Purinergic Stimulation Research Articles

Extracellular ATP and ADP Activate Transcription Factor NF-κB and Induce Endothelial Cell Apoptosis

Inflammation within the vasculature is associated with endothelial cell (EC) perturbation, loss of vascular ATP-diphosphohydrolase activity, and platelet microthrombus formation with release of ATP and ADP into the micro-environment. The nature and effects of purinergic stimulation of EC under these circumstances remain largely undetermined. ATP and ADP activated EC transcribed mRNA from certain transcription factor NF-κB target genes and expressed E-selectin protein on cell membranes. Band shift analysis and reporter assays confirmed the activation of NF-κB in response to both ATP and ADP. Apoptosis was shown to occur in response to purinergic signaling, potentially through the activation of P2z/P2x7 receptors. Induction of EC activation responses and apoptosis in response to stimulation with ATP and ADP is associated with activation of NF-κB.

Impaired Cl−/HCO3− exchanger activity, associated with defective Cl− conductance, can be restored by purinergic stimulation in a cystic fibrosis pancreatic duct cell line (CFPAC-1)

Impaired Cl−/HCO3− exchanger activity, associated with defective Cl− conductance, can be restored by purinergic stimulation in a cystic fibrosis pancreatic duct cell line (CFPAC-1)

Ca 2+ dependent purinergic regulation of p42 and p44 MAP kinases in astroglial cultured cells

Adenosine triphosphate (ATP) is a signaling molecule for brain cells including astrocytes. In these cells, it has been shown that ATP stimulates myelin basic protein (MBP) kinase activity which is believed to represent the Erk family of MAP kinases. Indeed, we show that ATP activates simultaneously MBP kinase activity and phosphotyrosine incorporation in p42 Erk2 and p44 Erk1. Maximal effect of ATP is obtained at 50 microM after 5 min and disappears after 60 min. Effect of ATP is mimicked by 2-methylthio-ATP whereas alpha beta-methyleneadenosine 5' triphosphate (AMP-CPP) and adenosine do not promote any effect. Uridine triphosphate (UTP) activates also p42 and p44 MAP kinases. These observations indicate that p42-p44 MAP kinases activation can be obtained through P2v and P2u receptors. Purinergic stimulation of Erk is insensitive to pertussis toxin which inactivates heterotrimeric Gi protein. It is not inhibited by a PLA2 inhibitor (4 bromophenacyl bromide [B phi B]) and the PI3 kinase inhibitor, wortmannin. In contrast, purinergic stimulation of Erk is partially inhibited by the PKC inhibitor. GF109203X, at 5 microM and suppressed when extracellular calcium is complexed by ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

Calcium-dependence of hydrogen peroxide-induced c-fos expression and growth stimulation of multicellular prostate tumor spheroids

Hydrogen peroxide (H 2O 2) in nanomolar concentrations (20–100 nM) stimulated the growth of small (diameter 100±30 μm) multicellular prostate cancer spheroids and increased c-fos expression. H 2O 2 transiently raised [Ca 2+] i by Ca 2+ release from intracellular stores as the transient persisted in low (10 nM) Ca 2+ solution but was abolished when intracellular Ca 2+ stores were depleted by thapsigargin or chelation of [Ca 2+] i with BAPTA. The H 2O 2-induced [Ca 2+] i transient was furthermore inhibited by the P 2-purinoreceptor antagonists suramin and basilen blue, indicating that H 2O 2 may act via purinergic receptor stimulation. Treatment of spheroids with either suramin, basilen blue or BAPTA inhibited the H 2O 2-induced growth stimulation and c-fos expression, indicating that the H 2O 2-mediated growth stimulation of multicellular spheroids is mediated via a Ca 2+-dependent pathway.

Membrane-specific Regulation of Cl− Channels by Purinergic Receptors in Rat Submandibular Gland Acinar and Duct Cells

Measurement of [Cl-]i and the Cl- current in the rat salivary submandibular gland (SMG) acinar and duct cells was used to evaluate the role of Cl- channels in the regulation of [Cl-]i during purinergic stimulation. Under resting conditions [Cl-]i averaged 56 +/- 8 and 26 +/- 7 mM in acinar and duct cells, respectively. In both cells, stimulation with 1 mM ATP resulted in Cl- efflux and subsequent influx. Inhibition of NaKCl2 cotransport had no effect on [Cl-]i changes in duct cells and inhibited only about 50% of Cl- uptake in acinar cells. Accordingly, low levels of expression of NaKCl2 cotransporter protein were found in duct cells. Acinar cells expressed high levels of the cotransporter. Measurement of Cl- current under selective conditions revealed that acinar and duct cells express at least five distinct Cl- channels; a ClCO-like, volume-sensitive, inward rectifying, Ca2+-activated and CFTR-like Cl- currents. ATP acting on both cell types activated at least two channels, the Ca2+-activated Cl- channel and a Ca2+-independent glibenclamide-sensitive Cl--current, possibly cystic fibrosis transmembrane regulator (CFTR). Of the many nucleotides tested only 2'-3'-benzoylbenzoyl (Bz)-ATP and UTP activated Cl- channels in SMG cells. Despite their relative potency in increasing [Ca2+]i, BzATP in both SMG cell types largely activated the Ca2+-independent, glibenclamide-sensitive Cl- current, whereas UTP activated only the Ca2+-dependent Cl- current. We interpret this to suggest that BzATP and UTP largely activate Cl- channels residing in the membrane expressing the receptor for the active nucleotide. The present studies reveal a potentially new mechanism for transcellular Cl- transport in a CFTR-expressing tissue, the SMG. Coordinated action of the P2z (luminal) and P2u (basolateral) receptors can mediate part of the transcellular Cl- transport by acinar and duct cells to determine the final electrolyte composition of salivary fluid.

Characterization and Localization of P2 Receptors in Rat Submandibular Gland Acinar and Duct Cells

[Ca2+]i and the Cl- current were measured in isolated submandibular gland acinar and duct cells to characterize and localize the purinergic receptors expressed in these cells. In both cell types 2'-3'-benzoylbenzoyl (Bz)-ATP and ATP increased [Ca2+]i mainly by activation of Ca2+ influx. UTP had only minimal effect on [Ca2+]i at concentrations between 0.1 and 1 mM. However, a whole cell current recording showed that all nucleotides effectively activated Cl- currents. Inhibition of signal transduction through G proteins by guanyl-5'-beta-thiophosphate revealed that the effect of ATP on Cl- current was mediated in part by activation of a G protein-coupled and in part by a G protein-independent receptor. BzATP activated exclusively the G protein-independent portion, whereas UTP activated only the G protein-dependent portion of the Cl- current. Measurement of [Ca2+]i in the microperfused duct showed that ATP stimulated a [Ca2+]i increase when applied to the luminal or the basolateral sides. BzATP increased [Ca2+]i only when applied to the luminal side, whereas UTP at 100 microM increased -Ca2+-i only when applied to the basolateral side. The combined results suggest that duct and possibly acinar cells express P2z receptors in the luminal and P2u receptors in the basolateral membrane.

KATP channels contribute to beta- and adenosine receptor-mediated pulmonary vasorelaxation.

ATP-sensitive K+ (KATP) channels have been implicated in the regulation of vasomotor tone in aortic, mesenteric, and pulmonary vascular smooth muscle. Several investigators have described an association between KATP channels and isoproterenol (Iso)-stimulated relaxation responses. To study the relationship between receptor-dependent pulmonary vasorelaxation and KATP channels, we examined the response to agonists that generate adenosine 3',5'-cyclic monophosphate at two distinct levels of the signal transduction pathway after inhibition or activation of KATP channels in isolated rat pulmonary artery rings. Cumulative concentration responses to beta-adrenergic receptor stimulation (Iso), purinergic receptor stimulation [adenosine (Ado)], and direct stimulation of adenylate cyclase [forskolin (FSK)] were studied with and without concurrent inhibition of KATP channels (glibenclamide or tolbutamide). In addition, the effect of direct KATP channel activation (cromakalim) on the response to beta-adrenergic and purinergic receptor stimulation was determined. Last, we investigated the influence of KATP channel inhibition on endothelium-dependent and -independent mechanisms of pulmonary vasorelaxation linked to guanosine 3',5'-cyclic monophosphate production. KATP channel inhibition impaired the response to Iso and Ado. Activation of KATP channels caused a leftward shift in the dose responses of Iso and Ado, with a significant decrease in the 50% effective concentration for each agent. KATP channel inhibition did not impair the pulmonary arterial vasorelaxation response to FSK, acetylcholine, or sodium nitroprusside. KATP channels appear to contribute to beta-adrenergic and purinergic receptor-stimulated vasorelaxation in rat pulmonary arteries.

Purinergic agonists, but not cAMP, stimulate coupled granule fusion and Cl- conductance in HT29-Cl.16E.

Cl- conductance and capacitance were simultaneously measured in the mucin-secreting cell line, HT29-Cl.16E (Cl.16E), and its sister cell line, HT29-Cl.19A (Cl.19A), which lacks mucin granules. Purinergic stimulation by extracellular ATP transiently increased Cl- conductance in both cell lines with similar peak increases of 0.92 and 1.00 nS/pF in Cl.16E and Cl.19A cells, respectively (baseline of 0.08 nS/pF). Cell capacitance increased only in Cl.16E cells (17% above baseline of 22 pF in Cl.16E and 1% above baseline of 18 pF in Cl.19A cells). Wortmannin inhibited the purinergically activated Cl- conductance and capacitance increases in Cl.16E by 50 and 80%, respectively, but had no effects in Cl.19A cells. In Cl.16E cells, adenosine 3',5'-cyclic monophosphate (cAMP) signaling increased Cl- conductance from 0.08 to 0.52 nS/pF without changing capacitance. Cl- secretion in Cl.16E monolayers was additive in response to supramaximal stimulation of purinergic receptors and adenylyl cyclase, even though granule fusion is nine times greater with purinergic than adenylyl cyclase stimulation. In conclusion, 1) wortmannin does not directly inhibit activation of Cl- conductance, 2) at least 50% of purinergically activated Cl- conductance in Cl.16E is associated with granule fusion, and 3) cAMP-activated Cl- conductance is not associated with granules.

Elevated Cytoplasmic Sodium and Chloride Is Associated With Purinergic Receptor Stimulation of Rat Ventricular Myocytes: Implications For Myocardial Ischemia

During myocardial ischemia, several factors have been identified which contribute to the development of cardiac arrhythmias. These include membrane depolarization, increased cytosolic Na+ and decreased intracellular pH. This increase in intracellular Na+ has been shown to be associated with the development of ventricular arrhythmias. Changes in intracellular Cl- may also be involved in development of cardiac arrhythmias since decreasing extracellular Cl- concentration in the perfusion medium can prevent development of arrhythmias during myocardial ischemia in vitro. Extracellular ATP is known to increase in the heart during myocardial ischemia. ATP activation of p2 purinergic receptors on cardiac myocytes has been proposed to contribute to the initiation of the arrhythmias and ventricular fibrillations which characterize myocardial ischemia. Activation of p2 purinergic receptors results in membrane depolarization and decreased intracellular pH, thus reproducing some of the changes that occur during ischemia. We recently showed that activation of p2 purinergic receptors in both quiescent and electrically stimulated ventricular myocytes triggers spontaneous oscillatory contractions and Ca2+ transients.

Effects of P2 purinergic receptor stimulation in brown adipocytes.

Sympathetic stimulation of brown adipocytes plays a major role in body energy homeostasis by activating energy-wasting pathways. Sympathetic neuronal input initiates a variety of metabolic, developmental, and membrane responses in brown fat cells. Many of these actions are mediated by adrenergic pathways mobilized by released norepinephrine. However, since sympathetic stimulation may also release vesicular ATP, we tested brown fat cells for ATP responses. Micromolar concentrations of extracellular ATP had a number of effects on brown adipocytes. We have shown previously that ATP elicits substantial (average of approximately 30%) increases in cell membrane capacitance (P. A. Pappone and S. C. Lee, J. Gen. Physiol. 108: 393-404, 1996). Here, we show that cytosolic calcium levels were increased by ATP, both through release from intracellular stores and through influx, as assessed by fura 2 imaging. In addition, ATP indirectly activated a nonselective cation conductance that was independent of cytosolic calcium levels in patch voltage-clamped brown fat cells. Similar calcium, conductance, and capacitance responses could be activated by 2-methylthio-ATP and ADP, consistent with mediation by a P2 type purinergic receptor. Calorimetric measurements from cell suspensions showed that ATP increased basal heat production of isolated brown fat cells by approximately 40% but had no effect on the greater than fivefold increase in heat production seen with maximal adrenergic stimulation. These myriad responses to extracellular ATP suggest that P2 receptor-mediated signaling is important in brown adipocyte physiology and that sympathetic stimulation may normally activate purinergic as well as adrenergic pathways in brown fat.

Measurement of adenylylcyclase activity in the AV nodal region of the canine heart: evidence for inhibition by adenosine and acetylcholine.

Although it is essential to cardiac conduction, little is known about the biochemistry underlying postreceptor adrenergic, cholinergic and purinergic processes in the AV node. To study these mechanisms, we adapted a new and highly sensitive fluorometric assay for cyclic adenosine monophosphate (AMP) to characterize regional adenylylcyclase activity (cyclic AMP production in pmol/min/mg of protein) in membrane preparations made from 20-50 pieces of freeze-dried, 20-microm thick, microdissected samples of tissue from canine right atrium, the AV nodal region, and left ventricle. Basal and NaF-stimulated adenylylcyclase activity (mean +/- SEM, n = 6) were 7.2 +/- 0.4 and 72.4 +/- 7.5 in atrial, 15.6 +/- 1.3 and 58.8 +/- 4.7 in AV nodal, and 6.4 +/- 0.9 and 66.7 +/- 5.0 in ventricular tissues, respectively. Isoproterenol (10(-7)-10(-4) M) increased adenylylcyclase activity in a dose-dependent fashion in three different regions. The isoproterenol (10(-6) M)-stimulated adenylylcyclase activity (n = 6) was 14.4 +/- 1.3 in atrial, 21.9 +/- 1.6 in AV nodal and 13.4 +/- 1.4 in ventricular tissues. Adenosine (10(-3) M) and carbachol (10(-5) M) inhibited isoproterenol (10(-6) M)-stimulated adenylylcyclase activity to 10.1 +/- 1.1, 12.9 +/- 1.3 in atrial, 15.1 +/- 1.6, 15.5 +/- 1.2 in AV nodal, and 7.5 +/- 0.7, 11.9 +/- 1.2 in ventricular tissues, respectively. The results demonstrate that there are regional differences in adenylylcyclase activity under basal conditions and after adrenergic, purinergic, and cholinergic stimulation in the heart. Unlike adenosine, the inhibitory effects of cholinergic stimulation appear to be more specific for the AV node.

Evidence for a role of intracellular-calcium release in nitric oxide-stimulated relaxation of the rabbit corpus cavernosum.

Erection is mediated by relaxation of the smooth muscle elements within the sinusoids of the corpus cavernosum. Although cavernosal relaxation can be mediated by a variety of mechanisms including purinergic stimulation, prostoglandins, and beta-adrenergic stimulation the major mechanism involves the stimulated release of nitric oxide (NO) and subsequent relaxation of the corporal smooth muscle. Experimentally, NO can be released both by direct stimulation of NO-containing nerves (using field stimulation) and indirectly via cholinergic stimulation of NO release from the endothelium (using bethanechol). Preliminary studies have indicated that NO release and/or NO-stimulated relaxation of corporal smooth muscle is an active process involving both an increase in cytosolic calcium and an increase in metabolic energy utilization. Ryanodine is a pharmacological agent that can inhibit calcium-stimulated calcium release from the sarcoplasmic reticulum. The results of the current study demonstrated that ryanodine inhibited both field-stimulated relaxation and bethanechol-stimulated relaxation but did not affect relaxation induced by adenosine triphosphate (ATP) or nitroprusside. These studies strongly support the hypothesis that NO-stimulated relaxation is mediated, in part, by calcium release from the sarcoplasmic reticulum through ryanodine-sensitive channels.

An arginine analog inhibits responses of both the endothelium and smooth muscle of canine in situ vein grafts

Increased shear, pressure, and oxygen tension in vein grafts may alter production of endothelium-derived vasoactive and anti-mitogenic factors such as nitric oxide which subsequently affect development of neointimal hyperplasia. This study was designed to determine whether or not nitric oxide mediates endothelium-dependent responses in femoral in situ vein grafts. Non-reversed, canine femoral vein grafts were placed bilaterally to bypass a ligated segment of the femoral artery in dogs. After 6 weeks, the grafts were removed, cut into rings, and suspended in organ chambers for measurement of isometric force. In some rings the endothelium was removed deliberately. In the presence of indomethacin, the synthetic analog of l-arginine, l-N G-monomethyl-arginine (10 −4 M; l-NMMA) did not cause a significant change in baseline tension of rings with endothelium. l-NMMA reduced only contractions of rings with endothelium to the alpha-adrenergic agonist UK 14,304. The analog also reduced maximal relaxations to the calcium-ionophore, A23187 in rings with endothelium. In addition, l-NMMA reduced relaxations of rings without endothelium to adenosine diphosphate by 35%. Positive immunostaining for nitric oxide synthase was present in both the myointima and media of histological sections of grafts. In conclusion, these results suggest that in situ vein grafts exhibit two unique properties which are unlike unoperated arteries or veins: (i) alpha 2-adrenergic receptors may be coupled to the release of contractile endothelium-derived factors associated with production of nitric oxide; and (ii) nitric oxide may be released by the smooth muscle in response to purinergic stimulation. The presence of nitric oxide synthase throughout the wall of the graft may result in production of nitric oxide in response to adenosine diphosphate released by platelets and to circulating catecholamines. © 1997 The International Society for Cardiovascular Surgery.

An Arginine Analog Inhibits Responses of Both the Endothelium and Smooth Muscle of Canine in situ Vein Grafts

Increased shear, pressure, and oxygen tension in vein grafts may alter production of endothelium-derived vasoactive and anti-mitogenic factors such as nitric oxide which subsequently affect development of neointimal hyperplasia. This study was designed to determine whether or not nitric oxide mediates endothelium-dependent responses in femoral in situ vein grafts. Non-reversed, canine femoral vein grafts were placed bilaterally to bypass a ligated segment of the femoral artery in dogs. After 6 weeks, the grafts were removed, cut into rings, and suspended in organ chambers for measurement of isometric force. In some rings the endothelium was removed deliberately. In the presence of indomethacin, the synthetic analog of L-arginine, L-NG-monomethyl-arginine (10−4 M: L-NMMA) did not cause a significant change in baseline tension of rings with endothelium. L-NMMA reduced only contractions of rings with endothelium to the alpha-adrenergic agonist UK 14,304. The analog also reduced maximal relaxations to the calcium-ionophore, A23187 in rings with endothelium. In addition, L-NMMA reduced relaxations of rings without endothelium to adenosine diphosphate by 35%. Positive immunostaining for nitric oxide synthase was present in both the myointima and media of histological sections of grafts. In conclusion, these results suggest that in situ vein grafts exhibit two unique properties which are unlike unoperated arteries or veins: (i) alpha2-adrenergic receptors may be coupled to the release of contractile endothelium-derived factors associated with production of nitric oxide; and (ii) nitric oxide may be released by the smooth muscle in response to purinergic stimulation. The presence of nitric oxide synthase throughout the wall of the graft may result in production of nitric oxide in response to adenosine diphosphate released by platelets and to circulating catecholamines.

Electrophysiological consequences of purinergic receptor stimulation in isolated rat pulmonary arterial myocytes.

Neither the electrophysiological effects of purinergic receptor stimulation nor the role of ATP in regulating the tone of pulmonary arterial smooth muscle has been determined. Therefore, we investigated the effects of purine nucleotides on acutely dissociated smooth muscle cells from rat small pulmonary arteries using the patch-clamp recording technique. Extracellular application of ATP activated a fast transient inward current (which decayed in the continued presence of the nucleotide) and produced sustained periodic oscillations of predominantly inward current. Pharmacological and anion substitution experiments revealed that the transient inward current was carried by the movement of cations. In contrast, the periodic oscillations of current were due primarily to a Ca(2+)-activated Cl- current (ICl,Ca) dependent on the release of Ca2+ from intracellular stores. Experiments using ATP analogues revealed the following order of potency for activation of the fast transient inward current: 2-methylthio ATP (2-meSATP) > ATP > alpha,beta-methylene ATP (alpha,beta-meATP) > > ADP > UTP = adenosine. Cross desensitization was seen between applications of ATP, alpha,beta-meATP, and 2-meSATP, suggesting that these agonists act via a common site. The order of potency for activation of ICl,Ca was UTP = ATP > > ADP > or = 2-meSATP > alpha,beta-meATP = adenosine. Both the fast transient inward current and ICl,Ca evoked by ATP and its analogues were abolished by the nonselective P2 purinoceptor antagonist suramin. These results show the existence of P2x and P2U purinoceptor subtypes in pulmonary arterial smooth muscle cells. Stimulation of these receptors results in activation of a fast transient inward cation current and ICl,Ca, respectively. It is likely that ATP acts via these receptor subtypes to regulate pulmonary arterial tone under physiological or pathological conditions.

Protein kinase C induced calcium influx and sustained enhancement of ciliary beating by extracellular ATP

The major purpose of this work was to determine protein kinase C (PKC) influence on ciliary beat frequency (CBF) and to assess participation of PKC in purinergic ciliary stimulation. The experiments were performed by simultaneous measurement of [Ca 2+] i and CBF on tissue culture of frog esophagus epithelium. The PKC activators TPA and DiC 8 produced significant elevation of [Ca 2+] i and strong frequency enhancement. The calcium elevation was inhibited by lowering the extracellular calcium level, or by La 3+, but was unaffected by verapamil and the phospholipase C inhibitor U-73122, suggesting that Ca 2+ influx was via non-voltage-operated calcium channels. The inhibition of [Ca 2+] i elevation resulted in corresponding inhibition of CBF enhancement. The effect of TPA was blocked by the selective PKC inhibitor chelerythrine, calphostin C, and GF109203X, and by the enzyme downregulation. The downregulation of PKC, or the enzyme inhibitors did not affect the immediate response to extracellular ATP but caused rapid decay of initially stimulated [Ca 2+] i and CBF to the basal level. These results suggest that PKC produces CBF enhancement via activation of calcium influx through non-voltage-operated calcium channels. This calcium influx seems to be responsible for the duration of ciliary stimulation produced by the extracellular ATP.

Purinergic stimulation of rabbit ciliated airway epithelia: control by multiple calcium sources.

1. Simultaneous measurements of average intracellular calcium concentration ([Ca2+]i) and ciliary beat frequency (CBF) were carried out on ciliated rabbit tracheal cells in order to determine quantitatively the role of calcium in the regulation of mucus-transporting cilia. 2. Extracellular ATP caused a rapid increase in both [Ca2+]i and CBF in the 0.1-1000 microM concentration range. The rise in [Ca2+]i levelled off to an elevated [Ca2+]i plateau while the cilia remained in a high activation state. The magnitude of the rise in [Ca2+]i and CBF as well as the value of the elevated [Ca2+]i plateau and the value of the sustained CBF were dependent on the concentration of ATP in the solution. 3. No correlation was found between the mean values of [Ca2+]i and CBF at rest but a sigmoidal relationship was found to exist between the maximal rises of these parameters following excitation with extracellular ATP. This sigmoidal correlation incorporated the experiments where [Ca2+]i rise was induced by depletion of internal calcium stores with thapsigargin or by entry of calcium induced by ionomycin. 4. Extracellular ATP caused both the release of calcium from internal stores and calcium influx from the extracellular solution. The release of calcium was identified as originating from a thapsigargin-sensitive and a thapsigargin-insensitive calcium store. It is suggested that the release of calcium from these stores induces the initial rise in CBF. 5. The sustained activation of the cilia and elevated calcium plateau were found to be the result of the extracellular ATP-induced calcium influx. This calcium influx was insensitive to the voltage-gated calcium channel inhibitors verapamil and diltiazem, but was completely eliminated by lowering the extracellular calcium concentration to 0.1 microM. 6. We propose that the initial jump in the CBF is mediated by the calcium released from a thapsigargin-insensitive calcium store adjacent to the cilia, while the later, and longer, rise in CBF is the result of the calcium emanating from the thapsigargin-sensitive store which is positioned further away from the cilia within the cell cytoplasm. The calcium influx that follows is responsible for sustaining the cilia at a high level of excitation.

Purinergic receptor stimulation increases membrane trafficking in brown adipocytes.

Stimulation of brown adipocytes by their sympathetic innervation plays a major role in body energy homeostasis by regulating the energy-wasting activity of the tissue. The norepinephrine released by sympathetic activity acts on adrenergic receptors to activate a variety of metabolic and membrane responses. Since sympathetic stimulation may also release vesicular ATP, we tested brown fat cells for ATP responses. We find that micromolar concentrations of extracellular ATP initiates profound changes in the membrane trafficking of brown adipocytes. ATP elicited substantial increases in total cell membrane capacitance, averaging approximately 30% over basal levels and occurring on a time scale of seconds to minutes. The membrane capacitance increase showed an agonist sensitivity of 2-methylthio-ATP > or = ATP > ADP > > adenosine, consistent with mediation by a P2r type purinergic receptor. Membrane capacitance increases were not seen when cytosolic calcium was increased by adrenergic stimulation, and capacitance responses to ATP were similar in the presence and absence of extracellular calcium. These results indicate that increases in cytosolic calcium alone do not mediate the membrane response to ATP. Photometric assessment of surface-accessible membrane using the dye FM1-43 showed that ATP caused an approximate doubling of the amount of membrane actively trafficking with the cell surface. The discrepancy in the magnitudes of the capacitance and fluorescence changes suggests that ATP both activates exocytosis and alters other aspects of membrane handling. These findings suggest that secretion, mobilization of membrane transporters, and/or surface membrane expression of receptors may be regulated in brown adipocytes by P2r purinergic receptor activity.

Purinergic stimulation of rat cardiomyocytes induces tyrosine phosphorylation and membrane association of phospholipase Cγ: a major mechanism for InsP3 generation

Phospholipase C gamma (PLC gamma) expression and activation by a purinergic agonist were investigated in adult rat cardiomyocytes. PLC gamma is expressed in isolated cardiomyocytes. Stimulation of cells with extracellular ATP induces a rapid increase in membrane-associated PLC gamma immunoreactivity most probably due to redistribution of the lipase from the cytosol to the membrane. The purine triggers a significant phosphorylation on tyrosine residues of a cytosolic pool of PLC gamma with a time course that correlates with that of translocation. Extracellular ATP also increases intracellular Ins(1,4,5)P3 content. All these events (translocation and phosphorylation of PLC gamma, InsP3 formation) are blocked by genistein, a tyrosine kinase inhibitor. The purinergic effect on both PLC gamma translocation and phosphorylation are Ca-sensitive. We thus propose that the purinergic stimulation activates a non-receptor tyrosine kinase that phosphorylates PLC gamma in the presence of an increased Ca level and induces PLC gamma redistribution to the membrane. There, PLC gamma becomes activated leading to the hydrolysis of phosphatidylinositol diphosphate and in turn Ins(1,4,5)P3 formation. This cascade of events may play a significant role in the induction of arrhythmogenesis by purinergic agonists.

Differences in the responses to purinergic nerve stimulation and applied ATP in the guinea-pig vas deferens

The responses to sympathetic nerve stimulation and to applied ATP in the guinea-pig vas deferens were compared. Nifedipine (10 μ) markedly reduced the non-adrenergic neural contraction but only partially blocked the contractions produced by bath-applied ATP. Suramin (300 μM) also markedly reduced the contractile responses produced by nerve stimulation, but had no significant effect on the contractions produced by bath-applied ATP. Using intracellular recording techniques, nerve stimulation was shown to produce an excitatory junction potential which was abolished by suramin (1 μM). Ionophoretic application of ATP and bath-applied ATP also produced a depolarization. Suramin (1 μ) failed to abolish the response to bath-applied ATP and enhanced the ionophoretically induced depolarization. These results suggest either that ATP is not a transmitter in the vas deferens or that two classes of purinoceptor are present, one suramin-sensitive receptor which produces a contraction via the opening of voltage-dependent Ca z+ channels, and another which is suramin-resistant and produces a contraction by another means.