Differential alterations in responsiveness in particulate and soluble guanylate cyclases in pregnant guinea pig myometrium.
OBJECTIVE
The mechanism underlying myometrial quiescence during pregnancy is unknown. Our group has previously shown that during pregnancy myometrial cyclic guanosine monophosphate content rises to several hundred times the nonpregnant levels, only to abruptly decline days before the onset of labor. Cyclic guanosine monophosphate plays an integral role in the relaxation of smooth muscle. The aim of this investigation was therefore to determine the effects of pregnancy on both soluble and particulate guanylate cyclase enzymatic activities and messenger ribonucleic acid expressions.


METHODS
Myometrium was obtained from randomly cycling adult nonpregnant guinea pigs and near-term (50-60 days' gestation) pregnant guinea pigs of similar chronologic age. Subcellular fractions were prepared by differential ultracentrifugation. Guanylate cyclase activity was determined by the conversion of guanosine triphosphate to cyclic guanosine monophosphate under basal or stimulated conditions in either the soluble guanylate cyclase or particulate guanylate cyclase fraction. A nitric oxide donor, S-nitroso- N-penacillamine, was used to activate soluble guanylate cyclase (n = 10 animals in each group). Several natriuretic peptides (atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide) and uroguanylin were used to stimulate the different particulate guanylate cyclase isoforms guanylate cyclase A, guanylate cyclase B, and guanylate cyclase C, respectively, in pregnant (n = 8) and nonpregnant (n = 6) animals. Cyclic guanosine monophosphate content was measured by radioimmunoassay, and enzymatic activity was expressed as picomoles of cyclic guanosine monophosphate per milligram of protein per minute. Total guanylate cyclase represented the sum of soluble guanylate cyclase and particulate guanylate cyclase activities for a tissue. To investigate whether the observed changes in guanylate cyclase activity were paralleled by changes in receptor expression, messenger ribonucleic acid levels of the genes for guanylate cyclase A and guanylate cyclase B isoforms were quantified by ribonuclease protection assay (n = 5 animals in each group).


RESULTS
Under basal conditions particulate guanylate cyclase represented 78% (nonpregnant state) to 88% (during pregnancy) of the total guanylate cyclase activity in the guinea pig myometrium. Pregnancy further reduced myometrial soluble guanylate cyclase (both basal and stimulated by nitric oxide) relative to the nonpregnant state. Pregnancy selectively increased atrial natriuretic peptide-stimulated particulate guanylate cyclase activity (attributed to guanylate cyclase A), although it did not change basal myometrial particulate guanylate cyclase activity in general. Guanylate cyclase B (particulate guanylate cyclase stimulated by C-type natriuretic peptide) and guanylate cyclase C (particulate guanylate cyclase stimulated by uroguanylin) activities were unaltered by pregnancy. The selective increase in responsiveness of particulate guanylate cyclase to atrial natriuretic peptide during pregnancy was not paralleled by an increased in level of messenger ribonucleic acid for the gene for guanylate cyclase A.


CONCLUSIONS
Pregnancy reduced the in vitro responsiveness of the myometrial soluble guanylate cyclase to nitric oxide while increasing the responsiveness of the particulate isoform to atrial natriuretic peptide and brain natriuretic peptide through a mechanism independent of any change in receptor expression.
Buhimschi
I A
IA
Department of Obstetrics, Gynecology and Reproductive Sciences, University of Maryland School of Medicine, Baltimore 21201-1559, USA.
San Martin-Clark
O
O
Aguan
K
K
Thompson
L P
LP
Weiner
C P
CP
D000818:Animals; D005260:Female; D006162:Guanylate Cyclase / Q000378:metabolism*; D006168:Guinea Pigs; D007527:Isoenzymes / Q000378:metabolism; D009215:Myometrium / Q000187:drug effects / Q000201:enzymology*; D045283:Natriuretic Agents / Q000494:pharmacology; D009569:Nitric Oxide / Q000502:physiology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D011247:Pregnancy; D011270:Pregnancy, Animal / Q000378:metabolism*; D012016:Reference Values; D026423:S-Nitroso-N-Acetylpenicillamine; D012995:Solubility
D007527:Isoenzymes; D045283:Natriuretic Agents; D020030:Nitric Oxide Donors; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D006162:Guanylate Cyclase; D010396:Penicillamine