Biomedical subjects

L Hertz

Publications and source records attributed to L Hertz.

At least 145 records · Page 8Linked to original sources

PubMed1985

Functional role of the potassium-induced stimulation of oxygen uptake in brain slices studied with cesium as a probe.

The potassium-induced stimulation of oxygen consumption in brain slices has a threshold value of 15-20 mM potassium, and it reaches its maximum at 35-50 mM. Although this phenomenon now has been known for almost 50 years, its physiological role remains undetermined. One reason for this may be that the high concentrations of potassium that are required for this response also have many other consequences, e.g., a depolarization of the cells, and that the different effects to some extent may mask each other. For this reason this investigation studied the effects of cesium, which evokes a maximal stimulation of oxygen consumption already at 15 mM. Like potassium, concentrations of cesium that stimulate oxygen consumption also lead to an enhanced swelling. Unlike potassium, the sodium content is affected very little by these concentrations of cesium, whereas cesium and chloride contents are increased. On this basis it is concluded that the cesium-induced stimulation of oxygen uptake is a metabolic manifestation of an active uptake of cesium and chloride, which secondarily leads to an uptake of water, i.e., the cesium-induced swelling. Analogously, it is suggested that the potassium-induced stimulation of oxygen uptake represents an active accumulation of potassium and chloride.

Animals↗

PubMed1985

Developmental change of endogenous glutamate and gamma-glutamyl transferase in cultured cerebral cortical interneurons and cerebellar granule cells, and in mouse cerebral cortex and cerebellum in vivo.

The developmental change of endogenous glutamate, as correlated to that of gamma-glutamyl transferase and other glutamate metabolizing enzymes such as phosphate activated glutaminase, glutamate dehydrogenase and aspartate, GABA and ornithine aminotransferases, has been investigated in cultured cerebral cortex interneurons and cerebellar granule cells. These cells are considered to be GABAergic and glutamatergic, respectively. Similar studies have also been performed in cerebral cortex and cerebellum in vivo. The developmental profiles of endogenous glutamate in cultured cerebral cortex interneurons and cerebellar granule cells corresponded rather closely with that of gamma-glutamyl transferase and not with other glutamate metabolizing enzymes. In cerebral cortex and cerebellum in vivo the developmental profiles of endogenous glutamate, gamma-glutamyl transferase and phosphate activated glutaminase corresponded with each other during the first 14 days in cerebellum, but this correspondence was less good in cerebral cortex. During the time period from 14 to 28 days post partum the endogenous glutamate concentration showed no close correspondence with any particular enzyme. It is suggested that gamma-glutamyltransferase regulates the endogenous glutamate concentration in cultured neurons. The enzyme may also be important for regulation of endogenous glutamate in brain in vivo and particularly in cerebellum during the first 14 days post partum. Gamma-glutamyl transferase in cultured neurons and brain tissue in vivo appears to be devoid of maleate activated glutaminase.

Age Factors↗

PubMed1985

Ontogenetic development of glutamate metabolizing enzymes in cultured cerebellar granule cells and in cerebellum in vivo.

The ontogenetic development of the enzymes phosphate activated glutaminase (PAG), glutamate dehydrogenase (GLDH), glutamic-oxaloacetic-transaminase (GOT), glutamine synthetase (GS), and ornithine-delta-aminotransferase (Orn-T) was followed in cerebellum in vivo and in cultured cerebellar granule cells. It was found that PAG, GLDH, and GOT exhibited similar developmental patterns in the cultured neurons compared to cerebellum. PAG showed, however, a more pronounced phosphate activation in the cultured granule cells compared to in vivo. The activity of GS remained low in the cultured neurons compared to the increasing activity of this enzyme found in vivo. On the other hand Orn-T exhibited an increase in its specific activity in the cultured cells as a function of time in culture in contrast to the non-changing activity of this enzyme in vivo. Compared to cerebellum the cultured neurons exhibited higher activities of GLDH, GOT, and Orn-T whereas the activity of PAG was only slightly higher in the cultured cells. The activity of GS in the cultured neurons was only 5-10% of the activity in cerebellum in vivo. It is concluded that cultured cerebellar granule cells represent a reliable model system by which the metabolism and function of glutamatergic neurons can be conveniently studied in a physiologically meaningful way.

Animals↗

PubMed1985

Plasticity of astrocytes in primary cultures: an experimental tool and a reason for methodological caution.

Astrocytes in primary cultures constitute an exceedingly useful preparation for studies of astroglial development and function. These cells, however, demonstrate a pronounced plasticity in their reactions to culturing conditions. Thus, species and spatiotemporal region of CNS chosen for source of cells, dissociation procedures used, cell density in culture, culture medium chosen, type and/or concentration of serum used (if any) and exposure to dibutyryl cyclic AMP (dBcAMP) may all markedly affect the epigenotype of the cells. This provides an experimental tool for studies of astroglial development and function, but is at the same time a reason for caution in the interpretation of data when such cultured cells are used as models of their in vivo counterparts. About 95% of the cells in cultures used by the authors are positive for astrocyte-specific markers (glial fibrillary acidic protein and glutamine synthetase), and the cells possess presumably astrocytic characteristics such as high potassium permeability. This latter characteristic may be drastically altered by minor changes in culturing conditions. It is likely that an overwhelming majority of the astrocytes in such cultures are protoplasmic in nature. Addition of dBcAMP to the culture medium results in a pronounced morphological and a more modest functional differentiation.

Animals↗

PubMed1984

Astrocytes in primary cultures: membrane potential characteristics reveal exclusive potassium conductance and potassium accumulator properties.

Mouse astrocytes in homogenous primary cultures were used to study the membrane potential response, measured with intracellular microelectrodes, to alterations of external ion composition and to certain drugs, and the observations were correlated with radiotracer measurements of equilibrated K+ content. The membrane potential was--92 mV at 3 mM K+, and reduction of external Na+ and Cl-concentration, as well as addition of furosemide, which acts on Cl- and Na+ fluxes, had no effect, showing that there is no other ion than K+ which significantly contributes to membrane conductance. Addition of ouabain showed that there is no electrogenic component of the membrane potential at either 'resting' conditions or during stimulation. Nevertheless, the behavior of the membrane potential was found to deviate from a Nernst potential for K+ when extracellular K+ was changed (the slope was linear from 1.5 to 100 mM K+ with 51 mV/10-fold change compared with 61 mV for a Nernstian behavior at 37 degrees C). The radioisotope measurements at different external K+ showed that this was due to accumulation of intracellular K+. We conclude that these astrocytes have spatial buffer and active accumulator properties towards the K+ ion.

Animals↗

PubMed1984

Sodium transport in astrocytes.

Sodium transport in astrocytes in homogeneous primary cultures from mouse brain cortex were investigated with radiotracer (22Na) and electrophysiological methods. The equilibrated Na+ content was 190 nmol X mg-1 protein and the influx and efflux rates were identical at about 560 nmol X mg-1 X min-1. No significant change was observed in Na+ efflux or influx when external K+ was raised from 5.4 to 12 or 54 mM, but the Na+ content decreased. Intracellular Na+ loading, evoked by previous exposure to ice-cold K+-free medium, double the Na+ efflux. Ouabain, a Na+-K+ exchange inhibitor, exerted a small, nonsignificant inhibition of Na+ efflux at both 5.4 and 12 mM K+ and caused a large increase in Na+ content. At 5.4 mM K+, amiloride, a Na+-H+ exchange inhibitor, decreased both influx and efflux of Na+ and caused an increase in Na+ content. Furosemide, an inhibitor of a cation-Cl- carrier, decreased both content and influx of Na+ slightly but had no significant effect on Na+ efflux. The effects of amiloride or furosemide on Na+ influx were abolished at elevated (12 and 54 mM) K+. Attempts to stimulate the Na+-K+ pump with elevated external K+ or internal Na+ produced no electrogenic component of the membrane potential, probably owing to the high K+ permeability. Based on the present results and earlier experiments on K+ influx, it is concluded that 1) the Na+-K+ pump of astrocytes under normal conditions transports more K+ than Na+; 2) intracellular Na+ loading increases Na+ efflux; 3) some Na+-H+ exchange and cotransport of Na+ and Cl- seem to occur at 5.4 mM K+; and 4) neither of the latter two transport mechanisms is enhanced at elevated K+ concentrations.

Animals↗

PubMed1984

Metabolic fate of [14C]-glutamine in mouse cerebral neurons in primary cultures.

The metabolic fate of L-[14C]-glutamine was followed in cerebral cortical neurons in primary cultures, a GABAergic preparation. Part of the glutamine was converted to GABA (0.3 nmol/min per mg protein), which is consistent with the presence of glutaminase and glutamate decarboxylase activity in the cells and with findings by other authors in vivo or in brain slices. However, an even larger part (1.8 nmol/min per mg protein) was converted to CO2 and succinate via an oxidative deamination to alpha-ketoglutarate. This is not consistent with the concept that transfer of glutamine from astrocytes to neurons should replenish neuronal GABA stores quantitatively after release of GABA and its partial accumulation into astrocytes, but it is well compatible with the recent demonstration of a net glutamine uptake by the brain.

Absorption↗

PubMed1984

Receptor expression in primary cultures of neurons or astrocytes.

beta-Adrenergic receptors are abundant on astrocytes which contain mainly the beta 1 subtype. These cells also possess alpha 1 and alpha 2 receptors. Neurons display fewer beta-adrenergic receptor sites and those present are of the beta 2 subtype. Dopaminergic receptors are found on astrocytes and show a regional variation. Serotonergic receptors have been demonstrated on astrocytes but are expressed to a larger extent in neurons. Astrocytes display muscarinic receptors but have no receptor sites for the transmitter amino acids glutamate and GABA. GABA receptors are present on cerebellar granule cells, are induced by the presence of GABA agonists in the medium and mediate an evoked release of glutamate. Glutamate receptors are found on cultured cortical interneurons. Some, but not all, peptide hormones have receptors on astrocytes and/or neurons in primary cultures.

Animals↗

PubMed1984

Barium-induced inhibition of K+ transport mechanisms in cortical astrocytes--its possible contribution to the large Ba2+-evoked extracellular K+ signal in brain.

Homogenous mouse astrocytes in primary cultures were used to investigate the action of different Ba2+ concentrations on 42K transport, membrane potential and Na+,K+-adenosine triphosphatase activity. Five millimolar Ba2+ reduced total K+ influx and efflux (each by 83%) and ouabain-sensitive net K+ uptake (by 80%); it decreased the K+ content, depolarized the membrane potential reversibly and completely inhibited the Na+,K+-adenosine triphosphatase activity. The concentration dependence of these effects was biphasic. Concentrations between 2 and 20 microM affected only the passive K+ fluxes (IC50: 6 microM). Concentrations between 50 microM and 5 mM inhibited the Na+,K+-adenosine triphosphatase and had no further effect on passive fluxes, but inhibited the ouabain-sensitive net uptake of K+ (IC50: 3.1-0.6 mM). It is suggested that the large evoked extracellular K+ increase in the brain observed in Ba2+-treated preparations in vivo or in brain slices to a large extent is due to the impairment of passive and active K+ clearance by glial cells.

Animals↗

PubMed1984

Intense furosemide-sensitive potassium accumulation in astrocytes in the presence of pathologically high extracellular potassium levels.

An intense K+ accumulation in primary cultures of astrocytes, occurring when external K+ was increased from 5.4 to 54 mM, was investigated. This increase resulted in a doubling of the K+ content within 10 s. Thirty percent of the accumulation was inhibited by furosemide (2 mM). This drug had no effect on the unidirectional influx of K+ at 5.4 mM K+, but when the extracellular K+ concentration was increased, there appeared to be a furosemide-sensitive component of the influx. This component increased with increasing external K+ levels, reaching 44% of the total influx at 72 mM. These results show that astrocytes exhibit an intense furosemide-sensitive K+ accumulation which is activated by K+ levels resembling those occurring in the extracellular compartment during pathological events. Previous studies on a furosemide-sensitive Cl- pump in cultured astrocytes suggest that this accumulation might be via KCl cotransport, which in other systems is involved in volume control.

Animals↗

PubMed1984

Influence of pathological concentrations of ammonia on metabolic fate of 14C-labeled glutamate in astrocytes in primary cultures.

Rates of glutamine formation and of carbon dioxide production (as an indication of oxidative deamination of glutamate) were determined in primary cultures of astrocytes exposed to 50 microM labeled glutamate in the absence or presence of added ammonia (0.1-3 mM). Glutamine formation (1.7 nmol/min/mg protein) was unaffected by all concentrations of added ammonia. This probably reflects the presence of a low content of ammonia (0.1-0.2 mM), originating from degradation of glutamine, in the cells even in the absence of added ammonia, and it shows that pathophysiological concentrations of ammonia do not increase the formation of glutamine from exogenous glutamate. The carbon dioxide production rate was 5.9 nmol/min/mg protein, i.e., three to four times higher than the rate of glutamine formation. It was significantly reduced (to 3.5 nmol/min/mg protein) in the presence of 1 mM or more of ammonia. This is in keeping with suggestions by others that toxic levels of ammonia affect oxidative metabolism.

Ammonia↗

PubMed1984

Flunitrazepam binding to intact and homogenized astrocytes and neurons in primary cultures.

[3H]Flunitrazepam binds to intact and homogenized mouse astrocytes and neurons in primary cultures. In intact cells, the binding is to a single, high-affinity, saturable population of benzodiazepine binding sites with a KD of 7 nM and Bmax of 6,033 fmol/mg protein in astrocytic cells and a KD of 5 nM and Bmax of 924 fmol/mg protein in neurons. After homogenization, the Bmax values decrease drastically in both cell types, but most in astrocytes. The temperature and time dependency are different for the two cell types, with a faster association and dissociation in astrocytes than in neurons and a greater temperature sensitivity in the astrocytes. Moreover, flunitrazepam binding sites on neuronal and astrocytic cells have different pharmacological profiles. In intact astrocytic cells, Ro 5-4864 (Ki = 4 nM) is the most potent displacing compound, followed by diazepam (Ki = 6 nM) and clonazepam (Ki = 600 nM). In intact neurons, the relative order of potency of these three compounds is different: diazepam (Ki = 7 nM) is the most potent, followed by clonazepam (Ki = 26 nM) and Ro 5-4864, which has little effect. After homogenization the potency of diazepam decreases. We conclude that both neuronal and astrocytic cells possess high-affinity [3H]flunitrazepam binding sites. The pharmacological profile and kinetic characteristics differ between the two cell types and are further altered by homogenization.

Animals↗

PubMed1984

Alterations in uptake and release rates for GABA, glutamate, and glutamine during biochemical maturation of highly purified cultures of cerebral cortical neurons, a GABAergic preparation.

This study demonstrates that virtually homogenous cultures of mouse cerebral neurons, obtained from 15-day-old embryos, differentiate at least as well as cultures which in addition contain astrocytes. This was indicated by glutamate decarboxylase activity which within 2 weeks rose from a negligible value to twice the level in the adult mouse cerebral cortex, and by a gamma-aminobutyric acid (GABA) uptake rate which quadrupled during the second week in culture and reached higher values than in brain slices. Within the same period, the GABA content increased four to five times to 75 nmol/mg protein, and a potassium-induced increase in [14C]GABA efflux became apparent. Although the development was faster than in vivo, optimum differentiation required maintenance of the cultures beyond the age of 1 week. Uptake and release rates for glutamate and glutamine underwent much less developmental alteration. At no time was there any potassium-induced release of radioactivity after exposure to [14C]glutamate, and the glutamate uptake was only slightly increased during the period of GABAergic development. This indicates that exogenous glutamate is not an important GABA precursor. Similarly, glutamine uptake was unaltered between days 7 and 14, although a small potassium-induced release of radioactivity after loading with glutamine suggests a partial conversion to GABA.

Animals↗

PubMed1983

Comparison between fluxes of potassium and of chloride in astrocytes in primary cultures.

Transport processes operating in astrocytes were examined by measuring unidirectional fluxes of 42K and 36Cl in primary cultures of mouse astrocytes, at steady-state with respect to ion composition. The total K+ uptake rate was 2025 nmol X mg-1 protein X min-1. This rate was not influenced by furosemide (2 mM), an inhibitor of Cl- uptake and K+-K+ exchange, or acetazolamide (0.1 mM), a carbonic anhydrase inhibitor. Ouabain (1 mM) inhibited the uptake rate by 541 nmol X mg-1 X min-1. The equilibrated K+ content was determined to be 696 nmol X mg-1. The rate constant for efflux was 2.76 min-1. This equals an efflux rate of 1921 nmol X mg-1 X min-1, i.e. a similar value as the influx. Furosemide and ouabain did not inhibit the efflux. The equilibrated Cl- content was found to be 167 nmol X mg-1 and it decreased in furosemide-treated cells to 68.1 nmol X mg-1. The total Cl- uptake was 35 nmol X mg-1 X min-1 and it was inhibited by furosemide or bumetanide by 27 nmol X mg-1 X min-1. The mean resting membrane potential was found to be -77.4 mV. From these data we conclude: (1) that the K+ uptake rates are high, as can be expected from estimates based on literature data for K+ conductance in mammalian glial cells in situ; and (2) that the cells possess a very low relative Cl- permeability.

Animals↗

PubMed1983

Acute and chronic effects of antidepressant drugs on beta-adrenergic function in astrocytes in primary cultures: an indication of glial involvement in affective disorders?

Acute and chronic effects of the antidepressant drugs tranylcypromine, a monoamine oxidase inhibitor, and amitriptyline, a monoamine uptake inhibitor, were studied on beta-adrenergic receptor function in mouse astrocytes in primary cultures. In clinically relevant concentrations, acute administration of either antidepressant drug had a direct inhibitory effect on the binding of the beta-adrenergic ligand dihydroalprenolol and on the isoproterenol-induced accumulation of cyclic AMP. However, in the absence of isoproterenol, these drugs enhanced the formation of cyclic AMP in the astrocytes. Chronic exposure to amitriptyline or tranylcypromine led to a decrease in isoproterenol-induced accumulation of cyclic AMP, and the time course for the development of this phenomenon was similar to that reported for whole brain in vivo. These findings suggest that these antidepressant drugs act as a partial agonists at beta-adrenergic receptors on astrocytes, and that the down-regulation of beta-adrenergic activity that occurs in vivo after chronic administration of antidepressant drugs may, to a large extent, take place in astrocytes and may result from the partial beta-agonist nature of the drugs.

Affective Disorders, Psychotic↗

PubMed1983

Ion dependency of uptake and release of GABA and (RS)-nipecotic acid studied in cultured mouse brain cortex neurons.

The influence of sodium ions on GABA uptake into neurons cultured from cerebral cortex was investigated at sodium concentrations ranging from 16 to 151 mM. Sodium did not affect the Km for GABA uptake but the uptake rates as a function of the sodium concentration was sigmoid at all GABA concentrations studied (1-200 microM). Hill plots of these curves exhibited straight lines with slopes of 1.7-2.1, suggesting that the coupling ratio between sodium and GABA is at least 2. (RS)-Nipecotic acid, a GABA analogue, was transported with high affinity (Km 8.0 +/- 2.1 microM) like GABA, but the Vmax for nipecotic acid was lower than found for GABA (0.664 +/- 0.035 versus 0.955 +/- 0.059 nmol X min-1 X mg-1), suggesting a microheterogeneity of the GABA transport sites. Neurons loaded with the respective tritiated compounds exhibited a pronounced K+-stimulated, calcium-dependent release of both GABA and nipecotic acid indicating that these cultured cells have developed functionally active GABA-ergic synapses.

Animals↗

PubMed1983

Intracellular ion changes of astrocytes in response to extracellular potassium.

Intracellular changes of K+, Na+, and Cl- were investigated by the aid of radiotracers in primary cultures of astrocytes when extracellular K+ was (1) increased from 3 to 12 mM and subsequently again decreased to 3 mM; and (2) increased from 5.4 to 54 mM with subsequent decrease to 5.4 mM. In both situations the K+ content increased by 50% within seconds, and it doubled within 1-2 min. The increase must be carrier mediated, because keeping the K X Cl product (Donnan equilibrium) constant did not lower the K+ accumulation rates. The Na+ content decreased when K+ was increased to 12 mM, but the decrease corresponded only to 10% of the accumulated K+. When K+ was increased to 54 mM, the Na+ content increased transiently. Cl- increased by about 15-25% of the accumulated K+. Return of extracellular K+ to original levels evoked a very fast K+ release, reversing all ion changes. The Na+ content increased transiently during the release process. For an interpretation of these observations, it is necessary to postulate endogenous production of an anion and of H+, which in turn is partly exchanged with Na+.

Animals↗

PubMed1983

The effects of antidepressant drugs on adenylyl cyclase linked beta adrenergic binding sites on mouse astrocytes in primary cultures.

It has been widely reported that the chronic administration of antidepressant drugs induces a down regulation of beta receptors in the brains of experimental animals with a time course that parallels the therapeutic improvement seen in depressed patients given these drugs. It has been tacitly assumed that these beta receptors are located on neurons. All classes of antidepressant drugs tested, various monoamine uptake inhibitors, a monoamine oxidase inhibitor, or novel drugs lacking either of these actions, reduced the retention of dihydroalprenolol by intact astrocytes in primary cultures. The drug concentrations altering this retention by astrocytes (Ki) are in the same range as those reported by other investigators using homogenates of glioma cells or whole brain. The isoproterenol-induced stimulation of cyclic AMP formation by astrocytes in primary cultures is reduced acutely by the antidepressants amitriptyline, tranylcypromine and doxepin. Following washout of the antidepressant drug, isoproterenol stimulation of adenylyl cyclase is reduced in astrocytes exposed in culture to amitriptyline or tranylcypromine for 12-14 days or longer but is not altered in astrocytes exposed to the antidepressants for only 5 days. This indicates that the chronic exposure of astrocytes in culture to antidepressant drugs, down regulates astrocyte beta receptors with a time course that parallels the beta down regulation seen in vivo in animal brain homogenates and the therapeutic improvement seen in depressed patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗