Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The extent to which pituitary explants secrete GH requires clarification. In the present study GH release from ectopic pituitary tissue was assessed using the reverse hemolytic plaque assay. In addition, the effects of this tissue on eutopic GH and PRL release were simultaneously investigated. Anterior pituitary glands from adult female donor rats were grafted beneath the kidney capsule of adult male hosts. Four weeks later, this ectopic pituitary tissue and the eutopic pituitary glands of the host animals were removed, dispersed with trypsin, and subsequently assayed for GH and PRL release. Contrary to expectations, we found that 37% of the ectopic pituitary cells were GH secretors. Furthermore, these GH cells remained highly responsive to GRF (10(-7) M), which evoked a 7-fold increase (P less than 0.05) in mean GH plaque area. By comparison, only 28% of these ectopic cells secreted PRL, and this release was not consistently augmented by exposure to TRH (10(-7) M). In the second half of this study we found that the presence of ectopic pituitary tissue severely compromised the secretory capacity of the eutopic pituitary cells. More specifically, GH-releasing factor induced a 3-fold increase in GH secretion from the eutopic pituitary cells of sham-operated control animals, but it enhanced the release from cells of host animals by only 2-fold. Moreover, the response to TRH by the eutopic PRL cells from explant-bearing animals was curtailed to such an extent that it was not significantly greater (P greater than 0.05) than basal release in that group. We conclude that the potential of pituitary explant cells to release GH is far greater than previously believed and that this ectopic hormone production may inhibit hormone release from the eutopic pituitary cells.
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PMID:Pituitaries transplanted under the renal capsule contain functional growth hormone (GH) secretors and suppress GH and prolactin release from individual eutopic pituitary cells. 251 Sep 89

Plasma PRL levels in male rats are highest during the peripubertal period. We previously reported that the posterior pituitary (PP) contains a potent PRL-releasing factor (PRF), a trypsin-insensitive small peptide which is distinct from known PRL secretagogues. The objectives were to determine the ontogeny of PRF activity in the PP as well as age-related alterations in anterior pituitary responsiveness to PRF. We also explored if the PP contains a nondopaminergic PRL-inhibiting factor (PIF). PRF/PIF activities were assessed by the ability of PP extracts to alter PRL release from cultured anterior pituitary cells. The PP were extracted with perchloric acid and lyophilized, thus eliminating endogenous dopamine. PRF activity in PP extracts from 10- and 20 day-old (d) rats was very low, increased gradually in 30d and 40d rats, and remained unchanged in adult (90d) rats. In a second experiment, age-related changes in anterior pituitary responsiveness to PP extracts from adult rats and to TRH were determined. The responsiveness of anterior pituitary cells from 10d rats to PRF was low, increased dramatically in cells from 20d rats, and was reduced in cells from 30d and adult rats. The responsiveness to TRH was highest in cells from 10d rats. In a third experiment, anterior pituitary responsiveness to age-matched PP extracts was assessed. Only PIF activity was observed when PP extracts from 10d rats were incubated with anterior pituitary cells from 10d rats. In contrast, PP extracts from 20d, 30d and adult rats exhibited only PRF activity when incubated with age-matched cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ontogeny of prolactin releasing and inhibiting activities in the posterior pituitary of male rats. 251 64

Immunoreactive PRL (IR-PRL) has been identified in many areas of the rat brain. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses we have recently shown that the primary IR-PRL protein in the rat brain has an apparent mol wt (Mr) of 24,000, which was identical to that of pituitary PRL. In these studies, brain and pituitary 24,000 Mr PRL were compared by peptide mapping and lectin chromatography. PRL-enriched fractions were prepared from the pituitary, hypothalamus, hippocampus, and pons-medulla and labeled with 125I. This material was further purified by immunoprecipitation, and immunopurified 24,000 Mr PRL was isolated by sodium dodecyl sulfate-gel electrophoresis. Cleavage of 125I-labeled 24,000 Mr IR-PRL prepared from the pituitary and the three brain regions with chymotrypsin resulted in identical peptide maps with two primary labeled peptide fragments (5,500 and 6,000 Mr), and approximately five less intense fragments. Similarly, trypsin cleavage of brain and pituitary 24,000 Mr IR-PRL resulted in the production of two major fragments (6,200, and 5,200 Mr), and three less intense fragments. Cleavage of the 24,000 Mr IR-PRL with Staphylococcus V8 protease resulted in identical fragment patterns with two primary peptide fragments (14,000 and 6,200 Mr). When the pituitary and brain 24,000 Mr PRL were applied to Concanavalin-A-Sepharose columns no 24,000 Mr PRL was absorbed. The similarity of the peptide fragments obtained from the cleavage of the 24,000 Mr IR-PRL from the brain and pituitary clearly indicate that the IR-PRL found in the brain has an amino acid sequence that shares a high degree of structural homology with pituitary PRL.
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PMID:Comparison of brain and pituitary immunoreactive prolactin by peptide mapping and lectin affinity chromatography. 279 95

An improved rat anterior pituitary primary cell culture technique for studying GH-releasing activity of human pancreatic GH-releasing factor (hpGRF) and its analogs is described. Male pituitaries, dispersed by a combination of trypsin digestion and mechanical agitation, were plated at a density of 200,000 cells per well and cultured for 4 days. The attached cells were then stimulated with synthetic hpGRF which was comprised of the first 29 residues of the larger, originally isolated forms and which was amidated at the C-terminal (hpGRF-29). Analogs of hpGRF-29 which were modified in positions 1, 2, 3, or 7, and other secretagogues were similarly tested. Medium was collected after 3 h, and secreted hormone was measured by RIA. The cells were extremely sensitive to hpGRF-29 stimulation, and this effect was specific. The minimal effective dose of hpGRF-29 was an unprecedented 0.4 X 10(-15)M. No stimulation of LH, FSH, or PRL by hpGRF-29 was observed. Bombesin and vasoactive intestinal peptide were ineffective in stimulating GH release. [D-Trp6]LHRH (a potent LHRH agonist), also did not release GH but did stimulate secretion of LH and FSH at doses ranging from 0.4 X 10(-10)M to 1.0 X 10(-9)M. Responses of the cells to hpGRF-29 analogs were characterized by distinct heterologous dose-response curves. [D-Ala2]hpGRF-29 was 50 times more active than its parent 29-amino-acid peptide. [D-Thr7]hpGRF-29, another analog that differed from hpGRF-29 by the insertion of a D-isomer for the naturally occurring L-residue, was about 10,000 times less effective in stimulating GH secretion than was hpGRF-29 itself. Potencies of these and other analogs with respect to GH release in vitro were similar to those estimated in vivo. Thus, this primary cell culture provides an extremely sensitive, selective, and reproducible system for studying hpGRF structure-activity relationships. Further, such tremendous sensitivity to hpGRF can provide a system to study changes in pituitary sensitivity to hpGRF during different physiological states.
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PMID:An extremely sensitive in vitro model for elucidating structure-activity relationships of growth hormone-releasing factor analogs. 285 74

The role of PRL in the control of the mechanisms of regulation of testicular function is still unclear. Indeed, hyperprolactinemia is usually associated with male hypofertility. Several studies have demonstrated that human seminal plasma (HSP) contains radioimmunoassayble PRL and than testis and prostate possess membrane receptors for PRL (Charreau et al. 1977; Aragona et al. 1977). Recently Demoulin and Franchimont have observed, in HSP, the presence of a substance capable of inhibiting PRL secretion by isolated rat pituitary cells (Demoulin et al. 1978). They abbreviated it SPIF (seminal plasma PRL inhibiting factor). The inhibition of PRL secretion was significant for concentrations equal to or higher than 4.2 l of HSP/ml of culture medium. This non steroidal substance is thermostable and trypsin resistant; the molecular weight is less than 10,000 Daltons. In this work, we have compared the SPIF activity with basal levels of LH, FSH, PRL, Testosterone and, after stimulation, in the serum of patients affected with various fertility problems.
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PMID:Prolactin (PRL) inhibiting substance in seminal plasma of infertile patients. Preliminary results. 287 54

Reverse hemolytic plaque assays were used to compare the responsiveness of cells from different pituitary regions to the modulatory effects of human pancreatic GH-releasing factor (GRF), TRH, and dopamine (DA). Tissues from the peripheral rim (outer zone) and the central region (inner zone) of adenohypophyses from day 10 lactating rats were dispersed with trypsin, and the cells were placed into culture. On the following day, these cells were subjected to GH plaque assays (conducted in the presence or absence of GRF) and PRL plaque assays (performed with or without TRH and DA). Cells from both zones responded similarly to GRF with a rapid acceleration of GH plaque formation. However, the rate of PRL plaque formation in response to TRH and DA differed between cells from these regions. For outer zone cells, plaque development increased greatly with TRH treatment, but was only moderately affected by DA. Plaque formation from inner zone cells was influenced slightly by TRH, but markedly inhibited by DA. These results suggest that PRL, but not GH, cells from these pituitary regions are differentially responsive to at least two hypothalamic secretagogues. We then performed fixed sequential plaque assays to determine whether the proportions of cells that released PRL only (classical mammotropes) or those that released both GH and PRL (mammosomatotropes) also differed between the inner and outer zones. Using this approach, we found that the outer zone contained a much larger proportion of dual hormone secretors than did the inner zone. These results, when taken together with the responsiveness differences discussed above, raise the possibility that the release of PRL from mammotropes and mammosomatotropes is regulated differently and that the ratio of these two cell types may dictate, in part, the manner in which a specific region of the pituitary responds to hypothalamic input.
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PMID:Functional variations among prolactin cells from different pituitary regions. 310 Feb 86

We previously reported that the rat posterior pituitary contains a potent PRL-releasing factor (PRF) which is distinct from oxytocin (OT), TRH, and angiotensin II (AII). The objectives of this study were 1) to examine whether posterior pituitary extracts stimulate PRL release in the presence of dopamine (DA), 2) to determine the chemical nature of PRF, and 3) to estimate its mol wt. Perifused anterior pituitary cells were used to assess PRF activity. Posterior pituitaries and medial basal hypothalamus (MBH) fragments were extracted with perchloric acid and lyophilized. Subsequent to various treatments, samples were reconstituted in the perifusion medium and introduced to the cells in short pulses. Fractions were collected and analyzed for hormone content by RIA. During a constant infusion of DA (50 nM), PRL secretion was inhibited by 75%, yet the posterior pituitary extract retained its ability to rapidly stimulate PRL release. Studies using proteolytic enzymes showed that posterior pituitary PRF was resistant to inactivation by trypsin, whereas the PRF activity of AII was abolished. Both chymotrypsin and proline-specific endopeptidase significantly reduced the PRF activity in the posterior pituitary. The PRL-releasing activity of TRH was not affected by chymotrypsin. Immunoreactive vasoactive intestinal polypeptide was undetectable in posterior pituitary extracts. Oxidation of posterior pituitary extracts with performic acid caused only a modest reduction of their PRF activity, while the ability of OT to stimulate PRL release as well as immunoreactive OT was abolished. Studies using ultrafiltration membranes showed that the PRF activity in the posterior pituitary was less than 5,000 mol wt. Furthermore, posterior pituitary PRF partitioned in nearly equal amounts across 1K membranes, as did AII and OT. In contrast, about 80% of the PRF activity in the MBH and all of the synthetic TRH passed through the 1K membranes. We conclude that 1) posterior pituitary PRF can stimulate PRL secretion from perifused anterior pituitary cells in the presence of physiological concentrations of DA; 2) PRF is a small peptide(s) of less than 5,000, and perhaps closer to 1,000, mol wt; 3) PRF is resistant to inactivation by trypsin and to oxidation by performic acid, but is hydrolyzed by both chymotrypsin and proline-specific endopeptidase; and 4) these data further distinguish posterior pituitary PRF from known PRL secretagogues.
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PMID:Characterization of prolactin-releasing factor in the rat posterior pituitary. 313 Nov 18

The factors that regulate the release of human placental lactogen (hPL) are poorly understood. To determine whether hPL is regulated by a factor(s) in pregnancy serum, placental explants were exposed for up to 9 h to a pool of serum samples from 50 women in the third trimester. In static explant cultures, the addition of the serum (0.6-10.8 mg protein/ml) caused a dose-dependent and reversible increase in hPL release during a 6-h period. The maximum release by the explants exposed to pregnancy serum was 200-250% greater than that of control explants, and the half-maximal dose was 2-3 mg/ml. Perifusion of placental explants with 15% pregnancy serum (final concentration, 10.5 mg protein/ml) also caused a significant release in hPL within 15 min, which reached a maximum of 200-225% above control levels. Two other pools of pregnancy serum samples as well as individual samples from four pregnant women also stimulated hPL release. Although pregnancy serum significantly stimulated hPL release, there was no increase in either the release of hCG or trichloroacetic acid-precipitable 35S-labeled proteins. Serum from nonpregnant women and men, as well as bovine serum, also stimulated hPL release, but their potencies were only 20-25% that of pregnancy serum. Chicken and porcine serum (10.8 mg/ml each) caused only small (less than 10%) increases in hPL release, and purified human albumin and ovalbumin had no effect. Dialysis or ultrafiltration of pregnancy serum using membranes with mol wt exclusions of 10K daltons caused no loss of activity. Delipidation of pregnancy serum with acetone-ethanol or acid-charcoal also caused no loss of activity, but treatment with trypsin caused greater than 95% loss of activity. Purification of the stimulatory activity by successive chromatographies on Sephadex G-150, Cibacron blue, and Sephadex G-75 resulted in an approximately 800-fold increase in specific activity. Approximately 90% of the total activity eluted from Sephadex G-75 with an apparent mol wt of 31,000, the remainder eluted in the void volume. Although partially purified pregnancy serum stimulated hPL release, the active fractions did not affect the release of rat LH, FSH, or GH from rat pituitary cells or the release of PRL from human decidual explants. Incubation of placental explants in calcium-deficient medium blocked the stimulatory effect of the partially purified pregnancy serum by greater than 90%. These studies indicate that human serum contains a protein(s) that causes a specific, rapid, dose-dependent, and reversible increase in hPL release.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization and partial purification of a serum protein which stimulates the release of human placental lactogen in vitro. 372 25

Developmental changes and sexual differences in the lactotrope population were studied with a reverse hemolytic plaque assay for detection and measurement of PRL secretion from individual cells in conjunction with PRL immunocytochemistry (ICC). Pituitary glands from both sexes at different ages were monodispersed with 0.1% trypsin. Freshly dispersed cells were incubated in Cunningham chambers for measurement of the fraction of plaque-forming cells and the size of plaques formed, or attached to glass slides for measurement of the fraction of cells staining for PRL by ICC. The percentage of plaque-forming cells in both sexes gradually increased with age from about 5% of the total anterior pituitary cell population at 5 days of age to the adult levels of about 54% in the females at proestrus and about 37% in the males. Sexual differences in the percentage of plaque-forming cells were consistently seen at 40 days of age and thereafter. The mean size of plaques in both sexes also increased with age from about 1,100 microns 2 at 5 days of age to a peak level of about 15,600 microns 2 in the females at proestrus and about 6,700 microns 2 in the males at 60 days of age; then fell to the adult levels of about 5,500 microns 2 in the females at proestrus and about 3,200 microns 2 in males. Sexual differences first appeared at 40 days of age and were estrogen-dependent. The results from ICC closely matched those from the plaque assay, except at 5 days of age and in the adult males. At 5 days, the fraction of cells staining for PRL was twice that of cells forming plaques. In adult male rats there were about 47% immunostained PRL cells but only about 39% plaque-forming cells. This difference, however, disappeared after estrogen treatment. These results demonstrate that sexual differences in the lactotrope population develop around puberty in rats and appear to be estrogen dependent.
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PMID:Postnatal development of pituitary lactotropes in the rat measured by reverse hemolytic plaque assay. 378 May 61

Evidence obtained in human breast cancer cell lines in culture suggests that estradiol stimulates the synthesis of secretory proteins which may, in turn, mediate its mitogenic effect. We questioned whether a similar mechanism could mediate the growth-promoting effect of PRL in the N-nitrosomethylurea-induced rat mammary tumor grown in soft agar, where PRL exerts a dose-dependent colony-stimulating effect. Conditioned medium obtained from PRL-treated tumors, but not from control tumors, was found to exert a significant dose-dependent colony-stimulating effect when added to N-nitrosomethylurea-induced mammary tumors plated in soft agar under serum-free medium conditions. The growth-promoting action of conditioned medium from PRL-treated tumors was abolished by pretreatment with heat, trypsin, and Concanavalin-A, suggesting the possible glycoprotein nature of the oPRL-induced growth factor(s). These results provide support for the novel hypothesis that estradiol and PRL may support the growth of hormone-responsive breast cancer through a similar mechanism.
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PMID:Autocrine stimulation by prolactin of hormone-responsive breast cancer growth in culture. 404 74


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