Glutamine uptake and metabolism to N-acetylaspartylglutamate (NAAG) by crayfish axons and glia.

We have proposed that N-acetylaspartylglutamate (NAAG) or its hydrolytic product glutamate, is a chemical signaling agent between axons and periaxonal glia at non-synaptic sites in crayfish nerves, and that glutamine is a probable precursor for replenishing the releasable pool of NAAG. We report here, that crayfish central nerve fibers synthesize NAAG from exogenous glutamine. Cellular accumulation of radiolabel during in vitro incubation of desheathed cephalothoracic nerve bundles with [3H]glutamine was 74% Na(+)-independent. The Na(+)-independent transport was temperature-sensitive, linear with time for at least 4 h, saturable between 2.5 and 10 mM L-glutamine, and blocked by neutral amino acids and analogs that inhibit mammalian glutamine transport. Radiolabeled glutamine was taken up and metabolized by both axons and glia to glutamate and NAAG, and a significant fraction of these products effluxed from the cells. Both the metabolism and release of radiolabeled glutamine was influenced by extracellular Na(+). The uptake and conversion of glutamine to glutamate and NAAG by axons provides a possible mechanism for recycling and formation of the axon-to-glia signaling agent(s).

Properties of glutaminase of crayfish CNS: implications for axon-glia signaling.

Glutaminase of crayfish axons is believed to participate in recycling of axon-glia signaling agent(s). We measured the activity and properties of glutaminase in crude homogenates of crayfish CNS, using ion exchange chromatography to separate radiolabeled product from substrate. Crayfish glutaminase activity is cytoplasmic and/or weakly bound to membranes and dependent on time, tissue protein, and glutamine concentration. It resembles the kidney-type phosphate-activated glutaminase of mammals in being stimulated by inorganic phosphate and alkaline pH and inhibited by the product glutamate and by the glutamine analog 6-diazo-5-oxo-L-norleucine. During incubation of crayfish CNS fibers in Na(+)-free saline containing radiolabeled glutamine, there is an increased formation of radiolabeled glutamate in axoplasm that is temporally associated with an increase in axonal pH from about 7.1 to about 8.0. Both the formation of glutamate and the change in pH are reduced by 6-diazo-5-oxo-L-norleucine. Our results suggest that crayfish glutaminase activity is regulated by cellular changes in pH and glutamate concentration. Such changes could impact availability of the axon-glia signaling agents glutamate and N-acetylaspartylglutamate.

Involvement of K+ channels in adenosine A2A and A2B receptor-mediated hyperpolarization of porcine coronary artery endothelial cells.

This study investigated the effects of the following adenosine agonists: 5;-ethylcarboxamidoadenosine (NECA), N6-cyclopentyadenosine (CPA) 2-[p-(2-carboxyethyl)]phenylamino-5;N-ethylcarboxamidoadenosine (CGS-21680), and 2-chloroadenosine (CAD) and its antagonist, 4-(2-[7-amino-2-[2-furyl]]1,2,4-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385), a selective A2A adenosine receptor antagonist, and the involvement of the K+ATP and KCa channels on the resting membrane potential (RMP) of confluent monolayers of cultured porcine coronary artery endothelial cells (PCAECs). Adenosine agonists and K+ATP channel openers (pinacidil, cromakalim) hyperpolarized cultured PCAECs. The average RMP was -32.31 +/- 1.2 mV. Adenosine agonists at 10-5 M caused a significant increase in RMP to -65.0 +/- 1.5 mV for CAD (a nonselective adenosine receptor agonist) to -75.9 +/- 1.6 mV for CGS-21680 (a selective A2A receptor agonist) and to -87.0 +/- 3.5 mV for NECA (a nonselective A1/A2A/A2B receptor agonist). Pinacidil and cromakalim at 10 microM increased the membrane potential to -76.2 +/- 1.2 mV and -75.22 +/- 0.12 mV, respectively. The hyperpolarization induced by adenosine receptor agonists and KATP openers was inhibited by an application of the K+ATP channel blocker glibenclamide (10 microM), indicating the involvement of the K+ATP channel in the adenosine-mediated hyperpolarization of PCAECs. Moreover, 1-EB10, a selective opener of the maxi-KCa channel, hyperpolarized PCAECs, and the effect of 1-EB10 was completely blocked by a selective, irreversible blocker of the high conductance KCa (maxi-K) channels (penitrem A), but it only partially blocked the effect of NECA. ZM-241385 has no effect on hyperpolarization elicited by K+ATP and KCa channel openers. However, ZM-241385 significantly blocked the hyperpolarization effect of CAD and CGS-21680. ZM-241385 partially blocked the hyperpolarizing effect of NECA, and a combination of ZM-241385 and penitrem A further blocked the hyperpolarizing effect of NECA. These results further support the involvement of K+ channels in adenosine A2A and A2B receptor-mediated hyperpolarization of PCAECs.

Mechanisms for clearance of released N-acetylaspartylglutamate in crayfish nerve fibers: implications for axon-glia signaling.

Crayfish nerve fibers incubated with radiolabeled glutamate or glutamine accumulate these substrates and synthesize radioactive N-acetylaspartylglutamate (NAAG). Upon stimulation of the medial giant nerve fiber, NAAG is the primary radioactive metabolite released. Since NAAG activates a glial hyperpolarization comparable to that initiated by glutamate or axonal stimulation through the same receptor, we have proposed that it is the likely mediator of interactions between the medial giant axon and its periaxonal glia. This manuscript reports investigations of possible mechanisms for termination of NAAG-signaling activity. N-acetylaspartyl-[(3)H]glutamate was not accumulated from the bath saline by unstimulated crayfish giant axons or their associated glia during a 30-min incubation. Stimulation of the central nerve cord at 50 Hz during the last minute of the incubation dramatically increased the levels of radiolabeled glutamate, NAAG, and glutamine in the medial giant axon and its associated glia. These results indicate that stimulation-sensitive peptide hydrolysis and metabolic recycling of the radiolabeled glutamate occurred. There was a beta-NAAG-, quisqualate- and 2-(phosphonomethyl)-pentanedioic acid-inhibitable glutamate carboxypeptidase II activity in the membrane fraction of central nerve fibers, but not in axonal or glial cytoplasmic fractions. Inactivation of this enzyme by 2-(phosphonomethyl)-pentanedioic acid or inhibition of N-methyl-D-aspartate (NMDA) receptors by MK801 reduced the glial hyperpolarization activated by high-frequency stimulation. These results indicate that axon-to-glia signaling is terminated by NAAG hydrolysis and that the glutamate formed contributes to the glial electrical response in part via activation of NMDA receptors. Both NAAG release and an increase in glutamate carboxypeptidase II activity appear to be induced by nerve stimulation.

N-acetylaspartylglutamate (NAAG) is the probable mediator of axon-to-glia signaling in the crayfish medial giant nerve fiber.

Glial cell hyperpolarization previously has been reported to be induced by high frequency stimulation or glutamate. We now report that it also is produced by the glutamate-containing dipeptide N-acetylaspartylglutamate (NAAG), by its non-hydrolyzable analog beta-NAAG, and by NAAG in the presence of 2-(phosphonomethyl)-pentanedioic acid (2-PMPA), a potent inhibitor of the NAAG degradative enzyme glutamate carboxypeptidase II. The results indicate that NAAG mimics the effect of nerve fiber stimulation on the glia. Although glutamate has a similar effect, the other presumed product of NAAG hydrolysis, N-acetylaspartate, is without effect on glial cell membrane potential, as is aspartylglutamate (in the presence of 2-PMPA). The hyperpolarization induced by stimulation, glutamate, NAAG, beta-NAAG, or NAAG plus 2-PMPA is completely blocked by the Group II metabotropic glutamate receptor antagonist (S)-alpha-ethylglutamate but is not altered by antagonists of Group I or III metabotropic glutamate receptors. The N-methyl-D-aspartate receptor antagonist MK801 reduces but does not eliminate the hyperpolarization generated by glutamate, NAAG or stimulation. These results, in combination with those of the preceding paper, are consistent with the premise that NAAG could be the primary axon-to-glia signaling agent. When the unstimulated nerve fiber is treated with cysteate, a glutamate reuptake blocker, there is a small hyperpolarization of the glial cell that can be substantially reduced by pretreatment with 2-PMPA before addition of cysteate. A similar effect of cysteate is seen during a 50 Hz/5 s stimulation. From these results we suggest that glutamate derived from NAAG hydrolysis appears in the periaxonal space under the conditions of these experiments and may contribute to the glial hyperpolarization.

Synthesis and release of N-acetylaspartylglutamate (NAAG) by crayfish nerve fibers: implications for axon-glia signaling.

Early physiological and pharmacological studies of crayfish and squid giant nerve fibers suggested that glutamate released from the axon during action potential generation initiates metabolic and electrical responses of periaxonal glia. However, more recent investigations in our laboratories suggest that N-acetylaspartylglutamate (NAAG) may be the released agent active at the glial cell membrane. The investigation described in this paper focused on NAAG metabolism and release, and its contribution to the appearance of glutamate extracellularly. Axoplasm and periaxonal glial cell cytoplasm collected from medial giant nerve fibers (MGNFs) incubated with radiolabeled L-glutamate contained radiolabeled glutamate, glutamine, NAAG, aspartate, and GABA. Total radiolabel release was not altered by electrical stimulation of nerve cord loaded with [(14)C]glutamate by bath application or loaded with [(14)C]glutamate, [(3)H]-D-aspartate or [(3)H]NAAG by axonal injection. However, when radiolabeled glutamate was used for bath loading, radiolabel distribution among glutamate and its metabolic products in the superfusate was changed by stimulation. NAAG was the largest fraction, accounting for approximately 50% of the total recovered radiolabel in control conditions. The stimulated increase in radioactive NAAG in the superfusate coincided with its virtual clearance from the medial giant axon (MGA). A small, stimulation-induced increase in radiolabeled glutamate in the superfusate was detected only when a glutamate uptake inhibitor was present. The increase in [(3)H]glutamate in the superfusion solution of nerve incubated with [(3)H]NAAG was reduced when beta-NAAG, a competitive glutamate carboxypeptidase II (GCP II) inhibitor, was present.Overall, these results suggest that glutamate is metabolized to NAAG in the giant axon and its periaxonal glia and that, upon stimulation, NAAG is released from the axon and converted in part to glutamate by GCP II. A quisqualate- and beta-NAAG-sensitive GCP II activity was detected in nerve cord homogenates. These results, together with those in the accompanying paper demonstrating that NAAG can activate a glial electrophysiological response comparable to that initiated by glutamate, implicate NAAG as a probable mediator of interactions between the MGA and its periaxonal glia.

[Synthesis and release of N-acetylaspartyl glutamate (NAAG) in medial giant axons in crayfish]. / Sintez i osvobozhdenie N-acetylaspartylglutamate (NAAG) v sredinnykh gigantskikh aksonakh raka.

Studies of crayfish Medial Giant nerve Fiber suggested that glutamate (GLU) released from the axon during action potential generation initiates metabolic and electrical responses of periaxonal glia. This investigation sought to elucidate the mechanism of GLU appearance extracellularly following axon stimulation. Axoplasm and periaxonal glial sheath from nerve fibers incubated with radiolabelled L-GLU contained radiolabeled GLU, glutamine (GLN), GABA, aspartate (ASP), and NAAG. Total radiolabel release was not altered by electrical stimulation of nerve cord loaded with [14C]-GLU by bath application or loaded with [14C]-GLU, [3H]-D-ASP, or [3H]-NAAG by axonal injection. However, radioactivity distribution among GLU and its metabolic products in the superfusate was changed, with NAAG accounting for the largest fraction. In axons incubated with radiolabeled GLU, the stimulated increase in radioactive NAAG in the superfusate coincided with the virtual clearance of radioactive NAAG from the axon. The increase in [3H]-GLU in the superfusion solution that was seen upon stimulation of nerve bathloaded with [3H]-NAAG was reduced when beta-NAAG, a competitive NAALADase inhibitor, was present. Together, these results suggest that some GLU is metabolized to NAAG in the giant axon and its periaxonal glia and that, upon stimulation, NAAG is released and converted to GLU by NAALADase. A quisqualate-, beta-NAAG-sensitive NAALADase activity was detected in nerve cord homogenates. Stimulation or NAAG administration in the presence of NAALADase inhibitor caused a transient hyperpolarization of the periaxonal glia comparable to that produced by L-GLU. The results implicate N-acetylaspartylglutamate (NAAG) and GLU as potential mediators. of the axon-glia interactions.

Uptake and metabolism of glutamate at non-synaptic regions of crayfish central nerve fibers: implications for axon-glia signaling.

In crayfish and squid giant nerve fibers, glutamate appears to be an axon-glia signaling agent. We have investigated glutamate transport and metabolism by crayfish central nerve fibers in order to identify possible mechanisms by which glutamate could subserve this non-synaptic signaling function. Accumulation of radiolabeled L-glutamate by desheathed cephalothoracic nerve bundles was temperature and Na(+) dependent, linear with time for at least 8h and saturable at about 0.5-1mM L-glutamate. Most accumulated radiotracer was associated with the periaxonal glial sheath and remained as glutamate. Compounds known to block glutamate transport in invertebrate peripheral nerves or mammalian brain slices or cell cultures were also effective on crayfish central nerve fibers. Tissue radiotracer levels were only 3% of control levels when 1mM p-chloromercuriphenylsulfonate was present, and 13%, 20%, 26%, 38% and 42% of control levels, respectively, when L-cysteate, L-cysteine sulfinate, L-aspartate, D-aspartate or DL-threo-beta-hydroxyaspartate was present. L-Glutamine, GABA, N-methyl-DL-aspartate, alpha-aminoadipate and D-glutamate were without inhibitory effect on tissue tracer accumulation. Radiolabeled D-aspartate was an equivalent non-metabolized substitute for radiolabeled L-glutamate. D-Aspartate, p-chloromercuriphenylsulfonate and GABA had comparable effects on isolated medial giant nerve fibers.These studies indicate that L-glutamate is taken up primarily by the periaxonal glia of crayfish central nerve fibers by a low-affinity, saturable, Na(+)-dependent transport system and is retained by the fibers primarily in that form. Our results suggest that the glia are not only the target of the glutamate signal released from non-synaptic regions of the crayfish medial giant axon during high-frequency stimulation, but that they are also the primary site of its inactivation.

Acute and chronic regulation of Na+/K+-ATPase transport activity in the RN22 Schwann cell line in response to stimulation of cyclic AMP production.

Na+/K+-ATPase-dependent Rb+ uptake of RN22 Schwann cells was stimulated by cholera toxin (0.25 microg/ml), forskolin (2 mM), or 8-bromo cAMP (1 mM). At 2 h Rb+ uptake was increased by 162+/-6% (cholera toxin), 151+/-14% (forskolin), and 207+/-15% (8-bromo cAMP). Cholera toxin or 8-bromo cAMP treatment for 12-24 h resulted in a second peak of Na+/K+-ATPase-dependent Rb+ transport activity of 186+/-12 and 265+/-9% of control, respectively. Cholera toxin also transiently stimulated the activity of the Na+, K+, 2Cl- -cotransporter with a peak at 2 h (179+/-9%), returning to basal levels by 24 h. Inhibition of the Na+,K+,2Cl- -cotransporter by bumetanide (0.1 mM) or by reduction of the Na+ gradient (10 mM veratridine treatment) prevented the early peak in ATPase activity but not the second peak. These results indicated that the early transient stimulation of Na+/K+ ATPase activity by cholera toxin was due to an increase in cellular Na+, secondary to stimulation of Na+,K+,2Cl -cotransport activity. Western blot analysis of cellular homogenates and purified membrane fractions showed that the second peak of Rb+ uptake activity was a result of translocation of transport protein from an intracellular microsomal pool to the plasma membrane. Rb+ uptake by dominant negative protein kinase A mutants of the RN22 cell was not stimulated by cholera toxin treatment (acute or chronic) confirming the cAMP/protein kinase A dependency of both acute and long-term regulation of transport activity. In the absence of a change in Michaelis constants or of an increase in total transport protein of cellular homogenates, neither a change in enzyme kinetics nor an increase in de novo synthesis of transport protein could account for the increase in transport activity.

Effect of ouabain on adenosine receptor-mediated hyperpolarization in porcine coronary artery smooth muscle.

We investigated the effect of inhibitors of endothelium-derived nitric oxide and sodium-potassium (Na(+)-K+) pumps on adenosine receptor-mediated hyperpolarization of porcine coronary artery smooth muscle with and without endothelium. The average resting membrane potential (RMP) in porcine coronary artery smooth muscle was -51.5 +/- 0.2 and -50.7 +/- 0.2 mV, in the presence and absence of endothelium, respectively. Neither ouabain, N-nitro-L-arginine methyl ester (L-NAME) nor ouabain and L-NAME in combination significantly affected the resting membrane potential in the absence of vasodilator agonists. Adenosine agonists, 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine at 10(-5) M caused a significant increase in RMP with intact endothelium and caused a smaller but significant increase in RMP in the absence of endothelium. Ouabain (10(-5) M) in the absence of L-NAME significantly reduced hyperpolarization due to 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine in the presence of endothelium. However, in the absence of endothelium, its inhibitory effect was not significant. When ouabain plus L-NAME (10(-5) M) were given simultaneously, the hyperpolarization caused by adenosine agonists was significantly further attenuated nearly to the RMP level. Attenuation of the response to 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine by ouabain was not reversed by the nitric oxide precursor, L-arginine (10(-5) M) both in the presence and absence of endothelium. These results suggest that hyperpolarization of vascular smooth muscle of the porcine coronary artery by adenosine agonists is at least partly endothelium dependent and possibly involves the Na(+)-K+ pump and the release of nitric oxide.

Glutamine cycle enzymes in the crayfish giant nerve fiber: implications for axon-to-glia signaling.

Two of the key enzymes involved in glutamate metabolism, glutaminase and glutamine synthetase, were quantitatively localized to axons and glia of the crayfish giant nerve fiber by immunocytochemistry and electron microscopy of antibody-linked gold microspheres. In Western blots, rabbit antisera for glutamine synthetase and glutaminase specifically recognized crayfish polypeptides corresponding approximately in size to subunits of purified mammalian brain enzymes. Glutamine synthetase immunoreactivity was found to be 11 times greater in the adaxonal glial cells than in the axon. Glutaminase immunoreactivity was found in somewhat greater concentration (2.5:1) in glia as compared to axoplasm. Glutamate immunoreactivity also was evaluated and found to be present in high concentration in both glia and axons, as might be expected for an important substrate of cellular metabolism. Using radiolabeled substrates it was demonstrated that glutamine and glutamate were interconverted by the native enzymes in the intact crayfish giant nerve fiber and that the formation of glutamine from glutamate occurred in the axoplasm-free nerve fiber, the cellular component of which is primarily periaxonal glia. The results of this investigation provide immunocytochemical and metabolic evidence consistent with an intercellular glutamine cycle that modulates the concentration of periaxonal glutamate and glutamine in a manner similar to that described for perisynaptic regions of the vertebrate central nervous system. These findings further corroborate previous electrophysiological evidence that glutamate serves as the axon-to-glial cell neurochemical signal that activates glial cell mechanisms responsible for periaxonal ion homeostasis.

Intracellular pH regulation in rat Schwann cells.

We examined H+ and HCO3- transport mechanisms that are involved in the regulation of intracellular pH of Schwann cells. Primary cultures of Schwann cells were prepared from the sciatic nerves of 1-3-day-old rats. pHi of single cells attached to cover slips was continuously monitored by measuring the absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein incorporated intracellularly. The average pHi of neonatal Schwann cells bathed in HEPES mammalian solution was 7.17 +/- 0.02 (n = 32). In the nominal absence of HCO3-, pHi spontaneously recovered from an acute acid load induced by exposing the Schwann cells to 20 mM NH4+ (NH4+ prepulse). This pHi recovery from the acute acid load was totally inhibited in the absence of external Na+ or in the presence of 1 mM amiloride. In both cases, the pHi recovery was readily restored upon readdition of external Na+ or removal of amiloride. In the steady-state, addition of amiloride caused a small and slow decrease in pHi which was readily reversed upon removal of amiloride. In the presence of HCO3-, removal of external Cl- caused pHi to rapidly and reversibly increase by 0.23 +/- 0.03 (n = 15) and the initial rate of alkalinization was 20.6 +/- 2.7 x 10(-4) pH/sec. In the absence of external Na+, removal of bath Cl- still caused pHi to increase by 0.15 +/- 0.02 and the initial rate of pHi increase was not significantly altered. In the nominal absence of HCO3-, removal of bath Cl- caused pHi to increase very slightly (0.05 +/- 0.01) with an initial dpHi/dt of only 4.4 +/- 0.2 x 10(-4) pH/sec (n = 4). Addition of 100 microM DIDS did not inhibit the pHi increase caused by removal of bath Cl-. These data indicate that 1) Rat Schwann cells regulate their pHi via an Na-H exchange mechanism which is moderately active at steady-state pHi. 2) In the presence of HCO3-, there is a Na-independent Cl-HCO3 (base) exchanger which also contributes to regulation of intracellular pH in Schwann cells.

Mechanisms of glutamate activation of axon-to-Schwann cell signaling in the squid.

Membrane potentials from Schwann cells associated with giant axons of the small squid (Alloteuthis and Loliguncula) and the large squid (Loligo) were monitored with glass microelectrodes following 100 Hz/15 s axonal stimulation, or the application of 10(-7) M glutamate and ion substitutions, in the presence or absence of 10(-7) M d-tubocurarine. Glutamate or stimulation caused the membrane of the Schwann cell to depolarize to approximately -32 mV. This was rapidly replaced by a transient hyperpolarization to approximately -55 mV; the potential returning to the resting level (-40 mV) in approximately 7 min. In the presence of d-tubocurarine only the initial depolarization was evident. Nominally zero [Na+]o or treatment with 10(-7) M tetrodotoxin (in normal [Na+]o) blocked the stimulation- and glutamate-induced depolarization while low Clo- hyperpolarized the Schwann cell without effect on the glutamate- or stimulation-induced depolarization. Nao+ depletion or pretreatment with tetrodotoxin in normal Nao+ did not affect the development of the Schwann cell hyperpolarization. These results do not support the hypothesis that the glutamate-induced depolarization is the trigger leading to the Schwann cell hyperpolarization. Preliminary experiments to test the possibility that inositol phosphate second messenger and an increase in [Ca2+]i are triggered by glutamate receptor activation showed that nominally 0 Cao2+/75 mM Mgo2+ only slightly reduced the hyperpolarizing response to stimulation or glutamate while intracellular Bapta (20-30 microM) blocked the hyperpolarization but not the depolarization. [3H]Myoinositol incorporation into axon-Schwann cell plasma membranes was high.(ABSTRACT TRUNCATED AT 250 WORDS)

Axon-glia interactions in the crayfish: glial cell oxygen consumption is tightly coupled to axon metabolism.

Oxygen consumption (QO2) of single isolated axons and their associated glial cell sheath was investigated under a variety of conditions to determine the contribution of each cell type to whole tissue QO2. It was found that the QO2 of the sheath, in the absence of a functional axon, represented approximately 30% of the total tissue QO2. When the axon was injected with carboxyatractyloside, an inhibitor of mitochondrial oxidative phosphorylation that is membrane impermeant, electrophysiological properties of the axon were not affected and glial sheath respiratory activity was stimulated by 1.7 to 2.7 times the untreated control level. These results suggest that glial cell metabolic activity is regulated by the metabolic activity of the axon. Depending on the experimental conditions the glial sheath accounts for 30% to nearly 100% of the QO2 of axon-glial cell tissue. On the basis of these and morphometric measurements we estimate that in a normally functioning axon-glial cell system the glial sheath accounts for 90% of the tissue QO2.

Determination of the membrane potential of cultured mammalian Schwann cells and its sensitivity to potassium using a thiocarbocyanine fluorescent dye.

The membrane potential of cultured rat sciatic nerve Schwann cells was determined with conventional microelectrode and voltage-sensitive fluorescent dye, Di-S-C3(5), optical techniques. The value for membrane potential obtained with microelectrodes was -42.1 +/- 4.7 mV (n = 8). Using optically determined fluorescent intensity changes caused by changes in external potassium ion concentration, in the presence or absence of valinomycin (null point method), the membrane potential was estimated at -45.7 +/- 6.2 mV (n = 7); with a gramicidin and valinomycin double ionophore method it was -52.2 +/- 9.1 (n = 4). The membrane potential of Schwann cells was found to be potassium sensitive at and above the physiological range of [K+] at 27.5 mV/10x delta[K+], which is approximately half the Nernstian value. This result suggests that other ion permeabilities strongly influence the resting membrane potential of cultured Schwann cells. Since Na+ had little effect on the membrane potential, it is concluded that Cl- is a likely candidate for the other permeant ionic species. The optical method has been shown to be a useful tool for the systematic study of the membrane potential of Schwann cells in culture and for the characterization of its ionic basis and regulation.

Effect of extracellular potassium on ouabain-sensitive consumption of high-energy phosphate by crayfish giant axons: a study of the energy requirement for transport in the steady state.

Crayfish axons exposed to a high or low extracellular K+ concentration ([K+]o) maintain intracellular Na+ and K+ concentrations constant, for up to 3 h, by adjusting both the Na+/K+ transport "coupling ratio" and turnover rate in compensation for changes in ion fluxes due to altered electrochemical gradients. These findings give rise to the prediction that the steady-state consumption of high-energy phosphate (approximately P) [ATP and phospho-L-arginine (Arg-P)] is inversely proportional to the [K+]o, i.e., directly proportional to the product of membrane conductance and magnitude of the transmembrane electrochemical gradients for Na+ and K+. This investigation was designed to test this hypothesis. The [K+]o did not influence total approximately P consumption (Q approximately P) of the axon. For a [K+]o between 0.5 and 21.6 mM, Q approximately P averaged 52.8 +/- 4.7%/h (n = 44) of the initial [ATP] + [Arg-P]. Unlike total Q approximately P, the ouabain-sensitive portion of Q approximately P was markedly influenced by [K+]o. In 0.5 mM K+o, ouabain poisoning reduced Q approximately P to 8%/h, a result indicating that 85% of the total Q approximately P was ouabain sensitive. For 1.35 mM K+o, the ouabain-sensitive portion was 66%; at 5.4 mM K+o, 45%; and at 13.5 mM K+o, 41%. There was a small but significant increase in the ouabain-sensitive Q approximately P at 21.6 mM K+o, compared with Q approximately P at 5.4 mM K+o. The pattern of effect of [K+]o on Q approximately P was similar to its effect on the electrical power content of the Na+ and K+ electrochemical gradients. In contrast to the generally accepted Na+ flux (JNa)/approximately P stoichiometry of 3, an actual ratio of JNa/approximately P stoichiometry of approximately 33:1 was calculated for the experiments reported here, a result suggesting that cells in a zero-membrane current steady state utilize efficient energy conservation mechanisms that may not operate under non-steady-state conditions.

High potassium selective permeability and extracellular ion regulation in the glial perineurium (blood-brain barrier) of the crayfish.

Selective ion permeability, ion transport properties, and electrical resistance of the perineurial barrier, as they relate to interstitial ion regulation, where studied and characterized electrophysiologically in ion substitution experiments. In high external [K+] a transient spike-like voltage was generated across the perineurial barrier which fell over 1-2 min to a slowly decaying voltage. The glial perineurium had at least a 10 times greater permeability to K+ than Cl-, and was effectively impermeant to Na+. The potential, in high external [K+], was determined by the K+ and Cl- gradients and their relative permeabilities across the sheath. For other cations the selectivity sequence of the perineurial barrier, as determined from electrophysiological measurements, was K+ greater than or equal to Rb+ much greater than NH4+ greater than Cs+ greater than Li+ greater than Na+ corresponding most closely to the Eisenman sequence IV. The perineurium had a resistance of 260 +/- 23 omega cm2 in crayfish physiological solution. In high [K+]0 the resistance fell by over half during the transient spike potential and then recovered towards resting levels as the voltage decayed. In the intact nerve cord interstitial [K+] rose to only 10-20 mM during a 2-min exposure to 100 mM K0+. K influx and efflux were related to the change in barrier permeability and an increased selectivity to K+ which, in these studies, was determined primarily by its electrochemical gradient across the perineurial barrier. The results suggest that the crayfish perineurium is a leaky epithelium capable of a high degree of ion regulation. Trans-perineurial barrier potential and conductance in high external [K+] are primarily functions of passive processes of the perineurial glial cell membranes and of the paracellular conductance channels driven by the electrochemical gradient for the K+. Accordingly, the mass transport of [K+] showed the same quantitative relationship in both directions.