The Signaling Mechanism of Contraction Induced by ATP and UTP in Feline Esophageal Smooth Muscle Cells

P2 receptors are membrane-bound receptors for extracellular nucleotides such as ATP and UTP. P2 receptors have been classified as ligand-gated ion channels or P2X receptors and G protein-coupled P2Y receptors. Recently, purinergic signaling has begun to attract attention as a potential therapeutic target for a variety of diseases especially associated with gastroenterology. This study determined the ATP and UTP-induced receptor signaling mechanism in feline esophageal contraction. Contraction of dispersed feline esophageal smooth muscle cells was measured by scanning micrometry. Phosphorylation of MLC₂₀ was determined by western blot analysis. ATP and UTP elicited maximum esophageal contraction at 30 s and 10 μM concentration. Contraction of dispersed cells treated with 10 μM ATP was inhibited by nifedipine. However, contraction induced by 0.1 μM ATP, 0.1 μM UTP and 10 μM UTP was decreased by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, chelerythrine, ML-9, PTX and GDPβS. Contraction induced by 0.1 μM ATP and UTP was inhibited by Gαi₃ or Gαq antibodies and by PLCβ₁ or PLCβ₃ antibodies. Phosphorylated MLC₂₀ was increased by ATP and UTP treatment. In conclusion, esophageal contraction induced by ATP and UTP was preferentially mediated by P2Y receptors coupled to Gαi₃ and G q proteins, which activate PLCβ₁ and PLCβ₃. Subsequently, increased intracellular Ca²⁺ and activated PKC triggered stimulation of MLC kinase and inhibition of MLC phosphatase. Finally, increased pMLC₂₀ generated esophageal contraction.     

Analysis of purine- and pyrimidine-induced vascular responses in the isolated rat cerebral arteriole

Effects of extraluminal UTP were studied and compared with vascular responses to ATP and its analogs in rat cerebral-penetrating arterioles. UTP, UDP, 2-methylthio-ATP, and alpha,beta-methylene-ATP dilated arterioles at the lowest concentration and constricted them at high concentrations. Low concentrations of ATP dilated the vessels; high concentrations caused a biphasic response, with transient constriction followed by dilation. Endothelial impairment inhibited ATP- and UTP-mediated dilation and potentiated constriction to UTP but not to ATP. ATP- and 2-methylthio-ATP- but not UTP-mediated constrictions were inhibited by desensitization with 10(-6) M alpha,beta-methylene-ATP or 3 x 10(-6) M pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). PPADS at 10(-4) M abolished the UTP-mediated constriction and induced vasodilation in a dose-dependent manner but did not affect the dilation to ATP. These results suggest that in rat cerebral microvessels 1) ATP and 2-methylthio-ATP induce transient constriction via smooth muscle P(2X1) receptors in the cerebral arteriole, 2) UTP stimulates two different classes of P(2Y) receptors, resulting in constriction (smooth muscle P(2Y4)) and dilation (possibly endothelial P(2Y2)), and 3) ATP and UTP produce dilation by stimulation of a single receptor (P(2Y2)).     

Investigation of the functional expression of purine and pyrimidine receptors in porcine isolated pancreatic arteries

Receptors for purines and pyrimidines are expressed throughout the cardiovascular system. This study investigated their functional expression in porcine isolated pancreatic arteries. Pancreatic arteries (endothelium intact or denuded) were prepared for isometric tension recording and preconstricted with U46619, a thromboxane A₂ mimetic; adenosine-5′-diphosphate (ADP), uridine-5′-triphosphate (UTP) and MRS2768, a selective P2Y₂ agonist, were applied cumulatively, while adenosine-5′-triphosphate (ATP) and αβ-methylene-ATP (αβ-meATP) response curves were generated from single concentrations per tissue segment. Antagonists/enzyme inhibitors were applied prior to U46619 addition. ATP, αβ-meATP, UTP and MRS2768 induced vasoconstriction, with a potency order of αβ-meATP > MRS2768 > ATP ≥ UTP. Contractions to ATP and αβ-meATP were blocked by NF449, a selective P2X1 receptor antagonist. The contraction induced by ATP, but not UTP, was followed by vasorelaxation. Endothelium removal and DUP 697, a cyclooxygenase-2 inhibitor, had no significant effect on contraction to ATP but attenuated that to UTP, indicating actions at distinct receptors. MRS2578, a selective P2Y₆ receptor antagonist, had no effect on contractions to UTP. ADP induced endothelium-dependent vasorelaxation which was inhibited by MRS2179, a selective P2Y₁ receptor antagonist, or SCH58261, a selective adenosine A_2A receptor antagonist. The contractions to ATP and αβ-meATP were attributed to actions at P2X1 receptors on the vascular smooth muscle, whereas it was shown for the first time that UTP induced an endothelium-dependent vasoconstriction which may involve P2Y₂ and/or P2Y₄ receptors. The relaxation induced by ADP is mediated by P2Y₁ and A_2A adenosine receptors. Porcine pancreatic arteries appear to lack vasorelaxant P2Y₂ and P2Y₄ receptors.     

ATP stimulates Ca2+ oscillations and contraction in airway smooth muscle cells of mouse lung slices

In airway smooth muscle cells (SMCs) from mouse lung slices, > or =10 microM ATP induced Ca2+ oscillations that were accompanied by airway contraction. After approximately 1 min, the Ca2+ oscillations subsided and the airway relaxed. By contrast, > or =0.5 microM adenosine 5'-O-(3-thiotriphosphate) (nonhydrolyzable) induced Ca2+ oscillations in the SMCs and an associated airway contraction that persisted for >2 min. Adenosine 5'-O-(3-thiotriphosphate)-induced Ca2+ oscillations occurred in the absence of external Ca2+ but were abolished by the phospholipase C inhibitor U-73122 and the inositol 1,4,5-trisphosphate receptor inhibitor xestospongin. Adenosine, AMP, and alpha,beta-methylene ATP had no effect on airway caliber, and the magnitude of the contractile response induced by a variety of nucleotides could be ranked in the following order: ATP = UTP > ADP. These results suggest that the SMC response to ATP is impaired by ATP hydrolysis and mediated via P2Y(2) or P2Y(4) receptors, activating phospholipase C to release Ca2+ via the inositol 1,4,5-trisphosphate receptor. We conclude that ATP can serve as a spasmogen of airway SMCs and that Ca2+ oscillations in SMCs are required to sustain airway contraction.     

Involvement of Rho-kinase in P2Y-receptor-mediated contraction of renal glomeruli

The involvement of Rho-kinase in P2Y-receptor induced contraction of isolated rat renal glomeruli was investigated. The contraction effects have been investigated based on changes in the intracapillary volume of isolated glomeruli. ATP was found to induce time- and concentration-dependent contraction of isolated glomeruli. Other tested nucleotides (ADP, UTP) and ATP analogues (beta,gamma-methylene-ATP, 2-methylothio-ATP) contracted glomeruli in similar magnitude whereas AMP had no effect. Furthermore, the contractive effect of ATP was prevented in the presence of an antagonist of P2Y-receptors, reactive blue 2. However, a selective antagonist of A1-receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), had no effect. Contraction induced by ATP, ADP, and UTP, in contrast to 2-methylothio-ATP and beta,gamma-methylene-ATP, was prevented in the presence of Rho-kinase's inhibitor, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632). These findings suggest the involvement of Rho-kinase pathways in P2Y-induced contraction of isolated glomeruli.     

Microvascular oxygen tension in the rat mesentery

Extracellular purines and pyrimidines have major effects on cardiac rhythm and contraction. ATP/UTP are released during various physiopathological conditions, such as ischemia, and despite degradation by ectonucleotidases, their interstitial concentrations can markedly increase, a fact that is clearly associated with arrhythmia. In the present whole cell patch-clamp analysis on ventricular cardiomyocytes isolated from various mammalian species, ATP and UTP elicited a sustained, nonselective cationic current, I(ATP). UDP was ineffective, whereas 2'(3')-O-(4-benzoylbenzoyl)-ATP was active, suggesting that P2Y(2) receptors are involved. I(ATP) resulted from the binding of ATP(4-) to P2Y(2) purinoceptors. I(ATP) was maintained after ATP removal in the presence of guanosine 5'-[gamma-thio]triphosphate and was inhibited by U-73122, a PLC inhibitor. Single-channel openings are rather infrequent under basal conditions. ATP markedly increased opening probability, an effect prevented by U-73122. Two main conductance levels of 14 and 23 pS were easily distinguished. Similarly, in fura-2-loaded cardiomyocytes, Mn(2+) quenching and Ba(2+) influx were significant only in the presence of ATP or UTP. Adult rat ventricular cardiomyocytes expressed transient receptor potential channel TRPC1, -3, -4, and -7 mRNA and the TRPC3 and TRPC7 proteins that coimmunoprecipitated. Finally, the anti-TRPC3 antibody added to the patch pipette solution inhibited I(ATP). In conclusion, activation of P2Y(2) receptors, via a G protein and stimulation of PLCbeta, induces the opening of heteromeric TRPC3/7 channels, leading to a sustained, nonspecific cationic current. Such a depolarizing current could induce cell automaticity and trigger the arrhythmic events during an early infarct when ATP/UTP release occurs. These results emphasize a new, potentially deleterious role of TRPC channel activation.     

Multiple P2Y receptors couple to calcium-dependent,chloride channels in smooth muscle cells of the rat pulmonary artery

Background

Uridine 5''-triphosphate (UTP) and uridine 5''-diphosphate (UDP) act via P2Y receptors to evoke contraction of rat pulmonary arteries, whilst adenosine 5''-triphosphate (ATP) acts via P2X and P2Y receptors. Pharmacological characterisation of these receptors in intact arteries is complicated by release and extracellular metabolism of nucleotides, so the aim of this study was to characterise the P2Y receptors under conditions that minimise these problems.

Methods

The perforated-patch clamp technique was used to record the Ca²⁺-dependent, Cl^- current (I_Cl,Ca) activated by P2Y receptor agonists in acutely dissociated smooth muscle cells of rat small (SPA) and large (LPA) intrapulmonary arteries, held at -50 mV. Contractions to ATP were measured in isolated muscle rings. Data were compared by Student''s t test or one way ANOVA.

Results

ATP, UTP and UDP (10^-4M) evoked oscillating, inward currents (peak = 13–727 pA) in 71–93% of cells. The first current was usually the largest and in the SPA the response to ATP was significantly greater than those to UTP or UDP (P < 0.05). Subsequent currents tended to decrease in amplitude, with a variable time-course, to a level that was significantly smaller for ATP (P < 0.05), UTP (P < 0.001) and UDP (P < 0.05) in the SPA. The frequency of oscillations was similar for each agonist (mean≈6–11.min^-1) and changed little during agonist application. The non-selective P2 receptor antagonist suramin (10^-4M) abolished currents evoked by ATP in SPA (n = 4) and LPA (n = 4), but pyridoxalphosphate-6-azophenyl-2'',4''-disulphonic acid (PPADS) (10^-4M), also a non-selective P2 antagonist, had no effect (n = 4, 5 respectively). Currents elicited by UTP (n = 37) or UDP (n = 14) were unaffected by either antagonist. Contractions of SPA evoked by ATP were partially inhibited by PPADS (n = 4) and abolished by suramin (n = 5). Both antagonists abolished the contractions in LPA.

Conclusion

At least two P2Y subtypes couple to I_Cl,Ca in smooth muscle cells of rat SPA and LPA, with no apparent regional variation in their distribution. The suramin-sensitive, PPADS-resistant site activated by ATP most resembles the P2Y₁₁ receptor. However, the suramin- and PPADS-insensitive receptor activated by UTP and UDP does not correspond to any of the known P2Y subtypes. These receptors likely play a significant role in nucleotide-induced vasoconstriction.     

Effect of cholinergic agonists on muscular tonus of the lizard small intestine and esophagus.

The underlying mechanisms of acetylcholine-induced intestinal relaxation in the lizard Liolaemus tenuis tenuis are still unknown. By using a classical model of intestinal recording of isometric contraction and relaxation in conjunction with specific pharmacological tools, this article studies the possible influence of EDRF/NO and nicotinic ganglionar receptors on the Ach-induced relaxation in an effort to elucidate the probable mechanisms involved in ACh effect. It was observed that the relaxation of the lizard intestine elicited by ACh (10(-7) - 4 x 10(-4) M) was not affected by hexametonium (5 x 10(-4) M) or tetrodotoxin (10(-6) M). Nicotine (10(-7) to 10(-4) M) induced relaxation was significantly antagonized by hexametonium; however, it was not influenced by tetrodotoxin. These results allow us to discard a neuronal pathway in cholinergic-induced relaxation, suggesting a more direct cholinergic effect on the smooth muscle, perhaps mediated by an unknown substance released by some specialized tissue. N-nitro-L-arginine, used to block NO-synthase and NO production, induced no changes in ACh-induced relaxation. Methylene blue, a soluble guanylate cyclase inhibitor, induced no changes in ACh-induced relaxation. These results allow us to discard a probable role of EDRF/nitric oxide in the ACh-induced relaxation of lizard small intestine, providing evidence that this mechanism could be different from that reported in other species.     

Adenosine selectively inhibits noncholinergic transmission in guinea pig bronchi

The neuromodulatory action of adenosine and ATP was investigated in isolated guinea pig bronchial strip chain preparations contracted with electrical field stimulation. The tissues were placed in organ baths containing physiological salt solution and stimulated at 8-Hz frequency, 0.5-ms pulse duration, and 30 V (approximately 100 mA) for 5 s. Electrical field stimulation evoked a biphasic contraction of bronchial muscle, consisting of an initial contraction followed by a sustained contraction, which was mediated by intramural cholinergic and noncholinergic nerve stimulations, respectively. Adenosine, at concentrations greater than M, caused a concentration-dependent inhibition in the height of the noncholinergically mediated contraction, accompanied by a very weak inhibition on the cholinergically mediated response. ATP (10(-5) to 3 x 10(-3) M) also produced a similar inhibitory effect on the noncholinergically mediated contraction, but the inhibitory potency was less than that of adenosine. The inhibitory response to adenosine was enhanced by the pretreatment with dipyridamole (2 x 10(-6) M) but antagonized with aminophylline (10(-5) M). Contractions of bronchial muscle evoked by exogenous acetylcholine (2 x 10(-6) M) or substance P (2 x 10(-7) M) were significantly inhibited by the adenosine (3 x 10(-4) M) pretreatment. These data suggest that in isolated guinea pig bronchi adenosine selectively inhibits noncholinergic neurotransmission through prejunctional P1-purinoceptors.     

Aalpha,beta-methylene ATP enhances P2Y4 contraction of rabbit basilar artery

Interactions between different selective P2 receptor agonists have been used as tools to identify different P2 receptor subtypes. In the present study, we examined the P2 receptor subtypes and the mechanisms of potentiation of UTP contraction (P2Y contraction) by alpha,beta-methylene ATP [(2-carboxypiperazin-4-yl)propyl-1-phosphanoic acid (CPP), a P2X agonist] using isometric tension in the denuded rabbit basilar artery. We made the following observations: 1). a predominant P2X receptor contraction was observed in the rabbit ear artery by the rank order of CPP > 2-methylthioATP > ATP > UTP; 2). functional P2Y receptors were observed in the rabbit basilar artery by the rank order of UTP > ATP = CPP = 2-methylthioATP; 3). CPP potentiated UTP-, ATP-, and ATPgammaS-induced contractions, possibly by activation of P2Y4 receptors because ATPgammaS does not activate P2Y6 receptors; and 4). ectonucleotidase did not play a predominant role in the potentiative effect of CPP because Evans blue, Ca(2+)-free medium, or divalent cation Ni(2+) did not affect the effect of CPP. Evans blue potentiated the contraction by UTP but not by ATP or ATPgammaS. We conclude that CPP enhanced P2Y4-mediated contraction in the rabbit basilar artery, and the influence by ectonucleotidases on CPP-potentiation remains unclear.     

Extracellular nucleotide inhibits cell proliferation and negatively regulates Toll-like receptor 4 signalling in human progenitor endothelial cells

Extracellular nucleotides mediate a wide range of physiological effects by interacting with plasma membrane P2 purinergic receptors. P2 receptors are expressed in certain kinds of stem cells, and function to induce cytokine expression and to modulate cell proliferation. We have analysed the expression and the function of P2 receptors in human umbilical cord blood-derived EPCs (endothelial progenitor cells). EPCs expressed P2X4,6,7 and P2Y2,4,11,13,14 receptors and extracellular ATP inhibited EPCs proliferation. As in a previous study, EPCs expressed functional TLR4 (Toll-like receptor 4) and activation of TLR4 by LPS (lipopolysaccharide) evoked a pro-inflammatory immune response. When human EPCs were stimulated with LPS and nucleotides, ATP or UTP inhibited the expression of pro-inflammatory cytokines including MCP-1 (monocyte chemoattractant protein-1), IFNα (interferon α), TNFα (tumour necrosis factor α) and adhesion molecule VCAM-1 (vascular cell adhesion molecule 1) induced by LPS. ATP and UTP also down-regulated the gene expression of TLR4, CD14 and MyD88 (myeloid differentiation factor 88), a TLR adaptor molecule, and protein expression of CD14 and MyD88. Moreover, the phosphorylation of NF-κB (nuclear factor κB) p65 induced by TLR4 activation was inhibited partly by ATP or UTP at concentrations of 1-5 μM. These results suggest that extracellular nucleotides negatively regulate EPCs proliferation and TLR4 signalling.     

Do ATP and UTP involve cGMP in positive inotropism on rat atria?

ATP and UTP induced a dual inotropic effect in rat left atria: first a decrease and then an increase in contractile tension were observed. PPADS, an antagonist of P2X receptors, inhibited positive inotropism induced by ATP and alpha,beta-meATP. Chiefly, we investigated intracellular mechanisms responsible for the positive inotropism. We tested cromakalim and glibenclamide, an activator and an inhibitor, respectively, of ATP-sensitive K(+) channels. These compounds did not influence the effects of ATP. IBMX, a phosphodiesterase inhibitor, and H-7, an inhibitor of protein kinase C and cAMP-dependent protein kinase, did not modify the inotropic effects of ATP. Instead, H-8, an inhibitor of cAMP- and cGMP-dependent protein kinases, strongly inhibited the positive effects of both ATP and UTP, suggesting the possible involvement of cGMP in the inotropism. Also, LY 83583, an inhibitor of cGMP production, reduced positive inotropism by alpha,beta-meATP, ATP and UTP. Moreover, 8-Br-cGMP (50 microM), a stable analogue of cGMP, inhibited positive inotropism by all nucleotides. Lastly, we determined intracellular cGMP levels by RIA; the cyclic nucleotide increased during positive inotropism induced by ATP and UTP. The results regarding positive inotropism suggest that: (a) ATP acts through P2X receptors, while UTP may act by P2X, but also through PPADS-insensitive receptors; and (b) changes in intracellular cGMP concentration are involved in this inotropic effect.     

P2Y(11), a purinergic receptor acting via cAMP, mediates secretion by pancreatic duct epithelial cells

Pancreatic duct epithelial cells (PDEC) mediate the exocrine secretion of fluid and electrolytes. We previously reported that ATP and UTP interact with P2Y(2) receptors on nontransformed canine PDEC to increase intracellular free Ca2+ concentration ([Ca2+](i)) and stimulate Ca2+-activated Cl- and K+ channels. We now report that ATP interacts with additional purinergic receptors to increase cAMP and activate Cl- channels. ATP, 2-methylthio-ATP, and ATP-gamma-S stimulated a 4- to 10-fold cAMP increase with EC(50) of 10-100 microM. Neither UTP nor adenosine stimulated a cAMP increase, excluding a role for P2Y(2) or P1 receptors. Although UTP stimulated an (125)I(-) efflux that was fully inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), ATP stimulated a partially resistant efflux, suggesting activation of additional Cl- conductances through P2Y(2)-independent and Ca2+-independent pathways. In Ussing chambers, increased cAMP stimulated a much larger short-circuit current (I(sc)) increase from basolaterally permeabilized PDEC monolayers than increased [Ca2+](i). Luminal ATP and UTP and serosal UTP stimulated a small Ca2+-type I(sc) increase, whereas serosal ATP stimulated a large cAMP-type I(sc) response. Serosal ATP effect was inhibited by P2 receptor blockers and unaffected by BAPTA-AM, supporting ATP activation of Cl- conductances through P2 receptors and a Ca2+-independent pathway. RT-PCR confirmed the presence of P2Y(11) receptor mRNA, the only P2Y receptor acting via cAMP.     

Acetylcholine Releases ATP from Varicosities Isolated from Guinea Pig Myenteric Plexus

The effects of cholinergic agonists and antagonists on the release of ATP from isolated myenteric varicosities were studied using a firefly luciferin-luciferase technique. In a previous study, acetylcholine and nicotine released ATP from isolated myenteric varicosities, whereas the muscarinic agonist bethanechol did not. In the present study, release of ATP by acetylcholine was shown to be Ca2+ dependent. d-Tubocurarine competitively antagonized the release of ATP by either acetylcholine or nicotine. Maximal release of ATP by acetylcholine (10(-3) M) was approximately 24% that observed with the depolarizing drug veratridine (5 X 10(-5) M), suggesting either that not all of the varicosities capable of releasing ATP possess nicotinic receptors or that acetylcholine does not depolarize the varicosities to the degree that veratridine does. Tetrodotoxin slightly but significantly reduced ATP release induced by acetylcholine or nicotine, indicating some involvement of Na+ channels in the release process. Finally, 6-hydroxydopamine pretreatment produced a 48% reduction in the acetylcholine-evoked release of ATP, suggesting that much, but possibly not all, of the ATP release occurs from noradrenergic varicosities present in the preparation.     

Multiple dilator pathways in skeletal muscle contraction-induced arteriolar dilations

To determine whether nitric oxide (NO), adenosine (Ado) receptors, or ATP-sensitive potassium (K(ATP)) channels play a role in arteriolar dilations induced by muscle contraction, we used a cremaster preparation in anesthetized hamsters in which we stimulated four to five muscle fibers lying perpendicular to a transverse arteriole (maximal diameter approximately 35-65 microm). The diameter of the arteriole at the site of overlap of the stimulated muscle fibers (the local site) and at a remote site approximately 1,000 microm upstream (the upstream site) was measured before, during, and after muscle contraction. Two minutes of 4-Hz muscle stimulation (5-15 V, 0.4 ms) produced local and upstream dilations of 19 +/- 1 and 10 +/- 1 microm, respectively. N(omega)-nitro-L-arginine (10(-4) M; NO synthase inhibitor), xanthine amine congener (XAC; 10(-6) M; Ado A(1), A(2A), and A(2B) receptor antagonist), or glibenclamide (Glib; 10(-5) M; K(ATP) channel inhibitor) superfused over the preparation attenuated the local dilation (by 29.7 +/- 12.7, 61.8 +/- 9.0, and 51.9 +/- 14.9%, respectively), but only XAC and Glib attenuated the upstream dilation (by 68.9 +/- 6.8 and 89.1 +/- 6.4%, respectively). Furthermore, only Glib, when applied to the upstream site directly, attenuated the upstream dilation (48.1 +/- 9.1%). Neither XAC nor Glib applied directly to the arteriole between the local and the upstream sites had an effect on the magnitude of the upstream dilation. We conclude that NO, Ado receptors, and K(ATP) channels are involved in the local dilation initiated by contracting muscle and that both K(ATP) channels and Ado receptor stimulation, but not NO, play a role in the manifestation of the dilation at the upstream site.     

Evidence of histamine receptor function in isolated horse penile dorsal arteries

The effect of histamine (10(-9)-10(-3) M) on horse penile dorsal artery was evaluated. Precontracted vessels showed a biphasic response (relaxation-contraction) to histamine, while at basal tone, histamine only induced a contractile effect. The H1 receptor agonist, 2-pyridylethylamine (PEA) (10(-9)-10(-3) M), induced concentration-dependent relaxation in precontracted rings and provoked vasoconstriction at basal tone. Mepyramine (10(-9)-10(8) M), an H1 receptor antagonist, competitively antagonized the relaxant response to histamine (pA2 = 9.7) and PEA (pA2 = 9.2). At basal tone, mepyramine (10(-10)-10(-8) M) also caused a rightward shift in the histamine contraction curve (pA2 = 10.1). Mepyramine (10(-9)-10(-8) M)/PEA Schild plots for resting vessels yielded a pA2 value of 9.4. A regulatory role for H2 and H3 receptors was precluded since there was no response to their agonists (dimaprit (10(-9)-10(-3) M), (R)-alpha-methylhistamine (10(-10)- 3 x 10(-4) M)), and antagonists (cimetidine (10(-5) M), thioperamide (10(-6) M)) did not affect control curves. Removal of the endothelium abolished the relaxant component causing a leftward shift in the contractile component in precontracted rings, with no effect on maximum contraction. Inhibitors of nitric oxide (NO) synthesis, L-NAME (3 x 10(-4) M) and L-NOARG (3 x 10(-4) M), modified the relaxant response while contraction was unaffected. L-Arginine (3 x 10(-4) M) potentiated maximum relaxation but did not affect contraction in precontracted rings. Effects of a prostanoid and K+ channels were ruled out. The biphasic response of precontracted vessels persisted in the presence of indomethacin (3 x 10(-6) M), tetraethylammonium (10(-3) M) and gliblenclamide (10(-5) M). L-NAME plus indomethacin, or this combination plus TEA or glibenclamide produced similar effects as isolated treatments. In resting vessels, histamine contraction was also unaffected by the lack of endothelium, or L-NAME, L-arginine or indomethacin pretreatment. The biphasic response to histamine is probably mediated by H1 receptors with a partial role for NO in the relaxant response in precontracted vessels. In the absence of tone, the contractile effect may be mediated by direct action on smooth muscle.     

The relaxation induced by indole and nonindole 5-HT agonists in the molluscan smooth muscle

1. The abilities of two indole agonists and some nonindole agonists to induce relaxation of catch contraction and the influence of the agonists on cyclic AMP (cAMP) levels in the anterior byssus retractor muscle (ABRM) of Mytilus were investigated. 2. 5-MeOT (5-methoxytryptamine) and 5-MeODMT (5-methoxy-N,N-dimethyltryptamine) dose-dependently relaxed the contraction. 3. TFMPP (m-trifluoromethylphenyl piperazine), PAPP (p-amino-phenyl TFMPP) and mCPP (1-(3-chlorophenyl)piperazine dose-dependently relaxed the contraction, but 2MPP (1-(2-methylphenyl) piperazine and quipazine did not. 4. 5-MeOT (10(-6)M), 5-MeODMT (10(-6)M), TFMPP (10(-4)M), 2MPP (10(-4)M), quipazine (10(-4)M) and 8-OH-DPAT (3 x 10(-5) M) significantly reduced the cAMP levels, but PAPP (3 x 10(-4)M) and mCPP (10(-4)M) did not have any effect on cAMP levels. 5. These findings indicate that the pharmacological properties of 5-HT1-like receptors in the ABRM are similar to those of 5-HT1A receptors in mammalian tissues, and that the changes in cAMP levels induced by the agonists used are unlikely to be directly linked to the relaxation induced by them.     

Up4A stimulates endothelium-independent contraction of isolated rat pulmonary artery

Extracellular nucleotides, such as ATP, UDP, and UTP, regulate pulmonary vascular tone through P2X and P2Y receptors. Recently, uridine adenosine tetraphosphate (Up(4)A) was reported as a novel endothelium-derived vasoconstrictive factor. Up(4)A contains both purine and pyrimidine moieties, which potentially activate P2X and P2Y receptors. The present study examined the effect of Up(4)A on contractility of isolated rat pulmonary artery. Up(4)A at 1-100 microM stimulated contraction in a concentration-dependent manner. Up(4)A was equipotent as UTP and UDP in the endothelium-denuded artery while much more effective than UTP and UDP in endothelium-intact preparations. The vasoconstrictor effect of Up(4)A was inhibited by suramin but not IP(5)I or desensitization of P2X receptors with alpha,beta-methylene-ATP (alpha,beta-Me-ATP). Up(4)A-induced contraction was also inhibited by pretreatment with thapsigargin, nitrendipine, or EGTA but unaffected by H1152. Furthermore, unlike ATP and UTP, Up(4)A did not induce relaxation of endothelium-intact preparations precontracted with phenylephrine. These results suggest that Up(4)A is a potent vasoconstrictor, but not a vasodilator, of the rat pulmonary artery. Up(4)A likely acts through a suramin-sensitive P2Y receptor. The contractile effect of Up(4)A involves the entry of extracellular Ca(2+) and release of Ca(2+) from intracellular stores but not Ca(2+) sensitization via the RhoA/Rho kinase pathway. Up(4)A, therefore, potentially plays an important role in the regulation of pulmonary vascular tone.     

On the Role of Extracellular ATP in the Induction of Long-Term Potentiation in the Hippocampus

Abstract: The involvement of a purinergic system in the mechanisms of ATP- and electrically induced long-term potentiation (LTP) has been investigated in mouse hippocampal slices. Extracellular ATP (500 n M ) and its slowly hydrolyzable analogue adenosine 5'- O -(3-thiotriphosphate) (ATP-γ-S; 2.5 µ M ) amplified permanently the magnitude of the population spike. This effect was antagonized by adenylimidodiphosphate (AMPPNP), a non-hydrolyzable analogue of ATP. AMPPNP, other ATP analogues [2-methylthioadenosine triphosphate (2-MeSATP) and α,β-methyleneadenosine 5'-triphosphate (α,β-methyleneATP)], or a purinergic receptor antagonist (Cibacron Blue 3G) tested in the concentration range of 3–40 µ M did not exert agonistic activity similar to that of ATP or ATP-γ-S, suggesting that ATP hydrolysis is required to exert this effect. All the tested nonhydrolyzable analogues reduced or prevented the establishment of stable, nondecremental LTP without blocking the short-lasting increase in the magnitude of the population spike immediately after electrical stimulation (short-term potentiation). These results indicate that ATP released by high-frequency stimulation contributes to the maintenance of stable LTP. The underlying mechanism operating in this process may involve a new type of ATP receptors or hydrolysis by ecto-ATPase. However, the findings that ATP-γ-S is less potent than ATP and that other ATP analogues known to act as agonists of purinergic receptors did not induce LTP but rather inhibited its maintenance are more consistent with the possibility that ecto-protein kinase, using extracellular ATP as a cosubstrate, plays a role in mechanisms underlying synaptic plasticity.