Abstract
Cytoskeleton proteins play important roles in regulating vascular smooth muscle (VSM) contraction and relaxation. We tested the hypotheses that the expression levels of several of these proteins change significantly during the course of development, and that these changes contribute to age-related changes in contractile responses. In cerebral arteries from 95-day (d) gestation and 140-d fetus, newborn lambs, and adult sheep, by Western immunoblot (n= 5 for each age) we quantified the relative expression of alpha-actin, alpha-tubulin, cyclophilin A, and proliferating cell nuclear antigen (PCNA). In addition, we examined middle cerebral artery tension responses to phenylephrine (PHE) stimulation in the absence or presence of cytochalasin D (3 x 10(-7)m) and nocodazole (3 x 10(-6)m), inhibitors of alpha-actin and alpha-tubulin polymerization, respectively. The expression levels of alpha-actin and cyclophilin A varied little during the course of development. In contrast, alpha-tubulin expression was approximately 2.5-fold greater in both fetal age groups as compared to adult. Also, as compared to adult and as expected, expression of PCNA was several-fold greater in cerebral arteries of the 95-d fetus (x8), 140-d fetus (x 5), and newborn (x 3). In both adult and fetal middle cerebral artery, cytochalasin D-induced inhibition of actin polymerization decreased PHE-induced contraction, to approximately 60 and approximately 40% of control, respectively (despite no significant change in expression level). In contrast, alpha-tubulin inhibition by nocodazole showed little effect on PHE-induced tension (in spite of the age-related decrease in expression). In conclusion, expression levels of alpha-actin, a thin filament protein involved in contraction, remained relatively constant during the course of development, as did the effects of inhibition of its polymerization on contractility. In contrast, alpha-tubulin, important in intracellular protein trafficking, showed a significant age-related decrease in expression and played a relatively minor role in contractility. The present studies suggest that other cytoskeletal structural proteins and/or elements of pharmaco-mechanical coupling are important to developmental differences in cerebrovascular contractility. In addition, the relatively constant expression levels of alpha-actin and cyclophilin A with development, suggest that these are useful internal standards for studies of cytosolic protein expression.