Various cellular parameters were measured with regard to their usefulness as criteria of viability of isolated cells. Stainability by trypan blue and release of lactate dehydrogenase indicate only severe irreversible damage of cells. Neither endogenous respiration nor even the ATP/ADP ratio is a sensitive criterion of viability. On aging of cells, the ATP/ADP ratio remains high, even though the membrane potential, the intracellular K® concentration and the content of adenine nucleotides decrease considerably. A sensitive, easily performed test is the stimulation of cellular respiration by ImM succinate. Only a damaged Vitalitätskriterien für isolierte LeberzellenZusammenfassung: Leberparenchymzellen der Ratte wurden in Gegenwart von Ca 2 ® isoliert. Vorgeschädigte Zellen wurden durch Rotation der Zellsuspension in einem Kolben fast völlig zerstört.Nach-Zentrifugalfiltration wurden Zellvolumen, Membranpotential und intrazelluläre Na®-und K®-Konzentrationen gemessen. Für frisch isolierte Zellen fanden wir ein Membranpotential von 36.4 ± 3.4 mV (n = 5), eine intrazelluläre K®-Konzentration von 109.0 ± 9. ImM und eine intrazelluläre Na®-Konzentration von 47.0 ± 13.4mM.
Voltage-activated Ca(2+) channels comprise complexes of a pore-forming Ca(V)alpha(1) and auxiliary subunits Ca(V)beta, Ca(V)alpha(2)delta and sometimes Ca(V)gamma. The intracellular Ca(V)beta subunit assists in trafficking and surface expression of the Ca(V)alpha(1) subunit and can modulate biophysical properties of the Ca(2+) channel. Four genes, Ca(V)beta1-4, exist which confer different properties to Ca(2+) currents through the various Ca(V)alpha(1) subunits. Ca(2+) currents in cochlear inner (IHC) and outer hair cells (OHC) serving synaptic transmission flow predominantly through the L-type Ca(V)alpha(1) subunit Ca(V)1.3, but associated Ca(V)beta subunits are unknown. In the organ of Corti, we found mRNA and protein for all four Ca(V)beta subunits including Ca(V)beta2, but clear assignment of the Ca(V)beta1-4 immunolabelling with hair cells or nerve fibers was difficult. We analyzed Ca(V)beta3 knockout (Ca(V)beta3(-/-)) and Ca(V)beta4 mutant mice (Ca(V)beta4(lh/lh)), which had normal hearing. Recording voltage-activated Ba(2+) currents from hair cells of the two mouse models revealed distinct significant changes of cell size and Ba(2+) current properties compared with their wild-type controls. Neonatal Ca(V)beta4(lh/lh) IHCs showed reduced membrane capacitances and changes in the voltage dependence and kinetics of current activation, whereas mature IHCs had reduced peak currents compared with Ca(V)beta4(wt), altogether indicating the presence of Ca(V)beta4 in IHCs. Ba(2+) currents of Ca(V)beta3(-/-) OHCs showed largely reduced amplitudes, changes in the voltage dependence and kinetics of Ba(2+) current activation, and increased inactivation compared with Ca(V)beta3(wt), pointing to a role of Ca(V)beta3 for OHCs. These results indicate that neither Ca(V)beta3 nor Ca(V)beta4 are indispensable for hair cell Ca(2+) currents but contribute to the overall current properties.
(Received Septcmber 1 Deccmbcr 7, 19x2) -EJH 59591. The ability of external ATP to induce calcium uptake in isolated rat liver cells was further characterized. Stimulation of calcium uptake was specific for ATP, other nucleotides or ATP metabolites had no comparable effect. ATP was dephosphorylated while stimulating calcium uptake, but there was no stoichiometry between ATP hydrolysis and calcium uptake nor did dephosphorylation depend on calcium concentration. ATP acted from outside and was dephosphorylated by an ccto-ATPase of the cells.2. In addition to its direct action, ATP enhanccd succinate-dependent calcium uptake in a coopcrativc fashion. This is best explained by different sites of action. ATP increases cell membrane permeability while succinatc stimulates uptake into mitochondria.3. ATP was able to lower Na' and K t gradients and the pH gradient between cells and incubation medium. Increasing calcium concentration countcracted this effect though calcium uptake was then stimulated.4. Succinate alone did not affect monovalent cation gradients but raised the pH gradient. It partially coiinteractcd the ATP effects on these gradients.5. Since catecholamine-like actions of ATP may be mediated by an increase in cytoplasmic calcium concentration, the action of extracellular ATP can be taken as a model t o study the role of calcium as a transmitter of hormone actions. From interdependence between ATP-stimulated and succinatc-stimulated calcium uptake, conclusions can be drawn on the resulting cytoplasmic calcium concentration and its effect on plasma membrane permeability.In a previous study, isolated liver cells proved to be a useful model to study the delicately poised equilibrium of calcium distribution across the various cellular compartments [l]. This was especially true for disclosing the role of cellular mitochondria in this interplay. Respiratory substrates, pH, inorganic phosphate, and the redox grade of pyridine nucleotides were found important in triggering calcium movements at the mitochondria1 site, whereas external ATP and La3+ induced calcium uptake at the plasma membrane site [I. 21.In this report, thc action of added ATP is further characterizcd. The responses of functional parameters such as cation distribution and the pH gradient between cells and incubation medium are compared with those seen after succinate addition. Relations between the different responses and the probablc changes in cytoplasmic calcium concentrations are discussed. This is of interest because external ATP was previously reported to exert a catecholamine-like action on the plasma membrane permeability [3, 41 and to prcvent the stimulatory effect of insulin on hexose transport and oxidation [S -71. As r-adrenergic stimulation generally results in changes in both calcium and potassium distribution (for review see [8]) the study of ATP action might shed light on the relcvance of various cellular parameters in a state similar to that during a-adrenergic action. MATERIALS AND METHODS Radiochmicals.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.