Sustained negative inotropism mediated by alpha-adrenoceptors in adult mouse myocardia: developmental conversion from positive response in the neonate.

1. Inotropic responses to alpha-adrenoceptor stimulation and the effects of antagonists were examined in isolated ventricular preparations from neonatal and adult mice. 2. Phenylephrine, in the presence of propranolol, produced positive inotropic responses in neonates up to 1 week after birth, while it produced negative inotropic responses in mice older than 3 weeks. 3. Both positive and negative responses to phenylephrine in neonates and adults, respectively, were antagonized by prazosin, WB4101 (2-([2,6-dimethoxyphenoxyethyl]aminomethyl)-1,4-benzodioxane) and 5-methylurapidil, but not by atropine, yohimbine or chlorethylclonidine. 4. Noradrenaline (NA) produced positive inotropic responses both in the neonate and adult; the responses were observed in a lower concentration-range in the neonate than in the adult. WB4101 produced a significant leftward shift of the concentration-response curve for noradrenaline in adult preparations while only a slight rightward shift was observed in the neonate. 5. Our results demonstrate the presence of alpha-adrenoceptor-mediated inotropic responses in the mouse ventricular myocardia. The response to phenylephrine changes from a positive to a negative effect during postnatal development. The responses are mediated by alpha 1-adrenoceptors, and modulate the overall inotropic response to NA in the adult.     

Characterization of angiotensin II antagonism displayed by Ib, a novel nonpeptide angiotensin AT(1) receptor antagonist

The pharmacologic profile of Ib, 5-n-butyl-4-{4-[2-(1H-tetrazole-5-yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6-dichloridephenyl)-3H-1,2,4-triazol-3-one, a novel nonpeptide angiotensin AT(1) receptor antagonist, was investigated by receptor-binding studies, functional in vitro assays with rabbit and rat aorta, and in vivo experiments in rats. Ib inhibited [(125)I] angiotensin II binding to AT(1) receptors in rat liver membranes (K(i)=2.5+/-0.5 nM) and did not interact with AT(2) receptors in bovine cerebellar membranes. In functional studies with rat and rabbit aorta, Ib inhibited the contractile response to angiotensin II (pD(2)' value: 7.43 and 7.29, respectively) with a significant reduction in the maximum. In pithed rats, Ib inhibited the angiotensin II induced pressor response in a dose-related manner. After intravenous administration, Ib produced a dose-dependent antihypertensive effects in spontaneously hypertensive rats and renal hypertensive rats. These results suggest that Ib is a potent angiotensin AT(1) selective receptor antagonist with a mode of insurmountable antagonism.     

Blockade of adenosine receptors unmasks a stimulatory effect of ATP on cardiac contractility.

1. The effects of ATP, alpha,beta-methylene ATP and beta,gamma-methylene ATP on the contractile tension of guinea-pig isolated left atria were evaluated. 2. ATP (1-100 microM) produced a concentration-dependent negative inotropic effect; this response was converted to a positive inotropic effect in the presence of the antagonist of adenosine A1 receptors, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 0.1 microM), and in the presence of 8-phenyltheophylline (10 microM), an antagonist of A1 and A2 receptors. 3. The positive inotropic effect of ATP was antagonized by the P2 receptor antagonist, suramin (500 microM). Reactive blue 2 (30-500 microM), a putative P2y receptor antagonist, concentration-dependently reduced and finally abolished the effect of ATP. 4. In the presence of 8-phenyltheophylline, the stable analogues of ATP, alpha,beta-methylene ATP and beta,gamma-methylene ATP (1-30 microM), produced a concentration-dependent increase in atrial contractility of a lesser degree than that induced by ATP. 5. The results suggest that when inhibitory adenosine receptors are blocked, ATP produces a positive inotropic effect, probably mediated by P2y receptor stimulation.     

Pharmacological characteristics of the positive inotropic effect of angiotensin II in the rabbit ventricular myocardium.

1. In order to elucidate the mechanism underlying the positive inotropic effect (PIE) of angiotensin II (AII), we measured changes in phosphoinositide hydrolysis and contractile force induced by AII in the rabbit ventricular myocardium. 2. AII (1.0 nM-3 microM) produced a PIE in a concentration-dependent manner in the presence of bupranolol (0.3 microM) and prazosin (0.1 microM), the maximal response being about 40% of that to isoprenaline and the EC50 30 nM. 3. The PIE of AII was associated with a concentration-dependent increase in the total duration of contraction; the time to peak force and the relaxation time were prolonged. 4. AII (10 nM-30 microM) elicited an accumulation of [3H]-inositol monophosphate in a concentration-dependent manner in rabbit ventricular slices prelabelled with myo-[3H]-inositol. 5. The PIE and the accumulation of [3H]-inositol monophosphate induced by AII were inhibited by a non-selective AII receptor antagonist, saralasin (10 nM-1 microM) and by a selective AT1 receptor antagonist, losartan (10 nM-1 microM), but not a selective AT2 receptor antagonist, PD 123319 (1 microM). 6. A tumour-promoting phorbol ester, phorbol 12,13-dibutyrate (PDBu, 10-100 nM), inhibited the AII-induced PIE and [3H]-inositol monophosphate accumulation in a concentration-dependent manner. 7. These results suggest that AII exerts a PIE through activation of AT1 receptors and subsequent acceleration of phosphoinositide hydrolysis. Activation of protein kinase C by PDBu may inhibit the AII-induced stimulation of phosphoinositide hydrolysis and thereby the PIE of AII in the rabbit ventricular myocardium.     

Influence of a Na+-H+ exchange inhibitor ethylisopropylamiloride, a Na+-Ca2+ exchange inhibitor KB-R7943 and their combination on the increases in contractility and Ca2+ transient induced by angiotensin II in isolated adult rabbit ventricular myocytes

In rabbit, ventricular myocytes loaded with indo-1/AM, angiotensin II (0.1 nM-0.1 microM) exerted a positive inotropic effect with a significant increase in the amplitude of Ca2+ transients. For a given increase in cell shortening, the increase in Ca2+ transients induced by angiotensin II was less than that induced by elevation of extracellular Ca2+ concentration ([Ca2+]0) or isoprenaline, an indication that both the increase in mobilization of intracellular Ca2+ ions and myofibrillar sensitivity to Ca2+ ions contribute to the positive inotropic effect of angiotensin II. The effects of angiotensin II on Ca2+ transients and cell shortening were inhibited by the AT1 receptor antagonist losartan. A Na+ -H+ exchange inhibitor EIPA [5-(N-ethyl-N-isopropyl)amiloride] at 1 and 3 microM did not affect the Ca2+ transients and cell shortening, but it inhibited the angiotensin-II-induced responses in a concentration-dependent manner more effectively than the responses to elevation of [Ca2+]0, indicating that EIPA elicited a selective inhibitory action on the effects of angiotensin II. The observation that EIPA at 10 microM abolished the positive inotropic effect of angiotensin II without a significant depression of the inotropic response to elevation of [Ca2+]0 supports the selective action of EIPA at the high concentration on the response to angiotensin II. A novel selective Na+ -Ca2+ exchange (reverse mode) inhibitor KB-R7943, 2-[2-[4-(-nitrobenzyloxy)phenyl]ethyl] isothiourea methanesulphonate, at 0.3 and 1 microM inhibited also the responses to angiotensin II more effectively than the response to elevation of [Ca2+]0; however, over the same concentration range it suppressed significantly the amplitude of Ca2+ transients and cell shortening. Combination of EIPA (3 microM) and KB-R7943 (0.3 microM), each of which attenuated partially the angiotensin-II-induced responses, abolished the positive inotropic effect and the increase in Ca2+ transients induced by angiotensin II with much less depressant effect on the responses to elevation of [Ca2+]0. Thus, these ion exchange inhibitors exerted selective actions on the respective targets. The results with these selective inhibitors indicate that the activation of Na+ -H+ exchanger and subsequent modulation of the activity of Na+ -Ca2+ exchanger may be responsible for the increase in [Ca2+]i and the myofilament Ca2+ sensitization induced by stimulation of AT1 receptors by angiotensin II in rabbit ventricular myocytes.     

Increased sensitivity of neonatal mouse myocardia to autonomic transmitters

1 Chronotropic and inotropic responses to noradrenaline and acetylcholine were examined in isolated right atrial and ventricular preparations from neonatal and adult mice. 2 Noradrenaline and acetylcholine produced positive and negative chronotropic responses, respectively, in the atria from both ages. Noradrenaline produced positive inotropic responses in ventricular preparations from both ages. In all cases, the sensitivity, expressed in terms of pD₂ values, was higher in neonatal preparations. 3 In the ventricle, desipramine produced a leftward shift of the concentration–response curve for noradrenaline in the adult, but no such shift was observed in the neonate. The sensitivity to isoprenaline of ventricular preparations was higher in the neonate than in the adult. 4 Our results demonstrated developmental decreases in sensitivities to autonomic transmitters in mouse myocardia. As for the inotropic response to noradrenaline of ventricular muscle, both pre- and postjunctional mechanisms were responsible for the developmental decrease in sensitivity.     

Developmental regulation of hippocampal excitatory synaptic transmission by metabotropic glutamate receptors

The aims of this study were, to use agonists selective for the 3 mGlu receptor groups to identify developmental changes in their effects, and to assess the usefulness of proposed selective antagonists as pharmacological tools. Hippocampal slices (400 microm) were prepared from neonate (9 - 14 days) and young adult (5 - 7 weeks) Sprague-Dawley rats. Field excitatory postsynaptic potentials (fEPSP) were recorded from CA1. DHPG (100 microM), a group I agonist, produced a slowly developing enhancement of fEPSP slope in slices from adults. In slices from neonates, DHPG (75 microM) depressed fEPSP slope. DCG-IV (500 nM), a group II agonist, did not affect the fEPSP recorded from slices from adults whereas perfusion in neonate slices produced a sustained depression. The group III agonist L-AP4 (50 microM) was ineffective in adult slices but depressed fEPSP slope in slices prepared from neonates. DHPG-induced depression of fEPSP slope was inhibited by 4-CPG (400 microM), a group I antagonist, but was unaffected by MCCG (500 microM) and MAP4 (500 microM), group II and III receptor antagonists respectively. MCCG but not MAP4 antagonized the effects of DCG-IV with 4-CPG producing variable effects. The effect of L-AP4 was unaffected by MCCG, blocked by MAP4, and enhanced by 4-CPG. The results show that the effects of the agonists for all groups of mGlu receptors are developmentally regulated. Furthermore, MCCG and MAP4 behave as effective and selective antagonists for group II and group III mGlu receptors respectively, whereas the usefulness of 4-CPG as a group I antagonist may be limited.     

A unique xanthine derivative KMCP-98 with activation of adenosine receptor subtypes.

KMCP-98 is a newly synthesized adenosine receptor agonist by alkylation at the 7-position of the xanthines nucleus. We first investigated the pharmacological activities of KMCP-98 under in vivo and in vitro conditions. Acute intravenous injection of KMCP-98 (1.0, 2.0 and 3.0 mg/kg) produced a temporary fall in blood pressure and heart rate, followed by a sustained fall in heart rate in pentobarbital-anesthetized Wistar rats. The hypotensive and bradycardiac responses were inhibited by pretreatment with an A(1) adenosine receptor antagonist 8-phenyltheophylline (8-PT, 0.5 mg/kg). Both KMCP-98 and adenosine (0.3-100 microM) produced negative inotropic activity in isolated guinea pig left atria. The negative inotropic activity of KMCP-98 was significantly blocked by pretreatment with A(1) receptor antagonists 8-PT (10 microM) and xanthine amine congener (XAC, 10 microM), a nonselective adenosine antagonist theophylline (10 microM), a K(+) channel blocker tetraethylammonium (TEA, 10 mM) and a K(ATP) channel blocker glibenclamide (1 microM). KMCP-98 (0.03-30 microM) produced concentration-dependent relaxations in carbachol (1 microM) precontracted guinea pig tracheal smooth muscle. The trachea relaxant response of KMCP-98 was markedly inhibited by A(2), A(2a) and A(2b) adenosine receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX, 10 microM), 8-(3-chlorostyryl)caffeine (CSC, 10 microM) and alloxazine (10 microM), respectively, the nitric oxide synthase (NOS) inhibitor L-NAME (100 microM) and also by TEA and glibenclamide. In addition, KMCP-98 (0.03-30 microM) elicited relaxant response in norepinephrine (3 microM) precontracted rat thoracic aorta in a concentration-dependent manner. The thoracic aorta relaxant response of KMCP-98 was also significantly inhibited by DMPX, CSC, alloxazine, L-NAME, TEA and glibenclamide. Furthermore, the binding characteristics of KMCP-98, adenosine and 5'-N-ethylcarboxaminoadenosine (NECA) were evaluated in [(3)H]DPCPX and [(3)H]CGS 21680 binding to rat cortex and striatum, respectively. The K(i) values of KMCP-98 for predominate A(1) and A(2) adenosine receptor sites were 3908+/-952 and 158+/-10 nM, respectively. In conclusion, KMCP-98 was found to be a xanthine-based adenosine receptor agonist associated cardiac depression, tracheal and aortic smooth muscle relaxations.     

Dual effect of endothelin 1 on angiotensin II-potentiated purinergic neurotransmission in prostatic rat vas deferens

Angiotensin and endothelin are vasoactive peptides with neuromodulatory effect, however their interactions in facilitating neurotransmission are largely unknown. In the present study, effort was made to examine how endothelin 1 modulates angiotensin II-potentiated purinergic neurotransmission in prostatic rat vas deferens. Both peptides facilitated field-stimulated muscle contraction in a concentration-dependent manner with Kd values of 16.97+/-6.47 and 2.46+/-0.83 nM for angiotensin II and endothelin 1, respectively. Hill plot analysis gave Hill constants of 0.91+/-0.15 and 0.97+/-0.26 for angiotensin II and endothelin 1, respectively. Correlation analysis indicated that the extent of potentiation by angiotensin II, but not endothelin 1, was proportional to the basal field-stimulated muscle contraction. In the presence of low concentrations of endothelin 1 (< or = 3 nM), angiotensin II-potentiated field-stimulated contraction was further enhanced by endothelin. However, in the presence of high concentrations of endothelin 1 (> or = 10 nM), a much increased basal field-stimulated contraction was observed, and the addition of angiotensin II did not elicit any further enhancement in the contractile response. Intriguingly, after prolonged exposure of prostatic rat vas deferens to a high concentration of endothelin 1, the addition of angiotensin II induced a refractory response to field-stimulation. Taken together, our result indicated that endothelin 1 augmented angiotensin II-facilitated purinergic neurotransmission in prostatic rat vas deferens at low concentrations, but inhibited gradually at high concentrations.     

Losartan inhibits the angiotensin II-induced stimulation of the phosphoinositide signalling system in vascular smooth muscle cells.

2-n-Butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)bip hen yl-4-yl)methyl]imidazole, potassium salt (Losartan) (previous name, DuP 753 or MK 954) is a nonpeptide angiotensin II receptor antagonist. This study was performed to investigate the ability of Losartan to inhibit the angiotensin II-induced stimulation of the phospoinositide signalling system and the angiotensin II-induced hypertrophy in aortic vascular smooth muscle cells of normotensive Wistar-Kyoto rats. 10(-7) M Losartan abolished the angiotensin II-induced formation of inositol 1,4,5-trisphosphate in vascular smooth muscle cells. 10(-6) M Losartan completely abolished the angiotensin II-induced elevation of the intracellular free Ca2+ concentration ([Ca2+]i). 10(-6) M Losartan lacked effects on the [Arg8]vasopressin-induced elevation of [Ca2+]i. In addition, 10(-6) M completely inhibited the angiotensin II-induced stimulation of Na+/H+ exchange in the vascular smooth muscle cells. 10(-10) to 10(-6) M Losartan inhibited the angiotensin II-induced cell protein synthesis in a concentration-dependent manner, yielding to an effective concentration (ED50) of 6.2 +/- 1.8 x 10(-8) M (n = 4). Losartan did not affect the platelet-derived growth factor-BB-induced increase in cell protein. These results show that Losartan is a highly specific angiotensin II receptor antagonist which inhibits angiotensin II-induced cell growth and thus may have beneficial effects on the development and regression of vascular hypertrophy.     

Positive chronotropic and inotropic effects of angiotensin II in the dog heart

The effects of angiotensin II on sinus rate and atrial contractility were investigated in 17 isolated canine atria and 5 isolated paced ventricular preparations perfused with arterial blood conducted from a heparinized donor dog. When angiotensin II was injected into the cannulated sinus node artery, positive chronotropic responses were dose-dependently produced starting from the 0.01 microgram dose although inotropic responses to angiotensin II were not consistently induced. Angiotensin II produced a similar inotropic response pattern in the paced ventricular preparation. Moreover, when angiotensin II was given into the jugular vein of the donor dog, similar positive chronotropic and inotropic responses were also shown in the isolated atrium. Angiotensin II-induced positive chronotropic and slight inotropic effects were not influenced by treatment with the beta-adrenoceptor blocking agents, propranolol and carteolol, but significantly suppressed by saralasin which has been reported to be a competitive antagonist of angiotensin II. From these results, it is suggested that angiotensin II induced a positive chronotropic and slight positive inotropic effect via angiotensin II receptors in the dog heart.     

Differential inhibition by TAK-044 of the inotropic effects of endothelin-1 and endothelin-3

The influence of a nonselective antagonist of endothelin receptors, TAK-044 (cyclo-[d-alpha-aspartyl-3-[(4-phenylpiperazin-1-yl)carbonyl]-l-alanyl-l-alpha-aspartyl-d-2-(2-thienyl)glycyl-l-leucyl-d-tryptophyl] disodium), on the positive inotropic effect of endothelin-1 and endothelin-3 was investigated in isolated rabbit myocardium. While TAK-044 produced a concentration-dependent rightward shift of the concentration-response curve for endothelin-1 and endothelin-3, the effect of endothelin-3 was hundred times more sensitive to TAK-044 than that of endothelin-1. The combination of FR139317 ([2-(R)-[2(R)-[2(S)-[[1-(hexahydro-1H-azepinyl)]carbonyl]amino-4-methylpentanoyl]amino-3-[3-(1-methyl-1H-indolyl)]propionyl] amino-3-(2-pyridyl)propionic acid]) and BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-l-gamma-methylleucyl-d-1-methoxycarbonyltryptophanyl-d-norleucine) mimicked the inhibitory action of TAK-044 on the positive inotropic effect of endothelin-3 but enhanced the effect of endothelin-1. In a receptor-binding assay, TAK-044 was four times more potent in antagonizing the specific binding of endothelin-1 than that of endothelin-3. Endothelin-1 may activate receptor subtypes that trigger both positive and negative inotropic effects, the latter being more susceptible to the antagonistic action of TAK-044, which may explain in part the differential antagonistic action of TAK-044 on the inotropic effect of endothelin-1 and endothelin-3.     

Angiotensin II type 1 receptor-mediated increase in cytosolic Ca(2+) and proliferation in mesothelial cells

We investigated the Ca(2+) signaling pathways of the response to angiotensin II in pleural mesothelial cells and the role of these Ca(2+) signaling pathways in mesothelial cell proliferation. Rat pleural mesothelial cells were maintained in vitro, and the Ca(2+) movement to angiotensin II was evaluated using the fluorescent Ca(2+) indicator fura 2. Furthermore, proliferation of mesothelial cells was assessed using a spectrophotometric 3-(4, 5-dimethylthazol-2-yl)-2,5-diphenyl-2H-tetrasodium bromide (MTT) assay. Angiotensin II (1 pM-100 microM) induced in mesothelial cells a biphasic elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) that consisted of a transient initial component, followed by a sustained component. Neither removal of extracellular Ca(2+) nor inhibition of Ca(2+) influx by 1 microM nifedipine affected the angiotensin II-induced initial transient elevation of [Ca(2+)](i) in mesothelial cells. Nifedipine did not block angiotensin II-induced sustained elevation of [Ca(2+)](i). Angiotensin II (1 pM-100 microM) had a proliferative effect on mesothelial cells in a dose-dependent manner. Angiotensin II type 1 (AT(1)) receptor antagonist ([Sar(1), Ile(8)]angiotensin II) inhibited both angiotensin II-induced elevation of [Ca(2+)](i) and proliferation of mesothelial cells. Pertussis toxin did not affect angiotensin II-induced responses. These results suggest that angiotensin II-induced responses to mesothelial cells are extremely dependent on the angiotensin AT(1) receptor coupled with pertussis toxin-insensitive G protein.     

Insurmountable angiotensin AT1 receptor antagonists: the role of tight antagonist binding.

Angiotensin II increased the inositol phosphates production (EC50 = 3.4+/-0.7 nM) in Chinese hamster ovary (CHO) cells expressing the cloned human angiotensin AT1 receptor (CHO-AT1 cells). Coincubation with angiotensin AT1 receptor antagonists produced parallel rightward shifts of the concentration-response curve without affecting the maximal response. The potency order is 2-ethoxy-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]-1H-benz imidazoline-7-carboxylic acid (candesartan) > 2-n-butyl-4-chloro-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]i midazole-5-carboxylic acid (EXP3174) > 2-n-butyl-4-spirocyclopentane-1-[(2'-(1H-tetrazol-5-yl)biphe nyl-4-yl)methyl]2-imidazolin-5-one (irbesartan)> of 2-n-butyl-4-chloro-5-hydroxymethyl-1-(2'-(1H-tetrazol-5-yl)bipheny l-4-yl)methyl]imidazole (losartan). Additionally, preincubation with these antagonists depressed the maximal response, i.e., 95%, 70%, 30% of the control response for candesartan, EXP3174 and irbesartan and not detectable for losartan. Increasing the antagonist concentration or prolonging the preincubation time did not affect this depression. Furthermore, these values remained constant for candesartan and EXP3174, when the angiotensin II incubation time varied between 1 and 5 min. Our data indicate that antagonist-receptor complexes are divided into a fast reversible/surmountable population and a tight binding/insurmountable population at the very onset of the incubation with angiotensin II.     

L-163,491 is a partial angiotensin AT(1) receptor agonist in the hindquarters vascular bed of the cat

Responses to the nonpeptide angiotensin II agonist 5, 7-Dimethyl-2-ethyl-3-[[2'-([butyloxycarbonyl) aminosulfonyl]-5'-(3-methyoxybenzyl)-[1, 1'-biphenyl]-4-yl]methyl]-3H-imidazo[4,5-b]pyridine (L-163,491) were investigated and compared with responses to angiotensin II, angiotensin IV and norepinephrine in the hindquarters vascular bed of the cat under constant-flow conditions. Injections of L-163,491 into the hindquarter perfusion circuit caused dose-related increases in hindquarters perfusion pressure. In relative terms, angiotensin II was more potent than norepinephrine, which was more potent than angiotensin IV and L-163,491 in increasing hindlimb vascular resistance. The slope of the dose-response curve for L-163,491 was flat, while the apparent affinity of the compound for angiotensin AT(1) receptors was slightly greater than angiotensin IV. Responses to L-163,491 were inhibited by the angiotensin AT(1) receptor antagonist DuP 532 (2-propyl-4-pentafluoroethyl-1-[2'-(1H-tetrazol-5-yl)bipheny l-4-yl)me thyl]imidazole-5-carboxylic acid) and were not altered by the angiotensin AT(2) receptor antagonist PD123,319 (S(+)-1-[[4-(Dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl+ ++) -4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditribluoroacetate). However, the increase in hindlimb perfusion pressure in response to angiotensin II and angiotensin IV was significantly decreased following injection of L-163,491. These data suggest that the nonpeptide angiotensin analog L-163,491 has partial agonist activity, which is dependent on the stimulation of angiotensin AT(1) receptors in the hindquarters vascular bed of the cat.     

Pharmacological characterization of TA-0201, an endothelin receptor antagonist,with recombinant and human prostate endothelin receptors

The pharmacological profile of N-(6-(2-(5-bromopyrimidine-2-yloxy)ethoxy)-5-(4-methylphenyl)pyrimidin-4-yl)-4-(2-hydroxy-1,1-dimethylethyl) benzensulfonamide sodium salt sesquihydrate (TA-0201), a new antagonist of endothelin receptors, was examined, using human recombinant and prostate endothelin receptors. In binding experiments with [125I]endothelin-1, TA-0201 showed extremely high affinity for recombinant endothelin ET(A) receptors (pK(i)=10.7), as compared with that for recombinant endothelin ET(B) receptors (pK(i)=7.8). Endothelin ET(A) and ET(B) receptors coexisted in human prostate with different proportions (endothelin ET(A) receptor: approximately 70%), which were distinguished by TA-0201 in the same manner as with recombinant receptors. Human prostate strips contracted in response to endothelin-1 and sorafotoxin S6c, but the maximum contraction induced by endothelin-1 was approximately three times greater than that induced by sarafotoxin S6c. The response to endothelin-1, but not to sarafotoxin S6c, was inhibited by TA-0201 and cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ123) (endothelin ET(A) receptor antagonist) but not by BQ788 (endothelin ET(B) receptor antagonist). These results suggest that TA-0201 is a highly selective endothelin ET(A) receptor antagonist and will be useful for understanding the physiological and pathological roles of the endothelin ET(A) receptor in human prostate and other tissues.     

Negative inotropic effect of endothelin-1 in the mouse right ventricle

Effects of endothelin-1 on the contraction and cytosolic Ca(2+) concentrations (?Ca(2+)(i)) of the mouse right ventricle were investigated. Endothelin-1 (1-300 nM) elicited a negative inotropic effect in a concentration-dependent manner. The endothelin-1-induced negative inotropy was antagonized by a selective endothelin ET(A) receptor antagonist, BQ-123 (cyclo ?Asp-Pro-Val-Leu-Trp-; 3, 10 microM). Endothelin-1 reduced the peak amplitudes of both the ?Ca(2+)(i) transient and contraction without changing inward Ca(2+) current. The relationship between peak amplitude of ?Ca(2+)(i) and peak force generated by changing the extracellular Ca(2+) concentration (?Ca(2+)(o)) was not affected by endothelin-1. In addition, the trajectory of the ?Ca(2+)(i)-contraction phase plane diagram obtained at 2 mM ?Ca(2+)(o) in the absence of endothelin-1 was superimposable on that obtained at 4 mM ?Ca(2+)(o) in the presence of endothelin-1 (300 nM). Endothelin-1 (300 nM) translocated protein kinase C from cytosol to membrane, suggesting activation of protein kinase C. Further, a selective protein kinase C inhibitor, bisindolylmaleimide I (10 microM), inhibited the endothelin-1-induced negative inotropy. These results suggest that endothelin-1 elicits negative inotropy by reducing the amplitude of the ?Ca(2+)(i) transient without changing inward Ca(2+) current through the activation of the endothelin ET(A) receptor followed by protein kinase C activation in the mouse right ventricle.     

Effects of YM358, an angiotensin II type 1 (AT1) receptor antagonist, and enalapril on blood pressure and vasoconstriction in two renal hypertension models

The effects of the angiotensin II type 1 receptor antagonist YM358 on blood pressure were compared to those of the angiotensin converting enzyme inhibitor enalapril in one-kidney, one-clip renal hypertensive rats (1K1C RHR), two-kidney, one-clip renal hypertensive rats (2K1C RHR) and normotensive rats (NTR). Additionally, the local drug actions in peripheral tissues were investigated using isolated mesenteric arteries from these rats. In 2K1C RHR, YM358 and enalapril produced a long-lasting hypotensive effect in a dose-dependent manner. In 1K1C RHR, YM358 (30 mg/kg) also produced an antihypertensive effect, whereas enalapril (30 mg/kg) had no effect. Administration of YM358, but not enalapril, to 1K1C RHR, 2K1C RHR and NTR did not affect heart rate. In isolated mesenteric arteries from 1K1C RHR and 2K1C RHR, angiotensin II (Ang II), angiotensin I (Ang I) and tetradecapeptide (TDP), a physiologically active renin substrate, produced concentration-dependent vasoconstriction. YM358 (10(-7) M) inhibited the vasoconstricting responses to Ang II, Ang I and TDP in isolated mesenteric arteries. In contrast, enalaprilat (10(-7) M), an active metabolite of enalapril, did not completely inhibit the response to Ang I and TDP. These results indicate that YM358 has higher efficacy than enalapril for the treatment of hypertension.     

Different mechanisms involved in the positive inotropic effects of benzimidazole derivative UD-CG 115 BS (pimobendan) and its demethylated metabolite UD-CG 212 Cl in canine ventricular myocardium.

UD-CG 115 BS produced a positive inotropic effect in a concentration-dependent manner (EC50 = 9.2 x 10(-5) M, efficacy = 0.65) in isolated canine ventricular muscle. UD-CG 212 Cl also elicited a positive inotropic effect (EC50 = 1.9 x 10(-7) M, efficacy = 0.23); its potency was higher, but its efficacy was much less than that of UD-CG 115 BS. Although the effect of UD-CG 115 BS was not altered by a beta-adrenoceptor antagonist, bupranolol (3 x 10(-7) M), the response to UD-CG 212 Cl in high concentrations became transient in the presence of bupranolol: After reaching a peak, the force decreased gradually to the control level at greater than or equal to 10(-4) M. Both UD-CG 115 BS and UD-CG 212 Cl elevated the cyclic AMP level, but to a much smaller extent than other newly developed cardiotonic agents such as amrinone, milrinone, enoximone, and piroximone. Carbachol (3 x 10(-6) M) abolished the accumulation of cyclic AMP produced by these agents while it suppressed the maximum contractile response to UD-CG 115 BS by only 30%. The positive inotropic effect of UD-CG 212 Cl was converted to a negative effect by carbachol. Both UD-CG 115 BS and UD-CG 212 Cl produced a leftward shift in the concentration-response curve for the positive inotropic effect of isoproterenol. These results suggest that an elevation of cyclic AMP levels owing to cyclic AMP phosphodiesterase inhibition may be predominantly responsible for the positive inotropic effect of UD-CG 212 Cl but that a cyclic AMP-independent mechanism may contribute significantly to the positive inotropic effect of UD-CG 115 BS. UD-CG 212 Cl (greater than 3 x 10(-6) M) elicits a negative inotropic effect that is unmasked by beta-adrenoceptor blockade.     

Cardiovascular effects of L-158,809, a new angiotensin type 1 receptor antagonist,assessed using the halothane-anesthetized in vivo canine model

L-158,809 is a new angiotensin II type 1 receptor antagonist. We simultaneously assessed its antagonistic potency and cardiovascular effects with the halothane-anesthetized in vivo canine model (n = 5). L-158,809 was intravenously infused over 10 min at escalating doses of 0.03, 0.3 and 3 mg/kg. Angiotensin II (0.1 microg/kg, i.v.)-induced vasopressor and negative inotropic responses were significantly suppressed from the low dose L-158,809. Meanwhile, L-158,809 did not affect any of the cardiovascular parameters except that QTc was slightly shortened after the high dose administration. These results support the previous in vitro knowledge that L-158,809 is a highly selective angiotensin II receptor antagonist.