Prenatal cocaine exposure induces an increased F-actin level which is normalized by protein phosphatase treatment without alteration in phosphorylation state of actin.

<p>(<b>a</b>) Synaptosomes obtained from the frontal cortices (FCX) of P21, prenatal cocaine- or saline-treated rats were treated with alkaline phosphatases, alkaline phosphatases (1 mg/ml) plus phosphatase inhibitors or cytochalasin D (10 µM) <i>in vitro</i>. The reactions were terminated, synaptosomes solubilized, and actin-containing proteins extracted using biotin-conjugated anti-actin antibodies and placed into streptavidin-coated plates. The level of F-actin was determined by rhodamine-conjugated phalloidin and the fluorescence intensity of phalloidin was measured using Beckman multimode plate reader, DX880. <i>n</i> = 6. Data are means ± s.e.m. of the fluorescence intensity. The statistical significance was evaluated by Newman-Keuls multiple comparisons that followed one-way ANOVA. *p<0.01 compared to native GRIP1 from saline-treated group. #p<0.01 compared to dephosphorylated GRIP in cocaine-treated group. +p<0.01 compared with native GRIP1 from respective group. (<b>b</b>) The phosphorylation state of actin was evaluated in synaptosomes derived from the FCX of P21, prenatal cocaine- or saline-treated rats. Total actin was purified by immunoprecipitation with anti-actin and the levels of phosphor-serine (pS), -threonine (pT) and -tyrosine (pY) in the anti-actin immunoprecipitate was determined by Western blotting using phosphoepitope-specific antibodies. <i>n</i> = 4. Data are means ± s.e.m. of the ratios of pS-, pT- or pY-actin to total actin optical intensities. There was no discernible difference noted in any of the actin phosphoepitopes in FCX of the prenatal cocaine- comparing to saline-exposed rats.