Effects of second messengers on gap junctional intercellular communicationof ovine luteal cells throughout the estrous cycle

Corpora lutea (CL) from Days 5, 10, and 15 after superovulation were enzyma tically dispersed, and a portion of the cells were elutriated to obtain fra ctions enriched with small or large luteal cells. Mixed, small, and large l uteal cell fractions were incubated with no treatment or with agonists or a ntagonists of cAMP (dbcAMP or Rp-cAMPS), protein kinase C (PKC; TPA or H-7) , or calcium (A23187, EGTA, or A23187 + EGTA). The rate of contact-dependen t gap junctional intercellular communication (GJIC) was evaluated by laser cytometry. Media were collected for progesterone (P-4) radioimmunoassay, an d luteal cells cultured with no treatment were fixed for immunocytochemistr y or frozen for Western blot analysis. Luteal cells from each stage of the estrous cycle exhibited GJIC. The dbcAMP increased (P < 0.05) GJIC for all cell types across the estrous cycle. The Rp-cAMPS decreased (P < 0.05) GJIC for small luteal cells on Day 5 and for all cell types on Days 10 and 15. The TPA inhibited (P < 0.01), but H-7 did not affect, GJIC for all cell typ es across the estrous cycle. The A23187 decreased (P < 0.05) GJIC for large luteal cells touching only small or only large luteal cells, whereas A2318 7 + EGTA decreased (P < 0.05) GJIC for all cell types across the estrous cy cle. For the mixed and large luteal cell fractions, dbcAMP increased (P < 0 .05), but TPA and A23187 + EGTA decreased (P < 0.05), P-4 secretion. The A2 3187 alone decreased (P < 0.05) P-4 secretion by large, but not by mixed, l uteal cells. For all days and cell types, the rate of GJIC and P-4 secretio n were correlated (r = 0.113-0.249; P < 0.01). Connexin 43 was detected in cultured luteal cells by immunofluorescence and Western immunoblotting. Thu s, intracellular regulators like cAMP, PKC, or calcium appear to regulate G JIC, which probably is an important mechanism for coordinating function of the ovine CL.