Gareth W. John

Increased resistance to ischaemic injury in the isolated perfused atherosclerotic heart of the cholesterol-fed rabbit

OBJECTIVEnIn isolated, Langendorff-perfused hearts in the early stages of atherosclerosis from rabbits exposed to hypercholesterolaemia induced by 2% cholesterol feeding for 6 weeks (n = 23), and age-matched normal controls fed standard chow (n = 12), we studied baseline cardiac haemodynamics and the susceptibility of these hearts to 30 min global, normothermic ischaemia and 90 min reperfusion.nnnMETHODSnSpontaneously beating hearts were perfused with oxygenated Krebs buffer (pH 7.4) at constant pressure, and were enclosed in a thermostated water jacket at 37 degrees C. Isovolumetric left ventricular (LV) pressure was measured by means of a balloon placed in the LV cavity. An electromagnetic flow probe placed around the perfusion cannula determined coronary flow. At the end of an initial 30 min stabilisation period, several baseline cardiodynamic variables were measured, just before subjecting the hearts to 30 min ischaemia. Recovery of mechanical function and lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) activities in the coronary effluent were recorded throughout 90 min reperfusion.nnnRESULTSnBaseline spontaneous heart rate, LV developed pressure (LVDP), coronary flow and pressure-rate index (PRI) were all significantly lower in hearts from cholesterol-fed rabbits (CFR) than in age-matched controls (P < 0.01). Although large differences in several baseline haemodynamic parameters in hearts from CFR and controls were evident before ischaemia, no statistically significant differences were discernible in these parameters between the two groups from 60 min reperfusion onwards (p = NS). Furthermore, CPK and, to a lesser extent, LDH release during reperfusion was attenuated in hearts from CFR compared to controls.nnnCONCLUSIONSnHearts from CFR exhibited markedly improved recovery upon reperfusion compared to age-matched controls, strongly suggesting increased myocardial resistance to ischaemic injury.

Intrinsic activity of the non‐prostanoid thromboxane A2 receptor antagonist, daltroban (BM 13,505), in human platelets in vitro and in the rat vasculature in vivo

1 We evaluated the effects of daltroban on (i) human platelet shape change and aggregation in vitro, and (ii) mean systemic and pulmonary arterial pressures (MAP and MPAP, respectively) as well as haematocrit, in anaesthetized, open‐chest Sprague‐Dawley rats, compared with those of a chemically distinct prostanoid thromboxane A2 (TxA2) receptor antagonist, SQ 29,548, and agonist, U‐46619. 2 In human platelets in vitro, daltroban (10 nM‐100 μM; n = 6 per group) concentration‐dependently induced shape change, attaining at 50 μM, a maximum amplitude of 0.83 ± 0.09 mV representing 46.4 ±4.8% of that evoked by U‐46619 (1.78 ± 0.20 mV at 0.2 μM; n = 9); and inhibited U‐46619‐induced platelet aggregation with an IC50 of 77 (41–161)nM. SQ 29,548 (10 nM‐100 μM; n = 6 per group) failed to evoke any platelet shape change, but potently inhibited U‐46619‐induced platelet aggregation with an IC50< 10nM. 3 In anaesthetized rats in vivo, daltroban (10–2500 μg kg−1, i.v. infused over 2 min; n = 4–8 per group) produced a bell‐shaped dose‐response curve for MPAP and haematocrit, and evoked maximal increases of 12.7 ± 2.1 mmHg and 5.8 ± 1.5% at 80 μg kg−1 (n = 6) and 630 μg kg−1 (n = 8), respectively (both P<0.05) with ED50S of 20 (16–29) and 217 (129–331) μg kg−1, respectively. By comparison, U‐46619 (0.16–20 fig kg−1, i.v.), induced dose‐dependent increases in MPAP and haematocrit (25.4 ± 1.0 mmHg and 16.1 ±2.9% at the highest dose; n = 12, both P<0.01), with ED50s of 1.8 (1.3‐2.5) and 3.9 (3.5‐5.4) fig kg−1, respectively. Daltroban dose‐dependently increased MAP with a maximum amplitude of 42.2 ±4.4 mmHg at a dose of 80 μkg−1 [ED50 = 94 (64–125) fig kg1], similar to that induced by U‐46619 (41.3 ±9.6 mmHg) at a dose of 0.63 fig kg−1 [ED50 = 0.22 (0.13‐0.24) fig kg−1]. SQ 29,548 (10–2500 μg kg−1, i.v.; n = 4 per group) failed to modify significantly any of these parameters. 4 Our results clearly demonstrate that daltroban, in a similar manner to the TxA2 analogue, U‐46619, but unlike the TxA2 receptor antagonist, SQ 29,548, exhibits significant intrinsic activity in human platelets in vitro and in the rat vasculature in vivo, possibly through TxA2 receptor activation.

Veratrine-induced tetanic contracture of the rat isolated left atrium

SummaryAn investigation has been made of the putative direct myocardial protective effects of the α1-adrenoceptor antagonists, prazosin and WB 4101, against tetanic contractures of rat isolated left atria following modified Na+ channel function and consequent Ca2+ loading elicited by veratrine.Veratrine evoked concentration-dependent, reversible, tetanic contractures which were critically dependent upon the external Ca2+ concentration. Tetrodotoxin (TTX), prazosin, WB 4101 and R 56865 (0.1–10 μM) prevented tetanic contracture elicited by veratrine (100 μg/ml) at concentrations which were significantly lower than those which decreased active tension development. The apparent Hill coefficients (nH) obtained for TTX, prazosin, WB 4101 and R 56865 were comparable (range 0.79–0.93), and are consistent with a single site of action. In contrast, the class 1 antiarrhythmic agents, quinidine and lidocaine, elicited no significant inhibition of veratrine-induced contracture at 30 μM, but almost completely abolished the contractures at 100 μM. The nH values for quinidine and lidocaine were found to be significantly greater than unity (3.1 and 2.6, respectively). The L-type Ca2+ channel blockers, diltiazem, nicardipine, nifedipine and verapamil only weakly prevented tetanic contracture, whilst markedly, and concentration-dependently, decreasing active tension development. Neither atropine (10 μM) nor propranolol (1 μM) significantly modified either veratrine-induced contractures or active tension development.In conclusion, evidence is presented of novel, direct protective effects of prazosin and WB 4101 against tetanic contracture following modified Na+ channel function and Ca2+ loading provoked by veratrine. The precise mechanisms involved are unclear at present, but appear to be distinct from blockade of atrial α1-adrenoceptors or L-type Ca2+ channels. A possible involvement of a TTX-sensitive, quinidine-insensitive site on, or associated with the Na+ channel is suggested.

Influence of endothelium and nitric oxide on the contractile response evoked by 5HT1D receptor agonists in the rabbit isolated saphenous vein

1. We investigated whether contractile responses evoked by 5-HT1D receptor agonists were influenced by the endothelium (E) and nitric oxide (NO) in the rabbit isolated saphenous vein. 2. Saphenous vein rings were set up for isometric tension recording in oxygenated (5% CO2 in O2) Krebs solution (pH 7.4) containing (10(-6) M): idazoxan (1), indomethacin (10), ketanserin (0.1), prazosin (10), and N(omega) nitro-L-arginine methyl ester (L-NAME; 0 or 10), a NO synthase inhibitor. In some experiments, the E was removed mechanically. 3. 5-Hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT) and sumatriptan (Sum) contracted rabbit saphenous vein rings in the potency order (pD2 range) of 5-CT(7.2-7.6) > 5-HT(6.2-7.1) > Sum(5.0-5.8), irrespective of the presence or absence of the E or L-NAME (n = 9-37 per group) indicating that the potencies of the 3 agonists were not significantly affected by either the E or L-NAME. 4. Efficacy, as assessed by the maximal contractile response (Emax), was significantly greater for Sum compared to 5-HT and 5-CT with intact E irrespective of the presence (77 +/- 3, 62 +/- 3, and 50 +/- 3 mN respectively; P < 0.05 Sum versus 5-HT and 5-CT) or absence (26 +/- 3, 14 +/- 4, and 13 +/- 2 mN respectively; P < 0.05 Sum versus 5-HT and 5-CT) of L-NAME. In E-denuded rings, the Emax values were all higher than in E-intact rings and did not differ between the 3 agonists (36 +/- 4, 37 +/- 4, and 36 +/- 5 mN for Sum, 5-HT and 5-CT, respectively; P > 0.5 between the 3 agonists) indicating that an endothelium-derived relaxing factor (EDRF) counteracted the constrictor activities of the 5-HT1D receptor agonists and raising the possibility that a component of the Sum-induced contractile responses was E-dependent. Without E, the presence of L-NAME did not significantly affect the Emax values of the 3 agonists (41 +/- 4, 41 +/- 5, and 41 +/- 4 mN for Sum, 5-HT, and 5-CT respectively; P > 0.5 between the 3 agonists) indicating that the NO synthase inhibited was of endothelial origin. 5. Potentiation of the Emax of the 3 agonists by L-NAME was significantly albeit partially reversed by L-arginine (10(-2) M) indicating that NO synthase was indeed inhibited by L-NAME. Furthermore, in the presence of E, potentiation of Emax of the 3 agonists by L-NAME was mimicked by methylene blue (10(-5) M) providing further evidence that NO was involved in the attenuation by the E of the contractile responses induced by the 5-HT1D receptor agonists. 6. In the presence of an intact E and L-NAME, contractile responses elicited by 5-HT and Sum were competitively antagonized by the non-selective 5-HT1D receptor antagonist, methiothepin (pA2: 9.4 and 8.8; slopes: 0.66 and 0.81, respectively) and the highly selective 5-HT1D receptor antagonist, GR 127935 (pA2: 9.0 in each case; slopes: 1.04 and 0.93, respectively) indicating that contractions were mediated through activation of a single population of 5-HT1D receptors. Contractile responses elicited by 5-CT were also competitively antagonized by methiothepin and GR 127935, but non parallel rightward shifts of the concentration-response curves were observed suggestive of the involvement of additional but as yet unidentified receptors in mediating the 5-CT-induced responses. 7. In conclusion, the efficacy, but not the potency, of 5-HT, 5-CT and Sum in evoking 5-HT1D receptor-mediated contractile responses are subject to a substantial inhibitory influence of the E and of an EDRF (probably NO).

Prevention by specific chemical classes of α1-adrenoceptor antagonists of veratrine-contractures in rat left atria independently of α1-adrenoceptor blockade

1 The putative direct protective effects of a series of chemically diverse α1‐adrenoceptor antagonists against veratrine alkaloid‐induced tetanic contractures in rat isolated left atria have been investigated. 2 Atria were mounted in organ baths containing normal, oxygenated physiological salt solution (20 ml, pH 7.4), for isometric tension recording. Atria were electrically driven at 4 Hz and were maintained at 34°C. Veratrine (100 μg ml−1) was applied to the atria to elicit tetanic (diastolic) contracture. 3 Concentration‐dependent protective effects against veratrine‐contractures, in the absence of negative inotropic responses, were observed with the quinazoline congeners, prazosin and doxazosin and with the benzodioxane‐related compounds, WB 4101 and its thio analogue, benoxathian. IC50 concentrations and apparent Hill coefficients of all four drugs ranged from 0.27 to 0.93 μm, and from 0.86 to 1.09, respectively, and are consistent with interaction at a single site. 4 In contrast, no protective activity versus veratrine‐contractures was observed with corynanthine, 5‐methyl‐urapidil, phenoxybenzamine, phentolamine or chloroethylclonidine (10 μm). 5 Contractures were prevented by prazosin at concentrations 2–3 log units higher than those which antagonized methoxamine‐evoked inotropic responses. In addition, concommitant α1‐adrenoceptor occupancy by high concentrations of methoxamine (100 μm), phentolamine (10 μm, inactive per se in preventing contracture), or both drugs together, failed, in each case, to modify significantly the protective effects of prazosin or WB 4101 against veratrine‐contractures. 6 Our findings demonstrate that α1‐adrenoceptor antagonists which prevent veratrine‐contractures belong to specific chemical classes of the quinazoline‐ and benzodioxane‐type. The mechanism by which these drugs afford protection is apparently independent of an interaction with defined α1‐adrenoceptors.