P382Cyclic guanosine monophosphate compartmentation in vascular smooth muscle cells

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Abstract

Cyclic nucleotide-gated (CNG) channels are usually expressed in photoreceptors and in olfactory neurons sensory. They open by the direct binding of cyclic nucleotides, cAMP and cGMP. The cyclic guanosine monophosphate (cGMP) synthesis is controlled by two types of guanylyl cyclases (GC) that differ in their cellular location and activation by specific ligands: a particular CG present at the plasma membrane, which is activated by natriuretic peptides such as atrial natriuretic pepetide (ANP); and a soluble GC present in the cytosol and activated by nitric oxide (NO). NO and ANP use cGMP as second messenger, there are many instances in which activation of particular and soluble GC leads to different functional effects, one explication for the different functional effects is that cGMP rises in specifc subcellular locations, regulating different targets in different parts of the cell. Recent studies performed by our group in HUA smooth muscle cells demonstrated that ANP increases cGMP levels by activating particulate GC and NO increases cGMP levels by activating soluble GC. In this sense, the aim of this work is to analyze the compartmentalization of the cGMP at the vascular level using cells infected with an adenovirus containing the CNG channels gene. The whole cell configuration of patch clamp technique was used to measure the signal activation of CNG channels, which are activated by cGMP. HUA smooth muscle cells were infected with WT CNGA2 encoding adenovirus. Some drugs were used to analyze the compartmentalization: ANP, SNP (sodium nitroprusside), IBMX (3-isobutyl-1-methylxanthine) (nonselective phosphodiesterase inhibitor), To-156 (specific phosphodiesterase 5 inhibitor), cilostamide (specific phosphodiesterase 3 inhibitor) and Sp-8 (an analogue of the natural signal molecule cyclic GMP). ANP and SNP stimulated, with different intensity, the CNG current. The particulate cGMP pool induced by ANP seems to be controled by the PDE5 and PDE3. Moreover, the administration of SNP seems to created two separated polls, one localized next to the plasma membrane, that is controlled by the PDE5 and PDE3, and other poll that is localized inside (in the middle) of the cells and is regulated by PDE3. Differential spatial and temporal distributions of cGMP may therefore contribute to the specific effects of natriuretic peptides and NO donors on vascular function.

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