Agents increasing cyclic GMP amplify 5-HT4-elicited positive inotropic response in failing rat cardiac ventricle

Abstract

Activation of 5-HT(4) receptors in failing ventricles elicits a cAMP-dependent positive inotropic response which is mainly limited by the cGMP-inhibitable phosphodiesterase (PDE) 3. However, PDE4 plays an additional role which is demasked by PDE3 inhibition. The objective of this study was to evaluate the effect of cGMP generated by particulate and soluble guanylyl cyclase (GC) on the 5-HT(4)-mediated inotropic response. Extensive myocardial infarctions were induced by coronary artery ligation in Wistar rats, exhibiting heart failure 6 weeks after surgery. Contractility was measured in left ventricular preparations. Cyclic GMP was measured by EIA. In ventricular preparations, ANP or BNP displayed no impact on 5-HT(4)-mediated inotropic response. However, CNP increased the 5-HT(4)-mediated inotropic response as well as the β(1)-adrenoceptor (β(1)-AR)-mediated response to a similar extent as PDE3 inhibition by cilostamide. Pretreatment with cilostamide eliminated the effect of CNP. Inhibition of nitric oxide (NO) synthase and soluble GC by L-NAME and ODQ, respectively, attenuated the 5-HT(4)-mediated inotropic response, whereas the NO donor Sin-1 increased this response. The effects were absent during PDE3 inhibition, suggesting cGMP-dependent inhibition of PDE3. However, in contrast to the effects on the 5-HT(4) response, Sin-1 inhibited whereas L-NAME and ODQ enhanced the β(1)-AR-mediated inotropic response. cGMP generated both by particulate (NPR-B) and soluble GC increases the 5-HT(4)-mediated inotropic response in failing hearts, probably through inhibition of PDE3. β(1)-AR and 5-HT(4) receptor signalling are subject to opposite regulatory control by cGMP generated by soluble GC in failing hearts. Thus, cGMP from different sources is functionally compartmented, giving differential regulation of different G(s)-coupled receptors.

Fig. 1

Concentration–response curves of inotropic responses to 5-HT₄ receptor stimulation in the absence (control) or presence of natriuretic peptides; 1 μM ANP, 1 μM BNP or 300 nM CNP. Natriuretic peptides were added 20 min before 5-HT. Ordinate: inotropic response expressed as increase in (dF/dt)_max in percent above basal. Vertical bars represent SEM of maximal inotropic responses. Abscissa: concentration of the agonist. Horizontal bars represent SEM of –logEC₅₀. Double asterisks: p < 0.01 vs. control

Fig. 2

a Concentration–response curves of inotropic responses to 5-HT₄ receptor stimulation in papillary muscles in the absence (control) or presence of 300 nM CNP or in combination with PDE inhibitors (Cil, 1 μM cilostamide; Rol, 10 μM rolipram). b Concentration–response curves of inotropic responses (normalized) to β₁-adrenoceptor stimulation by (−)-noradrenaline (50 nM ICI118551 present) in absence (control) or presence of CNP or in combination with PDE inhibitors (Cil, 1 μM cilostamide; Rol, 10 μM rolipram) in left ventricular strips. CNP and PDE inhibitors were added 20 and 45 min before the agonist, respectively. Ordinate: inotropic response expressed as increase in (dF/dt)_max in percent above basal (a) or as percent of maximum (normalized) (b). Vertical bars represent SEM of maximal inotropic responses. Abscissa: concentration of the agonist. Horizontal bars represent SEM of –logEC₅₀. Double asterisks: p < 0.01 CNP vs. control (for maximal response in a, and –logEC₅₀ in b). Single crosses: p < 0.01 CNP/Rol vs. CNP (for maximal response in a, and –logEC₅₀ in b). Double crosses: p < 0.01 –logEC₅₀ CNP/Rol vs. CNP

Fig. 3

Cyclic GMP levels in left ventricular strips in absence (control) or presence of 100 μM l-NAME (NOS inhibitor), 10 μM ODQ (sGC inhibitor) or 300 μM Sin-1 (NO donor). l-NAME and ODQ were added 45 min before and Sin-1 20 min before the ventricular strips were snap-frozen. Number of experiments “n” inside the bars; mean±SEM. Asterisk: p < 0.05 vs. control. Number sign: p = 0.16 vs. control (after Bonferroni correction)

Fig. 4

a, b Concentration–response curves of inotropic responses to 5-HT₄ receptor stimulation in the absence (control) and presence of 100 μM l-NAME (NOS inhibitor), 10 μM ODQ (sGC inhibitor), 300 μM Sin-1 (NO donor) or in combination with PDE inhibitors (Cil, 1 μM cilostamide; Rol, 10 μM rolipram) in papillary muscles. All the inhibitors were added 45 min before 5-HT, whereas Sin-1 was added 20 min before 5-HT. Ordinate: inotropic response expressed as increase in (dF/dt)_max in percent above basal. Vertical bars represent SEM of inotropic response. Abscissa: concentration of the agonist. Horizontal bars represent SEM of –logEC₅₀. Asterisk: p < 0.05 vs. control. Number sign: p = 0.08 vs. control (after Bonferroni correction)

Fig. 5

Concentration–response curves of inotropic responses (normalized) to β₁-adrenoceptor stimulation by (−)-noradrenaline (50 nM ICI118551 present) in absence (control) or presence of 100 μM l-NAME (NOS inhibitor), 10 μM ODQ (sGC inhibitor) or 300 μM Sin-1 (NO donor) in left ventricular strips. l-NAME and ODQ were added 45 min before and Sin-1 20 min before the (−)-noradrenaline. Ordinate: inotropic response expressed as increase in (dF/dt)_max in percent of maximum (normalized). Abscissa: concentration of the agonist. Horizontal bars represent SEM of –logEC₅₀. Double asterisks: p < 0.01 vs. control

Fig. 6

Illustration of the proposed functional compartmentation of 5-HT₄ receptor, β₁-adrenoceptor and cyclic nucleotides in cardiac myocytes. The inotropic response to 5-HT₄ receptor and β₁-adrenoceptor stimulation is mediated by cAMP and regulated by PDE3. 1: cGMP generated by NPR-B inhibits PDE3 and increases the 5-HT₄ receptor- and β₁-adrenoceptor-mediated inotropic response. 2: cGMP generated by NPR-A has no access to the PDE3 regulating inotropic response. 3: A pool of cGMP generated by sGC increases the 5-HT₄-mediated inotropic response by inhibiting PDE3. 4: A pool of cGMP generated by sGC has no access to the PDE3 regulating the β₁-adrenoceptor-mediated inotropic response, but inhibits it by some other mechanism, possibly through PKG