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Regulation of intracellular calcium by androgens in stromal cells derived from the human prostate
thesisposted on 14.02.2017, 00:17 by Oliver, Victoria Louise
The prostate stroma plays a large role in the pathophysiology of two common diseases afflicting the human male: prostate cancer and benign prostatic hyperplasia. Both diseases are highly dependent on testicular androgens, yet develop at an age when circulating testosterone levels decline. The primary site of androgen action in the prostate is the stroma. Given that intracellular calcium ([Ca2+]i) regulates a variety of cellular processes implicated in the stromal response to disease, this study investigated the effects of androgens on [Ca2+]i in human cultured prostatic stromal cells (HCPSC). Chapter 3: Approximately 55% of cells exhibited spontaneous [Ca2+]i elevations with an average frequency of approximately 1 every 2 minutes. Oscillations were dependent on extracellular calcium influx, release of calcium from inositol triphosphate (IP3) sensitive intracellular stores and cyclic adenosine monophosphate (cAMP). Neither chronic (3 days) nor acute (5 mins) testosterone treatment had any effect on calcium oscillations (height, frequency or incidence). Cells exhibiting oscillations were smooth muscle α-actin positive and oscillation cycles were unaffected by neighbouring cells. Chapter 4: Cells cultured in low (3 nM), but not mid (30 nM) or high (300 nM) concentrations of testosterone had significantly elevated basal [Ca2+]i. The testosterone-induced elevation of [Ca2+]i was dependent on aromatase and estrogen receptors (ERs) indicating that this effect was mediated by estradiol. Indeed, cells cultured directly with estradiol also had elevated basal [Ca2+]i, an effect that appeared to be mediated through the ER-β but not ER-α subtype. Culture with the potent androgen, dihydrotestosterone (DHT), decreased basal [Ca2+]i. The overall calcium elevating effect of 3 nM testosterone was most likely due to an up-regulation of aromatase and thus an elevated estradiol to DHT ratio. Chapter 5: DHT, but not testosterone or estradiol, caused an acute, transient elevation of [Ca2+]i. This effect was mediated through androgen receptors and largely dependent on the release of calcium from intracellular IP3 sensitive pools, and also, to a lesser extent, influx of calcium through L-type channels. Responses were blocked by the epidermal growth factor receptor (EGFR) inhibitor, AG1478. These findings are indicative of phospholipase C activation, which most likely occurs through the EGFR. Responses to DHT were diminished after culture with testosterone (300 nM) or DHT (30 nM) through an as yet unexplained mechanism. Acute responses to DHT were down-regulated by endogenous transforming growth factor-β. These studies show that androgens and estrogens have complex effects on [Ca2+]i in HCPSC. Importantly, the data presented here suggest that declining levels of testosterone, akin to those associated with aging, may induce significant alterations in prostatic stromal cells which contribute to their ability to regulate normal and pathological processes in the prostate.