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)

Tracheal smooth muscle cells (TSMCs) were isolated from dog trachea in order to analyze the direct effects of growth factors and hormones on cell proliferation and muscarinic receptor (mAchR) expression. Dissection and dissociation of tracheal smooth muscle tissue with a collagenase I, deoxyribonuclease I and elastase IV mixture resulted in high yield and viability of TSMCs. A screen of growth factors, hormones, and serum concentration for the stimulation of cell growth, revealed that insulin-like growth factor, basic fibroblast growth factor, epidermal growth factor, insulin, transferrin, or hydrocortisone alone at the concentration used was not necessary or sufficient to stimulate growth of TSMCs in the primary culture with DMEM/F-12 containing 1% FBS. The regulation of cell surface mAchR expression in response to serum and cell growth in primary culture of TSMCs has been examined. In the presence of 1% serum, TSMCs withdraw from the cell cycle and express high levels of cell surface mAchRs. Exposure of quiescent TSMCs to 10% serum results in a loss of surface mAchRs. In addition, insulin-like growth factor, insulin or transferrin could stimulate the expression of mAchRs on TSMCs cultured in DMEM/F-12 containing 1% FBS. The results demonstrated that low serum concentration culture system may provide a useful model to elucidate the expression of mAchRs in the culture of TSMCs.
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PMID:Muscarinic receptor expression in the primary culture of tracheal smooth muscle cells. 207 1

Intrarenal administration of cholinergic agents produces diuresis. However, neither cholinergic innervation or specific cholinergic receptors have been shown to be present in the kidney. Recently, we have demonstrated that carbachol, a cholinergic agent, stimulates phosphoinositide hydrolysis in the inner medullary collecting duct (IMCD) cells. The effect was blocked by atropine (a cholinergic antagonist), suggesting that phosphoinositide hydrolysis occurs through the interaction of carbachol with specific cholinergic receptors in these cells. Therefore, we examined the cholinergic receptors in IMCD cells by measurement of radioligand binding of a cholinergic receptor antagonist, I-quinuclidinyl (phenyl-4-3H)benzilate([3H]QNB). The IMCD cells were prepared from rabbit kidneys by incubating the inner medullary slices with collagenase and treating the isolated cells with hypotonic solution to lyse cells other than IMCD cells. Binding of [3H]QNB to IMCD cells was measured at 37 degrees C for 60 min in the absence (total binding) and the presence (nonspecific binding) of 100 microM atropine (a muscarinic receptor antagonist). The specific binding (the difference between total and nonspecific binding) of [3H]QNB to IMCD cells was saturable with a Bmax (maximum binding sites) of 27.5 fmol/mg of protein and Kd (dissociation constant) of 0.27 nM. Atropine, but not hexamethonium (a nicotinic antagonist), was able to displace [3H]QNB from IMCD cells with a Ki of 0.1 microM. It is, therefore, concluded that specific high affinity muscarinic receptors are present in IMCD cells. These receptors may play a role in producing the pharmacologic actions of cholinergic agents on the kidney.
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PMID:Cholinergic receptors in renal medullary collecting duct cells. 253 24

1. Single dispersed cells obtained by collagenase treatment of longitudinal muscle of rabbit small intestine were voltage clamped with low-resistance patch pipettes and membrane current was measured. 2. In cells held at -20 or -30 mV, a discharge of spontaneous transient outward currents (STOCs) was usually seen; these are believed to represent the sporadic release of calcium from storage sites in the cell in relation to TEA-sensitive, 4 AP-resistant, calcium-activated potassium channels. 3. Caffeine (20 mM) externally applied, accelerated and then abolished STOCs; carbachol (0.1 mM) had similar effects; the initial burst of STOCs was often carried on a large, temporary, outward current which could occur alone. This was suggested to be caused by the rapid release of stored calcium in relation to calcium-activated potassium channels. 4. If STOCs were abolished by caffeine (or carbachol) then carbachol (or caffeine) did not evoke outward current indicating that these drugs act on the same calcium store but by different pathways. Inclusion of ryanodine (10(-8)-10(-4) M) in the patch pipette abolished STOCs soon after establishing whole-cell recording mode; afterwards, outward current to caffeine or to carbachol could not be evoked. 5. STOCs were quickly abolished in cells patched with pipettes filled with GTP gamma S (0.1-1 mM) or Gpp(NH)p (0.1-1 mM) but were large or normal in size in cells where GDP beta S (0.1-1 mM) was included in the pipette. GTP gamma S or Gpp(NH)p in the cell abolished outward current to caffeine or to carbachol, but had no effect on calcium-activated potassium channel activity in isolated patches or on a TEA-sensitive, 4-AP-resistant, outward potassium current evoked in single cells by stepping positively from a -20 mV holding potential. These results suggest that the effect of guanine nucleotide analogues are on the calcium store rather than on calcium-activated potassium channels. 6. The effects of GTP gamma S or Gpp(NH)p could be explained if they depleted calcium stores via a G-protein mechanism; this effect may involve activation of phospholipase C enzyme (PLC) and D-myo-inositol 1,4,5-trisphosphate (IP3) production as well as a direct effect on stores. However a separate G-protein-independent pathway of activation of PLC by muscarinic receptor activation may exist as calcium release by carbachol was large or normal in cells filled with GDP beta S.
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PMID:Properties of calcium stores and transient outward currents in single smooth muscle cells of rabbit intestine. 258 96

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 loss of [3H]quinuclidinyl benzilate ([3H]QNB) binding following high-energy radiation was used to compare the muscarinic receptor size on single smooth muscle cells isolated by collagenase digestion from the canine stomach and on plasma membranes derived from intact gastric smooth muscle without exposure to exogenous proteolysis. Radiation inactivation of galactose oxidase (68 kdaltons), yeast alcohol dehydrogenase (160 kdaltons), and pyruvate kinase (224 kdaltons) activities were used as molecular-weight standards. Radiation inactivation of [3H]QNB binding to rat brain membranes, which gave a target size of 86 kdaltons, served as an additional control. In isolated smooth muscle cells, the calculated size of the muscarinic receptor was 80 +/- 8 kdaltons. In contrast, in a smooth muscle enriched plasma membrane preparation, muscarinic receptor size was significantly smaller at 45 +/- 3 kdaltons. Larger molecular sizes were obtained either in the presence of protease inhibitors (62 +/- 4 kdaltons) or by using a crude membrane preparation of gastric smooth muscle 86 +/- 7 kdaltons).
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PMID:Muscarinic receptor size on smooth muscle cells and membranes. 374 Feb 62

We have previously shown that inositol-1,4,5-trisphosphate (IP3) releases Ca2+ from an intracellular calcium store in permeabilized acinar cells of rat pancreas (H. Streb et al., 1983, Nature (London) 306:67-69). This observation suggests that IP3 might provide the missing link between activation of the muscarinic receptor and Ca2+ release from intracellular stores during stimulation. In order to localize the intracellular IP3-sensitive calcium pool, IP3-induced Ca2+ release was measured in isolated subcellular fractions. A total homogenate was prepared from acinar cells which had been isolated by a collagenase digestion method. Endoplasmic reticulum was separated from mitochondria, zymogen granules and nuclei by differential centrifugation. Plasma membranes and endoplasmic reticulum were separated by centrifugation on a sucrose step gradient or by precipitation with high concentrations of MgCl2. IP3-induced Ca2+ release per mg protein in the total homogenate was the same as in leaky cells and was sufficiently stable to make short separation procedures possible. In fractions obtained by either differential centrifugation at 7000 X g, sucrose-density centrifugation, or MgCl2 precipitation there was a close correlation of Ip3-induced Ca2+ release with the endoplasmic reticulum markers ribonucleic acid (r = 0.96, 1.00, 0.91, respectively) and NADPH cytochrome c reductase (r = 0.63, 0.98, 0.90, respectively). In contrast, there was a clear negative correlation with the mitochondrial markers cytochrome c oxidase (r = -0.64) and glutamate dehydrogenase (r = -0.75) and with the plasma membrane markers (Na+ + K+)-ATPase (r = -0.81) and alkaline phosphatase (r = -0.77) in all fractions analyzed. IP3-induced Ca2+ release was distributed independently of zymogen granule or nuclei content of the fractions as assessed by electron microscopy. The data suggest that inositol-1,4,5-trisphosphate releases Ca2+ from endoplasmic reticulum in pancreatic acinar cells.
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PMID:Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas. 633 62

Smooth muscle cells from the guinea pig gastric fundus were isolated by successive collagenase digestions. Tritiated quinuclidinyl benzilate [( 3H]QNB) was used to study the binding characteristics of the muscarinic cholinergic receptors on these cells. Each cell bound 8.3 X 10(-19) mol of QNB, and a concentration of QNB of 0.19 nM was required to label one-half of the binding sites. This suggests a concentration of about 500,000 muscarinic cholinergic receptors per smooth muscle cell. The muscarinic cholinergic receptor antagonists atropine and scopolamine inhibited QNB binding with a 50% inhibiting concentration (IC50) in the nanomolar range, whereas the agonists acetylcholine (ACh), oxotremorine, and carbamylcholine had IC50S in the micromolar range. Hill coefficients (nH) for antagonists approached unity, but agonists displayed fractional nH. Exposure of cells to cholinergic muscarinic agonists resulted in dose-dependent decreases in cell length. The concentration of agonist required to induce half-maximal contractions (ED50) was 8.3 X 10(-12) M for ACh and 6.3 X 10(-13) M for oxotremorine. Atropine (10(-9) M) decreased the sensitivity to ACh, increasing the ED50 for ACh-induced contractions to 1.2 X 10(-10) M. These results suggest the existence of muscarinic receptor heterogeneity for cholinergic agonists but not for antagonists.
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PMID:Contraction and [3H]QNB binding in collagenase isolated fundic smooth muscle cells. 688 50

The similarity of porcine and human physiology and the availability of slaughterhouse tissues suggests the use of porcine parotid cells as a model for amylase secretion. A procedure is described for the isolation of porcine parotid cells by collagenase-P/dispase digestion of the tissue. The preparation consisted of individual cells and small aggregates that were maintained in primary culture, during which the cells formed aggregates that firmly attached to the plastic substrate. The amylase content of the cultured cells remained adequate for assay of secretory activity during culture for one week after isolation. Depending upon variations in experimental treatments, the cultured cells secreted approx. 35-65% of cellular amylase in response to a carbachol challenge. The cells were slightly responsive to long exposures to isoproterenol, and were unresponsive to nicotine, elevated extracellular K+ or substance P. Secretion induced by carbachol required extracellular Ca2+, was inhibited by atropine and occurred with a nearly linear response over a 30-min period. The Ca2+ ionophore A23187 was also a potent secretagogue for amylase secretion, producing levels of secretion equal to that induced by carbachol. The ease of preparation and the retention of amylase during primary culture suggests that the preparation will be useful in studies on muscarinic receptor-mediated control of amylase secretion.
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PMID:Amylase secretion by cultured porcine parotid cells. 754 48

We previously reported a simple method of acutely preparing dissociated smooth muscle cells from urinary bladder tissue, but the feasibility of this method has not been well ascertained. In the present study, we assessed whether this method is applicable for measuring muscarinic receptor function in intestinal smooth muscle cells. Single smooth muscle cells were prepared from the longitudinal muscle tissue of guinea pig colon by the enzymatic dissociation with papain and hyaluronidase, followed by collagenase digestion. Muscarinic responses in the isolated smooth muscle cells were measured by intracellular Ca(2+) mobilization and extracellular acidification through Fura-2 fluorometry and Cytosensor microphysiometry, respectively. A single, viable population of colon longitudinal smooth muscle cells (approximately 6 x 10(6) cells/animal) was obtained. In these cells, carbachol (muscarinic agonist) induced Ca(2+) mobilization and extracellular acidification over the concentration range similar to that previously reported to produce contraction of the intact colon muscle strips. Atropine (nonselective muscarinic antagonist) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, M(3)-selective antagonist) inhibited the Ca(2+) mobilization with potencies approximately 3 log units greater than that for methoctramine (M(2)-selective antagonist). For extracellular acidification, the potency differences between these antagonists was approximately 2 log units. In addition, the carbachol-induced extracellular acidification was inhibited by 5-[N-ethyl-N-isopropyl]-amiloride, a selective inhibitor of the Na(+)/H(+) exchanger. These findings indicate that in isolated colonic smooth muscle cells, M(3) receptors are predominantly involved in Ca(2+) mobilization, while a mixed population of M(2) and M(3) receptors seems to contribute to extracellular acidification. Our results further suggest the role of the Na(+)/H(+) exchanger in muscarinic-mediated extracellular acidification. Consequently, our method produces viable isolated colonic smooth muscle cells that display physiologically appropriate responses to muscarinic receptor activation, and the method may be applicable for several types of nonvascular smooth muscle tissues.
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PMID:A method for measurement of muscarinic receptor-mediated responses in dissociated single colon longitudinal smooth muscle cells. 1175 83

Cultured cells of the human urinary bladder smooth muscle are useful for investigating bladder function, but methods for culturing them are not well developed. We have now established a novel enzymic technique. The smooth muscle layer was separated out and incubated with 0.2% trypsin for 30 min at 37 degrees C. The samples were then minced and incubated with 0.1% collagenase for 30 min and centrifuged at 900 g. The pellets were resuspended in RPMI-1640 medium containing 10% fetal calf serum (FCS) and centrifuged at 250 g. The smooth muscle cells from the supernatant were cultured in RPMI-1640 containing 10% FCS. The cells grew to confluence after 7-10 days, forming the "hills and valleys" growth pattern characteristic of smooth muscle cells. Immunostaining with anti-alpha-actin, anti-myosin, and anti-caldesmon antibodies demonstrated that 99% of the cells were smooth muscle cells. To investigate the pharmacological properties of the cultured cells, we determined the inhibitory effect of muscarinic receptor antagonists on the binding of [3H]N-methylscopolamine to membranes from cultured cells. The pKi values obtained for six antagonists agreed with the corresponding values for transfected cells expressing the human muscarinic M2 subtype. Furthermore, carbachol produced an increase in the concentration of cytoplasmic free Ca2+ an action that was blocked by 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3 selective antagonist. This result suggests that these cells express functional M3 muscarinic receptors, in addition to M2 receptors. The subcultured cells therefore appear to be unaffected by our new isolation method.
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PMID:A new enzymic method for the isolation and culture of human bladder body smooth muscle cells. 1183 27


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