STAUROSPORINE DOES NOT PREVENT ADRENERGIC-INDUCED SITUS-INVERSUS, BUTCAUSES A UNIQUE SYNDROME OF DEFECTS IN RAT EMBRYOS GROWN IN CULTURE

Staurosporine, an alkaloid isolated from Streptomyces species, is comm only used as a protein kinase C (PKC) inhibitor in animal investigatio ns. In the present study, we used this compound to determine whether a lpha(1)-adrenergic stimulation-induced situs inversus in rats is media ted by PKC. Embryos were explanted at 8 A.M. on day 9 of gestation. Th ose with a neural groove but with no visible neural folds (Stage 11a) were selected and were cultured in medium containing various concentra tions of staurosporine with or without 50 mu M of phenylephrine, an al pha(1)-adrenergic agonist. At 10 A.M. on day 11 of gestation, embryos were examined for situs inversus and other abnormalities. Staurosporin e, tested at 0.0001, 0.001, 0.01, 0.1, 0.375, and 0.5 mu M (lethal con centration), did not block phenylephrine-induced situs inversus at any concentration. However, staurosporine alone produced situs inversus a t concentrations above O. 1 mu M. At 0.5 and 1.0 mu M, staurosporine a lso caused cyst-like lesions projecting dorsally from the mesencephalo n that we named ''mesencephalic vesicles'' and the formation of second ary somites. To confirm and further examine these unique effects of st aurosporine both grossly and histologically, we conducted additional e xperiments using staurosporine from another source. Embryos were expla nted between 6 A.M. and 9 P.M. on day 9 of gestation and were placed i n 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 c ultured with various concentrations of staurosporine. Those cultured f rom Stage 11a exhibited similar lesions to those seen in the initial e xperiment but at somewhat higher concentrations of staurosporine. Embr yos cultured from Stage 10b showed a similar pattern of lesions as see n at Stage 11a, except that higher concentrations of staurosporine wer e required to cause mesencephalic vesicles and secondary somites forma tion. Embryos cultured from stage 11b showed similar effects to those cultered from younger stages except that maximum incidences of situs i nversus were much lower. Those cultured from Stage 11c showed similar dose-response to those cultured from Stage 11b except that the inciden ce of secondary somites formation was much higher. In addition, in app roximately 40% (n = 25) of embryos treated with greater than 1.0 mu M of staurosporine, the growing end of the allantois did not reach the c horion and remained unattached in the exocoelomic cavity. Embryos cult ured from Stage 12/s1-2 showed similar effects to those cultured from Stage 11c except that incidences of failure of chorion-allantois fusio n were somewhat higher. Embryos cultered from Stages 12/s3-4 and 12/s5 -6 were not viable after exposure to 1.0 mu M staurosporine. At 0.5 mu M, approximately 15% of embryos showed some kind of abnormality but n o mesencephalic vesicles were observed. Histological examinations on t he embryos cultured from Stage 11b with 1.5 mu M staurosporine reveale d that the first sign of the mesencephaiic vesicles was abnormal varia ble thickness and convolutions of the neuroepithelium lateral to the n eural groove. These results suggest that situs inversus induced by sti mulation of alpha 1-adrenergic receptors is not mediated through PKC. They also show that staurosporine causes situs inversus and various ki nds of unique abnormalities in a dose-dependent and stage-specific man ner. (C) 1994 Wiley-Liss. Inc.