Isolated Rabbit Ear Artery Research Articles

Effects of some sympathomimetic amines on the response of the rabbit isolated ear artery to noradrenaline and electrical stimulation.

Effects of some sympathomimetic amines on the response of the rabbit isolated ear artery to noradrenaline and electrical stimulation.

Structure–activity relationships of isoeugenol‐based chlorophenylpiperazine derivatives on serotonergic/adrenergic receptor, platelet aggregation, and lipid peroxidation

AbstractThree isoeugenol‐based eugenosedin chlorphenylpiperazine derivatives, Eu‐A, Eu‐B, and Eu‐C, were synthesized and tested for their serotonergic, adrenergic antagonist, antioxidant, and anti‐aggregation activities. In radioligand binding assays, all three agents displayed significant binding affinities on α1, α2, β1, 5‐HT1B, and 5‐HT2A receptors. In human platelet, they inhibited epinephrine and 5‐HT‐induced aggregation, and in human platelet with α2 and 5‐HT2A receptors they had a competitive binding effect. Eu‐B and Eu‐C were more potent than Eu‐A. All compounds had antioxidant effects derived from aryloxypropanolamine. Eu‐ A, Eu‐B, or Eu‐C (1, 3, 5 mg/kg iv) given to normotensive Wistar rats produced a dose‐dependent decrease in mean arterial blood pressure and heart rate and when injected into the cisternum, Eu‐A, Eu‐B, or Eu‐C (0.3, 0.03 µmol) increased blood pressure within 15 min. Pretreatment with any of the three agents inhibited clonidine (38 pmol)‐induced hypotension. In vitro experiments, Eu‐A, Eu‐B, or Eu‐C (1, 10, and 100 µM) competitively antagonized norepinephrine‐, clonidine‐, and 5‐HT (10−8–10−4 M)‐induced vasocontraction in isolated rat aorta, and competitively antagonized isoproterenol (10−8–10−4 M)‐induced positive inotropic effects in a concentration‐dependent manner in the isolated rat left atrium. In isolated rabbit ear arteries sensitized with 16 mM K+, all three agents antagonized 5‐nonyloxytryptamine‐ and 5‐HT‐induced vasocontractions. These findings show that Eu‐A, Eu‐B, and Eu‐C possess functional α1, α2, β1, 5‐HT1B, and 5‐HT2A receptor blocking activities. In conclusion, the changes in the position of chloride at phenylpiperazine influenced the serotonergic receptor, adrenoceptor antagonistic activities, but not anti‐aggregation and antioxidant activities. Drug Dev Res 71:1–9, 2010. © 2010 Wiley‐Liss, Inc.

Hypotensive Effects of Eugenosedin-A with Serotonin, Alpha- and Beta-Adrenoceptor Antagonistic Activities in Spontaneously Hypertensive and Normotensive Rats

Eugenosedin-A is a newly synthesized compound with special serotonergic, α- and β₁-adrenergic blocking actions. Intravenous injection of eugenosedin-A significantly caused dose-dependent decreases in the mean arterial blood pressure and heart rate in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). The effects of eugenosedin-A-decreased blood pressure and heart rate in SHR were more potent than in WKY. In in vitro experiments, eugenosedin-A competitively antagonized the serotonin-, norepinephrine- and clonidine-induced vasocontraction in a concentration-dependent manner in isolated thoracic aorta of WKY and SHR. We also observed that eugenosedin-A competitively antagonized the isoproterenol-induced positive inotropic effects in a concentration-dependent manner in the isolated left atrium of WKY and SHR. These findings clearly suggested that eugenosedin-A possesses α₁/α₂, β₁ and 5-HT_2A receptor-blocking activities. The order of pA₂ values in isolated tissues of WKY was 5-HT_2A > α₁/α₂ > β₁. However, the order of pA₂ values in isolated tissues of SHR was α₁/α₂ >5-HT_2A > β₁. Similarly, we found that the in vitro functional activity of eugenosedin-A is quite different between WKY and SHR. On the other hand, in the isolated rabbit ear artery sensitized with 16 mmol/l K⁺, eugenosedin-A antagonized 5-nonyloxytryptamine- and serotonin-induced vasocontractions, indicating that it also blocked 5-HT_1B and 5-HT_2A receptors. In radioligand binding experiments, eugenosedin-A had significant binding affinities on α₁/α₂, β₁, 5-HT_1B and 5-HT_2A receptors. Finally, we suggest that the hypotensive effects of eugenosedin-A can be attributed to its multiple actions on the blockade of 5-HT_1B, 5-HT_2A, α and β₁ receptors in both WKY and SHR strains.

Identification of alpha-1L adrenoceptor in rabbit ear artery.

The alpha-1L adrenoceptor (AR) was identified in rabbit ear artery by both functional and ligand binding studies. In functional studies using arterial rings, the contractile response to NS-49 [(R)-(-)-3'-(2-amino-1-hydroxyethyl)-4'-fluorometh-anesulfonanilide hydrochloride] (alpha-1A and alpha-1L AR-selective agonist) was competitively antagonized with low affinities by prazosin, RS-17053 [N-[2-(2-cyclopropylmethoxyphenoxy) ethyl]-5-chloro-alpha,alpha-dimethyl-1H-indole-3-ethamine hydrochloride], and 5-methylurapidil but with high affinities by tamsulosin and KMD-3213 [(-)-1-(3-hydroxypropyl)-5-[(2R)-2-([2-[(2,2,2-trifluoroethoxy)phenoxy]ethyl]amino)propyl]-2,3-dihydro-1H-indole-7-carboxamide]. In contrast, the response to noradrenaline (nonselective alpha-1 AR agonist) was inhibited noncompetitively by these antagonists (except 5-methylurapidil) with Schild slopes different from unity. These results suggest that the response to NS-49 was mediated predominantly via alpha-1L ARs, whereas the response to noradrenaline was produced through two distinct alpha-1 AR subtypes (presumably alpha-1B and alpha-1L ARs). In binding studies with intact segments of rabbit ear artery, [3H]KMD-3213 bound with high affinity (pKD=9.7) to alpha-1 ARs, which were subdivided by prazosin, RS-17053, and 5-methylurapidil into two subtypes (alpha-1A and alpha-1L ARs). In contrast, [3H]prazosin binding sites in ear artery segments (pKD = 9.8) were identified as alpha-1A and alpha-1B ARs. In conventional binding studies using isolated rabbit ear artery microsomal membranes, [3H]KMD-3213 binding sites were identified as alpha-1A ARs with high affinities for prazosin, RS-17053, and 5-methylurapidil. Our study indicates that an alpha-1L AR having a unique pharmacological profile coexists with alpha-1A and alpha-1B ARs in rabbit ear artery and can be identified either functionally or by binding studies using intact tissues but not microsomal membrane preparations.

Mechanism of action of hypotensive prostaglandins in patients with essential hypertension.

Despite extensive investigation, the biological mechanisms causing essential hypertension (EHT) remain unclear. To clarify the means by which hypotensive prostaglandins (Hypo-PGs, mainly PGE1 and PGE2) act in patients with EHT, the interaction between intravenously infused Hypo-PGs and pressor substances such as an adrenergic neurotransmitter, noradrenaline (NA) and angiotensin II (AII) was examined both in patients with EHT and in perfused isolated rabbit ear artery preparations. In patients with EHT, Hypo-PGs were shown to reduce the pressor responses to intravenously infused NA or AII, although no significant difference was found between the pressor responses to NA under basal conditions and the responses during intravenous infusion of Hypo-PGs. Animal studies were undertaken to investigate the inhibitory action of Hypo-PGs on the vasoconstrictive responses to electrical stimulation of the perivascular sympathetic nerves (VSNS) and to exogenous NA at pre- and postjunctional sites in blood vessel walls. The suppressive action of Hypo-PGs on the response to VSNS was shown to be more potent than that to their action on the response to exogenous NA. Thus, it was concluded that the hypotensive action of intravenously infused Hypo-PGs in patients with EHT may be more dependent on prejunctional sites than on the postjunctional sites in the walls of blood vessels.

Effects of dermatan and dextran sulfates on arginine amidase activity released from isolated rabbit arteries.

We examined the levels of arginine amidase secreted from isolated rabbit arteries treated with dermatan and dextran sulfates, and the relation between the secretion of arginine amidase activity and the concentration of dermatan sulfate added to these arteries. The results showed that while dextran sulfate tended to accelerate the release of arginine amidase activity from the isolated rabbit ear artery, the induction was not significant. There was a significant increase in the level of arginine amidase released from the lower portion of isolated rabbit aorta (p<0.05), but no significant change in the upper portion of the aorta. In contrast, the addition of dermatan sulfate significantly increased the level of arginine amidase activity released from the isolated rabbit ear artery and the upper and lower portions of the aorta (p<0.05). Linear dose-response relationships were observed between the level of arginine amidase activity released from the isolated rabbit ear artery and aorta and the concentration of dermatan sulfate added.

Halothane Attenuates Myogenicity in the Rabbit Ear Artery

The aim of this study was to test the hypothesis that halothane interferes with the myogenic response to an increase in intraluminal pressure. Myogenic responsiveness refers to the intrinsic property of vascular smooth muscle to dilate and then constrict in response to an increase in intraluminal pressure, in an attempt to maintain vessel diameter. Vessel segments taken from the rabbit central ear artery were cannulated, pressurized to 60 mm Hg, and perfused with and suspended in Krebs solution. After exposure to extraluminal l-norepinephrine, vessels contracted to an initial diameter (Di) and were subjected to intraluminal pressure increases to 100 mm Hg. Myogenic reactivity was assessed by measurement of the extent of dilatation after the pressure increase from Di to a maximal diameter (Dm) and then the constriction and recovery (against the pressure increase) to a final (Df) diameter. Myogenicity was further assessed by determining the rate of return of the vessel diameter (angle of recovery) and vessel recovery (defined as Dm - Df/Dm - Di) and expressed as a percentage. Myogenicity was determined before and after exposure to halothane in concentrations of between 1-5%. Halothane significantly attenuated the myogenic response at all concentrations studied. The effect of halothane was maximal at a concentration of 5% where there was virtual abolition of the myogenic response with recovery assessed at 6+/-2.7% (SEM), compared with control (98+/-2.5%, P < 0.05). The angle of recovery was likewise attenuated. These data suggest that halothane, in a dose-dependent manner, attenuates myogenicity in the isolated rabbit ear artery preparation. Blood pressure is controlled partially by the myogenic response. This refers to the capacity of arteries to dilate and then constrict in response to pressure increase. Using arteries from rabbits, we have shown that administration of halothane reduces or abolishes this response. This observation may be a contributing factor to hypotension caused by halothane.

Halothane Attenuates Myogenicity in the Rabbit Ear Artery

The aim of this study was to test the hypothesis that halothane interferes with the myogenic response to an increase in intraluminal pressure. Myogenic responsiveness refers to the intrinsic property of vascular smooth muscle to dilate and then constrict in response to an increase in intraluminal pressure, in an attempt to maintain vessel diameter. Vessel segments taken from the rabbit central ear artery were cannulated, pressurized to 60 mm Hg, and perfused with and suspended in Krebs solution. After exposure to extraluminal l-norepinephrine, vessels contracted to an initial diameter (Di) and were subjected to intraluminal pressure increases to 100 mm Hg. Myogenic reactivity was assessed by measurement of the extent of dilatation after the pressure increase from Di to a maximal diameter (Dm) and then the constriction and recovery (against the pressure increase) to a final (Df) diameter. Myogenicity was further assessed by determining the rate of return of the vessel diameter (angle of recovery) and vessel recovery (defined as Dm − Df/Dm − Di) and expressed as a percentage. Myogenicity was determined before and after exposure to halothane in concentrations of between 1–5%. Halothane significantly attenuated the myogenic response at all concentrations studied. The effect of halothane was maximal at a concentration of 5% where there was virtual abolition of the myogenic response with recovery assessed at 6 ± 2.7% (SEM), compared with control (98 ± 2.5%, P < 0.05). The angle of recovery was likewise attenuated. These data suggest that halothane, in a dose-dependent manner, attenuates myogenicity in the isolated rabbit ear artery preparation. Implications Blood pressure is controlled partially by the myogenic response. This refers to the capacity of arteries to dilate and then constrict in response to pressure increase. Using arteries from rabbits, we have shown that administration of halothane reduces or abolishes this response. This observation may be a contributing factor to hypotension caused by halothane.

Effect of dextran derivatives on arginine amidase activities released from isolated rabbit arteries.

In the present research we examined the levels and types of arginine amidase activities that were released from isolated rabbit arteries treated with heparin or chondroitin sulfate. Heparin accelerated the release of arginine amidase activity from the isolated rabbit ear artery, the induction was not significant; a slight increase in activity was observed in the level of arginine amidase released from isolated rabbit aorta, but no significant difference was observed. On the other hand, it was revealed that the addition of chondroitin sulfate, accelerated this release from isolated rabbit ear artery with 5% significant differences. After the addition of chondroitin sulfate, the arginine amidase activity released from isolated rabbit arteries was analyzed using various affinity adsorption methods. This analysis confirmed the presence of two types of fibrinolytic enzymes: plasminogen/plasmin activity and plasminogen activators, but no thrombin was detected.

Effect of inhibition of tyrosine phosphatases on voltage-operated calcium channel currents in rabbit isolated ear artery cells.

1. The effect of increasing cellular tyrosine phosphorylation by inhibiting endogenous tyrosine phosphatases was examined on voltage-operated calcium channel currents in vascular smooth muscle cells. 2. In single ear artery smooth muscle cells of the rabbit, studied by the whole cell voltage clamp technique, intracellular application of the tyrosine phosphatase inhibitors, sodium orthovanadate (100 microM) and peroxyvanadate (100 microM orthovanadate + 1 mM H2O2) increased voltage-operated calcium channel currents by 56% and 83%, respectively. 3. Bath application of two other membrane permeant tyrosine phosphatase inhibitors, phenylarsine oxide (100 microM) and dephostatin (50 microM) also increased voltage-operated calcium channel currents by 48% and 52%, respectively. 4. The selective tyrosine kinase inhibitor, tyrphostin-23 (100 microM) reduced calcium channel currents by 41%. Pre-incubation with tyrphostin-23 abolished the effects of peroxyvanadate, phenylarsine oxide and dephostatin on calcium channels. 5. Western blot analysis of rabbit ear artery cell lysates showed increased tyrosine phosphorylation of several endogenous proteins following treatment with peroxyvanadate. 6. These results indicate that a number of structurally dissimilar inhibitors of tyrosine phosphatases increase voltage-operated calcium channel currents in arterial smooth muscle cells presumably due to increased tyrosine phosphorylation.

Synthesis, vasodilating and antithrombotic activity of pyridyl-substituted silylisoxazolines

3 - (2-Pyridyl) - 5 - phenyldimethylsilylisoxazoline, 3-(3-pyridyl) - 5 - phenyldimethylsilylisoxazoline and 3-(4-pyridyl) - 5 - phenyldimethylsilylisoxazoline were obtained by the [2+3] cycloaddition reaction of pyridyl nitrile oxides to phenyldimethylvinylsilane. The condensation of 3-pyridyl-substituted 5-triethoxysilylisoxazolines with triethanolamine afforded 3 - (2-pyridyl) - 5 - silatranylisoxazoline, 3 - (3 - pyridyl)-5-silatranylisoxazoline and 3-(4-pyridyl)-5-silatranylisoxazoline (12). In experiments in vivo and in vitro the vasodilating, antiarrhythmic and antithrombotic properties of pyridyl-substituted silylisoxazolines, their influence on the haemodynamic parameters in anaesthetized animals and their acute toxicity have been studied. It has been found that pyridyl-substituted silylisoxazolines possess vasodilating and antithrombotic properties. In experiments on the noradrenaline-preconstricted isolated rabbit ear artery, 3-(2-pyridyl)- and 3- (4- pyridyl) - 5 - phenyldimethylsilylisoxazoline exhibited pronounced vasodilating activity. 3 - (2 - Pyridyl) - and 3 - (3 - pyridyl) - 5 phenyldimethylsilylisoxazoline and 3-(2-pyridyl)-5-silatranylisoxazoline prolonged blood coagulation time

Pharmacological classification of α1‐adrenoceptors mediating contractions of rabbit isolated ear artery: comparison with rat isolated thoracic aorta

1. The present study attempted to classify pharmacologically the alpha 1-adrenoceptor subtype(s) present in two isolated, vascular ring preparations, the rabbit ear artery and rat thoracic aorta. 2. In the ear artery, the agonist effects of phenylephrine were antagonized by 5-methyl urapidil (pA2 = 7.90; Schild slope = 0.85) and BMY 7378 (pA2 = 6.11; Schild slope = 0.80) but not in a simple competitive manner. The shallow Schild slopes are consistent with the activation of a heterogeneous receptor population. Indeed the 5-methyl urapidil data set could be fitted to a two-receptor model yielding a high antagonist affinity (pKBH) estimate of 7.85 and a low affinity (pKBL) estimate of 6.03. 3. The effects of clonidine in the ear artery were competitively antagonised by 5-methyl urapidil (pKB = 7.91) and BMY 7378 (pKB = 5.53). These data are consistent with contractions to clonidine being mediated by a single receptor subtype. 4. In the aorta, the effects of phenylephrine were antagonized by 5-methyl urapidil (pA2 = 7.95; Schild slope = 1.11) and BMY 7378 (pA2 = 9.08; Schild slope = 0.73). Neither data set was consistent with a simple competitive interaction. The BMY 7378 data suggested again, that phenylephrine was acting at a heterogeneous receptor population. Subsequent analysis by the two-receptor model yielded a high affinity (pKBH) estimate of 8.95 and a low affinity (pKBL) estimate of 7.00. 5. The alkylating agent, chloroethylclonidine (CEC) elicited concentration-dependent contractions in the ear artery with a potency (p[A]50) of 5.57. Pretreatment of this tissue with CEC (5 microM, 30 min incubation) had no effect on subsequent responses to phenylephrine. In contrast, in the aorta, CEC demonstrated no agonism but pretreatment with this agent (5 microM, 15 min incubation) caused a rightward shift and depression of subsequent phenylephrine concentration-effect curves. 6. The affinity of clonidine in the rabbit ear artery (pKA = 6.17) was found to be significantly different to its affinity in the rat thoracic aorta (pKA = 7.12) suggesting that this agonist activates different alpha 1-adrenoceptor subtypes in the two tissues. 7. These results suggest that heterogeneous populations of alpha 1-adrenoceptors are present in both tissues. In the ear artery, the profile of antagonist and agonist activity is most consistent with alpha 1A-adrenoceptors being the predominant receptor subtype. The second receptor population does not appear to correspond to any of the recognized alpha 1-adrenoceptor subtypes. In the aorta alpha 1D-adrenoceptors appear to predominate, with alpha 1A-adrenoceptors being the most likely candidate for the second receptor population.

Effect of platelet‐derived growth factor on voltage‐operated calcium channels in rabbit isolated ear artery cells

1. Platelet derived growth factor (PDGF), AB and BB isoforms (100 pM) increased calcium channel currents measured by whole cell voltage clamp technique in single vascular smooth muscle cells isolated from rabbit ear arteries. 2. Tyrphostin-23 (100 microM) a selective inhibitor of protein tyrosine kinases, reduced calcium channel currents. Pre-incubation with tyrphostin-23 prevented PDGF-AB induced increase in calcium channel currents. However, in these same cells 10 nM (+)-202791, a dihydropyridine calcium channel agonist, did increase calcium channel currents. 3. Bistyrphostin (10 microM), a selective inhibitor of epidermal growth factor (EGF)-kinase also reduced calcium channel currents and inhibited PDGF-AB-induced increases in calcium channel currents. 4. Genistein (100 microM) a selective inhibitor of tyrosine kinases, structurally unrelated to the tryphostins, also inhibited calcium channel currents and pre-incubation with genistein prevented the PDGF-AB-induced rise in calcium channel currents. 5. These results indicate that PDGF increases calcium channel currents in vascular smooth muscle. This action of PDGF probably involves a tyrosine kinase.

Substrate- and inhibitor-specificity of a nonendothelial enzyme which forms [Met 5]-enkephalin from [Met 5]-enkephalin-Arg 6, Phe 7 in isolated rabbit ear artery: Pharmacological characterization

The captopril-inhibited enzyme which forms [Met 5]-enkephalin from [Met 5]-enkephalin-Arg 6, Phe 7 in isolated rabbit ear artery was characterized further by using various natural substrate candidates/analogues ([Met 5]-enkephalin-Arg 6, Phe 7 and its amide, [Met 5]-enkephalin, angiotensin I and bradykinin), peptidase inhibitors such as captopril, enalaprilate and thiorphan and by endothel removal. 10 −5 and 10 −4 M but not 10 −6 M captopril reduced the effectiveness of [Met 5]-enkephalin-Arg 6,Phe 7 and potentiated the effect of bradykinin but did not affect markedly the action of the other peptides. Of the inhibitors, enalaprilate was less effective than captopril, and thiorphan had no effect. The [Met 5]-enkephalin-Arg 6,Phe 7 →[Met 5]-enkephalin conversion was not affected by endothel removal. The substrate and inhibitor spectrum of this non-endothelial enzyme activity bears no relationship in other, hitherto characterized dipeptidylcarboxypeptidases/endopeptidases known to be involved in the metabolism of the tested peptides.

Role of cyclic AMP in stimulation-evoked release of 3H-noradrenaline from rabbit isolated ear artery.

The possible involvement of cyclic AMP in the stimulation-evoked 3H-overflow from rabbit isolated ear artery preloaded with 3H-noradrenaline was studied. Cyclic AMP (10(-5)-3 x 10(-4)M), 8-bromo-cyclic AMP (3 x 10(-4)M) and adenosine (10(-5)-3 x 10(-4)M) enhanced stimulation-evoked 3H-overflow. Dibutyryl-cyclic AMP (10(-5)-3 x 10(-4)M) had no effect. Theophylline (3 x 10(-5)M) and the phosphodiesterase inhibitor AH 21-132 (3 x 10(-5)M) did not alter the enhancement of 3H-overflow caused by either cyclic AMP or adenosine. Forskolin (3 x 10(-6)M) and the phosphodiesterase inhibitors ICI 63 197 (10(-4)M) and AH 21-132 (3 x 10(-6)-3 x 10(-5)M) increased stimulation-evoked 3H-overflow. Forskolin (10(-6)M) enhanced the effect of ICI 63 197 (3 x 10(-5)M) but it did not alter the effect of AH 21-132. It is concluded that cyclic AMP is involved in the stimulation-evoked release of noradrenaline from postganglionic sympathetic nerves in the rabbit ear artery.

Increase in tone and intracellular Ca2+ in rabbit isolated ear artery by platelet-derived growth factor.

1. The effect of platelet-derived growth factor (PDGF-AB) on tone and intracellular Ca2+ ([Ca2+]i) was examined in rabbit isolated ear arteries. Arteries were mounted in a myograph and loaded with the Ca(2+)-sensitive fluorescent indicator, fura-2, for concurrent measurements of isometric force and [Ca2+]i. 2. PDGF-AB contracted rabbit ear artery in a concentration-dependent manner. PDGF-AB induced tone was associated with a rise in [Ca2+]i. In the presence of noradrenaline, PDGF-AB induced a similar rise in [Ca2+]i but contraction in response to PDGF-AB in the presence of noradrenaline was increased compared with PDGF-AB alone. 3. PDGF-AB-induced rise in [Ca2+]i and tone were abolished by removal of extracellular Ca2+ (with addition of BAPTA, a Ca2+ chelator), and by preincubation with a dihydropyridine calcium channel blocker, (-)-202,791. Bistyrphostin, a selective inhibitor of tyrosine kinases, also inhibited PDGF-AB-induced tone, but had no effect on noradrenaline- or potassium-induced tone. 4. PDGF-AB contracts rabbit ear artery by increasing Ca2+ entry through voltage-operated calcium channels. This effect involves activation of a tyrosine kinase.

Pharmacological and biochemical analysis of FPL 67156, a novel, selective inhibitor of ecto‐ATPase

1. FPL 67156 (6-N,N-diethyl-beta, gamma-dibromomethylene-D-ATP), is a newly synthesized analogue of ATP. 2. In a rabbit isolated tracheal epithelium preparation, measuring P2U-purinoceptor-dependent chloride secretion, FPL 67156 was discovered to potentiate the responses to UTP but not those to ATP-gamma-S. UTP agonist-concentration effect (E/[A]) curves were shifted to the left by 5-fold in the presence of 100 microM FPL 67156. The differential effect of FPL 67156 on UTP and ATP-gamma-S was hypothesized to be due to the greater susceptibility of UTP to enzymatic dephosphorylation and the ability of FPL 67156 to inhibit this process. 3. FPL 67156 was tested as an ecto-ATPase inhibitor in a human blood cell assay, measuring [gamma 32P]-ATP dephosphorylation. The compound inhibited [gamma 32P]-ATP degradation with a pIC50 of 4.6. 4. FPL 67156 was then tested for its effects on ATP and alpha, beta-methylene-ATP responses at P2X-purinoceptors in the rabbit isolated ear artery. In the concentration range 30 microM-1 mM, the compound potentiated the contractile effects of ATP but not those of alpha, beta-methylene-ATP. At 1 mM, FPL 67156 produced a 34-fold leftward shift of ATP E/[A] curves. 5. The effects of FPL 67156 on ATP E/[A] curves in the rabbit ear artery were analyzed using a theoretical model (Furchgott, 1972) describing the action of an enzyme inhibitor on the effects of a metabolically unstable agonist. This analysis provided an estimate of the pKi for FPL 67156 as an ecto-ATPase inhibitor of 5.2. 6. Using appropriate assays, FPL 67156 was shown to have weak antagonist effects at P2X- and P2T-purinoceptors (pA2 ~ 3.3 and 3.5 respectively), and weak agonist effects at P2u-purinoceptors(p[A 50]~ 3.5).7. The degree of potentiation of ATP and UTP effects elicited by FPL 67156 confirms previous results concerning the influence that ecto-ATPase has on the position of E/[A] curves for metabolically unstable agonists. The magnitude of this influence is predicted to have a major effect on the agonist potency orders currently used to designate purinoceptors.8.This study indicates FPL 67156 to be a potentially valuable probe in studies on the action of nucleotides and in the classification of purinoceptors.

FPL 66096: a novel, highly potent and selective antagonist at human platelet P2T‐purinoceptors

1. ADP-dependent platelet aggregation is mediated by the P2T-purinoceptor and is specifically inhibited by ATP, which is a competitive P2T-purinoceptor antagonist. However, ATP functions as an agonist at other P2-purinoceptor subtypes in other tissues and is, therefore, non-selective. This paper describes the effects of the novel ATP analogue, FPL 66096 (2-propylthio-D-beta,gamma-difluoromethylene ATP), on ADP-induced and ADP-independent aggregation of human washed platelets and in standard preparations containing P2X- (rabbit ear artery) and P2Y-purinoceptors (guinea-pig aorta). 2. In suspensions of human washed platelets, FPL 66096 (1-100 nM) produced concentration-dependent rightward displacement of concentration-effect (E/[A]) curves obtained for ADP-induced platelet aggregation. Logistic fitting of E/[A] data indicated that the effect of FPL 66096 was consistent with simple competition with a pKB value of 8.66. FPL 66096 (10-1000 nM) had no effect on aggregation produced by the thromboxane A2-mimetic, U46619 (0.1-10 microM) when the response to this agent was rendered ADP-independent by inclusion of the non-selective P2-purinoceptor antagonist, suramin (100 microM). 3. The anti-aggregatory potency of FPL 66096 was not influenced by increasing the incubation time from 2 to 15 min nor by inclusion of the P1-purinoceptor antagonist 8-sulphophenyltheophylline at a concentration (300 microM) that produced a 68 fold rightward displacement of the anti-aggregatory E/[A] curve for the P1-purinoceptor agonist, 5'-N-ethylcarboxamidoadenosine (0.1-1000 microM). 4. FLP 66096 behaved as a weak (pA" 3.68) but full P2x-purinoceptor agonist in preparations of the rabbit isolated ear artery and as a weak, competitive antagonist (apparent pKB 4.71) at P2Y purinoceptors in the guinea-pig isolated aorta, indicating a selectivity of at least 9000 fold for the P2t-subtype. In the latter preparation, non-specific relaxations were produced by concentrations of FPL 66096 >10M gM.5. These results indicate that FPL 66096 is a P2-purinoceptor antagonist of unprecedented potency and selectivity and that its effects are consistent with simple competition at the P2-purinoceptor. Therefore,FPL 66096 represents a novel pharmacological tool in the classification of P2-purinoceptors and in the elucidation of the mechanisms involved in activation of platelets by ADP.

Cellular mechanisms of the steroid-induced vascular responses in the rabbit ear artery

1. In anesthetized rabbits, topical application of dexamethasone to the ear produced an initial vasodilation followed by a vasoconstriction with long delay (120 min). Diphenhydramine inhibited the former, but not the latter. 2. In isolated rabbit ear arteries, dexamethasone reduced the amplitude of contractions of smooth muscles produced by nerve stimulation, noradrenaline and high-K solution, only at high concentrations (>10 −5M). 3. The initial vasodilation induced by topical application of dexamethasone may be related to endogenous histamine, while the delayed constriction response may not be direct actions of steroid to smooth muscles.

Methoxamine enhances the release of endogenous noradrenaline from rabbit ear artery: possible involvement of ATP.

The effect of methoxamine, an alpha 1-adrenoceptor agonist, on the electrically-evoked release of endogenous noradrenaline was examined in the isolated rabbit ear artery. Noradrenaline was quantified by high performance liquid chromatography-electrochemical detection. The release of adenine nucleotides and nucleosides by methoxamine was examined using high performance liquid chromatography-fluorescence detection. The release of noradrenaline evoked by electrical field stimulation (EFS) at 4 Hz was reduced by tetrodotoxin 0.3 mumol/l and clonidine 1 mumol/l by approximately 80% and 50%, respectively. On the other hand, methoxamine at 10 but not 1 mumol/l enhanced the release of noradrenaline to approximately twice the control, and the enhancement was prevented by prazosin 1 mumol/l. The facilitatory action of methoxamine was also abolished after desensitization of P2-purinoceptors by alpha,beta-methylene ATP 30 mumol/l as well as by the presumed P2-purinoceptor antagonist suramin given at 10 mumol/l. Exogenous ATP 10 mumol/l significantly enhanced the EFS-evoked release of noradrenaline, and the enhancement was abolished by alpha,beta-methylene ATP and suramin. None of the drugs changed the spontaneous outflow of noradrenaline. These results indicate that endogenous ATP, acting at prejunctional purinoceptors, may participate in the facilitatory effect of methoxamine. Indeed, methoxamine 10 mumol/l significantly enhanced the spontaneous outflow of ATP and, less so, ADP. The methoxamine evoked release of ATP and ADP was antagonized by prazosin 1 mumol/l. It is concluded that methoxamine releases endogenous ATP from postjunctional sites which then, via prejunctional purinoceptors, facilitates action potential-evoked release of noradrenaline in rabbit ear artery.