Isolation, stages of differentiation, and long term maintenance of adult rat myocytes

Although a variety of techniques have been developed to isolate myocytes from adult hearts, the long term viability of such cells has only recently been investigated. In addition, relatively little is known about the stages of differentiation such cells proceed through following isolation. In the present study myocytes were isolated using two techniques, one involving retrograde perfusion via the aorta, and the other involving mechanical "shearing." In addition, several modifications were made to minimize the trauma normal associated with isolating myocytes from adult hearts. Both techniques yielded a high percentage of rod-shaped, quiescent myocytes, although myocytes isolated using the "shearing" method were less likely to remain viable for more than 24 hours. With both techniques those cells which remained viable for more than 24 hours proceeded through an identical pattern of differentiation leading to stable, attached cells which remained viable for up to four weeks. These results demonstrate that with the appropriate isolation techniques it is possible to maintain adult myocardial cells in culture for lengthy periods of time.     

EFFECT OF CYANIDE AND ELECTRICAL STIMULATION ON PHOSPHOINOSITIDE METABOLISM IN LOBSTER NERVES

The contents of phosphoinositides, ATP, glucose and lactate in leg and claw nerves of the lobster were determined. Nerves were also analysed after cyanide poisoning, after electrical stimulation, and 1 h after removing the leg from the lobster. Cyanide poisoning decreased the levels of ATP and glucose and increased the content of lactate but did not alter the levels of phosphoinositides. Nerves left in situ for 1 h after disconnection from the central nervous system exhibited a decrease in the content of tri-phosphoinositides (TPI) of 50 per cent, without changes in ATP, glucose or lactate. The TPI change was reversed after incubation for 1 h in oxygenated seawater. Nerves labelled in vivo with ³²P were removed and stimulated at 50 Hz for 5 min. The turnover of TPI phosphorus increased on stimulation in both normal and cyanide-poisoned nerves. In contrast, turnover of ATP increased after stimulation in normal nerves but not in cyanide-treated nerves. We sought to determine whether polyphosphoinositides play a greater role in resting metabolism of the nerve or in the conducting mechanisms. Our results make more likely the involvement of TPI in permeability changes of neural membranes during excitation.     

THE BINDING OF BOTULINUM TOXIN TO MEMBRANE LIPIDS: SPHINGOLIPIDS, STEROIDS AND FATTY ACIDS

A number of lipids known to be constituents of nerve-ending membranes were tested for their ability to inactivate botulinum toxin. Inactivation of the toxin by a lipid was taken as presumptive evidence that the lipid might be the in vivo receptor for the toxin. Several sphingolipids (sphingosine, galactosylceramide, glucosylceramide, lactosylceramide, cytolipin K and cytolipin R), steroids (cholesterol and deoxycholic acid) and fatty acids (palmitic acid, stearic acid, prostaglandin E₁) did not affect the potency of botulinum toxin, and thus were discounted as potential toxin receptors. However, the gangliosides did inactivate botulinum toxin rapidly (in less than 5 min), within a temperature range of 2°-40°C, and at ionic strengths of 0.05-0.40. Inactivation diminished as pH fell below 6. The activity of gangliosides in suppressing the potency of botulinum toxin was a function of the number of sialic acid residues in the lipid. Thus, the data suggest that a molecule containing sialic acid may be the receptor for the toxin.     

THE FINE STRUCTURE OF SHEEP MYOCARDIAL CELLS; SARCOLEMMAL INVAGINATIONS AND THE TRANSVERSE TUBULAR SYSTEM

An electron microscope study of sheep myocardial cells has demonstrated the presence of a transverse tubular system, apparently forming a network across the cell at each Z band level. The walls of these tubules resemble the sarcolemma in consisting of two dense layers—plasma membrane and basement menbrane; continuity of the tubule walls with the sarcolemma can be seen when longitudinal sections of a cell are obtained between two subsarcolemmal myofibrils and at the same time perpendicular to the cell surface. The demonstration of communication between the lumen of the transverse tubular system and the extracellular space appears to be more definite in this study than in any work hitherto published. It provides anatomical evidence of a possible direct pathway for transmission of the activating impulse from the sarcolemma to the myofibril Z bands.