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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)

To clarify the characteristics of cellular ATP synthesis in individual nephron segments for assessing nephrotoxicity of chemicals, cellular ATP content was measured by the luciferin/luciferase system under various conditions using intact nephron segments isolated from male Sprague-Dawley rats. Increasing the duration of collagenase treatment of kidney slices significantly lowered the cellular levels of ATP newly synthesized from 2 mM glutamine in PST at 37 degrees C over 30 min (p less than 0.01). The tubular incubation time significantly affected the cellular ATP content in the early and middle portions (S2) of the proximal tubule (p less than 0.05 and p less than 0.01, respectively) over 20 min and in the late proximal tubule over 10 min. Among numerous substrates tested, such as D-glucose, glutamine, pyruvate, DL-lactate, and beta-hydroxybutyrate, the substrate utilization for maintaining cellular ATP content was entirely variable according to each nephron segment. Pyruvate and glutamine were the best substrates in the proximal tubule. On the other hand, ATP production from glutamine was less than that from the other substrates in the distally located nephron segments: medullary and cortical thick ascending limbs of Henle's loop (MAL and CAL, respectively), distal tubule, cortical and medullary collecting tubules (CCT and MCT, respectively). In general, glucose, pyruvate, and lactate appear to be equivalent in maintaining ATP content in the distal segments of renal tubules. A monovalent cation ionophore, monensin, at 10 micrograms/ml decreased the cellular ATP content in MAL, CAL, and MCT significantly. Mercuric chloride (HgCl2) was used as a model compound to study nephrotoxicity by investigating its effects on cellular ATP metabolism in microdissected nephron segments. HgCl2 at 1 x 10(-6) M significantly decreased ATP content only in S2 (p less than 0.05), clearly demonstrating S2 to be the most sensitive segment within the nephron. These results indicate that measurement of cellular ATP content would be a useful method forecasting the intrarenal toxic site and potency of possible nephrotoxic chemical compounds.
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PMID:Nephrotoxicity assessment by measuring cellular ATP content. I. Substrate specificities in the maintenance of ATP content in isolated rat nephron segments. 255 Oct 74

Rat renal proximal tubular cells were isolated by successive EGTA and collagenase perfusions and purified by filtration and isopycnic centrifugation. The method is rapid and provides a much higher fraction of proximal tubular cells (90-95%) than comparable methods. The yield of viable (97 +/- 3%) cells is high (30 X 10(6) cells/g kidney). The intracellular ATP was 16 +/- 2 nmol/mg protein and remained essentially constant for at least 3 hr. The cells were characterized for transport of organic ions and glucose. Glucose transport was studied by alpha-[14C]methylglucose uptake; apparent Km and Vmax values were 3.4 +/- 0.5 mM and 4.1 +/- 0.9 nmol/min.mg protein, respectively. This transport could almost be completely inhibited by phloridzin, indicating that the uptake is mediated by the brush border glucose carrier.
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PMID:Isolated proximal tubular cells from rat kidney as an in vitro model for studies on nephrotoxicity. I. An improved method for preparation of proximal tubular cells and their functional characterization by alpha-methylglucose uptake. 255 15

We investigated the effect of changes in perfusate substrate and Ca content on the quality and yield of isolated adult rat heart cells. When 1 mM Ca was added to the recirculating perfusate 15 min after collagenase addition, the ATP level of cells in the heart 15 min later, and their morphology in histological section, was no different from when no Ca was added back. The cells subsequently isolated were of similar yield, but a greater percentage were rod-shaped, compared with cells isolated without Ca restoration to the perfusate. Increased yield could be obtained by including substrates in the perfusate in addition to glucose. Either fatty acids or amino acids were effective. We conclude that: (1) all cells in the heart are Ca tolerant at the end of enzyme perfusion; (2) the presence of substrates in addition to glucose can help cells survive the isolation process.
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PMID:Some determinants of quality and yield in the isolation of adult heart cells from rat. 273 Dec 28

To clarify the nephrotoxic site and potency of ochratoxin A (OCTA), we measured cellular ATP contents in nine nephron segments incubated with or without OCTA in vitro. Cellular ATP contents of nephron segments isolated under stereomicroscopic observation after treatment of renal slices with 0.1% collagenase were measured by the microchemiluminescence method. OCTA decreased cellular ATP content in a dose-dependent manner. A concentration-response study of OCTA showed that the minimum concentration of OCTA needed to cause a significant decrease in ATP was 10(-8) M in the middle portion of the proximal tubule (S2; p less than 0.05) and 5 x 10(-4) M in the medullary collecting tubule (MCT; p less than 0.01). Among nine nephron segments, OCTA at 5 x 10(-5) M significantly decreased cellular ATP content in only S2 and the terminal portion of the proximal tubule (S3; p less than 0.01). ATP synthesis in mitochondria isolated from the renal cortex was significantly inhibited by 10(-6) M OCTA (p less than 0.05). Probenecid at 4 x 10(-4) M protected against the OCTA-induced cellular ATP decrease. These results suggest that OCTA might enter the plasma membrane in S2 and S3 through the organic anion transport pathway and inhibit mitochondrial oxidative phosphorylation. This newly established method would be applicable to evaluation of the intrarenal toxic site and potency of various chemical compounds.
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PMID:Nephrotoxicity assessment by measuring cellular ATP content. II. Intranephron site of ochratoxin A nephrotoxicity. 278 64

The mechanism of the periportal (p.p.) toxicity of allyl alcohol (AlOH) was investigated in p.p. and perivenous (p.v.) hepatocytes isolated by digitonin-collagenase perfusion. The distinct origin of the cell preparations was confirmed by the p.p./p.v. ratios of alanine aminotransferase (p.p./p.v. = 1.8), lactate dehydrogenase (1.3) and glutamine synthetase (0.10). The activity of alcohol dehydrogenase (ADH) was not markedly different in p.p. and p.v. cells. Both types of cells oxidized AlOH at a high but equal rate of about 3 mumol/(min.g cells). Concomitantly with rapid oxidation of 0.7 mM AlOH, glutathione (GSH) was depleted by about 95% and its secretion was completely inhibited in both cell types. Although the GSH content was partially restored during a subsequent 3-h incubation, cellular ATP and K+ content gradually decreased and the leakage of lactate dehydrogenase increased in both types of cells. However, the p.p. cells tended to resist AlOH in vitro better, probably due to their 26% higher GSH content after preincubation with L-methionine. Altering the partial pressure of oxygen in physiological range had no effect on the toxicity of AlOH. The results are contrary to the suggestions that the p.p. location of AlOH liver injury is caused by higher ADH activity or higher oxygen tension in the p.p. zone. Rather, the regiospecificity of the injury may be due to rapid uptake and oxidation of AlOH in the p.p. region.
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PMID:Allyl alcohol cytotoxicity and glutathione depletion in isolated periportal and perivenous rat hepatocytes. 283 85

The zonal distribution of GSH metabolism was investigated by comparing hepatocytes obtained from the periportal (zone 1) or perivenous (zone 3) region by digitonin/collagenase perfusion. Freshly isolated periportal and perivenous cells had similar viability (dye exclusion, lactate dehydrogenase leakage and ATP content) and GSH content (2.4 and 2.7 mumol/g respectively). During incubation, periportal cells slowly accumulated GSH (0.35 mumol/h per g), whereas in perivenous cells a decrease occurred (-0.14 mumol/h per g). Also, in the presence of either L-methionine or L-cysteine (0.5 mM) periportal hepatocytes accumulated GSH much faster (3.5 mumol/h per g) than did perivenous cells (1.9 mumol/h per g). These periportal-perivenous differences were also found in cells from fasted rats. Efflux of GSH was faster from perivenous cells than from periportal cells, but this difference only explained 10-20% of the periportal-perivenous difference in accumulation. Furthermore, periportal cells accumulated GSH to a plateau 26-40% higher than in perivenous cells. There was no significant difference in gamma-glutamylcysteine synthetase or glutathione synthetase activity between the periportal and perivenous cell preparations. The periportal-perivenous difference in GSH accumulation was unaffected by inhibition of gamma-glutamyl transpeptidase or by 5 mM-glutamate or -glutamine, but was slightly diminished by 2 mM-L-methionine. This suggests differences between periportal and perivenous cells in their metabolism and/or transport of (sulphur) amino acids. Our results suggest that a lower GSH replenishment capacity of the hepatocytes from the perivenous region may contribute to the greater vulnerability of this region to xenobiotic damage.
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PMID:Glutathione replenishment capacity is lower in isolated perivenous than in periportal hepatocytes. 290 50

Preparations of distinct nephron segments were obtained from dog kidneys by collagenase treatment. Four morphologically different tissues were isolated: glomeruli, proximal tubules, thick ascending limbs, and papillary collecting ducts. Each segment possessed a characteristic assay of membrane-bound and cytoplasmic enzymes. Specific metabolic characteristics also were found: gluconeogenesis and ammoniagenesis in proximal tubules, glycolytic aerobic metabolism in thick ascending limbs, and glycolytic anaerobic metabolism in papillary collecting ducts. The assay of Na+ -K+ ATPase, H+ -ATPase, and Ca2+ -ATPase activities in these nephron segments demonstrated a specific enrichment of Na+ -K+ ATPase in thick ascending limbs, and of H+ -ATPase in proximal tubules and papillary collecting ducts. Tubular respiration in the absence or presence of ouabain, 1,3-dicyclohexylcarbodiimide, or furosemide demonstrated that the respiration of each segment could be correlated to the activity of specific ion motive ATPases. Furthermore, a tight coupling between ion transport, ATP turnover, and substrate oxidation was demonstrated. These isolated tubular structures are thus viable and capable of transepithelial transport. Our preparation provides large amounts of defined population of tubules and are thus useful for the study of biochemical and functional heterogeneity along the nephron.
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PMID:Characterization and metabolism of canine proximal tubules, thick ascending limbs, and collecting ducts in suspension. 297 51

The K+ ionophore valinomycin, in concentrations as low as 0.1 microM, induces an inhibition of thyroid-stimulating hormone (TSH)-stimulated cAMP formation in cat and pig thyroid slices and isolated, trypsin-collagenase-dispersed beef thyroid cells. Valinomycin was also shown to inhibit histamine and prostaglandin E1 stimulation of thyroid cAMP formation. The inhibitory effect of valinomycin could be partially overcome by elevated (81 mM) K+ concentrations. In the absence of valinomycin, the ability of TSH to stimulate thyroid cAMP formation was dependent on extracellular K+. Chronic removal or addition of K+ to medium bathing thyroid sections was accompanied by inhibition of TSH-stimulated cAMP formation. Maximum TSH stimulation was observed at an extracellular K+ of 2.7 mM. Valinomycin had no significant effect on thyroid ATP content but did reduce the ATP-to-ADP ratio. However, chronic removal of K+ had no effect on either ATP or the ATP-to-ADP ratio. Varying extracellular Na+ from 26 to 144 mM or addition of tetrodotoxin did not affect TSH action. Valinomycin addition to thyroid slices was associated with a reduction in iodide transport as measured by the ratio of tissue to extracellular iodide concentrations. The effect of valinomycin on iodide transport was accompanied by an increase in iodide efflux that was not greater than that observed with perchlorate ion, suggesting a reduced recirculation of released iodide in valinomycin-treated tissue. These findings suggest that alterations in thyroid cell K+ permeability or intracellular K+ concentration may be accompanied by changes in TSH-induced stimulation of thyroid cAMP formation.
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PMID:Effect of valinomycin on thyroid iodide transport and TSH-stimulated cAMP formation. 300 1

To clarify the nature of the noradrenaline (NA)-induced contraction, the effects of NA on inositol phospholipid metabolism and the actions of inositol 1,4,5-trisphosphate (InsP3) on skinned muscle of the rabbit mesenteric artery were investigated. NA, in concentrations over 1 nM, reduced the amount of phosphatidylinositol 4,5-bisphosphate (PI-P2) and increased the amount of phosphatidic acid (PA). The maximum reduction in the amount of PI-P2 and the maximum increase in the amount of PA were observed in the presence of 1 microM-NA. With prolonged application of NA, the PI-P2 was gradually restored to near the control level, but with repeated applications of NA separated by rinses with Krebs solution, there was a consistent reduction of PI-P2. The NA-induced PI-P2 breakdown was inhibited by the alpha 1-adrenoceptor blocking agent, prazosin. After incubation of the tissue with radioactive inositol-containing solution, NA transiently increased the amount of radioactive InsP3 which was followed by increases in the amount of inositol 1,4-bisphosphate (InsP2) and inositol monophosphate (InsP). After accumulation of Ca by saponin-treated muscle cells of the dog mesenteric artery dispersed by collagenase, InsP3 released Ca stored in cells but InsP2 did not. A23187 (5 microM) but not InsP3 (up to 10 microM), depleted Ca accumulated in the presence of ATP. In saponin-treated skinned muscle tissues, InsP3 in concentrations over 0.3 microM, produced contraction following accumulation of Ca into the store site. InsP3 released Ca from the same source as caffeine. The release of Ca by InsP3 appeared in a concentration-dependent manner and this release also depended on the amount of Ca stored in cells (the median effective dose (ED50) was 3.0 microM in 0.1 microM-Ca and 1.0 microM in 0.3 microM-Ca). We concluded that NA activates alpha 1-adrenoceptors, thus hydrolysing PI-P2 and synthesizing InsP3. This product can release Ca stored in cells as estimated from the contraction in skinned muscle tissues, and also reduces the residual amount of Ca stored in skinned dispersed muscle cells. Contraction evoked by NA through pharmacomechanical coupling can be explained as a function of InsP3.
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PMID:Inositol 1,4,5-trisphosphate activates pharmacomechanical coupling in smooth muscle of the rabbit mesenteric artery. 300 48

The action of procaine on pharmaco-mechanical coupling activated by application of acetylcholine (ACh) was investigated using collagenase-treated dispersed intact and skinned smooth muscle cells and intact muscle tissues of the porcine coronary artery. ACh reduced stored 45Ca2+, and this action was prevented by procaine in intact dispersed cells. The maximum reduction in the level of stored 45Ca induced by caffeine (25 mM) or inositol 1,4,5-trisphosphate (InsP3; 3 microM) was also prevented by procaine in the skinned muscle cells in the presence or absence of ATP. However, inhibitions of the latter required higher concentrations of procaine than the former. Release by 10 microM ACh of Ca2+ from its store site in the presence or absence of extracellular Ca2+ was also inhibited by procaine and was detected using the quin2 fluorescence method. In these smooth muscle tissues, ACh (above 10 nM) reduced the amount of phosphatidylinositol 4,5-bisphosphate (PI-P2) and dose dependently increased the amount of phosphatidic acid. Procaine inhibited the hydrolysis of PI-P2 activated by ACh, thus reducing the amount of InsP3 and the release of Ca2+ from the store site. It is concluded that procaine has multiple actions on the porcine coronary artery, and one of the actions related with pharmacomechanical coupling appears through inhibition of hydrolysis of PI-P2 induced by ACh.
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PMID:Effects of procaine on pharmaco-mechanical coupling mechanisms activated by acetylcholine in smooth muscle cells of porcine coronary artery. 303 48


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