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)

H+ extrusion by the isolated skins of two amphibia, Rana ridibunda and Bufo bufo, was studied in order to test for the presence of exchange mechanisms of the type Na+/H+ and Cl-/HCO3-, which have been described in several epithelial structures. The preparations were mounted in chambers of the Ussing type, so that the short-circuit current could be used as a function of Na+ transport and the pH-stat techinique was utilize to determine the rates of H+ extrusion under different experimental conditions. The conditions were either the withdrawal of the ions intervening the mentioned exchanges (Cl- or Na+), or the addition of drugs with well-known effects on Na+ up-take and transport (antidiuretic hormone and amiloride). In the frog skin, H+ excretion was detected in solutions containing either Cl- or SO4-2-, with identical rates. Again, Na+ substitution by Mg-2+ had no effect on H+ excretion rates, neither did the suppression of Na+ influx by amiloride or its stimulation by antidiuretic hormone. These experiments were repeated with similar results in gland-free preparations of the epidermis of frog skin separated from the corion by the action of collagenase. Experiments in toad skin that H+ excretion could not be detected whan Cl- was present in the outer medium, but became apparent if an impermant anion, SO4-2-, was used. This observation is compatible with the existence of an exchange mechanism of the type Cl-/HCO3-. Secondly, in these preparations H+ extrusion increased after stimulation with antidiuretic hormone and decreased when amiloride was used or when Na+ was substituted by Mg+, suggesting that a least a fraction of the total H+ efflux is linked to Na+ influx. In the isolated frog skin this mechanism does not seem to be operative.
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PMID:The excretion of hydrogen ion by the isolated amphibian skin: effects of antidiuretic hormone and amiloride. 23 91

1. Carotid body chemoreceptors were removed intact from adult rats and subjected to protease and collagenase enzymatic digestion of connective tissue. 2. Recordings from the sinus nerve demonstrated that chemotransduction remains intact for at least 2-3 h after isolation, enzyme exposure, and suspension in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline at room PO2. 3. After mechanical dissociation, the interrelationship between changes in extracellular PO2 and pH and relative changes in intracellular calcium (Ca2+i) were observed in glomus cells with the use of fluo-3 and confocal microscopy. 4. Brief (60-s) decreases in PO2 from 150 mmHg to near 0 mmHg, at nadir, caused a marked reduction in Ca2+i (peak delta F/F0 = -32 +/- 3%, mean +/- SE, n = 43), which rapidly recovered after reoxygenation. The decrease was reproducible from trial to trial and was also observed in HCO3(-)-buffered Ringer solution. 5. Superfusion with Ca(2+)-free HEPES saline with 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) blocked the hypoxia-induced increase in afferent chemoreceptor activity in vitro. Superfusion of the same solution over isolated cells for 15 min caused a large decrease in Ca2+i (-34 +/- 7%, n = 16). 6. In the presence of Ca(2+)-free HEPES, reoxygenation caused calcium fluorescence to increase. This suggests that the Ca2+ decrease during hypoxia is due, at least partially, to binding to an intracellular site. 7. Extracellular cobalt (1 mM, 15 min) also reversibly blocked the chemoreceptor response to hypoxia, in vitro, and caused a reduction in Ca2+i (delta F/F0 = -37 +/- 8%, n = 11).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Hypoxia decreases intracellular calcium in adult rat carotid body glomus cells. 162 63

The effect of cholinergic stimulation on the magnitude of anion loss from collagenase-isolated rat parotid acini suspended in a Krebs-Ringer bicarbonate buffer was characterized by means of 36Cl. It was found that on stimulation, the acinar Cl- concentration decreases from 62.3 mM to 22.6 mM in about 10 seconds after addition of carbachol. Concomitant with the dramatic net loss of Cl- a transient fall in the intracellular pH (pHi) of 0.1 pH-units occurred and that was interpreted as being due to acinar loss of HCO3-. The magnitude of the HCO3- loss was estimated in two different ways. From calculation of the intracellular buffer capacity at the unstimulated pHi of 7.28 it was found that the stimulation-induced fall of 0.1 pH-units is due to an efflux of about 5.5 mM from the acini. Another estimate of the HCO3- loss was based on the assumption that the ratio of HCO3- distribution on the internal and external sites of the plasma membranes is distributed according to the ratio for Cl-. By this calculation a HCO3- loss of 5.6 mM was found, which is in good accordance with the value obtained from the change in pHi. It was concluded that on cholinergic stimulation the acinar cells within seconds produce an isotonic fluid containing Cl- as the predominant anion and a small amount of HCO3-.
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PMID:Cholinergic-induced HCO3- loss from rat parotid acini. 253 64

Fourier transform infrared spectroscopy (FTIR) was used to characterize the organic and mineral phases present during the induction of mineral formation by collagenase-released matrix vesicles (CRMV) during incubation in a synthetic cartilage lymph in vitro. CRMV mineralization, which occurs in the absence of alkaline phosphatase organic phosphate substrates, is characterized by an initial short lag period of limited Ca2+ accumulation, followed by a period of rapid Ca2+ uptake, and finally, by a plateau period during which Ca2+ accumulation continued at a slower rate. FTIR spectra taken at timed intervals during the induction of mineralization revealed the presence of absorptions characteristic of protein, phospholipid, and mineral components in the CRMV. These became progressively more intense with time. To reveal underlying changes occurring during the successive stages of Ca2+ accumulation, FTIR spectra of nascent (or demineralized) CRMV were computer-subtracted from subsequent spectra, nulling on the C-H stretch modes characteristic of the lipid acyl chains. These difference spectra showed little change during early Ca2+ loading, revealing that mineral ions initially accumulated in a form similar to that present in nascent matrix vesicles (MV). During the period of rapid Ca2+ uptake prior to appearance of crystalline mineral, difference spectra revealed subtle changes in the carbonyl and amide nitrogen stretch modes indicative of protein conformational changes. The first definable mineral phase appeared late in the rapid Ca2+ uptake period and was a distinct, crystalline octacalcium phosphate (OCP)-like phase. With time, the OCP-like precursor became more apatitic in character. There was no evidence that any amorphous calcium phosphate phase formed during the MV mineralization sequence. The mature MV mineral phase closely resembled hydroxyapatite formed via an OCP precursor and was similar to other biological apatites that show a substantial incorporation of carbonate.
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PMID:Fourier transform infrared characterization of mineral phases formed during induction of mineralization by collagenase-released matrix vesicles in vitro. 284 33

Isolated hepatocytes from the rat were used to assess the maintenance of liver cell function in relation to the composition of the preservation medium. After separation by collagenase, they were incubated in Krebs-Ringer-Bicarbonate medium (KRB), Euro-Collins (EC), or University of Wisconsin (UW) solutions. Potassium influx, cell volume, and transaminase release were measured in cells freshly separated from control livers or from livers preserved in vitro up to 12 h in these media or having undergone orthotopic liver transplantation (OLT). While ion exchange levels were retained in all media, cells shrank significantly in UW but were able to restore their volume after 3 h of liver preservation. With regard to in vivo conditions, UW appears to be the best medium to prevent edema and to maintain more stable potassium exchange and enzyme production. These results are of value for liver transplantation in humans.
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PMID:Potassium uptake and water content in hepatocytes isolated from rat livers preserved in Euro-Collins and UW solutions and after transplantation. 783 53

The biostability of the Vascugraft arterial prosthesis, a porous synthetic graft made by a novel spinning process from a unique poly(ester urethane) polymer, has been studied by means of an in vitro enzyme incubation technique. Samples of the Vascugraft were exposed to buffered solutions of collagenase and pancreatin, as well as the buffer solutions alone, for periods of up to 100 days at 37 +/- 1 degrees C. On removal and after cleaning, a number of different analytic methods, including X-ray photoelectron spectroscopy for chemical analysis (ESCA), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), size exclusion chromatography (SEC), scanning electron microscopy (SEM), interference microscopy, moisture content and contact angle measurements, were used to examine the changes in chemical structure and surface morphology of the samples. During incubation in both enzymes the molecular weight of the polyurethane appeared to decrease in the presence of enzyme but increase in the presence of buffer. Further microphase separation in the polyurethane material developed during incubation in buffer solutions. Such changes in microstructure were associated with increased surface hydrophilicity, increased moisture content and a significant improvement in the extent of order and preferred orientation of the hard segment domains within the fibres. In the sampling depth of about 5 nm, both enzymes decreased the carbonate group content at the surface of the prosthesis to as little as 40% of their original values. The results from ATR-FTIR and DSC demonstrated that this phenomenon was limited primarily to the soft segment phase. While the Vascugraft prosthesis did exhibit some limited chemical modifications on exposure to concentrated enzyme solutions, nevertheless such changes were confined to the surface layer of the polyurethane microfibres. The importance and significance of those results will be more adequately determined by in vivo investigation.
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PMID:In vitro exposure of a novel polyesterurethane graft to enzymes: a study of the biostability of the Vascugraft arterial prosthesis. 789 15

1. This investigation set out to use 23Na n.m.r. spectroscopy to measure changes in intracellular levels of sodium in isolated suspensions of rat proximal tubules. The effects of temperature, an inhibitor of the sodium pump and known natriuretic drugs on intracellular sodium content of such tubular preparations were measured and compared with calcium channel antagonists where action at this level is unclear. 2. Rat kidneys were perfused with collagenase, roughly chopped, subjected to mechanical dispersion and washed to remove all traces of the enzyme. The proximal tubules were then purified and concentrated by Percoll density gradient centrifugation and then resuspended in buffer containing dysprosium tripolyphosphate shift reagent. 3. Distinct peaks corresponding to intracellular and extracellular sodium signals were observed when the tubules were placed into the n.m.r. spectrometer. As the temperature of the suspension rose to 37 degrees C, there was an exponential decrease in sodium content, with a decay constant of 0.15 +/- 0.02 min-1, which reached a stable level within 20 to 25 min. Addition of ouabain, 10(-3) M, resulted in a significant (P < 0.01) 30% increase in intracellular sodium content within 5 min which peaked at 70% 20 min later. Although acetazolamide (10(-3) M) significantly (P < 0.01) increased intracellular sodium content by 45%, amlodipine (10(-4) M) had no effect. 4. These data show that changes in the activity of the Na+/K+/ATPase have a considerable influence on the intracellular levels of sodium in proximal tubule cells. Inhibition of carbonic anhydrase activity resulted in a rise in intracellular sodium content which is compatible with its action to reduce the turnover rate of the Na+/(HCO3-)3 symporter. The lack of effect of amlodipine was consistent with the suggestion that it does not have a direct action on the sodium handling processes at the level of the proximal tubule.
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PMID:The influence of acetazolamide and amlodipine on the intracellular sodium content of rat proximal tubular cells. 792 16

The duodenum, in contrast to the jejunum, actively secretes HCO3- at a high rate, a process that protects the mucosa from acid/peptic injury. Our purpose was to define the mechanisms involved in HCO3- transport by studying the acid-base transport processes in isolated duodenal enterocytes. Individual rat duodenocytes, isolated by a combination of Ca2+ chelation and collagenase, attached to a collagen matrix were loaded with the pH-sensitive fluoroprobe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), and intracellular pH was monitored by microfluorospectrophotometry. To identify Na(+)-H+ transport, cells in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid 1) were pulsed with NH4Cl (40 mM) in the absence and presence of amiloride and 2) were removed of Na+. To examine Cl(-)-HCO3- exchange, Cl- was removed from Ringer-HCO3- superfusate in the presence and absence of dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2DIDS). The NaHCO3 cotransporter was studied by addition and subtraction of Na+ to amiloride-treated and Cl(-)-depleted enterocytes perfused with Na(+)- and Cl(-)-free Ringer-HCO3- buffer with and without H2DIDS. Mammalian duodenocytes contain at least three acid-base transporters: an amiloride-sensitive Na(+)-H+ exchanger that extrudes acid, a DIDS-sensitive Cl(-)-HCO3- exchanger that extrudes base, and a NaHCO3 cotransporter, also DIDS sensitive, that functions as a base loader. These acid-base transporters likely play a key role in duodenal mucosal HCO3- secretion.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proximal duodenal enterocyte transport: evidence for Na(+)-H+ and Cl(-)-HCO3- exchange and NaHCO3 cotransport. 823 51

Paraquat (PQ) induces lung, liver and kidney damage. Since PQ mainly is eliminated by the kidney, the kidney damage is of particular importance to the outcome of PQ poisoning. The exact toxic mechanism of PQ is still unclear but it is assumed to involve redox cycling and formation of reactive oxygen species. In this study, further investigations on the toxic mechanism and metabolic effects of PQ were performed using isolated renal proximal tubules from rabbits. Proximal tubules were isolated using a combined iron perfusion and collagenase method. Suspended tubules were incubated for varying periods and concentrations of PQ at 25 or 37 degrees C in Krebs-Ringer phosphate buffer or HCO3-/CO2 buffer. The cytotoxic effect of PQ was evaluated by (1) markers of oxidative stress: status of glutathione (GSH/GSSG) and formation of malondialdehyde (MDA); and (2) markers of tubular metabolism: oxygen consumption (QO2), transport of 14C-p-aminohippuric acid (PAH) and 14C-tetraethylammonium (TEA). Using 0.5 and 5 mM PQ, the GSH/GSSG ratio decreased whereas formation of MDA increased indicating oxidative stress. PQ reduced the accumulation of PAH and TEA, the basal QO2 and the ouabain sensitive QO2 indicating inhibition of the Na/K-ATPase. Nystatin-stimulated QO2 was reduced by PQ, excluding inhibition of Na+ entry as a possible cytotoxic mechanism and suggesting mitochondrial injury. This was confirmed by measuring FCCP-uncoupled QO2. Thus high concentrations of PQ appear to disrupt mitochondrial electron chain transfer resulting in reduction of metabolic functions.
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PMID:The cytotoxic effect of paraquat to isolated renal proximal tubular segments from rabbits. 927 8

The ability of rainbow trout liver cells to regulate their intracellular pH (pHi) was studied using two methods on hepatocytes isolated by collagenase digestion: (i) by monitoring pHi with the fluorescent dye BCECF-AM, and (ii) by measuring the amiloride-sensitive uptake of 22Na, which represents Na+/H+ exchange. In low-Na+ medium (&frac34;16mmoll-1), Na+ uptake was reduced by approximately 70% in the presence of amiloride derivatives (DMA or MPA, 10(-4)moll-1). Changing separately either the extracellular pH (pHe) or the intracellular pH (pHi, clamped by treating the cells with nigericin in the presence of 140mmoll-1 K+) between 6 and 8 induced an increase in the rate of Na+ uptake when pHe was raised or when pHi was reduced. When transferred to hypertonic medium, hepatocytes shrank to nearly 72% of their initial volume, and thereafter a slow and partial regulatory volume increase phase was observed, with an increase in the amiloride-sensitive rate of Na+ uptake and an increase in intracellular pH. As DIDS-sensitive Cl- uptake was concomitantly enhanced, it is suggested that hypertonic stress activates Na+/H+ and Cl-/HCO3- exchange.
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PMID:Na+/H+ exchange and osmotic shrinkage in isolated trout hepatocytes 932 Feb 88


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