Cross-Talk between CB1, AT1, AT2 and Mas Receptors Responsible for Blood Pressure Control in the Paraventricular Nucleus of Hypothalamus in Conscious Spontaneously Hypertensive Rats and Their Normotensive Controls

Figures

• 

  Figure 1. A representative photograph of the microinjection site in the paraventricular nucleus of hypothalamus (PVN) coupled with the matching slide from the Paxinos and Watson rat brain atlas [24]. One mm thick brain slice with the injection site shown by Evans blue dye. The drawn outline represents the confines of PVN.

• 

  Figure 2. Effects of AM251 and A-779 on basal systolic (SBP), diastolic (DBP) and mean (MBP) blood pressure in conscious Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). CB1 receptor antagonist AM251 was injected intravenously (a) or into the paraventricular nucleus of hypothalamus (PVN) (b). Mas receptor antagonist A-779 was injected into the PVN. Results are presented as mean ± SEM (n = 21–27 for A; n = 10 for B; n = 6–7 for (c) and (d). * p < 0.05 compared to WKY; ## p < 0.01, ### p <0.001 compared to A-779 in the absence of AM251.

• 

  Figure 3. Representative original traces of the pressor effects of angiotensin II (a), angiotensin 1–7 (b) and CP55940 (after i.v. administration of AM251) (c) injected into the paraventricular nucleus of hypothalamus (PVN) on blood pressure (BP) and mean blood pressure (MBP) in conscious Wistar Kyoto (WKY) and spontaneously in hypertensive rats (SHR). Arrows show the moment of application of the particular agonist.

• 

  Figure 4. Effect of angiotensin II (AT1/2 receptor agonist) (a,b) and angiotensin 1–7 (Mas receptor agonist) (c,d) on systolic (SBP), diastolic (DBP) and mean (MBP) blood pressure, and their interaction with AT1, AT2 and Mas receptor antagonists (losartan, PD123319 and A-779, respectively) injected into the paraventricular nucleus of hypothalamus (PVN) in conscious Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Results are presented as mean ± SEM (n = 7–8) and calculated as change in basal values determined before agonist injection. # p < 0.05, ## p < 0.01, ### p < 0.001 compared to the corresponding control group; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to WKY.

• 

  Figure 5. Influence of the CB1 receptor antagonist AM251 on the pressor effect of angiotensin II (AT1/2 receptor agonist) (a,b) and angiotensin 1–7 (Mas receptor agonist) (c,d) injected into the paraventricular nucleus of hypothalamus (PVN) in conscious Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Results, presented as mean ± SEM (n = 7–8), are calculated as change in basal values determined before agonist/antagonist injection. # p < 0.05, ### p < 0.001 compared to the corresponding control group. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to WKY.

• 

  Figure 6. Influence of AT1 and AT2 (losartan and PD123319; (a,b), and Mas (A-779; (c,d) receptor antagonists on the pressor effect of the CB1 receptor agonist CP55940 injected into the paraventricular nucleus of hypothalamus (PVN) in conscious Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). All experiments were performed after i.v. injection of the CB1 receptor antagonist AM251. Results, presented as mean ± SEM (n = 5–7), are calculated as the change in basal values determined before agonist injection. # p < 0.05, ## p < 0.01, ### p < 0.001 compared to the corresponding control group; * p < 0.05 compared to WKY.

• 

  Figure 7. Fold change of cannabinoid CB1 (a), AT2 (b) and Mas (c) receptors and representative Western blots of the paraventricular nucleus of hypothalamus (PVN), rostral ventrolateral medulla (RVLM) and nucleus tractus solitarii (NTS) in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). β-actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control. Results presented as mean ± SEM (n = 6). ** p < 0.01, *** p < 0.001 compared to WKY. Uncropped Western blot images can be seen in Supplementary Materials, file Figure S1.

• 

  Figure 8. Mechanisms involved in the pressor response induced by Ang II and CP55940. Ang II, via AT1 receptors, leads to endocannabinoid formation in the paraventricular nucleus of hypothalamus (PVN). Endocannabinoids in turn inhibit GABA release by activation of CB1 receptors. Since little GABA is released, much glutamate (Glu) can be released, also since facilitatory AT1 receptors on the glutamatergic neuron further increase Glu release. The extents of Glu release and of the pressor response are correlated. The pressor response in SHR increased since AT1 and CB1 receptor density in their PVN increased. On the other hand, the pressor response induced by Ang II can be attenuated by CB1 receptor antagonist AM251. In addition, the pressor response induced by the CB1 receptor agonist CP55940 can be antagonized by AT1 receptor antagonist losartan.