Selective AT1 Receptor Research Articles

Pharmacological characterization of the contractile responses to angiotensin analogues in guinea‐pig isolated longitudinal muscle of small intestine

1. The contractile responses to angiotensin II, angiotensin III and two synthetic analogues, [Lys2]angiotensin II and [Sar1]angiotensin II, in the guinea-pig isolated longitudinal muscle preparation of small intestine have been characterized in vitro. 2. Tachyphylaxis to the angiotensin analogues was reduced by use of a Krebs-Henseleit solution containing a raised (sub-contractile) concentration of potassium (11.2 mM). Under these conditions. reproducible cumulative concentration-response curves to all agonists were established. The pD2 estimates for angiotensin II, [Lys2]angiotensin II, angiotensin III and [Sar1]angiotensin II were 9.15 +/- 0.14, 7.42 +/- 0.06, 7.69 +/- 0.18 and 9.50 +/- 0.15 respectively and the maximum responses achieved were not significantly different. 3. The contractile responses to angiotensin II, angiotensin III and [Sar1]angiotensin II were reduced by greater than 80% by tetrodotoxin (TTX; 0.1 microM). However, the responses to [Lys2]angiotensin II were reduced by only 63 +/- 5%. Atropine (0.1 microM) also reduced the responses to angiotensin II, angiotensin III and [Lys2]angiotensin II, although its effect was less than that produced by TTX. Furthermore, while responses to these agonists were not significantly modified by the NK1 receptor antagonist (+/-)-CP-96,345 (30 nM) alone, the combined pre-incubation with both atropine and (+/-)-CP-96,345 reduced maximum agonist responses to a level not significantly different from those produced by TTX. 4. Indirect and direct contractile responses to angiotensin II and [Lys2]angiotensin II (in the presence of TTX) respectively were characterized by use of the selective AT1 receptor antagonist, losartan and the AT2 receptor antagonist, PD123177. Losartan produced parallel rightward displacement of the concentration-response curve to angiotensin II and [Lys2]angiotensin II, with an estimated pKB of 8.56(8.42-8.68) and 9.18 (8.63-9.50) respectively. The AT2 receptor antagonist, PD123177 (3 microM) failed to modify the contractile responses to either angiotensin II or [Lys2]angiotensin II.5. We conclude that two populations of angiotensin II receptors exist in the guinea-pig longitudinal muscle of small intestine, one located neuronally mediating the release of both acetylcholine and substance P and the other located on the smooth muscle mediating direct contractile responses. The neuronal component provides the major contribution to the agonist responses. Both receptor populations are of the AT1 receptor subtype.

Characterization of the angiotensin II-receptor subtype in the longitudinal smooth muscle of the rat portal vein.

The purpose of the present study was to identify the angiotensin II-receptor subtype involved in the enhancement of the amplitude of the phasic contractions by angiotensin II in the isolated rat portal vein preparation. At an extracellular Ca2+ concentration of 0.9 mmol/l and a K+ concentration of 4 mmol/l, angiotensin II induced concentration-dependent increases in the amplitude of the phasic contractions. The enhancement of phasic contraction amplitude caused by angiotensin II was not significantly altered by pretreatment of the rat portal vein with indomethacin 10(-5) mol/l or nitro-L-arginine 10(-4) mol/l, indicating that neither prostaglandins nor the endothelium derived-relaxing factor (NO) are involved. Losartan (DuP 753), a nonpeptide selective AT1-receptor antagonist, concentration-dependently shifted the concentration-response curve for the effect of angiotensin II on the amplitude of the contractions to the right, without reducing the maximal response (pA2 = 8.6, slope = 0.98), thus suggesting competitive antagonism at the level of AT1-receptors. By contrast, PD 123,177, a nonpeptide selective AT2-receptor antagonist, even at 10(-5) mol/l, caused no significant change of the phasic myogenic response to angiotensin II, indicating the absence of AT2-receptor involvement. Dithiothreitol, a disulfide-reducing agent which is known to inactivate AT1-receptors in various tissues, markedly inhibited (3 mmol/l) or even abolished (5 mmol/l) the contractile response of the rat portal vein to angiotensin II, supporting the conclusion that these receptors can be classified as AT1-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

P-317 - Pharmacological properties of YM358, a novel, selective and competitive AT1 receptor antagonist.

P-317 - Pharmacological properties of YM358, a novel, selective and competitive AT1 receptor antagonist.

Effects of alprazolam and triazolam on the synaptosomal calcium levels and glutamate release

Contribution of renal angiotensin II type I receptor to gene expressions in hypertension-induced renal injury. Recent evidence indicates that transforming growth factor-β1 (TGF-β1) plays an important role in renal fibrosis via stimulation of extracellular matrix synthesis. The present study was undertaken to investigate the role of angiotensin II type I receptor (AT1 receptor) in hypertension-induced renal injury. Twenty-two-week-old stroke-prone spontaneously hypertensive rats (SHRSP), which had established hypertension and moderate renal damage, were orally given TCV-116, a selective non-peptide AT1 receptor antagonist (0.1, 1 or 10 mg/kg/day), enalapril (10 mg/kg/day) or vehicle once a day for 10 weeks. At the end point of the treatment, we examined renal function, the gene expressions of TGF-β1 and extracellular matrix components in the interstitium [collagen types I (COI) and III (COIII), fibronectin (FN)] and the basement membrane (COIV and laminin), and renal microscopic morphology in rats aged 32 weeks. In vehicle-treated 32 week-old SHRSP with renal dysfunction and nephrosclerosis, renal mRNA levels for TGF-β1, COI, COIII, FN, COIV were all several-fold higher than in WKY. Thus, renal TGF-β1 gene expression was enhanced in SHRSP, which may contribute to the increased renal expressions of COI, COIII, FN, COIV in SHRSP. Treatment with TCV-116 (0.1 mg/kg/day) in SHRSP, in spite of no reduction of blood pressure, decreased renal mRNA levels for TGF-β1, COI, COIII, FN, COIV, being accompanied by the significant decrease in urinary protein and albumin excretion, blood urea nitrogen and plasma creatinine. Treatment with TCV-116 (10 mg/kg/day) in SHRSP decreased mRNAs for TGF-β1, COI, COIII, FN and COIV to almost the same levels as WKY, being associated with normalization of urinary protein and albumin excretion and the prevention of nephrosclerosis, as judged by microscopic histological observations. On the other hand, the effects of enalapril (10 mg/kg/day) on the above mentioned mRNA levels, renal function and renal morphology were weaker than those of TCV-116 (10 mg/kg/day) and were as much as TCV-116 (1 mg/kg/day). These results suggest that independently of hypotensive action, AT1 receptor antagonist has a potent renal protective effect by inhibiting the gene expression of renal TGF-β1 and extracellular matrix components.

The angiotensin hexapeptide 3–8 fragment potently inhibits [ 125I]angiotensin II binding to non-AT 1 or -AT 2 recognition sites in bovine adrenal cortex

In the present studies, ligand competition experiments were conducted to examine the ability of angiostensin II peptide agonists and nonpeptide AT 1- and AT 2-selective receptor antagonists to inhibit the binding of [ 125I]angiostensin II to bovine adrenal cortical membranes. Angiostensin II, angiostensin III, the All-(3–8) hexapeptide fragment of angiotensin II, and the AT 1-selective receptor antagonist L-158,809, inhibited [ 125I]angiostensin II binding in a biphasic fashion indicative of a ligand interaction at more than one recognition site. Approximately 20% of low affinity [ 125I]angiostensin II binding was inhibited only by high micromolar concentrations of L-158,809. RG 13647 (1(-1,4-benzodioxan-2-methyl)-5-diphenylacetyl-4,5,6,7-tetrahydro-1H-imidazo-[4,5,c]- pyridine-6-carboxylic acid) represents a potent and AT 2-selective analog of PD 123177 and showed weak activity in competing for [ 125I]angiostensin II binding with an IC 50 value of 100 μM. When subsequent competition studies were conducted in the presence of 1 μM L-158,809 to block [ 125I]angiotensin II to the AT 1 receptor subtype, the angiotensin II agonists produced monophasic inhibition curves with AII-(3–8) showing the greatest activity (IC 50 = 6 nM) followed by angiotensin III (IC 50 = 15 nM) ⪢ angiotensin II (IC 50 = 110 nM). RG 13647 was not found to significantly inhibit this portion of [ 125I]angiotensin II binding. These data demonstrate that bovine adrenal cortex contains both the AT 1 receptor subtype, as well as, a novel class of [ 125I]angiotensin II recognition sites which may be analogous to the recently described angiotensin IV (AT 4) receptor.

Modulation of net outward current in cultured neurons by angiotensin II: involvement of AT 1 and AT 2 receptors

In this study we have used whole-cell, voltage-clamp procedures to determine the effects of angiotensin II (AII) on net outward current (I no) in neurons co-cultured from the hypothalamus and brainstem of 1-day-old rats. I no is the sum of all inward and outward membrane currents (minus Na +, which is blocked by tetrodotoxin) which occur during the repolarization phase of the action potential. We have determined that AII elicits two separate effects on I no in cultured neurons. AII caused a reversible and concentration (0.1 nM–10 μM)-dependent increase in I no. This effect is inhibited by the AT 2 receptor-selective antagonists, PD123177 and PD123319 (both 100 nM), but not by the AT 1-selective receptor blocker, DuP753 (Losartan; 100 nM), and so it is mediated by AT 2 receptors. In a smaller number of neurons AII induced a reversible and concentration (0.01 nM–10 μM)-dependent decrease in I no that was blocked by Losartan (100 nM) but not by PD123177 (100 nM). Thus the decrease in I no is mediated by AT 1 receptors. Additionally, some neurons displayed both AT 1- and AT 2 receptor-mediated effects on I no. Our results demonstrate two distinct actions of AII on membrane ionic currents in cultured neurons, effects that are mediated by different AII receptor subtypes.

Effects of a non-peptide angiotensin receptor antagonist on drinking and blood pressure responses to centrally administered angiotensins in the rat

Both angiotensin II (ANG II) and angiotensin III (ANG III) administered centrally produce drinking and increases in blood pressure. The recent characterization of two subtypes for the ANG II receptor, the AT1 and the AT2, raises the questions of whether drinking and pressor responses to ANG II can be separated pharmacologically and whether ANG III acts via the same receptor subtype. Therefore, the current study examined drinking and blood pressure responses to ANG II and ANG III administered centrally in adult male Sprague-Dawley rats in the presence or absence of a selective AT1 receptor antagonist. Blockade of the AT1 receptor abolished both drinking and pressor responses to ANG II and ANG III. However, drinking to the cholinergic agonist, carbachol, was unaffected. These results demonstrate that centrally administered ANG II and ANG III increase both water intake and blood pressure via the AT1 receptor subtype.

Angiotensin-II Receptor Subtypes in Median Eminence and Basal Forebrain Areas Involved in Regulation of Pituitary Function

We used quantitative autoradiography to characterize angiotensin-II (ANG-II) receptor subtypes in areas of the rat basal forebrain involved in pituitary control. ANG binding was totally displaced by the selective AT1 receptor subtype antagonist DuP 753 and was inhibited by dithiothreitol in median eminence, infundibular stem, paraventricular nucleus, subfornical organ, median preoptic nucleus, anterior pituitary, and the forebrain ANG-II receptor band, which extends from the subfornical organ to the vascular organ of the lamina terminalis, including the lamina terminalis and a continuous band of receptors reaching the paraventricular nucleus. In these areas, binding was not affected by the selective AT2 competitors PD 123177 or CGP 42112 A, indicating the presence of AT1 and the absence of AT2 receptors. AT1 binding was higher in the external layer and lateral parts of the median eminence. Conversely, ANG binding in the dura mater encapsulating the pituitary, in the vessels adjacent to this part of the dura mater, presumably branches of the hypophyseal arteries, and in anterior cerebral arteries was displaced by PD 123177 and CGP 42112 A, and was unaffected by dithiothreitol or DuP 753, indicating the presence of AT2 receptors in these structures. Our results implicate AT1 receptors in the central neural regulation of pituitary function. AT2 receptors may play a role in the regulation of blood flow to the basal forebrain and the pituitary gland.

Effect of angiotensin II on cytosolic free calcium in neonatal rat cardiomyocytes

The effect of angiotensin II (ANG II) on cytosolic free Ca2+ concentration ([Ca2+]i) was studied in cultured neonatal rat ventricular myocytes. [Ca2+]i was estimated in groups of one to three cells by dual-wavelength microfluorometry or in cell populations using conventional fluorometry. ANG II (10(-8) M) produced an acute short-lived increase over the control basal diastolic [Ca2+]i and increased the frequency of the [Ca2+]i transients. The amplitude of the [Ca2+]i transients was decreased to 64.4% of basal values. The effect of ANG II on [Ca2+]i was blocked by the selective AT1 receptor subtype antagonist Du Pont 753 but not by the AT2 antagonist PD 123319. Removal of extracellular Ca2+ or blockade of voltage-gated Ca2+ channels in cells cultured for 5-7 days abolished the [Ca2+]i transients, but only partially diminished the effect of ANG II on [Ca2+]i. Thapsigargin, an inhibitor of sarcoplasmic reticulum Ca(2+)-Mg(2+)-ATPase, reduced or abolished the [Ca2+]i response to ANG II. Phorbol 12-myristate 13-acetate (PMA), 10(-6) and 10(-7) M, also decreased the amplitude of the Ca2+ transients similar to ANG II. Pretreatment with 10(-6) M PMA or 10(-6) M 1-oleoyl-2-acetyl-glycerol (OAG) inhibited the initial rise in [Ca2+]i and the Ca2+ transients. Thus ANG II produces an acute rise in [Ca2+]i which is derived predominantly from sarcoplasmic reticulum intracellular stores. This acute effect is followed by a significant reduction in the amplitude for the Ca2+ transient and may be mediated by activation of protein kinase C.