Neuroblastoma-glioma hybrid cells (NG108-15) in suspension accumulate the permeant lipophilic cation [3Hltetraphenylphosphonium (TPP+) against a concentration gradient. The steady-state level of TPP+ accumulation is about twice as great in physiological media of low K+ concentration (i.e., 5 mM K+/135 mM Na+) than in a medium of high K+ concentration (i.e., 121 mM K+/13.5 mM Na+ Many of the basic neurophysiological properties that underlie nervous system function have been described and analyzed in neuroblastoma-glioma hybrid cell line NG108-15, thereby establishing this line as an appropriate model system for studies of the nervous system. These properties include: (i) generation of action potentials (1); (ii) synthesis, storage, and release of the neurotransmitter acetylcholine (2, 3); (iii) synapse formation (1, 4); and (iv) presence of specific plasma membrane receptor sites for neurotransmitters and neuromodulators, such as acetylcholine, opiates, adenosine, and prostaglandins, which regulate adenylate cyclase (5)(6)(7)(8)(9). Because many of these phenomena are clearly related to alterations in the electrical potential (AI) across the plasma membrane, correlations between changes in AI and specific biochemical functions take on fundamental importance. Traditionally, measurement of AI in nervous tissue relies on techniques that require impaling selected cells with microelectrodes. However, because this technique is limited by the size of the cells and cannot be applied to populations of cells at large, it is apparent that alternative methods would be of value.This communication describes the application of a biochemical method for determining AI in cultured neuroblastoma-glioma hybrid cells. The technique involves the use of the permeant lipophilic cation tetraphenylphosphonium (TPP+) which has been used to measure AI in mitochondria (10), Ehrlich's ascites cells (11), and bacteria and bacterial membrane vesicles.$ The use of "lipophilic ions" to measure AI across biological membranes was introduced by Skulachev and Liberman (for review, see ref. 12) and their coworkers in the early 1970s, and this class of compounds has been applied to a number of prokaryotic and eukaryotic systems (10-16, ¶). The ions are constructed in such a fashion that they are sufficiently lipophilic to enter the hydrophobic core of the membrane, and they also have the propensity for charge delocalization which allows passive equilibration with AM (17). It should be noted, however, that in no case have AA determinations with lipophilic cations been validated quantitatively by direct electrophysiological measurements. The results presented here provide a strong indication that TPP+ distribution can be used to determine AI in populations of neuroblastoma-glioma hybrid cells. METHODSCells. The mouse neuroblastoma-rat glioma hybrid clone NG108-15 was used and was grown and maintained as described (18). Harvesting was accomplished by shaking the cells off the culture flask and centrifuging at 250-500 X g for 5 min at room temperat...

Inhibition of the adenylate cyclase activity in homogenates of mouse neuroblastoma-glioma hybrid cells (NG108-15) by the opioid peptide [-Ala2,Metsjenkephalin amide (AMEA) requires the presence of Na+ and GTP. In this process, the selectivity for monovalent cations is Na+ > Li+ > K+ > choline+; ITP will replace GTP but ATP, UTP, or CTP will not. The apparent Km for Na+ is 20 mM and for GTP it is I .&M. Under saturating Na+ and GTP conditions, the apparent Ki for AMEA-directed inhibition is 20 nM for basal and 100 nM for prostaglandin E1-activated adenylate cyclase activity. For both cyclase activities, maximal inhibition is only partial (i.e., -55% of control in each case). In intact viable NG108-15 cells, the decrease in basal and prostaglandin El-stimulated intracellular cyclic AMP concentrations by AMEA is also dependent upon extracellular Na+. Thee ephalin-directed reductions in cyclic AMP co riss are at least 75%. The specificity of the monovalent cation requirement for enkephalin action on intact cells is the same as for enkephalin regulation of homogenate adenylate cyclase activity. Based on these data, a model is presented in which the transfer of information from opiate receptors to adenylate cyclase requires active separate membrane components, which correspond to the sites of action of Na+ and GTP in this process. Opiates decrease the intracellular cyclic AMP (cAMP) concentrations of intact mouse neuroblastoma-rat glioma hybrid cells (NG108-15) by inhibiting the activity of the adenylate cyclase of these cells (1-3). This regulation of cAMP requires occupation of highly specific receptors by opiate agonists and is blocked by opiate antagonists. Recently, these opiate receptors were found to be sensitive to guanine nucleotides (4, 5) and Na+ (4-7). The effects of Na+ and nucleotides on NG108-15 opiate receptors parallel the effects of Na+ (8-14) and nucleotides (15, 16) on brain opiate receptors. Although nucleotides and Na+ have nonspecific effects on NG108-15 and brain opiate receptors, the actions of ions and nucleotides can be used to discriminate between agonist and antagonist interactions with opiate receptors.The selective nucleotide and ion regulation of agonist interactions with opiate receptors is reminiscent of the welldocumented guanine nucleotide regulation of agonist interaction with hormonal receptors that mediate activations of adenylate cyclase (17-20). In the latter case, agonist occupation of the receptor can take place without any nucleotides present; however, in order for the agonist-receptor complex to cause an increase in adenylate cyclase activity, the presence of a guanine nucleotide is required. Biochemical (21), genetic (22, 23), cell fusion (24, 25), and reconstitution (26-37) studies have shown that the receptor and catalytic units of adenylate cyclase are separate proteins and that another membrane component(s) (here termed the "coupler") is actually required for receptordirected activation of the catalytic unit.The published observations of Na+ and nucleotide sen...

Addition of the ionophore monensin to mouse neuroblastoma-rat glioma hybrid NG108-15 cells leads to a 20 to 30-mV increase in the electrical potential across the plasma membrane as shown by direct intracellular recording techniques and by distribution studies with the lipophilic cation 13H-tetraphenylphosphoniumt (TPP+) [Lichtshtein, D., Kaback, H. R. & Blume, A. J. (1979) Proc. NatL Acad. Sci. USA 76, 650-6541.The effect is not observed with cells suspended in high K medium, is dependent upon the presence of Na+ externally, and the concentration of monensin that induces half-maximal stimulation of TPP+ accumulation is approximately 1 pM. The ionophore also causes rapid influx of Na+, a transient increase in intracellular pH, and a decrease in extracellular pH, all of which are consistent with the known ability of monensin to catalyze the transmembrane exchange of H+ for Na+. Although ouabain has no immediate effect on the membrane potential, the cardiac glycoside completely blocks the increase in TPP+ accumulation observed in the presence of monensin. Thus, the hyperpolarizing effect of monensin is mediated apparently by an increase in intracellular Na+ that acts to stimulate the electrogenic activity of the Na+,K+-ATPase. Because monensin stimulates TPP± accumulation in a number of other cultured cell lines in addition to NG10815, the techniques described may be of general use for studying the Na+,K+ pump and its regulation' in situ.The Na+,K+-activated ATPase (Na+,K+'pump) (ATP phosphohydrolase, EC 3.6.1.3) is present in the membranes of excitable (1) and nonexcitable (2-4) tissues and represents a major pathway for Na+ and K+ transport across the plasma membrane of eukaryotic cells. Moreover, hydrolysis of ATP by this membranous enzyme is often accompanied by the simultaneous movement of three equivalents of Na+ out and two equivalents of K+ into the cell. This inequality in ion movements confers an electrogenic activity to the enzyme. That is, its activity results in the net outward movement of a positive current, which may lead to the generation of a membrane potential (AI, interior negative).Although the resting Az in nerve is due primarily to a K+ diffusion gradient ([K+Iin > [K+Iout), electrogenic Na+,K+-ATPase activity makes a contribution to A+I in certain cells (5, 6). Moreover, enhanced activity of the pump may have important consequences, because hyperpolarization will result. Presynaptically, this will lead to reduced transmitter release and postsynaptically, to decreased sensitivity to excitatory synaptic stimulation.Evidence has been presented indicating that the activity (7, 8) and coupling ratio-i.e., Na+ efflux/K+ influx (9, 10)-of the Na+,K+-ATPase are not constant, but are subject to regulation. In liver cells, for example, catecholamines, prostaglandin E1, and glucagon inhibit the Na+,K+ pump in a manner that is blocked by insulin, and these effects are related to changes in the intracellular concentration of cyclic AMP (11-13). In contrast, in frog skeletal muscle (14), rat s...

Gamma-aminobutyric acid (GABA) levels in the CSF were measured in 9 normal individuals, 17 drug-free schizophrenic patients and 10 of these same schizophrenic patients after neuroleptic treatment. There was no significant difference between CSF level of GABA in the control group compared to those in schizophrenic patients; however, 6 of the 7 lowest GABA levels were from schizophrenic patients. There was a significant decline of 12 per cent in mean GABA levels in the CSF after a mean of two months of neuroleptic treatment.

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