Regulation of the cGMP-cPKG pathway and large-conductance Ca2+-activated K+ channels in uterine arteries during the ovine ovarian cycle

Abstract

The follicular phase of the ovine ovarian cycle demonstrates parallel increases in ovarian estrogens and uterine blood flow (UBF). Although estrogen and nitric oxide contribute to the rise in UBF, the signaling pathway remains unclear. We examined the relationship between the rise in UBF during the ovarian cycle of nonpregnant sheep and changes in the uterine vascular cGMP-dependent pathway and large-conductance Ca(2+)-activated K(+) channels (BK(Ca)). Nonpregnant ewes (n = 19) were synchronized to either follicular or luteal phase using a vaginal progesterone-releasing device (CIDR), followed by intramuscular PGF(2alpha), CIDR removal, and treatment with pregnant mare serum gonadotropin. UBF was measured with flow probes before tissue collection, and second-generation uterine artery segments were collected from nine follicular and seven luteal phase ewes. The pore-forming alpha- and regulatory beta-subunits that constitute the BK(Ca), soluble guanylyl cyclase (sGC), and cGMP-dependent protein kinase G (cPKG) isoforms (cPKG(1alpha) and cPKG(1beta)) were measured by Western analysis and cGMP levels by RIA. BK(Ca) subunits were localized by immunohistochemistry. UBF rose >3-fold (P < 0.04) in follicular phase ewes, paralleling a 2.3-fold rise in smooth muscle cGMP and 32% increase in cPKG(1alpha) (P < 0.05). sGC, cPKG(1beta), and the BK(Ca) alpha-subunit were unchanged. Notably, expression of beta(1)- and beta(2)-regulatory subunits rose 51 and 79% (P <or= 0.05), respectively. Increases in endogenous ovarian estrogens in follicular-phase ewes result in increases in UBF associated with upregulation of the cGMP- and cPKG-dependent pathway and increased vascular BK(Ca) beta/alpha-subunit stoichiometry, suggesting enhanced BK(Ca) activation contributes to the follicular phase rise in UBF.

Fig. 1.

Representative immunoblot for the 42-kDa protein α-actin that served as a loading control for studies of cGMP-dependent protein kinase G (cPKG_1α) and the large-conductance Ca²⁺-activated K⁺ channel (BK_Ca) β₁-subunit. There were no differences between luteal (n = 4) and follicular (n = 4) phase uterine arteries, P > 0.8 by nonpaired t-test on each immunoblot. Supplemental Fig. 1 shows the relationship with the proteins of interest.

Fig. 2.

Change in basal uterine blood flow during the luteal and follicular phases of the ovine ovarian cycle. Data are means ± SE; *P < 0.04, nonpaired t-test.

Fig. 3.

Comparison of cGMP contents in luteal (n = 3) and follicular (n = 3) phase uterine arteries during the ovine ovarian cycle. Data are means ± SE; *P < 0.02, nonpaired t-test.

Fig. 4.

Effect of the ovarian cycle on cPKG_1α and cPKG_1β expression in uterine arteries from luteal (n = 4) and follicular (n = 6) phase sheep. A: immunoblot analyses. B: densitometry in arbitrary units from the immunoblots for cPKG_1α (B1) and cPKG_1β (B2). Data are means ± SE; *P = 0.047, nonpaired t-test.

Fig. 5.

Effect of the ovarian cycle on BK_Ca subunit expression in the uterine artery during the luteal (n = 4) and follicular (n = 6) phases. A: immunoblot analyses for α-, β₁-, and β₂-subunits. B: densitometry in arbitrary units from the immunoblots for α (B1)-, β₁ (B2)-, and β₂ (B3)-subunits. Data are means ± SE; *P = 0.009 and **P = 0.05, nonpaired t-test.

Fig. 6.

Representative immunohistochemistry of BK_Ca α-, β₁-, and β₂-subunits in randomly selected uterine arteries during the luteal (A–D) and follicular (E–H) phases of the ovine ovarian cycle. Figures are at ×40 magnification. NIRS, control nonimmune rabbit serum; L, vessel lumen.