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P M Sluss

Publications and source records attributed to P M Sluss.

At least 37 records · Page 2Linked to original sources
PubMed1994

Development of a bioassay for FSH using a recombinant human FSH receptor and a cAMP responsive luciferase reporter gene.

FSH exerts its actions primarily by increasing cAMP levels via a G protein-linked transmembrane receptor. We report the development of a bioassay for FSH using a cell line that stably expresses the human FSH receptor and a cAMP responsive human glycoprotein hormone alpha subunit luciferase reporter construct. Receptor activation by FSH was measured by changes in luciferase activity. The cell line was shown to express 1.6 x 10(4) receptors per cell which bound FSH with high affinity (Kd 2.76 x 10[-9] M). Human pituitary FSH caused a dose-dependent increase in cAMP (ED50, 190 mIU/ml) and luciferase (ED50, 31.5 mIU/ml) activity. The sensitivity of the bioassay was less than 0.6 mIU/well. Postmenopausal serum, rat, ovine and bovine FSH elicited a dose-dependent increase in luciferase activity. There was no significant stimulation by highly purified human LH or recombinant human TSH. This cell line should be useful in the determination of bioactive FSH and characterization of serum FSH inhibitors.

Animals↗
PubMed1994

Characterization of unique binding kinetics of follistatin and activin or inhibin in serum.

Serum binding proteins (BPs) have been identified for several peptide and protein hormones, and their presence has significant implications for the biological action of the hormone. Follistatin (FS) has been identified as an activin- and inhibin-BP in tissues, serum, and follicular fluid of several species, including humans. In this study, the binding kinetics of FS for activin and inhibin were characterized in human serum using gel filtration chromatography and compared to those of pure recombinant hormones using chromatography and a new solid phase assay. When complexed with radiolabeled activin or inhibin, FS eluted at a volume corresponding to a mol wt range of 67,000-150,000, an elution volume identical to the lower mol wt BP peak observed in serum. Furthermore, kinetic analyses of recombinant FS binding to activin using a solid phase assay revealed that 1) the FS-activin interaction is of high affinity, similar to or exceeding that estimated for activin binding to its receptor; 2) binding to activin is essentially irreversible at physiological pH; and 3) the potency of inhibin is approximately 500- to 1000-fold lower than that of activin in the FS binding assay. The lack of FS-[125I]activin complex reversibility observed in the solid phase assay was confirmed using a modified gel filtration chromatography protocol. Thus, preincubation of pure FS or serum with unlabeled activin for 2 h eliminated all binding of subsequently added labeled activin despite a much longer incubation period. However, when labeled activin was incubated with FS for 2 h, subsequent addition of unlabeled activin or inhibin was unable to displace labeled activin from FS, again demonstrating a lack of reversibility. Finally, to map this high affinity interaction, overlapping synthetic peptides were used to compete with labeled activin for FS binding. Two potential contact sites between FS and activin were identified, one near the N-terminus (amino acids 15-29) and the other near the C-terminus (amino acids 99-116). Given its apparently irreversible nature, high affinity, and ability to neutralize activin's biological activity, FS is quite different from the typical hormone-BP. These unique properties of FS undoubtedly attest to the potency of activin in many physiological and developmental settings and, therefore, to the importance of BPs, such as FS for regulating activin's bioactivity, distribution, and/or clearance.

Activins↗
PubMed1994

Relatively low levels of dimeric inhibin circulate in men and women with polycystic ovarian syndrome using a specific two-site enzyme-linked immunosorbent assay.

The endocrine feedback role of dimeric inhibin on FSH secretion from the pituitary has been well established in many species; however, evidence that inhibin is an important endocrine regulator of FSH in the human is more tenuous. One potential explanation for the equivocal data may be that the inhibin immunoassay used most widely in the human is a heterologous assay with an antiserum that exclusively recognizes the inhibin alpha-subunit in both its monomeric form and in inhibin dimers. The aim of the present study was to quantify serum inhibin levels in a variety of fertile and infertile men and women using a new ultrasensitive enzyme-linked immunosorbent assay that is specific for the dimeric form (alpha/beta) only. The specificity of the present assay was demonstrated by the absence of significant cross-reactivity with Mullerian inhibiting substance, transforming growth factor-beta, activin, FSH, LH, hCG, TSH, and hCG alpha and with the alpha-subunit of inhibin. The assay was sensitive to 1 pg/mL, and serial dilutions of human male and female serum samples paralleled the recombinant 32-kilodalton (kDa) dimeric inhibin standard curve. Complete recovery of exogenous recombinant 32-kDa inhibin added to serum was obtained. Mean serum inhibin levels ranged from a low of 5.7 +/- 0.6 pg/mL in the early follicular phase to 49.0 +/- 11.2 pg/mL in the midluteal phase of the normal menstral cycle and were elevated during ovulation induction (1250 pg/mL) and pregnancy (500 pg/mL). Interestingly, mean levels of dimeric inhibin in women with polycystic ovarian syndrome (PCOS) were indistinguishable from normal follicular phase. The most striking observation was the extremely low mean inhibin levels (< 2 pg/mL) found in normal men, GnRH-deficient men before any pulsatile GnRH treatment, and men with Klinefelter's syndrome, all of which were indistinguishable from levels observed in postmenopausal women. These observations in the male raise the possibly that 1) some forms of circulating and bioactive inhibin in the human are not detected by this assay due to either their conformation or the presence of a unique binding protein for endogenous inhibin that does not bind to recombinant 32-kDa inhibin; or 2) dimeric inhibin is not a major endocrine regulator of FSH in the human male. In addition, the relatively high dimeric inhibin levels at midcycle and in the luteal phase of the normal menstrual cycle, after gonadotropin stimulation, and during pregnancy suggest that dimeric inhibin is predominantly produced by dominant follicles, corpora lutea, and placental tissues.

Adult↗
PubMed1994

Estradiol and progesterone production by cultured granulosa cells cryopreserved from in vitro fertilization patients.

Gonadotropin-stimulated steroidogenesis was studied in cultured human granulosa-lutein cells obtained from patients undergoing procedures for in vitro fertilization. The impact of cryopreservation on cell function in vitro was studied. Granulosa cells obtained from in vitro fertilization patients were cultured in serum-supplemented medium or cryopreserved at -135 degrees C for 2-22 months. Fresh (unfrozen) cells (10(5) produced estradiol at a rate of 1320 pmol/l (over 72 h) and progesterone at about 2500 nmol/l. Estradiol production by either fresh or cryopreserved granulosa cells in culture was unaffected by physiological concentrations of follicle-stimulating hormone (7 IU/l). Adding testosterone (10(-7) mol/l) to the medium increased estradiol secretion approximately sixfold. In contrast, progesterone production was not affected by follicle-stimulating hormone or testosterone. No significant differences were observed in cultures of cryopreserved granulosa cells compared to cultures of unfrozen cells with respect to estradiol secretion, the effects of follicle-stimulating hormone or testosterone on estradiol secretion, or progesterone production. Progesterone production by fresh and cryopreserved cells was stimulated by human chorionic gonadotropin. These data indicate that cryopreservation offers the potential to facilitate prospective studies utilizing large numbers of human granulosa-lutein cells in culture.

Aromatase↗
PubMed1993

Insulin-like growth factor I: action and receptor characterization in human prostate cancer cell lines.

The role of insulin-like growth factor I (IGF-I) in the growth and development of prostate cancer was studied using established human prostate cancer cell lines. Under steroid and growth factor-free culture conditions, IGF-I significantly stimulated the androgen-independent cell lines PC-3 and DU-145 to incorporate [3H]thymidine into DNA, while the androgen-dependent cell line, LNCaP, was not affected. However, in the presence of dihydrotestosterone (DHT), DNA synthesis of LNCaP cells was stimulated by IGF-I in a dose-dependent manner. None of the cell lines tested secreted an immunoreactive level of IGF-I into their conditioned medium. Characterization of receptors by ligand binding assays revealed that all prostate cancer cell lines tested express specific binding sites for IGF-I with similar dissociation constants (0.23-0.39 nM). Crosslinking studies supported the suggestion that 125I-IGF-I was bound to a receptor on these cells. The IGF-I receptor concentrations of androgen-independent cell lines were significantly higher than those of the androgen-dependent cell line. Androgen appeared to affect neither the expression of IGF-I receptors nor the secretion of IGF-I. The results suggest that IGF-I may play an important role in stimulating the growth and progression of prostate cancer.

Binding, Competitive↗
PubMed1993

Characterization of insulin-like growth factor I binding sites in human bladder cancer cell lines.

The role of insulin-like growth factor I (IGF-I) in the growth and development of bladder cancer cells was investigated using cultured human cell lines representing differentiated (RT-4, 5637) or undifferentiated (T-24, J-82, TCC-SUP) transitional cell carcinoma (TCC). In the presence of 2% serum, IGF-I significantly stimulated the growth of all cell lines. The proliferation of T-24, 5637, and RT-4 cells was more sensitive to IGF-I than that of J-82 and TCC-SUP cells. [125I]IGF-I binding to 5637 and J-82 cells was significantly higher than that to T-24 and TCC-SUP cells (P < 0.001). RT-4 cells possessed the lowest binding capacity among the cell lines tested. Scatchard analysis of [125I]IGF-I binding to four of the five cell lines indicated a single binding site for IGF-I, with apparent dissociation constants (Kd) of 1.27, 1.18, 1.34, and 1.39 nmol/l for TCC-SUP, J-82, 5637, and T-24, respectively. Therefore, the difference observed in [125I]IGF-I binding among the bladder cancer cell lines was attributed to the difference of IGF-I binding sites and not to a change in receptor binding affinity. Cross-linking studies supported the suggestion that [125I]IGF-I was bound to a receptor on these cells. The results indicate that cultured human bladder cancer cells contain functional IGF-I receptors. A differentiated cell line, RT-4, possesses significantly fewer IGF-I receptors than other cell lines. This suggests that the overexpression of IGF-I receptor may reflect the malignant potential of bladder cancer cells.

Binding Sites↗
PubMed1993

Multiple molecular weight forms of immunoreactive alpha-inhibin in human seminal plasma.

The purpose of these studies was to determine whether the total immunoreactive alpha-inhibin protein concentration in seminal plasma correlated with serum gonadotropin levels or semen characteristics and to identify the forms of alpha-inhibin present in seminal plasma. Thirty-eight serum samples from men being evaluated for infertility were selected for study based on their serum hormone profiles and semen parameters. Serum LH and testosterone levels were normal, but FSH levels ranged from normal to hypergonadotropic (> 20 IU/L). Most semen parameters were within normal ranges, but germ cell numbers ranged from normal to azoospermic. Thus, seminal plasma from these men provided a unique opportunity to examine the antigenic forms of alpha-inhibin in individuals in whom strong correlations between inhibin and FSH levels might be predicted because of the observed ranges of FSH levels and germ cell numbers. Seminal plasma alpha-inhibin was characterized by RIA or Western blotting, using an antiserum directed against the N-terminal of the alpha-subunit of mature [32,000 mol wt (M(r))] inhibin. The antiserum recognized the alpha-subunit of dimeric inhibin as well as free alpha-inhibin and alpha-inhibin precursor proteins. Total immunoreactive alpha-inhibin ranged from 8.21-43.99 nmol/L in seminal plasma. However, alpha-inhibin levels were not statistically correlated with serum FSH levels or any of the measured semen parameters (including germ cell number). In contrast, the immunoreactive alpha-inhibin concentration in seminal plasma was negatively correlated (P < 0.01) with the serum LH level. Western blot analyses revealed that multiple forms of immunoreactive alpha-inhibin are present in seminal plasma. The majority of immunoreactivity was associated with monomeric proteins (ranging from 58,000-95,000 M(r)) that were larger than the alpha-subunit (21,000 M(r)) predicted for mature dimeric human inhibin (32,000 M(r)). The relative amounts of individual forms of immunoreactive alpha-inhibin varied among the patients studied, but could not be correlated with other serum or seminal parameters measured. Our observations demonstrate that various monomeric alpha-inhibin proteins are present in human seminal plasma. It is unlikely that these proteins alone or combined with inhibin beta-subunit proteins have identical biological activities. Thus, until assays specific for each of the various forms of immunoreactive alpha-inhibin are developed, their role as well as that of inhibin in the endocrine or local modulation of testicular function cannot be deduced from RIA data alone.

Blotting, Western↗
PubMed1992

Changes in urinary magnesium, citrate, and oxalate levels due to cola consumption.

The serum concentration and urinary excretion of four potential stone constituents: calcium, oxalate, phosphate, and uric acid; and of two potential inhibitors of urinary stone formation, citrate and magnesium, were studied before and during ingestion of a carbonated cola beverage. Four participants, after baseline serum determinations, abstained for forty-eight hours from cola consumption and then attempted to drink 3 quarts per day of cola in the following forty-eight hours. One participant failed to drink more than two quarts per day of cola. In all 4 participants twenty-four-hour urinary excretion of magnesium decreased by an average of 2.6 mg. In the 3 participants who succeeded in drinking 3 quarts of cola per day, twenty-four-hour urinary excretion of oxalate increased an average of 8.3 mg and excretion of citrate decreased an average of 122 mg. These results demonstrate changes in urine constituents which could contribute to enhanced kidney stone formation in patients who drink large quantities of cola-flavored carbonated beverages.

Adult↗
PubMed1992

Suramin inhibits gonadotropin action in rat testis: implications for treatment of advanced prostate cancer.

Suramin is being evaluated for the treatment of metastatic prostate cancer based on its inhibition of growth factor action. In addition, suramin may inhibit the endocrine control of androgen production, which was explored herein. Adult Sprague-Dawley rats were injected (i.p.) daily with varying doses of suramin. At a cumulative dose of 200 mg., suramin significantly depressed serum testosterone (p less than 0.05), and follicle stimulating hormone (p less than 0.002) levels. In vitro studies showed that suramin-mediated suppression of androgen production might be secondary to inhibition of gonadotropin action. In MA-10 cell cultures, suramin inhibited a maximum stimulatory dose of human chorionic gonadotropin with an ED50 of 4.4 microM. Studies in rat Sertoli cell cultures showed that follicle stimulating hormone action was also inhibited by suramin, with an ED50 of 8.6 microM. Using receptor binding assays with calf testis membrane, we showed that suramin inhibited 125I-hFSH binding to receptor in a dose dependent fashion with an ED50 of 10.4 microM; comparable to the ED50 of suramin inhibition of follicle stimulating hormone action in Sertoli cell culture cells. Thus the mechanism of suramin's suppression of androgen production may involve multiple sites of action, including inhibition of gonadotropin binding to its receptor and suppression of pituitary gonadotropin levels in serum. This inhibition of androgen production may be useful in the treatment of advanced prostate cancer.

Animals↗
PubMed1992

Comparative effects of tin- and zinc-protoporphyrin on steroidogenesis: tin-protoporphyrin is a potent inhibitor of cytochrome P-450-dependent activities in the rat adrenals.

Synthetic metalloporphyins inhibit formation of bilirubin by the heme oxygenase system, an ability that is of considerable experimental and clinical interest for suppression of jaundice in the newborn. The present investigation compares the consequences of treatment with Sn- and Zn-protoporphyrin on hemoprotein-dependent enzymes of the rat adrenals and corticosterone production and defines Sn-protoporphyrin as a potent toxin to adrenal functions. Treatment of rats with Sn-protoporphyrin (two doses of 50 mumols/kg, in 7 d) resulted in a marked reduction of 30-40% in cytochrome P-450-dependent adrenal microsomal 21 alpha-hydroxylase and mitochondrial 11 beta-hydroxylase activities. In the serum, the levels of corticosterone were reduced to about 70% of the control value. In addition, the mitochondrial cytochrome P-450SCC activity was decreased by about 50%. This decrease, however, could not be attributed to a reduced total heme level or an accelerated heme degradatory activity. Disruption by Sn-protoporphyrin of adrenal hemoprotein-dependent functions was not restricted to steroidogenic activities and encompassed drug metabolism activity of the organ; benzo(a)pyrene hydroxylase activity of both the microsomal and the mitochondrial fractions, as well as the microsomal NADPH-cytochrome P-450 reductase activity, were significantly reduced. Zn-protoporphyrin did not cause significant alterations in the above measured parameters although it too was effective in inhibiting the hepatic microsomal heme oxygenase activity. In light of the presently defined adverse effects of Sn-protoporphyrin on adrenal steroidogenesis, we suggest Zn-protoporphyrin is the agent of choice for potential use in treatment of hyperbilirubinemia in humans.

Adrenal Glands↗
PubMed1992

Low molecular weight follicle-stimulating hormone receptor binding inhibitor in sera from premature ovarian failure patients.

Ovarian insensitivity to FSH, as observed in some patients suffering premature ovarian failure (POF), could conceivably involve abnormal regulation of local factors that modulate FSH action. Low molecular weight FSH receptor-binding inhibitor (FRBI) has been identified in ovarian follicular fluid and shown to be an antagonist of FSH action. Thus, we undertook these studies to test the hypothesis that elevated FRBI can account for the high serum levels of FSH as measured by RRA relative to RIA values in some POF patients. In order to accomplish this, 2 POF patients were selected from a group of 27 from whom serum FSH had been measured by RIA and RRA. Using a recently developed and validated RRA, FSH was 430 IU (second IRP-78/549)/L and 182 IU/L for serum from patients 1 and 2, respectively. FSH quantitated by RIA was 96 and 136 IU/L in these same serum samples. Thus, the RRA/RIA values for these patients were 4.48 and 1.34. These ratios are: 1) higher than observed for normal cycling women (0.62); 2) higher than observed for normal, postmenopausal women (0.65); and 3) at least 2 SD higher than the mean RRA/RIA ratio of the 27 patients screened. FRBI was separated from FSH in serum from both these patients. FRBI accounted for most of the elevated FSH measured in serum by RRA. The HPLC chromatographic behavior and binding inhibitory activity of FRBI isolated from a large volume of serum from patient 2 were virtually identical to previously observed characteristics of FRBI isolated from porcine follicular fluid. These observations demonstrate that FRBI can account for elevated FSH measured by RRA relative to that measured by RIA. Furthermore, the inhibitor can be biochemically separated from FSH and quantitated by RRA in order to study its postulated relationship to POF. Expanded studies to identify causal relationships between FRBI and ovarian insensitivity to FSH seem warranted at this time.

Adult↗
PubMed1991

Precursors of alpha-inhibin modulate follicle-stimulating hormone receptor binding and biological activity.

Although several forms of monomeric alpha-inhibin have been isolated from follicular fluid, no biological function has yet been ascribed to these posttranslationally processed forms of the alpha-subunit precursor protein. Moreover, previous studies of a FSH receptor binding competitor (FRBC) isolated and characterized from porcine follicular fluid (pFF) suggested certain biochemical similarities between this protein and alpha-inhibin precursors. We, therefore, investigated the hypothesis that alpha-inhibin and/or its precursors might represent autocrine and/or paracrine modulators of FSH action in the ovary, accounting for some of this FRBC activity and thereby exerting some degree of regulation over follicular maturation. Three separate sources of alpha-inhibin proteins were investigated for FRBC activity, including pFF, human FF (hFF), and a 293 cell line into which the full-length human alpha-inhibin cDNA had been stably transfected. Conditioned medium from these transfected cells contained several forms of alpha-inhibin precursors as well as mature alpha-inhibin, but no beta-subunit or intact inhibin. alpha-Inhibin proteins from all three sources, purified by a variety of methods, including immunoaffinity chromatography on an anti-alpha-inhibin column, inhibited FSH binding to both natural tissue FSH receptors as well as recombinant rat FSH receptors expressed in 293 cells. Furthermore, dimeric inhibin and activin, medium from untransfected 293 cells, and non-alpha-inhibin-containing purification fractions were inactive in either assay. In addition, purified recombinant alpha-inhibin proteins were partial in vitro FSH antagonists in a bioassay in which cAMP generation from 293 cells expressing the recombinant FSH receptor is used as an index of FSH biological activity. These same fractions of hFF containing FRBC activity did not bind to LH receptors, thereby demonstrating receptor specificity for this activity. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with alpha-inhibin or FRBC antisera, a 57,000 mol wt protein was identified in FRBC-active fractions from all three sources, suggesting that the active moiety was the full-length alpha-inhibin precursor protein or a large mol wt fragment, but not mature alpha-inhibin. Lastly, all FRBC activity from all three sources was extracted by an alpha-inhibin immunoaffinity column and was recoverable upon elution. These results demonstrate that proteins derived from the alpha-inhibin precursor modulate FSH binding to its receptor as well as its biological activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Cyclosporin-mediated depression of luteinizing hormone receptors and heme biosynthesis in rat testes: a possible mechanism for decrease in serum testosterone.

The toxic side-effects of the immunosuppressive drug cyclosporin (CsA) include testicular dysfunction and a decline in circulating testosterone. However, mechanisms for the consistently observed CsA-mediated depression of serum testosterone levels are unclear because of conflicting reports concerning circulating gonadotropin levels and incomplete studies of intratesticular steroidogenesis. To elucidate these mechanisms, endocrine-regulated testicular steroidogenesis and heme metabolic parameters were studied in male rats given sc injections of either 25 or 40 mg/kg.day CsA for 6 days and then killed on the seventh day. Consistent with earlier reports, CsA treatment dramatically suppressed serum testosterone levels (less than 20% of control at both CsA doses). Additionally, the intratesticular testosterone content declined with the higher CsA dose. Serum LH and FSH levels were elevated up to 2- to 4-fold after the higher CsA treatment regimen. Measurement of decreases in testicular receptors for LH revealed for the first time that CsA treatment significantly reduced the ability of the testes to respond to normal or elevated circulating levels of LH. In animals receiving higher dose of the drug, cytochrome P-450-dependent mitochondrial cholesterol side-chain cleavage activity, which is the rate-limiting step in steroidogenesis, was markedly reduced to a mere 30% of the control value. Additionally, the activity of the microsomal cytochrome P-450-dependent 17 alpha-hydroxylase was decreased to less than half of the control value. Biotransformation of the prototype drug, benzo(a)pyrene, as well as microsomal cytochrome P450 levels declined significantly after the higher CsA dose, suggesting that CsA has an adverse affect on testicular cytochromes P-450 in general. In addition, CsA treatment altered heme metabolic parameters; significant increases in the activity of uroporphyrinogen-I synthetase and total porphyrin content were noted. Conversely, the activity of ferrochelatase, the enzyme that incorporates iron into porphyrin to form heme molecule, decreased significantly, as did the total heme levels. The latter was reduced to only 61% of control values. The findings suggest the likelihood that the observed inhibition of heme formation may contribute substantially to the reduced levels of microsomal cytochromes P-450 and steroidogenic activities that depend on them. Taken collectively, these data suggest a plausible mechanism by which CsA may induce testicular dysfunction; as the result of a combination of reduction in the number of LH receptors and a suppression of heme formation, the hemoprotein-dependent steroidogenic enzymes activities are compromised, leading to an impairment of normal testicular function.

Animals↗
PubMed1991

Development of a radioligand receptor assay for measuring follitropin in serum: application to premature ovarian failure.

We have developed a radioligand receptor assay (RRA) with sufficient sensitivity and specificity for quantifying follitropin (FSH) in unextracted serum samples. Standard curves prepared by adding pituitary FSH to either buffer or gonadotropin-free serum were parallel and statistically indistinguishable in this assay, whereas gonadotropin-free serum alone had no activity. Cross-reactivity with related pituitary hormones was negligible. Pituitary FSH was calibrated with commonly used reference preparations so that RRA results could be compared with RIA results for identical standards. The patterns in daily blood samples in six normal menstrual cycles were similar by both methods. The mean RIA:RIA ratio in both the follicular and luteal phases was between 0.6 and 0.7, and at mid-cycle decreased to 0.48, suggesting an alteration of isohormone composition at mid-cycle. In 27 women with premature ovarian failure, RRA:RIA ratios ranged from below the RRA minimum detectable dose to 4.6, suggesting that immunoreactive FSH might not be capable of binding to the FSH receptor in some patients, whereas in patients with high RRA:RIA ratios, circulating inhibitors of FSH receptor binding might be present and perhaps contributing to the observed ovarian failure. Use of this RRA in conjunction with RIA and in vitro bioassays may better define the relative contribution of FSH isohormones, autocrine or paracrine modulators of FSH bioactivity, and FSH-receptor binding competitors to the "total FSH biological signal" as detected by the gonadal FSH receptor.

Adult↗
PubMed1990

Phospholipase C-mediated release of low molecular weight follicle-stimulating hormone receptor-binding inhibitor from testis membranes.

Low molecular weight inhibitors (FRBI) of FSH binding to receptor have been isolated from a variety of gonadal tissue extracts. Because of similarities noted in the composition of FRBI and that expected for polypeptides anchored to plasma membrane via a glycosyl-phosphatidylinositol linkage, we used bacterial phospholipase C to determine if FRBI could be released from calf testis membranes. FRBI was measured by use of radioligand-receptor assays and by a direct chemical method involving derivatization with dansyl chloride followed by HPLC. Phospholipase C treatment released FRBI from calf testis membranes in a time-dependent fashion. Phospholipase C-mediated release was blocked by O-phenanthroline, a specific inhibitor of phosphoinositol-phospholipase C (PI-PLC) activity. These data suggest that FRBI is anchored to testicular plasma membranes via a phospholipase C cleavable glycosyl-phosphatidylinositol anchor. The quantity of PI-PLC releasable FRBI in the testis and its FSH receptor-binding inhibitory potency suggest the possibility that endogenous regulation of FRBI release from testicular membranes could result in local attenuation of FSH action at the receptor level.

Animals↗
PubMed1990

cis-platinum-mediated decrease in serum testosterone is associated with depression of luteinizing hormone receptors and cytochrome P-450scc in rat testis.

Previously we had shown that cis-platinum decreases testosterone levels in rat serum and that hCG reverses this effect. The purpose of these studies was to determine the biochemical basis of cis-platinum-mediated effects on testicular testosterone production. In the testis of rats treated with cis-platinum (7 mg/kg, iv), the mitochondrial P-450scc concentration and side-chain cleavage activity were depressed by 40%. Also, the microsomal 17 alpha-hydroxylase activity and cytochrome P-450 concentration were decreased. Testicular binding capacity (in vitro) for [125I]hCG was decreased by 75-80%. On the other hand, FSH binding to Sertoli cell membrane receptors was not appreciably changed. hCG (25 IU/100 g daily) in treated rats caused complete occupancy of the remaining 20-25% LH receptors and caused a 20- to 30-fold increase in serum and testicular testosterone, a 2-fold increase in mitochondrial P-450scc, and a 5-fold acceleration of side-chain cleavage activity. 17 alpha-Hydroxylase activity and microsomal cytochrome P-450 were not increased over the control values. In addition to testicular functions, pituitary glycoprotein hormone production was assessed. Treatment of rats with cis-platinum (7 mg/kg, iv) did not change serum LH or FSH, but caused a 50% decrease in serum and testicular testosterone levels. A GnRH challenge test (1.5 micrograms/100 g, in 30 min) of treated rats caused prompt increases of 10- to 15-fold in serum LH and resulted in increases in serum and testicular testosterone. Thus, there was little evidence for cis-platinum effects at the level of hypothalamus or pituitary that could account for the decreased testosterone production. Reversal of the cis-platinum effect on steroidogenesis by hCG or GnRH appears to be due to the induction of suprasaturating levels of LH with full occupancy of remaining Leydig cell LH receptors. This, in turn, would reverse the diminished levels of mitochondrial side-chain cleavage activity and cytochrome P-450scc. These data suggest that cis-platinum causes a depression in serum testosterone, mainly by decreasing the number of LH receptors and inhibiting side-chain cleavage activity.

Animals↗
PubMed1989

Effect of bacterial lipopolysaccharide on growth of murine bladder cancer, MBT-2.

Intradermal palpable MBT-2 tumor responded with bacterial lipopolysaccharide to hemorrhagic necrosis (LPS) in C3H/HeN (endotoxin sensitive) mice. We have tested LPS fractions isolated from E. coli, Klebsiella pneumoniae, Salmonella minnesota, Pseudomonas aeruginosa and Serratia culture filtrates. All these LPS preparations showed tumor necrotizing activity accompanied by toxicity (body weight loss) in C3H/HeN mice. However, MBT-2 tumors grown in an endotoxin-resistant strain (C3H/HeJ) of mice did not respond to LPS, even at a very high dose. In vitro, the LPS showed no cytotoxic effect on MBT-2 cells. For comparison, systemic administration of tumor necrosis factor (cachexin ) did not affect the i.d. tumor growth. These data indicate that host reactions to LPS (endotoxicity) plays a pivotal role in the expression of tumor necrosis. Accordingly, comparisons of tumor response between endotoxin sensitive and resistant mice avoid potential overestimation of the therapeutic value of certain bacterial products and/or LPS contaminated agents.

Animals↗