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)

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

We tested the hypothesis that the adenylate cyclase system and Na+, K(+)-ATPase are reciprocally related in rat pancreatic islets. We studied the effect of theophylline, caffeine, and dibutyryl cyclic AMP on Na+, K(+)-ATPase activity in a membrane preparation from collagenase-isolated rat islets. Theophylline, caffeine, or dibutyryl cyclic AMP, in concentrations of 1 mM, all inhibited Na+, K(+)-ATPase activity (44,62, and 43%, respectively). Kinetic analysis indicated that theophylline and dibutyryl cAMP inhibit Na+, K(+)-ATPase by different mechanisms; theophylline decreased Vmax and decreased apparent Km (ATP), whereas dibutyryl cAMP decreased Vmax and increased apparent Km (ATP). Similar inhibition of Na+, K(+)-ATPase by theophylline or dibutyryl cAMP was noted in a particulate fraction from rat kidney and in a purified porcine brain Na+, K(+)-ATPase preparation. The adenylate cyclase system and Na+, K(+)-ATPase may act reciprocally in pancreatic islets and in other tissues. In the beta cell this relationship may be essential in coordinating consumption of ATP in the stimulated, as opposed to the rest, state.
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PMID:Relationships between adenylate cyclase and Na+, K(+)-ATPase in rat pancreatic islets. 215 93

Sarcolemmal vesicles were produced from adult mouse extensor digitorum longus muscle (EDL) by treating swollen muscle fibres with collagenase. Vesicles formed from dystrophic (C57BL/6J dy/dy) and phenotypically normal animals were patch clamped and the single channel activity was recorded. Three types of K+ channel were observed in excised patches taken from normal and dystrophic muscle. A large conductance (300 pS) Ca2(+)-dependent K+ channel (KCa) was the most frequently observed of the K+ channels in both types of muscle preparation. In a number of patches taken from dystrophic muscle the open probability-voltage relationship for the KCa channel was markedly different from that in normal muscle, suggesting a possible reduction in Ca2+ sensitivity. An ATP-sensitive K+ channel (90 pS) was common to both normal and dystrophic muscle vesicles and was present in a large number of patches. An inwardly rectifying K+ channel (40 pS) was also observed in both types of sarcolemmal vesicles. The properties of all three K+ channels types were broadly consistent with other observations of skeletal muscle K+ channels, though all had higher conductances than had previously been noted in other species.
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PMID:Potassium channel activity in sarcolemmal vesicles formed from skeletal muscle fibres of normal and dystrophic mice. 223 Aug 31

Intraluteal infusion of the prostaglandin (PG) synthesis inhibitor, sodium meclofenamate (Mec) causes premature luteolysis in rhesus monkeys. To evaluate further the actions of PG synthesis inhibitors in primate luteal function, we examined the in vitro effects of Mec and another inhibitor, flurbiprofen (Flur), on PG, cAMP, and progesterone (P) production by macaque luteal tissue obtained at midluteal phase of the menstrual cycle. First, collagenase dispersed luteal cells were incubated with 0-100 microM Mec or Flur, either alone or in the presence of 10 microM arachidonic acid (AA) to assess PGF2 alpha and PGE2 synthesis. Levels of both PGF2 alpha and PGE2 were stimulated (P less than 0.05) by AA (3.3- and 5.8-fold, respectively). Maximal suppression (P less than 0.01) of basal and AA-stimulated PGF2 alpha and PGE2 synthesis was elicited by 1 microM Mec and Flur. Second, adenylate cyclase activity, measured by the conversion of alpha 32P-ATP to alpha 32P-cAMP, was monitored in luteal homogenates exposed to increasing doses of Mec and Flur either alone or with maximal stimulatory doses of hCG, PGE2, or PGI2. Mec elicited a dose-dependent reduction (P less than 0.01) in control activity (incubated with 50 microM GTP), as well as inhibiting hCG- and PG-stimulated activity. The presence of 100 microM Mec suppressed (P less than 0.01) hCG-, PGE2- and PGI2-stimulated activity to control levels, but had no effect on activity stimulated by GMP-P(NH)P or forskolin. In contrast, Flur at any dose did not alter control activity or that stimulated by hormonal or nonhormonal activators. Third, P production by dispersed luteal cells was quantified during exposure to 0, 1, and 100 microM Mec or Flur alone or with maximal stimulatory doses of hCG, PGE2, PGD2, 6 beta PGI1, PGA2, or dibutyryl cAMP (dbcAMP). All hormones and dbcAMP stimulated (P less than 0.01) P synthesis 2-3 fold over basal levels, except PGA2, which had no effect. The presence of 100 microM Mec reduced (P less than 0.01) basal P production by 62% and abolished (P less than 0.05) hCG-, PG-, and dbcAMP-induced stimulation. Conversely, neither 1 microM Mec nor either dose of Flur affected P synthesis in the absence or presence of hormones or dbcAMP. These data indicate that: 1) Mec and Flur are potent inhibitors of PG synthesis in primate luteal cells in vitro and 2) higher doses of Mec suppress PG- and gonadotropin-sensitive adenylate cyclase activity and P production.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Disparate effects of the prostaglandin synthesis inhibitors, meclofenamate, and flurbiprofen on monkey luteal tissue in vitro. 230 10

A new procedure for separately isolating milligram quantities of rabbit renal proximal straight (PST) or convoluted (PCT) tubules is described, and the differential abilities of these segments to utilize glucose as a metabolic substrate are investigated. Separate dissection of the cortical cortices and the outer medullary stripe, followed by collagenase digestion and discontinuous Percoll centrifugation, provide enriched populations (greater than 98% pure) of PCT (37 mg) and PST (14 mg), respectively, per rabbit. The purity of PCT and PST fractions was quantitated morphologically and by comparing the enriched activity of the proximal tubular marker leucine aminopeptidase and deenriched activity of the distal marker hexokinase to previously published values reported from microdissection studies. To investigate glucose-dependent metabolic differences, PCT and PST suspensions (1 mg/ml) were preincubated in Dulbecco's modified Eagle's-Ham's F-12 medium for 1 h before being incubated for 30 min in buffer with or without glucose as the only available metabolic substrate. In glucose-containing buffer, PST segments maintained their oxygen consumption and ATP contents at levels significantly higher than PCT segments. These differential responses between PST and PCT were glucose-dependent because they were abolished when segments were incubated under glucose-free conditions. Because responses in PCT were glucose-independent, these results suggest that PCT cannot utilize glucose to support oxidative metabolism, whereas PST segments can oxidatively metabolize this substrate. These differences in glucose utilization do not correlate with the distribution of glycolytic enzyme activities, suggesting that differential metabolic regulation of these enzymes may determine the ability of each segment to utilize glucose.
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PMID:Bulk isolation of renal PCT and PST. I. Glucose-dependent metabolic differences. 237 89

Adenosine administration was tested in rats with carbon tetrachloride-induced hepatic fibrosis and was able to partially prevent the enlargement of liver and spleen induced by the toxin. This amelioration of the hepatomegaly was accompanied by a 50% reduction of the liver collagen deposition and preservation of content of glycosaminoglycans. A stimulated hepatic collagenase activity is apparently the mechanism for reduction of collagen accumulation. These effects were associated with a striking improvement in liver function. Adenosine treatment did not modify the late hepatotoxic effect of the carbon tetrachloride; however, the stimulatory effect of the nucleoside on energy state appeared to counteract the drastic decreases in adenine nucleotides, ATP, ATP/ADP ratio and energy charge elicited by the hepatotoxin. Moreover, a possible beneficial action of enhanced hepatic oxygenation caused by the vasodilator properties of adenosine cannot be ruled out. Regardless of the mechanism, adenosine seems to change the cellular response to the injury induced by the hepatotoxin.
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PMID:Adenosine partially prevents cirrhosis induced by carbon tetrachloride in rats. 239 Oct 66

The role of calcium in the parathyroid hormone-mediated increase in 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) production was evaluated using isolated proximal tubules from rats fed a low calcium diet (0.002% Ca) for 14 days. Tubules were prepared by collagenase digestion and centrifugation through Percoll. Tubules from rats fed a low calcium diet produced 1,25-(OH)2D3 at rates 10 times that of tubules from rats fed normal calcium diet (1.2% Ca). In vitro 1,25-(OH)2D3 biosynthesis was highly dependent upon extracellular calcium with inhibition in the absence of medium calcium and maximal production at 0.25 mM medium calcium (0.9 +/- 0.25 versus 15.1 +/- 2.3 nmol/mg protein/5 min, p less than 0.03). Inhibition of 1,25-(OH)2D3 production was partly due to depressed ATP content (0 versus 1.2 mM calcium, 6.8 +/- 0.6 versus 12.7 +/- 0.6 nmol/mg protein, p less than 0.006). EGTA reduced 1,25-(OH)2D3 synthesis and total cell calcium and ATP production. Ruthenium red blocked the inhibitory effects of EGTA on 1,25-(OH)2D3 production. Barium (1.0 mM) inhibited 1,25-(OH)2D3 production (7.2 +/- 0.5 versus 3.4 +/- 0.3, p less than 0.001) without altering ATP production. The calcium ionophore A23187 increased 1,25-(OH)2D3 production in a calcium-dependent manner. It is concluded that parathyroid hormone-mediated increases in 1,25-(OH)2D3 production, as during low calcium diet, require extracellular calcium. Extracellular calcium maintains mitochondrial calcium at optimal concentrations for normal ATP production, a requirement for 25-hydroxyvitamin D3-1-hydroxylase (25-OH-D3-1-hydroxylase) activity. Inhibition of 25-OH-D3-1-hydroxylase activity by barium without an alteration of ATP suggests calcium may also control 1,25-(OH)2D3 production independent of its effects on oxidative phosphorylation, perhaps through a direct interaction with one or more components of the 25-OH-D3-1-hydroxylase.
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PMID:Evidence for calcium-dependent control of 1,25-dihydroxyvitamin D3 production by rat kidney proximal tubules. 242 68

Taste discs were dissected from the tongue of R. ridibunda and their cells dissociated by a collagenase/low Ca/mechanical agitation protocol. The resulting cell suspension contained globular epithelial cells and, in smaller number, taste receptor cells. These were identified by staining properties and by their preserved apical process, the tip of which often remained attached to an epithelial (associated) cell. When the patch pipette contained 110 mM KCl and the cells were superfused with NaCl Ringer's during whole-cell recording, the mean zero-current potential of 22 taste receptor cells was -65.2 mV and the slope resistance 150 to 750 M omega. Pulse-depolarization from a holding voltage of -80 mV activated a transient TTX-blockable inward Na current. Activation became noticeable at -25 mV and was half-maximal at -8 mV. Steady-state inactivation was half-maximal at -67 mV and complete at -50 mV. Peak Na current averaged -0.5 nA/cell. The Ca-ionophore A23187 shifted the activation and inactivation curve to more negative voltages. Similar shifts occurred when the pipette Ca was raised. External Ni (5 mM) shifted the activation curve towards positive voltages by 10 mV. Pulse depolarization also activated outward K currents. Activation was slower than that of Na current and inactivation slower still. External TEA (7.5 mM) and 4-amino-pyridine (1 mM) did not block, but 5 mM Ba blocked the K currents. K-tail currents were seen on termination of depolarizing voltage pulses. A23187 shifted the IK(V)-curve to more negative voltages. Action potentials were recorded when passing pulses of depolarizing outward current. Of the frog gustatory stimulants, 10 mM Ca caused a reversible 5- to 10-mV depolarization in the current-clamp mode. Quinine (0.1 mM, bitter) produced a reversible depolarization accompanied by a full block of Na current and, with slower time-course, a partial block of K currents. Cyclic AMP (5 mM in the external solution or 0.5 microM in the pipette) caused reversible depolarization (to -40 to -20 mV) due to partial blockage of K currents, but only if ATP was added to the pipette solution. Similar responses were elicited by stimulating the adenylate cyclase with forskolin. Blockage of cAMP-phosphodiesterase enhanced the response to cAMP. These results suggest that cAMP may be one of the cytosolic messengers in taste receptor cells. Replacement of ATP by AMP-PNP in the pipette abolished the depolarizing response to cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Patch-clamp study of isolated taste receptor cells of the frog. 244 95

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

Effects of intracellularly perfused ATP, and extracellularly applied cyanide and 2-deoxy-D-glucose, on fast and slow Ca2+ channel currents of isolated single vascular smooth muscle cells were investigated by a whole-cell voltage-clamp method combined with an intracellular perfusion technique. Single smooth muscle cells were prepared by collagenase treatment from guinea pig small mesenteric arteries (diameter of less than 300 micron). With Cs+-rich solution in the pipette and isotonic Ba2+ solution (100 mM) in the bath, depolarizing pulses evoked two types of the Ca2+ channel current. Depolarizing pulses from the holding potential of -80 mV to over -30 mV evoked a fast Ca2+ channel current. This fast component was inhibited by shifting the holding potential in a positive direction. With a holding potential of -40 mV, the fast component was almost inhibited. In contrast, the slow current was evoked by command potentials to above -10 mV, and its full amplitude was preserved at the holding potential of -40 mV. Without ATP in the pipette, the fast current was dominant. Increase in the ATP concentration in the pipette (0.3 to 5 mM) enhanced the slow current but did not affect the fast current. Maximum enhancement of the slow current was observed at 5 mM ATP. Increase in ATP concentration, however, did not modify the shape of the current trace and the steady state inactivation curve of the slow current. Maximum amplitudes of the fast current and slow current recorded with 5 mM ATP averaged 17.4 pA (SD of 10.4 pA, n = 30; observed at -10 mV to +10 mV) and 141.8 pA (SD of 27.1 pA, n = 30; observed at +30 mV to +40 mV), respectively. Presence of CN- and 2-deoxy-D-glucose (without glucose) in the bath, and absence of ATP in the pipette, abolished the slow current within 10 minutes; in contrast, it took more than 10 minutes to depress the fast current. The inhibitory effect of CN- and 2-deoxy-D-glucose on the slow current was reduced by intracellular application of ATP. In summary, the activation of the slow Ca2+ channel required physiological concentration of ATP, whereas the fast channel current was preserved, even under ATP-free conditions. These results indicate that only the slow current is a metabolically dependent Ca2+ channel current in these vascular smooth muscle cells.
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PMID:ATP regulation of the slow calcium channels in vascular smooth muscle cells of guinea pig mesenteric artery. 253 96


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