP levels in the SK-N-MC cells. It is concluded that Y1 receptor stimulation is associated with both mobilization of intracellular calcium and inhibition of adenylate cyclase activity.     

Identification and characterization of adenosine 5'-tetraphosphate in human myocardial tissue

Endocrine functions of the human heart have been studied extensively. Only recently, nucleotidergic mechanisms have been studied in detail. Therefore, an isolation strategy was developed to isolate novel nucleotide compounds from human myocardium. The human myocardial tissue was fractionated by several chromatographic studies. A substance purified to homogeneity was identified as adenosine 5'-tetraphosphate (Ap(4)) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), post-source decay MALDI MS, and enzymatic cleavage analysis. Furthermore, Ap(4) was also identified in ventricular specific granules. In the isolated perfused rat heart, Ap(4) elicited dose-dependent vasodilations. Vasodilator responses were abolished in the presence of the P(2Y1) receptor antagonist MRS 2179 (1 microm) or the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (50 microm). After removal of the endothelium by Triton X-100, Ap(4) induced dose-dependent vasoconstrictions. Inhibition of P(2X) receptors by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (30 microm) or desensitization of P(2X) receptors by alpha,beta-methylene ATP (alpha,beta-meATP, 1 microm) diminished these vasoconstrictor responses completely. In the present study Ap(4) has been isolated from human tissue. Ap(4) was shown to exist in human myocardial tissue and was identified in ventricular specific granules. In coronary vasculature the nucleotide exerted vasodilation via endothelial P(2Y1) receptors and vasoconstriction via P(2X) receptors on vascular smooth muscle cells. Ap(4) acts as an endogenous extracellular mediator and might contribute to the regulation of coronary perfusion.     

Single GABAergic synaptic terminals from rat midbrain exhibit functional P2X and dinucleotide receptors, able to induce GABA secretion

GABAergic terminals from rat midbrain characterized by immunolocalization of glutamic acid decarboxylase and/or the vesicular inhibitory amino acid transporter respond to ATP or P(1),P(5)-di(adenosine-5') pentaphosphate (Ap(5)A) with an increase in the intrasynaptosomal calcium concentration measured by a microfluorimetric technique in single synaptic terminals. The ATP response is mediated through the activation of P2X receptors with an abundant presence of P2X(3) subunits. Ap(5)A, however, exerts its effects by acting through a different receptor termed the dinucleotide receptor. Both receptors, once activated in the presence of extrasynaptosomal calcium, induce a concentration-dependent GABA release from synaptosomal populations with EC(50) values of 16 and 20 microM for ATP and Ap(5)A, respectively. Specific inhibition of GABA release is obtained with pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (80 microM) on the ATP effect and with P(1),P(5)-di(inosine-5') pentaphosphate (100 nM) on the dinucleotide receptor.     

Catabolism of diadenosine 5',5"'-P1,P4-tetraphosphate in procaryotes. Purification and properties of diadenosine 5',5"'-P1,P4-tetraphosphate (symmetrical) pyrophosphohydrolase from Escherichia coli K12

Enzymatic activity which hydrolyzes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) yielding ADP has been identified in extracts of eubacteria, Escherichia coli and Acidaminococcus fermentans, and of a highly thermophilic archaebacterium, Pyrodictum occultum. Specific Ap4A (symmetric) pyrophosphohydrolase from Escherichia coli K12 has been purified almost 400-fold. The preparation was free of phosphatase, ATPase, phosphodiesterase, AMP-nucleosidase, and adenylate kinase. The Ap4A pyrophosphohydrolase molecular weight estimated by gel filtration is 27,000 +/- 1,000. Activity maximum is at pH 8.3. The Km value computed for Ap4A is 25 +/- 3 microM. The sulfhydryl group(s) is essential for enzyme activity. Metal chelators, EDTA, and o-phenanthroline, inhibit Ap4A hydrolysis; I0.5 values are 3 and 50 microM, respectively. Co2+ is a strong stimulator with an almost 100-fold increase in rate of Ap4A hydrolysis and a plateau in the range of 100-500 microM Co2+, when compared with the nonstimulated hydrolysis. Other transition metal ions, Mn2+, Cd2+, and Ni2+, stimulate by factors of 8, 3.5, and 3.5, respectively, with optimal concentrations in the range 200-500, 2-5, and 4-8 microM, respectively. Zn2+, Cu2+, and Fe2+, up to 30 microM, are without effect and they inhibit at higher concentrations. Mg2+ or Ca2+, in the absence of other divalent metal ions, are weak stimulators (1.5-fold stimulation occurs at 1-2 mM concentration), but act synergistically with Co2+ at its suboptimal concentrations. Stimulation in the presence of 10 microM Co2+ and either 1 mM MgCl2 or CaCl2 increases up to 75-fold. The same degree of synergy is found at 10 microM Co2+ and either 2-5 mM spermidine or 0.5-1.5 mM spermine. Besides Ap4A, bacterial Ap4A pyrophosphohydrolase hydrolyzes effectively Ap5A and Gp4G, and, to some extent, p4A, Ap6A, and Ap3A yielding in each case corresponding nucleoside diphosphate as one of the products.     

Dinucleotide polyphosphates contribute to purinergic signalling via inhibition of adenylate kinase activity

Dinucleoside polyphosphates are well described as direct vasoconstrictors and as mediators with strong proliferative properties, however, less is known about their effects on nucleotide-converting pathways. Therefore, the present study investigates the effects of Ap(4)A (diadenosine tetraphosphate), Up(4)A (uridine adenosine tetraphosphate) and Ap(5)A (diadenosine pentaphosphate) and the non-selective P2 antagonist suramin on human serum and endothelial nucleotide-converting enzymes. Human serum and HUVECs (human umbilical vein endothelial cells) were pretreated with various concentrations of dinucleotide polyphosphates and suramin. Adenylate kinase and NDP kinase activities were then quantified radiochemically by TLC analysis of the ATP-induced conversion of [(3)H]AMP and [(3)H]ADP into [(3)H]ADP/ATP and [(3)H]ATP respectively. Endothelial NTPDase (nucleoside triphosphate diphosphohydrolase) activity was additionally determined using [(3)H]ADP and [(3)H]ATP as preferred substrates. Dinucleoside polyphosphates and suramin have an inhibitory effect on the serum adenylate kinase [pIC(50) values (-log IC(50)): Ap(4)A, 4.67+/-0.03; Up(4)A, 3.70+/-0.10; Ap(5)A, 6.31+/-0.03; suramin, 3.74+/-0.07], as well as on endothelial adenylate kinase (pIC(50) values: Ap(4)A, 4.17+/-0.07; Up(4)A, 2.94+/-0.02; Ap(5)A, 5.97+/-0.04; suramin, 4.23+/-0.07), but no significant effects on serum NDP kinase, emphasizing the selectivity of these inhibitors. Furthermore, Ap(4)A, Up(4)A, Ap(5)A and suramin progressively inhibited the rates of [(3)H]ADP (pIC(50) values: Ap(4)A, 3.38+/-0.09; Up(4)A, 2.78+/-0.06; Ap(5)A, 4.42+/-0.11; suramin, 4.10+/-0.07) and [(3)H]ATP (pIC(50) values: Ap(4)A, 3.06+/-0.06; Ap(5)A, 3.05+/-0.12; suramin, 4.14+/-0.05) hydrolyses by cultured HUVECs. Up(4)A has no significant effect on the endothelial NTPDase activity. Although the half-lives for Ap(4)A, Up(4)A and Ap(5)A in serum are comparable with the incubation times of the assays used in the present study, secondary effects of the dinucleotide metabolites are not prominent for these inhibitory effects, since the concentration of metabolites formed are relatively insignificant compared with the 800 mumol/l ATP added as a phosphate donor in the adenylate kinase and NDP kinase assays. This comparative competitive study suggests that Ap(4)A and Ap(5)A contribute to the purinergic responses via inhibition of adenylate-kinase-mediated conversion of endogenous ADP, whereas Up(4)A most likely mediates its vasoregulatory effects via direct binding-mediated mechanisms.     

NPY, NPY receptors, and DPP IV activity are modulated by LPS, TNF-alpha and IFN-gamma in HUVEC

Since NPY increases endothelial cell (EC) stickiness for leukocytes, we studied the effects of LPS, TNF-alpha and IFN-gamma on its expression and action in HUVEC. Cytokines raised NPY and pro-NPY intracellular content and dipeptidyl peptidase IV (DPP IV) activity. Y1 and Y2 receptors were expressed in basal conditions, and LPS, TNF-alpha and IFN-gamma induced Y5 receptor expression with a concomitant extinction of Y2 receptor expression. NPY induced an intracellular calcium increase mainly mediated by Y2 and Y5 receptors in basal conditions. After stimulation with LPS, TNF-alpha and IFN-gamma, calcium increase was mainly caused by Y5 receptor. The modulation of the NPY system by LPS, TNF-alpha and IFN-gamma, and the NPY-induced calcium signaling suggest a role for NPY during the inflammatory response.     

Alpha 1-adrenergic modulation of metabotropic glutamate receptor-induced calcium oscillations and glutamate release in astrocytes

Astrocytic responses to activation of metabotropic glutamate receptors group I (mGluRs I) and alpha(1)-adrenoreceptors in cultured cells have been assessed using spectral analyzes and calcium imaging. Concentration-dependent changes were observed after stimulation with the mGluR I agonist (S)-3,5-dihydroxyphenylglycine (DHPG). These responses changed from a regular low frequency signal with sharp peaks at 1 microm to a pronounced stage of irregularity at 10 microm. After stimulation with 100 microm the signal was again homogenous in shape and regularity but occurred at a higher frequency. In contrast, the spectral properties after stimulation with the alpha(1)-adrenoreceptor agonist phenylephrine, exhibited considerable variation for all investigated concentrations. DHPG-induced increases in [Ca(2+)](i) were also associated with astroglial glutamate release, whereas no release was observed after noradrenergic stimulation. Both DHPG-mediated calcium signaling and glutamate release were inhibited by preincubation with 10 or 100 microm phenylephrine. Collectively, the present investigation provides new information about the spatial-temporal characteristics of astroglial intracellular calcium responses and demonstrates distinct differences between noradrenergic and glutamatergic receptors regarding intracellular calcium signaling and coupling to glutamate release. The noradrenergic modulation of DHPG-induced responses indicates that intracellular astroglial processes can be regulated in a bi-directional feedback loop between closely connected astrocytes and neurons in the central nervous system.     

ATP and its metabolite adenosine act synergistically to mobilize intracellular calcium via the formation of inositol 1,4,5-trisphosphate in a smooth muscle cell line.

Interactions between ATP and adenosine on the formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and mobilization of intracellular calcium were investigated in the smooth muscle cell line DDT1 MF-2. Activation of adenosine A1 receptors with adenosine or cyclopentyladenosine (CPA) or of nucleotide receptors with ATP increased both Ins(1,4,5)P3 formation and intracellular calcium concentrations. The A1 receptor-induced Ins(1,4,5)P3 formation (EC50 10 nM) was antagonized by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and by pretreatment of the cells with pertussis toxin (PTX). ATP-stimulated Ins(1,4,5)P3 formation (EC50 21 microM) was attenuated, but still present, after PTX treatment. ATP and CPA had supraadditive effects on Ins(1,4,5)P3 accumulation and CPA increased ATP-induced Ins(1,4,5)P3 accumulation in a concentration-dependent manner with an EC50 of 3 nM, a concentration which per se had little or no effect on Ins(1,4,5)P3 accumulation. ATP (EC50 4 microM) and CPA (EC50 4 nM) both increased intracellular calcium levels. The effect of ATP was partially sensitive to PTX treatment, whereas the effect of CPA was blocked both by PTX and by DPCPX. Concentrations of ATP and CPA that by themselves were insufficient to raise intracellular calcium were able to do so when combined. The synergy between ATP and CPA on the mobilization of intracellular calcium was abolished after treatment of cells with PTX or when DPCPX was included in the experiment. Since ATP was metabolized by ecto-enzymes to ADP, AMP, and adenosine, we also examined whether adenosine formed from ATP could enhance the ATP effects on Ins(1,4,5)P3 accumulation. Indeed, the addition of the A1 receptor antagonist DPCPX or removal of endogenous adenosine by inclusion of adenosine deaminase in the experimental medium significantly attenuated the ATP response, and the two treatments did not have additive effects. The present study thus demonstrates that in a clonal cell line two types of receptors increase phospholipase C activity, but via different pathways; nucleotide receptors appeared to act via partially PTX-insensitive, and A1 receptors via PTX-sensitive G-proteins. ATP and CPA are not only able per se to induce formation of Ins(1,4,5)P3 and mobilize intracellular calcium, but they also act synergistically. Finally, it is demonstrated that endogenous adenosine, possibly formed from the rapid breakdown of ATP, can significantly enhance some ATP effects.     

In human and rat lung membranes [35S]GTPgammaS binding is a tool for pharmacological characterization of G protein-coupled dinucleotide receptors.

The P2Y receptor family is activated by extracellular nucleotides such as ATP and UTP. P2Y receptors regulate physiological functions in numerous cell types. In lung, the P2Y2 receptor subtype plays a role in controlling Cl- and fluid transport. Besides ATP or UTP, also diadenosine tetraphosphate (Ap4A), a stable nucleotide, seems to be of physiological importance. In membrane preparations from human and rat lung we applied several diadenosine polyphosphates to investigate whether they act as agonists for G protein-coupled receptors. We assessed this by determining the stimulation of [35S]GTPgammaS binding. Stimulation of [35S]GTPgammaS binding to G proteins has already been successfully applied to elucidate agonist binding to various G protein-coupled receptors. Ap(n)A (n = 2 to 6) enhanced [35S]GTPgammaS binding similarly in human and rat lung membranes, an indication of the existence of G protein-coupled receptor binding sites specific for diadenosine polyphosphates. Moreover, in both human and rat lung membranes comparable pharmacological properties were found for a diadenosine polyphosphate ([3H]Ap4A) binding site. The affinity for Ap2A, Ap3A, Ap4A, Ap5A, and Ap6A was also comparable. 8-Diazido-Ap4A and ATP were less potent, whereas the pyrimidine nucleotide UTP showed hardly any affinity. Thus, we present evidence that different diadenosine polyphosphates bind to a common G protein-coupled receptor binding site in membranes derived either from human or rat lung.     

Extracellular ATP induces Ca2+ transients in cardiac myocytes which are potentiated by norepinephrine

Isolated rat ventricular cardiac myocytes loaded with the fluorescent calcium indicator fura2 showed significant changes in intracellular calcium concentrations upon exposure to greater than 1 microM ATP (EC50 = 7.4 +/- 1.3 microM, n = 4, SE), suggesting that extracellular ATP may have an important influence on myocardial contractility. The response was found to be highly ATP specific and required extracellular calcium. Furthermore, 30 s pretreatment of the cells with 0.2-1 microM norepinephrine decreased the concentration of ATP required for the Ca2+ transient, shifting the EC50 for ATP to 1.7 +/- 0.1 microM (n = 3, SE). beta-Propranolol (a beta 1-receptor antagonist) prevented potentiation, whereas phentolamine (an alpha 1-receptor antagonist) did not, indicating that regulation is through the beta 1-adrenergic receptor. ATP and norepinephrine released locally from sympathetic neurons may act in concert through the ATP and beta 1-adrenergic receptors to regulate myocardial calcium homeostasis.     

Calcium involvement in the muscarinic response of the gastric parietal cell

The influence of extracellular Ca2+ on the mediation of carbachol stimulation in isolated rabbit gastric parietal cells was studied. Removing Ca2+ from extracellular medium caused a 42% decrease of the aminopyrine accumulation due to carbachol with the same EC50 value (approximately 5 microM). A short time depletion in extracellular calcium suppressed the carbachol-dependent Ca2+ influx without affecting Ca2+ release from internal stores (fura-2 measurements). Similarly, the production of inositol phosphates under cholinergic stimulation was reduced by 29%. A rapid increase in Ins(1,4,5)P3 was obtained 5 s after carbachol stimulation, and this increase was not changed in Ca2(+)-depleted medium. In contrast, a 20 min incubation with carbachol caused a 50% reduction in both basal and carbachol-stimulated inositol phosphate accumulations. In conclusion, phospholipase C activation, intracellular Ca2+ release and aminopyrine accumulation were sequentially observed following carbachol stimulation of the isolated gastric parietal cell and extracellular calcium contributed to sustain this acid secretory response.     

5-Hydroxytryptamine2B receptors stimulate Ca2+ increases in cultured astrocytes from three different brain regions

The expression of 5-hydroxytryptamine-2B (5-HT2B) receptor mRNA has recently been shown in cultured astrocytes. Here the expression of functional 5-HT2B receptors has been studied in cultured astrocytes from rat cerebral cortex, hippocampus, and brain stem. Fluo-3- and fura-2-based microspectrofluorometry was used for measuring changes in intracellular free calcium concentrations ([Ca2+]i). The 5-HT2B agonist alpha-methyl 5-HT (40 nM) produced rapid transient increases in [Ca2+]i in astrocytes from all three brain regions studied, and these responses were blocked by the selective 5-HT2B antagonist rauwolscine (1 microM). The specificity of the responses to alpha-methyl 5-HT was further demonstrated by the failure of 4-(4-fluorobenzoyl)-1-(4-phenylbutyl)-piperidine oxalate (1 microM), a specific 5-HT2A/5-HT2C antagonist, to block these responses. The 5-HT2B-induced increases in [Ca2+]i persisted in Ca2+-free buffer, indicating that the increase in [Ca2+]i results from mobilization of intracellular Ca2+ stores. The expression of 5-HT2B receptors on astroglial cells was further verified immunohistochemically and by Western blot analysis. These results provide evidence of the existence of 5-HT2B receptors on astrocytes in primary culture.     

Stimulation of adenosine A1 and A2A receptors by AMP in the submucosal plexus of guinea pig small intestine

Actions of adenosine 5'-monophosphate (AMP) on electrical and synaptic behavior of submucosal neurons in guinea pig small intestine were studied with "sharp" intracellular microelectrodes. Application of AMP (0.3-100 microM) evoked slowly activating depolarizing responses associated with increased excitability in 80.5% of the neurons. The responses were concentration dependent with an EC(50) of 3.5 +/- 0.5 microM. They were abolished by the adenosine A(2A) receptor antagonist ZM-241385 but not by pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid, trinitrophenyl-ATP, 8-cyclopentyl-1,3-dimethylxanthine, suramin, or MRS-12201220. The AMP-evoked responses were insensitive to AACOCF3 or ryanodine. They were reduced significantly by 1) U-73122, which is a phospholipase C inhibitor; 2) cyclopiazonic acid, which blocks the Ca(2+) pump in intraneuronal membranes; and 3) 2-aminoethoxy-diphenylborane, which is an inositol (1,4,5)-trisphosphate receptor antagonist. Inhibitors of PKC or calmodulin-dependent protein kinase also suppressed the AMP-evoked excitatory responses. Exposure to AMP suppressed fast nicotinic ionotropic postsynaptic potentials, slow metabotropic excitatory postsynaptic potentials, and slow noradrenergic inhibitory postsynaptic potentials in the submucosal plexus. Inhibition of each form of synaptic transmission reflected action at presynaptic inhibitory adenosine A(1) receptors. Slow excitatory postsynaptic potentials, which were mediated by the release of ATP and stimulation of P2Y(1) purinergic receptors in the submucosal plexus, were not suppressed by AMP. The results suggest an excitatory action of AMP at adenosine A(2A) receptors on neuronal cell bodies and presynaptic inhibitory actions mediated by adenosine A(1) receptors for most forms of neurotransmission in the submucosal plexus, with the exception of slow excitatory purinergic transmission mediated by the P2Y(1) receptor subtype.