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)

Rat medial basal hypothalami (MBH) and sections of cerebral cortex (CC) were dissociated with trypsin to prepare single cells and subcellular fractions. They were then separated into four fractions on a discontinuous sucrose gradient. The small neurons in Fraction D were highly purified. Fraction A had synaptosomes, myelin and other cell particulates. Fraction B had glial cells, neurons and a few synaptosomes. Fraction C had large neurons and red blood cells. All four fractions contained LHRH, but most (62.5%) of this hormone was present in Fraction A. Dissociated cell suspensions were incubated with [3H]-steroids, with and without a 100-fold excess of unlabeled steroids, then separated on sucrose gradients. In most fractions the total uptake and specific uptake of [3H]-progesterone, [3H]-5 alpha-pregnane-3,20-dione (5 alpha-dihydroprogesterone) and [3H]-17 beta-estradiol were greater for the dissociated cells from the MBH than the CC. The dissociated cells and cell particulates in all four fractions from the MBH and CC metabolized progesterone, 5 alpha-dihydroprogesterone and 17 beta-estradiol. These results indicate that hypothalamic neurons contain small amounts of LHRH and retain the ability to take up and metabolize progesterone, 5 alpha-dihydroprogesterone and 17 beta-estradiol.
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PMID:Uptake and metabolism of female sex steroids by isolated small neurons and other cell fractions from the rat medial basal hypothalamus. 37 73

The anterior pituitary (AP) tissues were removed from adult male Sprague-Dawley rats by decapitation. The dispersed cells with high viability (greater than or equal to 95%) were prepared using trypsin digestion and mechanical dissociation, and then mixed with Bio-Gel P2, packed into the columns, and perfused continuously with M199 medium for more than 24 hours. Different doses of LHRH were administered by 6-min pulses at one hour intervals. A steady and detectable basal LH secretion was present in all columns during the experiment. LHRH stimulation pulse could induce LH secretion rapidly, and repeated LHRH pulses of same dose produced statistically equal LH pulses. The dose-response curve of LH secretion was linear within the range of 1 X 10(-10) to 1 X 10(-7) mol/L LHRH. These results indicate that continuous perfusion system of dispersed AP cells which offers significant advantages over other methodologies provides a very useful in vitro model for studying the mechanisms on LHRH regulation of LH secretion.
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PMID:[Continuous perfusion of dispersed anterior pituitary cells: a model for LHRH regulation of LH secretion]. 219 Mar 27

The fate of cell surface gonadotropin-releasing hormone (GnRH) receptors on pituitary cells was studied utilizing lysosomotropic agents and monensin. Labeling of pituitary cells with a photoreactive GnRH derivative, [azidobenzoyl-D-Lys6]GnRH, revealed a specific band of Mr = 60,000. When photoaffinity-labeled cells were exposed to trypsin immediately after completion of the binding, the radioactivity incorporated into the Mr = 60,000 band decreased, with a concomitant appearance of a proteolytic fragment (Mr = 45,000). This fragment reflects cell surface receptors. Following GnRH binding, the hormone-receptor complexes underwent internalization, partial degradation, and recycling. The process of hormone-receptor complex degradation was substantially prevented by lysosomotropic agents, such as chloroquine and methylamine, or the proton ionophore, monensin. Chloroquine and monensin, however, did not affect receptor recycling, since the tryptic fragment of Mr = 45,000 was evident after treatment with these agents. This suggests that recycling of GnRH receptors in gonadotropes occurs whether or not the internal environment is acidic. Based on these findings, we propose a model describing the intracellular pathway of GnRH receptors.
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PMID:Internalization and recycling of receptor-bound gonadotropin-releasing hormone agonist in pituitary gonadotropes. 282 11

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 characteristics of the steroidogenic response of reaggregated rat interstitial cells were examined in a perifusion system. Interstitial cells were isolated from 19-day-old rat testes by digestion with collagenase. The cells were cultured for 3 days as monolayers and were resuspended by brief treatment with trypsin. Constant gyratory shaking of the dispersed cells resulted in the formation of round and compact aggregates of 70-140 microns. The functional characteristics of these aggregates were examined by studying the output of cAMP, C19 steroids (testosterone and androstenedione), and C21 steroids (progesterone, 17 alpha-hydroxyprogesterone, 20 alpha-hydroxypregn-4-en-3-one) in a perfusion system. It is demonstrated that reaggregated interstitial cells maintain their responsiveness to LH, LHRH, and Leydig cell stimulatory factor(s) produced by Sertoli cells for at least 12 days. When exposed to low concentrations of LH (1 ng/ml), either in a continuous or in a pulsatile fashion, perifused aggregates maintain a constant output of steroids for more than 20 h. Under these conditions, LH-dependent differentiation of the steroidogenic machinery can be observed in vitro. In fact, although the sum of the measured steroids remains constant, C21 steroids progressively decrease whereas C19 steroid output increases during perifusion. When perifused with high concentrations of LH (10 ng/ml), desensitization becomes the predominant phenomenon. It is demonstrated that the steroid output of reaggregated interstitial cells considerably exceeds that of similarly treated cells maintained as monolayers. Moreover, perifusion of aggregates results in a 6-fold increase in steroid output as compared to static incubation and in a selective increase in androgen output. It is concluded that prepubertal interstitial cells allowed to reaggregate in suspension culture form functional multicellular structures. Perifusion of these aggregates is a useful tool in the study of the dynamics of the regulation of steroidogenesis.
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PMID:The dynamics of steroid and adenosine 3',5'-cyclic monophosphate output in perifused interstitial cell aggregates derived from prepubertal rat testes. 301 35

In the present study both the reverse hemolytic plaque assay for detecting luteinizing hormone (LH) secretion from single cells and LH immunocytochemistry (ICC) were applied to conduct quantitative studies on sexual differences in the gonadotrope population during postnatal development. Pituitary glands from both sexes at different ages were monodispersed with 0.1% trypsin. Freshly dispersed cells were incubated in Cunningham chambers in the presence of 10(-7) M gonadotropin-releasing hormone (GnRH) for measurement of the fraction of plaque-forming cells and the mean size of plaque formed, or attached to glass slides for measurement of the fraction of cells staining for LH by ICC. The percentage of immunostained LH cells increased with age in both sexes from about 5% of the total pituitary cell population at 5 days of age to a plateau of about 10% by 15 days and then fell to the adult level of about 5%. There were no significant sexual differences except at 30 and 40 days of age. In female rats the fraction of LH-secreting cells detected by plaque assay matched closely with that of LH-containing cells detected by ICC. However, there were significant sexual differences in the percentage of LH-secreting cells at day 15 through day 40. The mean LH output from individual cells of both sexes as indicated by the mean size of plaques also increased with age and reached a peak around 50 days. The sexual differences were first seen around 30 days of age with greater amounts in the female than in the male.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Sexual dimorphism of pituitary gonadotropes during postnatal development in the rat. 329 58

Extracts from bovine pituitary were found to contain an activity catalyzing the conversion of glutaminyl peptides such as [Gln1]gonadotropin-releasing hormone, [Gln1, Gly4]thyrotropin-releasing hormone (H-Gln-His-Pro-Gly-OH), and H-Gln-Tyr-Ala-OH to the respective pyroglutamyl peptides. The TRH precursor fragment H-Lys-Arg-Gln-His-Pro-Gly-Lys-Arg-OH and the D-glutaminyl stereoisomer of H-Gln-Tyr-Ala-OH did not react under the same conditions. The conversion products were identified by Edman degradation, amino acid analysis, and reversed-phase HPLC. That this activity was exhibited by an enzyme, glutaminyl cyclase, was concluded from the protein character of the activity (revealed by its abolition with trypsin and heat), the Michaelis-Menten relationship between substrate concentration and conversion rate, and the substrate specificity. It was determined that glutaminyl cyclase had a molecular weight of 43,000-50,000, a pH optimum at pH 8, and Km and Vmax values in the range of 60-130 microM and 390-690 pmol/microgram per hr, respectively. Glutaminyl cyclase was not observed to require ATP and could be inhibited with 1.0 M ammonium chloride, which increased the Km and decreased the Vmax value. The subcellular distribution of glutaminyl cyclase corresponded to the one of peptidylglycine alpha-amidating monooxygenase believed to catalyze C-terminal amidations during posttranslational precursor processing. It was also observed that the formation of pyroglutamyl from glutaminyl peptides occurred nonenzymatically; however, the enzymatic reaction carried out with crude extract was found to be approximately 70 times faster than the nonenzymatic reaction enhanced by phosphate. It is speculated that glutaminyl cyclase may participate in the posttranslational processing of hormonal precursors to pyroglutamyl peptides.
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PMID:Identification of a mammalian glutaminyl cyclase converting glutaminyl into pyroglutamyl peptides. 347 73

Procedures for cell dissociation and in vitro culture were validated to investigate secretion of luteinizing hormone (LH) from bovine anterior lobe (AL) pituitary cells. The concentration of trypsin used for dissociation affected cell yield, cell loss during preincubation, LH secretion, and response to gonadotropin-releasing hormone (GnRH). Optimum results were obtained with trypsin concentrations of 8-16 micrograms/mg fresh tissue. Duration of preincubation and of experimental culture markedly affected LH secretion and response to GnRH. Immediately after dissociation, cells contained relatively low quantities of LH, but they were able to release a substantial proportion of this LH. Basal release of LH and GnRH-induced release of LH were highly correlated with total quantities of LH, and all three parameters increased with time of preincubation until 24 hr. Experimental treatments of 2 hr duration were optimal for investigating GnRH stimulation of LH release, whereas longer treatments may be required to investigate effects of agents that inhibit the release of LH. Preincubation of dissociated AL cells with physiological concentrations of estradiol increased all three LH parameters. Progesterone had no effect either alone or in combination with estradiol. In conclusion, the procedures described for cell dissociation and culture of suspended cells provide a useful tool for studying release of LH from the bovine AL cell.
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PMID:LH release from dispersed bovine pituitary cells in culture: in vitro effects of estradiol and procedural variables. 350 88

The properties of gonadotropin-releasing hormone (GnRH) receptors were analyzed in isolated pituitary cells prepared by enzymatic dispersion with trypsin or collagenase-hyaluronidase. The initial impairment of LH responses to GnRH in isolated cells prepared by both methods was reversed during culture for 2 days in multiwell vessels. However, specific binding sites for GnRH, assayed by equilibration with [125I]iodi0[D-Ser(t-BU)6]des-Gly10-GnRH N-ethylamide (GnRH-A) were demonstrable in collagenase-dispersed cells, both initially and after 2 days in culture. Cellular uptake of GnRH-A was temperature dependent, with rapid and saturable binding to a limited number of specific receptor sites with high affinity for the labeled analog (Ka = 4.0 +/- 0.8 X 10(9) M-1). These sites showed common binding specificity for GnRH-A and the native GnRH peptide, with significantly lower affinity for the natural peptide (Ka = 2.3 X 10(7) M-1). Other protein and peptide hormones, including ovine LH, ovine PRL, hCG, TRH, somatostatin, and angiotensin II (up to 10(-6) M) did not inhibit binding of GnRH-A to its receptors. Cellular binding of GnRH-A was followed by increased cGMP production and LH release within 10 min. The analog was 50 times more potent than native GnRH in stimulating LH release. The Kact values derived for GnRH and GnRH-A were 0.5 and 0.01 nM, respectively, considerably lower than the Kd values of 50 and 0.25 nM derived from receptor binding analysis. These results indicate that GnRH receptors can be identified in isolated pituitary cells, in which peptide binding is followed by increased cGMP production and LH release. The impaired LH responses in acutely dispersed pituitary cells are not due to the loss of receptor sites but to a reversible postreceptor defect. Occupancy of about 20% of the GnRH-binding sites elicits a near-maximal LH response, indicating the nonlinearity of GnRH-receptor coupling to secretory responses in the cultured gonadotroph.
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PMID:Characterization of gonadotropin-releasing hormone receptors in cultured rat pituitary cells. 625 Jul 93

The interaction of gonadotropin-releasing hormone (GnRH) agonists and antagonists with pituitary membranes was studied by using 125I-labeled agonist, [D-Ser(t-Bu)6, des-Gly10-ethylamide]-GnRH, and antagonist [D-pGlu1, D-Phe2, Trp3,6]-GnRH. Their binding was affected differently by cations, and by pretreatment of membranes with proteolytic enzymes and sulfhydryl-blocking reagents. Monovalent cations at millimolar concentrations (10-100 mM) and divalent cations at lower concentrations (0.5-5 mM) reduced more significantly the binding of the agonist than that of the antagonist. Pretreatment of the membranes with trypsin and chymotrypsin abolished the specific binding of both agonist and antagonist, in a dose-response manner, with the former being less affected. Pretreatment of the membranes with sulfhydryl-blocking reagents did not alter the binding of the antagonist but enhanced the binding of the agonist. This enhancement in the specific binding was found to be due to an increase in the apparent affinity of the agonist. These results may suggest that GnRH agonists and antagonists bind differently to the same receptor.
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PMID:Some characteristics of GnRH receptors in rat-pituitary membranes: differences between an agonist and and antagonist. 626 24


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