Dual signaling pathways of arterial constriction by extracellular uridine 5'-triphosphate in the rat

We investigated actions of uridine 5'-triphosphate (UTP) in rat aorta, cerebral and mesenteric arteries, and their single myocytes. UTP (≥10 μM) elicited an inward-rectifying current strongly reminiscent of activation of P2X(1) receptor, and a similar current was also induced by α,β-methylene adenosine 5'-triphosphate (ATP) (≥100 nM). UTP desensitized α,β-methylene ATP-evoked current, and vice versa. The UTP-activated current was insensitive to G-protein modulators, TRPC3 inhibitors, or TRPC3 antibody, but was sensitive to P2-receptor inhibitors or P2X(1)-receptor antibody. Both UTP (1 mM) and α,β-methylene ATP (10 μM) elicited similar conductance single channel activities. UTP (≥10 μM) provoked a dose-dependent contraction of de-endothelialized aortic ring preparation consisting of phasic and tonic components. Removal of extracellular Ca(2+) or bath-applied 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) (30 μM) or nifedipine (10 μM) completely inhibited the phasic contraction while only partially reducing the tonic one. The tonic contraction was almost completely abolished by additional application of thapsigargin (2 μM). Similar biphasic rises in [Ca(2+)](i) were also evoked by UTP in rat aortic myocytes. In contrast to the low expression of TRPC3, significant expression of P2X(1) receptor was detected in all arteries by RT-PCR and immunoblotting, and its localization was limited to plasma membrane of myocytes as indicated by immunohistochemistry. These results suggest that UTP dually activates P2X(1)-like and P2Y receptors, but not TRPC3.     

Ionic interaction of amiloride and uridine 5'-triphosphate in nebulizer solutions

Combination therapy using nebulized amiloride hydrochloride and uridine-5'-triphosphate (UTP) trisodium salt aerosols has been investigated for the treatment of cystic fibrosis (CF). Amiloride in aqueous solution precipitates in the presence of UTP, reducing drug concentrations. Interactions between these drugs and NaCl in solution were studied using phase-solubility techniques monitored by UV spectrophotometry. Elemental analyses were employed for precipitate characterization. Amiloride solubility was reduced by more than 85% in saline. Amiloride solubility decreased with increasing UTP concentration, resulting in formation of a precipitated complex. The theoretical molar ratio of complexes range from 1-3 amiloride:1 UTP. At most concentrations only 3 amiloride:1 UTP complex was observed in precipitate. This is a reflection of low Ksp for the 3:1 complex of 2.92 x 10(-11) M4 compared with 2.09 x 10(-4) M2 for amiloride alone. Equilibration over excess bulk solid resulted in higher solubility estimates and different phase solubility diagrams than solubility studies utilizing precipitation technique. This may be explained by the absence of amiloride in the solid state and its impact on complex equilibria with UTP. The solubility suppressing effects of UTP and saline were largely additive. A number of ionic interactions increase complex solubility profile of amiloride hydrochloride in the presence of UTP and NaCl.     

Further evidence for involvement of adenosine-5'-triphosphate in non-adrenergic non-cholinergic relaxation of the isolated rat duodenum

The nature of the inhbitory non-adrenergic non-cholinergic (NANC) neurotransmitter responsible for neurogenic relaxation of rat duodenum was studied with in vitro techniques. Adenosine 5'-triphosphate (ATP)(1 mM), gamma-aminobutyric acid (GABA, 1 mM), dimethylphenylpiperazinium (DMPP, 0.1 mM) and field stimulation (60 V, 2 ms, 0.1 Hz) produced transient relaxation followed by rebound contraction. In contrast vasoactive intestinal polypeptide (VIP) (0.3 microM) and noradrenaline (1 microM) induced relaxation which set in more slowly and lasted longer. Tetrodotoxin (0.85 microM) abolished field stimulation-induced relaxation but not ATP-, VIP- or noradrenaline-induced relaxation. Nucleotide pyrophosphatase (0.25 U/ml), but not the proteolytic enzyme alpha-chymotrypsin (2 U/ml), selectively antagonized NANC relaxation. The rank order of potency of various adenine derivatives for inducing relaxation was adenosine-5'-triphosphate greater than adenosine-5'-diphosphate much greater than adenosine greater than adenosine-5'-monophosphate. ATP-induced relaxation was selectively antagonized by the putative P2 purinoceptor antagonist reactive blue 2, but unaffected by the selective P1 purinoceptor antagonist 8-phenyltheophylline. The duration of ATP- as well as beta-gamma-methylene adenosine-5'-triphosphate (a stable analogue of ATP)-induced relaxation was similar and was unaffected by indomethacin 10 microM (which abolished the rebound contraction). In those preparations whose contractile tone was increased by using a high-K+ medium the ability of ATP to elicit relaxation was markedly reduced, while GABA- and DMPP-induced relaxation was abolished. On the other hand, ATP-, GABA- and DMPP-induced relaxation of the tonic component of 5-hydroxytryptamine (5-HT)(0.1 mM)-induced contraction was similar to that observed in control conditions. These findings add further weight to the proposal that endogenous ATP is involved in determining NANC relaxation of rat duodenum.     

Contractile effects of 5-hydroxytryptamine and 5-carboxamidotryptamine in the equine jejunum

The use of human prokinetic drugs in colic horses leads to inconsistent results. This might be related to differences in gastrointestinal receptor populations. The motor effects of 5-hydroxytryptamine (5-HT; serotonin) on the equine mid-jejunum were therefore studied. Longitudinal muscle preparations were set up for isotonic measurement. 5-HT induced tonic contractions with superimposed phasic activity; these responses were not influenced by tetrodotoxin and atropine, suggesting a non-neurogenic, non-cholinergic pathway. The 5-HT receptor antagonists GR 127935 (5-HT(1B,D)), ketanserin (5-HT(2A)), SB 204741 (5-HT(2B)), RS 102221 (5-HT(2C)), granisetron (5-HT(3)), GR 113808 (5-HT(4)) and SB 269970 (5-HT(7)) had no influence on the 5-HT-induced response; the 5-HT(1A) receptor antagonists NAN 190 (pK(b)=8.13+/-0.06) and WAY 100635 (pK(b)=8.69+/-0.07), and the 5-HT(1,2,5,6,7) receptor antagonist methysergide concentration-dependently inhibited the 5-HT-induced contractile response. The 5-HT(1,7) receptor agonist 5-carboxamidotryptamine (5-CT) induced a contractile response similar to that of 5-HT; its effect was not influenced by tetrodotoxin and atropine, and SB 269970, but antagonised by WAY 100635. 8-OHDPAT, buspiron and flesinoxan, which are active at rat and human 5-HT(1A) receptors, had no contractile influence. These results suggest that the contractile effect of 5-HT in equine jejunal longitudinal muscle is due to interaction with muscular 5-HT receptors, which cannot be characterised between the actually known classes of 5-HT receptors.     

Direct and indirect effects of adenosine 5'-triphosphate on guinea-pig ileum.

1 The inhibitory effects of adenosine 5'-triphosphate (ATP) were compared on the responses to electrical stimulation, and to direct and indirect stimulation by drugs of the longitudinal smooth muscle of guinea-pig ileum before and after blocking nervous activity. 2 While the major inhibitory effect of ATP was an indirect one on the intramural excitatory nerves, there was also a small direct effect on the muscle. 3 ATP also had direct and indirect excitatory effects. The direct effect particularly was only seen with high concentrations of ATP, but the appearance of these excitatory effects may be affected by the inhibitory actions.     

Characterization and possible function of adenosine 5'-triphosphate receptors in activated rat microglia.

1. Purinoceptor agonist-induced currents in untreated (proliferating) and lipopolysaccharide (LPS; 100 ng ml-1)-treated (non-proliferating) rat microglial cells in culture were recorded by the whole-cell patch-clamp technique. These cells have two preferred resting membrane potentials, one at -35 mV and another one at -70 mV. 2. Most experiments were carried out in non-proliferating cells. ATP, ATP-gamma-S and alpha,beta-MeATP (1-1000 microM in all cases) evoked an inward current at a holding potential of -70 mV, followed, in some experiments, by an outward current. At -70 mV 2-methylthio ATP (1-1000 microM) evoked an inward current, whereas at -35 mV it produced an outward current only. 3. When K+ was replaced in the pipette solution by an equimolar concentration of Cs+ (150 mM), the main outward component of the ATP-gamma-S (10 microM) induced response disappeared. Instead, an inward current was obtained. Replacement of K+ by Cs+ did not affect the inward current evoked by 2-methylthio ATP (300 microM). 4-Aminopyridine (1-10 mM), however, almost abolished this current and unmasked a smaller outward current. 4. The rank order of agonist potency was 2-methylthio ATP > ATP > alpha,beta-MeATP. Adenosine and UTP were inactive. Suramin (300 microM) and reactive blue 2 (50 microM) antagonized the effect of 2-methylthio ATP (300 microM). 5. I-V relations were determined by delivering fast voltage ramps before and during the application of 2-methylthio ATP (300 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Contractile effects of tacrolimus in human and rat isolated renal arteries

1. We tested the vasoactive properties of the immunosuppressive drug FK 506 (tacrolimus) in preconstricted rat and human isolated renal arteries in vitro. 2. In rat renal arteries, tacrolimus (3, 10 microM) showed a direct and dose-dependent contractile effect by maximally 23 microm (10% of the noradrenaline effect), which was only observed in the presence of intact endothelium. Moreover, a lower concentration of tacrolimus (1 microM) potentiated pressor responses to the sympathetic neurotransmitter noradrenaline but not to ATP in this species. ATP- (0.01-10 microM) induced vasodilation was not affected by tacrolimus (1 microM). 3. In contrast, in human interlobar arteries, tacrolimus failed to induce direct vasoconstriction and did not significantly potentiate constrictor responses to noradrenaline. Acetylcholine-(1 microM) induced vasodilation was much smaller in human than in rat renal arteries suggesting the lack of functional endothelium in the human preparation. 4. The findings suggest that tacrolimus releases an endothelium-derived constricting factor in rat renal arteries to increase vascular tone and to potentiate pressor responses to noradrenaline. In human interlobar arteries, this effect of tacrolimus is not observed probably because of the absence of functional endothelium or the necessary mediator mechanism.     

Integrity of actin-network is involved in uridine 5'-triphosphate evoked store-operated Ca2+ entry in bovine adrenocortical fasciculata cells

Store-operated Ca(2+) entry channels (SOCs) play an important role in the regulation of diverse non-excitable cell functions. However, the precise mechanism of SOCs activation is still controversial. Uridine 5'-triphosphate (UTP) was shown to induce Ca(2+) entry in a dihydropyridines-insensitive manner and accelerated steroidogenesis in bovine adrenocortical fasciculata cells (BAFCs) via the Gq/11 protein-coupled P2Y(2) receptor. Therefore we investigated whether UTP is involved in SOCs activation and the mechanism of UTP-induced SOCs activation. Fura 2-loaded BAFCs were used for the measurement of intracellular concentration of Ca(2+) ([Ca(2+)](i)) mobilization. Extracellular UTP evoked Ca(2+) release from intracellular stores followed by an increase in Ca(2+) entry. The Ca(2+) influx elicited by UTP was inhibited not by nifedipine, but by Zn(2+), Cd(2+), and Ni(2+) (potency order: Zn(2+) > Cd(2+) > Ni(2+)), and the effect of UTP was also attenuated by a phospholipase C inhibitor (U73122). These results indicate that UTP activates SOCs in BAFCs. The increase in [Ca(2+)](i) by UTP was attenuated by ML-9, a myosin-light chain kinase inhibitor, and calmodulin inhibitors, W-7 and E6 berbamine, in a concentration-dependent manner. These reagents depolymerized actin filaments with rhodamine staining in BAFCs. Cytochalasin D also inhibited UTP-activated SOCs and depolymerized actin filaments. From these results, we proposed that calcium/calmodulin dependent myosin-light chain kinase is involved in the mobilization of actin filaments and the integrity of actin-network plays an important role in UTP-induced SOCs activation in BAFCs.     

Effects of adenosine 5'-triphosphate (ATP) and beta-gamma-methylene ATP on the rat urinary bladder.

1 High concentrations of adenosine 5'-triphosphate (ATP, 100 to 1000 micrometer) were required to cause contraction of the rat urinary bladder, while adenosine and adenosine 5'monophosphate (AMP, 1 to 50 micrometer) produced relaxation. 2 One hundred fold lower concentrations of beta-gamma-methylene ATP, which is resistant to degradation to AMP and adenosine, caused dose-dependent, phasic contractions which mimicked atropine-resistant responses to nerve stimulation. 3 Adenosine and AMP caused dose-dependent inhibition of carbachol-induced contractions; theophylline competitively antagonized this inhibition but not the contractile responses to beta-gamma-methylene ATP, ATP or atropine-resistant nerve stimulation. 4 These results suggest that the insensitivity of the rat bladder to ATP is due to its rapid degradation to AMP and adenosine and support the hypothesis that the bladder receives a purinergic excitatory innervation.     

Postjunctional synergism of noradrenaline and adenosine 5'-triphosphate in the mesenteric arterial bed of the rat

The ability of purines to modify responses to exogenous noradrenaline (NA) was investigated using the isolated perfused rat mesenteric bed. ATP, at subthreshold doses and above-threshold doses, produced a potentiation of vasoconstrictor responses to NA while adenosine was without effect. The stable analogue of ATP, alpha,beta-methylene ATP alpha,beta-meATP, at subthreshold and above-threshold doses also enhanced pressor responses to NA (to a greater extent than ATP). This potentiation caused a shift to the left of the dose-response curve, with no increase in the maximum response. Pressor responses to 5-hydroxytryptamine (5-HT) and to potassium chloride (KCl), however, were not affected by alpha,beta-meATP. Conversely, suprathreshold doses of NA potentiated contractions evoked by alpha,beta-meATP, but no potentiation was observed using subthreshold doses of NA. These results demonstrate a postjunctional synergistic action between NA and ATP which appeared to be specific for the alpha 1-adrenoceptors and the P2x-purinergic receptors since: (i) the potentiation of the contractile response to NA by ATP was mimicked by alpha,beta-meATP but not by adenosine and (ii) pressor responses to 5-HT or to KCl were not affected by alpha,beta-meATP. Possible mechanisms for this postjunctional synergism are discussed.     

Contractile effects of substance P and neurokinin A on the rat stomach in vivo and in vitro.

Substance P and neurokinin A (substance K) were infused into the coeliac artery of anaesthetized rats at doses of 0.06-20 nmol min-1. Both tachykinins caused contractions of the stomach, the threshold dose of neurokinin A being 10 times lower than of substance P. The dose-response curve for substance P was flatter than that for neurokinin A. On circular muscle strips from the rat gastric corpus in vitro, the dose-response curves for both tachykinins were parallel, neurokinin A being 10 times more potent than substance P. The contractions in response to 10 microM neurokinin A and to 30 microM substance P were 58 and 54%, respectively, of the maximal contraction to bethanechol (1 mM). The effect of substance P was reduced by atropine both in vivo and in vitro. In vitro, the contractions to substance P were also reduced by tetrodotoxin but left unaffected by methysergide. The action of neurokinin A was not affected by these drugs. It is concluded that neurokinin A contracts rat stomach by a direct action on the circular smooth muscle, whereas the action of substance P is mediated, at least in part, by cholinergic interneurones.     

Differential effects of uridine adenosine tetraphosphate on purinoceptors in the rat isolated perfused kidney

BACKGROUND AND PURPOSE

Purinergic signalling plays an important role in vascular tone regulation in humans. We have identified uridine adenosine tetraphosphate (Up₄A) as a novel and highly potent endothelial-derived contracting factor. Up₄A induces strong vasoconstrictive effects in the renal vascular system mainly by P2X₁ receptor activation. However, other purinoceptors are also involved and were analysed here.

EXPERIMENTAL APPROACH

The rat isolated perfused kidney was used to characterize vasoactive actions of Up₄A.

KEY RESULTS

After desensitization of the P2X₁ receptor by α,β-methylene ATP (α,β-meATP), Up₄A showed dose-dependent P2Y₂-mediated vasoconstriction. Continuous perfusion with Up₄A evoked a biphasic vasoconstrictor effect: there was a strong and rapidly desensitizing vasoconstriction, inhibited by P2X₁ receptor desensitization. In addition, there is a long-lasting P2Y₂-mediated vasoconstriction. This vasoconstriction could be blocked by suramin, but not by PPADS or reactive blue 2. In preparations of the rat isolated perfused kidney model with an elevated vascular tone, bolus application of Up₄A showed a dose-dependent vasoconstriction that was followed by a dose-dependent vasodilation. The vasoconstriction was in part sensitive to P2X₁ receptor desensitization by α,β-meATP, and the remaining P2Y₂-mediated vasoconstriction was only inhibited by suramin. The Up₄A-induced vasodilation depended on activation of nitric oxide synthases, and was mediated by P2Y₁ and P2Y₂ receptor activation.

CONCLUSIONS AND IMPLICATIONS

Up₄A activated P2X₁ and P2Y₂ receptors to act as a vasoconstrictor, whereas endothelium-dependent vasodilation was induced by P2Y_1/2 receptor activation. Up₄A might be of relevance in the physiology and pathophysiology of vascular tone regulation.     

Effects of Ro 5-4864 and PK 11195 in rat duodenum and vas deferens.

Ro 5-4864 and PK 11195 inhibit in a concentration-dependent manner carbachol-induced contractions in rat duodenum (IC50: 1.56 +/- 0.07 x 10(-5) M and 1.18 +/- 0.07 x 10(-5) M respectively). The antagonism is non-competitive and is not mediated by peripheral-type benzodiazepine receptors. The Ro 5-4864 effect is modulated by the calcium concentration of the Tyrode-Ringer solution. In the presence of 1 mM NaF/10 microM AlCl3, Ro 5-4864 and PK 11195 do not inhibit carbachol-induced contractions. Moreover, Ro 5-4864 and PK 11195 significantly relax AlF(4-)-induced contractions, with IC50 values of 2.01 +/- 0.12 x 10(-5) M and 1.28 +/- 0.11 x 10(-5) M respectively. This effect is also modulated by the calcium concentration of the medium. Pertussis toxin potentiates the antagonist effects of Ro 5-4864 and PK 11195 on carbachol-induced contractions, but cholera toxin does not affect them. Ro 5-4864 and PK 11195 inhibit 45Ca2+ uptake induced by KCl (120 mM) in rat vas deferens, but do not affect either basal 45Ca efflux or noradrenaline-induced 45Ca2+ efflux. Only high doses of PK 11195 (above 5 x 10(-5) M) are able to produce a slight reduction of the accumulation of inositol phosphates induced by methoxamine in rat vas deferens, while Ro 5-4864 has no significant effect. Finally, Ro 5-4864 and PK 11195 reduce calcium influx, but do not seem to be the only mechanism of the antagonistic effect on carbachol-induced contractions. An alteration of other second messengers, probably cyclic monophosphate nucleotides, may be involved.     

Comparison of drug effects on the isolated rat colon and duodenum]

Adrenaline and isoproterenol elicited nearly maximal relaxation of the colon even in small doses, whereas increase in the doses caused greater relaxation in the duodenum. In the colon, these drugs prevented, to a great extent the contraction induced by acetylcholine (ACh) and serotonin but in the duodenum were totally ineffective. Dibenamine and propranolol reduced adrenaline- and isoproterenol-induced relaxation in the duodenum, though propranolol decreased the relaxation caused by isoproterenol. Atropine prevented ACh-induced contraction in both the colon and duodenum in the same way. After 2-bromolysergic acid diethylamide, duodenal contraction caused by ACh or serotonin decreased by over 70%; however, the contraction of the colon was not significantly inhibited. Methysergide had similar effects, but to a lesser degree. In calcium-free bathing fluid without addition of Na2EDTA, ACh and prostaglandin E1 elicited contraction in the colon, but not in the duodenum.     

Evidence that adenosine 5'-triphosphate is the third inhibitory non-adrenergic non-cholinergic neurotransmitter in the rat gastric fundus

In the rat gastric fundus, non-adrenergic, non-cholinergic (NANC) relaxations are mediated by nitric oxide (NO), vasoactive intestinal polypeptide (VIP), and a third, as yet unidentified, neurotransmitter. The possible involvement of adenosine 5'-triphosphate (ATP) in the NANC relaxations was examined using pyridoxalphosphate-6-azophenyl-2',5'-disulphonic acid (PPADS), apamin and desensitization to alpha,beta-methylene ATP. NANC responses were studied in the absence and presence of N(G)-nitro-L-arginine methyl ester (NAME; 100 microM) and alpha-chymotrypsin (1 u ml(-1)), to inhibit responses to NO and VIP, respectively. PPADS (100 microM), apamin (1 microM) and desensitization to alpha,beta-methylene ATP (10 microM, three additions) all significantly (P<0.05) reduced NANC relaxations to electrical field stimulation (0.5 - 4 Hz, 30 s trains) in longitudinal strips of rat gastric fundus and almost abolished the residual relaxation remaining in the presence of NAME and alpha-chymotrypsin. PPADS had no effect on responses to the NO-donor, sodium nitroprusside (SNP), or VIP. Apamin slightly reduced relaxations to SNP, but did not affect those to VIP, whereas desensitization to alpha,beta-methylene ATP markedly reduced responses to both SNP and VIP. The effects of PPADS and apamin in this study provide strong evidence that the third inhibitory NANC neurotransmitter in the rat gastric fundus is ATP.     

The effects of vasoactive intestinal polypeptide and of adenosine 5'-triphosphate on the isolated anococcygeus muscle of the mouse.

1 Vasoactive intestinal polypeptide (VIP, 0.01- MicroM) produced dose-related relaxations of the mouse anococcygeus muscle. 2 Following incubation with indomethacin (2.8 microM 1 h) adenosine 5'-triphosphate (ATP, 0.5-10 mM) produced dose-related relaxations of the mouse anococcygeus. 3 Haemolysed blood reduced inhibitory responses of the mouse anococcygeus to field stimulation but had no effect on relaxations to VIP or ATP. 4 Apamin (0.5 microM) had no effect on the relaxation of mouse anococcygeus to field stimulation, VIP, or ATP. 5 2-2'-Pyridylisatogen tosylate (PIT, 50 microM) itself reduced muscle tone but it did not abolish inhibitory responses to field stimulation, VIP, or ATP. 6 During prolonged inhibitory nerve stimulation the relaxation of the mouse anococcygeus in response to VIP was reduced greatly while that to ATP was unaffected. 7 Bundles of VIP-immunoreactive sites were detected in sections of the mouse anococcygeus treated by the peroxidase-antiperoxidase (PAP) immunocytochemical technique. 8 The results suggest that the mechanisms underlying non-adrenergic, non-cholinergic inhibitory transmission in the mouse anococcygeus are similar to those in the bovine retractor penis and unlike those in the guinea-pig taenia caeci. 9 The possibility that VIP or ATP might be involved in inhibitory neurotransmission in the mouse anococcygeus is discussed.