Adrenomedullin gene is abundantly expressed and directly regulated by androgen in the rat ventral prostate.

A gene-expression screen, looking for androgen response genes in the rat ventral prostate, has identified adrenomedullin (AM), a 52-amino acid pluripotent peptide hormone, first isolated from pheochromocytoma. Northern blot analysis demonstrates that the level of expression in the prostate is reduced at least 25-fold by castration, with the majority of the decrease occurring in the first day, and that androgen replacement in seven-day castrated rats stimulates expression to supernormal levels, with the majority of the increase occurring within 14 h. The level of expression in the prostate is at least 50-fold higher than in the adrenal gland and cardiac atria, tissues previously reported to have the highest level of expression in the rat. In prostate organ culture, androgen was able to induce AM expression; and this induction resists protein synthesis inhibition, indicating that AM is a direct androgen response gene in the prostate. In situ hybridization of normal rat prostate tissue showed that AM expression is localized in the epithelial cells. Our analysis demonstrates that AM, a multifunctional peptide hormone, is abundantly expressed and directly regulated by androgen in the prostate epithelial cells. Thus, AM has the potential to play a crucial function in androgen action in the prostate.

Calreticulin: an intracellular Ca++-binding protein abundantly expressed and regulated by androgen in prostatic epithelial cells.

Calreticulin was identified in a screen for androgen-response genes in the rat ventral prostate. Northern blot and Western blot analyses in the rat model showed that both calreticulin messenger RNA and protein are down-regulated by castration and up-regulated by androgen replacement in the prostate. Northern blot analysis showed that calreticulin expression level in the prostate is much higher than that in seminal vesicles, heart, brain, muscle, kidney, and liver. The regulation of calreticulin expression by androgen is only observed in the prostate and seminal vesicles, two male secondary sex organs. The induction of calreticulin by androgen in prostate organ culture partially resists protein synthesis inhibition, suggesting that calreticulin is a direct androgen-response gene. In situ hybridization and immunohistochemistry studies showed that calreticulin is an intracellular protein in prostatic epithelial cells. Because calreticulin is a major intracellular Ca++-binding protein with 1 high-affinity and 25 low-affinity Ca binding sites, our observations suggest that calreticulin is a promising candidate that mediates androgen regulation of intracellular Ca++ levels and/or signals in prostatic epithelial cells. The expression of calreticulin is also regulated by androgen in the mouse and human prostate, suggesting that androgen regulation and function of calreticulin in the prostate are conserved evolutionarily.

Urinary tract infection in urology, including acute and chronic prostatitis.

Heightened awareness of patients with increased risk for severe or potentially severe UTIs is paramount for early diagnosis and treatment. Urologic assessment of these patients is frequently necessary for cure and to prevent significant sequelae. Unresolved infections are usually caused by resistant bacteria and are treated by modification of therapy based on antimicrobial sensitivity testing. When unresolved bacteriuria is caused by organisms sensitive to the initial antimicrobial therapy, azotemia or a large bacterial mass density should be suspected. Recurrent infections at close intervals or with the same organism are usually caused by a bacterial focus in an acquired or congenital abnormality of the urinary tract, such as infection stones. The bacterial focus must be removed to cure the recurrent infections. If the bacterial focus within the urinary tract cannot be removed, long-term, low-dose antimicrobial suppression will prevent the morbidity of recurrent infections. Reinfection requires careful bacteriologic monitoring and low-dose prophylactic, intermittent, or postintercourse antimicrobial therapy. In the setting of prostatitis syndrome, the patient must first be classified into one of three categories: bacterial prostatitis, nonbacterial prostatitis, or pelviperineal pain syndrome. Bacterial prostatitis frequently responds to appropriate antimicrobial agents, whereas nonbacterial prostatitis and pelviperineal pain require an empiric multimodal approach.

The ATP and Mg2+ dependence of Na(+)-K(+)-2Cl- cotransport reflects a requirement for protein phosphorylation: studies using calyculin A.

Na(+)-K(+)-2Cl- cotransport activity has previously been shown to depend on both intracellular ATP and Mg2+, but the mechanisms remain unknown. Cotransport in avian erythrocytes can be stimulated by a variety of agents including cAMP and permeant serine/threonine phosphatase inhibitors and is inhibited by prior depletion of either ATP with antimycin A, or mg2+ by incubation in A23187 plus EDTA. However, when cells were first stimulated with cAMP rather than calyculin A then subjected to either depletion strategy, a differential effect was found. The phosphatase-inhibitor-treated cells were resistant to subsequent ATP or Mg2+ depletion while cAMP-treated cells were sensitive to both treatments. Parallel examination of protein phosphorylation confirmed that ATP or Mg2+ depletion leads to dephosphorylation of membrane proteins in cAMP-treated but not in calyculin-A-treated cells. These results suggest that, for cotransport, ATP and Mg2+ are required primarily to maintain the system in a phosphorylated state rather than as direct modulators. The relative effectiveness of okadaic acid (EC50 approximately 630 nM) and calcyulin A (EC50 approximately 8 nM) in stimulating the cotransporter indicate that a type-1 protein phosphatase is probably responsible for dephosphorylating the system. Cells stimulated by hypertonicity were also resistant to ATP or Mg2+ depletion suggesting that the mechanism of shrinkage-induced cotransport stimulation might also involve protein phosphatase modulation.

The regulation of Na/K/2Cl cotransport and bumetanide binding in avian erythrocytes by protein phosphorylation and dephosphorylation. Effects of kinase inhibitors and okadaic acid.

The Na/K/2Cl cotransport system in the avian erythrocyte can be activated by agents that raise intracellular cAMP suggesting the involvement of cAMP-dependent protein kinase (cAMP-PK) in its regulation. Another group of stimuli including fluoride and hypertonicity stimulate cotransport via cAMP-independent means. To further investigate the role of phosphorylation in these processes, we examined the effects of protein kinase inhibitors of 8 (p-Cl-phenylthio)-cAMP (cpt-cAMP), fluoride and hypertonic activation of cotransport in duck red cells, and [3H]bumetanide binding to isolated membranes. Preincubation of cells with the kinase inhibitors K-252a (Ki approximately 1.6 microM) and H-9 (Ki approximately 100 microM) blocked cpt-cAMP activation of bumetanide-sensitive 86Rb influx and bumetanide binding. These inhibitors also led to a rapid deactivation of cotransport and decrease in bumetanide binding when added to cells maximally stimulated by cpt-cAMP. K-252a and H-9 inhibited cotransport activation by cAMP-independent stimuli, but 10-fold higher concentrations were required, implying the involvement of a cAMP-independent phosphorylation process in the mechanism of action of these agents. Removal of stimuli that elevate cAMP leads to a rapid reversal of cotransport indicating the presence of active protein phosphatases in these cells. The protein phosphatase inhibitor okadaic acid (OA, EC50: 630 nM) stimulated both Na/K/2Cl cotransport and bumetanide binding to membranes. As with fluoride and hypertonic stimulation, the OA effect was inhibited only at relatively high concentrations of K-252a. Phosphorylation of the membrane skeletal protein goblin (Mr 230,000) at specific cAMP-dependent sites was used as an in situ marker for the state of activation of cAMP-PK. Goblin phosphorylation at these sites was increased by norepinephrine and cpt-cAMP and rapidly reversed by K-252a and H-9, confirming that both inhibitors do block cAMP-PK activity. While OA markedly increased overall phosphorylation of many erythrocyte membrane proteins, including goblin, it did not affect goblin phosphorylation at specific cAMP-dependent sites. These results implicate a cAMP-independent protein kinase in the mediation of the OA effect on cotransport and bumetanide binding. The bumetanide-binding component of the avian erythrocyte cotransporter, an Mr approximately 150,000 protein that can be photolabeled with the bumetanide analog [3H]4-benzoyl-5-sulfamoyl-3-(3-thenyloxy)-benzoic acid was found to be a phosphoprotein. These results strongly support the hypothesis that phosphorylation and dephosphorylation, possibly of the Na/K/2Cl cotransporter itself, regulates the activity of

[3H]bumetanide binding to avian erythrocyte membranes. Correlation with activation and deactivation of Na/K/2Cl cotransport.

The relationship between Na/K/2Cl cotransport activation in duck erythrocytes and binding of the diuretic [3H]bumetanide to isolated membranes from stimulated cells has been assessed. Cotransport was activated by either cAMP-dependent (norepinephrine) or -independent (fluoride, hypertonicity) pathways. Membranes isolated from unstimulated cells possessed no specific bumetanide binding. In the presence of norepinephrine, cotransport and saturable binding rose in parallel, reaching a maximum after 5-7 min. In membranes from maximally stimulated cells the K1/2 and Bmax for bumetanide binding were 100 nM and 1.7 pmol/mg protein, respectively. The diuretic binding properties of these membranes were characteristic of interactions of ligands with the Na/K/2Cl cotransporter: specific binding required the presence of all three cotransported ions (Na, K, and Cl), and the rank order of potency for diuretic competition with bumetanide for binding sites was benzmetanide greater than bumetanide greater than furosemide. The appearance of specific bumetanide binding was also seen in membranes from erythrocytes activated by non-cAMP-dependent stimuli, with an excellent temporal correlation between cotransport activation and diuretic binding. On removal of all stimuli both cotransport and bumetanide binding declined in parallel. Duck erythrocytes treated with norepinephrine in a solution containing 15 mM K+ swell to a new stable cell volume after 60 min, during which time cotransport becomes inoperative. Bumetanide binding to both whole cells and isolated membranes paralleled the decline in cotransport activity. It is concluded that bumetanide binding to isolated membranes faithfully reflects the state of activation of the Na/K/2Cl cotransporter in intact cells under a variety of conditions.