Behavioral Characterization of Alcohol-Tolerant and Alcohol-Nontolerant Rat Lines and an F2 Generation

The Alcohol Tolerant (AT) and Alcohol Nontolerant (ANT) rats, selectively bred for ethanol-induced ataxia on the inclined plane at ALKO in Finland, were moved to the University of Colorado in 1998. The selection phenotype was tested on generation 60 animals in Colorado. In week one, ataxia was measured on the inclined plane 30 minutes after an intraperitoneal dose of 2 g/kg 15% w/v ethanol. Differences in ethanol-induced ataxia between the AT and ANT lines at the University of Colorado were similar to those in the original lines in Finland. In week two, ataxia was measured on the inclined plane at 5 and 30 minutes, and tolerance was measured as the time to regain the original angle of sliding. The AT rats rapidly developed tolerance to 2 g/kg ethanol on the inclined plane; tolerance development was significantly slower in the ANT rats. In week three, the animals were tested for the duration of loss of righting reflex (LORR) and blood ethanol concentration at regain of the righting reflex (BEC_RRR) following a dose of 3.5 g/kg. The AT rats had a significantly higher BEC_RRR than did the ANT rats, but did not differ in LORR. A separate experiment with previously untreated rats demonstrated that naïve animals of the two lines did not differ in BEC_RRR or LORR. AT and ANT rats were genotyped for the mutation that occurs in the gene for the α6 subunit of the GABA_A receptor, a natural mutation that is known to affect benzodiazepine responses. All ANT animals tested carried the mutant allele, whereas some AT families carried the mutation and others were wild type. There was no effect of the mutation in AT rats for any of the phenotypes that were tested. After several generations of brother–sister mating, the AT and ANT lines were more than 90% inbred as determined by genotyping. One AT (wild-type) line and one ANT (mutant) line were selected for breeding an F₂ intercross generation of 1200 animals. They were phenotyped for sensitivity and tolerance to ethanol on each of three consecutive weeks. Order of testing had a modest effect on some of the phenotypes: when tested during the third week as compared to weeks one or two, BEC_RRR was increased, 30-minute sensitivity was increased, and development of acute tolerance was increased. Statistically significant correlations were found between tolerance and sensitivity at both 5 and 30 minutes, and between LORR and BEC_RRR. The smaller (or absence of) significant correlations between others of the phenotypes indicate(s) that they are most likely controlled by different sets of genes.     

Strong linkage disequilibrium of a HbE variant with the (AT)9(T)5 repeat in the BP1 binding site upstream of the β-globin gene in the Thai population

A binding site for the repressor protein BP1, which contains a tandem (AT)_x(T)_y repeat, is located approximately 530 bp 5 to the human -globin gene (HBB). There is accumulating evidence that BP1 binds to the (AT)₉(T)₅ allele more strongly than to other alleles, thereby reducing the expression of HBB. In this study, we investigated polymorphisms in the (AT)_x(T)_y repeat in 57 individuals living in Thailand, including three homozygotes for the hemoglobin E variant (HbE; 26Glu->Lys), 22 heterozygotes, and 32 normal homozygotes. We found that (AT)₉(T)₅ and (AT)₇(T)₇ alleles were predominant in the studied population and that the HbE variant is in strong linkage disequilibrium with the (AT)₉(T)₅ allele, which can explain why the ^E chain is inefficiently synthesized compared to the normal ^A chain. Moreover, the mildness of the HbE disease compared to other hemoglobinopathies in Thai may be due, in part, to the presence of the (AT)₉(T)₅ repeat on the HbE chromosome. In addition, a novel (AC)_n polymorphism adjacent to the (AT)_x(T)_y repeat (i.e., (AC)₃(AT)₇(T)₅) was found through the variation screening in this study.MIM and accession numbers and URLs for data presented herein are as follows: Online Mendelian Inheritance of Man (OMIM), (for HBB [MIM 141900]). GenBank, (accession number [NG_000007.2] reference sequence information).     

Pharmacokinetics and pharmacodynamics of candesartan cilexetil in patients with normal to severely impaired renal function

Objective: We studied the pharmacokinetics and pharmacodynamics of single and multiple doses of candesartan cilexetil 8 mg per day in hypertensive patients with different degrees of renal function impairment. Candesartan is an angiotensin II subtype 1 (AT1) receptor antagonist that is administered orally as candesartan cilexetil which is converted in the active compound. Methods: Twenty-three patients were included, divided into groups according to creatinine clearance (cr cl. group A >60 nl · min⁻¹ · 1.73 m⁻², group B 30–60 ml · min⁻¹ · 1.73 m⁻² and group C 15–30 ml · min⁻¹ · 1.73 m⁻²). Results: Trough serum concentrations of candesartan were higher in group C compared with group A. The values did not increase after multiple dosing, indicating absence of accumulation. There was a significant negative correlation between the area under the concentration-time curve extrapolated to time infinity (AUC_inf) and the glomerular filtration rate (GFR) indicating a lower renal clearance of candesartan in patients with impaired renal function. The onset of haemodynamic and hormonal effects was gradual. During the single-dose study blood pressure as well as plasma renin activity (PRA) and angiotensin II were unchanged at peak. At day 5 of the multiple-dose study blood pressure was lower and both PRA and angiotensin II were higher compared with baseline. Conclusion: Although serum trough levels increased during repeated administration and half-life was higher in patients with impaired renal function, candesartan cilexetil at a dose of 8 mg per day does not lead to drug accumulation in these patients. This dose is effective in lowering blood pressure and appears to be suitable for patients with renal function impairment. Received: 3 August 1998 / Accepted in revised form: 19 October 1998     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: MOLECULAR MECHANISMS OF ANGIOTENSIN II (AT1a) RECEPTOR ENDOCYTOSIS

• 1 Angiotensin II (AngII) initiates a variety of cellular responses through activation of type 1 (AT₁; with subtypes AT_1a and AT_1b) and type 2 (AT₂) cell surface angiotensin receptors. Both AT₁ and AT₂ receptors couple to heterotrimeric guanyl nucleotide binding proteins (G-proteins) and generate intracellular signals following recognition of extracellular AngII, but only AT₁ is targeted for the rapid ligand-stimulated endocytosis (internalization) typical of many plasma membrane receptors.
• 2 AT₁ endocytosis proceeds through clathrin-coated pits and is independent of G-protein coupling which predicts that the AngII-AT₁ receptor complex attains a conformation necessary for interaction with the endocytotic machinery, but separate from receptor signalling activation.
• 3 The function of AT₁ endocytosis and the reason for the disparity between AT₁ and AT₂ endocytosis is not fully appreciated, but the latter probably reflects differences in the primary amino acid sequence of these two receptor types.
• 4 For many receptors that undergo internalization, it has been established that internalization motifs (2–6 amino acids, often incorporating crucial tyrosine and hydrophobic amino acids) within the cytoplasmic regions of the receptor mediate the selective recruitment of activated receptors into clathrin-coated pits and vesicles.
• 5 Mutagenesis studies on the AT_1a receptor, aimed at identifying such motifs, reveal that sites within the third cytoplasmic loop and the cytoplasmic carboxyl terminal region are important for AngII-stimulated AT_1a receptor endocytosis.

     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: ROLE OF AT1 RECEPTORS IN THE CENTRAL CONTROL OF SYMPATHETIC VASOMOTOR FUNCTION

• 1 In a number of species, high concentrations of angiotensin II (AngII) receptors have been found in the rostral ventrolateral medulla (RVLM) in the hindbrain, which is an important region involved in the modulation of sympathetic vasomotor tone. The present review describes studies in which the contribution of angiotensin receptors in the brainstem to cardiovascular regulation, in particular sympathetic vasomotor reflexes, has been examined in conscious and anaesthetized rabbits.
• 2 In conscious rabbits, fourth ventricular infusions of AngII produced dose-dependent pressor responses as doses 400 times less than equipressor intravenous doses. Chronic baroreceptor denervation increased the sensitivity to AngII by 1000-fold. Administration of prazosin i.v. blocked the pressor response, suggesting that the mechanism involved sympathetic vasoconstriction.
• 3 The pattern of haemodynamic changes in response to AngII injected into the fourth ventricle (4V) involved decreased total peripheral conductance and mesenteric conductance, but a rise in hindlimb conductance. Sinoaortic denervation changed the hindlimb fall in conductance to an increase, suggesting that muscle vasomotor pathways were particularly inhibited by baroreceptor feedback mechanisms.
• 4 In anaesthetized rabbits, infusion of AngII into the RVLM increased blood pressure and transiently increased resting renal sympathetic nerve activity. The renal sympathetic baroreflex curves were shifted to the right and the upper plateau of the sympathetic reflex increase was markedly increased.
• 5 The pressor actions of 4V AngII were blocked by administration of a peptide antagonist injected into the RVLM or by the angiotensin AT₁ antagonist losartan injected into the 4V. These results suggest that mainly AT₁ receptors are involved and that the RVLM is a likely candidate site for the modulation of the renal sympathetic baroreflex.
• 6 Losartan administration into the 4V in conscious rabbits increased resting renal sympathetic tone and enhanced renal sympathetic baroreflex and chemoreflexes.
• 7 Our studies suggest that there are sympathoexcitatory AT₁ receptors in the RVLM accessible to AngII from the cerebrospinal fluid. In addition, an AT₁ receptor pathway normally inhibits the sympathoexcitation produced by baroreceptor unloading or chemoreceptor activation. The effect of losartan suggests that there is greater tonic activity within the sympathoinhibitory pathways. These two actions suggest that angiotensin receptors in the brainstem modulate sympathetic responses to specific afferent inputs, thus forming part of a potentially important mechanism for the integration of characteristic autonomic response patterns.

     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: PHYSIOLOGICAL ACTIONS OF ANGIOTENSIN II MEDIATED BY AT1 AND AT2 RECEPTORS IN THE BRAIN

• 1 Autoradiographic binding studies have shown that the AT₁ receptor is the predominant angiotensin II (AngII) receptor subtype in the central nervous system (CNS). Major sites of AT₁ receptors are the lamina terminalis, hypothalamic paraventricular nucleus, the lateral parabrachial nucleus, rostral and caudal ventrolateral medulla, nucleus of the solitary tract and the intermediolateral cell column of the thoraco-lumbar spinal cord.
• 2 While there are differences between species, AT₂ receptors are found mainly in the cerebellum, inferior olive and locus coeruleus of the rat.
• 3 Circulating AngII acts on AT₁ receptors in the subfornical organ and organum vasculosum of the lamina terminalis (OVLT) to stimulate neurons that may have a role in initiating water drinking.
• 4 Centrally administered AngII may act on AT₁ receptors in the median preoptic nucleus and elsewhere to induce drinking, sodium appetite, a sympathetic vasoconstrictor response and vasopressin secretion.
• 5 Recent evidence shows that centrally administered AT₁ antagonists inhibit dipsogenic, natriuretic, pressor and vasopressin secretory responses to intracerebroventricular infusion of hypertonic saline. This suggests that an angiotensinergic neural pathway has a role in osmoregulatory responses.
• 6 Central angiotensinergic pathways which include neural inputs to the rostral ventrolateral medulla may use AT₁ receptors and play a role in the function of sympathetic pathways maintaining arterial pressure.

     

氯沙坦对自发性高血压大鼠脑组织肾素血管紧张素系统的影响

目的　探讨氯沙坦对自发性高血压大鼠 (SHR)脑组织血管紧张素Ⅰ型受体 (AT1 R)mRNA的表达及脑组织局部肾素 血管紧张素系统 (RAS)活性的影响。方法　 6wk龄♂WKY和SHR各 1 6只分别随机分为氯沙坦用药组和生理盐水对照组 ,喂养 1 8wk后运用RT PCR法检测脑组织AT1 RmRNA的表达 ;放免法测定肾素活性 (RA)和血管紧张素Ⅱ(AngⅡ )的水平 ;紫外分光光度法测定血管紧张素转换酶活性 (ACEA)。结果　SHR组各部位脑组织AT1 RmRNA和血浆、下丘脑RA、AngⅡ、血清ACEA表达均高于WKY组(P <0 0 5) ;氯沙坦可降低各部位脑组织AT1 RmRNA的表达但升高血浆RA、AngⅡ ,对下丘脑RA、AngⅡ、ACEA则无明显影响。结论　SHR脑组织AT1 RmRNA的表达及循环和组织RAS活性增加可能与高血压的发生有关 ,氯沙坦可能通过降低脑组织AT1 RmRNA表达发挥脑保护作用     

氯沙坦对血管紧张素II致培养的牛脑微血管内皮细胞损伤的保护作用

目的观察氯沙坦对血管紧张素II(Ang II)致牛脑微血管内皮细胞(BCMECs)损伤的保护作用。方法用分光光度计测定培养的BCMECs乳酸脱氢酶(LDH)的漏出量,流式细胞仪测定BCMECs细胞间粘附分子-1(ICAM-1)的表达量,硝酸还原酶法和放射免疫分析法分别测定BCMECs上清液中一氧化氮(NO)和内皮素-1(ET₁)的含量。结果Ang II呈剂量依赖性增加BCMECs LDH漏出、NO和ET₁释放及ICAM-1表达,氯沙坦对此均有明显抑制作用。结论氯沙坦抑制Ang II致体外培养BCMECs的损伤。