Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Involvement of ATP in the regulation of slow (L-type) Ca2+ channels of vascular smooth muscle cells was investigated by recording single Ca2+ channel currents (single-channel conductance of 18 pS) using a patch clamp technique. In the cell-attached configuration, intracellular composition was modified by permeabilizing the cell membrane with mechanical disruption at one end of the cell. Single cells were freshly isolated from guinea-pig portal vein by collagenase treatment. For the channel recordings, the pipette solution contained 100 mM Ba2+ and the bath contained K+-rich solution (with 5 mM EGTA) to depolarize the membrane to near 0 mV. The channel activity decreased usually within 3 min after permeabilizing the cell end and exposure to ATP-free bath solution. If ATP (1-5 mM) was applied to the bath (access to cell interior) before complete disappearance of channel activity, channel activity was partially recovered. ATP did not change the current amplitude (i) or the mean open time of the channels, whereas the number of channels available for opening and/or the probability of their being open (NPo) were increased by ATP. A non-hydrolyzable analogue of ATP, AMP-PNP, did not exert an ATP-like effect; ATP-gamma-S had a weak effect. With 1 microM Bay-K-8644 (Ca2+ channel agonist) in the pipette, the activity of the Ca2+ channel was high; such activity persisted for more than 10 min after permeabilizing the cell and exposing to ATP-free solution containing KCN (1 mM) and 2-deoxy-D-glucose (10 mM).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Modulation of single slow (L-type) calcium channels by intracellular ATP in vascular smooth muscle cells. 247 13

To further understand the mechanism of PTH effects on bone and bone cells, we have analyzed the effect of PTH on specific protein phosphorylation in cells isolated from neonatal mouse calvaria. Four populations of cells (I-IV), isolated by sequential digestion with chromatographically purified bacterial collagenase isozymes and neutral proteinase, were cultured overnight. Alkaline phosphatase activity was greater than acid phosphatase activity in all four populations. PTH stimulated cyclic AMP production in all four populations, although the effect was greatest in populations II and III. Cultured cells were treated with PTH for up to 15 minutes. Cytosolic and membrane fractions were obtained and assayed for in vitro protein phosphorylation. No hormonal effects were found in membrane fractions. In cytosol fractions, treatment of the population II cells for 10-15 minutes with 0.1 microM PTH decreased the subsequent protein phosphorylation of an 85,000 Mr protein. In contrast, PTH treatment increased in vitro phosphorylation of both the 85,000 and 35,000 Mr proteins in population III cells. Phosphorylation of the 35,000 Mr protein was cyclic AMP-dependent. All of the phosphoproteins appeared to be phosphorylated solely on serine or threonine residues except the 85,000 Mr protein which may also contain significant amounts of phosphotyrosine. Therefore, some of the effects of PTH are cyclic AMP-mediated and other effects may be mediated through tyrosine phosphorylation. These data indicate that PTH has differential effects on in vitro protein phosphorylation in two separable populations of isolated neonatal mouse calvarial cells and support a hypothesis that multiple osteoblastlike cells exist in vivo.
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PMID:Differential effects of parathyroid hormone on protein phosphorylation in two osteoblastlike cell populations isolated from neonatal mouse calvaria. 249 26

Mixed bone cell cultures obtained by sequential collagenase-trypsin digestion of newborn chick, rat, and mouse calvaria responded to calcitonin gene-related peptide (CGRP) with a dose-dependent increase in cyclic AMP formation. The amplitude of response to CGRP in each species was less than that to parathyroid hormone (PTH). The CGRP effect was not the result of an action as a weak calcitonin agonist, since in most instances a calcitonin effect was not observed. Only in early digests of mouse calvarial cells were consistent stimulatory effects of calcitonin on cyclic AMP noted, and these were always considerably less in amplitude than those to CGRP. It is concluded that chick, rat, and mouse bones contain cells in osteoblast-rich populations that respond specifically to CGRP with a rise in cyclic AMP.
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PMID:Effects of calcitonin gene-related peptide on cyclic AMP formation in chicken, rat, and mouse bone cells. 254 86

Proximal tubules were prepared from rat kidney cortex by collagenase digestion and purified by Percoll gradient centrifugation. Their enrichment was estimated by comparing the specific activities of various cell-specific enzymes in homogenates of renal cortex and of the isolated tubules. The tubules were cultured in a 50:50 mixture of Dulbecco's modified Eagle's and Ham's F12 media supplemented with insulin, transferrin, epidermal growth factor, hydrocortisone, and prostaglandin E1. After 2 to 3 d an extensive outgrowth of epithelial cells developed from the attached tubules. After 5 to 7 d near confluent monolayers were obtained. Hormonal responsiveness, marker enzyme activities, and transport properties were determined to further characterize the primary cultures. The cultured cells exhibited increased cyclic AMP production in response to parathyroid hormone but not calcitonin or vasopressin, consistent with the absence of cells derived from distal and collecting tubules. The cells also retained significant levels of 25-hydroxyvitamin D3-1 alpha-hydroxylase, alkaline phosphatase, and gamma-glytamyl-transpeptidase, three enzymes that are primarily associated with the proximal tubule. The cultured epithelial cells also exhibit a Na+-dependent phosphate and glucose transport systems. Therefore, the cells retain many functional properties that are characteristic of proximal tubules. Thus, the primary cultures should be suitable for the study of processes that occur specifically within this segment of the rat nephron.
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PMID:Characterization of primary cell cultures derived from rat renal proximal tubules. 254 89

1. Catecholamines, adenosine, gonadotrophins, vasoactive intestinal peptide (VIP) and E-series prostaglandins all elicit K+ currents in follicle-enclosed Xenopus oocytes. Evidence suggests that cyclic nucleotides act as intracellular messengers in the activation of this K+ conductance. Muscarinic agonists and some divalent cations (e.g. Co2+, Mn2+, Ni2+ and Cd2+) elicit slow oscillatory Cl- currents, which are activated through hydrolysis of inositol phospholipids and mobilization of intracellular calcium by inositol phosphates. 2. We investigated whether these membrane current responses were generated in the oocyte itself or in enveloping follicular cells which are coupled to the oocyte by gap junctions. Oocytes were defolliculated, either enzymatically using collagenase, or by manual dissection combined with rolling over poly-L-lysine-coated slides. Removal of follicular cells was checked using scanning electron microscopy. Membrane current responses of defolliculated oocytes were compared with responses seen in follicle-enclosed oocytes taken from the same ovary. 3. The K+ responses evoked by all the various hormones/neurotransmitters were either drastically reduced (greater than 90%) or abolished by defolliculation. K+ currents generated by the adenylate cyclase activator forskolin and by intraoocyte injection of adenosine 3',5'-cyclic monophosphate (cyclic AMP), or guanosine 3',5'-cyclic monophosphate were similarly reduced in defolliculated oocytes. In contrast, oscillatory Cl- currents to acetylcholine and divalent cations were selectively preserved through defolliculation. 4. Injection of cyclic AMP (1-20 pmol) into defolliculated oocytes had little or no effect on oscillatory Cl- currents elicited by ACh. However, the calcium-dependent transient Cl- current, activated by depolarization of the oocyte membrane, was consistently potentiated (100-900%) by injections of cyclic AMP (1-10 pmol). 5. These experiments suggest that cyclic nucleotide-activated K+ currents arise essentially in follicular cells and are monitored within the oocyte through electrical coupling by gap junctions. Oscillatory Cl- responses evoked by ACh and divalent cations are produced largely or wholly in the oocyte itself.
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PMID:Effects of defolliculation on membrane current responses of Xenopus oocytes. 255 77

Sequential incubations with pronase and collagenase of pig gastric mucosa resulted in single cell preparations containing 10-20% parietal cells, which could be enriched further to 85-95% purity by density-gradient centrifugation followed by elutriation. Acid production of the isolated cells was measured by means of aminopyrine accumulation in their acid compartments. When small pieces of the mucosa were pretreated for 1 h in the presence of either histamine, pentagastrin or carbachol before preparation of cells, the ability of the subsequently isolated cells to produce acid was increased. In parietal cells isolated from resting (not pretreated) mucosa pentagastrin, carbachol and also adrenaline increased the histamine-stimulated aminopyrine accumulation (50-90% increase). Adrenaline alone had no significant effect on the aminopyrine accumulation. In the presence of 10(-4) M histamine the apparent EC50 for adrenaline was 5 X 10(-7) M. Adrenaline, histamine, forskolin and isobutylmethylxanthin (IBMX) increased the formation of cAMP in purified parietal cells. The three 'classical' secretagogues histamine, pentagastrin and carbachol, but also IBMX and forskolin, increased the cytosolic free Ca2+ from approximately 1.5 X 10(-7) M to 2.2-3.5 X 10(-7) M but adrenaline and dibutyryl cyclic AMP did not. Thus the present results indicate that there are - in addition to histaminergic H2 receptors - specific cholinergic, gastrinergic and adrenergic receptors on the plasma membrane and that there are separate cAMP and Ca2+-dependent stimulatory pathways in the parietal cell.
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PMID:Mechanisms of stimulation of acid production in parietal cells isolated from the pig gastric mucosa. 283 97

Smooth muscle cells from the gastric antrum of the rabbit were isolated using collagenase and pronase. We examined the characteristics of muscarinic receptors that control contraction of the muscle cell: kinetics, stoichiometry and specificity of both contractile response to muscarinic agents and binding of labeled N-methyl-scopolamine. Cells contracted in the presence of muscarinic agents after a short time (30 sec) while binding of (3H)-NMS reached a plateau after 10 min exposure. Specific binding was saturable and Scatchard analysis revealed a single class of high-affinity binding sites (Kd: 0.5 nM). Oxotremorine was the most potent agonist with an ED50 of 0.6 pM; acetylcholine and carbachol were 10 times less potent. Muscarinic antagonists competed with (3H)-NMS for binding with IC50 values in the same range (nanomolar or less) than those obtained for inhibition of acetylcholine-induced contractions. Pirenzepine antagonized contractile effect of muscarinic agonists with EC50 in a micromolar range. Intracellular levels of cyclic AMP were lowered by muscarinic agonists. Monoclonal anti-muscarinic receptor antibodies M-35 displayed agonist-like activities triggering contraction and lowering cyclic AMP levels of the cells. However, although the antagonist inhibits M-35-induced contractions and cAMP decrease, M-35 had no effect on binding of the antagonist to the muscarinic receptor. These data revealed the presence of an M2-muscarinic receptor subtype involved in the contractile response of the isolated smooth muscle cell.
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PMID:Muscarinic receptors in isolated smooth muscle cells from gastric antrum. 283 78

The secretion of prostaglandins (PGs) by bovine corpora lutea was investigated. Corpora lutea from the early, early-mid and late-mid stages of the luteal phase were dissociated by collagenase treatment and cultured in monolayer in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum. Treatment with either LH (100 ng/ml) or dibutyryl cyclic AMP (dbcAMP; 1 mmol/l) had no effect on progesterone secretion by early luteal phase cells but stimulated progesterone secretion two- to fourfold by cells from the latter stages. The secretion rates, per microgram cell protein, of 6-keto-PGF1 alpha, PGE2 and PGF2 alpha were substantially greater in cells from the early luteal phase than in those from the latter stages, however, all changes in PG secretion in response to treatments were qualitatively similar between cells from the three stages of the luteal phase. The secretion rate of 6-keto-PGF1 alpha was greater than that of PGE2 or PGF2 alpha and was inhibited by treatment with indomethacin (28 mumol/l) but unaltered by treatment with LH, dbcAMP or butyrate (1 mmol/l). Secretion of PGE2 was inhibited by indomethacin but stimulated two- to threefold by treatment with either dbcAMP or butyrate. Secretion of PGF2 alpha was minimal and not inhibited further by treatment with indomethacin, but was stimulated 10- to 40-fold with dbcAMP. Indomethacin treatment inhibited the stimulatory effect of dbcAMP; butyrate had no effect on PGF2 alpha secretion. Treatment with LH had no effect on any of the PGs measured. In these experiments the secretion of progesterone appeared unrelated to any changes in the secretion of PGs.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Secretion of progesterone and prostaglandins by cells of bovine corpora lutea from three stages of the luteal phase. 284 28

Monolayer cultures of human thyroid cells derived from thyroid adenoma were utilized for the assay of thyroid stimulating substances such as thyrotropin (TSH), cholera toxin and thyroid stimulating immunoglobulin (TSI) in patients with Graves' disease. Adenoma cells were treated with 0.1% collagenase or 2000 unit/ml dispase to thyrocytes. The cells were cultured in MEM containing 10% fetal calf serum under an atmosphere of 5% CO2 in air. Within 24 hours, the cells attached themselves to the plastic surface and formed a monolayer. Cyclic AMP responses to TSH, cholera toxin or Graves' IgG were tested in a medium (PBS) containing 0.5 mM IBMX. The cyclic AMP responses to TSH were generally maximal on the 3rd day of culture and declined thereafter. The response was dose-dependent, and 10 microU/ml of TSH produced a significant increase of cellular cyclic AMP. The response by 1 microU/ml of TSH was 28 approximately 57 fold above the basal. The response was also a function of the incubation period. The maximal response was attained after 1 h incubation. When the cultures were washed after exposure to TSH, the cellular cyclic AMP levels rapidly declined, suggesting that removal of receptor-bound TSH results in a prompt cessation of cyclic AMP production. The thyroid cells in monolayer also responded to cholera toxin. The response was dose-dependent, and cholera toxin as low as 1 ng/ml was able to increase cyclic AMP production. In contrast to the observations in TSH, the cyclic AMP responses induced by cholera were hardly affected by washing the cultures after exposure to cholera toxin. Treatment of the cells with cholera toxin for only 3 min resulted in a continuous stimulation of cyclic AMP production for more than 4 hours. Confirming recent observations by others, most of Graves' IgG stimulated cyclic AMP production in a dose-dependent manner, but some of them inhibited the response at high concentrations. IgG derived from normal subjects did not increase cellular cyclic AMP. The time course in the cyclic AMP responses induced by Graves' IgG was variable among the IgG preparations from different patients. In some patients, the maximal responses were attained after 4 hours of incubation. A significant difference was noted between TSH and Graves' IgG in the stimulation of cyclic AMP production after washing the cultures. When the cultures were treated with Graves' IgG for 30 min, washed and then incubated without Graves' IgG, cellular cyclic AMP levels remained at the levels which were almost equivalent to those observed in the continuous presence of the IgGs.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:[The effects of TSH, cholera toxin and Graves' IgG on cAMP production in cultured human thyroid adenoma cells in monolayer]. 286 66

It is currently believed that the thyroid stimulating immunoglobulin (TSI) of Graves' disease is involved in the pathogenesis of hyperthyroidism through the stimulation of the adenylate cyclase-cyclic AMP system. To evaluate this mechanism, TSI in the serum of patients with Graves' disease was determined by its ability to generate cyclic AMP (cAMP) in monolayer cells prepared from a normal thyroid gland. The thyroid tissue was digested with collagenase, and the liberated follicles were collected from the supernatant and cultured for 7 days. One gram of thyroid tissue yielded more than 1 X 10(7) monolayer cells which were stored in aliquots at -80C. Cells (1 approximately 2 X 10(4)/0.28 cm2 microtiter well) were incubated for 4 hours in 0.2 ml Hanks solution poor in NaCl, with various amounts of bovine TSH (bTSH) or 1.5 mg/ml Graves' serum IgG extracted by polyethylene glycol. cAMP accumulated in medium and cells was measured by RIA. Total cAMP (both medium and cells) was about 4 times higher when NaCl was deleted from Hanks solution. Moreover, as more than 90% of the cAMP was released into the medium, it was possible to omit the measurement of cellular cAMP, which requires extraction. The increase in medium cAMP concentration was dependent upon the number of cells, incubation time, and dose of bTSH. Time course and dose response curves in medium cAMP stimulated by IgG from 3 Graves' patients paralleled those of bTSH equivalent units. Accordingly, TSI activity could be expressed in bTSH equivalent units (bTSH microUeq). The assay could detect 1.0 or 3.3 microU/ml of bTSH and was highly reproducible. TSI activity in all of 16 IgGs from normal subjects was under 3.3 bTSH microUeq/ml, while it was greater than 3.3 bTSH microUeq/ml in IgGs from 33 of 37 (89%) untreated patients with Graves disease. Of the 13 patients followed for 2 to 7 months while on antithyroid drugs, 12 had greater than 3.3 bTSH microUeq/ml and, with the exception of one, all showed a decrease in their TSI activity. Moreover, 5 of 12 patients treated continuously for more than 1 year were TSI negative (less than 3.3 bTSH microUeq/ml), and except for one case, all had TSI values below 8 bTSH microUeq/ml (a value found in only 25% of untreated patients). This in vitro bioassay for TSI is simple and sensitive. It detects the presence of TSI in virtually 90% of untreated patients with Graves' disease. TSI activity showed a clear decrease during the course of antithyroid drug therapy.
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PMID:[Thyroid stimulating immunoglobulin bioassay using cultured normal human thyroid cells]. 286 82


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