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)

Isolated adrenal cells were prepared by collagenase digestion of guinea pig adrenal glands. Acetylcholine stimulates the secretion of catecholamines by these isolated adrenal cells. Acetylcholine-stimulated catecholamine secretion is inhibited by cholinergic blocking agents (atropine and hexamethonium) and by local anaesthetics (tetracaine), and is dependent upon the concentration of Ca2+ in the incubation medium. In the presence of Ca2+, catecholamine secretion is also stimulated by two divalent cation ionophores, A23187 and X-537A. Cyclic nucleotides and 5'-nucleotides cause a small, non-specific stimulation of catecholamine secretion. These results indicate that isolated adrenal cells are a useful system in which to study catecholamine secretion, and support the hypothesis that increased Ca2+ entry into chromaffin cells is a sufficient stimulus for catecholamine secretion.
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PMID:Catecholamine secretion by isolated adrenal cells. 17 36

1. The kinetics of tubocurarine inhibition were studied at the post-synaptic membrane of frog skeletal muscle fibres. Acetylcholine (ACh) and (+)-tubocurarine were ionophoresed from twin-barrel micropipettes, and the membrane potential of the muscle fibre was recorded intracellularly. Tubocurarine-receptor binding was measured by decreases in the response to identical pulses of ACh. 2. The responses to both ACh and tubocurarine had brief latencies and reached their maxima rapidly. It is suggested that under these conditions the kinetics of tubocurarine action are not slowed by diffusion in the space outside the synaptic cleft. 3. After a pulse of tubocurarine, recovery from inhibition proceeds along a roughly exponential time course with a rate constant, 1/tau off approximately equal to 0.5 sec-1. This rate constant does not depend on the maximal level of inhibition and varies only slightly with temperature (Q10 = 1.25). 4. After a sudden maintained increase in tubocurarine release, the ACh responses decrease and eventually reach a new steady-state level. Inhibition develops exponentially with time and the apparent rate constant, 1/tau on, is greater than 1/tau off. When the steady-state inhibition reduces the ACh response to 1/n of its original level, the data are summarized by the relation, 1/tau on = n(1/tau off). 5. When the ACh sensitivity is reduced with cobra toxin, both 1/tau on and 1/tau off increase. Thus, the kinetics of tubocurarine inhibition depend on the density of ACh receptors in the synaptic cleft. 6. After treatment with collagenase, part of the nerve terminal is displaced and the post-synaptic membrane is exposed directly to the external solution. Under these circumstances, 1/tau off increases more than tenfold. 7. Bath-applied tubocurarine competitively inhibits the responses to brief ionophoretic ACh pulses with an apparent equilibrium dissociation constant, K = 0.5 microM. 8. In denervated frog muscle fibres, extrasynaptic receptors have a lower apparent affinity for tubocurarine. After a pulse of tubocurarine, inhibition decays tenfold more rapidly at these extrasynaptic sites than at the synapse. 9. It is suggested that each tubocurarine molecule binds repeatedly to several ACh receptors before escaping from the synaptic from the synaptic cleft and that the probability of this repetitive binding is enhanced because the nerve terminal presents a physical barrier to diffusion out of the cleft. Consequently, the receptor transiently buffer the concentration of tubocurarine in the cleft, and the macroscopic kinetics of inhibition are much slower than the molecular binding rates for tubocurarine.
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PMID:The kinetics of tubocurarine action and restricted diffusion within the synaptic cleft. 22 14

1. Quantitative ionophoresis at the neuromuscular junction is possible when (a) the drug is released from appropriate distances (15--20 micrometer for most drugs), (b) the topology of receptors is known and (c) high resistance drug pipettes (100--200 M omega) are sued. 2. With this method, drug concentration-endplate conductance relations were determined in voltage-clamped end-plates of the frog for the agonists ACh, carbamylcholine (CCh) and suberyldicholine (SubCh). 3. Based on the co-operative and independent model, theoretical dose-response curves were computed using as parameters the Hill coefficient nH, maximum conductance gmax., and apparent dissociation constant K. It was found that the co-operative model fitted the data much better than the independent model. 4. Based on the co-operative model, the mean maximum conductance for ACh was gmax. = 169 nS/micrometer, equivalent to 9000 ionic channels/micrometer length of a nerve terminal which can be opened at high drug concentrations. 5. The maximum conductance for CCh at--80 mV membrane potential was, on the average, 78% of that for ACh measured at the same end-plates. This value is termed the relative efficacy of CCh. 6. The mean values for the apparent dissociation constant K were 27.8 micrometer for ACh, 336 micrometer for CCh and 18 micrometer for SubCh. 7. The inhibition of the acetylcholinesterase activity by edrophonium (3--10 micrometer) affected only the local ACh concentration at the receptor sites, but not gmax. and nH. 8. Dose-response curves measured before and after removal of single nerve terminals in collagenase-treated muscle fibres showed no change in the nH, gmax. and K. A slight increase in gmax. to a value of 218 nS/micrometer observed comparing collagenase-treated and untreated end-plate. 9. Desensitization of receptors may occur in the range of several tens of milli-seconds.
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PMID:Determination of dose-response curves by quantitative ionophoresis at the frog neuromuscular junction. 30 3

1. Adult rat flexor digitorum brevis muscles were dissociated by treatment with collagenase and trituration. Several hundred isolated fibres were obtained from each muscle. 2. Most isolated fibres appeared to be intact as judged by some morphological and physiological criteria, although resting membrane potentials were about -60 mV, which is somewhat lower than normal. 3. A small percentage of the muscle fibres were branched. 4. Acetylcholine sensitivity was measured iontophoretically. The sensitivity fell abruptly outside the margin of the end-plate. Extrajunctional sensitivty was detected on all fibres, and declined smoothly away from the end-plate to an undetectable level over a distance of about 200 micron. On a few fibres, ACh sensitivity was mapped circumferentially from the end-plate. It appeared to decline with distance in a manner similar to the longitudinal sensitivity gradient. 5. Fibres dissociated from muscles denervated a week earlier were sensitive to ACh everywhere on their surfaces.
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PMID:Physiological properties of dissociated muscle fibres obtained from innervated and denervated adult rat muscle. 91 32

1. Postsynaptic responses to acetylcholine released from nerve terminals and from iontophoretic micropipettes were investigated in skeletal twitch-muscle fibers of the snake. The preparation consists of thin sheets of muscle fibers in which details of the end plate, including the outlines of individual synaptic boutons, are clearly seen in the living state. After treatment with collagenase, the motor nerve and its terminal boutons can be removed to expose the intact subsynaptic membrane to direct application of ACh by iontophoretic pipettes. 2. The number of ACh molecules in a quanta was estimated to be fewer than 10,000. This was done by developing a sensitive bioassay to measure the output of ACh from iontophoretic pipettes needed to produce synaptic responses closely resembling nerve-released miniature postsynaptic potentials. 3. Postsynaptic receptors are not saturated by the ACh in a quantum, since the peak of the quantal response produced by an appropriate background concentration of ACh from a pipette. 4. When acetylcholine esterase is inhibited, two or more quanta can act upon partially overlapping postsynaptic membrane areas and potentiate each other's effects. This potentiation reveals itself as a prolongation of the synaptic current. Postsynaptic potentiation is a consequence of the nonlinear dose-response characteristics of ACh receptors and can also be demonstrated in a model system in which ACh micropipettes substitute for quantal release from the nerve. 5. With AChE fully active, however, each quantum is functionally isolated from its neighbors and no postsynaptic potentiation is seen. 6. It is suggested that postsynaptic potentiation between quantum may play a role in signaling at synapses which have nonlinear dose-response characteristics and where transmitter is not so rapidly inactivated as at the neuromuscular synapse.
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PMID:The number of acetylcholine molecules in a quantum and the interaction between quanta at the subsynaptic membrane of the skeletal neuromuscular synapse. 106 24

The involvement of external Ca2+ in channel activity of nicotinic acetylcholine (nACh) receptors was investigated in the dissociated skeletal muscle fibers of adult mice. Single cells were prepared from flexor digitorum brevis muscles by treatment with collagenase and trypsin. Acetylcholine receptor-channel currents were recorded at endplates in the cell-attached mode, using the patch-clamp technique. Opening frequency, opening pattern and channel conductance were measured at different concentrations of ACh and succinylcholine (SuCh), using a patch pipette. Bath-applied ACh (30 microM) and SuCh (3-30 microM) decreased the ACh (1 microM)-induced channel conductance (SuCh was more potent than ACh). Large concentrations of both ACh (100-1000 microM) and SuCh (30-300 microM), which were applied via a patch pipette, also decreased channel conductance in a concentration-dependent manner. Increasing the concentration of calcium [Ca2+]0 in the patch pipette from 0 to 10 mM CaCl2 reduced ACh (1 microM)- and SuCh (1 microM)-activated single channel conductances. The increase in [Ca2+]0 prolonged the mean open times by 34% for ACh and by 22% for SuCh and decreased the channel opening frequency by 50% and 60%, respectively. These results demonstrate that ACh receptor-channel properties are dependent on [Ca2+]0 and that the ACh- and SuCh-induced decrease in channel currents may be also related to intracellular concentration of Ca2+. The effect of SuCh was greater than that produced by ACh.
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PMID:External Ca2+ dependence of acetylcholine- and succinylcholine-induced changes in channel conductance, open time and frequency at endplates of single muscle cells of adult mice. 166 97

The electric organs of two species of skate have been examined morphologically, physiologically and biochemically. They can be easily dissociated into innervated or denervated component electrocytes by a Torpedo Ringer's solution containing 1% collagenase. Collagenase treatment did not, however, separate the Schwann cell cover capping the synaptosomes. Isolated electrocytes generate normal MEPP frequencies and show evoked responses for two days in Torpedo Ringer's. The nerve terminals retain excitability and transmitter release properties up to the time of separation. Since isolated terminals and denervated electrocytes show normal ultrastructural characteristics for up to 12 h, the skate electric organ provides several preparations which are not attainable with Torpedo tissue. Acetylcholine (ACh) content of supernatant fractions containing the synaptosomes was comparable to that found in Torpedo (sps.). Collagenase specifically eliminates the basal lamina associated with the synaptic junctional region. Neuronal cell death and synaptic terminal degeneration were also noted in the adult organs of both species. The skate electric organ is ideally suited for the study of cholinergic development and transmission.
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PMID:Morphological, physiological and biochemical observations on skate electric organ. 216 Nov 87

Recent reports suggesting that the actions of certain neuroenteric peptides may be mediated in part by the secretion of histamine and other mast cell contents could have important implications for gastrointestinal motility and secretion. However, evidence for a mast cell-hormonal interaction is based on studies using peritoneal or cutaneous mast cells. Because intestinal mucosal mast cells (MMC) differ functionally from peritoneal mast cells (PMC), we compared the effects of several neurotransmitters and intestinal hormones on histamine secretion from two mast cell types in the rat. MMC hyperplasia was induced in rats by infection with the nematode Nippostrongylus brasiliensis, and MMC were isolated from the small intestine by collagenase digestion. Substance P, somatostatin, vasoactive intestinal polypeptide (VIP), neurotensin, and bradykinin had a potent secretagogue effect on (10(-7) to 10(-4)M) PMC which was temperature-, energy-, and calcium-dependent. In contrast to PMC, MMC released significant amounts of histamine only when challenged with substance P. Acetylcholine, bombesin, motilin, and pentagastrin had no secretory effect on either PMC or MMC. The differences between PMC and MMC in responsiveness to peptides could not be attributed to the MMC isolation procedure because PMC treated similarly or mixed with MMC suspensions retained their responsiveness to these stimuli. Our results extend the concept of neurocrine control of mast cell function, but indicate that mast cells from different sites have distinct profiles of responsiveness to regulatory peptides.
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PMID:Mast cell heterogeneity: effects of neuroenteric peptides on histamine release. 240 46

1. Catecholamines, adenosine, gonadotrophins, vasoactive intestinal peptide (VIP) and E-series prostaglandins all elicit K+ currents in follicle-enclosed Xenopus oocytes. Evidence suggests that cyclic nucleotides act as intracellular messengers in the activation of this K+ conductance. Muscarinic agonists and some divalent cations (e.g. Co2+, Mn2+, Ni2+ and Cd2+) elicit slow oscillatory Cl- currents, which are activated through hydrolysis of inositol phospholipids and mobilization of intracellular calcium by inositol phosphates. 2. We investigated whether these membrane current responses were generated in the oocyte itself or in enveloping follicular cells which are coupled to the oocyte by gap junctions. Oocytes were defolliculated, either enzymatically using collagenase, or by manual dissection combined with rolling over poly-L-lysine-coated slides. Removal of follicular cells was checked using scanning electron microscopy. Membrane current responses of defolliculated oocytes were compared with responses seen in follicle-enclosed oocytes taken from the same ovary. 3. The K+ responses evoked by all the various hormones/neurotransmitters were either drastically reduced (greater than 90%) or abolished by defolliculation. K+ currents generated by the adenylate cyclase activator forskolin and by intraoocyte injection of adenosine 3',5'-cyclic monophosphate (cyclic AMP), or guanosine 3',5'-cyclic monophosphate were similarly reduced in defolliculated oocytes. In contrast, oscillatory Cl- currents to acetylcholine and divalent cations were selectively preserved through defolliculation. 4. Injection of cyclic AMP (1-20 pmol) into defolliculated oocytes had little or no effect on oscillatory Cl- currents elicited by ACh. However, the calcium-dependent transient Cl- current, activated by depolarization of the oocyte membrane, was consistently potentiated (100-900%) by injections of cyclic AMP (1-10 pmol). 5. These experiments suggest that cyclic nucleotide-activated K+ currents arise essentially in follicular cells and are monitored within the oocyte through electrical coupling by gap junctions. Oscillatory Cl- responses evoked by ACh and divalent cations are produced largely or wholly in the oocyte itself.
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PMID:Effects of defolliculation on membrane current responses of Xenopus oocytes. 255 77

The concentration-dependent actions of neostigmine, a carbamate anticholinesterase agent, were studied on the acetylcholine receptor channel complex in voltage-clamped twitch fibers of costocutaneous muscles of garter snakes. Low concentrations of neostigmine (10(-6) or 10(-5) M) increased miniature endplate current (MEPC) amplitude and the time constant of MEPC decay without changing the relationship between the MEPC decay time constant and membrane potential. Acetylcholine- or carbachol-induced endplate current fluctuation spectra were well fitted by a single Lorentzian curve with a characteristic frequency and single-channel conductance unaltered by low concentrations of neostigmine. Concentrations of neostigmine greater than 5 X 10(-5) M decreased MEPC amplitude and split the decay of MEPCs into two components, one faster and one slower than the control rate. These effects were both voltage and concentration dependent. Spectra of current fluctuations recorded in concentrations greater than or equal to 5 X 10(-5) M neostigmine required two time constants, one faster and one slower than the control. Two component spectra were also obtained with carbachol-induced current fluctuation spectra, indicating that these effects of neostigmine were direct and not a consequence of acetylcholinesterase inhibition. Similar results were also obtained in muscles pretreated with collagenase to remove junctional acetylcholinesterase. The fast and slow time constants obtained from current fluctuation spectra decreased and increased, respectively, with either increases in the concentration of neostigmine or membrane hyperpolarization when analyzed in the same fiber. The effects of neostigmine on channel lifetime were reversible with washing. These results indicate that the effects of neostigmine are concentration dependent. Concentrations greater than 2.5 X 10(-5) M exhibit direct effects on the endplate receptor channel complex which are unrelated to acetylcholinesterase inhibition. These actions include: a prolongation of the gating kinetics of the endplate receptor channel complex, the production of an altered state of the receptor channel complex evidenced by a high frequency component to current fluctuation spectra, and a direct action to block the acetylcholine receptor.
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PMID:Concentration-dependent effects of neostigmine on the endplate acetylcholine receptor channel complex. 257 18


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