Fig. 6.

A and B, histamine H₃- and H₄ receptor activation inhibits the increase in PKG activity elicited by BNP in PC12-H₃ and PC12 cells, respectively. C and D, H₃ and H₄ receptor activation synergizes with PKG inhibition in attenuating BNP-induced NE release in cardiac synaptosomes, respectively. A, PKG activity in PC12-H₃ cells treated with 8-Br-cGMP (1 μM; positive control) or BNP (100 nM) in the absence or presence of the PKG inhibitor Rp-8-Br-cGMPS (0.5 μM) or the H₃ receptor agonist methimepip (1 nM), either alone or in combination with the H₃ receptor antagonist JNJ5207852 (30 nM). Top band, representative immunoblot of PC12-H₃ cell lysate probed with antiphosphorylated VASP (p-VASP; a major PKG substrate) antibody. Bottom band, same immunoblot probed with anti-β-actin antibody. Bars represent mean quantitative values (± S.E.M.; n = 4). Significantly different from control: ***, P < 0.0001 by unpaired t test. Significantly different from BNP: †††, P < 0.001 by unpaired t test. Significantly different from BNP + H₃ receptor agonist + H₃ receptor antagonist: ###, P < 0.001 by unpaired t test. B, PKG activity in PC12 cells treated with 8-Br-cGMP (1 μM; positive control) or BNP (100 nM) in the absence or presence of the PKG inhibitor Rp-8-Br-cGMPS (0.5 μM) or the H₄ receptor agonist (4-methylhistamine; 20 μM), either alone or in combination with the H₄ receptor antagonist (A943931; 300 nM). The H₃ receptor agonist methimepip (1 nM; negative control) failed to modify the response to BNP in PC12 cells, which do not constitutionally express H₃ receptors. Top band, representative immunoblot of PC12 cell lysate probed with antiphosphorylated VASP antibody. Bottom band, same immunoblot probed with anti-β-actin antibody. Bars represent mean quantitative values (± S.E.M.; n = 4). Significantly different from control: ***, P < 0.001 and **, P < 0.01 by unpaired t test. Significantly different from BNP: ††, P < 0.01 by unpaired t test. Significantly different from BNP + H₄R agonist + H₄R antagonist: ##, P < 0.01 by unpaired t test. C, inhibition of BNP (100 nM)-induced NE release in cardiac synaptosomes by subthreshold concentrations of the PKG inhibitor Rp-8-Br-cGMPS (0.3 μM) and the H₃ receptor agonist methimepip (0.03 nM), administered either alone or in combination. D, inhibition of BNP-induced NE release in cardiac synaptosomes by subthreshold concentrations of the PKG inhibitor Rp-8-Br-cGMPS and the H₄ receptor agonist 4-methyl histamine (0.03 μM), administered either alone or in combination. Note in C and D that a significant attenuation of NE release occurs when the PKG inhibitor is combined either with the H₃ or the H₄ receptor agonist (*, P < 0.05 and **, P < 0.005 by unpaired t test). Bars, means ± S.E.M. (n = 8–18), represent the BNP-induced increase in NE release above the basal level of 232.1 ± 8.9 pmol/mg (n = 25).