Biomedical subjects

A M Nakhla

Publications and source records attributed to A M Nakhla.

At least 19 recordsLinked to original sources

PubMed2002

Sex hormone-binding globulin is synthesized in target cells.

Sex hormone-binding globulin (SHBG) is a multifunctional protein that acts in humans to regulate the response to steroids at several junctures. It was originally described as a hepatically secreted protein that is the major binding protein for sex steroids in plasma, thereby regulating the availability of free steroids to hormone-responsive tissues. SHBG also functions as part of a novel steroid-signaling system that is independent of the classical intracellular steroid receptors. Unlike the intracellular steroid receptors that are ligand-activated transcription factors, SHBG mediates androgen and estrogen signaling at the cell membrane by way of cAMP. We have reviewed the current state of knowledge on the SHBG gene and the role of SHBG in steroid signaling (we shall not address its function as a plasma-binding protein).

Androgens↗

PubMed2000

Estradiol causes a dose-dependent stimulation of prostate growth in castrated beagle dogs.

BACKGROUND: Previous studies have shown that chronic treatment of castrate dogs with androgen and estrogen results in significant prostate growth. Estrogen treatment of castrate dogs in the absence of androgen has resulted in conflicting data as reported by several authors. The purpose of this experiment was to evaluate the effect of a physiological dose of estradiol on prostate growth in dogs, using ultrasound to study size changes over time. METHODS: Dogs (n = 25) were randomly divided into groups (n = 5) and treated as follows: castration alone (CC), castration plus low dose estradiol (E(2) low), castration plus high estradiol (E(2) high), castration plus estradiol and androstanediol (E(2)A), or no treatment (normal controls, NC). Silastic implants containing 5alpha-androstan-3alpha-17beta-diol (3alphadiol), and/or 17beta-estradiol were used for continous delivery of steroids. Prostate volume was measured by transrectal ultrasonography, and blood was drawn for hormone and sex hormone binding globulin (SHBG) determinations. RESULTS: Results show that serum estradiol and SHBG levels were fairly constant over 12 weeks in all groups. Estradiol-treated groups had mean serum estradiol values of approximately 40 and 60 pg/ml, respectively. Initially, all groups had similar prostate volumes. Over 12 weeks the castrate dogs had a decline in prostate volume, whereas the intact dogs and those treated with E(2) and 3alpha-diol maintained a constant prostate volume. Estradiol treatment caused a large, late onset (week 7), dose-dependent increase in prostate volume relative to the intact group (P < 0.01). At 12 weeks, animals were euthanized and prostates weighed. The mean prostate weights in each group were: NC 14.8 +/- 2. 9, CC 2.4 +/- 0.5, E(2)A 9.7 +/- 2.0, E(2) low 21.7 +/- 4.3, and E(2) high 63.6 +/- 12.6 g (geometric mean +/- SEM). Histologically, prostates of estrogen-treated dogs showed metaplastic squamous epithelium. CONCLUSIONS: These results demonstrate that estradiol causes marked dose-dependent stimulation of prostate growth in the castrate dog.

Anabolic Agents↗

PubMed1999

Sex hormone-binding globulin receptor signal transduction proceeds via a G protein.

The plasma protein, sex hormone-binding globulin (SHBG) binds to a receptor (R(SHBG)) on cell membranes to form an SHBG-R(SHBG) complex. When an appropriate steroid binds to this complex, there is a rapid rise in intracellular cyclic adenosine monophosphate (cAMP). Although the system is moderately well characterized, the molecular cloning of R(SHBG) has not been accomplished and there is a paucity of evidence regarding the mechanism of transmission of the R(SHBG) signal. In this communication, we offer two independent lines of evidence that a G protein is involved in R(SHBG) signal propagation. Exposure of cell membranes containing R(SHBG) to a non-hydrolyzable analog of guanosine triphosphate (guanylyl-5'-imidodiphosphate) caused a substantive decrease in the binding of SHBG to R(SHBG). This behavior is typical of membrane receptors coupled to G proteins and has been used by others as evidence to support that relationship. Another set of experiments involved the assumption that, if R(SHBG)-induced increases in cAMP were diminished when the wild-type alpha subunit of a G protein was replaced with mutants that were inefficient/ineffective in signal transduction, then the idea that G proteins were involved in that signal would be buttressed. Hence, we infected COS-1 cells with a construct containing such mutants, along with a cAMP response element reporter, and demonstrated a marked decrease in R(SHBG)-engendered reporter activity, e.g. cAMP generation.

Cyclic AMP↗

PubMed1998

Sex hormone-binding globulin mediates prostate androgen receptor action via a novel signaling pathway.

Estradiol (E2) and 5alpha-androstan-3alpha,17beta-diol (3alpha-diol) have been implicated in prostate hyperplasia in man and dogs, but neither of these steroids bind to androgen receptors (ARs). Recently, we reported that E2 and 3alpha-diol stimulated generation of intracellular cAMP via binding to a complex of sex hormone-binding globulin (SHBG) and its receptor (R(SHBG)) on prostate cells. We speculated that this pathway, involving steroids normally found in the prostate, was involved in the indirect activation of ARs. Using the dog as a model to test this hypothesis in normal prostate, we investigated whether E2, 3alpha-diol, and SHBG stimulated the production of the androgen-responsive protein, arginine esterase (AE), the canine equivalent of human prostate-specific antigen. In cultured dog prostate tissue preincubated with SHBG, E2 and 3alpha-diol stimulated AE activity. These effects were blocked by hydroxyflutamide, an AR antagonist, and by 2-methoxyestradiol, a competitive inhibitor of E2 and 3alpha-diol binding to SHBG. In the absence of exogenous steroids and SHBG, AE also was significantly increased by treatment with forskolin or 8-Bromoadenosine-cAMP. These observations support the hypothesis that in normal prostate, E2 and 3alpha-diol can amplify or substitute for androgens, with regard to activation of the AR via the R(SHBG) by a signal transduction pathway involving cAMP. Because both E2 and 3alpha-diol are involved in the pathogenesis of benign prostatic hyperplasia in dogs and implicated in benign prostatic hyperplasia in man, antagonism of the prostatic SHBG pathway may offer a novel and attractive therapeutic target.

Animals↗

PubMed1997

Estradiol activates the prostate androgen receptor and prostate-specific antigen secretion through the intermediacy of sex hormone-binding globulin.

These experiments were designed to examine the relationship between the effects of steroid hormones mediated by classic intracellular steroid hormone receptors and those mediated by a signaling system subserved at the plasma membrane by a receptor for sex hormone-binding globulin. It is known that unliganded sex hormone-binding globulin (SHBG) binds to a receptor (RSHBG) on prostate membranes. The RSHBG.SHBG complex is rapidly activated by estradiol to stimulate adenylate cyclase, with a resultant increase in intracellular cAMP. In this paper we examine the effect of this system on a prostate gene product known to be activated by androgens, prostate-specific antigen. In serum-free organ culture of human prostates, dihydrotestosterone caused an increase in prostate specific antigen secretion. This event was blocked by the anti-androgens cyproterone acetate and hydroxyflutamide. In the absence of androgens, estradiol added to prostate tissue, whose RSHBG was occupied by SHBG, reproduced the results seen with dihydrotestosterone. Neither estradiol alone nor SHBG alone duplicated these effects. The estradiol.SHBG-induced increase in prostate-specific antigen was not blocked by anti-estrogens, but was blocked both by anti-androgens and a steroid (2-methoxyestradiol) that prevents the binding of estradiol to SHBG. Furthermore, an inhibitor of protein kinase A prevented the estradiol.SHBG-induced increase in prostate-specific antigen but not that which followed dihydrotestosterone. These data indicate that there is a signaling system that amalgamates steroid-initiated intracellular events with steroid-dependent occurrences generated at the cell membrane and that the latter signaling system proceeds by a pathway that involves protein kinase A.

Culture Techniques↗

PubMed1996

Stimulation of prostate cancer growth by androgens and estrogens through the intermediacy of sex hormone-binding globulin.

Sex hormone-binding globulin (SHBG) not only regulates the free concentration of certain steroid sex hormones in plasma, but is involved in a nongenomic mechanism of steroid hormone action. It binds to a receptor on prostatic cell membranes and is activated by an appropriate steroid to initiate the generation of intracellular cAMP. Using the human prostate cancer cell line ALVA-41, we show that in serum-free medium, both dihydrotestosterone and estradiol increase growth in the presence, but not the absence, of SHBG. The increase in growth also follows the addition of cAMP to the cells and is enhanced by inhibiting protein dephosphorylation with the protein phosphatase inhibitor, okadaic acid. We conclude that cAMP causes increased growth in this prostate cancer cell line, and that both SHBG-dihydrotestosterone and SHBG-estradiol can regulate intracellular cAMP, and hence growth, in these cells.

Cell Division↗

PubMed1995

5 alpha-Androstan-3 alpha,17 beta-diol is a hormone: stimulation of cAMP accumulation in human and dog prostate.

5 alpha-Androstan-3 alpha, 17 beta-diol (3 alpha-diol) is a well known metabolite of dihydrostestosterone (DHT) and the concentration of its glucuronide in serum is looked upon as a marker of DHT activity in skin and prostate. The data in this communication show that 3 alpha-diol is a potent agonist of the human and canine prostatic SHBG-[SHBG receptor]. It causes a robust rise in intracellular cAMP within minutes; the 1/2 max. response occurs at a concentration equal to its concentration in male plasma. Estradiol is the only other agonist in this system. All other steroids that bind to SHBG are antagonists.

Androstane-3,17-diol↗

PubMed1994

Estradiol causes the rapid accumulation of cAMP in human prostate.

Androgens are widely acknowledged to be central to the pathogenesis of benign prostatic hypertrophy (BPH). However, BPH increases in prevalence as men age, at precisely the stage of life when plasma androgens are decreasing. The decrease in total plasma androgens is amplified by an age-related increase in plasma sex hormone-binding globulin (SHBG) that results in a relatively greater decrease in free androgens than in total androgens. In addition, estrogens have long been suspected to be important in BPH, but a direct effect on the human prostate has never been demonstrated. We present data that are consistent with a role for estradiol, and for a decrease in androgens and an increase in SHBG, in the pathogenesis of BPH. We show that estradiol, but not dihydrotestosterone, acts in concert with SHBG to produce an 8-fold increase in intracellular cAMP in human BPH tissue. This increase is not blocked by an antiestrogen and is not provoked by an estrogen (diethylstilbestrol) that does not bind to SHBG, thus excluding the classic estrogen receptor as being operative in these events. Conversely, dihydrotestosterone, which blocks the binding of estradiol to SHBG, completely negates the effect of estradiol. Finally, we demonstrate that the SHBG-steroid-responsive second-messenger system is primarily localized to the prostatic stromal cells and not to the prostatic epithelial cells. Thus, we have shown a cell-specific, powerful, nontranscriptional effect of estradiol on the human prostate.

Cells, Cultured↗

PubMed1994

Calcitonin releases acid phosphatase from rat ventral prostate explants.

Inclusion of salmon calcitonin in the culture medium of rat ventral prostate explants diminished l-tartarate-sensitive acid phosphatase activity in the tissues with a concomitant increment of the enzyme activity in the medium. The effect of the hormone was dose-dependent for a dose range of 10(-12)-10(-6) M. Acid phosphatase activity in prostate explants decreased from 38.6 +/- 3.5 to 20.5 +/- 2.8, whereas it increased from 0.60 +/- 0.15 to 2.80 +/- 0.40 nmol p-nitrophenol liberated/mg protein/30 min in the culture medium. Tissues exposed to 10(-6) M salmon calcitonin had higher acetylcholinesterase activity (8.8 +/- 0.7) than non-exposed ones (6.2 +/- 0.5 mumol substrate hydrolyzed/g tissue/min). These results suggest that locally produced calcitonin causes a release for prostatic acid phosphatase from prostate tissues possibly through its interaction with the cholinergic system.

Acetylcholinesterase↗

PubMed1994

Characterization of ALVA-41 cells, a new human prostatic cancer cell line.

The ALVA-41 cell line was derived from a bony metastasis from a human prostatic carcinoma. The line has a number of distinct, advantageous properties that should make it useful as a tool for the study of prostate cancer. It grows rapidly and is easy to work with. It has receptors for androgens and glucocorticoids but not for estrogens. Its growth is enhanced by physiological concentrations of dihydrotestosterone. It does not secrete prostate specific antigen, but does secrete prostatic acid phosphatase. Further, the secretion of prostatic acid phosphatase is enhanced by dihydrotestosterone.

Acid Phosphatase↗

PubMed1991

Sex hormone-binding globulin: anatomy and physiology of a new regulatory system.

Sex hormone-binding globulin (SHBG) is a plasma glycoprotein that binds a number of circulating steroid hormones (testosterone, dihydrotestosterone and estradiol) with high affinity, thus regulating their free concentration in plasma. In addition to binding steroids, SHBG itself binds to receptor sites on plasma membranes with somewhat unusual kinetics. Both the off and on rates are quite slow. The steroid-binding and membrane-binding functions are intertwined in what is clearly an allosteric relationship. Occupation of SHBG's steroid-binding site by a steroid inhibits its ability to bind to its membrane receptor-binding site. This inhibition is not related to a steroid's biological activity. Metabolites of steroids without biological activity, e.g. 2-methoxyestradiol, actively inhibit SHBG's interaction with its membrane receptor. However, if unliganded SHBG is allowed to bind to its receptor on intact cells, and an appropriate steroid hormone then is introduced, adenylate cyclase is activated and intracellular cAMP increases. This function is specific for steroids with biological activity, 2-methoxyestradiol has no activity in this arena. These observations demonstrate a potentially important role for SHBG as a regulator of cell function. They also demonstrate an additional mode of action of steroid hormones, one that does not require that the steroid interact with a steroid receptor.

Amino Acid Sequence↗

PubMed1990

Biologically active steroids activate receptor-bound human sex hormone-binding globulin to cause LNCaP cells to accumulate adenosine 3',5'-monophosphate.

The binding of human sex hormone-binding globulin (SHBG) to a human prostatic cancer cell line (LNCaP) and the results of that binding were examined. Membranes derived from LNCaP cells bound unliganded SHBG on two sets of sites whose affinities were: Ka1 = 3.1 +/- 1.6 x 10(10) M-1 and Ka2 = 8.7 +/- 4.3 x 10(6) M-1. Intact cells also bound SHBG, but even after 6 h, less than 10% of specifically bound SHBG was internalized. This observation speaks against a role for the membrane binding of SHBG in steroid transport across cell membranes. When LNCaP cells were prebound with SHBG, addition of dihydrotestosterone or estradiol resulted in a dose-dependent increase in intracellular cAMP. SHBG in the absence of steroids or dihydrotestosterone in the absence of SHBG was without effect. 2-Methoxyestradiol, a steroid metabolite without biological activity, but which binds to SHBG more tightly than does estradiol, was also without effect. These observations demonstrate a potentially important role for SHBG as a regulator of cell function. They also demonstrate an additional mode of action of steroid hormones, one that does not require that the steroid interact with a steroid receptor.

2-Methoxyestradiol↗

PubMed1988

Induction of adenylate cyclase in a mammary carcinoma cell line by human corticosteroid-binding globulin.

Corticosteroid-Binding globulin (CBG) is a plasma protein that binds certain steroid hormones, mainly cortisol and progesterone. It has been demonstrated recently that specific binding sites for this protein exist on cell membranes. In this communication we establish that binding to these sites results in the induction of adenylate cyclase activity and the accumulation of cAMP in MCF-7 cells. These events are critically dependent upon a steroid being bound to CBG. These data are consistent with the hypothesis that CBG is a prohormone which is activated when cortisol is bound to it.

Adenylyl Cyclases↗

PubMed1988

Calcitonin stimulates adenylate cyclase activity, the accumulation of cyclic adenosine 3',5' monophosphate and the release of prostaglandin E2 in rat intestinal mucosa.

Calcitonin stimulates intestinal fluid and electrolyte secretion by an unclear mechanism. The present results show that synthetic salmon calcitonin significantly stimulates adenylate cyclase activity in the membranes of rat intestinal mucosal cells. The effect of the hormone was in a dose-dependent manner for a dose range of 10(-9)-10(-7) M. The stimulated enzyme activity was followed by a progressive accumulation of cyclic adenosine 3',5' monophosphate and release of prostaglandin E2 in these cells during a 20 min experimental period of time. These results are discussed, and suggest that the formation of cyclic adenosine 3',5' monophosphate possibly mediates the action of calcitonin on intestinal cell functions.

Adenylyl Cyclases↗

PubMed1988

Are corticosteroid-binding globulin and sex hormone-binding globulin hormones?

Because it no longer seemed reasonable to us that the sole function of the steroid-binding proteins in plasma was to serve as a buffer reservoir for steroid hormones, we conducted experiments which sought out other possibilities. Both CBG and SHBG bind to cell membranes, and this interaction partakes of the general characteristics of peptide hormone-membrane receptor systems. Additionally, human CBG has the ability to cause an increase in the activity of membrane-bound adenylate cyclase in MCF-7 cells, and this, in turn, results in an increase in cellular cAMP content. Thus, CBG appears to be a protein hormone. As a first consideration, one might presume that because CBG's half-life is measured in days, it would be counted among the hormones which, for the most part, are tonic in their effects, e.g., thyroid hormone. However, two important considerations tend to believe this presumption: (1) CBG which is unoccupied by steroid is not hormonally active (Figure 5): (2) Depending upon the time of day, circulating CBG is approximately 0-60% occupied in normal humans. These observations result in a circumstance in which a substantial portion of circulating CBG is available for activation by bursts of cortisol secretion. It seems prudent to speculate that, because steroids are essential for CBG's activity, the hormonal role of CBG may be entwined with, or complementary to the steroids which it binds. Finally, we should comment on the impact that our model of CBG as a hormone has on the view that only unbound steroid can be hormonally active. First, it should be stated that we have not addressed this question experimentally. Although there is evidence that CBG may be required for cortisol action, we feel that an obligate role for it is not documented adequately. At this time, we believe that CBG's hormonal role is compatible with a hypothesis that encompasses the view that unbound steroid hormones can diffuse into cells in some tissues and that both free and bound steroid can enter cells in others. Obviously, the final word on these important topics, as always, awaits the proper experiments.

Binding Sites↗

PubMed1988

Pharmacological doses of calcitonin reduce sex hormone levels in rat plasma.

Administration of a pharmacological dose of salmon calcitonin into rats (10 MRC U/kg body weight) significantly reduced plasma concentrations of testosterone and estradiol in a time-dependent fashion. This apparently was mediated by a diminished secretion of luteinizing hormone and follicle-stimulating hormone, the levels of which were also significantly reduced after 1 h of calcitonin administration.

Animals↗

PubMed1987

Calcitonin induces ornithine decarboxylase in various rat tissues.

A single dose of synthetic salmon calcitonin administered to rats (20 MRC U/kg body weight) stimulated the activity of ornithine decarboxylase in the brain, liver, kidney, testis and ovaries by 3-, 15-, 5-, 2- and 2-fold respectively after 4 h of the treatment. The increase in the enzyme activity in the brain, testis and ovaries was accompanied by a comparable increase in the enzyme protein content. Hepatic and renal ornithine decarboxylase concentration increased only by 2-fold. These results suggest that calcitonin influences polyamine biosynthesis through a tissue-specific regulation of the activity and/or the number of ornithine decarboxylase molecules.

Animals↗

PubMed1985

Transforming growth factor is a potent stimulator of testicular ornithine decarboxylase in immature mouse.

Administration of transforming growth factor, type alpha (TGF-alpha), to eight-day old mice resulted in 22 fold increase of testicular ornithine decarboxylase activity. Two to seven fold increases of enzyme activities by TGF-alpha were also observed in intestine, kidney, spleen, liver, and heart. The maximal enzymatic responses were reached 2-4 h after TGF-alpha administration in vivo. The induction of tissue ornithine decarboxylase activity was accompanied by an increase in new protein synthesis. These effects were found to be comparable to those in littermates administered with mouse epidermal growth factor but were significantly more pronounced than with bovine growth hormone. Daily administration of TGF-alpha to newborn mice also produced a 1.8 fold increase of testicular weight after 14 days. The present studies therefore show that TGF-alpha is an epidermal growth factor-like mitogen and is likely to be important for the testicular development of immature animal.

Animals↗