Abstract
An adrenal cGMP-stimulated phosphodiesterase (cGS-PDE) has been shown to mediate atrial natriuretic peptide (ANP)-induced reductions in aldosterone secretion and cAMP levels in primary bovine glomerulosa cells. High concentrations of cGS-PDE have been localized to the zona glomerulosa cell layer of the adrenal cortex using biochemical and immunological techniques. Immunoblot analysis using an affinity-purified, isozyme-specific antiserum revealed a single band that comigrated with a purified cGS-PDE (105 kDa) (1) and that was most highly concentrated in the outermost 1-2 mm of the cortex, representing the capsule and zona glomerulosa regions. Greater than 90% of the overall phosphodiesterase activity present in tissue extracts prepared from these regions was immunoprecipitated using a solid-phase monoclonal antibody reagent, indicating the cGS-PDE as the predominant phosphodiesterase isozyme. Immunohistochemical staining experiments of frozen thin sections of intact adrenal tissue revealed that the cGS-PDE present in this region was localized in the glomerulosa cells themselves. The role of this isozyme as a mediator of ANP-induced decreases in intracellular cAMP concentrations and aldosterone production was tested in primary cultures of bovine adrenal glomerulosa cells. In cells stimulated by ACTH, ANP treatment produced dose-dependent reductions in aldosterone secretion and cellular cAMP content over the same concentration range. Increases in aldosterone production elicited by three cell-permeable cAMP derivatives (8-bromo-cAMP, 8-p-chlorophenylthio-cAMP, and N6-2'-O-dibutyryl-cAMP) were antagonized by ANP, indicating a site of action distal to adenylate cyclase for this hormone. Because the relative magnitude of the ANP effect differed depending upon the derivative used, the three derivatives were compared with respect to their relative rates of in vitro hydrolysis by adrenal cGS-PDE. A positive correlation between their rates of hydrolysis and the degree to which the steroidogenic response produced by these derivatives was antagonized by ANP was demonstrated, further suggesting an ANP-induced activation of the cGS-PDE as being responsible for this effect. The possible contribution of an additional pathway mediated by an inhibitory guanine nucleotide binding regulatory protein (Gi) acting on adenylate cyclase was tested by pretreatment of primary glomerulosa cells with pertussis toxin. Levels of pertussis toxin sufficient to inhibit subsequent in vitro ribosylation did not significantly alter the ANP effect on aldosterone production, although a partial reduction in the ANP effect on cAMP levels was observed.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
(cGS-PDE) has been shown to mediate atrial natri- ation did not significantly alter the atrial natriuretic peptide (ANP) effect on uretic peptide (ANP)-induced reductions in aldoste- aldosterone production, a partial reduction in rone secretion and cAMP levels inprimary bovine the ANP effect on cAMP levels was observed
Immunohisto- Cyclic GMP-stimulated phosphodiesterase’ acchemical staining experiments of frozen thin sections tivities have been observed in a wide variety of tissues [3], of intact adrenal tissue revealed that the cGS-PDE with both soluble and particulate forms having been purified present in thisregion was localized in the glomerulosa to apparent homogeneity from several sources including the cells themselves
Distribution of a n Adrenal cGMP-stimulatedPhosphodiesterase-Using a novel technique in which hypotonic extracts were prepared from sequential slices of intact adrenal gland, phosphodiesterase isozyme content was examined in extracts
Summary
Distribution of a n Adrenal cGMP-stimulatedPhosphodiesterase-Using a novel technique in which hypotonic extracts were prepared from sequential slices of intact adrenal gland, phosphodiesterase isozyme content was examined in extracts. Frozen sections (16 pm) were preknown to contain a t least two other phosphodiesterase iso- pared from fixed adrenal tissue and incubated with affinity-purified zymes values that contribute to activity under these assay obtained in immunoprecipitation experimentswith condi the tpaidroirnmesna,arlysaencttiisoenra as (X described under "Experimental Procedures." 25 magnification) stained with 1:750 dilution a, of purified anti-cGMP-stimulated phosphodiesterase antibody showing cGS-PDE monoclonal antibody reagent were used in determining therelative amounts of this isozyme in thetwo tissues. Calculation of the relative phosphodiesterase levels was based onactivitiesobtained for tissueextracts,using a specific activity value of 105 pmol/min/mg as reported by Martins et al [1].This comparison indicates roughly a 70-fold higher concentration of the cGS-PDE in the outer adrenal regions relative to the heart and corresponds to an estimisaotzeydme concentration of 0.23 pM (for the monomeric protein) in the outer adrenal regions. CGMP-stimulated phosphodiesterase (Fig. 2b), or using antibodies specificfor other phosphodiesterase isozymes (data not presented)
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