Calcium-independent and cAMP-dependent Modulation of Soluble Guanylyl Cyclase Activity by G Protein-coupled Receptors in Pituitary Cells

Tatjana S Kostic,Melanija Tomić,Silvana A Andric,Stanko S Stojilkovic

doi:10.1074/jbc.m112439200

Abstract

It is well established that G protein-coupled receptors stimulate nitric oxide-sensitive soluble guanylyl cyclase by increasing intracellular Ca(2+) and activating Ca(2+)-dependent nitric-oxide synthases. In pituitary cells receptors that stimulated adenylyl cyclase, growth hormone-releasing hormone, corticotropin-releasing factor, and thyrotropin-releasing hormone also stimulated calcium signaling and increased cGMP levels, whereas receptors that inhibited adenylyl cyclase, endothelin-A, and dopamine-2 also inhibited spontaneous calcium transients and decreased cGMP levels. However, receptor-controlled up- and down-regulation of cyclic nucleotide accumulation was not blocked by abolition of Ca(2+) signaling, suggesting that cAMP production affects cGMP accumulation. Agonist-induced cGMP accumulation was observed in cells incubated in the presence of various phosphodiesterase and soluble guanylyl cyclase inhibitors, confirming that G(s)-coupled receptors stimulated de novo cGMP production. Furthermore, cholera toxin (an activator of G(s)), forskolin (an activator of adenylyl cyclase), and 8-Br-cAMP (a permeable cAMP analog) mimicked the stimulatory action of G(s)-coupled receptors on cGMP production. Basal, agonist-, cholera toxin-, and forskolin-stimulated cGMP production, but not cAMP production, was significantly reduced in cells treated with H89, a protein kinase A inhibitor. These results indicate that coupling seven plasma membrane-domain receptors to an adenylyl cyclase signaling pathway provides an additional calcium-independent and cAMP-dependent mechanism for modulating soluble guanylyl cyclase activity in pituitary cells.

Highlights

We focused investigations on two receptors signaling through a Gs pathway, growth hormone-releasing hormone (GHRH) and corticotropin-releasing factor (CRF) receptors; two receptors signaling through a Gq/G11 pathway, thyrotropin-releasing hormone (TRH) receptor [26] and the endothelin (ET)-A receptor that couples to a Gi/Go signaling pathway [27, 28]; and the
GHRH, CRF, and TRH induced a significant and PDEindependent increase in cAMP accumulation (Fig. 1, left panels and Table I). These data indicate that an agonist-induced rise in cGMP levels does not result from modulation of PDE activity but represents de novo cGMP production
In accordance with this hypothesis, earlier studies have indicated that sGC is expressed in pituitary cells as well as nNOS and eNOS, and that spontaneous electrical activity and calcium signaling provide an effective mechanism for activation of NOS and subsequent stimulation of sGC [18, 24]

Summary

Introduction

In normal and immortalized pituitary cells, calcium-sensitive nNOS and eNOS were detected (9 –15) The activity of these enzymes was confirmed by measurements of NO, NO2, and NO3 under different experimental paradigms [16, 17]. Basal sGC activity is partially dependent on spontaneous voltage-gated calcium influx [18]. These findings support a generally accepted view about the relevance of calcium in activation of the NOS/sGC signaling pathway by GPCRs. Recent studies have indicated that high [Ca2ϩ]i inhibits cGMP accumulation, an action presumably mediated by direct inhibitory effects of [Ca2ϩ]i on sGC activity [24, 25]

Methods

Results

Conclusion