Roles of collagenases and other proteolytic enzymes in the dispersal of animal tissues

Kôno, Tetsuro

doi:10.1016/0005-2744(69)90410-0

Cited by 76 publications

(29 citation statements)

References 11 publications

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“…Proteolytic enzymes have been used for dissociating tissues into individual, viable cells (19)(20)(21)(22). In the present study, single cells were obtained by incubation of turtle retina with papain in calcium-free Ringer's solution.…”

Section: Resultsmentioning

confidence: 99%

Biosynthesis of Acetylcholine in Turtle Photoreceptors

Lam

1972

Proc. Natl. Acad. Sci. U.S.A.

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For determination of possible neurotransmitters synthesized by photoreceptor cells, turtle retinas were dissociated into single cells with proteolytic enzymes. These cells were partially separated by velocity sedimentation to yield a fraction rich in photoreceptors. Individual photoreceptor cells were then sucked into a micropipette and incubated with labeled precursors of known or suspected neurotransmitters. After incubation, the radioactive products were analyzed by high-voltage electrophoresis. Of all the chemicals tested, turtle photoreceptor cells synthesized only acetylcholine, suggesting that these cells may be cholinergic.Identities of neurotransmitters in the central nervous system of vertebrates are known for only a few cell types (1). Thus, although we now have a fair understanding of the neuronal connections and physiological behavior of various retinal cell types (2-7), the chemistry of synapses in the retina is still unknown. A direct way of obtaining this information is by examination of the ability of single, intact, and identified cells to synthesize known or suspected neurotransmitters. The vertebrate retina is a convenient part of the central nervous system for such an analysis, because most retinal cell types can be readily identified even when they are isolated.The presence of several known or suspected neurotransmitters, such as' acetylcholine, 'y-aminobutyric acid, and dopamine,'has been suggested or demonstrated in some vertebrate retinas (8-11). There is, however, no direct evidence that any of these compounds are synthesized or used in a particular cell type. In order to assign possible chemical transmitters to individual cell types, single cells were dissociated by proteolytic enzymes, separated by velocity sedimentation, and analyzed by incubation of selected cells with labeled precursors of known or suspected neurotransmitters. Photoreceptors were chosen for this initial analysis because they are first-order sensory neurons of the visual pathway. Turtle photoreceptors were used because they are unusually large and contain brightly colored oil droplets that permit easy identification. In addition, their structure permits analysis of transmitter synthesis by the cell bodies in the absence' of the pedicles, which may contain postsynaptic fragments from horizontal and bipolar cells. Furthermore, the anatomy (12) and physiology (13) of turtle photoreceptors have been studied in detail. MATERIALS AND METHODSCell Dissociation. All media and Ringer's solutions were supplemented with 1000 units/ml of penicillin G and 0.5 mg/ ml of streptomycin sulphate (Microbiological Associates, 1987 Inc., Bethesda, Md.), and sterilized (18). For cell dissociation and separation, isotonic calcium-free Ringer's solution supplemented with ethyleneglycol bis(aminoethyl) tetraacetic acid (EGTA) was used (in g/liter: 7.3. NaCl-0.5 NaHCO3-0.07 NaH2PO4-0.25 KCl-0.1 MgCl2-2 glucose; 5 mM EGTA; pH 7.2).Fresh-water turtles (Pseudemy8 scripta elegans, with shells about 20 cm long) were adapted to darknes...

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Section: Resultsmentioning

confidence: 99%

Biosynthesis of Acetylcholine in Turtle Photoreceptors

Lam

1972

Proc. Natl. Acad. Sci. U.S.A.

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“…Unfortunately purified collagenase is unable to liberate adipocytes from fat tissue [4]. The batches of commercial crude collagenase contain variable types and amounts of enzymes with trypsin-like activities [5].…”

Section: Evaluation Of Experimental Conditionsmentioning

confidence: 99%

Hexose transport in human adipocytes: Factors influencing the response to insulin and kinetics of methylglucose and glucose transport

Pedersen

Gliemann

1981

Diabetologia

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Summary. Optimal experimental conditions were defined for measuring the initial uptake rate of the non-metabolizable sugar analogue 3-O-methylglucose in non-stimulated and insulin-stimulated human adipocytes. The permeability of the adipocyte plasma membrane for tracer methylglucose (100 ~tmol/l) was 2.9 • 10 -7 cm • s -1 at 37~ and slightly lower at 20~ At 37~ and pH 7.4 insulin (5nmol/l) increased the permeability about twofold (range 1.5 to fivefold) with half maximal effect at about 100 pmol/1. At pH 7.0 the dose response curve for the insulin effect on the uptake rate of methylglucose was shifted about 2.5-fold to the right. The permeability to L-glucose due to simple diffusion was estimated as 3.0 • 10-~~ cm • s-1 suggesting that uptake of methylglucose occurs almost exclusively by facilitated diffusion. The Km for methylglucose equilibrium exchange in insulin stimulated cells was about 4.8mmol/1. The initial uptake of tracer methylglucose in insulin-stimulated cells was inhibited by unlabelled methylglucose and by D-glucose with inhibition constants of about 3.8 and 7.7 mmol/ 1, respectively. Uptake of tracer 2-deoxyglucose (50 ~tmol/l) in insulin-stimulated adipocytes was linear from 10 s to 5 min whereas the rate of uptake in the presence of 3 mmol/l of D-glucose was markedly decreased suggesting that deoxyglucose uptake after a few minutes is mainly limited by hexokinase in the presence of glucose.

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“…Cultured myocytes isolated from embryonic hearts have been a matter of interest for a long time, especially from a developmental standpoint, but their sizes are so small that most of the electrophysiological studies have been conducted on sheet, strand, or cluster specimens. In 1969, KoNO [31] isolated single adult rat ventricular cells through the collagenase treatment; since then many methods for preparing viable cardiac cells have been reported, but few physiological studies were done using these single cells until POWELL, TERRAR, and TWIST [49,50] and ISENBERG and KLOCKNER [21] demonstrated the usefulness of the single cell for physiological study.…”

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confidence: 99%

Electrophysiology of single cardiac cells.

Irisawa

1984

Jpn.J.Physiol

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The importance of using single myocytes isolated from the adult mammalian heart has lately received wide recognition, and many investigations on ionic currents using single ventricular cells have appeared from various laboratories. Cultured myocytes isolated from embryonic hearts have been a matter of interest for a long time, especially from a developmental standpoint, but their sizes are so small that most of the electrophysiological studies have been conducted on sheet, strand, or cluster specimens. In 1969, KoNO [31] isolated single adult rat ventricular cells through the collagenase treatment; since then many methods for preparing viable cardiac cells have been reported, but few physiological studies were done using these single cells until POWELL, TERRAR, and TWIST [49, 50] and ISENBERG and KLOCKNER [21] demonstrated the usefulness of the single cell for physiological study.The heart has a functional synticytium and the excitation of a single cell is conducted to the adjacent cells through low resistance nexus [64]. Much traditional electrophysiological work on heart muscle has been carried out with multicellular muscle strands, such as Purkinje fibers and the papillary strands. Using these preparations, most basic properties of excitation and conduction of the heart muscle have been established. Voltage clamp experiments in multicellular specimens [for review, see Refs. 4,9,42] are not ideal because of the difficulty of the spatial clamp and the desire of most cardiac physiologists to design their experiments using single cardiac cells. Thus the work of POWELL and TWIST [50] stimulated researchers in many fields of cardiac electrophysiology to develop a method of obtaining viable cardiac cells and to devise a way of recording electrical activity from single cells.Another development which triggered single cell physiology was the introduction of the patch clamp method [14], where single ionic channel activity and statistical analyses of channel activity became available. This newly developed method in the electrophysiology of the cardiac cell spurred increased interest among general physiologists and biophysicists, as well as cardiac physiologists.Single myocytes isolated from the adult heart have the following unique features.

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Roles of collagenases and other proteolytic enzymes in the dispersal of animal tissues

Cited by 76 publications

References 11 publications

Biosynthesis of Acetylcholine in Turtle Photoreceptors

Biosynthesis of Acetylcholine in Turtle Photoreceptors

Hexose transport in human adipocytes: Factors influencing the response to insulin and kinetics of methylglucose and glucose transport

Electrophysiology of single cardiac cells.

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