Natriuretic Peptides and Nitric Oxide Stimulate cGMP Synthesis in Different Cellular Compartments

Abstract

Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05–10 nM), whereas SNAP (0.01–100 μM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 μM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 μM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 μM SNAP; whereas 100 μM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide.

Synergy between Natriuretic Peptides and Phosphodiesterase 5 Inhibitors Ameliorates Pulmonary Arterial Hypertension

“…These findings fit well with previous reports revealing differential effects of pGC and sGC activation on functions within the same cells (26,27). Indeed, recent studies have provided the rationale for such discrepant effects of the two cGMP-generating pathways (28,29).…”

Phosphodiesterase inhibitors for the treatment of pulmonary hypertension

“…Increasing evidence suggests that the intracellular distribution of cGMP is not uniform, but rather is compartmentalised within cells, and this may underlie the differential effects of cGMP generated by soluble and particulate guanylyl cyclases [3]. The diverse effects of stimulating cGMP production in cardiovascular tissues are dependent on its binding to at least three groups of proteins: cGMPdependent protein kinase (or protein kinase-G; PKG), cGMP-regulated phosphodiesterases (PDEs) and cyclic nucleotide-gated ion channels [4].…”

Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors

“…FRETbased cGMP indicators are also limited in resolving possible compartmentalized intracellular cGMP signaling events using confocal microscopy. It was recently suggested that in VSM cells and cardiac myocytes cGMP signaling may be spatially segregated and that this functional compartmentalization may be the cause of the unique actions of ANP and NO (19)(20)(21)(22)(23). The goal of this study was to develop previously undescribed non-FRET biosensors suitable to monitor the temporal changes of [cGMP] i in response to low-nanomolar NO or ANP, and to investigate the spatial patterning of [cGMP] i , using real-time, confocal imaging techniques.…”