Staurosporine does not prevent adrenergic-induced situs inversus, but causes a unique syndrome of defects in rat embryos grown in culture.

Staurosporine, an alkaloid isolated from Streptomyces species, is commonly used as a protein kinase C (PKC) inhibitor in animal investigations. In the present study, we used this compound to determine whether alpha 1-adrenergic stimulation-induced situs inversus in rats is mediated by PKC. Embryos were explanted at 8 A.M. on day 9 of gestation. Those with a neural groove but with no visible neural folds (Stage 11a) were selected and were cultured in medium containing various concentrations of staurosporine with or without 50 microM of phenylephrine, an alpha 1-adrenergic agonist. At 10 A.M. on day 11 of gestation, embryos were examined for situs inversus and other abnormalities. Staurosporine, tested at 0.0001, 0.001, 0.01, 0.1, 0.375, and 0.5 microM (lethal concentration), did not block phenylephrine-induced situs inversus at any concentration. However, staurosporine alone produced situs inversus at concentrations above 0.1 microM. At 0.5 and 1.0 microM, staurosporine also caused cyst-like lesions projecting dorsally from the mesencephalon that we named "mesencephalic vesicles" and the formation of secondary somites. To confirm and further examine these unique effects of staurosporine both grossly and histologically, we conducted additional experiments using staurosporine from another source. Embryos were explanted between 6 A.M. and 9 P.M. on day 9 of gestation and were placed in one of the following groups according to their stage of development: 10b, 11a, 11b, 11c, 12/s1-2, 12/s3-4, and 12/s5-6. Embryos were then cultured with various concentrations of staurosporine. Those cultured from Stage 11a exhibited similar lesions to those seen in the initial experiment but at somewhat higher concentrations of staurosporine. Embryos cultured from Stage 10b showed a similar pattern of lesions as seen at Stage 11a, except that higher concentrations of staurosporine were required to cause mesencephalic vesicles and secondary somites formation. Embryos cultured from Stage 11b showed similar effects to those cultured from younger stages except that maximum incidences of situs inversus were much lower. Those cultured from Stage 11c showed similar dose-response to those cultured from Stage 11b except that the incidence of secondary somites formation was much higher. In addition, in approximately 40% (n = 25) of embryos treated with greater than 1.0 microM of staurosporine, the growing end of the allantois did not reach the chorion and remained unattached in the exocoelomic cavity.(ABSTRACT TRUNCATED AT 400 WORDS)

Receptor subtype and intracellular signal transduction pathway associated with situs inversus induced by alpha 1 adrenergic stimulation in rat embryos.

In previous studies, we have demonstrated that stimulation of alpha 1 but not alpha 2 or beta adrenergic receptors in rat embryos grown in culture interferes with normal development of the left/right body axis leading to situs inversus. In the present study, we aimed to determine the alpha 1 adrenergic receptor subtype and signal transduction pathway involved in this phenomenon. Rat embryos at Stage 11a by a modified Theiler's staging system were cultured for 50 hr in medium containing various compounds which are known to activate or inhibit different sites of the signal transduction pathways associated with alpha 1 adrenergic receptors. They were then examined to determine the sidedness of asymmetric body structures. WB4101, a selective antagonist of alpha 1A adrenergic receptor subtype, but not chlorethylclonidine, a selective antagonist of alpha 1B adrenergic receptor subtype, inhibited phenylephrine (an alpha 1 adrenergic agonist)-induced situs inversus. Neither the protein kinase C (PKC) activators phorbol 12-myristate 13-acetate and SC-9 nor the PKC inhibitor calphostin C caused situs inversus. Furthermore, calphostin C did not block phenylephrine-induced situs inversus. A23187, a Ca2+ ionophore, induced situs inversus; nifedipine, a L-type Ca2+ channel blocker, partially blocked phenylephrine-induced situs inversus. The calmodulin antagonists trifluoperazine, W-7, and W-13 blocked phenylephrine-induced situs inversus, although they did not cause situs inversus by themselves. KN-62, a Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) inhibitor, dose-dependently blocked phenylephrine-induced situs inversus. However, higher concentrations of this compound produced no block in the presence of phenylephrine and in its absence produced a 50% incidence of situs inversus. These results indicate that alpha 1 adrenergic stimulation-induced situs inversus is mediated by the alpha 1A adrenergic receptor subtype and that activation of CaM kinase II but not PKC may be involved.