Atrial natriuretic peptide induces breakdown of phosphatidylinositol phosphates in cultured vascular smooth‐muscle cells

Abstract

Discrepancies exist between extent of guanylate cyclase activation by atrial natriuretic peptide (ANP) in cell-free systems and ANP-stimulated levels of cyclic GMP in whole cells, and also between receptor affinity and dose effectiveness of ANP. Therefore, we have investigated whether, in addition to receptor-coupled guanylate cyclase activation, other second-messenger cascade systems may be involved in mediating both an increase in cyclic GMP and the physiological response to ANP. Equilibrium ¹²⁵I-ANP binding studies on cultured thoracic aorta smooth muscle cells revealed the existence of low-affinity (∼ 10⁻⁸ M, 84.5 fmol/10⁵ cells) and high-affinity (∼ 10⁻¹⁰ M, 12.5 fmol/10⁵ cells) binding sites. We confirm that ANP elevates intracellular cyclic GMP (EC₅₀∼ 10⁸ M) and inhibits agonist-(isoproterenol and forskolin)-induced increases in intracellular cyclic AMP (IC₅₀∼ 10⁻⁹ M). ANP also stimulated breakdown of phosphatidylinositol phosphates and generation of inositol phosphates with a half-maximally effective concentration of ∼ 10-¹⁰ M. The extent of phosphatidylinositol polyphosphate hydrolysis was small (120%) in comparison to that of phosphatidylinositol (Ptd-Ins) (200%). Ptd-Ins hydrolysis was paralleled by the appearance of glycerophosphoinositol, and there was also a close temporal relationship between these processes and the accumulation of intracellular cyclic GMP. Smooth muscle cells released [³H]arachidonic acid label in response to ANP (EC₅₀∼ 10⁻¹⁰ M). Taken together, the data suggest that the vasorelaxant hormone ANP has stimulatory effects on phosphoinositol lipid metabolism via both phospholipase C (generation of inositol phosphates) and phospholipase A₂ (generation of releasable [³H]arachidonic acid and indirectly glycerophosphoinositol). In contrast, stimulation of phosphatidylinositol phosphate breakdown by the vasoconstrictive hormone angiotensin II is not associated with glycerophosphoinositol formation, and neither cyclic GMP nor cyclic AMP levels were influenced by this hormone.