Differential regulation of androgen receptors in the separate rat prostate lobes: androgen independent expression in the lateral lobe

In order to understand the hormonal regulation of androgen receptors (AR) in the separate lobes of the rat prostate gland, the present study examined AR levels in the ventral, dorsal and lateral prostate lobes as a function of androgen withdrawal to complete prostatic regression and subsequent testosterone replacement. In the intact rat, the 3 prostate lobes contained significantly different amounts of androgen binding sites. Mean number of total cellular AR in the ventral, dorsal and lateral lobes was 7370, 1690, and 1015 fm/mg DNA, respectively. These receptors were primarily localized within the nuclear fraction of homogenized tissue: ventral, 86%; dorsal, 83%; and lateral, 100% nuclear localization. Androgen withdrawal was initiated via castration and rats were sacrificed 1, 2, 3, 5, 7, 10 and 14 days thereafter. Nuclear AR levels fell rapidly to 5, 24 and 30% of intact values by 48 h in the ventral, dorsal and lateral lobes, respectively. Levels of nuclear AR continued to decline in the ventral and dorsal lobes to undetectable levels by Day 10. In marked contrast, lateral lobe nuclear AR began to increase on Day 3 postcastration, reaching intact values by Day 7 and 133% intact levels by Day 14. Cytosolic AR in the ventral and dorsal lobes initially increased following castration, but subsequently declined to low levels by Day 14. Cytosolic AR were not detectable in the lateral prostate at any time point following castration. To determine the nuclear AR response to testosterone at this time, 14 day castrate rats were given 2 cm testosterone implants and sacrificed 1, 3, 5, 7, 10 and 14 days thereafter. As expected, nuclear AR rapidly returned in the ventral and dorsal lobes by Day 1 and reached a plateau by Day 5. A short term response to androgen exposure occurred in the lateral lobe where an immediate 9-fold increase in nuclear AR quantity was observed; however, these levels rapidly declined to pre-implant values by Day 5 and remained at that level despite continued exposure to testosterone. These f findings indicate that while nuclear AR levels in the ventral and dorsal prostate are primarily regulated by androgens, a testosterone-independent component exists within the lateral lobe.     

Effects of aging on growth factors gene and protein expression in the dorsal and ventral lobes of rat prostate

We hypothesize that various growth factors and their receptors gene and protein are modulated in dorsal and ventral lobes of aging prostate. To test this hypothesis, TGFbeta1, TGFbeta2 TGFbeta3, TGFbetaR-I, TGFbetaR-II, TGFalpha, EGF, EGFR, KGF and KGFR gene and protein expression were analyzed in dorsal and ventral lobes of aging rat prostates (1, 3, 6, 9, 12, 18, 24, and 28/30 months). KGF gene expression was very weak or absent in 1, 3, and 6 month old rat dorsal and ventral lobes of prostate whereas it re-expressed in 9, 12, 18, 24 and 30 month old rat prostate. All growth factors and their receptors expect KGF and EGFR were mainly localized in epithelium of ventral and dorsal lobes of aging rat prostates. EGF, TGFalpha, TGFbeta1, and TGFbetaR-I protein expression was lacking in stroma of dorsal and ventral lobes of 1, 3, 6, 9, 12/18 months old rat prostates. However, EGF, TGFbeta1 and TGFbetaR-I proteins re-expressed in stroma of 24 and 28 months old rat prostates. KGF protein expression was lacking in epithelium of dorsal and ventral lobes of all aging rat prostates. This is the first report to demonstrate differential gene and protein expression of growth factors in dorsal and ventral lobes is associated with aging rat prostate, suggesting their role in pathogenesis of prostatic diseases with aging.     

Affinity profiles of various muscarinic antagonists for cloned human muscarinic acetylcholine receptor (mAChR) subtypes and mAChRs in rat heart and submandibular gland.

A family of five subtypes of muscarinic acetylcholine receptors (mAChR) has been identified based on their molecular structures and second signal transduction pathways. In the present study, we examined the antagonist binding profiles of 9 muscarinic antagonists (atropine, 4-DAMP, pirenzepine, oxybutynin, tiquizium, timepidium, propiverine, darifenacin and zamifenacin) for human muscarinic acetylcholine receptor subtypes (m1, m2, m3, m4 and m5) produced by using a baculovirus infection system in Sf9 insect cells, and rat tissue membrane preparations (heart and submandibular gland). In a scopolamine methyl chloride [N-methyl-3H]- ([3H]NMS) binding assay, pirenzepine and timepidium displayed the highest affinities for the m1 and m2 subtypes, respectively, and both zamifenacin and darifenacin had the highest affinities for the m3 subtype, although the selectivities among the five subtypes were less than 10-fold. Propiverine showed a slightly higher affinity for the m5 subtype, whereas none of the drugs used in this study was uniquely selective for the m4 subtype. The binding affinities of muscarinic antagonists for rat heart and submandibular gland strong correlated with those for human cloned m2 and m3 subtypes, respectively. These data suggest that [3H]NMS binding studies using rat heart and submandibular gland might be useful methods which predict the affinities of test drugs for human muscarinic M2 and M3 receptor subtypes.     

Differential Regulation of Molecular Subtypes of Muscarinic Receptors in Alzheimer's Disease

Abstract: Molecular subtypes of muscarinic receptors (m1–m5) are novel targets for cholinergic replacement therapies in Alzheimer's disease. However, the status of these receptors in human brain and Alzheimer's disease is incompletely understood. The m1–m5 receptors in brains from control subjects and Alzheimer's disease patients were examined using a panel of specific antisera and radioligand binding. Quantitative immunoprecipitation demonstrated a predominance of the m1, m2, and m4 receptor subtypes in cortical and subcortical regions in control subjects. In Alzheimer's disease, normal levels of m1 receptors measured by radioligand binding contrasted with decreased m1 receptor immunoreactivity, suggesting that the m1 receptor is altered in Alzheimer's disease. The m2 immunoreactivity was decreased, consistent with the loss of m2 binding sites and the location of this receptor subtype on presynaptic cholinergic terminals. The m4 receptor was up-regulated significantly and may offer a target for new memory-enhancing drugs. Differential alterations of molecular subtypes of muscarinic receptors may contribute to the cholinergic component of Alzheimer's disease dementia.     

Diverse pre- and post-synaptic expression of m1-m4 muscarinic receptor proteins in neurons and afferents in the rat neostriatum

We have utilized subtype specific antibodies to determine the cellular and subcellular distributions of the muscarinic acetylcholine receptor subtypes that are highly expressed in the rat striatum (m1-m4). Each receptor is expressed in distinct populations of striatal neurons in the relative proportions predicted by their mRNAs. They concentrate at post-synaptic sites and each of the four subtypes are also transported to pre-synaptic sites. m2 appears to be the only presynaptic autoreceptor in the striatum, but it is also localized in non-cholinergic terminals. These distinct pre- and post-synaptic localizations suggest that muscarinic receptor subtype diversity evolved to enable increasingly complex responses to acetylcholine release.     

A difference in the in vitro accumulation and metabolism of testosterone -1,2-3H by the rat prostate gland following incubation with ovine or bovine prolactin

The $\text{in vitro}$ actions of ovine or bovine prolactin on the accumulation and metabolism of testosterone-1,2-³H by various lobes of the rat prostate gland were examined. Ovine prolactin at a concentration of 2 IU/ml of incubation media significantly increased the accumulation of total radiosteroid by the anterior and dorsal lobes of the rat prostate gland. Although the levels of both testosterone-1,2-³H and its principle metabolite 5α-dihydrotestosterone-1,2-³H were increased by this concentration of ovine prolactin, the ratio of the two steroids was not altered. Ovine prolactin did not alter either the accumulation or metabolism of testosterone-1,2-³H by the ventral or lateral lobes of the rat prostate gland.In contrast to ovine prolactin, bovine prolactin in a wide range of concentrations (0.02–8 IU/ml) significantly reduced the accumulation of total labeled radiosteroid by the dorsal lobe of the rat prostate gland. The reduction in total radiosteroid was reflected in concomitent decreases in both testosterone-1,2-³H and 5α-dihydrotestosterone-1, 2-³H. The anterior lobe responded with similar reductions but only at relatively high concentrations of bovine prolactin (4 and 8 IU/ml). No significant alterations were observed in either the ventral or lateral lobes as a result of incubation with bovine prolactin.     

Seasonal morphophysiological variations in the prostatic complex of the Tarabul’s gerbil (Gerbillus tarabuli)

Gerbillus tarabuli is a nocturnal Saharan rodent which has an annual reproductive cycle characterized by the reproductive activity in spring and a long phase of sexual quiescence in other seasons. We describe the morphology and hormonal regulation of the prostatic complex of this rodent in the two periods, based on anatomical, histological, morphometric, and immunohistochemical analyses. The organisation of this prostatic complex is similar to that reported for Meriones unguiculatus, but different from the prostate of Psammomys obesus, the rat, and the mouse. In addition to the anterior lobes, ventral lobes, and dorsal lobes, the prostatic complex of Gerbillus tarabuli, also includes dorsolateral lobe. Each lobe is composed of a fibro-muscular stroma surrounding a glandular epithelium. Dorsolateral lobes are easily distinguishable by their big volume. The prostate grows and regresses cyclically throughout the year. During the resting season, ventral lobes and anterior lobes showed atrophy, with a significant decrease in both epithelial height and supranuclear area size, and a strong thickening of the fibro-muscular compartment. In dorsal lobes, the epithelial and stromal compartments atrophied and regenerated simultaneously, whereas in dorsolateral lobe the thickness of the epithelium, the supranuclear zone and the stroma increased during resting period. Furthermore, seasonal variations were observed in the distribution and expression of both androgen receptors, and estrogens receptors. Expression patterns of all receptors were lobe-specific. In conclusion, both androgens and estrogens are involved in the homeostasis and regulation of the prostate in Gerbillus tarabuli. Dorsolateral lobe seems to be controlled by a different mechanism than other lobes.     

Morphological and immunohistochemical comparison of three rat prostate lobes (lateral, dorsal and ventral) in experimental hyperprolactinemia

The prolactin plays an important role in the regulation of growth and differentiation of prostate gland besides androgens. The goal of this study was to reveal the influence of elevated prolactin concentration on epithelial cells of prostate. We compared the morphology of epithelial cells of prostate dorsal, lateral and ventral lobes and expression of androgen receptors in these cells in rats with hyperprolactinemia and in control rats. We used sexually mature male Wistar rats. The experimental rats received metoclopramide; the control group received saline in the same way. The prostate dorsal, lateral and ventral lobes were collected routinely for light and electron microscopy. The intensity of immunohistochemical reaction of androgen receptor in epithelial cells of dorsal, lateral and ventral lobes was evaluated by measure of optical density with computer image analysis. The light and electron (transmission and scanning) microscopes were used for morphological observations. Results: In experimental rats twofold increase in prolactin and twofold decrease in testosterone found. In experimental group the expression of androgen receptor was lower in columnar epithelial cells of dorsal and ventral lobes but higher in lateral one. We observed morphological abnormalities in columnar epithelial cells of lateral and dorsal lobes. The columnar epithelial cells of ventral lobes didn't show any morphological changes in hyperprolactinemia.     

Ultrastructural and biochemical observations on the early changes in apoptotic epithelial cells of the rat prostate induced by castration

The present study describes the sequential ultrastructural changes in the apoptotic cells of the rat ventral and dorsal prostates during the early period of 1-3 days postcastration. The major morphological changes include: (1) condensation of heterochromatin along the nuclear envelope and fragmentation into crescent-shaped micronuclei; (2) formation of membrane-bound cytoplasmic spherical bodies, which contain various organelles and micronuclei, within the apoptotic cells; (3) formation of non-membrane-bound autolytic vacuoles by autolysis of cytoplasm; (4) focal rupture of outer mitochondrial membrane; and (5) phagocytosis of the fragmented cytoplasmic spherical bodies and apoptotic cells by macrophages. The occurrence of both cytoplasmic apoptotic bodies and autolytic vacuoles in apoptotic cells suggests that the cytoplasm of the apoptotic cells could be destroyed by different means. The responsiveness of different prostatic lobes to androgen withdrawal and the time course of the transitory apoptotic activity in different lobes were analyzed by counting the indices of the TUNEL-labeled apoptotic cells against the postcastration periods. The results showed that the ventral lobe responded more rapidly to castration than the lateral and dorsal lobes. The dorsal lobe was the slowest in response to castration among the three lobes. Analysis of protease activities by zymography has identified two Ca(2+)-independent proteases of apparent MW 20 and 24 kDa (expressed in both ventral and dorsolateral lobes), and one Ca(2+)-dependent protease of MW 66.5 kDa (expressed only in the dorsolateral lobe) which became activated at day 3 postcastration. Their expression patterns were different from that of CPP-3 in the castrated prostates, suggesting that the activated proteases were enzymes other than CPP-3. The association of their highest activities with the maximum apoptotic activity at day 3 postcastration and also their loss of activity at day 15 suggest that these protease activities might be related to apoptosis or glandular involution.     

Differential alterations in muscarinic receptor subtypes in Alzheimer's disease: implications for cholinergic-based therapies

Molecular subtypes of muscarinic receptors (m1-m5) are novel targets for cholinergic replacement therapies in Alzheimer's disease (AD). However, knowledge concerning the relative distribution, abundance and functional status of these receptors in human brain and AD is incomplete. Recent data from our laboratory have demonstrated a defect in the ability of the M1 receptor subtype to form a high affinity agonist-receptor-G protein complex in AD frontal cortex. This defect is manifested by decreased M1 receptor-stimulated GTPgammaS binding and GTPase activity and by a loss in receptor-stimulated phospholipase C activity. Normal levels of G proteins suggest that the aberrant receptor-G protein interaction may result from an altered form of the m1 receptor in AD. The combined use of radioligand binding and receptor-domain specific antibodies has permitted the re-examination of the status of muscarinic receptor subtypes in the human brain. In AD, normal levels of m1 receptor [3H]-pirenzepine binding contrasted with diminished m1 immunoreactivity, further suggesting that there is an altered form of the m1 receptor in the disease. Reduced m2 immunoreactivity was consistent with decreased numbers of m2 binding sites. Increased levels of m4 receptors were observed in both binding and immunoreactivity measurements. These findings suggest one possible explanation for the relative ineffectiveness of cholinergic replacement therapies used to date and suggest potential new directions for development of effective therapeutic strategies for AD.     

Plasminogen activator activities in the developing rat prostate

Plasminogen activator (PA) activities were measured in the rat prostatic complex and individual prostatic lobes during early postnatal and pubertal development and in sexually mature adult rats. There was no significant change in PA activity during postnatal prostate development. However, during sexual maturation with puberty, there was a decline in PA activity in the ventral (3-fold), dorsolateral (22-fold), and anterior (19-fold) prostate lobes when activity was expressed per unit protein. A decrease in activity of 25- and 11-fold was found for the dorsolateral and anterior lobes, respectively, when activity was expressed per unit DNA. There was no change in activity in the ventral lobe. The adult ventral prostate (and its secretion) have 3 broad bands of low molecular mass (approximately 23 and 26-32 kDa) plasminogen-independent protease activities. Proteases of these molecular sizes as well as an activity of 170 kDa were detected in the dorsolateral prostate. The former proteases in the ventral and dorsolateral lobes were first found at 21 days of age, whereas the 170 kDa protease was found in dorsolateral prostate immediately post-puberty (48 days). The low molecular mass plasminogen-independent proteases were also able to activate plasminogen (determined by zymography) and hence contribute to the total measured PA activity. Thus, at 21 days of age, the specific activity of plasminogen-dependent protease declined, since the total measured PA-specific activity did not change. Plasminogen-dependent activities in ventral, dorsolateral, and anterior prostate lobes of adult rats were found as doublets of approximately 57-59 kDa and 36-38 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat dorsal prostate is necessary for vaginal adhesion of the seminal plug and sperm motility in the uterine horns

The rat prostate comprises dorsal, ventral and lateral lobes that are morphologically and biochemically distinct. Lesions to these structures are expected to affect the quality of the ejaculate and male fertility. In experiment 1, we analyzed ejaculate parameters of males that had chemical lesions of the dorsal or ventral lobes. At pre-lesion and at 5 and 20 days post-lesion males were mated, and after ejaculation, seminal fluid and seminal plug were obtained from the mated females. In experiment 2, the ventral lobes were ablated, and the ejaculate was analyzed. In experiment 3, the fertility of males with chemically-lesioned dorsal lobes or ablation of the ventral lobes was evaluated. Chemical lesion of the dorsal lobe prevented the adhesion of the seminal plug to vaginal walls. When these males were tested at 5-days postlesion, no sperm were found in uterus, and at 20-days post-lesion, the few sperm encountered showed slow progressive motility. None of the females that mated with dorsal lobe-lesioned males became pregnant. However, chemical lesion or ablation of the ventral lobes did not affect ejaculate or fertility. Our results indicate that the dorsal prostatic lobes are indispensable for reproductive success in males, and define parameters of ejaculate with which fertility can be estimated.     

Muscarinic acetylcholine receptors: structural basis of ligand binding and G protein coupling

Muscarinic acetylcholine receptors (m1-m5) were studied by a combined molecular genetic/pharmacologic approach to elucidate the molecular characteristics of the ligand binding site and of the receptor domains involved in G protein coupling. Site-directed mutagenesis studies of the rat m3 muscarinic receptor suggest that the acetylcholine binding domain is formed by a series of hydrophilic amino acids located in the "upper" half of transmembrane domains (TM) III, V, VI, and VII. Moreover, we showed that mutational modification of a TM VI Asn residue (Asn507 in the rat m3 receptor sequence) which is characteristic for the muscarinic receptor family has little effect on high-affinity acetylcholine binding and receptor activation, but results in dramatic reductions in binding affinities for certain subclasses of muscarinic antagonists. The N-terminal portion of the third intracellular loop (i3) of muscarinic and other G protein-coupled receptors has been shown to play a central role in determining the G protein coupling profile of a given receptor subtype. Insertion mutagenesis studies with the rat m3 muscarinic receptor suggest that this region forms an amphiphilic alpha-helix and that the hydrophobic side of this helix represents an important G protein recognition surface. Further mutational analysis of this receptor segment showed that Tyr254 located at the N-terminus of the i3 loop of the m3 muscarinic receptor plays a key role in muscarinic receptor-induced Gq activation. The studies described here, complemented by biochemical and biophysical approaches, should eventually lead to a detailed structural model of the ligand-receptor-G protein complex.     

Morphological and functional heterogeneity in the rat prostatic gland.

Ductal morphogenesis and adult ductal branching patterns were examined in the rat prostate by a microdissection method. The rat prostate consists of paired (right and left) subdivisions which correspond in large part to the classically defined lobes: ventral prostate, lateral prostate, dorsal prostate, and coagulating gland. Of particular interest was the finding that the lateral prostate consists of two different ductal zones: (1) lateral type 1 prostate with 5-7 long main ducts (resembling miniature palm trees) that extend cranially towards both the seminal vesicle and dorsal prostate to arborize near the bladder neck, and (2) lateral type 2 prostate with 5-6 short main ducts that arborize caudal to the bladder neck and give rise to compact bushy glands. Both lateral prostatic groups had a ductal-acinar organization. The adult structure of the other rat prostatic lobes was also examined, and closely resembled their mouse counterparts. The ventral prostate, which had 2-3 pairs of slender main ducts per side, and the coagulating gland, which had 1 main duct per side, was completely ductal in structure. In contrast, the dorsal prostate, which had 5-6 pairs of main ducts per side, had a ductal-acinar structure. Ductal branching morphogenesis occurred at different rates in different lobes and was essentially complete in the prostate at the 30 days. Immunocytochemical studies with an antibody to DP-1, a major secretory protein of the rat dorsal prostate, revealed that secretory function was initiated at approximately 30 days after birth in the coagulating gland, the dorsal prostate, and lateral type 1 prostate. A consistent feature of the lateral type 2 prostate was the absence of DP-1. On Western blots, DP-1 was detected in the secretion of the coagulating gland, lateral type 1 and dorsal prostate, but not in the ventral and lateral type 2 prostate. Polyacrylamide gel electrophoresis confirmed this result and demonstrated that the lateral type 2 prostate expressed several low-molecular weight secretory proteins not found in the other lobes of the prostate. On the whole, the rat prostate exhibited considerable heterogeneity both between and within lobes in developmental processes, ductal patterning, histology, and functional expression.     

Cholinergic Deafferentation of Dorsal Hippocampus by Fimbria-Fornix Lesioning Differentially Regulates Subtypes (m1-m5) of Muscarinic Receptors

Abstract: Unilateral aspiration lesions of the rostral supracallosal stria/cingulum bundle and fimbria-fornix were performed on adult female rats. Ten and 24 days post lesioning, an elevation (17%; p<0.01) of total muscarinic receptors was observed in lesioned versus control hippocampi. By using antisera selective for each of the five molecularly defined subtypes (m1-m5) of muscarinic receptors, significant changes were observed in the levels of expression for at least four receptor proteins. Three receptor subtypes increased in density: m1 by 14% (from 943 to 1,078 fmol/mg); m3 by 77% (from 150 to 268 fmol/ mg); and m4 by 29% (from 220 to 285 fmol/mg). In contrast, a 22% decrease in the density of m2 receptors was found (from 220 to 173 fmol/mg). Detectable levels of m5 receptors were low in the hippocampus (∼1% of total receptors), and reliable measurements were not obtained. The directions of these changes are likely to be related to the pre- or postsynaptic localization of these receptor subtypes.     

Immunocytochemical localization of 5α-reductase type 1 in the prostate of normal and castrated rats

We immunohistochemically studied the localization of 5-reductase type 1 in combination with androgen receptor (AR) expression in individual lobes of the prostates of intact and castrated rats. In the normal rat prostate, 5-reductase was localized in the cytoplasm of most epithelial cells in the ventral, dorsal, and lateral type 1 (L1) lobes. Epithelial cells of lateral type 2 (L2) lobes were negative for 5-reductase. AR was present in the nuclei of all epithelial and stromal cells throughout the prostate. The number of 5-reductase-immunoreactive cells rapidly decreased in the ventral and L1 lobes after castration, whereas many positive cells remained in the dorsal lobe even at 4 weeks after castration. AR immunostaining was lost in the ventral, dorsal, and L1 lobes at 1 week after castration, but remained in the L2 lobe of 4-week-castrated rats. Electron microscopic immunocytochemistry showed that 5-reductase was exclusively localized in the rough endoplasmic reticulum membranes and that there were no distinct structural differences between the positively and negatively stained epithelial cells. These findings suggested that the expression of 5-reductase type 1 in the epithelial cell is heterogeneous within and among the individual lobes of the rat prostate, and does not correspond to AR expression.