Hemodynamic effects of angiotensin II and the influence of angiotensin receptor antagonists in pithed rabbits

We investigated the cardiovascular effects of angiotensin II (AII) and the influences of four angiotensin receptor antagonists: losartan, PD123177, BIBS 39, and BIBS 222 in the pithed rabbit preparation. AII (0.03-10 nmol/kg) elicited a dose-dependent increase in blood pressure (BP), left ventricular pressure (LVP), LV end-diastolic pressure (LVEDP), dP/dtmax, and heart rate (HR). The maximal hypertensive effect of AII is comparable to that of norepinephrine (NE), but its effects on LVEDP and HR are weaker than those of NE. On a molar base, AII is approximately 27 times more potent than NE. Propranolol (0.5 mg/kg i.v.) did not significantly influence the AII-induced increase in diastolic BP (DBP) and LVEDP, but it abolished AII-induced positive chronotropic effects over the entire dose range of angiotensin AII studied. Losartan, but not PD123177, shifted the dose-response curves for AII to the right in a parallel manner. BIBS 39 and BIBS 222 also caused rightward shifts of the AII dose-response curve. These experiments indicate that in propranolol-treated pithed rabbits AII causes vasoconstrictor effects in both resistance vessels and in the venous system, which are both mediated by AT1- but not by AT2-receptors. The AII-induced positive chronotropic effect is an indirect action mediated by the stimulation of postsynaptic beta 1-adrenoceptors. BIBS 39 and BIBS 222, two new nonpeptide angiotensin receptor blockers that have affinity for both AT1- and AT2-receptors are also potent antagonists of the cardiovascular effects of AII in pithed rabbits.     

Reciprocal modulation of the binding of angiotensin agonists and antagonists to angiotensin receptors in smooth muscle

1. Direct ligand binding studies have shown that the agonist 125I-[Sar1]Ang II and the antagonist 125I-[Sar1Ile8]Ang II bind to bovine uterus smooth muscle membranes in a time-dependent, reversible and saturable manner; both ligands had the same number of high affinity sites. 2. [Sar1Ile8]Ang II inhibited the binding of 125I-[Sar1]Ang II in a non-competitive manner by decreasing the number of high affinity sites without changing the binding affinity of the radioligand. 3. [Sar1]Ang II also inhibited the binding of 125I-[Sar1Ile8]Ang II in a non-competitive manner. 4. Dissociation of both radioligands from their receptor sites was fast enough that pseudo irreversible occupancy of the binding sites could not account for the observed non-competitive inhibition. 5. Displacement studies using 125I-[Sar1Ile8]Ang II as the radioligand provided evidence for the existence of two binding sites when the displacing ligand was [Sar1]Ang II but not when the displacing ligand was [Sar1Ile8]Ang II. 6. GTPS gamma S had no discernible effect on the binding of either 125I-[Sar1]Ang II or 125I-[Sar1Ile8]Ang II to bovine uterine membranes. 7. The present findings are consistent with an allosteric mechanism of antagonism for [Sar1Ile8]Ang II. The data are also consistent with a mechanism wherein agonist and antagonist ligands occupy different binding modes at the same receptor site and induce long-term conformational changes in the receptor which are idiosyncratic with respect to the nature of the ligand. An emerging relationship between the actions of angiotensin peptides and non-peptide mimetics of angiotensin is presented.     

In vivo pharmacology of an angiotensin AT1 receptor antagonist with balanced affinity for AT2 receptors

L-163,017 (6-[benzoylamino]-7-methyl-2-propyl-3-[[2'-(N-(3-methyl-1-butoxy) carbonylaminosulfonyl)[1,1']-biphenyl-4-yl]methyl]-3H-imidazo[4,5- b]pyridine) is a potent, orally active, nonpeptide angiotensin II receptor antagonist. Conscious rats and dogs were dosed p.o. and i.v.; in both species the plasma bioequivalents are similar at the angiotensin AT1 and AT2 receptor sites indicating balanced activity is maintained in vivo. L-163,017 prevents the pressor response to intravenous (i.v.) angiotensin II in the conscious rat, dog, and rhesus monkey. L-163,017 also significantly reduces blood pressure in a renin-dependent model of hypertension, similar to an angiotensin converting enzyme inhibitor (Enalapril) and an angiotensin AT1 receptor-selective antagonist (L-159,282). These studies indicate that neither the angiotensin AT2 receptor nor bradykinin is important in the acute antihypertensive activity of angiotensin converting enzyme inhibitors or angiotensin II receptor antagonists.     

The role of angiotensin AT1 receptors in the diuretic, natriuretic, kaliuretic and blood pressure responses induced by angiotensin II activation of the median preoptic nucleus in conscious rats

We determined the effects of two classical angiotensin II (ANG II) antagonists, [Sar1, Ala8]-ANG II and [Sar1, Thr8]-ANG II, and losartan (a nonpeptide and selective antagonist for the AT1 angiotensin receptors) on diuresis, natriuresis, kaliuresis and arterial blood pressure induced by ANG II administration into the median preoptic nucleus (MnPO) of male Holtzman rats weighing 250-300 g. Urine was collected in rats submitted to a water load (5% body weight) 1 h later. The volume of the drug solutions injected was 0.5 microliters over 10-15 s. Pre-treatment with [Sar1, Ala8]-ANG II (12 rats) and [Sar1, Thr8]-ANG II (9 rats), at the dose of 60 ng reduced (13.7 +/- 1.0 vs 11.0 +/0 1.0 and 10.7 +/0 1.2, respectively), whereas losartan (14 rats) at the dose of 160 ng totally blocked (13.7 +/- 1.0 vs 7.6 +/- 1.5) the urine excretion induced by injection o 12 ng of ANG II (14 rats). [Sar1, Ala8]-ANG II impaired Na+ excretion (193 +/- 16 vs 120 +/- 19), whereas [Sar1, Thr8]-ANG II and losartan block Na+ excretion (193 +/- 16 vs 77 +/- 15 and 100 +/- 12, respectively) induced by ANG II. Similar effects induced by ANG II on K+ excretion were observed with [Sar1, Ala8]-ANG II, [Sar1, Thr8]- ANG II, and losartan pretreatment (133 +/- 18 vs 108 +/- 11, 80 +/- 12, and 82 +/- 15, respectively). The same doses as above of [Sar1, Ala8]-ANG II (8 rats), [Sar1, Thr8]-ANG II (8 rats), and losartan (9 rats) blocked the increase in the arterial blood pressure induced by 12 ng of ANG II (12 rats) (32 +/- 4 vs 4 +/- 2, 3.5 +/- 1, and 2 +/- 1, respectively. The results indicate that the AT1 receptor subtype participates in the increases of diuresis, natriuresis, kaliuresis and arterial blood pressure induced by the administration of ANG II into the MnPO.     

Angiotensin antagonists and the adrenal cortex and medulla

Several analogs of angiotensin in which the phenylalanine in position 8 of the peptide chain was replaced by an aliphatic amino acid residue are specific antagonists of angiotensin in aorta, the adrenal medulla, and adrenal zona glomerulosa. In the adrenal cortex and medulla, all actapeptide analogs have more agonist activity than in aortic strips. In studies with N-terminally substituted analogs, it appears that adrenal degradation of the angiotensin molecule by aminopeptidase(s) does not occur or is not retarded by N-terminal mocifications such as sarcosine substitution. The decapeptide analog [Ile8]-angiotensin I and heptapeptide analog [des-Asp1, Ile8]-angiotensin II were excellent antagonists in the adrenal medulla and each peptide was devoid of intrinsic activity. These substituted homologs of angiotensin may offer a novel approach for the development of selective antagonists of angiotensin receptors. In the adrenal cotex, [des-Asp1, Ile8]-heptapeptide possessed greater receptor affinity than any of the angiotensin octapeptides studied. This C-terminally substituted heptapeptide does have significant intrinsic activity in the adrenal cortex which would limit the use of this compound as an antagonist of vascular responses to angiotensin II. In studies with [Ile8]-angiotensin II, [Sar1, Ile8]-angiotensin II, and [des-Asp1, Ile8]-angiotensin II, the pA2 values calculated indicate that the N-terminal residue is not important for receptor binding in the adrenal cortex but may be of significance in binding to adrenal medullary and aortic smooth muscle receptors. At the present time it appears unlikely that any single animal model or assay system can reliably predict the agoinst/antagonist activities of angiotensin analogs for all the various end organs which respond to the angiotensins.     

Structural evolution and pharmacology of a novel series of triacid angiotensin II receptor antagonists

cis-4-(4-Phenoxy)-1-[1-oxo-2(R)-[4-[(2-sulfobenzoyl)amino)-1H- imidazol-1-yl]octyl]-L-proline derivatives represent a novel class of potent nonpeptide angiotensin II (Ang II) receptor antagonists. These compounds evolved from directed structure-activity relationship (SAR) studies on a lead identified by random screening. Further SAR studies revealed that acidic modification of the 4-phenoxy ring system produced a series of triacid derivatives possessing oral activity in pithed rats. The most potent compound, cis-4-[4-(phosphonomethyl)phenoxy]-1-[1-oxo-2(R)-[4-[(2-sulfobenzoyl+ ++) amino]-1H-imidazol-1-yl]octyl]-L-proline (1e), inhibited the pressor response to exogenously administered Ang II for periods up to 8 h following oral dosing. The antihypertensive activity of 1e was evaluated in the Lasix-pretreated conscious spontaneously hypertensive rat (SHR) where it produced a dose-dependent fall in blood pressure following oral dosing lasting > 12 h. Antagonists such as 1e may serve as useful therapeutic agents for the treatment of hypertension as well as for studying the role of Ang II in various disease states.     

Comparison of inversion-recovery gradient- and spin-echo and fast spin-echo techniques in the detection and characterization of liver lesions

For several G protein-coupled receptors, amino acids in the seventh transmembrane helix have been implicated in ligand binding and receptor activation. The function of this region in the AT1 angiotensin receptor was further investigated by mutation of two conserved polar residues (Asn294 and Asn295) and the adjacent Phe293 residue. Analysis of the properties of the mutant receptors expressed in COS-7 cells revealed that alanine replacement of Phe293 had no major effect on AT1 receptor function. Substitution of the adjacent Asn294 residue with alanine (N294A) reduced receptor binding affinities for angiotensin II, two nonpeptide agonists (L-162,313 and L-163,491), and the AT1-selective nonpeptide antagonist losartan but not that for the peptide antagonist [Sar1, Ile8]angiotensin II. The N294A receptor also showed impaired G protein coupling and severely attenuated inositol phosphate generation. In contrast, alanine replacement of Asn295 decreased receptor binding affinities for all angiotensin II ligands but did not impair signal transduction. Additional substitutions of Asn295 with a variety of amino acids did not identify specific structural elements for ligand binding. These findings indicate that Asn295 is required for the integrity of the intramembrane binding pocket of the AT1a receptor but is not essential for signal generation. They also demonstrate the importance of transmembrane helices in the formation of the binding site for nonpeptide AT1 receptor agonists. We conclude that the Asn294 residue of the AT1 receptor is an essential determinant of receptor activation and that the adjacent Asn295 residue is required for normal ligand binding.     

Spinal muscarinic, glutamatergic and GABAergic receptor systems in cardiovascular regulation

The central administration of cholinergic agonists can produce a significant increase in arterial blood pressure by enhancing sympathetic vasomotor tone. The stimulation of spinal muscarinic receptors through intrathecal (i.t.) injection of carbachol in rats evoked a significant pressor response that returned to preinjection levels within 30 to 40 min. We investigated the roles of glutamatergic and GABAergic receptors in mediating the hypertensive response to i.t. injection of the muscarinic receptor agonist carbachol and in the maintenance of resting blood pressure and heart rate. The i.t. pretreatment with the N-methyl-D-aspartate (NMDA) receptor antagonists D-AP7 or MK801 maleate (dizocilipine) attenuated the pressor response to i.t. administration carbachol in a dose-dependent manner in conscious, freely moving rats. In contrast, i.t. pretreatment with 6-cyano-7-nitroquinoxaline-2,3-dione, a non-NMDA glutamate receptor antagonist, was not effective in this regard, indicating that the carbachol-evoked pressor response was not mediated through the quisqualate/kainate subtype of glutamate receptors. The i.t. pretreatment with the gamma-aminobutyric acid type B receptor agonist baclofen also inhibited the pressor response to i.t. injection of carbachol at doses that did not alter motor function. To determine whether the pressor response to stimulation of spinal muscarinic receptors required the participation of higher centers, rats received an intracisternal injection of either methylatropine or D-AP7 before the i.t. injection of carbachol. Both intracisternal pretreatments significantly reduced the expression of the pressor response to i.t. injection of carbachol. These findings are consistent with the presence of a powerful modulating spinobulbar muscarinic pressor system. Pharmacological activation of this system involves the participation of spinal and perhaps medullary glutamate-NMDA and gamma-aminobutyric acid type B receptor systems.     

Sympathoexcitation from the rostral ventrolateral medulla is mediated by spinal NMDA receptors

These studies examined the role of spinal N-methyl-D-aspartic acid (NMDA) receptors in mediating sympathoexcitation evoked by stimulation of neurons in the rostral ventrolateral medulla (RVLM). In urethane-anesthetized rats, blood pressure, heart rate, and splanchnic sympathetic nerve activity (SNA) were recorded. The NMDA receptor antagonist D-2-amino-7-phosphonoheptanoic acid (D-AP7) was administered to the spinal cord via intrathecal (IT) catheter. Blockade of spinal NMDA receptors reduced arterial blood pressure, heart rate, and SNA. Spinal administration of D-AP7 markedly attenuated the pressor and sympathoexcitatory responses evoked by L-glutamate stimulation of the RVLM. The small increases in heart rate evoked by stimulation of the RVLM were not affected by IT administration of D-AP7. These results indicate that NMDA receptors in the spinal cord mediate the pressor and sympathoexcitatory responses evoked by activation of a bulbospinal pathway originating from the RVLM. Moreover, these data suggest that excitatory amino acid neurotransmitters and NMDA receptors in the spinal cord play an important role in the maintenance and regulation of SNA and cardiovascular function.     

Cardiovascular effects of oxymetazoline and UK14,304 in conscious and pithed rats

Relatively selective alpha 2-adrenoceptor agonists have proven useful in a variety of therapeutic situations including hypertension, glaucoma and withdrawal from opiate addiction. In particular, oxymetazoline (OXY) and UK14,304 (UK) have been used in subclassifying alpha 2-adrenoceptors and imidazoline receptors. We evaluated the cardiovascular effects of OXY and UK in conscious and pithed rats in the presence and absence of efaroxan (EFA), idazoxan (IDA) and rauwolscine (RAU). Both OXY or UK (1, 5 and 10 micrograms/kg, i.v.) increased blood pressure (BP) and reduced heart rate (HR) in conscious rats. In pithed rats, OXY and UK each increased BP to a greater extent than that observed in conscious rats, but HR was not affected. BP increases following sympathetic nerve stimulation in the pithed rats were not affected by OXY but were reduced by UK at 0.1 Hz and 0.3 Hz. HR responses to nerve stimulation in pithed rats were reduced after OXY at all frequencies, but only at 0.1 Hz following UK. EFA, IDA and RAU inhibited the pressor responses of UK, with EFA being most potent. OXY-induced pressor responses were inhibited by all three antagonists, RAU being the least potent. HR responses to either OXY or UK were not affected by the antagonists. Taken together, the data suggest that: 1) alpha 2-adrenoceptors contribute less to the vascular response to OXY than to UK based upon the antagonistic effect of RAU; 2) prejunctional I1 receptors maybe more prevalent in the heart than in vascular tissue based upon the response to OXY in pithed rats. Thus, the heterogeneity among receptors mediating cardiac and vascular responses are complex.     

Cardiovascular effects produced by microinjection of angiotensins and angiotensin antagonists into the ventrolateral medulla of freely moving rats

In this study we determined the cardiovascular effects produced by microinjection of angiotensin peptides [Angiotensin-(1-7) and Angiotensin II] and angiotensin antagonists (losartan, L-158,809, CGP 42112A. Sar1-Thr8-Ang II, A-779) into the rostral ventrolateral medulla of freely moving rats. Microinjection of angiotensins (12.5-50 pmol) produced pressor responses associated to variable changes in heart rate, usually tachycardia. Unexpectedly, microinjection of both AT1 and AT2 ligands produced pressor effects at doses that did not change blood pressure in anesthetized rats. Conversely, microinjection of Sar1-Thr8-Ang II and the selective Ang-(1-7) antagonist, A-779, produced a small but significant decrease in MAP an HR. These findings suggest that angiotensins can influence the tonic activity of vasomotor neurons at the RVLM. As previously observed in anesthetized rats, our results further suggest a role for endogenous Ang-(1-7) at the RVLM. The pressor activity of the ligands for AT1 and AT2 angiotensin receptor subtypes at the RVLM, remains to be clarified.     

Eprosartan

Eprosartan is a nonpeptide angiotensin II receptor antagonist which has a high affinity for the AT1 receptor subtype. When administered at dosages of 400 to 800 mg/day (once or twice daily) for 13 weeks to patients with mild to moderate essential hypertension, eprosartan significantly reduced blood pressure compared with placebo. Eprosartan was at least as effective as enalapril 10 to 40 mg/day in a dose-titration study in patients with severe hypertension. Eprosartan is generally well tolerated; clinical trials have shown the drug to have a tolerability profile similar to that of placebo. As with other angiotensin II receptor antagonists, it does not cause cough. Eprosartan is not metabolised by the cytochrome P450 system and therefore has a low potential for drug interactions.     

AT1 receptor subtype mediates the inhibitory effect of central angiotensin II on cerebrospinal fluid formation in the rat

The effect of central administration of angiotensin II (AII) on cerebrospinal fluid (CSF) formation was studied in pentobarbital-anesthetized, artificially-ventilated rats. CSF production was measured by the ventriculocisternal perfusion method with Blue Dextran 2000 as the indicator. Baseline value of CSF production was 3.35 +/- 0.08 microliters/min. Intracerebroventricular (i.c.v.) infusion of AII at rates of 0.5 and 5 pg/min significantly lowered (P < 0.01) CSF formation by 23% and 16%, respectively. In comparison, high peptide doses (50 and 500 pg/min) did not alter this parameter. The inhibitory effect of low AII doses on CSF formation was blocked by the i.c.v. AT1 receptor subtype antagonists, losartan and SK&F 108566 (2.4 and 2.7 ng/min, respectively), but not by the AT2 receptor subtype-specific agent, PD 123319 (3.8 ng/min). Peptide AII antagonists, [Sar1,Ile8]AII (5 ng/min), which binds to both AT1 and AT2 receptors, had a similar effect to those of AT1-specific blockers. It is concluded that AII, by controlling CSF formation, may influence the water and electrolyte balance in the brain.     

Vascular and excretory effects of angiotensin II in the rat isolated perfused kidney: influence of an AT1 and a nonselective AT receptor antagonist

Angiotensin II (AII) is a potent vasoconstrictor which, at physiological plasma concentrations, produces antinatriuresis, whereas high intrarenal concentrations cause natriuresis and diuresis. We examined the effects of a selective AT1 receptor antagonist, losartan, and a nonselective AT receptor antagonist, Sar1Thr8AII, on the response to infusion of AII in the isolated rat kidney perfused at constant pressure with a recirculating modified Krebs-Henseleit buffer. AII increased renal vascular resistance (RVR), glomerular filtration rate (GFR) and urinary volume (UV) and sodium excretion (UNaV) without changing the fractional excretion of water or electrolytes. Thus, changes in GFR can account for the natriuresis/diuresis. Both AII receptor antagonists prevented the increase in RVR. However, losartan was without effect on angiotensin-induced increases in GFR, UV or UNaV, whereas Sar1Thr8 AII also prevented the increases in GFR, UV and UNaV. The angiotensin receptor mediating the increase in GFR can be dissociated from that mediating the increase in RVR, providing functional evidence of angiotensin receptor subtypes in the rat kidney.     

Principal modifications of the Duhamel procedure in the treatment of Hirschsprung''s disease. Analysis based on results of an international retrospective study of 2,430 patients

The present study was undertaken to investigate the effects of losartan, a non-peptide angiotensin II subtype 1 (AT1) receptor antagonist, on both the pressor responses elicited by stimulation of afferent vagal nociceptive fibres and the involvement of the sympathetic nervous system (evaluated by plasma levels of noradrenaline and its co-neurotransmitter neuropeptide Y) in dogs. Electrical stimulation of the afferent fibres of the vagus (1, 5, 10 and 20 Hz) elicited a frequency-dependent increase in blood pressure and heart rate. Plasma noradrenaline levels only increased after stimulation at frequencies of 10 and 20 Hz. Plasma neuropeptide Y levels did not change. Losartan (10 mg/kg i.v.) induced both a decrease in resting blood pressure and an increase in basal plasma levels of noradrenaline and neuropeptide Y. Losartan failed to modify the magnitude of the electrically-evoked pressor and positive chronotropic responses. The angiotensin AT1 receptor antagonist elicited a fall in plasma noradrenaline values after a 1 Hz stimulation and abolished the increase in plasma noradrenaline levels induced by the 10 (but not 20) Hz stimulation. The data suggest that angiotensin AT1 receptors are not directly involved in acute pressor responses induced by stimulation of afferent vagal fibres. Moreover, the results show that, besides its sympatho-inhibitory effect, losartan can exert a sympatho-excitatory action as shown by the increase in the plasma levels of both noradrenaline and its coneurotransmitter, neuropeptide Y.     

Synthesis and angiotensin II receptor antagonistic activities of benzimidazole derivatives bearing acidic heterocycles as novel tetrazole bioisosteres

The design, synthesis, and biological activity of benzimidazole-7-carboxylic acids bearing 5-oxo-1,2,4-oxadiazole, 5-oxo-1,2,4-thiadiazole, 5-thioxo-1,2,4-oxadiazole, and 2-oxo-1,2,3,5-oxathiadiazole rings are described. These compounds were efficiently prepared from the key intermediates, the amidoximes 4. The synthesized compounds were evaluated for in vitro and in vivo angiotensin II (AII) receptor antagonistic activities. Most were found to have high affinity for the AT1 receptor (IC50 value, 10(-6)-10(-7)M) and to inhibit the AII-induced pressor response (more than 50% inhibition at 1 mg/kg po). The 5-oxo-1,2,4-oxadiazole, 5-oxo-1,2,4-thiadiazole, and 5-thioxo-1,2,4-oxadiazole derivatives showed stronger inhibitory effects than the corresponding tetrazole derivatives, while their binding affinities were weaker. This might be ascribed to their improved bioavailability by increased lipophilicity. The 5-oxo-1,2,4-oxadiazole derivative 2 (TAK-536) and 5-oxo-1,2,4-thiadiazole derivative 8f showed efficient oral bioavailability without prodrug formation. This study showed that the 5-oxo-1,2,4-oxadiazole ring and its thio analog, the 5-oxo-1,2,4-thiadiazole ring, could be lipophilic bioisosteres for the tetrazole ring in nonpeptide AII receptor antagonists.     

Localization of AT2 angiotensin II receptor gene expression in rat brain by in situ hybridization histochemistry

To localize the gene expression of AT2 angiotensin II receptors in rat brain we performed in situ hybridization histochemistry using 35S-labeled antisense riboprobes. The AT2 receptor mRNA expression pattern was compared in consecutive brain sections, from 2 week old rats, with the receptor expression by means of [125I]Sar1-ANG II binding and displacement with AT2 selective ligands followed by autoradiography. Expression of AT2 receptor mRNA was found in several thalamic nuclei (ventral posterolateral, mediodorsal, central medial, paracentral, and paraventricular), the medial geniculate nuclei, the nucleus of the optic tract, the subthalamic nucleus, the interposed nucleus of the cerebellum, and in the inferior olive. In these areas the AT2 receptor gene expression corresponds well with [125I]Sar1-ANG II binding. In addition, AT2 receptor mRNA expression was found in the red nucleus where no [125I]Sar1-ANG II binding was present. No significant hybridization of the AT2 receptor antisense probe was found in septal nuclei, the locus coeruleus, the dorsolateral geniculate nucleus, or the cerebellar cortex, areas rich in [125I]Sar1-ANG II binding. Our results indicate that some brain regions may be involved in AT2 receptor formation, transporting the receptor protein to other brain areas. However, in most structures, both the formation and expression of receptors occur, suggesting the existence of local AT2 receptor circuits, or that of AT2 autoreceptors. Other structures express only the receptor protein, indicating that these AT2 receptors are produced elsewhere. Our present data are the basis for further studies on the clarification of AT2 receptor pathways in the brain.     

Differential effect of dithiothreitol and DuP 753 on angiotensin II receptor subtypes in bovine cerebellar cortex

Angiotensin II (AII) receptor subtypes were investigated in bovine cerebellar cortical membranes (BCCM) using agonists and antagonists. Differences were observed whether the tissue for the characterization was used fresh or frozen. In fresh BCCM, AII and angiotensin III (AIII) (not selective), p -aminophenylalanine6 -AII ([pNH2Phe6]AII) (AT2 selective), and DuP 753 (AT1 selective) interacted with two sites, at high and low affinity. In the presence of 10 mM dithiothreitol (DTT), the low-affinity site for AIII and [pNH2Phe6]AII and the high-affinity site for DuP 753 were no longer detectable. The same effect was obtained with 1 microM DuP 753, with only the low-affinity component of [pNH2Phe6]AII being resistant to this blocker. In frozen BCCM, DuP 753, PD 123319 (AT2 selective), and sarcosine1, isoleucine8-AII ([Sar1Ile8] AII) yielded monophasic curves. These data confirm the presence of multiple receptors (AT1 and AT2) for AII on BCCM and suggest the possibility that within the AT2 receptors, one subtype, DTT sensitive, may exist. Alternatively, a heterogeneity in the AT1 class, with respect to the sensitivity to DuP753, may be considered.     

Phenytoin blocks the reversal of a classically conditioned discriminative eyeblink response in rabbits

The aim of the present study was to assess the role of vascular alpha 1D-adrenoceptors in the sympathetic vasopressor response in vivo. Specifically, we evaluated the effect of a selective alpha 1D-adrenoceptor antagonist, BMY 7378 (8-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-8-azaspiro(4,5)dec ane-7,9- dione 2HCl), on the vasopressor response induced by preganglionic (T7-T9) sympathetic stimulation in the pithed rat. The vasopressor response was dose-dependently sensitive to inhibition by intravenous BMY 7378 (0.1, 0.31, 1 and 3.1 mg/kg), doses of 1 and 3.1 mg/kg being equally effective. Like BMY 7378, 5-methylurapidil (0.1, 0.31, 1 and 3.1 mg/kg) antagonized the vasopressor response to spinal stimulation; doses of 1 and 3.1 mg/kg were also equally effective. In combination experiments, BMY 7378 (1 mg/kg, i.v.) and the alpha 1A-adrenoceptor antagonist, 5-methylurapidil (1 mg/kg, i.v.), showed an additive effect. The present results demonstrate that the alpha 1D-adrenoceptor subtype plays an important role in the pressor response to sympathetic nerve stimulation in the pithed rat, and confirm the participation of the alpha 1A-adrenoceptor subtype in the same response.