Summary Growth of prostate cancer cells is dependent upon androgen stimulation of the androgen receptor (AR). Dihydrotestosterone (DHT), the most potent androgen, is usually synthesized in the prostate from testosterone secreted by the testis. Following chemical or surgical castration, prostate cancers usually shrink owing to testosterone deprivation. However, tumors often recur, forming castration-resistant prostate cancer (CRPC). Here, we show that CRPC sometimes expresses a gain-of-stability mutation leading to a gain-of-function in 3β-hydroxysteroid dehydrogenase type 1 (3βHSD1), which catalyzes the initial rate-limiting step in the conversion of the adrenal-derived steroid dehydroepiandrosterone to DHT. The mutation (N367T) does not affect catalytic function, but it renders the enzyme resistant to ubiquitination and degradation, leading to profound accumulation. Whereas dehydroepiandrosterone conversion to DHT is usually very limited, expression of 367T accelerates this conversion and provides the DHT necessary to activate the AR. We suggest that 3βHSD1 is a valid target for the treatment of CRPC.

Adrenal medullary chromaffin cells are a major peripheral output of the sympathetic nervous system. Catecholamine release from these cells is driven by synaptic excitation from the innervating splanchnic nerve. Acetylcholine has long been shown to be the primary transmitter at the splanchnic‐chromaffin synapse, acting through ionotropic nicotinic acetylcholine receptors to elicit action potential‐dependent secretion from the chromaffin cells. This cholinergic stimulation has been shown to desensitize under sustained stimulation, yet catecholamine release persists under this same condition. Recent evidence supports synaptic chromaffin cell stimulation through alternate transmitters. One candidate is pituitary adenylate cyclase activating peptide (PACAP), a peptide transmitter present in the adrenal medulla shown to have an excitatory effect on chromaffin cell secretion. In this study we utilize native neuronal stimulation of adrenal chromaffin cells in situ and amperometric catecholamine detection to demonstrate that PACAP specifically elicits catecholamine release under elevated splanchnic firing. Further data reveal that the immediate PACAP‐evoked stimulation involves a phospholipase C and protein kinase C‐dependant pathway to facilitate calcium influx through a Ni2+ and mibefradil‐sensitive calcium conductance that results in catecholamine release. These data demonstrate that PACAP acts as a primary secretagogue at the sympatho‐adrenal synapse under the stress response.

The neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) is a cotransmitter of acetylcholine at the adrenomedullary synapse, where autonomic regulation of hormone secretion occurs. We have previously reported that survival of prolonged metabolic stress in mice requires PACAP-dependent biosynthesis and secretion of adrenomedullary catecholamines (CAs). In the present experiments, we show that CA secretion evoked by direct high-frequency stimulation of the splanchnic nerve is abolished in native adrenal slices from male PACAP-deficient mice. Further, we demonstrate that PACAP is both necessary and sufficient for CA secretion ex vivo during stimulation protocols designed to mimic stress. In vivo, up-regulation of transcripts encoding adrenomedullary CA-synthesizing enzymes (tyrosine hydroxylase, phenylethanolamine N-methyltransferase) in response to both psychogenic and metabolic stressors (restraint and hypoglycemia) is PACAP-dependent. Stressor-induced alteration of the adrenomedullary secretory cocktail also appears to require PACAP, because up-regulation of galanin mRNA is abrogated in male PACAP-deficient mice. We further show that hypoglycemia-induced corticosterone secretion is not PACAP-dependent, ruling out the possibility that glucocorticoids are the main mediators of the aforementioned effects. Instead, experiments with bovine chromaffin cells suggest that PACAP acts directly at the level of the adrenal medulla. By integrating prolonged CA secretion, expression of biosynthetic enzymes and production of modulatory neuropeptides such as galanin, PACAP is crucial for adrenomedullary function. Importantly, our results show that PACAP is the dominant adrenomedullary neurotransmitter during conditions of enhanced secretory demand.

Background:The acute stress secretagogue PACAP leads to catecholamine release, but the source of calcium necessary for secretion is unknown. Results: PACAP stimulation increases LVA Ca v 3.2 influx in a PKC-dependent process. Conclusion: PACAP-mediated acute sympathetic stress functionally recruits a pool of latent Ca v 3.2 channels to supply calcium for secretion. Significance: Native sympathoadrenal stimulation elicits catecholamine release through a non-canonical mechanism.

The serpinopathies are a group of inherited disorders that share as their molecular basis the misfolding and polymerization of serpins, an important class of protease inhibitors. Depending on the identity of the serpin, conditions arising from polymerization include emphysema, thrombosis, and dementia. The structure of serpin polymers is thus of considerable medical interest. Wild-type ␣ 1 -antitrypsin will form polymers upon incubation at moderate temperatures and has been widely used as a model system for studying serpin polymerization. Using hydrogen/deuterium exchange and mass spectrometry, we have obtained molecular level structural information on the ␣ 1 -antitrypsin polymer. We found that the flexible reactive center loop becomes strongly protected upon polymerization. We also found significant increases in protection in the center of ␤-sheet A and in helix F. These results support a model in which linkage between serpins is achieved through insertion of the reactive center loop of one serpin into ␤-sheet A of another. We have also examined the heat-induced conformational changes preceding polymerization. We found that polymerization is preceded by significant destabilization of ␤-sheet C. On the basis of our results, we propose a mechanism for polymerization in which ␤-strand 1C is displaced from the rest of ␤-sheet C through a binary serpin/serpin interaction. Displacement of strand 1C triggers further conformational changes, including the opening of ␤-sheet A, and allows for subsequent polymerization.2 is a member of the serpin class of protease inhibitors. Serpins inhibit their target proteases via a unique mechanism ( Fig. 1) (1). Cleavage of a serpin's reactive center loop (RCL) by a protease triggers a massive conformational change in which the RCL inserts into the central ␤-sheet A, becoming a sixth strand. This process disrupts the protease active site and traps the acyl-enzyme intermediate, rendering the protease inactive. The active, loop-expelled form of ␣ 1 -AT is significantly less stable than the loop-inserted form, and as a result of this metastability, the serpin structure is readily disrupted by mutations that result in inappropriate conformational changes. It has been established that a number of serious diseases associated with serpin mutations are caused by the formation and accumulation of serpin polymers (2). The details of the polymerization mechanism are therefore of considerable interest.Serpin polymerization has been studied extensively by a variety of methods, including circular dichroism, fluorescence spectroscopy, electron microscopy, and x-ray crystallography (3-5). The most generally accepted model of pathological serpin polymerization is one in which the RCL of one serpin anneals between ␤-strands 3 and 5A of another (1). Two major lines of evidence support this model. The first is that RCLmimicking peptides have been shown to anneal between strands 3 and 5A and have also been shown to block polymerization in vitro (6). The second comes from fluorescence spectroscopy. Dist...

Previous studies have shown that catecholamine secretion from the adrenal medulla plays a critical role in chronic intermittent hypoxia (CIH)-induced alterations in cardiovascular function. In the present study we examined the cellular mechanisms associated with the effects of CIH on adrenal chromaffin cell catecholamine secretion. Experiments were performed on adult male mice (C57/BL6) that were exposed to 1-4 days of CIH or to normoxia. Perforated patch electrical capacitance recordings were performed on freshly prepared adrenal medullary slices that permit separating the chromaffin cell secretion from sympathetic input. CIH resulted in a significant increase in the readily releasable pool (RRP) of secretory granules, and decreased stimulus-evoked Ca 2+ influx. Continuous hypoxia (CH) either for 2.5 h (equivalent to hypoxic duration accumulated over 4 days of CIH) or for 4 days were ineffective in evoking changes in the RRP and Ca 2+ influx. CIH activated PKC in adrenal medullae as evidenced by increased phosphorylation of PKC at Thr 514 and PKC inhibitors prevented CIH-induced increases in the RRP and restored stimulus-evoked attenuation of Ca 2+ influx. CIH resulted in elevated thio-barbituric acid reactive substances (TBARSs, an index of oxidized proteins) and an antioxidant prevented CIH-induced changes in the RRP, suggesting the involvement of reactive oxygen species (ROS). These results demonstrate that CIH increases the RRP in adrenal chromaffin cells via ROS-mediated activation of PKC and suggest that CIH can directly affect the secretory capacity of chromaffin cells and contribute, in part, to elevated catecholamine levels.

We recently reported that adrenomedullary chromaffin cells (AMC) from neonatal rats treated with intermittent hypoxia (IH) exhibit enhanced catecholamine secretion by hypoxia (Souvannakitti D, Kumar GK, Fox A, Prabhakar NR. J Neurophysiol 101: [2837][2838][2839][2840][2841][2842][2843][2844][2845][2846] 2009). In the present study, we examined whether neonatal IH also facilitate AMC responses to nicotine, a potent stimulus to chromaffin cells. Experiments were performed on rats exposed to either IH (15-s hypoxia-5-min normoxia; 8 h/day) or to room air (normoxia; controls) from ages postnatal day 0 (P0) to P5. Quantitative RT-PCR analysis revealed expression of mRNAs encoding ␣ 3-, ␣5-, ␣7-, and ␤2-and ␤4-nicotinic acetylcholine receptor (nAChR) subunits in adrenal medullae from control P5 rats. Nicotine-elevated intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i) in AMC and nAChR antagonists prevented this response, suggesting that nAChRs are functional in neonatal AMC. In IH-treated rats, nAChR mRNAs were downregulated in AMC, which resulted in a markedly attenuated nicotine-evoked elevation in [Ca 2ϩ ]i and subsequent catecholamine secretion. Systemic administration of antioxidant prevented IH-evoked downregulation of nAChR expression and function. P35 rats treated with neonatal IH exhibited reduced nAChR mRNA expression in adrenal medullae, attenuated AMC responses to nicotine, and impaired neurogenic catecholamine secretion. Thus the response to neonatal IH lasts for at least 30 days. These observations demonstrate that neonatal IH downregulates nAChR expression and function in AMC via reactive oxygen species signaling, and the effects of neonatal IH persist at least into juvenile life, leading to impaired neurogenic catecholamine secretion from AMC.adrenal medullary chromaffin cells; nicotinic cholinergic receptors; recurrent apneas; reactive oxygen species; catecholamine secretion ADRENAL MEDULLARY CHROMAFFIN cells (AMC) are sensitive to hypoxia in neonates, and low O 2 stimulates catecholamine secretion (2,10,21,31,33,35,37). Hypoxia-evoked catecholamine secretion from AMC involves inhibition of various types of K ϩ channels, leading to depolarization (10,15,17,20,38) Acetylcholine released from the splanchnic nerve activates neuronal nicotinic acetylcholine receptors (nAChRs) on AMC and evokes catecholamine secretion. Although sympathetic innervation to target organs is incomplete in neonates (10, 34), previous studies reported nAChR expression in neonatal AMC (19,32). Given that nicotine is a potent excitatory stimulus to AMC (32), in the present study, we tested the hypothesis that exposing neonatal rats to IH enhances the AMC response to nicotine in a manner similar to that reported for hypoxia. MATERIALS AND METHODSExperimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Chicago. Experiments were performed on neonatal Sprague-Dawley rats between ages postnatal day 0 (P0) and P35.Exposure to IH. Rat pups (P0), along with their mothers, were exposed to...

Bis(monoacylglycero)phosphate (BMP) is an unusually shaped, negatively charged phospholipid found in elevated concentrations in the late endosomes. The unusual structure and stereochemistry of BMP are thought to play important roles in the endosome, including structural integrity, endosome maturation, and lipid/protein sorting and trafficking. We have utilized dynamic light scattering, fluorescence spectroscopy and transmission electron microscopy to characterize the morphology and size of BMP hydrated dispersions and extruded vesicles. We find that the morphology of hydrated BMP dispersions varies with pH, forming highly structured, clustered dispersions of 500 nm in size at neutral pH 7.4. However, at acidic pH 4.5, spontaneous hydrolysis of BMP occurs, altering the vesicle morphology to spherically shaped dispersions. BMP vesicles are also significantly smaller in diameter than palmitoyloleoylglycerophosphocholine (POPC) vesicles. In a stability assay using dynamic light scattering measurements to compare and monitor 30 nm extruded vesicles of BMP, POPC, and POPG over a 5 week period, we find that BMP vesicles do not fuse to form larger structures. BMP also forms lamellar vesicles evidenced by the fluorescence leakage assay studies. These results shed light on the possibility that the biosynthesis of BMP and the increasing acidity during the maturation process of late endosomes play an important role in the formation of intraendosomal vesicular bodies.