Temporal and sequential changes of glial cells and cytokine expression during neuronal degeneration after transient global ischemia in rats.

BACKGROUND: How glial cells and cytokines are associated with the progression of delayed neuronal death induced by transient global ischemia is still unclear. To further clarify this point, we studied morphological changes in glial cells (microglial cells and astrocytes), and cytokine protein levels, during the progression of neuronal cell loss in CA1 (Cornu Ammonis 1) of the hippocampus after transient global ischemia. METHODS: Morphological changes in glial cells were studied immuno-histochemically. Nine cytokines (IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ and TNF-α) were simultaneously measured by a multiplexed bead-based immunoassay from 6 h to day21 after transient four vessel occlusion (4VO) in rats. RESULTS: During the process of neuronal loss, we observed four distinct phases: (1) lag phase day0-2 (no NeuN+ cell loss observed), (2) exponential phase day2-7 (NeuN+ cells reduced in number exponentially), (3) deceleration phase day7-14 (reduction rate of NeuN+ cells became low), (4) stationary phase day14 onward (NeuN+ cell loss progressed no longer). In the lag phase, activated glial cells were observed in the entire hippocampus but later were gradually restricted to CA1. Cytokine protein levels in the lag and exponential phases were lower than in the deceleration and stationary phases. IL-1α, IL-1ß, IL-4, IL-6 and IFN-γ in 4VO were significantly higher in all four phases than in sham. Compared with sham level, GM-CSF was significantly high in the deceleration phase. TNF-α was significantly high in both the deceleration and stationary phases. CONCLUSION: Ischemic stress in 4VO activated glial cells in areas beyond CA1 in the lag phase. Pyramidal neurons were injured in CA1 from the end of the lag phase and then neuronal cells reduced in CA1 in the exponential phase. After neuronal death began, the influence of dead cells on glial cells and cytokine expression gradually became stronger than the influence by ischemic stress. Therefore, from the deceleration phase, changes in glial cells and cytokine production were likely caused by dead cells. Cytokine interaction in the microenvironment may determine the functions of IL-1α, IL-1ß, IL-4, IL-6 and IFN-γ in all four phases. The function of GM-CSF and TNF-α in the deceleration phase may be neurotrophic.

The effects of MPTP on the activation of microglia/astrocytes and cytokine/chemokine levels in different mice strains.

The effects of MPTP on two mouse strains with different MPTP sensitivities and immunological backgrounds were compared: MPTP-sensitive C57BL/6 mice (B6) with a propensity for Th1 and less MPTP-sensitive BALB/c mice (BALB) with a propensity for Th2. It was found that acute MPTP treatment induced behavioral dysfunction, activated microglia/astrocytes, and increased the levels of IL-10, IL-12(p40) IL-13, IFN-gamma, and MCP-1 in CSF in B6, but not in BALB. This suggests that variances in immunological backgrounds might be a major contributing factor in sensitivity differences to MPTP.

Oral administration of dihomo-gamma-linolenic acid prevents development of atopic dermatitis in NC/Nga mice.

Disorders of the metabolism of essential fatty acids (EFAs) are related to atopic dermatitis (AD). Concentrations of dihomo-gamma-linolenic acid (DGLA), an EFA, in the serum of AD patients are lower than those in healthy volunteers. Recently we developed a fermented DGLA oil, and examined whether oral administration of DGLA prevents development of dermatitis in NC/Nga mice, which spontaneously develop human AD-like skin lesions. NC/Nga mice were fed a diet either containing or not containing DGLA for 8 weeks under in air-uncontrolled conventional circumstances. Clinical skin severity scores were significantly lower in mice fed DGLA than in mice not fed it. Scratching behavior and plasma total IgE levels were also reduced in the DGLA group, in association with histological improvement. DGLA suppressed clinical severity of skin lesions dose-dependently, with an increase in DGLA contents in phospholipids of skin, spleen, and plasma. Discontinuation of DGLA administration resulted in the onset of dermatitis and a decrease in DGLA contents in skin, spleen, and plasma. These findings indicate that oral administration of DGLA effectively prevents the development of AD in NC/Nga mice, and that DGLA in phospholipids is a compound of key importance in the development and prevention of dermatitis.

Long-lasting reactive changes observed in microglia in the striatal and substantia nigral of mice after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Parkinson's disease (PD) is an age-related movement disorder that progresses over a period of 10 to 20 years. The existence of microglia in a long-lasting activated state, expressing MHC II, has been thought to play an important role in the progression of PD. PD mouse models, induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), exhibit only transient PD-like movement dysfunction in contrast to MPTP-intoxicated monkeys which show progressive and permanent movement dysfunction. To understand the reasons why the progression does not occur in MPTP-treated mice, we used immunohistochemical analyses to study whether activated microglia in the striatum and/or substantia nigra persist long after MPTP treatment. Microglial changes in the striatum and substantia nigra of mice at 2 days and 6 months after MPTP treatment (four intraperitoneal injections of 20 mg/kg MPTP at two hour intervals) were examined. C57BL/6 mice (which are highly sensitive to MPTP) displayed transient movement dysfunction and highly activated microglia were observed at day two. In contrast, BALB/c mice (which are less sensitive to MPTP) exhibited no movement dysfunction and only slightly activated microglia were observed at day two. After 6 months, the microglia in the striatum and substantia nigra pars compacta of the treated C57BL/6 mice were still more hypertrophic compared with the control, although less hypertrophic than those observed at day two. In the treated BALB/c mice, the microglia were also hypertrophic compared with the control after 6 months. MHC II-positive microglia were undetectable at any time after MPTP treatment in both mice. These data show that MPTP administration results in the existence of persistent activated microglia that are not MHC II-positive, and is independent of the MPTP sensitivity of the mouse strain. These results suggest that long lasting MHC II-positive microglia might be required for PD progression. In MPTP-intoxicated mice, the absence of MHC II-positive microglia might explain why there is no progression of PD-like dysfunctional symptoms.

Efficacy and safety of collagenase Clostridium histolyticum injection for Dupuytren's contracture in non-Caucasian Japanese patients (CORD-J Study): the first clinical trial in a non-Caucasian population.

To assess the efficacy, safety and pharmacokinetics of 0.58 mg collagenase Clostridium histolyticum injections for the treatment of Dupuytren's contracture in Japanese patients, we conducted a phase III, multicentre, uncontrolled, open-label clinical study in patients with Dupuytren's contracture. Of the 77 patients, 66 achieved clinical success in the primary treated joint (86%; 95% confidence interval: 76% to 93%), confirming the efficacy of collagenase Clostridium histolyticum injections. More improvement was seen in the metacarpophalangeal joints than in the proximal interphalangeal joints (94% versus 73%). The main adverse reaction was a local reaction in the injected hand. No tendon rupture or anaphylactic reactions were seen. The concentrations of collagenase Clostridium histolyticum were below the lower limit of quantification in plasma samples at all time points. As seen in global studies in Caucasian patients, a corrective effect on Dupuytren's contracture and good tolerance were observed in most non-Caucasian (Asian) Japanese patients. LEVEL OF EVIDENCE: Level 3.

The intra-arterial injection of microglia protects hippocampal CA1 neurons against global ischemia-induced functional deficits in rats.

In the present study, we have attempted to elucidate the effects of the intra-arterial injection of microglia on the global ischemia-induced functional and morphological deficits of hippocampal CA1 neurons. When PKH26-labeled immortalized microglial cells, GMIR1, were injected into the subclavian artery, these exogenous microglia were found to accumulate in the hippocampus at 24 h after ischemia. In hippocampal slices prepared from medium-injected rats subjected to ischemia 48 h earlier, synaptic dysfunctions including a significant reduction of synaptic responses and a marked reduction of long-term potentiation (LTP) of the CA3-CA1 Schaffer collateral synapses were observed. At this stage, however, neither significant neuronal degeneration nor gliosis was observed in the hippocampus. At 96 h after ischemia, there was a total loss of the synaptic activity and a marked neuronal death in the CA1 subfield. In contrast, the basal synaptic transmission and LTP of the CA3-CA1 synapses were well preserved after ischemia in the slices prepared from the microglia-injected animals. We also found the microglial-conditioned medium (MCM) to significantly increase the frequency of the spontaneous postsynaptic currents of CA1 neurons without affecting the amplitude, thus indicating that MCM increased the provability of the neurotransmitter release. The protective effect of the intra-arterial injected microglia against the ischemia-induced neuronal degeneration in the hippocampus was substantiated by immunohistochemical and immunoblot analyses. Furthermore, the arterial-injected microglia prevented the ischemia-induced decline of the brain-derived neurotrophic factor (BDNF) levels in CA1 neurons. These observations strongly suggest that the arterial-injection of microglia protected CA1 neurons against the ischemia-induced neuronal degeneration. The restoration of the ischemia-induced synaptic deficits and the resultant reduction of the BDNF levels in CA1 neurons, possibly by the release of diffusible factor(s), might thus contribute to the protective effect of the arterial-injection of microglia against ischemia-induced neuronal degeneration.

Comparison of drug-metabolizing activities in the livers of carcinogen-sensitive parent rats and carcinogen-resistant descendants.

Donryu strain albino rats were maintained on a diet containing 0.06% 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) for nine successive generations. Some rats in the fourth to eighth generations showed marked resistance to the carcinogenic action of 3'-Me-DAB. In the liver where we found tumors, their size and number are smaller than in the corresponding original strain of rats fed on a diet containing 3'-Me-DAB. No significant differences were found in the total cytochrome P-450 contents or epoxide hydrolase activities of the livers of the resistant variant and the original strain, but the benzo(a)pyrene hydroxylase activity which is mainly attributed to cytochrome P-448 and glutathione S-transferase activity of the resistant variant were lower. The inductions of hepatic cytochrome P-488 and benzo(a)pyrene hydroxylase on administration of polychlorinated biphenyls or 3-methylcholanthrene were also lower in the resistant rats. In the mutagenicity test on Salmonella typhimurium TA 98 the liver 9000 X g supernatant fraction from 3'-Me-DAB-resistant F7 rats did not fully induce the mutagenicities of 3'-Me-DAB and several other carcinogens. Thus the resistance of F7 rats to the chemical carcinogen may be related to the lower activities of some drug-metabolizing enzymes and the poor inducibility of cytochrome P-448 in their liver, although selection of resistant rats should be continued for further generations before coming to a definite conclusion on biochemical basis of apparent resistance to 3'-Me-DAB.

Decreased activation of carcinogens in the liver of carcinogen-resistant rats.

We have developed a new strain of rats (resistant rat) which exhibit resistance against the carcinogenic action of several carcinogenic compounds. In the present study, we compared the ability of resistant rat liver to activate 3'-methyl-4-dimethylaminoazobenzene and 2-acetylaminofluorene to highly reactive metabolites, which can covalently bind to DNA, with that of carcinogen-sensitive parent strain rats (sensitive rat), using in vitro DNA binding assay. The covalent binding of these carcinogens to calf thymus DNA, catalyzed by the hepatic 9000 x g supernatant fraction from resistant rats pretreated with 3-methylcholanthrene, was about one-half of that catalyzed by the 9000 x g supernatant from sensitive rats which had received the same treatment. These results suggest that the ability of resistant rat liver to activate the carcinogens decreases compared to sensitive rat liver. Further experiments revealed that this decreased activation of the carcinogens in resistant rat livers is due to a low inducibility of 3-methylcholanthrene-inducible forms of cytochrome P-450 mRNA. The hepatic cytosolic Ah receptor concentrations in resistant rats were shown to be significantly lower than those of sensitive rats. Scatchard plot analysis demonstrated, however, that there is no significant difference between the affinity of Ah receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin in these two strains. These data implicate that the hepatic Ah receptor level may be an important factor in determining carcinogen sensitivity in rats.

Simultaneous influence of erythrocyte deformability and macromolecules in the medium on erythrocyte aggregation: a kinetic study by a laser scattering technique.

The aggregation and sedimentation kinetics of human erythrocytes was studied by modifying the cellular properties and medium compositions simultaneously. Dextrans of average molecular weight 70400 and 494000 were used to provide suspending medium modifications, while diamide (diazene dicarboxylic acid bis(N,N-dimethylamide)) was used to alter the membrane structural properties. Laser scattering method was employed for this study, and it was compared with a kinetic method combined with a low-shear rheoscope and an image analyzer. From scattered light intensity profiles continuously obtained during aggregation of erythrocytes and sedimentation of the aggregates, characteristic kinetic parameters were computed. Kinetic parameters obtained from a phase of the one-dimensional aggregate formation and sedimentation corresponded well to the velocity of rouleaux formation obtained by the low-shear rheoscope technique. Dextrans accelerated the erythrocyte aggregation and the sedimentation, and diamide treatment suppressed the process by decreasing the erythrocyte deformability. The aggregating force by dextrans overcame the disaggregating force by the decreased deformability. However, the arrangement of erythrocytes as expressed in specific units for aggregates (i.e., rouleaux) became irregular by decreasing the erythrocyte deformability. In conclusion, the progression of erythrocyte aggregation and the structure of the aggregates were dependent on both erythrocyte properties and macromolecules in the medium.

Lipid peroxidation in the liver of carcinogen-resistant rats.

Recently, we developed a new strain of rats that exhibit marked resistance to the hepatotoxic and carcinogenic actions of 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) and some other carcinogens. In this work, we compared lipid peroxidation in the liver of these carcinogen-resistant (R) rats and the parental Donryu strain rats that are sensitive (S) to hazardous actions of these carcinogens. The liver microsomal fractions of the R group contained less amounts of polyunsaturated fatty acids. Microsomal lipid peroxidation in the presence of exogenous NADPH was much lower in R rats than in S rats. Liver microsomes of R rats were much less active than those of S rats also in producing 4-hydroxynonenal, carbonyl compounds and conjugated diene. The hepatic contents of ascorbic acid, glutathione, alpha-tocopherol and coenzyme Q in the R rats were similar to those in S rats. The activities of the free radical scavenger enzymes, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), in the two groups were also similar. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are both thought to function in disposal of these cytotoxic aldehydes. The liver microsomal and mitochondrial ALDH activities of the two groups were similar. The ADH activity of the liver cytosolic fraction of R rats was nearly twice that of S rats, as measured with 4-hydroxynonenal as substrate. The higher ADH activity may explain the decreased lipid peroxidation in R rats at least partly, if this enzyme is involved in lipid peroxidation.

Induction of a 23 kDa stress protein by oxidative and sulfhydryl-reactive agents in mouse peritoneal macrophages.

The synthesis of 23 kDa protein was enhanced when mouse peritoneal macrophages were exposed to oxidative agents such as hydrogen peroxide and menadione, or to sulfhydryl-reactive agents such as diethylmaleate, cadmium chloride and sodium arsenite. After 11 h exposure to these agents the 23 kDa protein was one of the actively synthesized proteins in the macrophages. Under similar conditions the 34 kDa protein previously identified as heme oxygenase, was induced and its synthesis preceded that of the 23 kDa protein. Neither the 23 kDa or the 34 kDa protein was induced by hyperthermia. Conversely, the various oxidative and sulfhydryl-reactive agents employed here did not induce the major heat shock proteins in the macrophages. When the macrophages were activated by bacterial lipopolysaccharide or other stimulants, many proteins are known to be induced, however, the 23 kDa and 34 kDa proteins were not induced. The 34 kDa protein, i.e., heme oxygenase, has been found to be stress-induced in various types of cell, but not the 23 kDa protein. This suggests that the 23 kDa protein is a stress protein predominantly expressed in macrophages.

Arundic acid (ONO-2506) ameliorates delayed ischemic brain damage by preventing astrocytic overproduction of S100B.

After focal cerebral ischemia, the infarct volume increases rapidly within acute infarct expansion (initial 12 to 24 h) and continues slowly during delayed infarct expansion (25 to 168 h). While acute infarct expansion represents progressive necrosis within the ischemic core, delayed infarct expansion starts as disseminated apoptotic cell death in a narrow rim surrounding the infarct border, which gradually coalesces to form a larger infarct. Discovery of a distinct correlation between reactive astrogliosis along the infarct border and delayed infarct expansion in the rodent ischemia model led us to investigate the possible causal relationship between the two events. Specifically, the calcium binding protein S100B exerts detrimental effects on cell survival through activation of various intracellular signaling pathways, resulting in altered protein expression. Arundic acid [(R)-(-)-2-propyloctanoic acid, ONO-2506] is a novel agent that inhibits S100B synthesis in cultured astrocytes. In the rodent ischemia model, this agent was shown to inhibit both the astrocytic overexpression of S100B and the subsequent activation of signaling pathways in the peri-infarct area. Concurrently, delayed infarct expansion was prevented, and neurologic deficits were promptly ameliorated. The results of subsequent studies suggest that the efficacy of arundic acid is mediated by restoring the activity of astroglial glutamate transporters via enhanced genetic expression.

Purification and properties of bovine liver gamma-glutamyltransferase.

gamma-Glutamyltransferase [EC 2.3.2.2] (gamma-GTP) was purified to a homogeneous state from bovine liver. It had an apparent molecular weight of about 110,000, as judged by polyacrylamide gel electrophoresis, and consisted of two non-identical glycopeptides with molecular weights of 68,000 and 27,000. The amino acid composition of the bovine liver enzyme was similar to those of other gamma-GTPs. The enzyme contained large amounts of neutral sugars, amino sugars and sialic acid, although the sialic acid content varied in different preparations. The Michaelis constant of the enzyme was estimated to be 0.8 mM for gamma-L-glutamyl-p-nitroanilide in the presence of glycylglycine and 1.25 mM in the absence of glycylglycine. Glutathione competitively inhibited the release of p-nitroaniline from gamma-L-glutamyl-p-nitroanilide with a Ki value of 0.3 mM. The specific activities of the enzyme for gamma-L-glutamyl-p-nitroanilide in the presence of glycylglycine (pH 8.6) and for glutathione, a natural substrate (pH 7.4), were comparable to those reported for gamma-GTPs from other mammalian sources. The bovine liver enzyme showed the same gamma-glutamyl group acceptor specificity as other gamma-GTPs from normal mammalian tissues. The phosphate-independent glutaminase activity of the enzyme was much lower than that of the rat kidney enzyme both in the presence and absence of maleate.

Relative contributions of sulfur atoms of dietary cysteine and methionine to rat liver glutathione and proteins.

The relative contributions of sulfur atoms of dietary L-Cys and L-Met to the syntheses of proteins and GSH in rat liver were examined. 1) When the amount of sulfur-containing amino acids in L-Trp-deficient diets was fixed at 0.36%, incorporation of L-[35S]Cys into GSH was proportional to the amount of L-Cys administered, in the presence of L-Met in the diet. Incorporation of 35S from L-Met into GSH was lower than that from the same amount of L-Cys and became negligible in the presence of a large amount of L-Cys. 2) When rats were given L-Trp-deficient diet, more L-Cys was always incorporated into liver GSH than into proteins. But, when rats were given L-Trp-fortified diet containing more L-Met than L-Cys, more L-Cys was incorporated into liver proteins than into GSH. 3) Met-sulfur was preferentially incorporated into liver proteins with or without L-Trp. Its absolute incorporation into proteins was significantly greater in the presence of L-Trp than in its absence. 4) When the amount of L-Met in the diet was increased from 0.18 to 0.36 or 0.54%, incorporation of Met-sulfur into proteins increased proportionally, in the presence of 0.18% L-Cys. Unexpectedly, incorporation of L-Cys formed from L-Met into liver proteins was larger than that from L-[3H]Met itself. L-Cys formed from L-Met was incorporated into proteins more readily than L-Cys given as such. 5) When the amount of L-Met in the diet was increased from 0.18 to 0.54%, incorporation of Met-35S into GSH increased 8-fold. Even with a large excess of L-Met, L-Cys was invariably incorporated into GSH. 6) These results are consistent with the role of liver GSH as a reservoir of cysteine, as proposed by us.

Re-evaluation of protein-bound glutathione in rat liver.

The extent of intracellular glutathione binding to proteins through a disulfide linkage in rat liver was examined quantitatively. The content of glutathione associated with the acid-precipitable fraction and releasable on borohydride treatment was 0.024 +/- 0.016 mumol/g liver, which accounted for less than one per cent of the total glutathione (6-7 mumol/g liver) in the liver of fed rats. Most of the thiol (2-4 mumol/g liver) liberated from liver proteins into the acid-soluble fraction on borohydride reduction in the presence of guanidine hydrochloride was not glutathione but was proteinaceous in nature. The amounts of thiols liberated per g of liver were similar in fed, fasted, and dibutyryl-3',5'-cyclic AMP-treated rats.

Comparative study of the sugar chains of gamma-glutamyltranspeptidases purified from human hepatocellular carcinoma and from human liver.

The sugar chains of gamma-glutamyltranspeptidases, purified from human hepatoma and from normal human liver, were released quantitatively as oligosaccharides by hydrazinolysis. Comparative study of their structures revealed that the following structural alterations are induced by hepatocyte carcinogenesis: 1) high mannose type sugar chains are detected in the hepatoma enzyme but not in the normal liver enzyme; 2) abnormal biantennary sugar chains containing C-2,4 outer chain branches newly appeared; 3) the total amounts of tri- and tetraantennary sugar chains containing C-2,6 outer chain branches increased up to three times.

GABAergic synaptic current in dissociated nucleus basalis of Meynert neurons of the rat.

gamma-Aminobutyric acid (GABA)-mediated spontaneous inhibitory postsynaptic currents (IPSCs) were recorded from dissociated rat nucleus basalis of Meynert neurons which still had their synaptic boutons attached. The membrane currents were recorded by the whole-cell patch-clamp technique. Elevated extracellular K+ concentration and the addition of the calcium ionophore, A23187, enhanced the amplitude and frequency of spontaneous IPSCs. Ryanodine and Ca(2+)-free external solution containing EGTA or BAPTA markedly decreased the spontaneous IPSC activities. Spontaneous IPSC activities were reversibly reduced by baclofen and increased by phaclofen, indicating that the GABAB receptor regulates the release of GABA from nerve terminals and acts as a negative autoreceptor.

Ontogeny of neuropeptides in the nucleus ventromedialis hypothalami of the rat: an immunohistochemical analysis.

The ontogeny of leucine-enkephalin (ENK)-, cholecystokinin-8 (CCK)-, substance P (SP)- and neurotensin (NT)-like immunoreactive structures were examined in the nucleus ventromedialis hypothalami (vm) of the rat by means of an indirect immunofluorescence method. ENK- (ENKI), SP- (SPI) and CCK-like immunoreactive (CCKI) structures in the vm exhibited very similar ontogenetic developments, though no CCKI cells were detected. ENKI fibers as well as SPI fibers and neurons first appeared at gestational day 21, ENKI neurons at postnatal day 2, and CCKI fibers at postnatal day 1. These structures subsequently increased in number. The maximum content was reached at postnatal day 21 and was observed even in the adult rats (postnatal day 30). On the other hand, NTI fibers first appeared at postnatal day 1; they decreased in number and no or only a few NTI fibers were seen in the adult rats. No NTI cells were seen in the vm.

Regulation of cytosol-nucleus pH gradients by K+/H+ exchange mechanism in the nuclear envelope of neonatal rat astrocytes.

In order to study the subcellular heterogeneity of intracellular H+ concentration in reactive astrocytes, the pH in the nucleus and cytosol of cultured astrocytes was measured using a confocal laser scanning microscope (CLSM) and pH indicator dye, 5'(and 6')-carboxyseminaphthofluorescein (carboxy SNAFL-1). The change in intracellular pH was indexed by the fluorescence ratio (F535/F610) at an excitation wavelength of 514.5 nm. The in vitro fluorescence ratio increased as pH decreased. This ratio in the nucleus was significantly lower than that in the cytosol of astrocytes when perfused by HEPES-buffered Hanks' balanced salt solution (HHBSS) at pH 7.4. Acid stimulations of cells (pH 5.0) raised the fluorescence ratio in both nucleus and cytosol. However, the increase in the fluorescence ratio of the nucleus was less than that of cytosol. Treatment with a K+/H+ ionophore, nigericin (20 microM), reversibly nullified this cytosol-nucleus pH gradient. These findings suggest that a buffering mechanism(s) for maintaining of intracellular pH exists between the nucleus and cytosol, and a K+/H+ exchanger may act on the nuclear envelope to eventuate intranuclear pH maintenance in the living cells.

Acetylcholine receptors in dissociated nucleus basalis of Meynert neurons of the rat.

Electrical and pharmacological properties of acetylcholine (ACh)-induced currents in neurons dissociated from the nucleus basalis of Meynert (nBM) of immature (2-week-old) rats were investigated with the whole-cell mode of the patch-clamp technique. At a holding potential (VH) of -50 mV, ACh (10(-4)M) evoked a transient inward current mimicked by nicotine (InACh), followed by a sustained outward current mimicked by carbamylcholine (ImACh). The KD values were 1.2 x 10(-4) M for ImACh and 8.7 x 10(-7) M for ImACh. The reversal potential of ImACh was close to EK. The ImACh was determined to be elicited via the M2 muscarinic receptor, based on the differences in sensitivity to muscarinic antagonists such as pirenzepine and AF-DX-116.