JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2006, 57, 4, 627636 www.jpp.krakow.pl 1 1 2 S. LIPIÑSKA , A. ¯EBROWSKA-BADALLA, J. LIPIÑSKA OXYTOCIN RELEASE AFTER BLEEDING IN RAT: THE ROLE OF SYMPATHETIC AND RENIN-ANGIOTENSIN SYSTEM 1 Chair of Experimental and Clinical Physiology, Department of Clinical Physiology, 2 Medical University of Lodz II Chair of Pediatrics, Clinic of Pediatric Cardiology Medical University of Lodz The aim of this experiment was to compare the role of renin-angiotensin and sympathetic nervous system in post-haemorrhagic mechanism of oxytocin release. Oxytocin content in venous dialysates was determined by radioimmunoassay. In control rats the release of oxytocin into dialysates did not change during whole experiment. The injection of captopril induced 2-fold higher oxytocin release, but caused no change in oxytocin release after bleeding. Superior cervical ganglionectomy (SCGx) 20 days before, caused 5-fold higher increase in oxytocin release than in control group. Injection of captopril in rats after SCGx, did not decrease the high level of oxytocin in dialysate. However, bleeding increased oxytocin release and 1 hour after bleeding the highest - 14-fold increase, took place. In the contrary to 14-fold increase in oxytocin release in animals with superior cervical ganglia (SCG), bleeding after SCGx caused only 2-fold higher oxytocin release. When SCGx, bleeding and injection of captopril were done simultaneously, oxytocin release remained on the control concentration level. We assumed that blockade of renin angiotensin system and sympathetic dennervation prevent the increase in oxytocin release after bleeding. On basis of our present experiments, it can be assumed that, in posthaemorrhagic oxytocin release into the blood, sympathetic innervation derived from SCGx, as well as, renin-angiotensin system are involved. Key words: oxytocin, bleeding, superior cervical ganglion, renin-angiotensin system INTRODUCTION Oxytocin is a neurohormone synthesised not only in the magnocellular nuclei of the hypothalamus: paraventricular and supraoptic nuclei, but also in the microcellular neurons of the paraventricular nucleus innervates many structures of 628 the central nervous system, where regulation centers of the cardiovascular system are located. It is suggested, that oxytocin acts in regulation cardiovascular homeostasis (5-12). Oxytocin increases the hart rate during stress (6-9). It also strengthen baroreceptor reflex and decreases blood pressure by alpha-2-adrenergic system (10). Antagonist of oxytocin produces a mild pressor response (11). Not only vasopressin but also oxytocin stimultaneous release after bleeding is still discussed. Experiments on the rats showed that, oxytocin content in neurohypophysis after bleeding was decreased (12-14) and its release into the blood increased rapidly (15). Our previous studies showed, that in posthaemorrhagic mechanism of vasopressin release peripheral noradrenergic (sympathetic) projection to the hypothalamic-pituitary axis, derived from the SCG, as well as, renin-angiotensin system, were involved (16). We showed also, that after SCGx oxytocin content in posterior pituitary lobe was lower (12). The aim of the present study was to investigate what was the role of the renin- angiotensin and sympathetic system in post-haemorrhagic oxytocin release. MATERIAL AND METHODS Animals The experiments were performed on male rats, weighing 300-320 g, 5-9 months old F1 generation cross-strains of male August and female Wistar, from the Institute of Oncology in Warsaw. The animals in surgical experiments were anaesthetised by an i.p. injection of a solution containing 6 mg of chloralose (Roth) and 60 mg of urethane (Flucka Ah, CH-9470 Bucks) per 100 g b.w. In chronic experiments the animals were anaesthetized by i.p. injection by hexobarbitane 80 mg/kg b.w. All procedures were carried out according to EU directives and reviwed by local Ethical Committee. Experimental groups: 1. control (n = 10); 2. injection of captopril i.v. half hour before dialysis (5mg/100g b.w.), (n = 8); 3. injection of captopril and bleeding (1% b.w.), (n = 8); 4. 20 days after superior cervical ganglionectomy, injection of captopril (n = 10); 5. after superior cervical ganglionectomy, injection of captopril and bleeding (n= 10); 6. 20 days after superior cervical ganglionectomy (n = 10); 7. 20 days after superior cervical ganglionectomy and bleeding (n = 10); 8. bleeding (1% b.w.), (n = 10). Exposure and superior cervical ganglionectomy The salivary glands were exposed through a ventral incision in the neck. After that, they were retracted, to expose the strap muscles. Each SCG was identified at the bifurcation of the common carotid artery. The ganglia were totally removed from both sides. Care was taken to administer and attain a surgical stage of anaesthesia and to allow a rapid recovery of the animal. 629 Blood withdrawal from the inferior vena cava The inguinal region was infiltrated with 2% polocaine hydrochloride. The femoral vein was exposed and a thin polyethylene catheter filled with isotonic saline was introduced toward the vena cava. Heparin (100 UJ/mL in 0.2 mL) was injected through the catheter and blood volume, which was equivalent to 1% b.w. was withdrawn during 1-2 min. Dialysate blood sampling In order to obtain blood dialysate, one polyethylene cannula was inserted into the heart end of the internal maxillary vein, and the second into the maxillary vein in the vicinity of cavernous sinus of the sella turcica. Blood was drawn from the region of sella turcica through the polyethylene cannula to the minidialysator with the use of the peristaltic pump. After that blood was returned to the circulation through the cannula inserted into the heart end of the maxillary vein. At the beginning of the experiment 2 mL of Locks solution with heparin (400UJ/mL) was injected into the internal maxillary vein. The whole amount of dialysing fluid was exchanged every 30 min for 3 hrs, by draining it directly into a test tube. Six 1 mL samples of dialysate were obteined in that way. Before refilling the minidialysator with dialysing fluid, it was rinsed with Mc Ilwain-Rodnight solution. At the end of each experiment 1% solution of trypan blue was injected through the cannula inserted into the internal maxillary vein. Then the brain was removed from the skull and the dye in the posterior pituitary lobes was verified under a stereomicroscope. Into the results were included only, these dialysate samples, that are collected from animals, which showed the staining of the posterior pituitary lobe. Staining of the posterior pituitary lobe has proved proper insertion of the cannula into the vicinity of cavernous sinus of the sella turcica, and proper blood collection (17). Minidialysator characteristics Minidialysators have two tips for Louers needles, one to connect with cannula in vein and the other with a peristaltic pump. There are two other tips for Louers needles for exchange of the dialysing fluid. Minidialysators were tested in in vitro experiments. 60% of oxytocin amount was recovered to the dialysing fluid (18). Radioimmunoassay of oxytocin The oxytocin content in dialysates was assayed by radioimmunoassay and expressed in pg/mL/30 min. Statistical analysis Statistical analysis of the results was performed with a two-way factorial analysis of variance (ANOVA) followed by Duncans test. RESULTS The release of oxytocin into the dialysis fluid in control rats was stable during 180 min of the experiment and maintain at the mean level 16.7± 3.9 pg/mL/30 min (group 1; sample I-VI). The injection of captopril (group 2) caused 2-fold higher oxy release, but caused no increase in oxytocin release after bleeding 630 (group 3). SCGx 20 days earlier (group 6), caused a significant increase in oxytocin release, 5-fold higher than in control group. Injectjon of captopril in rats after SCGx (group 4), did not decrease the high level of oxytocin in dialysate. Bleeding (group 8) also increased oxytocin release. The highest, 14-fold increase, took place 1 hour after bleeding. Bleeding after SCGx (group 7) caused only 2- fold higher oxytocin release in the contrary to 14-fold increase of oxytocin release in animals with SCG. After SCG, the injection of captopril and bleeding (group 5) done together oxytocin release was abolished to the control concentration. The results are summarized on Figure 1 and Table 1 DISCUSSION Oxytocin release into the dialysate after bleeding Oxytocin release into the dialysate was stable during 3 hours of our present experiments. The method of blood dialysis applied in the present study allowed us to observe the dynamic changes in neurohormone release in the same animal. Blood collected from the cavernous sinus of sella turcica region, was that outflowing from the brain, as well as, from pituitary gland. That was the reason there were much more hormones than in peripheral blood (19). Grzegorzewski and co-authors postulated the possibility of oxytocin transfer from the cavernous sinus to arterial blood suppling the brain and hypophysis (20). Fig. 1. Oxytocin realese into the dialisate (pg/mL/30 min) 631 Table 1. Oxytocin release into the dialysate, pg/mL/30 min (means ±SE). sample No I II III IV V VI group 1 control 13.8 ± 4.0 19.7 ± 4.8 13.2 ± 3.3 16.9 ± 3.7 19.7± 3.8 17.1 ± 3.9 2 captopril 20.9 ± 1.8 22.3 ± 1.7 21.7 ± 1.6* 38.7 ± 6.6* 42.3 ± 8.2* 47.5 ± 9.2* 3 captopril 28.0 ± 3.7* 38.4 ± 8.6* 39.2 ± 6.1* 43.2 ±12.6* 55.5 ± 9.4* 33.5 ± 5.8* + bleeding 4 SCGx + 73.9 ± 12.8* 53.8 ± 6.8* 43.3 ±12.3* 80.8 ± 14.2* 87.2 ± 17.8* 83.0 ± 13.0* captopril 5 SCGx + captopril 9.7 ± 2.3 18.0 ± 2.0 13.5 ± 1.7 11.0 ±1.2 14.3 ± 2.4 15.0 ± 2.1 + bleeding 6 SCGx 60.2 ± 9.3* 82.2 ±10.2* 79.0 ± 13.1* 94.8 ± 15.3* 98.5 ± 15.2* 107.0 ± 16.8* 7 SCGx + 65.0 ± 5.3* 68.7 ± 5.3* 80.0 ± 8.8* 110.1 ± 12.8* 130.0 ± 15.6* 115.0 ± 18.3* bleeding 8 bleeding 18.4 ± 2.6 23.0 ± 2.2 60.5 ± 8.8* 109.0 ±13.8* 234.0 ± 28.0* 120.0 ± 13.8* *statistically significant: group 1, 5 vs.