Low-dose in utero effects of xenoestrogens in mice and their relevance to humans: an analytical review of the literature.

Several lines of evidence suggest that mouse embryos are sensitive to naturally-occurring and environmental estrogens. These include prostatic enlargement post-partum in male fetuses exposed in utero to low doses of estradiol, diethylstilbestrol (DES) or bisphenol A (BPA). The NIEHS/EPA Endocrine Disruptors Low Dose Peer Review Panel evaluated the relevant studies and concluded that while credible evidence exists for low dose effects of BPA, the effect had not been established as a "general and reproducible finding" based on the number and power of negative studies. The Panel suggested that the discrepancies in data were attributed to conditions, such as intrauterine position, environmental factors, and genetic factors. An issue that is potentially relevant to the health implications of low-dose xenoestrogen exposure in utero, and not previously addressed, is the comparative physiology of gestation in the mouse and human. These two species differ with regard to the extent of involvement and hormonal control of the corpus luteum, organs involved in progestin and estrogen secretion, the specific estrogens produced, and estrogen blood levels attained in the mother and embryo. On the basis of these species differences (particularly, the markedly higher estrogen levels attained in human pregnancy compared to the mouse), it would appear unlikely that low doses of BPA or other xenoestrogens produce adverse endocrine disruptive effects during human pregnancy.

Investigation of intracellular signals mediating the anti-apoptotic action of prolactin in Nb2 lymphoma cells.

Studies were undertaken to identify intracellular mediators of prolactin inhibition of glucocorticoid-induced apoptosis in Nb2 lymphoma cells. A short-term assay was implemented that quantitates fragmented DNA released from the genome by reaction with diphenylamine. Induction and inhibition of internucleosomal DNA cleavage (indicative of apoptosis) was verified by agarose gel electrophoresis of extracted cellular DNA. Synchronized Nb2 cells (G0/G1) exhibited increased DNA fragmentation after 4-hr incubation with dexamethasone (DEX) (25-100 nM) which was inhibited by ovine prolactin (oPRL) (0.1-1 ng/ml), the glucocorticoid receptor antagonist, RU486 (500 nM), and the nuclease inhibitor, aurintricarboxylic acid (100 microM). Signals previously implicated in prolactin induction of mitogenesis in Nb2 cells were investigated for their role in prolactin inhibition of apoptosis including: protein kinase C activation, arachidonic acid metabolism, polyamine production, tyrosine phosphorylation, and extracellular calcium. Protein kinase C agonists, phorbol-12-myristate-13-acetate, and 1,2-dioctanoyl-sn-glycerol, +/- the calcium ionophore, A23187 (200 nM), did not mimic oPRL inhibition of DEX-induced DNA fragmentation. Protein kinase C inhibitors, gossypol and quercetin, did not block prolactin action. Arachidonic acid did not mimic prolactin protection against DEX-induced DNA fragmentation. Inhibitors of arachidonic acid metabolism, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and indomethacin did not block prolactin action. The polyamine, spermine, inhibited DEX-induced DNA fragmentation at 1.5 to 2.5 mM. However, inhibition of polyamine synthesis with alpha-difluoromethyl ornithine or methylglyoxal bis(guanylhydrazone) did not inhibit prolactin action. Prolactin action was not blocked by inhibitors of tyrosine kinase activation, genistein and tyrphostin-47. On the other hand, pervanadate, a potent tyrosine phosphatase inhibitor, consistently inhibited DEX-induced DNA fragmentation. Prolactin action and DEX-induced apoptosis both occurred in calcium-free PBS. In summary, protein kinase C activation and eicosanoid production do not appear to mediate this prolactin action. Although spermine could block DNA fragmentation, blockade of the polyamine cascade did not inhibit prolactin action, suggesting that polyamines do not mediate this prolactin effect. While inhibitors of tyrosine kinase activation did not block prolactin action, tyrosine phosphatase inhibition in the presence of basal tyrosine kinase activity mimicked prolactin action, suggesting tyrosine phosphorylation participation in the anti-apoptotic effect. Extra-cellular calcium was not required for prolactin or DEX action.

Comparison of glucocorticoid-induced effects in prolactin-dependent and autonomous rat Nb2 lymphoma cells.

Cultured Nb2 node rat lymphoma cells require lactogenic hormone for their proliferation. We reported previously that dexamethasone (Dex) inhibits prolactin (PRL)-induced mitogenesis and, in the absence of mitogen, induces apoptosis of Nb2 cells. Both antiproliferative and cytolytic effects of Dex on Nb2 cells appear to involve glucocorticoid (Type II) receptor mediation. In this study, we compared Dex effects in PRL-dependent Nb2 cells (Nb2) with SFJCD1 (SF), a clone of Nb2 cells that proliferates independently of exogenous PRL. Proliferative assays involved a 72-hr incubation in a chemically defined, serum-free medium where ovine PRL (1 ng/ml) was added to Nb2 cells but not to SF cells. Both cell lines were responsive to the antiproliferative effects of Dex in a dose (6.25-200 nM)-dependent fashion of comparable sensitivity and magnitude. Co-incubation with the glucocorticoid receptor antagonist, RU 486, prevented the antiproliferative effect of Dex in both cell lines. In the same medium devoid of PRL, Dex was cytolytic to Nb2 cells and fragmented DNA in a fashion reflective of apoptosis, but was ineffective in SF cells. A dual chamber incubation system revealed no evidence that SF cells produced cytokines that were mitogenic or anticytolytic to Nb2 cells. Both Nb2 and SF cells fragmented DNA in a fashion indicative of apoptosis in the presence of the Ca2+ ionophore, A23187 (1 microM). These studies reveal a basic difference in glucocorticoid responsiveness between the PRL-dependent Nb2 cell line and its PRL-independent subclone, SF. While both cell lines exhibit functional glucocorticoid receptors and the necessary intranuclear machinery for apoptosis, the pathway mediating the latter is inhibited or dysfunctional in SF cells.

Glucocorticoid-prolactin interactions in Nb2 lymphoma cells: antiproliferative versus anticytolytic effects.

The interaction between the immunosuppressive effects of glucocorticoids and the mitogenic effects of prolactin (PRL) were examined in Nb2 lymphoma cells, a pre-T cell line. The synthetic glucocorticoid, dexamethasone (Dex), caused a concentration-dependent (6.25-200 nM) inhibition of basal and ovine PRL (oPRL)-stimulated Nb2 cell proliferation. Although Dex was antiproliferative, the steroid had no effect on cell viability in the presence of PRL. However, when PRL was omitted from the medium, Dex increased the proportion of dead Nb2 cells by 24 hr in a concentration (25-200 nM)-dependent fashion without affecting total cell number. The antiproliferative and cytolytic effects of Dex were mimicked by other corticosteroids (cortisol, corticosterone, aldosterone, and deoxycorticosterone) in the expected order of glucocorticoid potency, but not by other steroids (17-beta-estradiol, progesterone, testosterone, 5-alpha-dihydrotestosterone, and dehydroepiandrosterone) or triiodothyronine. In addition, the antiproliferative and cytolytic effects of glucocorticoids were antagonized by the glucocorticoid receptor antagonist RU 486. Since corticosteroid-induced cytolysis was apparent only in the absence of mitogen, the anticytolytic effects of oPRL were tested. In the presence of Dex (100 nM), oPRL (25-1600 pg/ml) caused a concentration-dependent inhibition of cytolysis without changing cell number. Other lactogenic hormones (human growth hormone, human placental lactogen, rat PRL), but not trophic nonlactogenic hormones (rat growth hormone, human chorionic gonadotropin, ACTH), also inhibited Dex (100 nM)-induced cytolysis. Agarose gel electrophoresis of DNA extracted from Nb2 cells revealed that within 12 hr, 100 nM Dex induced DNA fragmentation, indicative of programmed cell death or apoptosis. Coincubation of cells with Dex and oPRL (1 ng/ml) inhibited Dex-induced fragmentation of Nb2 cell genomic DNA. These studies reveal a complex interaction between glucocorticoids and PRL in Nb2 cells. Although a glucocorticoid receptor-mediated antiproliferative effect is evident, PRL (at concentrations that usually stimulate cell proliferation) has the capacity to protect the cell against glucocorticoid-receptor-mediated induction of apoptosis.

Effects of oral erythrosine (2',4',5',7'-tetraiodofluorescein) on the pituitary-thyroid axis in rats.

Erythrosine (FD&C Red Dye No.3) is a tetraiodinated derivative of fluorescein. Rats fed a 4% erythrosine diet for 30 months beginning in utero have an increased incidence of thyroid adenomas and adenocarcinomas. These tumors may be secondary to increased stimulation of the thyroid gland by TSH. This study was undertaken to determine if dietary erythrosine disrupts the pituitary-thyroid axis thereby altering serum thyroid hormone levels. TSH levels, or the pituitary's response to TRH. Rats were fed diets containing erythrosine (0.5, 1.0, 4.0%), sodium iodide (0.16%), or fluorescein (1.6%) for 3 weeks after which TRH testing was performed in vivo. Erythrosine produced a dose-dependent increase in serum T4 levels. With the 4% erythrosine diet, serum T4 and T3 levels and the free-T4 index were significantly increased, whereas the free-T3 index were significantly increased, whereas the free-T3 index was unchanged. Rats fed the 4.0% erythrosine diet had an exaggerated TSH response to TRH; 10 min after the TRH injection, serum TSH levels were 80% greater than TSH levels of control rats. Short-term administration of erythrosine to rats decreased hepatic T3 production by decreasing its conversion of T4 to T3, indicating that erythrosine decreases hepatic 5'-deiodinase activity. These data demonstrate that dietary ingestion of 4% erythrosine disrupts the pituitary-thyroid axis as evidenced by an increased TSH response to TRH. This effect is mediated by erythrosine or an iodinated metabolite, since ingestion of its fluorescein nucleus had no effect. Erythrosine's effects were not likely mediated by iodide, because serum T4 and T3 levels were elevated and iodide administration did not increase the TSH response to TRH. These data suggest that erythrosine increases the pituitary's TSH response to TRH by altering thyrotroph cell conversion of T4 to T3. Chronic erythrosine ingestion may promote thyroid tumor formation in rats via chronic stimulation of the thyroid by TSH.

A critical analysis of the relationship between parental smoking and pulmonary performance in children.

To date, at least 24 epidemiologic research papers, of essentially similar design, have been published on the effects of current or lifetime parental smoking on pulmonary function parameters in children. In these studies, parental smoking and other data obtained from standardized questionnaires and spirometric measurements in children were compared statistically according to the smoking status of the parents. A survey of these reports reveals a number of inconsistencies in the association between parental smoking status and pulmonary function parameters (FEV1 or FEV0.75, FEF25-75, FVC, and Vmax50%) in the child. A number of factors should be considered when interpreting the results of these studies, particularly in light of the observed inconsistencies and the fact that children were classified solely on the basis of questionnaire data. Among these are sources of misclassification bias, which could either underestimate or overestimate parental smoking effect, socioeconomic status, other variables, and genetic factors. Also, effects of maternal smoking in utero or on lactation, as well as exposure of the child to environmental tobacco smoke (ETS), need to be considered as possible causes of any apparent decrement in pulmonary function in children.

The effect of small increases in dietary iodine on thyroid function in euthyroid subjects.

Dietary iodine intake in the United States is greater than that considered necessary for the maintenance of normal thyroid function. The administration of pharmacologic quantities of iodine (10 to 1,000 mg daily) to euthyroid subjects results in small decreases in the serum T4 and T3 concentrations and a compensatory increase in the basal and TRH-stimulated serum TSH concentrations. Studies were carried out to determine whether a far smaller increase in iodine intake would also affect thyroid function. Normal volunteers received 1,500, 500, or 250 micrograms supplemental iodine daily for 14 days. Following the administration of 1500 micrograms iodine daily, there were small but significant decreases in the serum T4 and T3 concentrations and a small compensatory increase in the serum TSH concentration and the serum TSH response to TRH. In contrast, no changes in pituitary-thyroid function occurred during the administration of 500 or 250 micrograms iodine daily. These findings indicate that a small increase in dietary iodine can induce subtle changes (all values remaining within the normal range) in pituitary-thyroid function, probably by inhibiting thyroid hormone release. The smaller iodine supplements of 500 and 250 micrograms daily, quantities that may easily be achieved under normal conditions, did not, however, affect thyroid function.

Effects of oral erythrosine (2',4',5',7'-tetraiodofluorescein) on thyroid function in normal men.

Erythrosine (Er), a tetraiodinated derivative of fluorescein, is a coloring agent widely used in foods, cosmetics, and pharmaceutical products. Because of its high iodine content and previous reports demonstrating an inhibitory effect of erythrosine on hepatic 5'-monodeiodination, we studied the effects of this compound on thyroid function and serum and urinary iodide concentrations in normal subjects. Thirty normal men, equally divided into three treatment groups, each received a 14-day course of oral Er in doses of 20, 60, or 200 mg/day. Serum thyroxine (T4), triiodothyronine (T3), reverse T3 (rT3), thyroid stimulating hormone (TSH), protein-bound iodide (PBI), and total iodide concentrations, serum T3-charcoal uptake, and 24-hour urinary iodide excretion were measured on Days 1, 8, and 15. Thyrotropin-releasing hormone (TRH) tests were performed on Days 1 and 15. There were no significant changes in serum T4, T3, rT3, and T3-charcoal uptake values at any dose. In men receiving 200 mg Er/day, the mean basal serum TSH concentration increased significantly from 1.7 +/- 0.1 (SE) on Day 1 to 2.2 +/- 0.1 microU/ml on Day 15 (p less than 0.05), and the mean peak TSH increment after TRH increased from 6.3 +/- 0.5 to 10.5 +/- 1.0 microU/ml (p less than 0.05). There were no significant changes in basal or peak TSH responses in the men receiving 20 or 60 mg Er/day. Significant dose-related increases in serum total iodide and PBI concentrations occurred during all three doses, and significant dose-related increases in urinary iodide excretion occurred during the 60 and 200 mg/day Er doses. These data suggest that the increase in TSH secretion induced by Er was related to the antithyroid effect of increased serum iodide concentrations, rather than a direct effect of Er on thyroid hormone secretion or peripheral metabolism.

Biological, immunological and biochemical characterization of cleaved prolactin generated by lactating mammary gland.

We have previously shown that rat prolactin is proteolytically cleaved in its loop by peripheral tissues of the rat. Of the tissues examined to date, lactating mammary gland exhibits the highest prolactin-cleaving activity. The objective of this study was to characterize cleaved prolactin, biologically, immunologically and chemically. By modifying an established analytical method, we were able to generate large (micrograms) amounts of cleaved rat prolactin from cell fractions of rat mammary gland which could then be assayed for biological and immunological activity relative to intact hormone. The cleaved product showed no significant difference relative to the intact rat prolactin when assayed for its ability to compete with 125I-labelled ovine prolactin for the prolactin receptor and for its ability to stimulate the proliferation of rat Nb2 lymphoma cells. Cleaved rat prolactin, however, did show a 50-60% reduction in activity relative to intact rat prolactin when assayed by radioimmunoassay. Using Edman degradation and partial amino acid analysis, we determined that the second N-terminus of the cleaved rat prolactin begins at amino acid 149. The divergence of biological and immunological activity produced by proteolytic cleavage in the loop of rat prolactin suggests that biological and immunological sites differ in location. The possible physiological implications of a cleaved rat prolactin molecule generated by target tissue with maintained biological activity and reduced immunological activity are discussed.

Proteolytic modification of rat prolactin by subcellular fractions of the lactating rat mammary gland.

The current study explored prolactin proteolysis by rat lactating mammary gland. 125I-labelled rat prolactin was incubated with tissue fractions of lactating mammary gland and the extent of prolactin degradation and fragment formation was visualized and densitometrically quantitated from autoradiographs derived from SDS-polyacrylamide gel electrophoresis under reducing conditions. At pH 4.5, the 25 000 X g pellet of mammary gland converted intact prolactin (23 kDa band) to proteolytic fragments (8-16 kDa bands) in a time- and tissue concentration-dependent fashion similar to that reported previously for rat ventral prostate. The prolactin-degrading and -fragmenting activity in lactating mammary gland was 5-10-times that observed for ventral prostate, the most active male tissue. This activity at acid pH was also demonstrable in other fractions of mammary gland but appeared to predominate in the cytosol. The above activities in mammary gland virtually disappeared at pH 7.4, appeared sensitive to aspartate and sulfhydryl proteinase inhibitors, and insensitive to serine and metalloenzyme proteinase inhibitors. The distribution of this activity could not be correlated with a particular enzyme marker. These characteristics of mammary gland activity differed significantly from those reported previously for prostate. When electrophoresis was conducted under non-reducing conditions, prolactin proteolysis in prostate and mammary gland was primarily associated with the formation of a more slowly migrating product (24 kDa band) with little spontaneous 8-16 kDa fragment formation. Re-electrophoresis of the 24 kDa band under reducing conditions resulted in the appearance of the 8 and 16 kDa fragments. In conclusion, prolactin is proteolytically modified by prostate and lactating mammary gland to a variant of intact hormone (24 kDa band) with a cleavage site in its large loop, by two or more widely distributed, acid-dependent proteinases. Lactating mammary gland, the principal target for prolactin, has the capacity to cleave the hormone in its loop at rates higher than any other tissue examined to date.

Modulation of prolactin binding sites in vitro by membrane fluidizers. IV. Differential effects on plasma membrane and Golgi fractions of male prostate and female liver in the rat.

In vitro treatment of crude particulate fractions of male rat ventral prostate and female rat liver with membrane fluidizers (aliphatic alcohols) has been previously reported by us to increase prolactin (PRL) receptor levels, presumably by unmasking cryptic prolactin receptors. The objective of this study was to determine if similar in vitro treatment of purified plasma membrane- and Golgi-rich fractions of male rat prostate and female rat liver with ethanol produced differential effects on prolactin binding in these two subcellular fractions. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. 125I-PRL specific binding to Golgi-rich fractions of male ventral prostate and female liver was approximately 4-fold higher than that observed in plasma membrane-rich fractions. The microviscosity parameter, inversely related to lipid fluidity, was consistently lower in Golgi-rich fractions than that in plasma membrane-rich fractions in both prostate and liver. In vitro ethanol treatment of prostatic and hepatic plasma membrane fractions produced a dose-related increase and then decline in prolactin binding and a maximal (60-75%) increase in prolactin binding was observed at 4.8% and 2.0% ethanol in prostatic and hepatic membranes, respectively. This in vitro treatment also produced a significant increase in apparent lipid fluidity of plasma membrane-rich fractions of prostate gland and liver. However, similar in vitro ethanol treatment of Golgi fractions of both prostate gland and liver exhibited little increase in prolactin binding without changing microviscosity. Our observations are consistent with the direct relationship between membrane fluidity and prolactin receptor levels. The changes in prostatic and hepatic plasma membrane fractions following in vitro ethanol treatment suggest that prolactin receptors located on the plasma membranes may be modulated (via membrane lipid microviscosity changes) in vivo to a greater extent by various physiological agents than those located within the Golgi fraction.

Prolactin binding in minor salivary gland tumors.

An immunohistochemical study of 15 minor salivary gland tumors was initiated to determine if prolactin binding occurred in these tissues. Eight benign mixed tumors (BMT) and 7 adenoid cystic carcinomas (ACC) were selected at random from the surgical biopsy service of the MCV/VCU School of Dentistry, Department of Oral Pathology. The specimens were cut and mounted on slides along with sections of rat pituitary and rat ventral prostate which served as methodologic controls. Experimental specimens were incubated for 24 hours with varying concentrations of highly purified (iodination grade) rat prolactin; controls were incubated with vehicle. Following incubation the specimens were stained according to the Sternberger peroxidase-antiperoxidase method. Results showed dose-dependent staining for prolactin binding sites in 7 of 8 BMTs and 5 of 7 ACCs. The staining was wider in distribution than we observed in normal human minor salivary gland tissue. Binding was confined primarily to cells of duct origin in both types of tumor. In individual cells, staining was observed in diffuse cytoplasmic and perinuclear locations as well as in nuclei and apical regions. We conclude that two minor salivary gland neoplasms (BMT and ACC) exhibit prolactin binding at different cellular locations and in a more widespread pattern than was observed in normal minor salivary gland.

Prolactin increases lipid fluidity and prolactin binding of rat prostatic membranes.

The objective of these studies was to determine whether prolactin could modify the lipid fluidity of rat ventral and dorsolateral prostate membranes and subsequently modify the availability of prolactin receptors. Additional studies were also undertaken to determine the effects of prolactin on serum lipid fluidity. Adult male rats were injected with 0, 100, or 400 micrograms ovine prolactin/day subcutaneously for a period of 5 days. Serum and prostatic membrane lipid fluidity was measured by a fluorescence polarization method using a lipid probe 1,6-diphenylhexatriene. Prolactin binding in dextran-coated charcoal-pretreated prostatic membranes was determined by radioreceptor assay. This pretreatment has been reported by us to remove the endogenous substances that interfere with prolactin binding assay (J. R. Dave and R. J. Witorsch, Endocrinology 111: 2144-2146, 1982). Prolactin binding increased by approximately 44 and 72% in dorsolateral prostate and 16 and 39% in ventral prostate in 100- and 400-micrograms groups, respectively. Membrane fluidity increased by approximately 16 and 19% in dorsolateral prostate and 10 and 13% in ventral prostate in 100- and 400-micrograms groups, respectively. Serum lipid fluidity increased 50 and 79% in 100- and 400-micrograms groups, respectively.

Modulation of prolactin binding sites in vitro by membrane fluidizers. Effects on male prostatic and female hepatic membranes in alcohol-fed rats.

The objectives of this study were (i) to determine if in vivo administration of ethanol to rats produced changes in apparent lipid fluidity and prolactin binding capacity of male prostatic and female hepatic membranes and (ii) to compare the effects of membrane fluidizers (aliphatic alcohols) in vitro on prolactin binding of prostatic and hepatic membranes in control and alcohol-fed animals. In vitro ethanol has been shown by us previously to increase prolactin receptor levels presumably by unmasking cryptic prolactin receptors. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Adult male and female rats were given either water or 4% ethanol as the sole source of drinking fluid for a period of 6 weeks. No significant changes in plasma prolactin were observed between control and ethanol-treated groups of either sex. However, the microviscosity parameter, inversely related to lipid fluidity, was increased approx. 34% and 40%, respectively, in male prostatic and female rat hepatic membranes after ethanol feeding. Furthermore, 125I-prolactin binding capacity was decreased approx. 30% and 26%, respectively, in prostatic and hepatic membranes of alcohol fed animals. In vitro treatment with aliphatic alcohols had no effect on either microviscosity or prolactin binding in hepatic or prostatic membranes from ethanol-fed rats, but both fluidized and increased prolactin binding in the same membrane preparations from control rats. Our observations are consistent with the direct relationship between membrane fluidity and prolactin receptor levels. The changes in prostatic and hepatic membranes after alcohol feeding, namely decreased prolactin receptor levels, decreased fluidity and increased resistance to the fluidizing effects of in vitro aliphatic alcohols may reflect a fundamental membrane defect.

Endocrine-mediated parallel changes in hepatic glucocorticoid and prolactin receptors.

The objective of this study was to determine whether in female rat liver any relationship existed between prolactin and glucocorticoid receptors after hormonal manipulation. Bromocryptine (CB-154) treatment of adult SD female rats (80-100 days old) for 48 h decreased prolactin binding to hepatic membranes 49% and dexamethasone binding in hepatic cytosol 40% below control values. Administration of rat prolactin along with bromocriptine prevented these changes. In another study, prolactin binding to hepatic membranes increased 53% and dexamethasone binding in hepatic cytosol increased 113% above sham-control values, 3 days after adrenalectomy. On the other hand, hydrocortisone treatment of sham-operated rats reduced prolactin binding by 57% and dexamethasone binding by 76%. Scatchard analyses of the prolactin or dexamethasone binding data indicated that these manipulations changed the number of prolactin or dexamethasone binding sites rather than their apparent affinity constants. In vitro treatment of rat whole liver homogenate with various doses (10(-9) - 10(-5) M) of dexamethasone and corticosterone for 15 min at 22 degrees C resulted in a dose-dependent decrease in prolactin binding activity. However, direct addition of dexamethasone to a hepatic 15 000 X g to 100 000 X g membrane preparation exhibited no significant effects on prolactin binding. In conclusion, these studies show that (a) there is a parallel in vivo modulation of rat liver prolactin and glucocorticoid receptors under various experimental conditions and (b) in vitro exposure of whole liver homogenate to glucocorticoids inhibits the prolactin binding activity.

Prolactin binding in normal human minor salivary gland tissue: an immunohistochemical study.

The breast and salivary gland exhibit some similarities in the biochemical and cellular spheres. There are also some tumors that are common to both organs. The protein hormone prolactin seems to play a role in the health and disease of the mammary gland, and an investigation was launched to see if this hormone exhibited any binding activity in normal human minor salivary gland. With the use of immunohistochemical methods, dose-dependent staining for prolactin binding was demonstrated in the striated and collecting ducts in four of six specimens of normal human minor salivary gland (two males and two females). The hormone concentrations used ranged from 6.25 ng/ml to 500 ng/ml of highly purified rat prolactin. A review of known functions of prolactin in man and other animals is presented, and it was concluded that normal human minor salivary gland tissue possesses binding sites for prolactin, which suggests that prolactin may play a role in the metabolic functioning of the salivary duct cells.

Proteolytic degradation and modification of rat prolactin by subcellular fractions of the rat ventral prostate gland.

We previously showed that at pH 7.4, the cytosol and a low speed pellet (3300 X g) of rat ventral prostate degraded rat PRL, whereas a high speed pellet (25,000 X g) was inactive. The current study further explores PRL degradation by ventral prostatic tissue. Enzyme markers indicated that the low speed pellet was mitochondria enriched (cytochrome c oxidase), and the high speed pellet was lysosome enriched (acid phosphatase), although there was considerable cross-contamination between the two fractions. Proteolysis of hormone was examined by incubating 125I-labeled rat iodo-PRL with tissue fractions, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioautography to identify products and quantify the extent of PRL degradation. Acidification of the incubation medium (pH 4-5) to optimize lysosomal protease activities inhibited PRL degradation in cytosol and activated the process in both pellets consistent with the distribution of acid phosphatase. Under acidic incubation conditions, both low and high speed sediments produced the same electrophoretic pattern of PRL degradation, indicating the generation of peptide fragments from the hormone with a molecular mass of approximately 16,000-8,000 daltons. Cytosol (pH 7.4) also produced peptide fragments in this weight range, although at much lower rates and in relatively lower proportions. Peptide fragment formation from PRL by the low speed pellet (pH 7.4) was minimal. On a protein basis, PRL degradation by the high speed sediment (pH 4.5) was 3-10 times that by the low speed sediment (pH 7.4). Enzyme inhibitor analysis indicated differences in each of the cell fractions with regard to the types of proteases involved in PRL degradation. When compared with other tissues of the male rat, ventral prostate was the most active in degrading PRL and generating peptide fragments. In addition, kidney, spleen, and lung were among the more active tissues, whereas liver, was deferens, and dorsolateral prostate were relatively less active in degrading hormone. Our studies indicate that qualitatively different processes degraded PRL in each of the active subcellular fractions and that the generation of peptide fragments from PRL predominates in a lysosome-rich fraction of ventral prostate. The possible significance of subcellular variations in PRL processing and the generation of peptide fragments of the hormone by peripheral tissue are discussed.

Modulation of prolactin binding sites in vitro by membrane fluidizers. II. Age-dependent effects on rat ventral prostatic membranes.

The objectives of this study were to determine (i) if the age-related changes in 125I-labeled ovine prolactin specific binding of rat ventral prostate was correlated with changes in membrane lipid microviscosity and (ii) if membrane fluidizers produced age-dependent effects on prolactin binding of prostatic membranes. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Membrane preparations of ventral prostate glands obtained from immature (24-25 days old), young-adult (80-90 days old) and aged (550-610 days old) male rats were used for prolactin binding and membrane lipid microviscosity measurements. Relative to immature rats, prostatic prolactin binding decreased approximately 50% in young-adult rats and 75% in aged rats. Membrane lipid microviscosity, relative to immature rats, was increased 72% in young-adult rats and 140% in aged rats. Prostatic membranes obtained from immature animals exhibited no significant effects of in vitro alcohol treatment on prolactin binding, whereas, those obtained from aged animals exhibited maximal increase in prolactin binding. The value of the microviscosity parameter, after in vitro alcohol exposure, exhibited no significant changes in immature animals, whereas, this parameter was decreased approximately 15% in young-adults and approximately 30% in aged animals. These data suggest that in vitro fluidization of prostatic membrane exhibits an age-dependent modification of prolactin binding.

Indomethacin decreases both luteinizing hormone binding and fluidity of testicular microsomal membranes in rat.

The objective of these studies was to examine the effects of in vivo treatment with indomethacin, a prostaglandin synthesis inhibitor, on luteinizing hormone receptors and fluidity in microsomal membrane preparations of rat testis. Adult male rats were treated with vehicle or either of two doses of indomethacin (3.8 micrograms/g body weight or 7.5 micrograms/g body weight) every 8 h for 1, 2 and 3 days. Indomethacin treatment decreased human luteinizing hormone (hLH) binding in rat testis in a time-dependent fashion. The lower dose of indomethacin decreased hLH binding by 19%, 35% and 50%, respectively, after 1, 2 and 3 days of treatment. The higher dose of indomethacin decreased hLH binding at these respective times by 21%, 44% and 56%. Scatchard analysis of the hLH binding of testicular membrane preparations indicated that indomethacin treatment decreased the number of hLH binding sites rather than the apparent affinity constant. Testicular membrane fluidity was estimated by a fluorescence polarization technique. The effects of indomethacin on apparent fluidity of microsomal membrane paralleled that of LH binding. The values of apparent microviscosity, inversely related to the fluidity, also increased in a time-dependent fashion. These simultaneous effects of indomethacin on testicular LH binding and fluidity suggest that these phenomena are interrelated and that prostaglandins are involved in the maintenance of LH receptors in vivo.