Down-regulation of neutral sphingomyelinase in androgen-dependent smooth muscle.

BACKGROUND: During puberty, proliferation of guinea pig seminal vesicle smooth muscle (SVM) is mediated by androgen-induced basal release of norepinephrine (NE), signaling through the post-junctional alpha1-adrenoceptor. In the adult, the SVM is terminally differentiated, such that cell number is androgen resistant. Sphingomyelinase activation generates second messenger ceramides, which in vascular smooth muscle have been reported to counter the alpha1-adrenoceptor-mediated contractile response and activate apoptosis. Accordingly, we hypothesized that SVM sphingomyelinase down-regulation by androgen may facilitate NE-induced proliferation and subsequent transition to the terminally differentiated state of the adult. MATERIALS AND METHODS: Pre-pubertal and adult guinea pigs were orchiectomized and treated+/-dihydrotestosterone (DHT). SVM was harvested free of epithelium, frozen, and stored for enzymatic analyses. Using radioactive sphingomyelin substrate, optimized reaction conditions for both neutral and acidic sphingomyelinase were established and used to assay the enzymes. RESULTS: Although acidic sphingomyelinase was stimulated by androgen in both the proliferative and amitotic phases of smooth muscle development, neutral sphingomyelinase was irreversibly reduced 35% at the time of DHT-induced proliferation. CONCLUSIONS: Decreased concentrations of a second messenger ceramide attenuate apoptosis and increase sensitivity to alpha(1)-adrenoceptor-mediated mitogenic signaling. Therefore, DHT-dependent suppression of neutral sphingomyelinase activity may reduce ceramide concentrations and facilitate NE-dependent smooth muscle growth.

Androgen-induced norepinephrine release mediating guinea pig seminal vesicle smooth muscle proliferation: potential role of pre-synaptic alpha2-adrenoceptors.

BACKGROUND: Recent studies from our laboratory have demonstrated that androgen-induced basal norepinephrine (NE) release is responsible for the onset of proliferation in seminal vesicle smooth muscle (SVM) cells during early puberty. With subsequent sexual maturation, SVM was irreversibly differentiated to an androgen-resistant-amitotic state in which basal NE release remained elevated and resistant to androgen withdrawal or repletion. Based on these findings, we hypothesized that this irreversible elevation of basal NE release during pubertal development is caused, at least in part, by the down-regulation of pre-synaptic NE feedback inhibition, secondary to irreversible reduction in the expression of neuronal (pre-synaptic) alpha(2)-adrenoceptors. Functional alpha(2)-adrenoceptors are selectively localized to pre-synaptic sites in SVM. METHODS: To test this hypothesis, we employed ligand binding techniques with [(3)H]RX821002, an antagonist which labeled all alpha(2)-adrenoceptor sub-types. Initial experiments focused on analysis of competitor specificity to identify the predominant alpha(2)-adrenoceptor sub-type in SVM. Subsequently, we quantified the changes in the receptor concentration (B(max)) for [(3)H]RX821002 at the point of maximal dihydrotestosterone (DHT)-induced change in basal NE release. RESULTS: Based on competitor specificity for [(3)H]RX821002, the alpha(2D)-adrenoceptor sub-type predominated in SVM. We treated pre-pubertal castrate animals with DHT for 7 days, which was previously demonstrated to maximally induce basal NE release. This treatment reduced the pre-synaptic alpha(2)-adrenoceptor B(max) 4-fold. In animals which had been castrated as adults, the B(max) for [(3)H]RX821002 remained irreversibly suppressed. CONCLUSIONS: The DHT-dependent reduction in the alpha(2)-adrenoceptor concentration is consistent with the developmental pattern of increased basal NE release. These findings support the hypothesis that the down-regulation of pre-synaptic NE feedback is mechanistically involved in the irreversible elevation of basal NE release. NE mediates proliferation in SVM in early pubertal development. Thus, the androgen-dependent pubertal growth of smooth muscle cells may be indirectly controlled at the level of neurotransmission.