The Central Effects Of Urocortin3 On Cardiovascular Regulation And The Interaction With NOS-NO System In Rats

Objectives: To investigate the changes of urocortin 3 and its selective receptor, Corticotropin-releasing factor receptor type 2 (CRF₂), as well as nitric oxide synthase (NOS) expression in hypothalamus and medulla oblongata, and test the central effects of urocortin3 in cardiovascular activity and its roles in stress-induced hypertension as well as the interaction between the urocortin3 and nitric oxide.Methods: the stress-induced hypertensive model was established by stimuli of electric foot shock combined with buzzer noise twice dayly (2h×2) and continued for 10 days. To detect the changes of urocortin3, CRF₂ and NOS in the rostral ventrolateral medulla (rVLM) and paraventricular nucleus(PVN) of hypothalamus in stress-induced hypertensive rats, real time-polymerase chain reaction (Realtime-PCR) and immunohistochemistry(IHC) methods were used. In addition, we tested the central cardiovascular effect and its potential mechanism of urocortin3 in the PVN and the rVLM by microinjection, iontophoretic, extracellular single-unit recordings and micro-dialysis methods.Results: (1) Consecutive stress for 10 days induced a significant increase in SBP, from 114±6 mmHg of basal level to 138±7mmHg(P<0.01, n=7) and maintained at a high level for up to 10 days, which meant that the stress-induced hypertension model was established successfully; (2) With Realtime-PCR method, we observed that the mRNA expression of urocortin3 was significantly up-regulated in the PVN, but not in the rVLM, of stressed rats compared with that of control rats(P<0.05, n=8), while no significant expression changes of CRF₂, the selective receptor of urocortin3, were observed in the PVN and rVLM; (3) nNOS and iNOS-like immunoreactivities were distributed in the PVN of the hypothalamus and in the rVLM. In the PVN, high labled cells were found in both meganocellular and parvocellular parts, with no area disparity. In the rVLM, the nNOS-like immuneoreactivities were restricted to the rostral and medial parts with clear positive cells and fibers, while the distribution of iNOS-like immuneoreactivities in the rVLM was more scattered. Foot shocks and noises stress for 10 days significantly increased nNOS-like immunoreactivity in thePVN and rVLM compared with in the control group (P<0.01, n=8) , whereas decreased inducible nitric oxide synthase (iNOS)-like immunoreactivity in the rVLM(P<0.05, n=8) ; (4) Unilaterally microinjection of urocortin3 (0.5 nmol, 0.1μl) into the rVLM and 0.1 nmol, 0.3nmol urocortin3 into the PVN induced no significant changes of SBP in normotensive rats. However, application of 0.5 nmol urocortin3 in the PVN of normotensive rats leaded to a significantly elevated SBP (from 110.29 ± 5.4 mmHg to 131.25±4.2 mmHg, P <0.01, n=15), with a culmination after 15min of the microinjection. Within the next 20min, the elevated SBP recovered gradually. This hypertensive effect of 0.5 nmol urocortin3 in the PVN was completely eliminated by ASV30, a selective CRF₂ antagonist, microinjection in the same site 5min in advancee. Likewise, microinjection of 0.5 nmol urocortin3 into the PVN of stress-induced hypertensive rats increased SBP significantly compared with that of control group rats (P<0.01, n=7); (5) At the meantime of the increases of SBP in normaltensive rats induced by microinjection of 0.5 nmol urocortin3 into the PVN, the integral of renal nerve discharge augmented from the 100% of basal level to 136%(P<0.05, n=9) within 10 min, then declined slowly to 127% within 20 min and resumed to the normal level within 30 min; (6) Microinjection of 0.5 nmol urocortin 3 into PVN increased the level of glutamate (P<0.05, n=12) while decrease the level of taurine(P<0.05, n=12) in the dialysate, an inhibitory amino acid neurontransmitter in the rVLM during the first 10 min of microinjection; (7) By using extracellular recording and iontophoresis methods, we recorded 38 spontaneously firing neurons in the PVN, with basal firing rate of 8.6±3.1 spikes/s. Of the 38 neurons recorded, 21 neurons showed excitatory response to the iontophoretical appliciation of urocortin3 in a current dependent manner, their firing rate increasing to 10.3±3.6 (P<0.05), 13.8±3.4 (P<0.05) and 17.5±4.4 spikes/s (P<0.05) at the current of 20, 40 and 60 nA, respectively. Once the injection current stopped, their firing rate restored to basal level. The iontophoretic application of ASV30 (40nA) significantly attenuated the excitation induced by urocortin3, but there were no obvious effect when iontophoretically applied alone. Furthermore, during the inotophoresis course of 7-NiNa (20 nA), a selective nNOS inhibitor, urocortin3 still exerted its excitatory effect in the PVN significantly and increased the firing rate to 133% (P<0.05); (8) We tested 17 barosensitive neurons synchronized to heart rate in the rVLM (basal level, 11.5±4.1 spikes/s). All these neurons (n=17) showed current dependently suppressive response to iontophoresisi of 7-NiNa, the firing rate decreasing to 28.7%, 46.1% and 74.8%(P<0.05) of the basal level at 10, 20 and 40 nA, respectively. The mean basal firing rate of another 13 neurons was 10.2±3.7 spikes/s, application of aminoguanidine (AG), a selective iNOS inhibitor, at 10, 20 and 40 nA increased their firing rate to 18.6%, 49.0% and 87.3% (P<0.05) from 11.5±4.1 spikes/s, respectively.Conclusions: (1) In hypertensive rats induced by chronic stimulation of foot shocks combined with buzzer noises, the mRNA expression of urocortin3 in the PVN of hypothalamus was significantly increased. As to the NOS, the expression of nNOS significantly increased in the PVN of hypothalamus and the rVLM, while the expression of iNOS decreased in the rVLM of stress-induced hypertensive rats. (2) As a novel member of CRF family, maybe urocortin3 exerted hypertensive effect in PVN by binding to its selective receptor—CRF₂ on the PVN neurons, increase the sympathetic output through increasingd release of Glu and decreased release of Tau in the rVLM. (3) In the PVN, NO derived from nNOS could inhibit the urocortin3 sensitive neurons so as to antagonize the hypertensive effect induced by urocortin3. While in the rVLM, NO derived from iNOS weakened the hypertensive of urocortin3 by the inhibition to Glu sensitive neurons.