The effects of aspirin and paracetamol on the aggregation of equine blood platelets

The responses of equine blood platelets in citrated platelet-rich plasma to arachidonic acid, U44069 (prostaglandin endoperoxide analogue), adenosine 5'-diphosphate, platelet-activating factor or collagen were investigated by turbidimetric aggregometry. Pre-treatment of the platelets with aspirin (1 mmol/1) or Paracetamo1 d-3 mmol/1) abolished shape change and aggregation in response to arachidonic acid; decreased the rate of aggregation in response to collagen, with no separate effect on shape change; had no marked effect on aggregation caused by the other agonists; but in no case transformed irreversible aggregation to reversible aggregation. We conclude that thromboxane A₂ generation is of minor importance in the aggregation of equine platelets, and in particular that thromboxane A₂ is not a significant mediator of irreversible aggregation.     

Characterisation of the response of equine digital arteries and veins to substance P

Substance P (SP), a potent vasodilator, has been detected in equine digital sensory-motor nerves. The aim of the study was to characterise the functional responses of equine digital blood vessels to exogenous SP. Pre-constricted equine digital arteries (EDA) and veins (EDV) vasodilated in a biphasic, endothelium- and concentration-dependent manner to SP. A nitric oxide (NO) synthase inhibitor Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME; 300 microm) inhibited both phases of the relaxation response curve of EDAs to SP by >70%. In EDVs, the first relaxant phase to SP was largely L-NAME-resistant, whereas the second phase was inhibited by 60%. Both L-NAME and a cyclo-oxygenase inhibitor (ibuprofen; 10 microm) were required to inhibit EDV relaxation to SP by > or =80%. Experiments determining the receptor mediated responses to physiological concentrations of SP (1 nm) revealed that the relaxant responses of both EDA and EDV were inhibited by a neurokinin-1 (NK1) receptor antagonist (CP-96 345; 10 nm). In conclusion, SP is an endothelium-dependent vasodilator of both EDA and EDV. NO is the predominant pathway activated in EDA, whereas both prostacyclin and NO pathways are involved in EDVs. NK1 receptors appear to mediate responses to low concentrations of SP.     

Equine coronary artery responds to 5-hydroxytryptamine with relaxation in vitro

Isolated equine coronary arteries responded to 5-hydroxytryptamine (5-HT) with relaxations in both endothelium-dependent and endothelium-independent mechanisms. Experiments were designed to characterize the 5-HT receptor sub type mediating these relaxations. Both 5-HT and alpha-methyl-5-HT (α-Me-5- HT; 5-HT₂ agonist) produced concentration-dependent relaxations in equine coronary arteries precontracted with a thromboxane A₂ derivative (0N011113). The degree of the maximal relaxation induced by α t-Me-5-HT was about one-half of that induced by 5-HT. In the coronary arteries without endothelium, α -Me-5-HT produced no relaxation, but 5-HT caused relaxation, which was inhibited by a 5-HT₁ antagonist (methysergide, mianserin and methiothepin), but was inhibited neither by ketanserin (5-HT₂ antagonist) nor by MDL72222 (5-HT₃ antagonist). In the coronary arteries with endothelium, however, the relaxation induced by α -Me-5-HT was inhibited by ketanserin, L-nitro-arginine (NO synthase inhibitor) and methylene blue (soluble guanylate cyclase inhibitor). These results suggest that the relaxation induced by 5-HT in equine coronary arteries depends mainly on the stimulation of both 5-HTi receptor subtype on smooth muscle cells directly, and 5-HT₂ receptor subtype on endothelial cells indirectly by liberating endothe-lium-derived NO.     

Characterization and comparison of the responses of equine digital arteries and veins to endothelin-1

OBJECTIVE: To compare the responses of equine digital arteries (EDAs) and equine digital veins (EDVs) to endothelin-1 (ET-1) and determine the role of the endothelium and type of receptors involved in the modulation and mediation of those responses, respectively. SAMPLE POPULATION: 5 to 9 palmar digital vessels/experiment from 28 healthy horses. PROCEDURE: Rings of dissected vessels were mounted under tension between force transducer wires in organ baths containing Krebs-Henseleit solution at 30 degrees C. Responses of EDAs and EDVs (with intact [+e] or denuded [-e] endothelium) to cumulative concentrations of ET-1 (10(-10) to 3 X 10(-7) M) were compared. For (+e)EDAs and (+e)EDVs precontracted with a thromboxane-mimetic (U44069; 10(-8) M) and (-e)EDAs and (-e)EDVs, responses to an ETB receptor agonist (S6c; 10(-10) to 3 X 10(-7) M) were evaluated. Responses to ET-1 (10(-7) M) in (-e)EDAs and (-e)EDVs were evaluated after incubation with an ETA receptor antagonist (BQ-123; 3 X 10(-7) M), an ETB receptor antagonist (BQ-788; 3 X 10(-7) M), or vehicle solution. RESULTS: Endothelin-1 induced a concentration-dependent contraction of endothelium-intact and -denuded EDAs and EDVs; EDVs were more sensitive. Neither vessel type relaxed in response to S6c, although 2 of the (-e)EDAs contracted mildly. Whereas BQ-123 inhibited the (-e)EDA and (-e)EDV responses to ET-1, BQ-788 had no effect. CONCLUSIONS AND CLINICAL RELEVANCE: Endothelin-1 induced digital vasoconstriction (marked constriction in veins). This action was unaffected by endothelium and mediated predominantly by ETA receptors. These findings suggest ET-1 can induce selective digital venoconstriction.     

Effects of stimulation of adenosine A2A receptors on lipopolysaccharide-induced production of reactive oxygen species by equine neutrophils

OBJECTIVE: To assess the anti-inflammatory effects of an adenosine analogue on lipopolysaccharide (LPS)-stimulated equine neutrophils. SAMPLE POPULATION: Neutrophils obtained from 10 healthy horses. PROCEDURES: An adenosine analogue (5'-N-ethylcarboxamidoadenosine [NECA]) was tested for its ability to inhibit production of reactive oxygen species (ROS) in LPS-stimulated equine neutrophils. Selective adenosine receptor antagonists were used to identify the receptor subtype responsible for effects. To assess the mechanism of action of NECA, cAMP concentrations were measured, and effects of dibutyryl cAMP (a stable analogue of cAMP) and rolipram (a type 4 phosphodiesterase inhibitor) were investigated. RESULTS: NECA elicited concentration-dependent inhibition of ROS production that was inhibited by ZM241385, a selective adenosine A(2A) receptor antagonist; this effect of NECA was not affected by the adenosine A(2B) receptor antagonist MRS1706. Also, ZM241385 blocked NECA-induced increases in cAMP concentrations, whereas MRS1706 did not alter this effect of NECA. Rolipram potentiated NECA-induced inhibition of ROS production, and dibutyryl cAMP also inhibited ROS production. CONCLUSIONS AND CLINICAL RELEVANCE: Activation of adenosine A(2A) receptors inhibited ROS production by LPS-stimulated equine neutrophils in a cAMP-dependent manner. These results suggest that stable adenosine A(2A) receptor agonists may be developed as suitable anti-inflammatory drugs in horses.     

Alpha-adrenoceptors in equine digital veins: Evidence for the presence of both alpha1 and alpha2-receptors mediating vasoconstriction

Rings of equine digital vein examined under conditions of isometric tension recording constricted to alpha-adrenoceptor agonists with an order of potency of 5-bromo-6-[2-imidazolin-2-yl-amino]-quinoxaline bitartrate (UK 14304) = noradrenaline > 6-Allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo-(4,5-d) azepine (BHT-920) > phenylephrine > dopamine > methoxamine. The maximum force generated was greatest for the non-selective agonist noradrenaline and lowest for the alpha₂-selective agonist BHT-920 with the other agonists between these two extremes. Selective inactivation of alpha₁-adrenoceptors (achieved by treating yohimbine-protected tissues with phenoxybenzamine) reduced the maximum responses of all agonists, the EC₅₀ values of UK 14304, BHT-920 and noradrenaline and increased the EC₅₀ values of phenylephrine and methoxamine. Prazosin (30 n M ) had no inhibitory effect on responses to low concentrations of BHT-920 and UK 14304 and caused competitive inhibition of responses to phenylephrine and noradrenaline giving pK_b values of 8.49 ± 0.18 and 8.23 ± 0.14, respectively. Yohimbine (0.1 μ M ) caused significant competitive inhibition of responses to BHT-920 and noradrenaline with calculated pK_b values of 8.43 ± 0.11 for BHT-920 and 7.43 ± 0.31 for noradrenaline and non-competitive inhibition of responses to UK 14304. 2-[2-methoxy-1,4-benzodioxan-2-yl]-2-imidazoline (RX 821002; 10 n M ) caused competitive inhibition of responses to BHT-920 (pK_b 9.04 ± 0.27) and dopamine (pK_b 8.2 ± 0.2). These data indicate that equine digital veins possess both post-synaptic alpha₁ and alpha₂-adrenoceptors.     

Antagonism of adenosine receptors by caffeine and caffeine metabolites in equine forebrain tissues

OBJECTIVE: To determine the presence of adenosine receptor subtypes A1 and A2a in equine forebrain tissues and to characterize the interactions of caffeine and its metabolites with adenosine receptors in the CNS of horses. SAMPLE POPULATION: Brain tissue specimens obtained during necropsy from 5 adult male research horses. PROCEDURE: Membrane-enriched homogenates from cerebral cortex and striatum were evaluated by radioligand binding assays with the A1-selective ligand [3H]DPCPX and the A2a-selective ligand [3H]ZM241385. Functional responses to adenosine receptor agonists and antagonists were determined by a nucleotide exchange assay using [35S]-guanosine 5'-(gamma-thio) triphosphate ([35S]GTPgammaS). RESULTS: Saturable high affinity [3H]DPCPX binding (A1) sites were detected in cerebral cortex and striatum, whereas high-affinity [3H]ZM241385 binding (A2a) sites were detected only in striatum. Caffeine and related methylxanthines had similar binding affinities at A1 and A2a sites with rank orders of drug binding affinities (theophylline > paraxanthine > or = caffeine > theobromine) similar to other species. [35S]GTPgammaS exchange revealed that caffeine and its metabolites act as pure adenosine receptor antagonists at concentrations that correspond to A1 and A2a receptor binding affinities. CONCLUSIONS AND CLINICAL RELEVANCE: Results of our study affirm the presence of guanine nucleotide binding protein linked adenosine receptors (ie, high-affinity A1 and A2a adenosine receptors) in equine forebrain tissues and reveal the antagonistic actions by caffeine and several biologically active caffeine metabolites. Antagonism of adenosine actions in the equine CNS by these stimulants may be responsible for some central actions of methylxanthine drugs, including motor stimulation and enhanced racing performance.     

Adenosine A2A receptor agonists inhibit lipopolysaccharide-induced production of tumor necrosis factor-alpha by equine monocytes

Adenosine is an endogenous nucleoside that regulates many physiological processes by activating one or more adenosine receptor subtypes, namely A1, A2A, A2B and A3. The results of previous studies indicate that adenosine analogues inhibit lipopolysaccharide (LPS)-induced production of reactive oxygen species (ROS) by equine neutrophils primarily through activation of A2A receptors. Because peripheral blood monocytes produce cytokines that are responsible for many of the deleterious effects of LPS, the current study was performed to evaluate the effects of an array of novel adenosine receptor agonists on LPS-induced production of tumor necrosis factor-alpha (TNF-alpha), and to assess the selectively of these agonists for equine adenosine A2A over the A1 receptor. Radioligand binding studies performed with equine tissues expressing adenosine A1 and A2A receptor subtypes yielded a rank order of affinity for the equine A2A receptor of ATL307>ATL309 approximately ATL310 approximately ATL313>ATL202 approximately ATL361 approximately ATL376>ATL372>CGS21680>NECA. Co-incubation of equine peripheral blood monocytes with LPS and these agonists resulted in inhibition of TNF-alpha production with a rank order of potency that strongly correlated with their binding affinities for equine adenosine A2A receptors. Results of experiments performed with one of the adenosine receptor agonists (ATL313) and selective adenosine receptor antagonists confirmed that inhibition of LPS-induced production of TNF-alpha occurred via stimulation of A2A receptors. Although incubation of monocytes with IB-MECA, a compound purported to act as an adenosine A3 receptor agonist, reduced LPS-induced TNF-alpha production, this effect of IB-MECA was inhibited by the A2A selective antagonist ZM241385 but not by the A3 receptor antagonist MRS1220. These results indicate that the adenosine receptor subtype responsible for regulation of LPS-induced cytokine production by equine monocytes is the A2A receptor. To address the signal transduction mechanism responsible for the anti-inflammatory effects of ATL313 in equine monocytes, production of cAMP was compared in the presence and absence of either the adenosine A2A receptor antagonist ZM241385 or the adenosine A2B receptor antagonist MRS1706. In the absence of the antagonists, ATL313 increased production of cAMP; ZM241385 inhibited this effect of ATL313, whereas MRS1706 did not. Furthermore, incubation of monocytes with either the stable analogue of cAMP, dibutyryl cAMP, or forskolin, an activator of adenylyl cyclase, also inhibited LPS-induced production of TNF-alpha production by equine monocytes. Collectively, the results of the current study indicate that adenosine analogues inhibit LPS-induced production of TNF-alpha by equine monocytes primarily via activation of adenosine A2A receptors and do so in a cAMP-dependent manner. The results of this study indicate that stable adenosine analogues that are selective for adenosine A2A receptors may be suitable for development as anti-inflammatory drugs in horses.     

Evaluation of beta3-adrenoceptor-mediated relaxation in intact and endotoxin-treated equine digital veins

OBJECTIVE: To investigate the functional expression of beta3-adrenoceptors (beta3-ARs) in equine digital veins (EDVs) and to examine whether beta3-AR relaxation was altered in EDVs incubated with endotoxin. SAMPLE POPULATION: Forelimbs obtained from 30 horses. PROCEDURE: Forelimbs were obtained from horses in an abattoir. Equine digital veins were carefully removed from distal portions of the forelimbs. Rings of dissected EDVs were mounted in 5-mL organ baths to record isometric tension in the presence of various beta3-AR agonists (SR 58611A, ZD 2079, and ZM 215001). RESULTS: In intact EDVs, isoprenaline, SR 58611A, ZD 2079, and ZM 215001 induced concentration-dependent relaxation. Isoprenaline and SR 58611A-induced relaxations were reduced or unaffected by nadolol, respectively. In intact EDVs, SR 58611A-induced relaxation was significantly reduced in the presence of 2 microM ZM 215001 (used as a beta3-AR antagonist). In endothelium-denuded EDVs or intact EDVs in the presence of a nitric oxide synthase inhibitor, isoprenaline and SR 58611A-induced relaxations were significantly decreased. The endothelium-independent relaxation to SR 58611A was significantly inhibited in the presence of ZM 215001. In endotoxin-treated EDV, isoprenaline- and SR 58611A-induced relaxations were significantly reduced. In these conditions, cycloheximide (a protein synthesis inhibitor) and ibuprofen (a cyclooxygenase inhibitor) restored the relaxant response to SR 58611A. CONCLUSIONS AND CLINICAL RELEVANCE: Beta3-adrenoceptors are functionally expressed in EDVs. Incubation in the presence of endotoxin, used as an in vitro model of laminitis, induced an alteration of beta-AR-mediated relaxations in EDVs, which could be the consequence of cyclooxygenase induction and subsequent prostanoid production.     

Pharmacologic characterization of novel adenosine A2A receptor agonists in equine neutrophils

OBJECTIVE: To evaluate anti-inflammatory effects of several novel adenosine receptor agonists and to determine their specificity for various adenosine receptor subtypes on neutrophils, cells heterologously expressing equine adenosine receptors, or equine brain membranes. SAMPLE POPULATION: Neutrophils isolated from 8 healthy horses. PROCEDURES: Radioligand binding experiments were performed to compare binding affinities of adenosine receptor agonists to equine adenosine A(1), A(2A), and A(3) receptor subtypes. Effects of these agonists on endotoxin-induced production of reactive oxygen species (ROS) by equine neutrophils and roles of specific adenosine receptor subtypes and cAMP production in mediating these effects were determined. RESULTS: Radioligand binding experiments yielded a ranked order of affinity for the brain equine A(2A) receptor on the basis of 50% inhibitory concentrations (IC(50)) of the agonists as follows: ATL307 (IC(50) = 1.9nM) and ATL313 > ATL309 and ATL310 > ATL202 > 2-([p-2- carboxyethyl] phenylethylamino)-5'-N-ethylcarboxyamidoadenosine > 5'-N-ethylcarboxamidoadenosine. Furthermore, ATL313 had approximately 100-fold greater selectivity for A(2A) over A(1) and A(3) receptors. In functional assays with equine neutrophils, the compounds inhibited endotoxin-induced ROS production and stimulated production of cAMP with the same ranked order of potency. Results of experiments performed with selective adenosine receptor antagonists indicated that functional effects of ATL313 were via stimulation of A(2A) receptors. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that activation of A(2A) receptors exerted anti-inflammatory effects on equine neutrophils and that stable, highly selective adenosine A(2A) receptor agonists may be developed for use in management of horses and other domestic animals with septic and nonseptic inflammatory diseases.     

Characterization of muscarinic receptor subtype mediating contraction and relaxation in equine coronary artery in vitro

In coronary arterial rings isolated from horses, 10-^-8-10^-6 mol/l acetylcholine (ACh) induced concentration-dependent contractions which were potentiated by the removal of endothelium and by pretreatment with I,-nitro-arginine (LNAG) or methylene blue (MB). Relatively lower concentrations of Ach 10-¹⁴-10-⁸ mol/l) induced relaxation when the coronary rings were contracted by phenylephrine (PE). ACh-induced contractions in the coronary rings without endothelium were competitively inhibited by each muscarinic subtype selective antagonist in the following order of potency: 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) > pirenzepine ≥ parafluoro-hexahydrosiladiphenidol (pFHHSiD) > methoctramine. ACh-induced relaxation in the rings with endothelium was inhibited by LNAG or MB, and by each selective antagonist in the following order of potency: 4-DAMP < pFHHSiD ≥ pirenzepine ≥ methoctramine. These results suggest that the ACh-induced contraction and relaxation in equine coronary arteries are mediated mainly by an M₃-receptor located on the smooth muscle cells and endothelial cells, respectively, and that the stimulation of the M₃-receptor on the endothelial cells liberates nitric oxide.     

Cloning and pharmacological characterization of the equine adenosine A2A receptor: a potential therapeutic target for the treatment of equine endotoxemia

The aim of the current study was to clone the equine adenosine A(2A) receptor gene and to establish a heterologous expression system to ascertain its pharmacologic profile via radioligand binding and functional assays. An eA(2A)-R expression construct was generated by ligation of the eA(2A) cDNA into the pcDNA3.1 expression vector, and stably transfected into human embryonic kidney cells (HEK). Binding assays identified those clones expressing the eA(2A)-R, and equilibrium saturation isotherm experiments were utilized to determine dissociation constants (K(D)), and receptor densities (B(max)) of selected clones. Equilibrium competition binding revealed a rank order of agonist potency of ATL > CV-1808 > NECA > 2-CADO > CGS21680, and a rank order of antagonist potency as ZM241385 > 8-phenyltheophylline > p-sulfophenyltheophylline > caffeine. Furthermore, adenylate cyclase assays using selective A(2A)-R agonists revealed that the eA(2A)-R functionally coupled to Galpha(s) as indicated by an increase in intracellular [(3)H]cAMP upon receptor activation. Finally, NF-kappaB reporter gene assays revealed a CGS21680 concentration-dependent inhibition of NF-kappaB activity. These results indicate that the heterologously expressed eA(2A)-R has a pharmacological profile similar to that of other mammalian A(2A) receptors and thus can be utilized for further characterization of the eA(2A)-R to ascertain whether it can serve as a suitable pharmacological target for equine inflammatory disease.     

Plasma pharmacokinetics of ranitidine HCl in adult horses

Plasma pharmacokinetics of ranitidine HCl were investigated after intravenous (i.v.) and oral (p.o.) administration of 2.2 mg/kg drug to six healthy adult horses. Concentrations of ranitidine were determined using normal-phase, high-performance liquid chromatography. Plasma concentrations of ranitidine HCl declined from a mean of 5175 ng/mL at 5 min to 37 ng/mL at 720 min after i.v. administration. A three-exponent equation, C_p= A₁· e^–k1t+ A₂· e^–k2t+ A₃· e^–k3t, best described data for all horses. Mean values for model-independent values calculated from the last quantifiable time point were: apparent volume of distribution (Vd_ss) = 1.07 L/kg; area under the curve ( AUC ) = 231,000 ng · min/mL; area under the moment curve ( AUMC ) = 26,900,000 ng · min²/mL; mean residence time ( MRT ) = 113 min; and clearance (Cl) = 9.8 mL/min.kg. Following p.o. administration, a two-exponent equation, C_p= A₁· e^–k1t+ A₂· e^–k2t, best described the data for five horses; data for the remaining horse were best described by a three-exponent equation. Mean values of pharmacokinetic values from the p.o. study include: AUC = 59,900 ng · min/mL; AUMC = 10,600,000 ng · min²/mL; mean absorption time ( MAT ) = 58.9 min; T _max= 99.2 min; C _max= 237 ng/mL; and F = 27%.     

The role of thromboxane A2 in regulating porcine basilar arterial tone

The aim of the present study was to clarify the participation of endogenous arachidonic acid (AA) metabolites in regulating porcine basilar, coronary, pulmonary and mesenteric arterial tones in vitro . A cyclooxygenase inhibitor, indomethacin, relaxed basilar artery but not other arteries examined. Quinacrine (a phospholipase A₂ inhibitor), OKY-046 (a thromboxane (TX) A₂ synthetase inhibitor) and ONO-3708 (a TXA₂/prostaglandin H₂ receptor antagonist) produced relaxation in basilar arteries with intact endothelium. Nordihydroguaiaretic acid (a lipoxygenase inhibitor) had no effect on the tone. The amount of TXB₂ (a stable metabolite of TXA₂) spontaneously released from porcine basilar arteries was 6–10 fold more than those from other arteries. Indomethacin and OKY-046 mostly inhibited the production of TXB₂. Endothelial denudation decreased indomethacin-induced relaxation and the amount of TXB₂. These results suggest that a vasoconstricting substance(s) is released from endothelial cells and possibly smooth muscle cells in porcine basilar arteries in vitro . The main constricting substance is proposed to be TXA₂. On the other hand, several arteries from peripheral vascular beds did not release this vasoconstricting substance.     

Effects of adenosine on bacterial lipopolysaccharide- and interleukin 1-induced nitric oxide release from equine articular chondrocytes

OBJECTIVE: To determine whether adenosine influences the in vitro release of nitric oxide (NO) from differentiated primary equine articular chondrocytes. SAMPLE POPULATION: Articular cartilage harvested from the metacarpophalangeal and metatarsophalangeal joints of 11 horses (3 to 11 years old) without history or clinical signs of joint disease. PROCEDURE: Chondrocytes were isolated, plated at a high density (10(5) cells/well), and treated with adenosine, the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), bradykinin, or other agents that modify secondary messenger pathways alone or in combination with bacterial lipopolysaccharide (LPS) or recombinant human interleukin-1alpha (rhIL-1alpha). Nitric oxide release was measured indirectly by use of the Griess reaction and was expressed as micromol of nitrite in the supernatant/microg of protein in the cell layer. Inducible nitric oxide synthase (iNOS) activity was determined by measuring the conversion of radiolabeled arginine to radiolabeled citrulline. RESULTS: Treatment of chondrocytes with adenosine alone had no significant effect on NO release. However, adenosine and NECA inhibited LPS- and rhIL-1alpha-induced NO release. This response was mimicked by forskolin, which acts to increase adenylate cyclase activity, but not by the calcium ionophore A23187 Treatment of chondrocytes with phorbol myristate acetate, which acts to increase protein kinase C activity, potentiated LPS-induced NO release. Adenosine treatment also significantly inhibited the LPS-induced increase in iNOS activity. CONCLUSIONS AND CLINICAL RELEVANCE: Adenosine and the nonspecific adenosine receptor agonist NECA inhibited inflammatory mediator-induced release of NO from equine articular chondrocytes. Modulation of adenosine receptor-mediated pathways may offer novel methods for treatment of inflammation in horses with joint disease.     

Cloning and pharmacological characterization of the equine adenosine A3 receptor

The aim of this study was to establish a heterologous expression system for the equine adenosine A(3) receptor (eA(3)-R) in an effort to ascertain its pharmacologic profile. Initially, radioligand binding assays identified clones expressing the eA(3)-R in human embryonic kidney cells (HEK) based on the specific binding of [(125)I]AB-MECA. Subsequently, adenylate cyclase assays were utilized to demonstrate functional coupling of the eA(3)-R to the G-protein/adenylate cyclase system. Equilibrium competition binding assays were then performed using selective and non-selective A(3) agonists and antagonists. Results from these experiments revealed a rank order of agonist potency to be IB-MECA > NECA > CGS21680, and an antagonist potency of MRS1220 > ZM241385 > 8-p-sulphophenyltheophylline; these rank orders were in agreement with that of other mammalian A(3)-R's. Lastly, NF-kappaB reporter gene assays revealed an IB-MECA concentration-dependent inhibition of TNFalpha-stimulated NF-kappaB activity. These results indicate that the heterologously expressed eA(3)-R is functional, has a pharmacological profile similar to that of other mammalian A(3) receptors, and its activation has an inhibitory effect on a key regulatory pathway in the inflammatory response. Thus, the eA(3)-R may serve as a pharmacological target in the treatment of equine inflammatory disease.     

Pharmacological characterization of alpha1-adrenoceptors in equine digital veins

Alpha-adrenoceptors mediate contractile responses in equine digital veins (EDVs) and arteries. Vascular smooth muscle alpha(1)-adrenoceptor subtypes have been implicated in a number of conditions, such as acute equine laminitis, and are therapeutic targets for the treatment of this condition. Digital veins, rather than arteries, were investigated in the present study because they have been specifically implicated in the pathophysiology of acute laminitis. The order of potency of a series of alpha(1)-adrenoceptor-selective agonists and antagonists was determined in isolated rings of EDVs under conditions of isometric tension. A61603 was the most potent agonist, with a higher potency (76-fold greater) than phenylephrine (PHE), suggesting the presence of the alpha(1A)-adrenoceptor subtype. Prazosin (30 nm) caused competitive inhibition of the responses to A61603 and PHE, with pK(b) values of 8.05 +/- 0.28 and 8.20 +/- 0.27, respectively. In addition, the alpha(1A)-adrenoceptor antagonist, WB4101 (10 nm), also caused competitive inhibition of the responses to the two agonists, with pK(b) values of 8.37 +/- 0.16 and 8.54 +/- 0.23, respectively, confirming the presence of the alpha(1A)-adrenoceptor subtype in EDVs. The selective alpha(1D)-adrenoceptor antagonist, BMY7378 (100 nm) did not cause a significant change in the response to the agonists, giving lower pK(b) values (6.97 +/- 0.27 and 6.88 +/- 0.17 vs. A61603 and PHE, respectively). Chloroethylclonidine dihydrochloride (45 microm, 30 min), used to produce selective inactivation of alpha(1B)-adrenoceptors, caused noncompetitive inhibition of the response to PHE, but was without effect on the response to A61603. These findings indicate that EDVs possess at least two different alpha(1)-adrenoceptor populations, which are predominantly of the alpha(1A) and alpha(1B) subtypes. These data may assist in the development of more selective antagonists for therapeutic use in horses.     

Relaxation of equine tracheal muscle in vitro by different adrenoceptor drugs

Strips of tracheal smooth muscle from 12 horses were contracted by carbachol in tissue baths under isometric conditions. This contraction (≅50% of maximum: EC₅₀) was relaxed completely with adrenoceptor drugs. The only exception was clenbuterol, where the degree of relaxation was ≅90%. In all horses the EC₅₀-value for isoprenaline (mean 1.6 × 10⁻⁸M) was less than that for adrenaline (mean 9.6 × 10^{− 8}M) and noradrenaline (mean 1. 8 × 10_{- 6}M). The potency ratio was 1 < 6 < 110 which indicates that the β₂-subtype dominates among the β-adrenoceptors of equine airways. All preparations were also very sensitive to the specific and potent β₂-receptor agonists clenbuterol (mean 5.7 × 10^{− 9}M) and procaterol (mean 3.6 × 10⁻¹⁰M). No differences in EC₅₀-values due to age, sex and breed were observed in this material. The standard deviation of the mean EC₅₀-values seems to be larger for the specific β₂-adrenoceptor agonists than for the unspecific. A reason for this could be differences in the pattern of the β-adrenoceptor population.     

Characterization of bradykinin-induced endothelium-independent contraction in equine basilar artery

We investigated the effect of bradykinin (BK) on isolated equine basilar arterial rings with and without endothelium. BK induced concentration-dependent contraction of resting arterial rings and no relaxation when the rings were precontracted by prostaglandin F_2α. The maximal response and pD₂ value were 161.2 ± 28.1% (to 60 m m KCl-induced contraction) and 8.24 ± 0.25 respectively. The cumulative concentration–response curve for BK was not shifted to the right by des-Arg⁹-[Leu⁸]-BK (a B₁-receptor antagonist), HOE140 (a B₂-receptor antagonist) or NPC567 (another B₂-receptor antagonist). In four of six basilar arteries, NPC567 induced concentration-dependent contraction. Indomethacin (a cyclooxygenase inhibitor), nordihydroguaiaretic acid (a lipoxygenase inhibitor), quinacrine (a phospholipase A₂ inhibitor), tetrodotoxin (a selective blocker of Na⁺ channels), guanethidine (a nor-adrenergic neuron blocking drug), phentolamine (an α-adrenoceptor antagonist), Nω-nitro- l -arginine ( l -NNA, a nitric oxide (NO) synthase inhibitor) and endothelial denudation did not affect the BK-induced contraction. l -NNA and indomethacin induced contraction and relaxation under resting vascular tone respectively. These results suggest that endothelial cells are not involved in BK-induced contraction and that the contraction is not mediated via activation of known B₁ and B₂ receptors. Arachidonic acid metabolites and neurotransmitters like norepinephrine and NO might not play a role in BK-induced contraction in equine basilar artery.     

Affinity of detomidine, medetomidine and xylazine for alpha-2 adrenergic receptor subtypes

α₂-Adrenergic receptor agonists are widely used in veterinary medicine as sedative/hypnotic agents. Four pharmacological subtypes of the α₂-adrenergic receptor (A, B, C and D) have been identified based primarily on differences in affinity for several drugs. The purpose of this study was to examine the affinities of the sedative agents, xylazine, detomidine and medetomidine at the four α₂-adrenergic receptor subtypes. Saturation and inhibition binding curves were performed in membranes of tissues containing only one subtype of a₂-adrenergic receptor. The K_D for the α₂-adrenergic receptor radioligand, [³H]-MK-912, in HT29 cells (α_2A-), neonatal rat lung (α_2B-), OK cells (α_2C-) and PC12 cells transfected with RG20 (α_2D-) were 0.38 ± 0.08 n m , 0.70 ± 0.5 n m , 0.07 ± 0.02 n m and 0.87 ± 0.03 n m , respectively. Detomidine and medetomidine had approximately a 100 fold higher affinity for all the α₂-adrenergic receptors compared to xylazine but neither agonist displayed selectivity for the α₂-adrenergic receptor subtypes. These data suggest that available sedative/hypnotic α₂-adrenergic receptor agonists can not discriminate between the four known α₂-adrenergic receptor subtypes.