Up-regulation of nitric oxide synthase and modulation of the guanylate cyclase activity by corticotropin-releasing hormone but not urocortin II or urocortin III in cultured human pregnant myometrial cells

Abstract

The biological actions of corticotropin-releasing hormone (CRH) in the human myometrium during pregnancy and labor are unknown. We hypothesized that CRH may modulate the nitric oxide system, and influence myometrial relaxation/contractility. Incubation of myometrial cells with CRH, but not urocortin II or urocortin III, for 8-16 h significantly induced mRNA and protein expression of endothelial and brain but not inducible nitric oxide synthase (NOS) isoforms. This action resulted in increased activity of soluble guanylate cyclase (GC(s)), demonstrated by the enhanced cGMP-producing capacity of the NO donor, sodium nitroprusside. CRH also caused acute activation of the membrane-bound GC, shown by increased basal or atrial natriuretic peptide (ANP)-stimulated cGMP production. These effects appeared to be mediated via the R1 receptors because the CRH receptor antagonists, astressin and antalarmin but not anti-sauvagine 30, could block them. The acute effects of CRH were significantly reduced by inhibition of protein kinase A (PKA) activity, suggesting it is partially PKA dependent. Activation of protein kinase C (PKC) resulted in significant inhibition of both ANP-and CRH-stimulated cGMP production, suggesting a direct effect of PKC on membrane-bound GC. In conclusion, CRH appears to have a dual effect on myometrial NOS/GC pathway, a short term effect predominantly mediated by PKA, and a long-term effect increasing constitutive NOS expression, mediated by a PKA-independent mechanism. This mechanism could potentially be active during human pregnancy, and, because cGMP stimulates myometrial relaxation, these findings further suggest that during pregnancy CRH primarily activates intracellular signals that contribute to the maintenance of myometrial quiescence.

Figure 1

Quantitative analysis of CRH receptor mRNA expressed in human pregnant myometrial tissue and myometrial cells by using the real time PCR. Similar results were obtained from three independent myometrial tissue biopsies/cell preparations.

Figure 2

NOS isoforms detection by immunofluorescence in human pregnant myometrial cells. Antibodies specific for brain (a and b), endothelial (c and d), and inducible (e and f) NOS isoforms were used in the absence or presence of blocking serum (b, d, and f). Expression of NOS isoforms is indicated by the FITC staining in the cell-cytoplasm. The nuclei were stained blue with 4′,6-diamidino-2-phenylindole (DAPI). (Double exposure; magnification, ×400.) Identical results were obtained from three independent myometrial cell preparations.

Figure 3

Changes in myometrial cell NOS isoform protein or mRNA expression or detected by immunostaining or fluorescent in situ hybridization (FISH). (a–d, i, and j) Cells treated without or with CRH for 16 h before staining for eNOS (a and b) or iNOS (e and f) with specific antibodies. Also, cells were treated without or with CRH for 16 h in the presence of antalarmin (100 nM; i) or anti-sauvagine 30 (1 μM; j). (e–h) Cells treated without or with CRH for 16 h before staining for eNOS (e and f) or iNOS (g and h) with specific oligonucleotide probes. (Magnification, ×400.) Identical results were obtained from five independent cell preparations.

Figure 4

Effect of CRH pretreatment on GC_s activity in human pregnant myometrial cells. cGMP levels of cells pretreated for 16 h with (a) different concentrations of CRH or (b) 100 nM CRH in the presence or absence of astressin (1 μM), or (c) 100 nM CRH in the presence or absence of myristoylated protein kinase A inhibitor fragment 14–22 (10 μM) or antalarmin (100 nM) or anti-sauvagine 30 (1 μM), before stimulation with SNP (0.1 mM) with or without l-NAME (0.1 mM). Results are representative of three independent cell preparations, and each point is the mean ± SE of three determinations. *, P < 0.05, compared with basal values; +, P < 0.05, compared with SNP-stimulated cGMP values.

Figure 5

(a) Acute CRH effects on GC_s activity in human pregnant myometrial cells. cGMP levels of cells pretreated for 30 min with vehicle alone, l-NAME (0.1 mM), or ODQ (10 μM) before stimulation with SNP (0.1 mM) in the presence or absence of 100 nM CRH. Results are representative of three independent cell preparations, and each point is the mean ± SE of three determinations. *, P < 0.05, compared with basal values; +, P < 0.05, compared with SNP-stimulated cGMP values. **, P < 0.05, compared with values obtained from cells pretreated with vehicle alone followed by stimulation with CRH or CRH + SNP. (b) Autoradiograph of CRH-induced photolabeling (with [³²P]GTP-AA) of various Gα-proteins in human pregnant myometrial cells. Identical results were obtained from six independent cell culture preparations.

Figure 6

Acute CRH effects on GC_m activity in human pregnant myometrial cells. cGMP levels of cells incubated with (a) different concentrations of CRH, or (b) ANP (100 nM) plus CRH (0–100 nM; filled diamonds) in the absence or presence of antalarmin (100 nM; filled squares), or anti-sauvagine 30 (1 μM; open squares) or astressin (1 μM; open circles). (c) Cells preincubated for 30 min in the presence or absence of myristoylated protein kinase A inhibitor fragment 14–22 (10 μM), or ODQ (10 μM) before stimulation with ANP (100 nM), CRH (100 nM), forskolin(10 μM), or ANP (100 nM) plus either CRH (100 nM) or forskolin (10 μM). Results are representative of three independent cell culture preparations, and each point is the mean ± SE of three determinations. *, P < 0.05, compared with basal values; +, P < 0.05, compared with ANP-stimulated cGMP values. **, P < 0.05, compared with values obtained from cells pretreated with vehicle alone followed by stimulation with CRH (or forskolin) + ANP.

Figure 7

Effect of phorbol 12-myristate 13-acetate (PMA) on GC_s and GC_m activity in human pregnant myometrial cells. cGMP levels of cells preincubated with either PMA overnight or for 2 h with or without bisindolylmaleimide I (100 nM) before 2-h treatment with or without PMA (200 nM). Subsequently, cells were stimulated with (a) SNP (0.1 mM) or (b) ANP (100 nM), or ANP (100 nM) plus CRH (100 nM). The effects of overnight PMA or 2 h with bisindolylmaleimide I treatment on PKC activity or PKC isoform expression are shown in c. Results are representative of three independent cell preparations, and each point is the mean ± SE of three determinations. *, P < 0.05, compared with untreated values; +, P < 0.05, compared with ANP or ANP plus CRH-induced cGMP values in the absence of PMA.