REGIONAL DIFFERENCE OF ENDOGENOUS ATP RELEASE IN THE PULMONARY ARTERY OF RABBITS

1. The release of adenyl purines such as ATP, ADP, AMP and adenosine, from the pulmonary artery trunk (PAT), extra-pulmonary artery (EPA) and intrapulmonary artery (IPA) were compared. 2. The amount of basal overflow of adenyl purines from the PAT was significantly smaller than those from EPA and IPA. There was no significant difference between the amount of the overflow from EPA and PAT. 3. Methoxamine, an α₁-adrenoceptor agonist, significantly increased the overflow of adenine nucleotides from the PAT, EPA and IPA, but did not increase those of adenosine. Methoxamine-induced release of adenyl purines from IPA was significantly larger than those from EPA and PAT. 4. These results suggest that an α,-adrenoceptor-mediated mechanism for ATP-release is not homogeneously distributed in the pulmonary artery and a larger amount of ATP may be released in the peripheral part of the artery.     

Co-release of endogenous ATP and noradrenaline from guinea-pig mesenteric veins exceeds co-release from mesenteric arteries

1. The present study was designed to compare the overflow of sympathetic neurotransmitters of guinea-pig inferior mesenteric artery and mesenteric vein evoked by electrical field stimulation (EFS) with special emphasis on the simultaneous release of ATP and noradrenaline (NA). The stimulation-evoked overflow of ADP, AMP and adenosine was also evaluated. 2. Endothelium-denuded segments of inferior mesenteric arteries or veins were superfused in a small volume (200 microL)-chamber for EFS and subsequent detection of NA (HPLC- electrochemical detection) and adenine nucleotides and adenosine (HPLC-fluorescence detection) in samples of the superfusate. 3. Both arteries and veins responded to EFS (15 V, 4-16 Hz, 0.3 msec for 60 s) with overflow of ATP and NA in a tetrodotoxin (1 micromol/L)- and guanethidine (10 micromol/L)-sensitive manner. The EFS-evoked overflow of NA in veins exceeded the overflow of NA in arteries at all frequencies of stimulation, whereas the EFS-evoked overflow of ATP, ADP and AMP in veins exceeded the overflow of adenine nucleotides in arteries at 4 and 8 Hz but not at 16 Hz stimulation. The EFS-evoked overflow of adenosine was similar in arteries and veins. 4. Activation of alpha1-adrenoceptors with methoxamine (10 micromol/L) did not produce overflow of ATP. 5. Blockade of alpha1/alpha2-adrenoceptors with phentolamine (1 micromol/L) did not affect EFS-evoked overflow of ATP, ADP, AMP and adenosine. 6. It is concluded that overflow of ATP and NA from sympathetic nerves may constitute an effective mechanism in the complex balance between capacitance and resistance in splanchnic circulation.     

Participation by purines in the modulation of norepinephrine release by methoxamine

The alpha 1-receptor agonist methoxamine reduced, by a prazosin sensitive mechanism, the nerve stimulation evoked release of norepinephrine in the rat caudal artery. The effect of methoxamine was also antagonized by the purinoceptor antagonist 8-(p-sulfophenyl)theophylline suggesting an involvement of endogenous purines in this process. Indeed, methoxamine caused the release of adenine nucleotides and adenosine, an action which was blocked by prazosin. These results suggest that methoxamine releases ATP or a related purine which in turn decreases transmitter release by acting on prejunctional purinoceptors.     

BOTH EXTRACELLULAR ATP AND SHEAR STRESS REGULATE THE RELEASE OF NITRIC OXIDE IN RAT CAUDAL ARTERY

1. To elucidate the physiological role of nitric oxide (NO) in regulating vascular tone, the effects of NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, on the vasoconstrictor response to noradrenaline (NA) in rat caudal artery was examined. 2. NG-Nitro-L-arginine methyl ester significantly potentiated the NA-induced increase in perfusion pressure in the perfused caudal artery, but did not affect the NA-induced contraction in caudal artery ring preparations. In addition, an increase in perfusion pressure mechanically produced by a stepwise increase in flow rate was not affected by L-NAME. 3. Noradrenaline evoked a significant increase in the release of endogenous ATP and its metabolites from the perfused artery, whereas increased perfusion pressure as a result of increased flow rate did not evoke release of endogenous ATP. 4. In the presence of exogenously applied ATP, L-NAME significantly potentiated the increase in perfusion pressure produced by increased flow rate. 5. These results indicate that perfused vascular tone is regulated by endogenous NO and suggest that extracellular ATP may participate in the synthesis and release of NO by shear stress in endothelial cells in the rat caudal artery.     

Characterization of prejunctional purinoceptors on adrenergic nerves of the rat caudal artery

Summary The effects of a number of purinoceptor agents on the release of endogenous noradrenaline from the electrically stimulated rat caudal artery were determined. Noradrenaline was quantified by high performance liquid chromatography-electrochemical detection techniques. Both P₁-receptor and P₂-receptor agonists reduced the release of noradrenaline; the relative order of potency being 2-chloroadenosine > beta, gamma methylene ATP > ATP adenosine. The adenosine uptake inhibitor S-p-nitrobenzyl-6-thioguanosine potentiated the effects of adenosine but not those of the adenine nucleotides. This suggests that the nucleotides do not need to be converted to adenosine to produce a prejunctional inhibition of the release of noradrenaline. The P₁-receptor antagonist 8-(p-sulfophenyl) theophylline reduced the inhibitory effects of both P₁- and P₂-receptor agonists as did the photolysis of tissues with an intense light source. The findings indicate that prejunctional purinoceptors that mediate an inhibition of the release of noradrenaline from the adrenergic nerves of the caudal artery may not be adequately defined as either P₁- or P₂-receptors and thus appear to represent a unique receptor. We suggest referring to these receptors as P₃-purinoceptors.Supported by grants from the American Heart Association, the Bently Nevada Corp. and NIH Grant HL38126 Send offprint requests to D. P. Westfall at the above address     

Characterization of the facilitatory modulation of adrenergic neurotransmission via prejunctional purinoceptors in rabbit ear artery]

The purinergic modulation of the release of norepinephrine (NE) from sympathetic nerves in the rabbit ear artery was investigated. Methoxamine, an alpha 1-adrenoceptor agonist, enhanced the NE-release by electrical stimulation (ES) and released large amounts of adenyl purines (ATP, ADP, AMP and adenosine) from the endothelium. Both actions of methoxamine were blocked by prazosin. In addition, the enhancement of the NE-release by methoxamine was abolished by 8-sulfophenyl theophylline (8SPT), a P1-Purinoceptor antagonist. These findings indicated that the endogenous purines and purinoceptors participate in the facilitation of NE-release by alpha 1-adrenoceptor stimulation. P1-Purinoceptor agonists, such as adenosine and 2-chloroadenosine, and P2-purinoceptor agonists, such as ATP and beta,gamma-methylene ATP (beta gamma-mATP), enhanced the ES-evoked NE-release. This enhancement was antagonized not only by the P1-purinoceptor antagonist, 8SPT, but also by the P2-purinoceptor desensitizing agent, alpha,beta-methylene ATP. A phosphodiesterase inhibitor, Ro20-1724 potentiates the enhancement of NE-release by beta gamma-mATP. On the other hand, an adenylate cyclase inhibitor, SQ22536, inhibited the enhancement of NE-release by beta gamma-mATP. These findings suggested that prejunctional facilitatory purinoceptors exist on the adrenergic nerves of the rabbit ear artery. This receptor may be coupled to adenylate cyclase and is different from well-known P1 and P2 purinoceptors. In the rabbit ear artery, adrenergic neurotransmission may be regulated by endogenous ATP and its metabolites via prejunctional facilitatory purinoceptors, which were initiated by alpha 1-adrenoceptor stimulation; i.e. transsynaptic regulation of neurotransmission.     

NMDA receptors are involved in dithiothreitol-induced hypothermia

The effect of nicorandil, an ATP-sensitive K(+) channel opener, on the level of intracellular Ca(2+) ([Ca(2+)](i)) and on ATP release in endothelial cells of the rat caudal artery was examined using a fluorescent confocal microscopic imaging system and high-performance liquid chromatography (HPLC) with fluorescent detection, respectively. Nicorandil significantly increased [Ca(2+)](i) and the overflow of ATP and its metabolites. The former reaction was abolished in the absence of extracellular Ca(2+), but it did not change in the presence of thapsigargin or cyclopiazonic acid. The increase in the overflow of ATP and [Ca(2+)](i) induced by nicorandil was markedly suppressed by glibenclamide, an ATP-sensitive K(+) channel blocker. The increase of [Ca(2+)](i) induced by nicorandil was significantly and inversely correlated with the level of intracellular ATP in the endothelial cells, suggesting that activation of ATP-sensitive K(+) channels by nicorandil increases Ca(2+) influx in endothelial cells. The increase of [Ca(2+)](i) might be associated with ATP release.     

Effect of reserpine on adenosine uptake and metabolism,and subcellular transport of platelet adenosine triphosphate in washed rabbit platelets

There is a slow exchange of adenine nucleotides between the metabolically active (cytoplasmic) pool and the releasable amine storage organelle pool of blood platelets. Reserpine is known to inhibit serotonin uptake into platelet storage organelles. Therefore, we have determined whether reserpine also inhibits the uptake of adenine nucleotides from the cytoplasm into the storage organelles of rabbit platelets. Transport of adenine nucleotides from the metabolically active pool into the releasable amine storage granule pool was followed by labeling the metabolically active pool of adenine nucleotides by incubating the platelets with [¹⁴C]adenosine or [¹⁴C]adenine. Practically complete release of amine storage granule constituents was induced at various times in aliquots of the labeled platelet suspensions by treatment with a high concentration of thrombin (0.45 units/ml. The fraction of the total labeled [¹⁴C]ATP released was taken as a measure of ATP transport from the metabolically active pool into the releasable pool. Reserpine (0.2 and 2 μM) decreased the rate of ATP transport into the storage granules by about 50 per cent. Platelets obtained from rabbits that had received 5 mg/kg of reserpine intraperitoneally 18 hr prior to the collection of blood released less ATP and ADP than control platelets from animals that had not received any drugs. This was not due to inhibition of the release reaction by reserpine. Since reserpine reduces the amount of adenine nucleotides in the storage granules, we conclude that if it affects the rate of efflux of adenine nucleotides from the granules at all, this effect must be slight compared with the inhibition of the uptake into the granules. Reserpine was also found to decrease the incorporation of [8-¹⁴C]adenosine into platelet adenine nucleotides by inhibiting adenosine uptake into the platelets noncompetitively (K_i = 2 μM). Inosine uptake was also inhibited by reserpine. The effect of reserpine on adenosine uptake was reversible. In contrast, the effect of reserpine on ATP transfer from the metabolically active pool into the releasable pool was irreversible. This is in keeping with earlier observations that some reserpine binds to platelets reversibly and some binds irreversibly.     

MODULATORY EFFECTS OF TYPE-C NATRIURETIC PEPTIDE ON SYMPATHETIC COTRANSMISSION IN THE RAT ISOLATED TAIL ARTERY

1. Rat type-C natriuretic peptide (CNP) has been studied for its effects on the neurogenically induced overflow of adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), adenosine 5-monophosphate (AMP), adenosine (ADO) and noradrenaline (NA) in endothelium-free segments of rat isolated tail artery. The overflow of each was evoked by electrical field stimulation (EFS) of 0.5 ms pulses at 8 Hz for 3 min and the amount of ATP, ADP, AMP and ADO was quantified by high-performance liquid chromatography (HPLC)-fluorescent detection, while the amount of NA was quantified by HPLC-electrochemical detection. 2. Type-C natriuretic peptide (100 nmol/L) was found to cause a significant reduction of the overflow of all adenine purines and NA. However, at lower concentrations (1 and 10 nmol/L), CNP caused a significant reduction of the overflow of NA but did not change ATP overflow. 3. The overflow of ADP, AMP and ADO was significantly reduced by either concentration of CNP, so that the ratio ATP:ADP was diminished from 1:2 in controls to 1:1 after 1 nmol/L CNP and to 1:1.2 after 10 nmol/L CNP. 4. The production of inorganic phosphate (P_i) in response to the exogenous application of ATP was significantly reduced by 1,10 or 100 nmol/L CNP. 5. Type-C natriuretic peptide exerts neuromodulatory effects on the neurogenically induced release of the cotransmitters ATP and NA in rat tail artery, consisting of an inhibition of the release of both ATP and NA. This effect is accompanied by inhibition of the breakdown of ATP by ecto-ATPases. Either effect results in apparent CNP-induced differential modulation of the overflow of the cotransmitters ATP and NA.     

N-type and P/Q-type calcium channels regulate differentially the release of noradrenaline, ATP and beta-NAD in blood vessels

Using HPLC techniques we evaluated the electrical field stimulation-evoked overflow of noradrenaline (NA), adenosine 5'-triphosphate (ATP), and beta-nicotinamide adenine dinucleotide (beta-NAD) in the presence of low nanomolar concentrations of omega-conotoxin GVIA or omega-agatoxin IVA in the canine mesenteric arteries and veins. omega-conotoxin GVIA abolished the evoked overflow of NA and beta-NAD in artery and vein, whereas the evoked overflow of ATP remained unchanged in the presence of omega-conotoxin GVIA. omega-agatoxin IVA significantly reduced the evoked overflow of ATP and beta-NAD. The overflow of NA remained largely unaffected by omega-agatoxin IVA, except at 16Hz in the vein where the overflow of NA was reduced by about 50%. Artery and vein exhibited similar expression levels of the alpha(1B) (CaV2.2, N-type) subunit, whereas the vein showed greater levels of the alpha(1A) (CaV2.1, P/Q-type) subunit than artery. Therefore, there are at least two release sites for NA, beta-NAD and ATP in the canine mesenteric artery and vein: an N-type-associated site releasing primarily NA, beta-NAD and some ATP, and a P/Q-type-associated site releasing ATP, beta-NAD and some NA. The N-type-mediated mechanisms are equally expressed in artery and vein, whereas the P/Q-type-mediated mechanisms are more pronounced in the vein and may ensure additional neurotransmitter release at higher levels of neural activity. In artery, beta-NAD caused a dual effect consisting of vasodilatation or vasoconstriction depending on concentrations, whereas vein responded with vasodilatation only. In contrast, ATP caused vasoconstriction in both vessels. beta-NAD and ATP may mediate disparate functions in the canine mesenteric resistive and capacitative circulations.     

The alpha-adrenoceptor antagonist, zolertine, inhibits alpha1D- and alpha1A-adrenoceptor-mediated vasoconstriction in vitro

1. The antagonist effect of zolertine (4-phenyl-1-[2-(5-tetrazolyl)ethyl]piperazine trihydrochloride), on vascular contraction elicited by noradrenaline in aorta, carotid (alpha1D-adrenoceptors), mesenteric (alpha1A/D-adrenoceptors) and caudal arteries (alpha1A-adrenoceptors) from Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats and rabbit aorta (alpha1B-adrenoceptors), was investigated in endothelium-denuded arterial rings. 2. The selective alpha1D-adrenoceptor agonist, noradrenaline, elicited concentration-dependent contractions in all arterial rings from both species. Noradrenaline selectivity was: carotid = aorta > mesenteric = rabbit aorta > caudal arteries. 3. The contractile responses induced by noradrenaline were competitively antagonized by zolertine in rat carotid and aorta arteries, yielding pA2 values of WKY, 7.48 +/- 0.18; SHR, 7.43 +/- 0.13 and WKY, 7.57 +/- 0.24; SHR, 7.40 +/- 0.08, respectively. Zolertine was a non-competitive antagonist in some blood vessels as Schild plot slopes were lower than unity. The pKb estimates for zolertine were WKY, 6.98 +/- 0.16; SHR, 6.81 +/- 0.18 in the mesenteric artery, WKY, 5.73 +/- 0.11; SHR, 5.87 +/- 0.25 in the caudal artery and 6.65 +/- 0.09 in rabbit aorta. 4. Competition binding experiments using the alpha1-adrenoceptor antagonist [3H]prazosin showed a zolertine pKi of 6.81 +/- 0.02 in rat liver (alpha1B-adrenoceptors) and 6.35 +/- 0.04 in rabbit liver (alpha1A-adrenoceptors) membranes. 5. Zolertine showed higher affinity for alpha1D-adrenoceptors compared to alpha1A-adrenoceptors, while it had an intermediate affinity for alpha1B-adrenoceptors. The ability of the alpha1-adrenoceptor antagonist zolertine to block alpha1D-adrenoceptor-mediated constriction in different vessels of WKY and SHR rats may explain its antihypertensive efficacy despite its low order of potency.     

Characteristics of the P2 purinoceptor that mediates prostacyclin production by pig aortic endothelial cells

Release of prostacyclin was studied by superfusing small columns containing cells cultured on microcarrier beads. Transient dose-dependent stimulation of prostacyclin release by up to 500-fold was induced by adenosine 5'-triphosphate (ATP; 0.5-50 microM). Adenosine 5'-diphosphate (ADP) gave similar responses, whereas adenosine 5'-phosphate (AMP) and adenosine were essentially inactive. Of other natural nucleotides tested only uridine 5'-phosphate (UTP) was active. The L-enantiomers of ATP and ADP were inactive. 2-Cl-ATP was approximately 100 times more potent than ATP; 2-MeS-ATP was also more potent (threshold 0.05 microM) but its maximal effectiveness was less than 20% that of ATP; 2-EtS-ATP had a similar threshold to ATP but was even less effective than 2-MeS-ATP. Phosphorothioate nucleotide analogues of ATP or ADP were active, with no stereoselectivity between Rp and Sp diastereoisomers. No analogue tested showed antagonist activity. We conclude that ATP mediates endothelial prostacyclin release apparently via a P2Y receptor, although there are some striking differences from the previously described P2Y receptor mediating endothelium-dependent vasodilation in pig aorta.     

Potentiation of adenosine triphosphate-induced contractile responses of the guinea-pig isolated vas deferens by adenosine monophosphate and adenosine 5''-monophosphorothioate.

The effects of incubating the guinea-pig isolated vas deferens in the presence of adenine nucleotides (adenosine triphosphate, ATP; adenosine diphosphate, ADP; and adenosine monophosphate, AMP), or in the presence of their phosphorothioate analogues (adenosine 5'-O-(3-thiotriphosphate), ATP gamma S; adenosine 5'-O-(2-thiodiphosphate), ADP beta S; and adenosine 5'-monophosphorothioate, AMP alpha S), on contractile responses to ATP were compared. After challenge with a low (1 microM) or high (300 microM) concentration of ATP to obtain control responses, one vas deferens of a pair was incubated for 5 min with one of the adenine nucleotides, while the contralateral preparation was incubated with the corresponding phosphorothioate analogue. At the conclusion of the incubation the preparations were challenged again with ATP. Incubation with AMP or AMP alpha S resulted in a transient potentiation of responses to 1 microM and 300 microM ATP. The potentiation following incubation with AMP alpha S was larger than that produced by AMP. After incubation with ADP, ADP beta S, ATP and ATP gamma S, responses to 1 microM ATP were decreased, while those to 300 microM ATP were unaffected. Thus, incubation with AMP and AMP alpha S results in potentiation, rather than inhibition, of ATP-induced responses. On the other hand, 5'-diphosphate, 5'-triphosphate, 5'-O-(2-thiodiphosphate) and 5'-O-(3-thiotriphosphate) moieties on adenosine have no effect or cause autoinhibition. These results indicate that AMP exerts a potentiating effect on reactivity to exogenous ATP. AMP arising from the enzymatic degradation of ATP might modulate the level of response to ATP released endogenously as a cotransmitter.     

Nicorandil stimulates release of adenyl purines from porcine coronary artery

1. To clarify the mechanism of the cardioprotective effect of nicorandil (2-nicotinamidoethyl-nitrate ester), the effects of nicorandil and nitric oxide (NO) donors on the release of ATP, ADP, AMP and adenosine from arterial segments and cultured endothelial cells of the porcine coronary artery were examined. 2. Nicorandil significantly increased the release of total adenyl purines from arterial segments and from cultured endothelial cells. 3. Cromakalim, an ATP-sensitive K+ channel opener, did not affect the release of total adenyl purines from coronary artery segments. 4. s-Nitroso-N-acetyl-D,L-penicillamine and isosorbide dinitrate, NO donors, significantly increased the release of total adenyl purines from coronary artery segments. 5. These results demonstrate that nicorandil stimulates ATP release from the coronary artery by acting not as an ATP-sensitive K+ channel opener, but as a nitrate, thus suggesting the cardioprotective properties of nicorandil.     

Participation of ATP in cell volume regulation in the endothelium after hypotonic stress

1. The role of ATP in the regulatory volume decrease (RVD) after hypotonic cell swelling was examined in cultured endothelial cells isolated from the rat caudal artery. 2. Hypotonic stress increased [Ca2+]i in addition to increasing the overflow of ATP and cell volume. The hypotonicity induced increase in [Ca2+]i was prevented by pyridoxalphosphate-6-azophenyl-1-2',4'-disulphonic acid (PPADS; a P2 purinoceptor antagonist), U-73122 (a phospholipase C inhibitor) and thapsigargin (a Ca2+ pump inhibitor). However, the hypotonicity induced increase in cell volume was potentiated by PPADS, U-73122 and thapsigargin. 3. Similar changes were observed in cells treated with 2-methylthioATP, a P2Y purinoceptor agonist, but not by alpha,beta-methylene ATP, a P2X purinoceptor agonist. Thus, it appears that the responses observed following hypotonic stress are mediated by activation of P2Y purinoceptors. 4. On the basis of these findings, it is suggested that ATP, which is released by hypotonicity, may participate in the RVD as a substantial regulator or initiator via P2 purinoceptor-induced increases in [Ca2+]i.     

Subtypes of alpha 1-adrenoceptors involved in noradrenaline-induced contractions of rat thoracic aorta and dog carotid artery.

We tried to determine alpha 1-adrenoceptor subtypes involved in noradrenaline-induced contractions of rat thoracic aorta and dog carotid artery. Prazosin competitively antagonized the contractions induced by noradrenaline in both the arteries with a high pKB value (approximately 9.7). WB4101, benoxathian, phentolamine, HV723 and 5-methylurapidil also competitively antagonized the responses to noradrenaline in both the arteries: however, the affinities for the antagonists were significantly higher in the rat thoracic aorta than in the dog carotid artery. The affinities for the competitive antagonists were not changed by treatment with nifedipine. In the rat thoracic aorta, chlorethylclonidine (CEC) elicited either a persistent contraction with rhythmic activities before treatment with nifedipine or partial inactivation of alpha 1-adrenoceptors in the presence of nifedipine. On the other hand, CEC produced only inactivation of alpha 1-adrenoceptors in the dog carotid artery. These results suggest that noradrenaline-induced contractions of the rat thoracic aorta and dog carotid artery are respectively mediated through distinct alpha 1-adrenoceptor subtypes. According to the alpha 1A, alpha 1B subclassification, the alpha 1-adrenoceptor of dog carotid artery is like the alpha 1B subtype, while that of rat thoracic aorta is atypical. Both subtypes are also identified as a high affinity site for prazosin (alpha 1H subtype) in the alpha 1H, alpha 1L and alpha 1N subclassification.     

Inhibition of adrenergic neuroeffector transmission in rabbit pulmonary artery and aorta by adenosine and adenine nucleotides

The effect of adenosine and adenine nucleotides on sympathetic neuroeffector transmission in the rabbit isolated pulmonary artery and aorta was studied. Adenosine (10(-5)-3 x 10(-4)M) decreased the contractile response of pulmonary artery and aorta evoked by electrical-field stimulation. The decrease was reversible. No tachyphylaxis developed. Inhibition of either adenosine deaminase by deoxycoformycin (3.6 x 10(-6)M) or of adenosine transport by dilazep (3 x 10(-6)M) did not alter the inhibitory effect of adenosine on the neurogenic contractions in the pulmonary artery. However, deoxycoformycin plus dilazep markedly enhanced the inhibitory effect of adenosine. The calcium antagonists nifedipine (1.5 x 10(-8)M) and nimodipine (1.3 x 10(-8)M) had no effect on the adenosine-induced inhibition. This was also the case with theophylline (5 x 10(-5)M), atropine (10(-7)M), and the prostaglandin synthetase inhibitors indomethacin (5 x 10(-5)M and suprofen (3 x 10(-5)M). The contractile response of the pulmonary artery elicited by exogenous (-)-noradrenaline (NA; 10(-9)-3 x 10(-4)M) was essentially not altered by adenosine (10(-5)-3 x 10(-4)M). Adenosine (10(-4)M) did not alter the spontaneous 3H-outflow from rabbit aorta preloaded with 3H-(-)-noradrenaline (3H-NA). Adenosine (10(-5)-3 x 10(-4)M), ADP (10(-4)M), ATP (10(-5)M), and inosine (10(-4)M) diminished the overflow of tritium from pulmonary artery and aorta preloaded with 3H-NA. The spontaneous outflow of tritium from aorta preloaded with 3H-NA consisted of 3H-NA (17%), 3H-dihydroxyphenylglycol (3H-DOPEG; 30%), 3H-dihydroxymandelic acid (3H-DOMA, 4%), 3H-O-methylated and deaminated metabolites (3H-OMDA; 42%), and 3H-normethanephrine (3H-NMN; 2%). Adenosine (10(-5) and 10(-4)M) enhanced 3H-DOPEG and 2H-NMN, decreased 3H-NA, and did not alter 3H-DOMA and 3H-OMDA. The stimulation-evoked overflow of tritium for aorta preloaded with 3H-NA consisted of 3H-NA (31%), 3H-DOPEG (18%), 3H-DOMA (2%), 3H-ONDA (46%), and 3H-NMN (3%). Adenosine (10(-5) and 10(-4)M) enhanced 3H-NA and 3H-DOPEG, decreased 3H-OMDA and did not alter 3H-DOMA and 3H-NMN. Adeosine (10(-6)-10(-4)M) did not alter the accumulation of 3H-NA (10(-8)M) by aorta. It is concluded that adenosine inhibits vascular sympathetic neuroeffector transmission by diminishing the release of transmitter from the nerve terminals.     

Selective modulation of noradrenaline release by alpha 2-adrenoceptor blockade in the rat-tail artery in vitro

The effects of blocking alpha(2)-adrenoceptors on noradrenaline (NA) and adenosine 5'-triphosphate (ATP) release from postganglionic sympathetic nerves have been investigated in rat-tail artery in vitro. Continuous amperometry was used to measure NA release and intracellularly recorded excitatory junction potentials (e.j.p.'s) were used to measure ATP release. Application of the alpha(2)-adrenoceptor antagonist, idazoxan (1 microm), increased the amplitude of NA-induced oxidation currents evoked by trains of 10 stimuli at 1 and 10 Hz. In cells deep in the media, idazoxan (1 microm) had no effect on the amplitude of e.j.p.'s evoked by trains of 10 stimuli at 1 and 10 Hz. In cells close to the adventitial - medial border, idazoxan produced a small increase in the amplitude of e.j.p.'s evoked at the end of trains of 10 stimuli at 1 Hz. In tissues pretreated with the neuronal NA uptake inhibitor, desmethylimpramine (0.3 microm), idazoxan (1 microm) markedly increased the amplitude of e.j.p.'s in cells deep in the media. The alpha(2)-adrenoceptor agonist, clonidine (0.5 microm), produced similar reductions in the amplitudes of both NA-induced oxidation currents and e.j.p.'s evoked by 10 stimuli at 1 Hz. These effects of clonidine were reversed by the subsequent addition of idazoxan (1 microm). The release of both NA and ATP is inhibited to a similar extent by activation of prejunctional alpha(2)-adrenoceptors by clonidine. In contrast, endogenously released NA more markedly inhibits NA release. These findings provide further support for the differential modulation of NA and ATP release.     

Nucleotide-mediated relaxation in guinea-pig aorta: selective inhibition by MRS2179

The vasodilatory effects of nucleotides in the guinea-pig thoracic aorta were examined to determine the relationship between molecular expression and function of P2Y receptors. In aortic rings precontracted with norepinephrine, vasodilatory responses to purine nucleotides exhibited a rank-order of potency of 2-methylthio-ATP>ADP>ATP. Responses to UTP, but not UDP suggested a functional role for P2Y4 but not P2Y6 receptors. Aortic endothelial cells express at least four P2Y receptors; P2Y1, P2Y2, P2Y4 and P2Y6. In primary culture, these cells exhibit desensitizing transient calcium responses characteristic of P2Y1, P2Y2 and P2Y4, but not P2Y6 receptors. UDP had no effect on endothelial cell calcium. The pyrimidinergic receptor agonist UTP is capable of eliciting robust vasodilation in aortic rings and causing calcium responses in cultured guineapig aortic endothelial cells. These responses are equivalent to the maximum responses observed to ATP and ADP. Measurement of intracellular calcium release in response to ATP and 2-methylthio-ATP were similar, however only the 2-methylthio-ATP response was sensitive to the P2Y1 antagonist N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179). In aortic rings, vasodilatory responses to 2-methylthio-ATP, ATP and ADP were all blocked by pre-incubation of tissues with MRS2179. MRS2179 pretreatment had no effect of the ability of UTP to cause relaxation of norepinephrine responses in aortic rings or the ability of UTP to cause calcium release in aortic endothelial cells. We demonstrate robust effects of purine and pyrimidine nucleotides in guineapig aorta and provide functional and biochemical evidence that MRS2179 is a selective P2Y1 antagonist.     

P2 purinoceptor regulation of mucin release by airway goblet cells in primary culture.

1. The effects of adenine analogues on mucin release by airway goblet cells have been examined in a hamster primary tracheal epithelial cell culture. 2. Adenosine, a P1 receptor agonist, had no effect on mucin release even at 2 mM, while ATP, a P2 receptor agonist, stimulated mucin release in a dose-dependent fashion with an apparent EC50 of 20 microM. The relative potency order among adenine nucleotides was ATP greater than ADP greater than AMP = adenosine. 3. ATP gamma S, a non-hydrolyzable analogue of ATP, was equipotent with ATP in stimulating mucin release. The potency order among some ATP analogues was ATP greater than 2-methylthio ATP greater than alpha, beta-methylene ATP greater than beta, gamma-methylene ATP. Reactive blue 2, a putative P2 gamma-purinoceptor antagonist, did not block the ATP-induced mucin release. 4. The present results indicate that mucin release by airway goblet cells is stimulated by extracellular ATP via P2 receptor-mediated mechanism. We suggest that this mechanism may be important in the physiological regulation of airway goblet cell mucin release in vivo.