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)

A method is described, which allows adenylate cyclase activity measurement in single pieces of various nephron segments. Tubular samples of 0.5 to 2 mm length were isolated by microdissection from collagenase treated slices of rabbit kidney. A photograph of each piece was taken in order to measure its length. After a permeabilisation treatment involving preincubation in a hypoosmotic medium and a freezing step, each sample was incubated for 30 mm at 30 degrees C in a medium containing high specific (alpha-32-P)-ATP 3-10-4 M, final volume 2.5 mu 1. The (32P)-cAMP formed was separated from the other labelled nucleotides by filtering the incubate on a dry aluminium oxide microcolumn, 3H cAMP was added as a tracer for measuring cAMP recovery. The sensitivity of the method was found to be a few fentomoles (10-15 M) cAMP. cAMP generation increased linearly as a function of the incubation time up to more than 30 min, and as a function of the length of the segment used. Control and fluoride (5 mM) stimulated adenvlate cyclase activities were measured in the following segments of the nephron: early proximal convoluted tubule (PCT), pars recta of the proximal tubule (PR), thin descending limb of the loop (TDL), cortical portion of the thick ascending limb (CAL), distal convoluted tubule (dct), first branched portion of the collecting tubule (BCT), further cortical (CCT) and medullary (MCT) portions of the collecting tubule. Mean control adenylate cyclase activity varied from 7 (PR) to 75 (BCT) fmoles/mm/30 min. Flouride addition resulted in a 10 (BCT) to 50 (PR) fold increase in enzyme activity. Series of replicates gave a scatter equal to plus or minus 20% (S.D. as a per cent of the mean). The method described appears to be suitable to determine which nephron segments contain hormone-dependent adenylate cyclase.
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PMID:Adenylate cyclase activity along the rabbit nephron as measured in single isolated segments. 16 67

The application of scanning electron microscopy to the study of cell surfaces is limited in intact tissues, because extracellular material may often obscure the details of nonluminal surfaces. To remove connective tissue elements we have treated human skin and both kidney, and an autonomic ganglion of the rat with hydrochloric acid and collagenase. Regional variations in the basal surface of the nephron are noted following removal of the basement membrane. The basilar interdigitations of the cells of the proximal tubule appeared as parallel ridges encircling the tubule. Ridges on the parietal epithelium of Bowman's capsule were randomly arranged and alternated with smooth surfaces. The dermal surface of the human epidermis has an alveolar or honeycomb appearance due to the elevation of the epidermal ridges and numerous pits for the dermal pegs. At higher magnifications the basal surface of cells of the stratum germinativum possessed numerous and irregular projections. Neurons with their processes are evident in the autonomic ganglion. The soma of the neurons are enclosed by flattened satellite cells. Irregular spaces between opposed satellite cells are interpreted as regions for the passage of processes related to the ganglion cells. Nodes of Ranvier were clearly seen along nerve fibers. Some pitting of the nerve fibers was also noted. The HCl-collagenase method has the advantage of the removal of collagen and basement membrane while preserving the structural integrity of the cell surface.
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PMID:Scanning electron microscopy of cell surfaces following removal of extracellular material. 18 20

Renal gluconeogenesis was studied in suspended tubule fragments isolated by collagenase treatment of rat kidney cortices. Angiotensin II increased glucose formation from pyruvate, lactate, and to a lesser extent from oxoglutarate and glutamine, but not from other substrates such as malate, succinate, dihydroxyacetone or fructose. Stimulation was significant with peptide concentration exceeding 1 . 10(-8) M and was also shown with an 8-Sar derivative. Other peptides such as 4-Ala-8-Ile-angiotensin II, hexapeptide and bradykinin had no effect. The stimulatory action of angiotensin II was additive to that of L-lysine, and 3',5'-adenosine cyclic monophosphate, suggesting a different mechanism of action. In the presence of maximally stimulatory concentrations of oleate, phenylephrine and 3',5'-guanosine cyclic monophosphate, however, the stimulatory effect of angiotensin II was absent. Cyclic GMP levels, however, did not increase in tubules after angiotensin II and phenylephrine addition, making a messenger function of this nucleotide unlikely. Omission of Ca2+ from the medium markedly reduced basal gluconeogenesis but did not result in a complete loss of angiotensin II effect. Reduction of medium potassium to 2 mM, however, increased basal gluconeogenesis and blunted the peptide effect. 1 mM ouabain was also able to inhibit the stimulatory effect of angiotensin II. Therefore changes in intracellular potassium levels are discussed as a possible mechanism of angiontensin action, whereas calcium seems not to be specifically linked to this metabolic action of angiotensin on the proximal tubule.
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PMID:Stimulation of renal gluconeogenesis by angiotensin II. 45 78

The sensitivity to catecholamines of the adenylate cyclase (AC) activity contained in single tubule samples was investigated on 10 different well defined segments, isolated by microdissection from collagenase treated rabbit kidneys. No responsiveness to isoproterenol (10(-6) M) was observed in the proximal tubule (convoluted and straight portions), the thin descending and thick ascending limbs of the loop of Henle, and the first ("bright") portion of the distal convoluted tubule (DCTb); in contrast high responses (stimulation factors: 4 to 6 fold) were obtained in the second ("granular") portion of the distal convoluted tubule (DCTg), as well as in both the "granular" (CCTg) and the "light" (CCTl) portions of the cortical collecting tubule. In absolute value, however, the CCTl response was definitely lower than those measured in DCTg and CCTg, as is its control activity. In the medullary portion of the collecting tubule, the AC response to isoproterenol was rather poor both in absolute and relative terms. Dose-response curves measured on DCTg samples indicated a threshold response with an isoproterenol concentration below 10(-8) M; half maximal effect corresponded to about 3 x 10(-8) M. CCTl sensitivity to isoproterenol was of the same order of magnitude. Isoproterenol as well as norepinephrine effects in DCTg and CCTl were completely suppressed by 10(-4) M propranolol, indicating that the observed AC stimulation was mediated via receptors of the beta type. In beta blocked CCTl, 10(-6) M norepinephrine did not inhibit vasopressin-induced AC stimulation; in the presence of 10(-6) M norepinephrine, 10(-4) M phentolamine resulted in no additional AC stimulation in DCTg and CCTl; these data suggest the absence of alpha receptors inhibiting AC activity in these structures. In DCTg, AC stimulation induced either by 10(-6) M isoproterenol or by 1 U/ml PTH were observed to be additive when the two hormones were given together. The presence of catecholamine-dependent AC activity in three distal portions of the rabbit nephron is discussed in relation to its possible physiological implications.
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PMID:Catecholamine sensitive adenylate cyclase activity in different segments of the rabbit nephron. 123 46

PTH stimulates mammalian renal proximal tubule cell synthesis and secretion of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] by a Ca-dependent process. In the present study regulation of 1,25-(OH)2D3 secretion by PTH, phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the Ca ionophore A23187, and calcitonin was evaluated in perifused rat proximal tubule cells isolated by collagenase digestion and centrifugation through Percoll. Tubules from rats fed a low Ca diet secreted 1,25-(OH)2D3 at a rate 2.5 times that of tubule cells from rats fed a normal Ca diet. Perifusion of tubules with human PTH-(1-34) (10(-7) M) induced an immediate and sustained increase in 1,25-(OH)2D3 secretion. Perifusion with either A23187 or 12-O-tetradecanoylphorbol 13-acetate caused transient increases in hormone secretion, while both agents perifused simultaneously resulted in a sustained increase in 1,25-(OH)2D3 secretion. Perifusion of tubule cells with the protein kinase-C (PKC) inhibitor staurosporine blocked the PTH-induced increase in 1,25-(OH)2D3 secretion. Calcitonin had no effect on 1,25-(OH)2D3 secretion rates. The results of the present studies show that an activator of PKC increases 1,25-(OH)2D3 secretion by mammalian proximal tubule cells and suggest that the phospholipase-C/PKC signalling system may mediate PTH stimulation of 1,25-(OH)2D3 secretion.
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PMID:Evidence that activation of protein kinase-C can stimulate 1,25-dihydroxyvitamin D3 secretion by rat proximal tubules. 132 62

Hydrolysis of arginine into urea and ornithine (Orn) was observed to take place in several segments of the rat nephron including cortical and medullary pars recta of the proximal tubule (PST) and collecting duct (CD). This work was now extended to the adult mouse and rabbit. Representative nephron segments, obtained by microdissection of collagenase-treated kidneys, were incubated with L-[guanido-14C]arginine (216 microM). Addition of urease produced 14CO2 + 2 NH3 from the newly formed urea released in the incubate. 14CO2 was trapped in KOH and counted. In both species, as well as in the rat, the PST was the site of the highest urea + Orn production, with an intensity increasing from cortex to medulla. For other nephron segments, the pattern was not similar in all species. Significant production of urea + Orn was observed in the proximal convoluted tubule and the medullary thick ascending limb in the rabbit, but not in the CD of either the rabbit or the mouse. The functional significance of this urea + Orn production remains unclear. The total amount of urea generated intrarenally by this reaction does not seem sufficient to play a significant role in the urinary concentrating mechanism. It may be assumed that Orn could be further metabolized to polyamines and play a role in maintaining cell integrity and function in the PST, especially in its medullary part, exposed to hypertonicity and poor oxygen supply.
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PMID:Localization of urea and ornithine production along mouse and rabbit nephrons: functional significance. 144 76

Normal rat kidney proximal tubule epithelial cell cultures were obtained by collagenase digestion of cortex and studied for 10 days. To assess the purity of the seeding suspension, we histochemically demonstrated gamma-glutamyltranspeptidase in greater than 95% of the starting material. To identify cell types in cultures, we investigated several markers. Cells stained positively for lectin Arachis hypogaea (rat proximal tubule) and negatively for Lotus tetragonolobus (rat distal tubule). Intermediate filament expression of cytokeratin confirmed the epithelial differentiation of the cultured cells. Using indirect immunofluorescence, we found that cultures were negative for vimentin and Factor VIII. Cells exhibited activities of two brush border enzymes, gamma-glutamyltranspeptidase and leucine aminopeptidase, and Na(+)-dependent glucose transport activity. Multicellular domes were evident in the Week 2 of culture. Proliferation was studied by comparing growth factor-supplemented serum-free medium to cells grown in serum; growth enhancers included insulin, hydrocortisone, transferrin, glucose, bovine albumin, and epidermal growth factor. Cells proliferate best in medium with 5 or 10% serum and in serum-free medium supplemented with insulin, hydrocortisone, transferrin, glucose, and bovine albumin. Proliferation was assessed by determining cell number (population doublings). By light microscopy, the cells were squamous with numerous mitochondria, a central nucleus, and a rather well-defined homogeneous ectoplasm. By electron microscopy, the cells were polarized with microvilli and cell junctions at the upper surface and a thin basal lamina toward the culture dish. These data show that the proximal tubule epithelial cells retain a number of functional characteristics and that they represent an excellent model for studies of normal and abnormal biology of the renal proximal tubule epithelium.
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PMID:Primary cultures of normal rat kidney proximal tubule epithelial cells for studies of renal cell injury. 171 31

A method for studying the in vivo accumulation of inorganic mercury along the nephron of Sprague-Dawley rats pretreated with a radiolabelled 0.66 mumol/kg dose of mercuric chloride is described in this article. Forth-eight hr after rats received the radiolabelled dose of mercuric chloride intravenously the kidneys of the animals were perfused in situ with a collagenase solution in order to dissect and isolate various readily assessable segments of the nephron and collecting duct. Three different categories of tubular segments were isolated; proximal convoluted tubules, proximal straight tubules and combined segments of the distal nephron and collecting duct. A group of isolated tubular segments were measured in length, drawn up and placed in counting tubes, and placed in a gamma counter for the determination of the content of inorganic mercury that accumulated in them during the 48 hr subsequent to the administration of the dose of mercuric chloride. In a separate set of animals, the intrarenal distribution of inorganic mercury was determined 48 hr after the intravenous dose of mercuric chloride was administered. Inorganic mercury accumulated mainly in the renal cortex and outer stripe of the outer medulla. In addition, the concentration of inorganic mercury in the outer stripe of the outer medulla was twice that in the cortex. The findings obtained with the isolated tubular segments revealed that most of the accumulated inorganic mercury in the kidneys of the rats was in the proximal tubule.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Method for studying the in vivo accumulation of inorganic mercury in segments of the nephron in the kidneys of rats treated with mercuric chloride. 194 26

The effects of oxidative damage were assessed in rat proximal tubule fragments (isolated by collagenase perfusion) by monitoring lactate dehydrogenase release (LDH-R) to measure cell viability and thiobarbituric acid (TBA) reactive material to follow oxidative damage. Increasing the oxygen content in the incubation atmosphere from 10 to 95% significantly increased LDH-R and TBA reactants. Addition of butylated hydroxytoluene or deferoxamine (DF) to the medium prevented these changes, but ascorbic acid or mannitol had no positive effect. Lima bean trypsin inhibitor also reduced LDH leakage significantly when added to the medium, but not when added to the perfusion buffers. In contrast, adding DF to the perfusate during tubule isolation produced the most pronounced benefit; net LDH-R after 4 hr was about 10% in tubules prepared this way compared to 20% when DF was omitted. Basal oxygen consumption declined to approximately the same extent as LDH-R increased. Maintenance of nystatin-stimulated respiration, ATP/ADP, GSH content and total adenine nucleotides indicated good cell function. These results suggest that oxidative damage initiated during the tubule isolation procedure limits cell survival but this effect can be counteracted substantially by the addition of DF to the perfusion buffer.
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PMID:Stress initiated during isolation of rat renal proximal tubules limits in vitro survival. 196 72

Suspensions of renal proximal tubules (RPT) are the in vitro model for many biochemical and physiologic investigations. Inasmuch as there are numerous procedures for tubule isolation and the more commonly used enzymatic procedures may disrupt the basement membrane, there is a need for information comparing the influence of various isolation methods on RPT viability and function in long-term suspension. Rabbit RPT isolated a) enzymatically (ENZ) by in vitro collagenase digestion and Percoll size and density purification, and b) mechanically (MECH) by in vitro iron oxide perfusion and purification by sieving and magnetic removal of glomeruli were compared for viability, morphology, and functional stability during long-term suspension. RPT isolated by ENZ and MECH methods had excellent viability (less than 15% lactate dehydrogenase release), limited lipid peroxidation (less than 0.2 nmol MDA.mg protein-1), and stable nystatin-stimulated oxygen consumption (QO2) (38 and 36 nmol O2.mg protein-1.min-1) throughout 24 h of incubation. Basal QO2 was higher in ENZ than MECH tubules (27 and 19 nmol O2.mg protein-1.min-1, respectively), and was unchanged over 24 h in each preparation. The higher basal QO2 in ENZ tubules was ouabain-sensitive, suggesting an increased rate of Na+,K(+)-ATPase activity in these tubules. Total glutathione content (oxidized + reduced) in ENZ and MECH tubules increased over the 24-h incubation from 8 to 18 nmol.mg protein-1. gamma-Glutamyltranspeptidase (GGT) activity of the RPT homogenates was equivalent in both preparations and stable over time. The ratio of suspension GGT activity to homogenate GGT activity doubled (0.4 to 0.8) during the incubation period. MECH tubules retained their tubule structure during 24 h of incubation whereas the ENZ tubules had a striking loss of tubular morphology over time. These results show that ENZ- and MECH-isolated renal proximal tubule suspensions exhibit similar biochemical properties in long-term incubations but differ in ouabain-sensitive QO2 and the retention of tubular morphology. The loss of tubular morphology and the increase in the rate of Na+,K(+)-ATPase activity in ENZ tubules may be secondary to the disruption of the tubular basement membrane.
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PMID:Differences in enzymatic and mechanical isolated rabbit renal proximal tubules: comparison in long-term incubation. 197 32


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