Lipofectin and the effect of antisense odn for AT1 receptors on thirst in conscious rat

Chunlong Huang & Edward J Johns, Department of Physiology, University College Cork, Ireland

The renin-angiotensin system is one of the most important hormonal systems in the regulation of body fluid. Angiotensin II (AII) within the brain is able to elicit thirst mediated by angiotensin type 1 (AT1) receptors in virtually all species. Antisense oligodeoxy-nucleotides (ODN) for AT1 receptors have been designed to inhibit the production of the specific proteins and liposomes have been used for encapsulating antisense ODN for delivery in blood (Phillips, 1997). The aim of the study was to investigate firstly, the effect of intracerebroventricular (icv) administration of antisense ODN for AT1 receptors on experimentally induced thirst in conscious rats, and secondly the role of lipofectin in this effect.

Male Wistar rats, 260-290g, were anaesthetised with pentobarbital sodium (60 mg/kg ip). The left carotid artery and right jugular vein were cannulated for monitoring blood pressure (BP) and heart rate (HR), or giving drugs. A guide cannula was implanted into the right lateral cerebroventricle for icv administration of drugs. The animal was then allowed to recover from anaesthesia and surgery for at least three days, and move freely in the home cage. Water intake during the first 20 min immediately following icv administration of AII at 50μg was measured before and one day after icv administration of either antisense ODN (5’TAACTGTGGCTGCAA) at 50 µg (group I), a mixture of 25 μg antisense ODN and 20 μg lipofectin (group II), or a mixture of 25 μg sense ODN (5’TGGCAGGCACAGTTA) and 20 μg lipofectin (group III). Animals were killed with overdose anaesthesia at the end of experiment. Data (means ±SEM) were subjected to Student's t test and significance taken as P<0.05.

In group I rats (n=5), basal BP was 113±5 mmHg and HR 451±18 beats/min. Water intake induced by AII icv was 11.1±1.7 ml. While BP and HR were comparable to the baselines, the response of water intake to AII icv one day after icv administration of antisense ODN at the higher dose was 9.9±1.1 ml, similar to that obtained before the antisense administration. In group II rats (n=5), baselines of BP, HR and the experimentally induced water intake were similar to those obtained in group I. However, icv administration of the mixture of antisense ODN, at lower dose, and lipofectin caused significant reductions in AII-induced water intake by 47% (P<0.05) one day later, although there was no significant change in either BP or HR. The baselines of BP, HR and AII icv induced water intake in group III rats (n=5) were comparable to those observed in the other two groups. Icv administration of mixture of sense ODN and lipofectin, served as a vehicle control, didn’t cause any significant change in BP and HR measured one day later. Furthermore, the response of water intake to AII icv one day after the mixture was 11.3±0.3 ml, similar to that obtained in the absence of sense ODN and lipofectin.

The result showed that lipofectin enhanced the inhibitory action of icv administration of antisense ODN for AT1 receptors on water intake induced by AII icv in conscious rats.

Phillips , MI (1997). Hypertension 29: 177-187.