Effects of K+ channel inhibitors and antagonists on NS-004 evoked relaxations in guinea-pig isolated trachea.

The smooth muscle relaxant responses to NS-004, an activator of charybdotoxin-sensitive, large conductance Ca(2+)-dependent K+ channels (BKCa) were studied on the basal spontaneous tone in guinea-pig trachea in vitro. The sensitivity of these responses to a range of K+ channel inhibitors and antagonists were also evaluated. NS-004 (0.1-30 microM) evoked concentration-related relaxations (pIC50 5.48 +/- 0.13) on the spontaneous tone in guinea-pig tracheal rings, suspended in Krebs bicarbonate solution, with a maximum response not different to that to aminophylline (1 microM). Charybdotoxin (0.03 and 0.1 microM) or iberiotoxin (0.1 microM) significantly displaced the NS-004 concentration-response curve to the right of control with no change in maximum response. In contrast, glibenclamide (1.0 microM) apamin (0.1 microM) and dofetilide (1.0 microM) each failed to modify the responses to NS-004 on spontaneous tone in guinea-pig trachea. These results suggest that relaxations in guinea-pig tracheal smooth muscle to the substituted benzimidazolone, NS-004, involve the activation of BKCa channels.

Effects of ATP-dependent K+ channel modulators on an ischemia-reperfusion rabbit isolated heart model with programmed electrical stimulation.

The effects of glibenclamide and BRL-38227 were studied in isolated rabbit hearts subjected to ischemia and programmed electrical stimulation. Coronary artery occlusion over 24 min decreased the ventricular effective refractory period in the ischemic zone. BRL-38227 (0.1 microM) showed significant coronary vasodilator effects, but failed to modify the ventricular effective refractory period under these conditions. A higher concentration (5 microM) of BRL-38227 potentiated the ischemia induced ventricular effective refractory period shortening effects. Glibenclamide (0.1 and 1 microM) delayed the onset of the ischemia-induced ventricular effective refractory period shortening. Glibenclamide (1 microM) inhibited the potentiated ventricular effective refractory period shortening effects of BRL-38227 (5 microM) during ischemia, but failed to antagonise the coronary vasodilator effects of BRL-38227 (5 microM). A higher incidence of ventricular fibrillation was inducible when an extra beat was applied in the ischemic zone through programmed electrical stimulation. The incidence of programmed electrical stimulation induced ventricular fibrillation was increased by BRL-38227 (5 microM) and antagonised by glibenclamide (1 microM). The results suggest that high concentrations of KATP-activators can accentuate ischemia-induced decreases in refractory period and increase the susceptibility of hearts to ventricular fibrillation when an extra beat is applied to the ischemic myocardium. These effects did not occur at lower coronary vasodilating concentrations of BRL-38227.

An in vitro method for the evaluation of antiarrhythmic and antiischemic agents by using programmed electrical stimulation of rabbit heart.

A new model using isolated rabbit hearts perfused at constant flow in the Langendorff mode with the sinus node destroyed and under constant (2 Hz) pacing is described. Ventricular ischemia (24 min) was induced by ligation of the left ventricular branch of the coronary artery (LVB), followed by reperfusion (15 min). The programmed electrical stimulation (PES) technique was used to induce arrhythmias in the ischemic zone (IZ). Three agents with different mechanisms of action were tested to validate this model: dl-sotalol (10(-6) and 10(-5) M), oxfenicine (10(-6) M), and lidocaine (10(-5) M). These compounds were administered 15 min before the ligature and maintained until the end of the experiment. Ventricular effective refractory period (VERP), PES-induced ventricular fibrillation (VF), and coronary perfusion pressure (CPP) were monitored. PES-induced VF was only observed in ischemic tissue. Sotalol slightly reduced VF incidence only during reperfusion. Oxfenicine prevented PES-induced VF during the ischemia, but not during reperfusion, while lidocaine prevented VF during ischemia and throughout the reperfusion period. In conclusion, the rabbit heart model where PES is applied to normal and ischemic myocardium, appears useful to discern different mechanisms involved in ventricular arrhythmias. In addition, this model is considerably cheaper than equivalent dog models.

Alfuzosin, a selective alpha 1-adrenoceptor antagonist in the lower urinary tract.

1. Phenylephrine-induced contractions of rabbit isolated trigone and urethra were antagonized in a competitive manner by alfuzosin (pA2 7.44 and 7.30, respectively) and prazosin. 2. The characteristics of [3H]-prazosin binding to human prostatic adenoma tissue were evaluated. [3H]-prazosin was potently displaced by alpha 1-adrenoceptor specific agents including alfuzosin, its (+)- and (-)-enantiomers and prazosin (IC50 0.035, 0.023, 0.019 and 0.004 microM, respectively), but only weakly by alpha 2-adrenoceptor selective agents, for example, yohimbine (IC50 = 6.0 microM). 3. In the pithed rat, alfuzosin (0.03-0.3 mg kg-1, i.v.) markedly inhibited pressor responses produced by the alpha 1-selective agonist, cirazoline but inhibited only slightly responses to the alpha 2-selective agonist, UK 14,304. Alfuzosin (1 mg kg-1, i.v.) had minimal effects against responses mediated by stimulation of prejunctional alpha 2-receptors (UK 14,304-induced inhibition of sympathetic tachycardia). 4. In the anaesthetized cat, alfuzosin (0.001-1 mg kg-1, i.v.) and prazosin (0.001-0.3 mg kg-1, i.v.) produced dose-related inhibition of the increases in urethral pressure caused by stimulation of sympathetic hypogastric nerves. Prazosin was approximately 5 fold more potent than alfuzosin. When phenylephrine was employed to induce urethral and vascular alpha 1-mediated tone simultaneously, prazosin inhibited both stimuli with similar potency whereas alfusozin was 3-5 fold more potent against elevated urethral pressure. This functional uroselectivity of alfuzosin was more evident by the intraduodenal route, since doses of 0.03 and 0.1 mg kg-1 alfuzosin inhibited urethral pressure with minimal effects on arterial blood pressure. 5. Alfuzosin is a potent selective alpha1-adrenoceptor antagonist in tissues of the lower urinary tract including the human prostate. This provides a pharmacological basis for its use in the treatment of benign prostatic hypertrophy.

The pharmacology of RS-15385-197, a potent and selective alpha 2-adrenoceptor antagonist.

1. RS-15385-197 ((8aR, 12aS, 13aS)-5,8,8a,9,10,11,12,12a,13,13a-decahydro- 3-methoxy-12-(methylsulphonyl)-6H-isoquino [2,1-g][1,6]-naphthyridine) was evaluated in a series of in vitro and in vivo tests as an antagonist at alpha 2-adrenoceptors. 2. RS-15385-197 had a pKi of 9.45 for alpha 2-adrenoceptors in the rat cortex (pA2 in the guinea-pig ileum of 9.72), whereas the 8aS, 12aR, 13aR enantiomer, RS-15385-198, had a pKi of only 6.32 (pA2 6.47) indicating a high degree of stereoselectivity. The racemate RS-15385-196 had a pKi of 9.18. 3. RS-15385-197 showed unprecedented alpha 2 vs. alpha 1 adrenoceptor selectivity in vitro. In the rat cortex, RS-15385-197 had a pKi of 9.45 in displacing [3H]-yohimbine and 5.29 in displacing [3H]-prazosin (alpha 2/alpha 1 selectivity ratio in binding experiments > 14000). The compound had a pA2 of 9.72 as a competitive antagonist of the inhibitory effects of UK-14,304 in transmurally-stimulated guinea-pig ileum and 10.0 against BHT-920-induced contractions in dog saphenous vein (DSV); this latter value was unaltered by phenoxybenzamine. An apparent pKB of 5.9 was obtained against cirazoline-induced contractions in DSV, whilst a pA2 of 6.05 was obtained against phenylephrine-induced contractions in the rabbit aorta (alpha 2/alpha 1 selectivity ratio in functional experiments > 4000). 4. RS-15385-197 was highly selective for alpha 2-adrenoceptors over other receptors: the compound showed low affinity for 5-HT1A (pKi 6.50) and 5-HT1D (pKi 7.00) receptor subtypes, and even lower affinity (pKi < or = 5) for other 5-HT receptor subtypes, dopamine receptors, muscarinic cholinoceptors, beta-adrenoceptors and dihydropyridine binding sites. RS-15385-197 was devoid of affinity for the non-adrenoceptor imidazoline binding site, labelled by [3H]-idazoxan, which provides further evidence that these sites are not related to alpha 2-adrenoceptors. In the DSV, contractile responses to 5-hydroxytryptamine (5-HT) were unaffected by a concentration of 1 microM RS-15385-197. 5. RS-15385-197 was non-selective for the alpha 2A- and alpha 2B-adrenoceptor subtypes in that the pKi for the alpha 2A-adrenoceptor in human platelets was 9.90 and the pKi for the alpha 2B-adrenoceptor in rat neonate lung was 9.70. However, RS-15385-197 showed lower affinity for the alpha 2-adrenoceptor subtype in hamster adipocytes (pKi 8.38). 6. In anaesthetized rats, RS-15385-197 was a potent antagonist of the mydriasis response induced by UK-14,304 or clonidine (AD50 5 and 7 microg kg-1, i.v., respectively; 96 microg kg-1, p.o.) and of UK-14,304-induced pressor responses in pithed rats (AD50 7 microg kg-1, i.v.); the compound therefore is both centrally and orally active. Even at a high dose (10 mg kg-1, i.v.), RS-15385-197 did not antagonize pressor responses to cirazoline in pithed rats, indicating that the selectivity for alpha2 vs. alpha1-adrenoceptors was maintained in vivo.8 RS-15385-197 is therefore a very potent, selective, competitive alpha2-adrenoceptor antagonist, both in vitro and in vivo, is orally active and readily penetrates the brain. It will thus be a powerful pharmacological tool for exploring the various physiological roles of alpha2-adrenoceptors.

Haemodynamic effects of the calcium facilitator Bay K-8644 in rats following vascular calcium overload.

1. The haemodynamic effects of the Ca2+ facilitator Bay K-8644 (Bay) were studied in a model of calcinosis induced by acute treatment with vitamin D3 and nicotine administration over 4 days with 13 days of recovery. 2. Calcium content of the left ventricular myocardium increased 8-9 fold, while aortic Ca2+ levels increased up to 12-fold in treated animals. There were minimal changes in the ECG and no change in the level of plasma alpha-hydroxy-butyrate-dehydrogenase, a cardiac specific enzyme which increases during ischaemia. Significant increases in pulse pressure (PP) were seen in anaesthetized and conscious calcinotic rats, with no increase in cardiac output index (DABF) or systemic vascular resistance. However, aortic rigidity (AORI) was significantly elevated in the calcinotic group under anaesthesia. 3. In both control and calcinotic rats, pressor responses to i.v. Bay were exclusively mediated by an increase in aortic blood flow (DABF) as lower body vascular resistance (TLBVR) did not change. The increase in DABF at low doses (0.1-1 microgram kg-1) of Bay probably resulted from an increase in venous return induced by the agonist, as Bay had little effect on cardiac contractility over this dose range (as estimated by left ventricular dp/dtmax) and did not cause tachycardia. At higher doses (10-1000 micrograms kg-1), Bay significantly increased LV dp/dt. Bay caused dose-related increases in AORI in pithed calcinotic rats, but a decrease in AORI in control animals. 4. The calcinosis model, which incorporates a recovery period to obviate the acute effects of nicotine and/or vitamin D3 treatment, results in long-term tissue calcium accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of endothelin-1 on the vasorelaxant properties of benzopyran and non-benzopyran potassium channel openers.

1. The effects of endothelin-1 (ET-1) on the vasorelaxant properties of structurally different potassium channel openers (PCOs), BRL-38227, Ro 31-6930, SDZ PCO 400, EMD-52692, RP-49356 and pinacidil, were studied. 2. All PCOs evoked concentration-related relaxations of ET-1 (10 nM) or KCl (20 mM) contracted rat isolated aortic rings denuded of endothelium. BRL-38227, EMD 52692, SDZ PCO 400 and Ro 31-6930 were 11-42 times less potent in relaxing contractions to ET-1 than KCl. In contrast, this differential potency was not observed with RP-49356 or pinacidil. 3. BRL-38227 (0.06-3.0 microM), RP-49356 (0.3-3.0 microM) and pinacidil (0.3-3.0 microM) displaced KCl concentration-response curves to the right of controls, without modifying the maximum response. A subcontractile concentration of ET-1 (0.1 nM) prevented the inhibitory effects of low concentrations of BRL-38227 (0.06-0.1 microM) on KCl responses, but failed to modify those to RP-49356, pinacidil or high concentrations of BRL-38227 (0.3-3.0 microM). The inhibitory effects of BRL-38227 (0.1 microM) were also not changed by ET-3 (1.0 nM) or angiotensin II (0.1 nM). 4. In anaesthetized spontaneously hypertensive rats (SHR), cumulative bolus intravenous administrations of BRL-38227 (1-1000 micrograms kg-1, i.v.), Ro 31-6930 (1-1000 micrograms kg-1, i.v.), RP-49356 (10-1000 micrograms kg-1, i.v.) or nitrendipine (0.1-30 micrograms kg-1, i.v.) produced dose-dependent falls in diastolic blood pressure (DBP).ET-1 (i.v.) evoked a transient fall in DBP (1 pg kg- = 58 + 1 mmHg) which returnedto pre-administration levels within 4 min.5. Pretreatment of anaesthetized SHR with ET-l (1 pg kg-', i.v.) significantly increased the ED,5 (dose to evoke a 15% fall in DBP) values for BRL-38227 and Ro 31-6930. However, ET-l failed to modify the ED,5 values for RP-49356 or nitrendipine. The ED50 values for all of the vasodilators studied were not modified by ET-1.6. Infusion of BRL-38227 (2 pgkg-'min-', i.v.) or RP-49356 (4 pgkg-'min', i.v.) to anaesthetized SHR evoked dose-related falls in DBP, with a corresponding increase in descending aortic blood flow (DABF) and a decrease in total lower body vascular resistance (TLBVR). Pretreatment with ET-1 (1 ptg kg-', i.v.) significantly attenuated the decreases in DBP and TLBVR observed with low doses of BRL-38227, but not RP-49356 or high doses of BRL-38227. In contrast, ET-3 (3 pig kg-, i.v.) failed to modify the effects of BRL-38227 on DBP or TLBVR.7. In conscious SHR, the fall in DBP to BRL-38227 (30 pgkg-', p.o.) was significantly reduced following ET-1 (1 pig kg-', i.a.) treatment. ET-1 (1 pg kg-', i.a.) pretreatment, however, failed to modify the decrease in DBP induced by an equieffective oral dose of RP-49356 (1001pgkg-1).8. In conclusion, ET-1 selectively attenuated the vasorelaxant effects of the potassium channel opener,BRL-38227 and other substituted benzopyrans. The results are compatible with the hypothesis that benzopyran PCOs and ET-1 have affinity for a site that does not recognise RP-49356 or pinacidil. Thus,ET-l can differentiate between structurally unrelated potassium channel openers. The cardiovascular effects of some, but not all, PCOs might be radically modified in the clinical setting by elevated endogenous levels of ET-1 associated with certain diseased states.

An industrial approach to drug discovery: a pharmacologist's eyeview.

1. The pressures on the pharmaceutical industry to produce novel, safe and effective therapeutic agents have provoked management to rethink the way in which drug discovery is undertaken. 2. Advances in drug therapy are likely to come from the application of new technologies and leading-edge science, perhaps in collaboration with academia. 3. Close collaboration between biologists and medicinal chemists is a proven way to discover new products. 4. The complexity of the modern industrial discovery process, due to the interaction of many disciplines, requires matrix-style management. 5. The early demonstration of efficacy in human subjects is considered essential for providing feedback to the basic scientists about the validity of hypotheses, and the relevance of the methods utilized, as well as whether a decision should be allowed to advance to full development.

Alpha-adrenoceptor subtypes in dog saphenous vein that mediate contraction and inositol phosphate production.

1. Studies have been made of the contractile responses to the alpha-adrenoceptor agonists phenylephrine (Phen), cirazoline (Cir) or BHT-920 (BHT) in dog isolated saphenous vein (DSV) rings, using the antagonists yohimbine (Yoh), idazoxan (Idaz), prazosin (Praz), WB-4101 (WB) and nitrendipine or zero Ca2+ medium. 2. Contractile concentration-response curves to Phen or BHT were displaced to the right of controls by Yoh (0.01-3 microM) with mean apparent antagonist dissociation constants (pKBs) of 7.9 and 8.6 respectively. Yoh did not show simple competitive antagonism against either agonist, since the Schild plot slopes were significantly less than unity. Neither the antagonist affinity of Yoh against Phen, nor the slope of the Schild plot was modified in the presence of catecholamine uptake inhibitors, nor in the presence of alpha,beta-methylene ATP, which desensitizes P2-purinoceptors, suggesting that Phen does not release ATP, or noradrenaline to cause contraction in DSV. In the presence of Praz (0.3 microM) the antagonist potency of Yoh (mean pKB 7.4) against Phen was slightly decreased. Yoh had low potency against responses induced by Cir (pKB 6.3). 3. WB (0.001-1.0 microM) was a very potent antagonist of Phen-induced contractions, however, the biphasic Schild plot against Phen could be separated into two affinity sites, a high pKB of 9.3 (equivalent to that obtained using Cir as the agonist; pKB 9.6) and a lower affinity (pKB 8.6). WB showed an even lower antagonist affinity (pKB 7.4) against BHT-induced contractions, suggesting that these effects might be mediated by alpha 2A-adrenoceptors. Praz also appeared to identify two sites using Phen-induced contractions, a high pKB of 8.4 was equivalent to that obtained with Cir (pKB 8.2) and a lower affinity site (pKB 7.7; pA2 7.6; slope 1.1) at which Praz showed competitive antagonism. Higher concentrations of Praz were required to antagonize contractions to BHT (pKB 5.9). 4. Idaz was a weak partial agonist in this tissue with threshold contractile effects at concentrations in excess of 3 microM. Idaz (0.1-1 microM) competitively antagonized the contractile effects of BHT, but showed low antagonist affinity against Phen at these concentrations. 5. Contractions to Phen were slightly antagonized by nitrendipine (1 microM), with a 36% decrease in Emax. Contractions to Phen and Cir were also markedly attenuated in zero calcium medium (with EGTA), but maximum responses of 4.2 +/- 0.1 and 3.6 +/- 0.1 g, could be obtained with these agonists respectively. Only part of the contractile effects to Phen or Cir are therefore due to calcium influx (but L-type channels are not totally implicated), while the contractile effects of BHT were abolished in zero Ca2 + medium. Yoh (0.1 microm) retained its antagonist effects on Phen-induced responses in zero Ca2 + medium. 6. The formation of inositol phosphates (InsPs) in the presence of lithium (10mM) was measured after incubation of intact DSV strips with myo-2-[3H]-inositol. Phen (1-1OO0 microM) and Cir (O.O1-1O microm) induced concentration-dependent increases in total labelled InsP1_3, but BHT showed minimal InsP stimulation. InsPs were recovered after Phen (100,M) stimulation (10min) as labelled InsP1 (71%), InsP2 (25%) and InsP3 (4%). Phen (100 microM)-stimulated InsP1-3 formation was significantly antagonized by Praz (10nM), but was not fully inhibited even after Praz 1 microM. Yoh and Praz (0.1 and 1.0 microM) were equipotent inhibitors of this response, while Idaz (0.3 microM) showed no effects. 7. The receptors in DSV which are stimulated by Phen to cause contraction show characteristics of the alpha lA-adrenoceptor (high pM antagonist affinity for WB-4101 and extracellular calcium sensitivity) and the alpha lB-adrenoceptor (contraction in calcium-free medium, increase in InsP and low nm antagonist affinity of WB). The paradoxical results obtained with Yoh (potent antagonist effects on Phen-stimulated PI and pKB 7.9 on contraction) and Praz (low affinity competitive antagonist of Phen-induced contraction, pKB 7.7 and failure to inhibit completely the PI response at 1 microM), cannot fully exclude an alpha 2B-subtype characterization of these responses. These pharmacological differences suggest that the adrenoceptor involved in the contractile and in particular the second messenger effects of Phen in DSV is not typically an alpha lB-adrenoceptor.

Pulmonary and systemic haemodynamic effects of cromakalim in conditions of normoxia and hypoxia in dogs.

1. In anaesthetized-closed chest dogs, exposure to hypoxia (60 min) caused a sustained increase in arterial pulmonary pressures (diastolic: DPP, mean: MPP and effective capillary: EPCP by 132, 66 and 104% respectively) and increased total pulmonary vascular resistance (TPVR, 143%) with minimal changes in systemic haemodynamics. 2. Under normoxia, cromakalim infusion (2 micrograms kg-1 min-1 for 30 min) progressively decreased diastolic systemic arterial pressure (DBP, 30%) and total systemic vascular resistance (TSVR, 54%). In contrast, DPP was increased by 45%, due to an increase in cardiac output (CO: 55%), while TPVR was essentially unchanged. 3. Under hypoxic conditions, cromakalim (2 micrograms kg-1 min-1 for 30 min) induced changes in systemic haemodynamics comparable to those seen under normoxia. However, DPP was decreased by 37% at the end of infusion. The compound progressively decreased TPVR in a dose-related manner and PaO2 (55.9 mmHg) was maintained since cardiac output was increased. 4. Thus, under normoxia, cromakalim induced haemodynamic effects on the pulmonary circulation which were directly related to an increase in CO, but were clearly different from those observed under hypoxia. In the hypoxia-induced constricted pulmonary vascular bed, cromakalim effectively reduced the elevated pulmonary vascular resistance at low doses but showed comparable vasodilator effects on the systemic circulation under both conditions. The findings suggest that a vasodilator agent like cromakalim may be useful in patients with respiratory conditions associated with elevated pulmonary vascular resistance.

Vascular calcium overload produced by administration of vitamin D3 and nicotine in rats. Changes in tissue calcium levels, blood pressure, and pressor responses to electrical stimulation or norepinephrine in vivo.

Increased calcium content of cardiovascular tissues is a phenomenon common to natural aging and various pathological conditions such as hypertension and arteriosclerosis. We investigated an accelerated cardiovascular calcium overload model in young rats produced by treatment with a single dose of vitamin D3 (300,000 IU/kg, i.m.) followed by up to 4 days of twice daily doses of nicotine (25 mg/kg, p.o.). Large increases in the calcium content of the aorta, kidneys, and myocardium but not in the liver or brain were seen. The magnesium content of these tissues was not modified. On the day following the last nicotine injection, there was marked cardiovascular calcium overloading, the aortic calcium level increasing by up to nine times that of controls. The animals had lower body weights, however, and there was a significant degree of mortality (up to 42%). Signs of kidney failure were evident; the blood urea level, for instance, was doubled. If rats were allowed 13 or 180 days to recover, they showed normal growth and kidney function; aortic calcium overload was still pronounced: 16- and 7-fold increases, respectively. Cardiovascular function in recovery animals was characterized by a doubling of pulse pressure. Dose-response curves following noradrenergic stimulation were shifted to the right after 13 (but not after 180) days recovery. Arterial norepinephrine content doubled. The chronic effects of hypervitaminosis D plus nicotine may produce a useful model for the study of the physiological and/or pharmacological consequences of calcium overload.

Pulmonary vascular and renal effects of cromakalim in anaesthetized dogs.

1) CROM reduced hypoxia-induced pulmonary vasoconstriction and maintained PaO2 with an increase in cardiac output. 2) Low doses of CROM infused into the kidney caused pronounced renal vasodilation, marked natriuresis and a significant increase in cardiac output with minor changes in blood pressure. In addition to its previously described systemic antihypertensive effects, CROM also reduces hypoxia-induced pulmonary hypertension and shows marked renal vasodilation with natriuresis in anaesthetized dogs.

Studies on the mechanisms of the development of tolerance to the hypotensive effects of fenoldopam in rats.

In pentobarbital-anesthetized rats prepared for hemodynamic measurements with Doppler flow probes, intravenous (i.v.) infusions of fenoldopam (2.5 - 160.0 micrograms/kg/min during 15 min) decreased mean carotid artery blood pressure, total peripheral, hindquarter, renal, and mesenteric vascular resistances and increased renal blood flow strongly. The hypotensive effects attained a maximum within the first 3 min of infusion but waned by greater than 30% at the end of fenoldopam administration. This tolerance was observed for calculated total peripheral and hindquarter vascular resistances and to a lesser extent for mesenteric resistance. However, it was absent on the renal vascular bed. Pretreatment with either enalapril, pepstatine, or bilateral nephrectomy significantly increased the hypotensive response to fenoldopam and attenuated the development of tolerance. In conscious spontaneously hypertensive rats (SHR), enalapril potentiated strongly the small blood pressure-lowering activity of fenoldopam. The fall in blood pressure produced by fenoldopam was specifically blocked by SCH 23390, an antagonist of DA-1 dopamine receptors. In normotensive vasopressin-supported pithed rats given phenoxybenzamine plus propranolol, fenoldopam, like SCH 23390, blocked the vasodepressor effects of i.v. bolus injection of dopamine and fenoldopam. In pithed rats, fenoldopam evoked a pressor response that was significantly reduced by enalapril, SCH 23390, or bilateral nephrectomy. In conclusion, fenoldopam exerts DA-1 agonist and antagonist effects. The latter property, together with the activation of the renin-angiotensin system, appears to be responsible for the development of tolerance to the fenoldopam evoked-hypotension. The lack of a tolerance at the level of the renal vascular bed is possibly due to the existence of a large population of DA-1 receptors in this region.

Comparative analysis of beta-1 adrenoceptor agonist and antagonist potency and selectivity of cicloprolol, xamoterol and pindolol.

The partial beta adrenoceptor agonist properties of cicloprolol, xamoterol and pindolol have been compared in vivo (anesthetized catecholamine-depleted or pithed rats) and in vitro (guinea pig or rat right atria and guinea pig tracheal muscle preparations) conditions. All three compounds increased heart rate in the former preparations, and their intrinsic activities relative to isoproterenol were 0.7, 0.65 and 0.45, respectively. The positive chronotropic effects of cicloprolol or xamoterol were competitively antagonized by betaxolol or propranolol; however, part of those induced by pindolol were resistant to these beta adrenoceptor antagonists. None of these compounds increased the spontaneous beating rate of isolated guinea pig atria; however, xamoterol only increased heart rate in isolated rat atria, and its intrinsic activity with respect to isoproterenol was 0.4. Pindolol, xamoterol and cicloprolol behaved as competitive beta-1 adrenoceptor antagonists against isoproterenol-induced tachycardia in a pithed rat model. In order to mimic the intrinsic effects of the partial agonist drugs, control dose-response curves for isoproterenol were determined in pithed rats in which the base-line heart rate was elevated by thoracic spinal cord stimulation. In this in vivo preparation, xamoterol and pindolol were more potent beta-1 adrenoceptor antagonists than cicloprolol; however, cicloprolol and xamoterol, in contrast to pindolol, were selective for beta-1 adrenoceptors. In isolated spontaneously beating guinea pig right atria, cicloprolol and xamoterol were equipotent beta-1 adrenoceptor antagonists but were about 50 times less potent than pindolol. In isolated rat atria, the beta-1 adrenoceptor antagonist potency of xamoterol was greater (pA2 = 8.7) than in guinea pig atria (pA2 = 7.8). The potencies of cicloprolol and pindolol did not vary between these species. In catecholamine-depleted rats, high i.v. doses of cicloprolol had vasodilator activity that was partly mediated by beta-2 adrenoceptors. In carbachol-contracted guinea pig trachea, cicloprolol and xamoterol, in contrast to pindolol, were relatively inactive against isoproterenol-induced relaxation. In conclusion, cicloprolol and xamoterol, similarly to pindolol, behave as agonists and antagonists of beta-1 adrenoceptors. However, only cicloprolol and xamoterol show an elevated degree of selectivity toward the beta-1 adrenoceptor subtype.

Effect of acidosis on alpha 1- and alpha 2-adrenoceptor-mediated vasoconstrictor responses in isolated arteries.

We have investigated the effect of reducing the pH (from 7.5 to 7.0 by addition of HCl) on vasoconstrictor responses to noradrenaline in cat middle cerebral artery (in which responses are mediated almost entirely by alpha 2-adrenoceptors) and in rabbit pulmonary artery (in which responses are mediated by alpha 1-adrenoceptors). In the cerebral artery, a reduction in pH caused a pronounced inhibition of the responses to noradrenaline, and the antagonistic effect of idazoxan (100 nM) was increased 10-fold. In contrast, in the pulmonary artery, a reduction in pH had no effect on the responses to noradrenaline and the antagonistic effect of prazosin (100 nM) was not altered. We conclude that acidosis selectively reduces the vasoconstriction mediated by alpha 2-adrenoceptors in vitro.

Calcium channel receptor binding studies for diltiazem and its major metabolites: functional correlation to inhibition of portal vein myogenic activity.

Pharmacologically distinct but allosterically interacting calcium channel antagonist binding sites have recently been identified using radiolabeled dihydropyridine derivatives (e.g., [3H]nitrendipine) and the benzothiazepine [3H]diltiazem. Whereas the functional significance of the dihydropyridine calcium channel antagonist receptor is well documented, it remains to be established whether drug interactions with the recognition site for [3H]diltiazem within the slow calcium channel or the allosteric interaction of the diltiazem binding site with the dihydropyridine receptor are of physiological significance. In a study of structure-activity relationships, we therefore examined the effects of diltiazem and five of its analogs on the binding of [3H]diltiazem and [3H]nitrendipine to the rat cerebral cortex. In parallel, we studied the effects of these drugs on the spontaneous myogenic contractions of the rat portal vein, a functional test of calcium antagonism. The diltiazem analogs used in this study correspond to its major metabolites in humans, i.e., N-desmethyl-(MA, desacetyl-(M1), N-desmethyl, desacetyl-(M2), O-desmethyl, desacetyl (M4), N-desmethyl, O-desmethyl, desacetyl-diltiazem (M6). Unlabeled diltiazem inhibited [3H]diltiazem binding at 37 degrees C with a pIC50 [-log IC50 (M)] of 6.87. pIC50 values for M1, MA, M2, M4, and M6 were 6.72, 6.49, 6.03, 5.51, and 5.33, respectively. pIC50 values for these drugs on [3H]diltiazem binding were significantly correlated (p less than 0.01) with their pEC50 values for enhancement of [3H]nitrendipine binding to cerebral cortical membranes at 37 degrees C. Maximal enhancement of [3H]nitrendipine binding by diltiazem, M1, MA, M2, M4, and M6 was 73, 50, 9.7, 11, 12, and 52%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiovascular characterization of the DA2 dopamine receptor agonist quinpirole in rats.

In normotensive anesthetized rats, 15-min IV infusions of quinpirole (2.5-40.0 micrograms/kg/min) produced dose-related, rapidly appearing, and long-lasting decreases in mean carotid artery BP and HR. Hemodynamically, the hypotensive effects of quinpirole (10.0 micrograms/kg/min) were due to a fall in total peripheral vascular resistance inasmuch as CO did not undergo significant changes. Mesenteric, hindquarter, and renal blood flows were, respectively, reduced, unchanged, and increased by quinpirole; thus, the renal vascular resistance fell more than either the total peripheral or hindquarter vascular resistance. Biochemically, the hypotensive effects of quinpirole were accompanied by a decrease in the plasma level of norepinephrine and plasma renin activity. The peak fall in blood pressure produced by quinpirole was not significantly modified by atenolol, idazoxan, ranitidine, SCH 23390 (DA1 dopamine receptor antagonist), enalapril, or SK&F 100273 (V1 vasopressin receptor antagonist), but was entirely blocked by S-sulpiride or removal of autonomic nerve drive to the cardiovascular system with chlorisondamine. The effect of quinpirole on systemic and regional vascular resistances was antagonized by S-sulpiride. Furthermore, SK&F 100273 prevented the fall in mesenteric flow produced by quinpirole. Intracerebroventricular injection of quinpirole (10.0 micrograms/kg over 2 min) in saline- or SK&F 100273-pretreated rats produced the same hypotensive effects as an identical IV dose of the compound. In pithed rats, quinpirole (10 micrograms/kg/min IV over 15 min) decreased pressor responses to electrical stimulation of spinal cord outflow without affecting those to exogenously injected angiotensin II, B-HT 920, cirazoline, norepinephrine, or 5-hydroxytryptamine. This inhibitory effect was antagonized by S-sulpiride. The bradycardia produced by quinpirole in intact rats was mediated by the autonomic nervous system inasmuch as it was slightly modified by bilateral vagotomy, partly reduced by atenolol, and entirely prevented by pithing even when the low HR of the last preparation had been raised by IV infusion of isoprenaline. Furthermore, S-sulpiride, but not SCH 23390 or idazoxan, antagonized this effect. In pithed rats, quinpirole similarly inhibited the tachycardic responses elicited by electrical stimulation of either the spinal cord outflow (preganglionic) or postganglionic cardioaccelerator nerve fibers. This effect of quinpirole was susceptible to S-sulpiride but not idazoxan blockade. Finally, in conscious spontaneously hypertensive rats (SHR) but not in normotensive rats, quinpirole (10 micrograms/kg/min IA over 15 min) lowered blood pressure.(ABSTRACT TRUNCATED AT 400 WORDS)

Possible involvement of presynaptic alpha 1-adrenoceptors in the effects of idazoxan and prazosin on 3H-noradrenaline release from tail arteries of SHR.

The effects of several alpha-adrenoceptor antagonists have been examined on tritium release elicited by electrical stimulation from isolated perfused SHR tail artery preparations prelabelled with 3H-noradrenaline (3H-NA). Phentolamine and yohimbine potently facilitated the stimulation evoked release of tritium at low frequencies of stimulation, but the alpha 2-adrenoceptor antagonist idazoxan was only weakly active at 1 mumol/l, despite antagonising the clonidine-evoked inhibition of 3H-release at a lower concentration of 0.1 mumol/l. The alpha 1-adrenoceptor antagonists prazosin and corynanthine also increased stimulation evoked tritium release in this preparation, suggesting the presence of prejunctional alpha 1-adrenoceptors. Furthermore, the alpha 1-adrenoceptor agonist methoxamine (3 mumol/l) caused a significant inhibition of tritium-evoked release, an effect which was blocked by prazosin (10 nmol/l). When alpha 1-adrenoceptors were blocked in the presence of prazosin, idazoxan (0.1 mumol/l) produced a significant facilitatory effect on the electrically-evoked release of 3H-transmitter. On the other hand, when alpha 2-adrenoceptors were blocked in the presence of yohimbine, exposure to idazoxan (0.1 mumol/l) reduced significantly the stimulation-evoked release of tritium elicited by electrical stimulation. The results indicate that in the SHR tail arteries, idazoxan has a partial agonist inhibitory activity on transmitter release, which can mask the facilitatory effects due to blockade of presynaptic alpha 2-adrenoceptors. The inhibitory effects of idazoxan appear to involve presynaptic alpha 1-adrenoceptors, which when stimulated, reduce 3H-NA release in SHR tail arteries.

Differential effects of alpha-beta-methylene ATP on responses to nerve stimulation in SHR and WKY tail arteries.

The effects of alpha,beta-,methylene-adenosine triphosphate, (alpha,beta-methylene ATP, a P2-receptor desensitising agent) have been evaluated on vasoconstrictor responses elicited by exogenous agonists or electrical field stimulation in isolated perfused SHR or WKY tail arteries and on tritium release elicited by electrical field stimulation in SHR-tail arteries pre-labeled with 3H-noradrenaline. Exposure to alpha,beta-methylene ATP (0.1 mumol/l) significantly inhibited vasoconstrictor responses to electrical field stimulation in SHR tail arteries. These inhibitory effects were not further increased at a higher concentration of alpha,beta-methylene ATP (1 mumol/l). In WKY tail arteries, alpha,beta-methylene ATP (1 mumol/l) failed to significantly inhibit vasoconstrictor responses to electrical stimulation. In SHR tail arteries prelabelled with 3H-noradrenaline, alpha,beta-methylene ATP (1 mumol/l) did not inhibit the stimulation evoked release of tritium. However, at this concentration, alpha,beta-methylene ATP significantly antagonized the vasoconstrictor responses of SHR tail arteries induced by exogenous ATP (1 mumol/l), beta,gamma-methylene ATP (30 mumol/l), a stable agonist at P2-receptors, or 60 mmol/l KCl. These effects of alpha,beta-methylene ATP on contractile responses to KCl were not observed in WKY-tail arteries. In tail arteries obtained from reserpine pretreated SHR, despite a 85-95% decrease in endogenous noradrenaline tissue content, the vasoconstrictor responses induced by periarterial field stimulation were greatly diminished, but not abolished. These residual responses to periarterial field stimulation were not antagonized by prazosin (0.1 mumol/l), but were practically abolished by the addition of alpha,beta-methylene ATP (1 mumol/l). In tail arteries from WKY rats pretreated with reserpine, exposure to prazosin (0.1 mumol/l) further reduced the residual responses elicited by electrical field stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Reserpine-resistant responses to nerve stimulation in the cat nictitating membrane involve the release of newly synthesized noradrenaline.

Preganglionic sympathetic nerve stimulation in cats pretreated with reserpine resulted in significant frequency-dependent contractions of the nictitating membrane, despite a severe depletion of tissue noradrenaline content. These residual responses to sympathetic nerve stimulation were potentiated by cocaine or pargyline, and were antagonised by the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine. In contrast to the residual responses to sympathetic stimulation in the nictitating membrane, the tachycardia evoked by postganglionic cardiac nerve stimulation was totally abolished by pretreatment with reserpine, even after the administration of cocaine. The alpha-adrenoceptor antagonists phentolamine or prazosin reduced, but did not abolish the reserpine-resistant responses of the nictitating membrane, suggesting the nerve-mediated release of a co-transmitter. In addition to this co-transmitter, a neuronal pool of neurotransmitter which is reserpine-resistant and involves newly synthesised noradrenaline, contributes to the residual responses of the nictitating membrane, following depletion of the neurotransmitter stores by the administration of reserpine. administration of reserpine.