| Part 1 The roles of melanocortin 3/4 receptors in paraventricular nucleus in modulating sympathetic outflow and blood pressureBackgroundExcessive sympathetic activation plays an important role in the pathogenesis of hypertension. Paraventricular nucleus(PVN) is an important vasomotor center that controls sympathetic nerve activity and arterial pressure. Central melanocortin 3/4 receptors(MC3/4R) are known to regulate energy balance. Moderate MC3 R and high MC4 R expressions are found in the PVN. Intracerebroventricular administration of the MC3/4R agonist melanotan II(MTII) produced a dose-dependent sympathetic activation. However, whether the MC3/4R in the PVN are involved in regulating the sympathetic nerve activity and arterial pressure in rats is still unknown. This study was designed to determine the roles of MC3/4R in the PVN in modulating the sympathetic activity and blood pressure, and to investigate its downstream pathway.Objective1. To determine whether the MC3/4R in the PVN are involved in regulating the sympathetic activity and blood pressure.2. To determine whether the cyclic AMP(cAMP)- protein kinase A(PKA) pathway is involved in the MC3/4R- related sympathetic activation and pressor response.MethodsExperiments were carried out on male Sprague-Dawley rats weighing between 350 and 400 g, which were approved by the Experimental Animal Center of Nanjing Medical University. The rats were kept in a temperature-controlled room on a 12 h–12 h light–dark cycle with free access to standard chow and tap water. In anesthetized rats, renal sympathetic nerve activity(RSNA) and mean arterial pressure(MAP) were recorded by a Power Lab data acquisition system. The bilateral PVN microinjections were performed with stereotaxie instrument. The enzyme-linked immunosorbent assay was used to measure the c AMP level in the PVN.Protocols:1. Rats were randomly divided into 12 groups(n = 6 for each). The effect of the PVN microinjections of saline, three doses of MTII(0.05, 0.1 and 0.2 nmol), Ag RP(0.4 nmol), SHU9119(0.4 nmol), three doses of HS024(0.2, 1.0 and 5.0 nmol), db-c AMP(1.0 nmol), SQ22536(2.0 nmol), Rp-c AMP(1.0 nmol) or MTII(0.2 nmol) after PVN microinjection of Ag RP(0.4 nmol), SHU9119(0.4 nmol), three doses of HS024(0.2, 1.0 and 5.0 nmol), SQ22536(2.0 nmol) or Rp-c AMP(1.0 nmol) on the RSNA and MAP were determined.2. Rats were randomly divided into 2 groups(n = 6 for each). The effects of PVN microinjection of saline or MTII(0.2 nmol) on c AMP level in the PVN were determined.3. Rats were randomly divided into 2 groups(n = 6 for each). The effects of PVN or hypothalamic area(HA) microinjection of MTII(0.2 nmol) on the RSNA and MAP were determined.Results1. MC3/4R agonist MTII increased the RSNA and MAP. MC3/4R antagonist AgRP or SHU9119 decreased the RSNA and MAP. MC4 R antagonist HS024 had nosignificant effect on the RSNA and MAP. PVN pretreatment with Ag RP or SHU9119 completely, HS024 partly, blocked the increasing effects of MTII on the RSNA and MAP.2. The cAMP analogue, db-cAMP, increased the RSNA and MAP. Adenylate cyclase(AC) inhibitor, SQ22536, had no significant effect on the RSNA and MAP. The PKA inhibitor, Rp-c AMP, decreased the RSNA and MAP. Either SQ22536 or Rp-c AMP abolished MTII-induced RSNA and MAP increases.3. PVN microinjection of MTII increased the c AMP level in the PVN.Conclusion1. MC3/4R activation in the PVN increases the sympathetic outflow and blood pressure. MC3 R rather than MC4 R in the PVN exert a tonic excitatory effect on sympathetic activity.2. The c AMP-PKA pathway is involved in the MC3/4R activation induced sympathetic activation and pressor response.Part 2 The roles of melanocortin 3/4 receptors in the paraventricular nucleus in modulating adipose afferent reflexBackgroundObesity is a major medical problem whose prevalence is increasing. Sympathetic activity and arterial blood pressure are increased in obesity and most of them develop into hypertension. The adipose afferent reflex(AAR) can be induced by the stimulation of white adipose tissue(WAT) by chemicals such as capsaicin, bradykinin, adenosine or leptin, and results in the increases in sympathetic outflow and artery blood pressure. Enhanced AAR plays an important role in sympathetic activation of obesity induced hypertension.PVN is an important vasomotor center that controls sympathetic nerve activity and arterial pressure. Central MC3/4R are known to regulate energy balance. Moderate MC3 R and high MC4 R expressions are found in the PVN. Intracerebroventricular administration of the MC3/4R agonist MTII produced a dose-dependent sympathetic activation. Our recent study has shown that MC3/4R activation in the PVN increases the sympathetic activity and blood pressure. However, whether the MC3/4R in the PVN are involved in regulating the AAR in rats is still unknown. This study was designed to determine the roles of MC3/4R in the PVN in modulating the AAR, and to investigate its downstream pathway.Objective1. To determine whether the MC3/4R in the PVN are involved in regulating the AAR. 2. To determine whether the c AMP- PKA mediates the effects of MC3/4R activationon the AAR.MethodsExperiments were carried out on male Sprague-Dawley rats weighing between 350 and 400 g, which were approved by the Experimental Animal Center of Nanjing Medical University. The rats were kept in a temperature-controlled room on a 12 h–12 h light–dark cycle with free access to standard chow and tap water. In anesthetized rats, RSNA and MAP were recorded on a Power Lab data acquisition system. The AAR was evaluated by the RSNA and MAP response to inguinal white adipose tissue(i WAT) injection of capsaicin. The bilateral PVN microinjections were performed with stereotaxie instrument. The enzyme-linked immunosorbent assay was used to measure the c AMP level in the PVN.Protocols:1. Rats were randomly divided into 9 groups(n = 6 for each). The effects of the PVN microinjections of saline, three doses of MTII(0.1, 0.2 or 0.4 nmol), SHU9119(0.4 nmol), HS024(1.0 nmol), db-c AMP(1.0 nmol), SQ22536(2.0 nmol) or Rp-c AMP(1.0 nmol) on the AAR were determined.2. Rats were randomly divided into 5 groups(n = 6 for each). The effects of pretreatment with PVN microinjection of saline, SHU9119(0.4 nmol), HS024(1.0 nmol), SQ22536(2.0 nmol) or Rp-c AMP(1.0 nmol) on the AAR responses to MTII were determined.3. Rats were randomly divided into 2 groups(n = 6 for each).The effects of PVN microinjection of saline or MTII(0.2 nmol) on c AMP level in the PVN were determined.4. Rats were randomly divided into 2 groups(n = 6 for each). The effects of i WAT injection of saline or capsaicin on c AMP level in the PVN were determined.5. Rats were randomly divided into 2 groups(n = 6 for each). The effects of thePVN pretreatment with saline or SHU9119(0.4 nmol) on c AMP level changes induced by i WAT injection of capsaicin were determined.Results1. MC3/4R agonist MTII enhanced the AAR. MC3/4R antagonist SHU9119 or MC4 R antagonist HS024 attenuated the AAR. PVN pretreatment with SHU9119 or HS024 blocked the enhancing effects of MTII on the AAR.2. The c AMP analogue, db-c AMP, enhanced the AAR. Both the AC inhibitor, SQ22536 and the PKA inhibitor, Rp-c AMP, attenuated the AAR. Either SQ22536 or Rp-c AMP abolished MTII-induced AAR enhancement.3. Both PVN microinjection of MTII and i WAT injection of capsaicin increased the c AMP level in the PVN. SHU9119 in PVN abolished i WAT injection of capsaicin-induced c AMP level increase in the PVN.Conclusion1 MC4 R rather than MC3 R activation in the PVN enhances the AAR.2. The cAMP- PKA pathway mediates the effects of MC3/4R activation on the AAR. |
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