Antisecretory actions of a novel vasoactive intestinal polypeptide (VIP) antagonist in human and rat small intestine

Vasoactive intestinal peptide (VIP) has been demonstrated in intestinal mucosal neurones and elicits chloride secretion from enterocytes. These findings have led to the proposal that VIP is a secretomotor neurotransmitter. Confirmation of such a role may now be possible with the development of PG 97-269, a high-affinity, selective antagonist of VIP type 1 (VPAC1) receptor, which is expressed by gut epithelial cells. We have evaluated the VIP antagonism and antisecretory potential of this novel compound using in vitro and in vivo models of intestinal secretion. Monolayers of the human colonic cell line (T84) and muscle-stripped preparations of rat jejunum and human ileum were set up in Ussing chambers for recording of transepithelial resistance and short-circuit current. Ussing chambers were modified to allow electrical stimulation of mucosal neurones. Effects of PG 97-269 on enterotoxin-induced secretion were investigated in perfused rat jejunum in vivo. PG 97-269 competitively antagonised VIP in T84 monolayers. In rat jejunum and human ileum, responses to VIP were inhibited as were responses of rat jejunum to electrical stimulation of mucosal neurons. In perfused rat jejunum, PG 97-269 abolished the effects of VIP on fluid and electrolyte transport and attenuated cholera toxin and Escherichia coli heat labile toxin-induced net fluid and electrolyte secretion. PG 97-269 is a competitive antagonist of enterocyte VIP receptors and effectively inhibits responses of rat and human intestinal mucosa to VIP. Antagonism of secretory responses to electrical stimulation of mucosal neurons and lumenal application of enterotoxins imply a secretory role for VIP in these processes.     

The enteric purinergic P2Y1 receptor

Significant recent discoveries have shown that the P2Y(1) purinergic receptor subtype is expressed in the enteric nervous system and at intestinal neuromuscular junctions. Secretomotor neurons, which release vasoactive intestinal peptide at their junctions with intestinal secretory glands, express the P2Y(1) receptor. Synaptically released ATP acts at these P2Y(1) receptors to stimulate glandular secretion of electrolytes and H(2)O. Motor neurons in the enteric nervous system release ATP as an inhibitory neurotransmitter at neuromuscular junctions in the intestinal circular muscle coat; this action of ATP is mediated by the P2Y(1) receptor. The emerging evidence for significant involvement of P2Y(1) receptors in local enteric neural control and coordination of intestinal secretion and motility suggests that either the receptors themselves or steps in the post-P2Y(1) receptor signal transduction cascade might be potential therapeutic targets.     

Neurally released ATP mediates endothelium-dependent hyperpolarization in the circular smooth muscle cells of chicken anterior mesenteric artery

The object of the present study was to clarify the neurotransmitter(s) controlling membrane responses to electrical field stimulation (EFS) in the circular smooth muscle cells of first-order branches of chicken anterior mesenteric artery.EFS (five pulses at 20 Hz, 1 ms) evoked a hyperpolarization of amplitude--21.6+/-1.2 mV, total duration 21.8+/-1.2 s and latency 641.7+/-81.9 ms. The response was tetrodotoxin-sensitive and nonadrenergic noncholinergic (NANC) in nature.The NANC response was blocked by the nonspecific purinergic antagonist, suramin, indicating that the response is mediated by the neurotransmitter adenosine 5'-triphosphate (ATP).Either desensitization or blockade of P2Y receptor with its putative agonist 2-methylthioATP (1 microM for 30 min) or with its antagonist cibacron blue F3GA (10 microM), respectively, abolished the purinergic hyperpolarization. PPADS at concentrations up to 100 microM had no effect on the EFS-induced response, indicating that this response is mediated through P2Y, but not P2X, receptor. In addition, the response was completely abolished by two specific P2Y1 receptor antagonists, namely, MRS 2179 (300 nM) and A3P5PS (10 microM). Removal of the endothelium abolished the purinergic hyperpolarization, which was converted, in some preparations, to a small depolarization, indicating that the hyperpolarizing response is endothelium-dependent.The present study suggests that in first-order branches of chicken anterior mesenteric artery, ATP released from perivascular nerves may diffuse to the endothelium-activating P2Y1 receptor to induce release of an inhibitory substance that mediates hyperpolarization in the circular smooth muscle.     

An electrophysiological study of excitatory purinergic neuromuscular transmission in longitudinal smooth muscle of chicken anterior mesenteric artery

1. The object of the present study was to clarify the neurotransmitters controlling membrane responses to electrical field stimulation (EFS) in the longitudinal smooth muscle cells of the chicken anterior mesenteric artery. 2. EFS (5 pulses at 20 Hz) evoked a depolarization of amplitude 19.7+/-2.1 mV, total duration 29.6+/-3.1 s and latency 413.0+/-67.8 ms. This depolarization was tetrodotoxin (TTX)-sensitive and its amplitude was partially decreased by atropine (0.5 microM); however, its duration was shortened by further addition of prazosin (10 microM). 3. Atropine/prazosin-resistant component was blocked by the nonspecific purinergic antagonist, suramin, in a dose-dependent manner, indicating that this component is mediated by the neurotransmitter adenosine 5'-triphosphate (ATP). 4. Neither desensitization nor blocking of P2X receptor with its putative receptor agonist alpha,beta-methylene ATP (alpha,beta-MeATP, 1 microM) and its antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic (PPADS, up to 50 microM), had significant effect on the purinergic depolarization. In contrast, either desensitization or blocking of P2Y receptor with its putative agonist 2-methylthioATP (2-MeSATP, 1 microM) and its antagonist Cibacron blue F3GA (CBF3GA, 10 microM) abolished the purinergic depolarization, indicating that this response is mediated through P2Y but not P2X receptor. 5. The purinergic depolarization was inhibited by pertussis toxin (PTX, 600 ng ml(-1)). Furthermore, it was significantly inhibited by a phospholipase C (PLC) inhibitor, U-73122 (10 microM), indicating that the receptors involved in mediating the purinergic depolarization are linked to a PTX-sensitive G-protein, which is involved in a PLC-mediated signaling pathway. 6. Data of the present study suggest that the EFS-induced excitatory membrane response occurring in the longitudinal smooth muscle of the chicken anterior mesenteric artery is mainly purinergic in nature and is mediated via P2Y purinoceptors.     

Cannabinoids inhibit noradrenergic and purinergic sympathetic cotransmission in the rat isolated mesenteric arterial bed

BACKGROUND AND PURPOSE: Noradrenaline and ATP are sympathetic co-transmitters. In the rat perfused mesenteric bed cannabinoids have been shown to modify the overall response to sympathetic nerve stimulation. This study has assessed whether cannabinoid receptor activation modulates differentially the noradrenergic and purinergic components of sympathetic vasoconstriction. EXPERIMENTAL APPROACH: Rat mesenteric beds were perfused with physiological salt solution and the effects of cannabinoids on responses to nerve stimulation, or exogenous noradrenaline or alpha,beta-methylene ATP (alpha,beta-meATP; P2X receptor agonist) were determined after raising tone with U46619. The effects of cannabinoids on the noradrenaline and ATP components of sympathetic neurotransmission were assessed using the alpha 1-adrenoceptor antagonist, prazosin, or after P2X receptor desensitization with alpha,beta-meATP. KEY RESULTS: Anandamide, WIN 55,212-2 and CP55,940 attenuated sympathetic neurogenic vasoconstrictor responses. The inhibitory actions of anandamide and WIN 55,212-2 were blocked by LY320135, a CB1 receptor antagonist, but not by SR144528, a CB2 receptor antagonist. The inhibitory actions of CP55,940 were unaffected by LY320135 and SR144528. WIN 55,212-3, the inactive S(-) enantiomer of WIN 55,212-2, had no effect on sympathetic neurogenic responses. None of the cannabinoids affected contractile responses to exogenous noradrenaline or alpha,beta-meATP. Anandamide and WIN 55,212-2 inhibited both the noradrenaline and ATP components of the sympathetic neurogenic contractile responses, with effects on the ATP component being most marked. CONCLUSIONS AND IMPLICATIONS: These results indicate that prejunctional CB1-like receptors mediate the sympathoinhibitory action of anandamide and WIN 55,212-2, but not CP55,940, in the rat mesenteric bed. Cannabinoids inhibit both the noradrenergic and purinergic components of sympathetic neurotransmission.     

Potentiation of anaphylaxis in guinea pig ileal mucosa by a selective delta-opioid receptor agonist.

Immediate hypersensitivity reactions in the intestinal mucosa evoke active chloride secretion which enhances the elimination of luminal antigens. The prosecretory actions of histamine and other soluble mediators of anaphylaxis are mediated by submucosal neurons, as are the antisecretory actions of opioid antidiarrheal medications. We tested the hypothesis that the selective delta-opioid receptor agonist [D-Pen2, D-Pen5]enkephalin (DPDPE) alters anaphylaxis-associated ileal anion secretion in vitro. Sheets of ileal mucosa with attached submucosa from guinea pigs sensitized to cow's milk were mounted in Ussing chambers under short-circuit conditions. Mucosal sheets responded to the serosal application of the milk protein, beta-lactoglobulin, with a rapid rise in transepithelial short-circuit current (Isc); in contrast, the egg protein, ovalbumin, was without effect. Pretreatment of tissues with the neuronal conduction blocker, saxitoxin, or the H1 histamine receptor antagonist, diphenhydramine, but not the opioid receptor antagonist, naloxone, significantly reduced mucosal responses to antigen. [D-Pen-2, D-Pen5]enkephalin (0.1 microM, serosal addition) decreased baseline Isc, but potentiated mucosal responses to antigen; its effects were abolished in tissues pretreated with naloxone. These results suggest that immediate hypersensitivity reactions in the guinea pig ileal mucosa are mediated by submucosal neural circuits that are phasically modulated by both mast cell products and opioids.     

Inhibitory pathways in the circular muscle of rat jejunum

1. Conflicting data have been reported on the contribution of nitric oxide (NO) to inhibitory neurotransmission in rat jejunum. Therefore, the mechanism of relaxation and contribution to inhibitory neurotransmission of NO, adenosine 5'-triphosphate (ATP), vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) was examined in the circular muscle of Wistar-Han rat jejunum. 2. Mucosa-free circular muscle strips were precontracted with methacholine in the presence of guanethidine and exposed to electrical field stimulation (EFS) and exogenous NO, ATP, VIP and PACAP. All stimuli induced reduction of tone and inhibition of phasic motility. Only electrically induced responses were sensitive to tetrodotoxin (3 x 10(-6) m). 3. NO (10(-6)-10(-4) m)-induced concentration-dependent relaxations that were inhibited by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ; 10(-5) m) and the small conductance Ca(2+)-activated K(+)-channel blocker apamin (APA; 3 x 10(-8) m). 4. Relaxations elicited by exogenous ATP (10(-4)-10(-3) m) were inhibited by the P2Y purinoceptor antagonist reactive blue 2 (RB2; 3 x 10(-4) m), but not by APA and ODQ. 5. The inhibitory responses evoked by 10(-7) m VIP and 3 x 10(-8) m PACAP were decreased by the selective PAC(1) receptor antagonist PACAP(6-38) (3 x 10(-6) m) and APA. The VPAC(2) receptor antagonist PG99-465 (3 x 10(-7) m) reduced relaxations caused by VIP, but not those by PACAP, while the VPAC(1) receptor antagonist PG97-269 (3 x 10(-7) m) had no influence. 6. EFS-induced relaxations were inhibited by the NO-synthase inhibitor N(omega)-nitro-l-arginine methyl ester (3 x 10(-4) m), ODQ and APA, but not by RB2, PG97-269, PG99-465 and PACAP(6-38). 7. These results suggest that NO is the main inhibitory neurotransmitter in the circular muscle of Wistar-Han rat jejunum acting through a rise in cyclic guanosine monophosphate levels and activation of small conductance Ca(2+)-dependent K(+) channels.     

Pharmacological comparison of the effect of ibogaine and 18-methoxycoronaridine on isolated smooth muscle from the rat and guinea-pig

Ibogaine and 18-methoxycoronaridine are naturally occurring alkaloids reported to possess antiaddictive properties in several models of drug dependence. We have examined their effect at mu-opioid receptors regulating neurogenic contractions of several smooth muscle preparations and also against spontaneous contractions of the rat isolated portal vein. Ibogaine (pIC(50) 5.28) and 18-methoxycoronaridine (pIC(50) 5.05) caused a concentration-dependent inhibition of cholinergic contractions of the guinea-pig ileum which was not affected by the opioid receptor antagonist naloxone (1 microM). In the rat isolated vas deferens ibogaine and 18-methoxycoronaridine caused a concentration-dependent enhancement of purinergic contractions. Both agents (30 microM) caused a 3 - 5 fold rightward displacement of DAMGO-induced inhibition of purinergic contractions, but similar effects were observed for ibogaine against alpha(2)-adrenoceptor-mediated inhibition of neurogenic responses. In the guinea-pig isolated bladder both ibogaine (10 microM) and 18-methoxycoronaridine (10 microM) caused a 2 fold increase in the purinergic component of neurogenic contractions without significantly altering cholinergic contractions or responses to exogenous ATP. In contrast, ibogaine (1 - 30 microM), but not 18-methoxycoronaridine, caused a concentration-dependent enhancement of spontaneous contractions of the rat isolated portal vein. In summary, while ibogaine and 18-methoxycoronaridine modulated electrically-evoked contractions in the three preparations examined, we have no evidence for a selective interaction with pre-junctional mu-opioid receptors. The pronounced enhancement of purinergic contractions produced by both agents is a novel finding and worthy of further investigation.     

Effects of TAK-637 on NK(1) receptor-mediated mechanisms regulating colonic secretion

This study investigates the effect of a selective NK(1) receptor antagonist TAK-637 on enteric mechanisms involved in regulation of epithelial secretion in the colon. Mucosal sheets isolated from guinea-pig colon were placed in modified Ussing chambers and the net active transport of electrolytes was measured as short-circuit current (Isc). GR-73632, a selective NK(1) receptor agonist, induced an increase in basal Isc, which was inhibited by TAK-637 (IC(50) of 21 nM). The increase in Isc induced by GR-73632 was significantly attenuated by tetrodotoxin (TTX, 1 microM), indicating that TAK-637 inhibits neuronal NK(1) receptors. Moreover, TAK-637 reduced the TTX-resistant component of the response to GR-73632 suggesting that NK(1) receptors expressed by epithelial cells are inhibited by TAK-637. In separate experiments, TAK-637 partially inhibited the submaximal Isc induced by electrical field stimulation (EFS, 0.5 ms, 15 Hz) of enteric nerves or by activation of primary afferent fibers using capsaicin (50 microM). TAK-637 had no significant effect on the basal Isc or on responses induced by neurokinin A (NKA), senktide, or forskolin. The results imply that inhibition of peripheral NK(1) receptors may reduce autonomic epithelial secretion in response to activation of autonomic secretomotor pathways, while having no significant effect on basal epithelial transport.     

Enhancement of sympathetic purinergic neurotransmission in the guinea-pig isolated vas deferens by the novel ecto-ATPase inhibitor ARL 67156.

1. Field stimulation of the sympathetic nerves of the guinea-pig isolated vas deferens with trains of pulses of 20 s at 1-8 Hz produced characteristic biphasic contractions. The effect of the novel ecto-ATPase inhibitor, 6-N,N-diethyl-D-beta, gamma-dibromomethyleneATP (ARL 67156, formerly known as FPL 67156), on the magnitude of the initial, predominantly purinergic peak of this response was studied in order to determine the influence of enzymatic degradation of adenosine 5'-triphosphate (ATP) on its action as a neurotransmitter. 2. The peak magnitude of the response to nerve stimulation was significantly increased in a concentration-dependent manner by ARL 67156 (5-100 microM) and the size of the neurogenic response at 4 Hz was approximately doubled in the presence of ARL 67156 (100 microM). 3. ARL 67156 (100 microM) has a rapid onset of action. The enhancing effect on neurogenic contractions was maximal after 10 min, was well maintained for at least 30 min and was rapidly reversed, with responses returning to control levels 10 min after washout. 4. The neurogenic contraction in the presence of prazosin (0.1 microM) was purely purinergic, as it was abolished by the P2-purinoceptor antagonist, PPADS (100 microM). ARL 67156 (100 microM) produced a similar degree of enhancement of neurogenic responses in the absence and presence of prazosin, supporting the view that the enhancing effects of ARL 67156 on neurogenic contractions result from potentiation of the action of ATP. 5. Exogenous ATP and alpha, beta-methyleneATP produced rapid transient contractions. Responses to ATP were increased in magnitude and duration in the presence of ARL 67156 (100 microM), whereas those to the stable analogue, alpha, beta-methylene ATP were not significantly affected. 6. Contractions to exogenous noradrenaline (10 microM) and KCl (40 mM) were significantly enhanced by ARL 67156 (100 microM), but this potentiation was abolished by PPADS (100 microM). Therefore, this effect of the ecto-ATPase inhibitor may be due to a build up of endogenous ATP, increasing the sensitivity of the smooth muscle to other agonists. 7. It is concluded that ARL 67156 potentiates the action of ATP, and that when ATP acts as a neurotransmitter its postjunctional actions are greatly attenuated by enzymatic degradation.     

Antagonism by reactive blue 2 but not by brilliant blue G of extracellular ATP-evoked responses in PC12 phaeochromocytoma cells.

1. The effects of reactive blue 2 and brilliant blue G, which have been shown to block extracellular ATP-evoked responses, were investigated to discover whether these compounds act as P2-purinoceptor antagonists in PC12 phaeochromocytoma cells. 2. Reactive blue 2 (10 to 100 microM) suppressed the ATP-stimulated dopamine secretion from PC12 cells in a dose-dependent manner. The concentration-response curve for ATP was shifted to the right and the maximal response was decreased by reactive blue (30 and 100 microM). Brilliant blue G (up to 100 microM) did not significantly affect the secretion. 3. Reactive blue 2 (10 to 100 microM) suppressed the ATP-activated inward current recorded from the voltage-clamped cells in a concentration-dependent manner. Brilliant blue G (up to 100 microM) did not affect the current. 4. The results suggest that reactive blue 2 but not brilliant blue G is a P2-purinoceptor antagonist in PC12 cells. The purinoceptors in these cells may be the same type as those involved in ATP-evoked smooth muscle relaxation, judging from the antagonism by reactive blue 2.     

Endothelium-dependent relaxation evoked by ATP and UTP in the aorta of P2Y2-deficient mice

Based on pharmacological criteria, we previously suggested that in the mouse aorta, endothelium-dependent relaxation by nucleotides is mediated by P2Y1 (adenosine diphosphate (ADP)), P2Y2 (adenosine triphosphate (ATP)) and P2Y6 (uridine diphosphate (UDP)) receptors. For UTP, it was unclear whether P2Y2, P2Y6 or yet another subtype was involved. Therefore, in view of the lack of selective purinergic agonists and antagonists, we used P2Y2-deficient mice to clarify the action of UTP. Thoracic aorta segments (width 2 mm) of P2Y2-deficient and wild-type (WT) mice were mounted in organ baths to measure isometric force development and intracellular calcium signalling.Relaxations evoked by ADP, UDP and acetylcholine were identical in knockout and WT mice, indicating that the receptors for these agonists function normally. P2Y2-deficient mice showed impaired ATP- and adenosine 5'[gamma-thio] triphosphate (ATPgammaS)-evoked relaxation, suggesting that in WT mice, ATP and ATPgammaS activate predominantly the P2Y2 subtype. The ATP/ATPgammaS-evoked relaxation and calcium signals in the knockout mice were partially rescued by P2Y1, as they were sensitive to 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS2179), a P2Y1-selective antagonist.In contrast to ATP, the UTP-evoked relaxation was not different between knockout and WT mice. Moreover, the action of UTP was not sensitive to MRS2179. Therefore, the action of UTP is probably mediated mainly by a P2Y6(like) receptor subtype.In conclusion, we demonstrated that ATP-evoked relaxation of the murine aorta is mainly mediated by P2Y2. But this P2Y2 receptor has apparently no major role in UTP-evoked relaxation. The vasodilator effect of UTP is probably mediated mainly by a P2Y6(like) receptor.     

ATP participates in the regulation of microvessel permeability

We demonstrated previously that stimulation of the P2Y receptor enhanced the macromolecular permeability of cultured endothelial cell monolayers via the paracellular pathway. To determine whether the P2Y receptor participates in the regulation of permeability in intact microvessels, we have examined the effects of exogenous and endogenous ATP on the permeation of the surface tissue of perfused rat tail caudal artery using a fluorescein isothiocyanate-dextran (FD-4; MW 4400; 1.0 mg mL(-1)). The permeation of FD-4 was assessed by a confocal fluorescence imaging system. We found that 2-methylthioadenosine 5'-triphosphate, a P2Y receptor agonist, enhanced the fluorescence intensity of FD-4 in the surface of the rat caudal artery tissue and that it was inhibited by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, a P2 receptor antagonist. Also, noradrenaline, a sympathetic neurotransmitter, and bradykinin, an inflammatory autacoid, enhanced the fluorescence intensity of FD-4 in the surface tissue of the rat caudal artery. The enhancement by noradrenaline was significantly inhibited by the P2 receptor antagonist. In addition, noradrenaline and bradykinin caused the release of ATP, ADP, AMP and adenosine from the endothelium of the rat caudal artery. These results indicated that the exogenous and endogenous ATP increased the macromolecular permeability of blood capillaries via the P2Y receptor. Such purinergic regulation of endothelial permeability may function in physiological and pathophysiological conditions.     

Dissociation of potentiation of Leu31 Pro34 neuropeptide Y on adrenergic and purinergic transmission in isolated canine splenic artery

The present study observed the effects of an activation of neuropeptide Y (NPY) Y1 receptors on adrenergic and purinergic components of double-peaked vasoconstrictor responses to periarterial nerve stimulation in the isolated, perfused canine splenic arteries. The results showed that 3-30 nM Leu31 Pro34 neuropeptide Y (LP-NPY) produced a dose-dependent potentiation of double-peaked vasoconstrictor responses to trains of 30-s pulses at 1, 4 or 10 Hz of stimulation. The potentiation of LP-NPY of the nerve-stimulated vasoconstrictions were completely inhibited by subsequent blockade of alpha1-adrenoceptors or Y1 receptors with 0.1 microM prazosin or with 1 microM BIBP 3226 ((R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-argininami de), respectively. The remaining responses in the presence of LP-NPY and prazosin were abolished by P2X receptor desensitization with 1 microM alpha,beta-methylene ATP. Moreover, 30 nM LP-NPY failed to modify the vasoconstrictor responses to nerve stimulation after treatment with prazosin. A subsequent administration of alpha,beta-methylene ATP completely suppressed the remaining responses after prazosin and LP-NPY. The vasoconstrictions induced by 0.003-1 nmol noradrenaline and 0.003-1 micromol ATP were slightly, but not significantly enhanced by 30 nM LP-NPY. The observations indicated that activation of postjunctional NPY Y1 receptors may have an important role in the modulation of adrenergic rather than purinergic transmission of the sympathetic co-transmission.     

Potentiation by adenosine of ATP-evoked dopamine release via a pertussis toxin-sensitive mechanism in rat phaeochromocytoma PC12 cells.

1. The effects of adenosine on adenosine 5''-triphosphate (ATP)-evoked dopamine release from rat phaeochromocytoma PC12 cells was investigated to determine whether adenosine exerts a regulatory effect on the ATP-evoked response. Adenosine potentiated ATP (30 microM)-evoked dopamine release in a concentration-dependent manner over a concentration-range of 1 to 100 microM. Adenosine (100 microM) shifted the concentration-dependence of the ATP-evoked response to the left without affecting the maximal response. 2. Aminophylline, a non-selective adenosine receptor antagonist, and CP66713, a selective antagonist at the A2 subclass of adenosine receptors, abolished the adenosine-induced potentiation. Furthermore, 8-cyclopentyltheophylline, a selective antagonist at the adenosine A1 receptor partially inhibited the adenosine-evoked potentiation. CGS22492, a selective A2 receptor agonist, potentiated ATP-evoked dopamine release whereas N6-cyclohexyladenosine (CHA), a selective A1 receptor agonist, had no effect. 3. Pertussis toxin (PTX), a bacterial exotoxin which catalyzes the ADP-ribosylation of guanosine 5''-triphosphate (GTP)-binding proteins (G-proteins), inhibited the adenosine-induced potentiation of dopamine release. Dibutyryl cyclic AMP (db cyclic AMP), an analogue of cyclic AMP, had no effect on the release on the ATP-evoked response. 4. Adenosine potentiated the ATP-evoked rise in intracellular Ca2+ concentration ([Ca]i) in PC12 cells. This potentiation was also observed with CGS 22492 but not with CHA. PTX completely inhibited the adenosine-induced potentiation of the rise in [Ca]i. 5. On the basis of these findings, we suggest that the adenosine-induced potentiation of ATP-evoked dopamine release was due to an increase in [Ca]i in the cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic adenosine monophosphate-dependent vascular responses to purinergic agonists adenosine triphosphate and uridine triphosphate in the anesthetized mouse.

The mechanism by which purinergic agonist adenosine triphosphate (ATP) and uridine triphosphate (UTP) decrease systemic arterial pressure in the anesthetized mouse was investigated. Intravenous injections of adenosine triphosphate (ATP) and uridine triphosphate (UTP) produced dose-dependent decreases in systemic blood pressure in the mouse. The order of potency was ATP > UTP. Vasodilator responses to ATP and UTP were altered by the cyclic adenosine monophosphate (cAMP) phosphodiesterase inhibitor rolipram. The vascular responses to ATP and UTP were not altered by a nitric oxide synthase inhibitor, a cyclooxygenase inhibitor, a cGMP phosphodiesterase inhibitor, or a particular P2 receptor antagonist. These data suggest that ATP and UTP cause a decrease in systemic arterial pressure in the mouse via a cAMP-dependent pathway via a novel P2 receptor linked to adenylate cyclase and that nitric oxide release, prostaglandin synthesis, cGMP, and P2X1, P2Y1, and P2Y4 receptors play little or no role in the vascular effects of these purinergic agonists in the mouse.     

Inhibition of ATP-induced glutamate release by MRS2179 in cultured dorsal spinal cord astrocytes

It was reported that ATP, an excitatory chemical mediator, exerts its effects by activation of the P2X (ligand-gated cationic channels) and P2Y (G protein-coupled receptors) purinoceptors in the nervous system. In the present work, we used confocal laser scanning microscopy and high-performance liquid chromatography to assess the role of the P2Y1 receptor in ATP-evoked Ca2+ mobilization and glutamate release from cultured dorsal spinal cord astrocytes. ATP (0.01-100 micromol/l) produces a dose-dependent rise in the Ca2+ relative fluorescence intensity in cultured astrocytes. N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179, 0.01-100 micromol/l), a P2Y1-specific antagonist, could dose-dependently inhibit ATP-evoked Ca2+ mobilization. In addition, 100 micromol/l ATP caused glutamate efflux from cultured dorsal spinal cord astrocytes in a time-dependent manner. 100 micromol/l MRS2179 significantly inhibited the glutamate efflux induced by ATP, which suggests that P2Y1 receptor activation is responsible for the ATP-induced glutamate efflux from astrocytes. Taken together, our results demonstrate that P2Y1 receptor plays an important role in modulating the function of astrocytes, which raises the possibility that MRS2179, a potent P2Y1-specific antagonist, may become a potential drug in treating many chronic neurological diseases characterized by astrocytic activation in the nervous system.     

The regulation of veratridine-stimulated electrogenic ion transport in mouse colon by neuropeptide Y (NPY), Y1 and Y2 receptors

1 Neuropeptide Y (NPY) is a prominent enteric neuropeptide with prolonged antisecretory effects in mammalian intestine. Veratridine depolarises neurons consequently causing epithelial anion secretion across mouse colon mucosa. Our aim was to characterise functionally, veratridine-stimulated mucosal responses and to determine the roles for NPY, Y(1), and Y(2) receptors in modulating these neurogenic effects. 2 Colon mucosae (with intact submucous innervation) from wild-type mice (+/+) and knockouts lacking either NPY (NPY-/-), Y(1)-/- or Y(2)-/- were placed in Ussing chambers and voltage clamped at 0 mV. Veratridine-stimulated short-circuit current (I(sc)) responses in +/+, Y(1) or Y(2) antagonist pretreated +/+ colon, Y(1)-/- and NPY-/- colon were insensitive to cholinergic blockade by atropine (At; 1 microM) and hexamethonium (Hex; 10 microM). Tetrodotoxin (TTX, 100 nM) abolished veratridine responses, but had no effect upon carbachol (CCh) or vasoactive intestinal polypeptide (VIP)-induced secretory responses. 3 To establish the functional roles for Y(1) and Y(2) receptors, +/+ tissues were pretreated with either the Y(1) or Y(2) receptor antagonist (BIBO3304 (300 nM) or BIIE0246 (1 microM), respectively) and veratridine responses were compared with those from Y(1)-/- or Y(2)-/- colon. Neither BIBO3304 nor Y(1)-/- altered veratridine-induced secretion, but Y(1) agonist responses were abolished in both preparations. In contrast, the Y(2) antagonist BIIE0246 significantly amplified veratridine responses in +/+ mucosa. Unexpectedly, NPY-/- colon exhibited significantly attenuated veratridine responses (between 1 and 5 min). 4 We demonstrate that electrogenic veratridine responses in mouse colon are noncholinergic and that NPY can act directly upon epithelia, a Y(1) receptor effect. The enhanced veratridine response observed in +/+ tissue following BIIE0246, indicates that Y(2) receptors are located on submucosal neurons and that their activation by NPY will inhibit enteric noncholinergic secretory neurotransmission. 5 We also demonstrate Y(1) and Y(2) receptor-mediated antisecretory tone in +/+ colon and show selective loss of each in Y(1) and Y(2) null colon respectively. In NPY-/- tissue, only Y(1)-mediated tone was present, this presumably being mediated by endogenous endocrine peptide YY. Y(2) tone was absent from NPY-/- (and Y(2)-/-) colon and we conclude that NPY activation of neuronal Y(2) receptors attenuates secretory neurotransmission thereby providing an absorptive electrolyte tone in isolated colon.     

Anion secretion evoked by Pasteurella multocida toxin across rat colon

Stimulation of muscarinic receptors is known to have a biphasic effect on colonic Cl(-) secretion: a short-lasting activation, which is followed by a long-lasting inhibition. In order to find out, which role Gq proteins play in both processes, Pasteurella multocida toxin was used, a known activator of G alpha q. This toxin (1.5 microg/ml) had a dual action on short-circuit current (Isc) across rat distal colon: it stimulated transiently Isc and subsequently down-regulated the Isc evoked by Ca2+-dependent secretagogues such as acetylcholine or ATP. The inactive mutant (P. multocida toxin C1165S), which does not stimulate G alpha q), was ineffective. Cl(-) dependence and sensitivity against bumetanide, a blocker of the Na+-K+-2Cl(-) cotransporter, confirmed that the increase in Isc evoked by the toxin represented Cl(-) secretion. The effect of P. multocida toxin was suppressed by YM-254890 (10(-7) M), a blocker of G alpha q. Experiments with apically permeabilized tissues revealed that the secretory response to P. multocida toxin was concomitant with an increase in basolateral K+ conductance as it is observed for other agonists inducing Ca2+-dependent anion secretion. Consequently, these results suggest that Gq proteins are not only involved in the activation of secretion, e.g. after stimulation of muscarinic or purinergic receptors, but also play a central role in the long-term down-regulation of intestinal secretion after activation of these types of receptors.