Physiol. Res. 56: 383-391, 2007 Role of Brain Angiotensin AT1 Receptor in the Carbachol- Induced Natriuresis and Expression of nNOS in the Locus Coeruleus and Proximal Convoluted Tubule M. WANG, C.L. JIANG, C.Y. WANG, Q.Y. YAO Department of Physiology, Dalian Medical University, Dalian, China Received April 4, 2006 Accepted July 10, 2006 On-line available August 22, 2006 Summary Central administration of losartan effectively blocked the increase of blood pressure and drinking response induced by angiotensin II (Ang II) or carbachol. However, the relationship between angiotensin AT1 receptors and the natriuresis induced by brain cholinergic stimuli is still not clear. The purpose of the study is to reveal the role of brain angiotensin AT1 receptor in the carbachol-induced natriuresis and expression of neuronal nitric oxide synthase (nNOS) in the locus coeruleus (LC) and proximal convoluted tubule (PCT). Our results indicated that 40 min after intracerebroventricular (ICV) injection of carbachol (0.5 μg), urinary sodium excretion was significantly increased to 0.548±0.049 μmol·min-1·100 g-1. Immunohistochemistry showed that carbachol induced an increase of neuronal nitric oxide synthase immunoreactivity (nNOS-IR) in the LC and renal proximal tubular cells. After pretreatment with losartan (20 μg), carbachol-induced urinary sodium excretion was reduced to 0.249±0.067 μmol·min-1·100 g-1. The same was true for carbachol-induced increase of nNOS-IR in the LC and PCT. The present data suggest that ICV cholinergic stimulation could induce a natriuresis and upregulate the activity of nNOS in the LC and PCT. The blockade of AT1 receptors might downregulate the effects induced by carbachol in the LC and PCT. Consequently, we provide a new evidence that brain angiotensinergic pathway and NO-dependent neural pathway contribute to the natriuresis following brain cholinergic stimulation and thus play an important role in the regulation of fluid homeostasis. Furthermore, the final effect of nitric oxide on proximal tubular sodium reabsorption participated in the natriuresis induced by brain cholinergic stimulation. Key words AT1 receptor • Neuronal nitric oxide synthase • Carbachol • Locus coeruleus • Proximal convoluted tubule Introduction into the anteroventral third ventricle region (AV3V), paraventricular nucleus (PVN), SFO and MSA (Fitts and Injection of cholinergic agonists into the lateral Simpson 1986, De Arruda Camargo and Saad 1999, cerebral ventricle, medial septal area (MSA), subfornical Camargo and Saad 2001, Saad et al. 2002). Previous organ (SFO) was found to induce natriuresis (Fitts and studies have revealed that the central administration of Simpson 1986, Colombari et al. 1992a,b). Similar effects losartan, a specific AT1-receptor blocker, effectively were obtained after injection of angiotensin II (Ang II) blocked the increase of blood pressure and drinking PHYSIOLOGICAL RESEARCH ISSN 0862-8408 © 2007 Institute of Physiology, v. v. i., Academy of Sciences of the Czech Republic, Prague, Czech Republic Fax +420 241 062 164 E-mail: [email protected] http://www.biomed.cas.cz/physiolres 384 Wang et al. Vol. 56 response induced by Ang II or carbachol (Colombari et pressure, renal sodium and potassium excretion in al. 1992a,b, De Arruda Camargo and Saad 1999, response to ICV injection of hypertonic saline could be Thunhorst and Johnson 1993, Saad et al. 1997, 2004). completely or partially abolished by AT1 receptors These data implicated that the brain angiotensinergic blocker, losartan (Skott 2003). pathway interacted with the brain cholinergic pathway in Based on these findings, it could be speculated the control of drinking and blood pressure. However, it is that there is an interaction between central angioten- not clear whether the angiotensin AT1 receptor is related sinergic pathway and central NO-dependent neural to the natriuresis induced by brain cholinergic stimuli. pathway in the renal sodium excretion caused by brain Locus coeruleus (LC), an important integrated cholinergic stimuli. To investigate these, we tested 1) the site in the pons, regulates sympathetic nerve activity, effect of blocking brain angiotensin AT1 receptors by fluid balance and arginine vasopressin (AVP) release, losartan (20 μg) on renal sodium excretion after ICV affecting sodium excretion via renal nerves and water injection of carbachol (0.5 μg), and 2) the effect of excretion via AVP release at rest and during volume blocking brain angiotensin AT1 receptors by losartan challenges (De Luca et al. 1990, McCann et al. 1997). (20 μg) on the changes of nNOS-IR in the LC and kidney Cholinergic stimulation in the SFO, lateral preoptic area induced by ICV injection of carbachol. (LPO), ventromedial hypothalamus (VMH), LC and septal area resulted in natriuresis and diuresis (Fitts and Methods Simpson 1986, Colombari et al. 1992a,b, De Luca et al. 1990). Neuronal nitric oxide synthase (NOS)-positive Animals neurons were found in the LC (Xu et al. 1998) and NO- Healthy male Sprague-Dawley rats (Dalian dependent neural pathway was involved in the natriuresis Medical University Animal Center, China) weighing 250- induced by blood volume expansion (Li et al. 2003). 280 g were kept on a 12 h/12h light/dark schedule with a However, it is not clear whether NO-dependent neural free access to standard laboratory food and water at room pathway is involved in the renal sodium excretion temperature. induced by ICV injection of cholinergic agonist. The primary role of the kidney is to maintain Experimental protocols the volume and electrolyte composition of body fluids Forty-eight rats were anesthetized with ether. A and, in this way, to regulate the blood pressure. Recent 23 gauge guide cannula was implanted in the right lateral studies indicated that renal nitric oxide is an important ventricle using a stereotaxic apparatus. Coordinates for factor in the control of urinary sodium excretion. NO ICV injection were based on the atlas of Paxinos and might enhance natriuresis by inhibiting transport along Watson with the skull horizontal: posterior 1.0 mm from the nephron as well as by altering renal hemodynamics the bregma, lateral 1.5 mm from the midline, deep (Manning and Hu 1994, Krier and Romero 1998, Noonan 3.0 mm from the skull. The cannula was fixed to the skull and Banks 1999). Renal medullary infusion of 7-nitro- with super glue and dental cement. Another stylette indazole (7-NI), a specific inhibitor of nNOS, decreased (31 gauge) was placed in the guide cannula to prevent medullary nNOS activity by 37 % and increased arterial occlusion. The rats were housed individually and were blood pressure in Sprague-Dawley rats on high sodium allowed to recover for one week after the surgery. During intake but not on normal sodium intake (Manning et al. this time, stylettes were checked regularly and rats were 2001). This suggested that nNOS might enhance renal handled daily to avoid stress-induced expression on the sodium excretion and thus help to prevent salt-sensitive day of the experiment. hypertension. However, the role of renal nNOS in the natriuresis induced by brain cholinergic stimuli remains ICV injection protocols to be investigated. All ICV injections were made between 8:00 and ICV injection of nitro-L-arginine methyl ester 9:00 a.m. to exclude the interference of circadian rhythms (L-NAME), an inhibitor of NOS, antagonized drinking in of nNOS expression with the experiment. All injected response to ICV injection of Ang II, and this effect could solutions were made in the volume of 5 μl over a period be abolished by ICV injection of L-arginine, implicating of 2 min with an additional 2 min allowed to elapse prior that Ang II interacted with NO in the control of drinking to the removal of infusion needles and replacement of the (Mathai et al. 1998). The increase of mean arterial blood cannulas. 2007 The Role of Brain Angiotensin AT1 Receptor 385 Twenty-four rats for the experiment in vivo were USA) for 1 h. Thereafter the sections were incubated in divided randomly into four groups (n=6 per group): the primary antibody (nNOS-Ab, 1:100, Boster physiological saline (NS) + carbachol (CBC) group, Company, P.R. China) overnight at 4 °C. The sections losartan (Los) + CBC group, Los + NS group and NS + were then rinsed three times in PBS for 10 min and NS group. NS + CBC group received ICV injection of 0.9 incubated with 2 % BSA for 1 h. Subsequently, the % NaCl (5 μl), followed by ICV injection of 0.1 μg/μl sections were rinsed three times in PBS for 10 min, and carbachol (5 μl) 20 min later. The treatment of other further incubated in the biotinylated-second antibody groups was similar to the NS + CBC group. The total (Boster Company, P.R. China) at room temperature for injected volume was 10 μl (5 μl the first drug, followed 2 h. Finally, the sections were again rinsed three times in by 5 μl the second drug 20 min later). Another 24 rats for PBS for 10 min and incubated with the avidin-biotin immunohistochemistry were also divided into 4 groups complex ABC (Boster Company, P.R. China) at room and treated as described in the experiment in vivo. temperature for 2 h. Diaminobenzidine (DAB, Sigma Co., USA) was used for signal detection. The control sections Experiment in vivo were incubated with PBS instead of primary antibody. Animals were anesthetized with urethane The HPIAS series colorful pathology photographic (1 ml/100 g b.w., i.p.). The right external jugular vein, the system was used to analyze nNOS-IR positive neurons. left carotid artery and urinary bladder were cannulated The brain sections were observed in a 20x magnification. with a polyethylene catheter for intravenous (i.v.) drug The number and optical density of nNOS-IR positive administration, MAP recording and urine collection, neurons were calculated per area and per group.