Original article

S. Lipinska1, S. Forys2, J. Lipinska3


THE POST-HAEMORRHAGIC VASOPRESSIN RELEASE INTO THE BLOOD


1 Department of Experimental and Clinical Physiology, Institute of Physiology and Biochemistry, Medical University of Lodz, Poland.
2 Department for Diagnosis and Prophylaxis of Fetal Malformation, Institute “Polish Mother's Memorial Hospital”, Lodz, Poland.
3 Department of Paediatrics' Cardiology, Medical University of Lodz, Poland


  The aim of the present study was to compare the influence of the renin-angiotensin and sympathetic system in the process of post-haemorrhagic vasopressin release. A dialysis of the venous blood from the sella turcica region was performed in male rats under anaesthesia. The animals were divided into eight experimental groups: 1) control; 2) bleeding; 3) 20 days after superior cervical ganglionectomy; 4) 20 days after superior cervical ganglionectomy and bleeding; 5) injection of captopril; 6) injection of captopril and bleeding; 7) 20 days after superior cervical ganglionectomy and injection of captopril; 8) 20 days after superior cervical ganglionectomy, injection of captopril and bleeding. The content of vasopressin in dialysates was determined by radioimmunoassay. In control rats the release of vasopressin into dialysates was constant during 180 min of the experiment. Bleeding, as well as, superior cervical ganglionectomy caused an increase in vasopressin release. Captopril did not change vasopressin release in comparison to control group. Furthermore, vasopressin release after both, bleeding and sympathetic denervation performed simultaneously was significantly abolished. We conclude that renin-angiotensin, as well as, sympathetic nervous system are involved in the increased post-haemorrhagic vasopressin release.

Key words: vasopressin, superior cervical ganglion, renin-angiotensin system



INTRODUCTION

Vasopressin is released from the posterior pituitary lobe into the blood under nervous and humoral control. The fact, that haemorrhage stimulates the release of vasopressin from the neurohypophysis has been known for more than 60 years. It has been shown, that both, circulatory reflexes and renin-angiotensin system are engaged in the post-haemorrhagic release of vasopressin [1- 4].

After intravenous, intraarterial and also intraventricular infusion of angiotensin II an increase in vasopressin concentration in blood plasma took place. Angiotensin II is suggested to act as neurotransmitter or neuromodulator in the cholinergic mechanism of vasopressin release (5). After haemorrhage vasopressin concentration in plasma as well as renin plasma activity augmented [6-10]. The existence of a functionally relevant peripheral noradrenergic (sympathetic) innervation of the hypothalamo-hypophysial system, which derived from the superior cervical ganglia (SCG), has been proposed (11, 12). Bilateral superior cervical ganglionectomy (SCGx) brought about a decrease in noradrenaline (13,14) and vasopressin concentration in the posterior pituitary lobe (14-16). Our earlier studies showed, that the release of vasopressin into the blood was increased 20 days after superior cervical ganglionectomy (17).

The aim of the present study was the assessment of renin-angiotensin system and sympathetic system influence in the process of post haemorrhagic vasopressin release.

The preliminary data were presented at the 1st Congress of Polish Neuroendocrine Society, on September 22-25, 2002 in Lodz, Poland.


MATERIAL AND METHODS

Animals

The experiments were performed on male rats, weighing 300-320 g, 5-9 months old, the F1 generation of cross-strains of male August and female Wistar, from the Institute of Oncology in Warsaw. In surgical experiments the animals were anaesthetised by an i. p. injection of solution containing 6 mg of chloralose (Roth) and 60 mg of urethane (Flucka Ah, CH-9470 Bucks) per 100 g body weight. In chronic experiments an i.p. injection of hexabarbitane 80 mg/kg b.w. was used.

All procedures were carried out according to EU directives and reviewed by the local ethical committee.

Experimental groups:
  1. control;
  2. injection of captopril (5mg/100g b.w.) iv.; half hour before dialysis.
  3. injection of captopril and bleeding (1 % b. w.); immediately after collection of the first sample of the dialysis fluid;
  4. 20 days after superior cervical ganglionectomy and injection of captopril;
  5. 20 days after superior cervical ganglionectomy, injection of captopril and bleeding;
  6. 20 days after superior cervical ganglionectomy;
  7. 20 days after superior cervical ganglionectomy and bleeding;
  8. bleeding.
Exposure and superior cervical ganglionectomy

Care was taken to administer and to attain a surgical state of anaesthesia and to allow a rapid recovery of the animal. The salivary glands were exposed through a ventral incision in the neck. After that, salivary glands were retracted to expose the strap muscles and each SCG was identified at the bifurcation of the common carotid artery. The ganglia were totally removed from both sides.

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 and pushed 35 mm deep toward the vena cava. Heparin (100 UJ in 0.2 mL 0.9% NaCL) was injected through the catheter and 2 min later a blood volume, equivalent to 1% body weight was withdrawn during 1-2 min.

Dialysate blood sampling

In order to obtain blood dialysate samples from the vicinity of the pituitary, 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. From the sella turcica region blood was drawn, through the polyethylene cannula to the minidialysator with the use of a peristaltic pump. It was then returned to the circulation through the cannula inserted into the heart end of the maxillary vein.

At the beginning of the experiments 2 mL of Lock's solution with heparin (400 UJ) 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 obtained in this way. Before refilling, the minidialysator with dialysing fluid 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. Only these dialysate samples, which were collected from animals showing the staining of the posterior pituitary lobe, were included into the results. 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 (18).

Minidialysator characteristics

The minidialysators have been manufactured according to our design by EURO-SEP-Ltd Warsaw. They have two tips for Louer,s needles, one to connect with a cannula in a vein and the other with a peristaltic pump. There are two others tips for Louer's needles for the exchange of the dialysing fluid.

The minidialysators were tested in vitro experiments. Inulin clearance, inulin flow and ultrafiltration were elaborated elsewhere (18). 50 - 60% of vasopressin and oxytocin amount was recovered to the dialysing fluid (17).

Radioimmunoassay of vasopressin

The content of vasopressin 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 Duncan's test.


RESULTS

The release of vasopressin into the dialysing fluid in control rats (group 1) remained stable during 180 min of the experiment at the mean level 48.1 ± 11.6 pg/mL/30 min. Vasopressin release was dramatically increased by bleeding (1% b. w.) (group 8; sample II-VI, 344.7 ± 47.7 pg/mL/30 min). The highest 12-fold increase in vasopressin release was delayed in time and set in about 1 h after the haemorrhage at the level of 693.21 ± 65.8 pg/mL/30 min. During the second hour of the experiment, vasopressin concentration decreased to 6-fold the highest level in comparison to the control sample and was twice as low as its maximum concentration. SCGx done 20 days earlier caused 4,5-time higher level of vasopressin than in control group (group 6; 217 ± 38.5 pg/mL/30 min) and it remained constant during the experiment. Bleeding after superior cervical ganglionectomy (group 7) caused significantly lower increase in vasopressin release, in comparison to AVP release in animals with residuing SCGs (group 8). After inhibition of AII formation by the intravenous injection of captopril (group 2), vasopressin release was similar to the control group (48.2 ± 11 pg/mL/30 min.). Moreover, bleeding after the injection of captopril also did not induce higher release of vasopressin (group 3; 46.3 ± 14.7 pg/mL/30 min). After superior cervical ganglionectomy and injection of captopril no statistically significant change in vasopressin release was noted (group 4; 66 ± 14 pg/mL/30 min). The high release of vasopressin, caused by the absence of impulsation from SCGs, was decreased by the intravenous injection of captopril. Furthermore, SCGx and the injection of captopril diminished the posthaemorrhagic vasopressin release (group - 5; sample II-VI, 170 ± 22 pg/mL/30 min), in comparison with AVP release in animals with SCG after bleeding (group 8; sample II-VI, 344.7 ± 47.7 pg/mL/30 min) as well as in animals after SCGx (group - 7; sample II-VI, 346.6 ± 36.7 pg/mL/30 min).

Results of vasopressin concentration are presented in Table 1. and Fig. 1.

Table 1. Vasopressin release into the dialysate, pg/mL/30 min (means ±SE).
Statistically significant:
group 1, 2, 3, 4 vs. group 5 sample II-VI; p < 0.01
group 1, 2, 3, 4 vs. group 6; p < 0.01
group 1, 2, 3, 4 vs. group 7; p < 0.01
0.01group 1, 2, 3, 4 vs. group 8 sample II-VI; p < 0.01;
group 5 sample II-VI vs. group 8 sample III-VI; p < 0.05.
* statistically significant vs. sample I.

Fig. 1. Vasopresin realesse into the dialisate (pg/mL/30 min).


DISCUSSION

Vasopressin release into the dialysate after bleeding

The method of blood dialysis applied in the present study allows to observe the dynamic changes in vasopressin release into the blood in the same animal. It enables to avoid blood sampling which is of extreme importance especially in studies on vasopressin, because bleeding is the strongest stimulus releasing AVP into the blood (9). There is a higher concentration of vasopressin in blood of the sella turcica region than in peripheral blood (18). Blood collected from cavernous sinus (that is outflowing from the brain as well as from the pituitary gland) containing higher levels of hormones than peripheral blood.

It has been demonstrated that there is a possibility of transfer of neurohormone (oxytocin, ß-endorphin, RH-LH) from the cavernous sinus to arterial blood supplying the brain and hypophysis. (19-21) Moreover, neuropeptide concentration in blood dialysates from cavernous sinus of the sella turcica, obtained in the present experiment, resulted in the release of the neurohormone into blood and its uptake in the area of the rete mirabile.

The fact of the increased vasopressin release after bleeding observed in the present research has been known for a long time. Our earlier studies pointed out the extremely increased vasopressin release into the blood after bleeding (22). The delayed increase of vasopressin concentration, observed in present studies, was caused by the use of dialisators. It was related to the fact that dialysis in blood provided only an average over the collection time, since any sharp changes in peripheral release were blunted. Moreover, dialysis membrane limited the diffusion of neurohormones between the dialysing fluid and the blood. During the experiment 50% of whole vasopressin quantity diffused from the blood to the dialysing fluid. The process of diffusion requires certain time to equalize the concentration levels (17).

The mechanisms responsible for cardiovascular adaptation to hypotensive hypovolemia are still not well understood. Integrated neural, humoral and local mechanisms, which became activated in haemorrhagic shock conditions, resulted in the centralisation of circulation. Moreover, the redistribution of the circulating blood was mainly due to the increased activity of sympathetic nervous system, the secretion of vasopressin and the activation of renin-angiotensin system (23). Vasopressin is known to play an important role in central cardiovascular control, eliciting bradycardia and sympathoinhibition during hypotensive haemorrhage (24-26). It was found that both bradycardia and sympathoinhibition were absent in Bratleboro rats (AVP- deficient) and restored by intravenous infusion of vasopressin (27, 28). It has been observed that blockade of central V1 AVP receptors during hypotensive haemorrhage abolished bradycardia and attenuated hypotension in Wistar-Kyoto rats, but it was not effective in rats spontaneously hypertensive (26).

Influence of renin-angiotensin system on the vasopressin release

In the present study we indicated that in post haemorrhagic vasopressin release A II was involved. We demonstrated that the dose of captopril used in our study was not sufficient enough to influence basal vasopressin release, but sufficient enough to lower the induced release of AVP. The dose of captopril applied in our researches caused the entire abolition of the increased release of AVP after bleeding, which persisted 3 hours of the experiment. This finding could suggest that the total blockade of AII production took place. Nevertheless, the possible peripheral effects of captopril administered intravenously and the function of vasopressinergic neurons thereby altered (e.g., by modified baroreflex) were possible.

Some authors have observed that A II was involved in the regulation of vasopressin release into peripheral blood and central nervous system. It has been reported that the intravenous infusion of AII caused significant rise of vasopressin levels in plasma (5). An increase in vasopressin release was also observed after intra-arterial as well as intracerebroventricular administration of angiotensin II (29, 30). Microinjection of A II into the supraoptic and paraventricular nuclei produced potent antidiuresis by vasopressin release mediated through adrenergic and angiotensinergic receptor (31). Subsequent studies have provided the evidence that circulating angiotensin II inhibited baroreflex and stimulated the vasopressin release (32). A II from the blood penetrated through deprived of blood-brain barrier paraventricular organ and influenced the cardiovascular regulation as well as vasopressinergic neurons function. Blockade of V1 receptors in the brain after A II infusion decreased high blood pressure. The action of A II in the brain was mediated by AT1 and AT2 receptors (33, 34). The renin-angiotensin system also participated in the mechanism of the stress-induced high blood pressure. The effect of A II was mediated mainly by stimulating hypothalamic synthesis and release of vasopressin, which resulted in an increased blood pressure by activation of central V1 receptors (35). A II excitated the activity of the sympathetic nervous system, increased ganglion transmission and noradrenaline release from postganglionic fibers (36). Dual angiotensin/vasopressin receptors were activated by both, vasopressin and angiotensin II. That might activate the same intracellular mechanisms and cooperate in activation of catecholminergic system, which caused pressing reaction (37).

Vasopressin release after superior cervical ganglionectomy

Up to now the research showed that sympathetic innervation from SCG influenced neurohormone release from neurohypophysis. It has been demonstrated that in rats with removed SCG, the “miniature neurohypophysis” at the proximal end of the cut pituitary stalk was not formed, while in the hypothalamus there was a degeneration of neurosecretory neurons (39). Cardinali showed that 20 days after SCGx, a complete denervation of structures innervated by SCG took place (12, 13). Saavedra observed the 40-60 % decrease of NA and AVP content in median eminence in neurohypophysis of SCGx rats (14, 15).

Our previous experiments showed that AVP content in neurohypophysis was decreased 20 days after SCGx as well as after bleeding. Bleeding after SCGx did not cause further decrease of AVP in neurohypophysis, because SCGx itself lowers AVP neurohypophysial pool (16).

In the present experiments it was observed that 20 days after SCGx vasopressin release was higher in comparison with control rats. Moreover, captopril decreased the higher vasopressin release caused by the absence of impulsation from removed SCG. SCGx and injection of captopril diminished the posthaemorrhagic vasopressin release, but did not abolish it entirely. It could be caused by incomplete blockade of renin-angiotensin system or might suggest that also other mechanisms were involved in that process.

On the basis of present and previous research we postulated that, under physiological conditions, the release of neurohormone from the posterior pituitary lobe was probably continually inhibited by impulsation from SCG. SCGx, however, neutralized this inhibition and in consequence an increased neurohormone release into the blood and decreased content of AVP in the posterior pituitary lobe took place. In these conditions, the initial pool of AVP is lower than in physiological conditions, and in consequence, bleeding could release lower quantity of AVP into the blood.

The effect of SCG on the release of the posterior pituitary hormones, occurred both directly or via the pineal gland as the dependence between the pineal body and the release of the posterior pituitary neurohormone has been demonstrated (40, 41).

Conclusion

On basis of our study, it could be assumed that autonomic system as well as renin angiotensin system influenced the post-haemorrhagic vasopressin release. Furthermore, renin-angiotensin system is thought to play a pivotal role in posthaemorrhagic AVP release comparing with autonomic system. Removal of both, renin-angiotensin and sympathetic system influence, did not abolish entirely the vasopressin release after bleeding. On that basis, it could be supposed that also other mechanisms could be involved in posthaemorrhagic AVP release.

Acknowledgements: I would like to express my gratitude to Agnieszka Zebrowska-Badala M. Sc., for her technical assistance during the experiments, Monika Orlowska-Majdak, Ph.D., for supplying the anti-vasopressin antiserum and Jadwiga Kaczorowska-Skora, M. Sc., for performing vasopressin radioimmunoassay.
The study was supported by a grant Nr. 502-111-690 for Medical University of Lodz.



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R e c e i v e d : February 25, 2003
A c c e p t e d : February 3, 2004

Author’s address: Stanis³awa Lipinska, Ph.D., D.Sc. Department of Experimental and Clinical Physiology Institute of Physiology and Biochemistry, Medical University of Lodz, Mazowiecka 6/8, 92-215 Lodz, Poland, tel./fax. (48-42) 678-26-61