Blood-brain barrier and neuro-AIDS.

Neuro-AIDS is becoming a major health problem among AIDS patients who experience improved survival under combined antiretroviral therapy (cART). Neuronal injury and loss are the critical issues of neuro-AIDS that need the entry of HIV into the central nervous system (CNS) via peripheral infected monocyte/macrophage carriers or viral direct penetration of blood-brain barrier (BBB). The mechanisms of HIV enhancing BBB permeability and entering CNS and the effect of drug abuse in HIV traffic across BBB are discussed. In addition, the current anti-HIV drugs, although they are effective in reducing plasma viral level, cannot eradicate the viruses completely from CNS. The possible mechanism of BBB hindrance and anti-HIV drug efflux by transport proteins, and general methods used to deliver antiretroviral drugs into brain are also discussed.

Phosphorylated AKT inhibits the apoptosis induced by DRAM-mediated mitophagy in hepatocellular carcinoma by preventing the translocation of DRAM to mitochondria.

Increasing autophagy is beneficial for curing hepatocellular carcinoma (HCC). Damage-regulated autophagy modulator (DRAM) was recently reported to induce apoptosis by mediating autophagy. However, the effects of DRAM-mediated autophagy on apoptosis in HCC cells remain unclear. In this study, normal hepatocytes (7702) and HCC cell lines (HepG2, Hep3B and Huh7) were starved for 48 h. Starvation induced apoptosis and autophagy in all cell lines. We determined that starvation also induced DRAM expression and DRAM-mediated autophagy in both normal hepatocytes and HCC cells. However, DRAM-mediated autophagy was involved in apoptosis in normal hepatocytes but not in HCC cells, suggesting that DRAM-mediated autophagy fails to induce apoptosis in hepatoma in response to starvation. Immunoblot and immunofluorescence assays demonstrated that DRAM translocated to mitochondria and induced mitophagy, which led to apoptosis in 7702 cells. In HCC cells, starvation also activated the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which blocks the translocation of DRAM to mitochondria through the binding of p-AKT to DRAM in the cytoplasm. Inactivation of the PI3K/AKT pathway rescued DRAM translocation to mitochondria; subsequently, mitochondrial DRAM induced apoptosis in HCC cells by mediating mitophagy. Our findings open new avenues for the investigation of the mechanisms of DRAM-mediated autophagy and suggest that promoting DRAM-mediated autophagy together with PI3K/AKT inhibition might be more effective for autophagy-based therapy in hepatoma.

Characterization of neuronal cell death in normal and diabetic rats following exprimental focal cerebral ischemia.

We have studied the forms of cell death following ischemia/reperfusion, and the influence of diabetes mellitus (DM) as an additional factor. Based on the models of diabetes and middle cerebral artery occlusion (MCAO), characteristics of cell death after ischemia/reperfusion were evaluated synthetically by different methods: pathology, FCM, TUNEL and DNA agarose electrophoresis. The results showed that the occurrence of cerebral injury after ischemia/reperfusion was accompanied by cell necrosis and cell apoptosis. Cell apoptosis was mainly located in the ischemic penumbral (IP) zone around the densely ischemic focus. The ischemic core was characterized by cell necrosis. At the same time, the results showed that the process of ischemic cerebral injury worsened by DM was related to inducing cell apoptosis in IP and mid zone. In conclusion, there existed not only cell apoptosis but cell necrosis in brain damage following focal cerebral ischemia/reperfusion and showed a close, internal relationship between them. Brain damage following cerebral ischemia/reperfusion was worsened distinctly under diabetic conditions.

Early release of cytochrome C and activation of caspase-3 in hyperglycemic rats subjected to transient forebrain ischemia.

The mechanisms underlying the aggravating effect of hyperglycemia on brain damage are still elusive. The present study was designed to test our hypothesis that hyperglycemia-mediated damage is caused by mitochondrial dysfunction with mitochondrial release of cytochrome c (cyt c) to the cytoplasm, which leads to activation of caspase-3, the executioner of cell death. We induced 15 min of forebrain ischemia, followed by 0.5, 1, and 3 h of recirculation in sham, normoglycemic and hyperglycemic rats. Release of cyt c was observed in the neocortex and CA3 in hyperglycemic rats after only 0.5 h of reperfusion, when no obvious neuronal damage was observed. The release of cyt c persisted after 1 and 3 h of reperfusion. Activation of caspase-3 was observed after 1 and 3 h of recovery in hyperglycemic animals. No cyt c release or caspase-3 activation was observed in sham-operated controls while a mild increase of cyt c was observed in normoglycemic ischemic animals after 1 and 3 h of reperfusion. The findings that there is caspase activation and cyt c relocation support a notion that the biochemical changes that constitute programmed cell death occur after ischemia and contribute, at least in part, to hyperglycemia-aggravated ischemic neuronal death.

Protein ubiquitination in rat brain following hypoglycemic coma.

Hypoglycemic coma of 30 min duration selectively damages CA1 pyramidal neurons and the crest of dentate gyrus (DG) granule cells in hippocampus. Here, we show by high-resolution confocal microscopy and biochemical analysis that 30 min of hypoglycemic coma induces the ubiquitination and aggregation of several proteins in rat brain tissues. Protein ubiquitination and aggregation occurred in the CA1 and DG regions as early as the end of 30 min of hypoglycemic coma and lasted until neuronal death in the late recovery period after hypoglycemia. In comparison, the neurons surviving hypoglycemia were less affected. On western blots, ubiquitinated proteins (ubi-proteins) were present mainly in Triton-insoluble pellets, indicating that they are irreversibly aggregated. We conclude that proteins are ubiquitinated and aggregated in neurons after hypoglycemia prior to their death. We hypothesize that protein ubiquitination and aggregation may contribute to neuronal damage after hypoglycemia.

Alteration of cyclic adenosine monophosphate response element binding protein in rat brain after hypoglycemic coma.

In the current study, the temporal and regional changes of the transcription factor cyclic adenosine monophosphate response element binding protein (CREB) were investigated in rat brains subjected to 30 minutes of hypoglycemic coma followed by varied periods of recovery using Western blot and confocal microscopy. The total amount of CREB was not altered in any area examined after coma. The level of the phosphorylated form of CREB decreased during coma but rebounded after recovery. In the relatively resistant areas, such as the inner layers of the neocortex and the inner and outer blades of the dentate gyms (DG), phospho-CREB increased greater than the control level after 30 minutes of recovery and continued to increase up to 3 hours of recovery. In contrast, little or no increase of phospho-CREB was observed during the recovery period in the outer layers of the neocortex and at the tip of the DG, that is, regions that are selectively vulnerable to hypoglycemic insults. The current findings suggest that a neuroprotective signaling pathway may be more activated in the resistant regions than in the vulnerable ones after hypoglycemic coma.

Differential phosphorylation at Ser473 and Thr308 of Akt-1 in rat brain following hypoglycemic coma.

We analyzed both total Akt-1 and phosphorylation of Akt-1 at residues Ser473 and Thr308 (phospho-Akt-1(Ser474) and phospho-Akt-1(Thr308), respectively) in the outer and inner layers of cortex following 30 min of hypoglycemic coma by Western blot analyses and confocal microscopy. The total amount of Akt-1 was not altered in any area examined. Phospho-Akt-1(Ser474), however, increased significantly in both layers of cortex at 0 and 30 min of recovery, but returned to control level at 3 h of recovery. In the vulnerable area (outer layer of cortex), no upregulation of phospho-Akt-1(Thr308) was observed at any time points examined. In the resistant area like inner layer of cortex, however, phospho-Akt-1(Thr308) was significantly over the control level at 3 h of recovery. Confocal microscopy result indicates that most of phospho-Akt-1(Thr308) had already moved into nucleus at 3 h of recovery. Our results suggest that Akt-1, when phosphorylated at Thr308, may play a protective role for neurons in the resistant regions of the brain.

Is neuronal injury caused by hypoglycemic coma of the necrotic or apoptotic type?

In this study, we explored if a 30 minute period of hypoglycemic coma yields damage which shows some features associated with apoptosis. To that end, we induced insulin-hypoglycemic coma of 30 min duration, and studied brain tissues after the coma period, and after recovery period of 30 min, 3 h, and 6 h. Histopathological data confirmed neuronal damage in all of the vulnerable neuronal populations. Release of cytochrome c (cyt c), assessed by Western Blot, was observed in the neocortex and caudoputamen after 3 and 6 h of recovery. In these regions, the caspase-like activity increased above control after 6 h of recovery. By laser-scanning confocal microscopy, a clear expression of Bax was observed after 30 min of coma in the superficial layers of the neocortex, reaching a peak after 30 min of recovery. Punctuate immunolabeling surrounding nuclei in soma and dendrites in cortical pyramidal neurons likely represents mitochondria, which suggests that Bax protein assembled at the surface of mitochondria in vulnerable neocortical neurons. It is concluded that although previous morphological data have suggested that cells die by necrosis, neuronal damage after hypoglycemic coma shows some features of apoptosis.

Survival- and death-promoting events after transient cerebral ischemia: phosphorylation of Akt, release of cytochrome C and Activation of caspase-like proteases.

Release of cytochrome c (cyt c) into cytoplasm initiates caspase-mediated apoptosis, whereas activation of Akt kinase by phosphorylation at serine-473 prevents apoptosis in several cell systems. To investigate cell death and cell survival pathways, the authors studied release of cyt c, activation of caspase, and changes in Akt phosphorylation in rat brains subjected to 15 minutes of ischemia followed by varying periods of reperfusion. The authors found by electron microscopic study that a portion of mitochondria was swollen and structurally altered, whereas the cell membrane and nuclei were intact in hippocampal CA1 neurons after 36 hours of reperfusion. In some neurons, the pattern of immunostaining for cyt c changed from a punctuate pattern, likely representing mitochondria, to a more diffuse cytoplasmic localization at 36 and 48 hours of reperfusion as examined by laser-scanning confocal microscopic study. Western blot analysis showed that cyt c was increased in the cytosolic fraction in the hippocampus after 36 and 48 hours of reperfusion. Consistently, caspase-3-like activity was increased in these hippocampal samples. As demonstrated by Western blot using phosphospecific Akt antibody, phosphorylation of Akt at serine-473 in the hippocampal region was highly increased during the first 24 hours but not at 48 hours of reperfusion. The authors conclude that transient cerebral ischemia activates both cell death and cell survival pathways after ischemia. The activation of Akt during the first 24 hours conceivably may be one of the factors responsible for the delay in neuronal death after global ischemia.

Role and mechanisms of secondary mitochondrial failure.

Ischemia is accompanied by mitochondrial dysfunction, as assessed by measurements of mitochondrial respiratory activities in vitro. Following brief periods of ischemia, mitochondrial function is usually normalized during reperfusion. However, particularly after ischemia of longer duration, reperfusion may be accompanied by secondary mitochondrial failure. After short periods of ischemia this is observed in selectively vulnerable areas and, after intermediate to long periods of ischemia, in other areas as well. However, it has remained unsettled if the mitochondrial dysfunction is the result or the cause of cell death. Although it has been commonly assumed that such failure is secondary to cell injury by other mechanisms, recent results suggest that mitochondrial dysfunction may be the cause of cell death. Indirect evidence for this postulate is provided by experiments showing that cyclosporin A (CsA), when allowed to cross the blood-brain barrier, is a potent neuroprotectant. CsA is a virtually specific blocker of the mitochondrial permeability transition (MPT) pore, a voltage-gated channel allowing molecules and ions with a mass < 1500 Daltons to pass the inner mitochondrial membrane. Experiments on isolated cells in vitro demonstrate that cell calcium accumulation or oxidative stress triggers the assembly of an MPT pore, which leads to collapse of the mitochondrial membrane potential, to ATP hydrolysis, to enhanced production of reactive oxygen species (ROS), and to cell death. The beneficial effect of CsA could thus be related to its ability to block the MPT pore. Longer periods of ischemia, such as occurs after transient middle cerebral artery (MCA) occlusion, lead to pan-necrotic lesions (infarction). In the rat, recirculation following 2 h of MCA occlusion leads to partial normalization of the bioenergetic state but this is followed within 4-6 h by secondary bioenergetic failure. The latter seems unrelated to blockade of the microcirculation, but correlates to secondary mitochondrial failure. The brain damage incurred is ameliorated by the spin trap alpha-phenyl-N-butyl nitrone (PBN) and by the immunosuppressant FK506 even when given 1-3 h after the start of recirculation. The two drugs also prevent the secondary mitochondrial failure during early recirculation, suggesting that such failure is pathogenetically important. Probably, though, the mitochondrial dysfunction involves not only the assembly of an MPT pore but also other mechanisms. Since recirculation is associated with release of mitochondrial proteins it is not unlikely that such proteins, e.g. cytochrome c, trigger cascades of events leading to cell death.6.

A novel glycosulfopeptide binds to P-selectin and inhibits leukocyte adhesion to P-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin on leukocytes that binds selectins. The molecular features of PSGL-1 that determine this high affinity binding are unclear. Here we demonstrate the in vitro synthesis of a novel glycosulfopeptide (GSP-6) modeled after the extreme N terminus of PSGL-1, which has been predicted to be important for P-selectin binding. GSP-6 contains three tyrosine sulfate (TyrSO(3)) residues and a monosialylated, core 2-based O-glycan with a sialyl Lewis x (C2-O-sLe(x)) motif at a specific Thr residue. GSP-6 binds tightly to immobilized P-selectin, whereas glycopeptides lacking either TyrSO(3) or C2-O-sLe(x) do not detectably bind. Remarkably, an isomeric glycosulfopeptide to GSP-6, termed GSP-6', which contains sLe(x) on an extended core 1-based O-glycan, does not bind immobilized P-selectin. Equilibrium gel filtration analysis revealed that GSP-6 binds to soluble P-selectin with a K(d) of approximately 350 nM. GSP-6 (<5 microM) substantially inhibits neutrophil adhesion to P-selectin in vitro, whereas free sLe(x) (5 mM) only slightly inhibits adhesion. In contrast to the inherent heterogeneity of post-translational modifications of recombinant proteins, glycosulfopeptides permit the placement of sulfate groups and glycans of precise structure at defined positions on a polypeptide. This approach should expedite the probing of structure-function relationships in sulfated and glycosylated proteins, and may facilitate development of novel drugs to treat inflammatory diseases involving P-selectin-mediated leukocyte adhesion.

Effects of streptozotocin-induced hyperglycemia on brain damage following transient ischemia.

Hyperglycemia is known to aggravate ischemic brain damage. The present experiments were undertaken to explore whether hyperglycemia caused by streptozotocin-induced diabetes exacerbates brain damage following transient brain ischemia as it does in animals acutely infused with glucose. Experimental diabetes was induced by injection of streptozotocin in rats which were subjected to 10 min of forebrain ischemia either 1 week (1-wk) or 4 weeks (4-wk) after the induction of diabetes. Normoglycemic rats exposed to the same duration of ischemia and sham-operated diabetic rats served as controls. The animals underwent evaluation of clinical outcome and histopathological analysis of brain damage. Postischemic seizures developed in 35.3 and 42.1% of 1-wk and 4-wk diabetic hyperglycemic animals, respectively. The incidence of seizure was not different between the two groups. None of the diabetic animals with plasma glucose concentrations below 12 mM exhibited seizure activity. The extent and distribution of brain damage were similar between 1-and 4-wk diabetic animals. In the CA1 and in the subicular regions of hippocampus, both diabetic hyperglycemic and normoglycemic animals showed 70-80% cell death. Diabetic hyperglycemic animals had more severe neuronal necrosis in the parietal cortex than normoglycemic animals. In diabetic hyperglycemic animals, neuronal damage involved additional brain structures, e.g., cingulate cortex, thalamus nuclei, substantia nigra, pars reticulata, and the hippocampal CA3 sector, i.e., structures in which neurons were not affected in normoglycemic ischemic subjects at this duration of ischemia. These findings demonstrate that diabetic hyperglycemic animals frequently develop postischemic seizures and that streptozotocin-induced hyperglycemia results exacerbated postischemic brain damage of the same density and distribution as in acutely glucose-infused animals.

Molecular cloning and expression of human and mouse tyrosylprotein sulfotransferase-2 and a tyrosylprotein sulfotransferase homologue in Caenorhabditis elegans.

Tyrosine O-sulfation, a common post-translational modification in eukaryotes, is mediated by Golgi enzymes that catalyze the transfer of the sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate to tyrosine residues in polypeptides. We recently isolated cDNAs encoding human and mouse tyrosylprotein sulfotransferase-1 (Ouyang, Y. B., Lane, W. S., and Moore, K. L. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2896-2901). Here we report the isolation of cDNAs encoding a second tyrosylprotein sulfotransferase (TPST), designated TPST-2. The human and mouse TPST-2 cDNAs predict type II transmembrane proteins of 377 and 376 amino acid residues, respectively. The cDNAs encode functional N-glycosylated enzymes when expressed in mammalian cells. In addition, preliminary analysis indicates that TPST-1 and TPST-2 have distinct specificities toward peptide substrates. The human TPST-2 gene is on chromosome 22q12.1, and the mouse gene is in the central region of chromosome 5. We have also identified a cDNA that encodes a TPST in the nematode Caenorhabditis elegans that maps to the right arm of chromosome III. Thus, we have identified two new members of a class of membrane-bound sulfotransferases that catalyze tyrosine O-sulfation. These enzymes may catalyze tyrosine O-sulfation of a variety of protein substrates involved in diverse physiologic functions.

Tyrosylprotein sulfotransferase: purification and molecular cloning of an enzyme that catalyzes tyrosine O-sulfation, a common posttranslational modification of eukaryotic proteins.

Tyrosine O-sulfation is a common posttranslational modification of proteins in all multicellular organisms. This reaction is mediated by a Golgi enzyme activity called tyrosylprotein sulfotransferase (TPST) that catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to tyrosine residues within acidic motifs of polypeptides. Tyrosine O-sulfation has been shown to be important in protein-protein interactions in several systems. For example, sulfation of tyrosine residues in the leukocyte adhesion molecule P-selectin glycoprotein ligand 1 (PSGL-1) is required for binding to P-selectin on activated endothelium. In this report we describe the purification of TPST from rat liver microsomes based on its affinity for the N-terminal 15 amino acids of PSGL-1. We have isolated human and mouse TPST cDNAs that predict type II transmembrane proteins of 370 amino acid residues with almost identical primary structure. The human cDNA encodes a fully functional N-glycosylated enzyme with an apparent molecular mass of approximately 54 kDa when expressed in mammalian cells. This enzyme defines a new class of Golgi sulfotransferases that may catalyze tyrosine O-sulfation of PSGL-1 and other protein substrates involved in diverse physiologic functions including inflammation and hemostasis.

Grap is a novel SH3-SH2-SH3 adaptor protein that couples tyrosine kinases to the Ras pathway.

A human cytoplasmic signaling protein has been cloned that possesses the same structural arrangement of SH3-SH2-SH3 domains as Grb2. This protein is designated Grap for Grb2-related adaptor protein. The single 2.3-kilobase (kb) grap transcript was expressed predominantly in thymus and spleen, while the ubiquitously expressed grb2 gene produced two mRNA species of 3.8 and 1.5 kb. Grap and Grb2 consist of 217 amino acids and share 59% amino acid sequence identity, with highest homology in the N-terminal SH3 domain. The GrapSH2 domain interacts with ligand-activated receptors for stem cell factor (c-kit) and erythropoietin (EpoR). Grap also forms a stable complex with the Bcr-Abl oncoprotein via its SH2 domain in K562 cells. Furthermore, Grap is associated with a Ras guanine nucleotide exchange factor mSos1, primarily through its N-terminal SH3 domain. These results show that a family of Grb2-like proteins exist and couple signals from receptor and cytoplasmic tyrosine kinases to the Ras signaling pathway.

The influence of calcium transients on intracellular pH in cortical neurons in primary culture.

The objective of this study was to assess the influence of Ca2+ influx on intracellular pH (pHi) of neocortical neurons in primary culture. Neurons were exposed to glutamate (100-500 microM) or KCl (50 mM), and pHi was recorded with microspectrofluorometric techniques. Additional experiments were carried out in which calcium influx was triggered by ionomycin (2 microM) or the calcium ionophore 4-Br-A23187 (2 microM). Glutamate exposure either caused no, or only a small decrease in pHi (delta pH approximately 0.06 units). When a decrease was observed, a rebound rise in pHi above control was observed upon termination of glutamate exposure. In about 20% of the cells, the acidification was more pronounced (delta pH approximately 0.20 units), but all these cells had high control pHi values, and showed gradual acidification. Exposure of cells to 50 mM KCl consistently increased pHi. Since this increase was similar in the presence and nominal absence of HCO3-, it probably did not reflect influx of HCO3- via a Na(+)-HCO3- symporter. Furthermore, since it occurred in the absence of external Ca2+ (or a measurable rise in Cai2+) it seemed independent of Ca2+ influx. It is tentatively concluded that the rise in pHi was due to reduced passive influx of H+ along the electrochemical gradient, which is reduced by depolarization. In Ca(2+)-containing solutions, depolarization led to a rebound increase in pHi above control. This, and the rebound found after glutamate transients, may reflect Ca(2+)-triggered phosphorylation and upregulation of the Na+/H+ antiporter which extrudes H+ from the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of acid-base changes on the intracellular calcium concentration of neurons in primary culture.

The influence of changes in intra- and extracellular pH (pHi and pHe, respectively) on the cytosolic, free calcium concentration ([Ca2+]i) of neocortical neurons was studied by microspectrofluorometric techniques and the fluorophore fura-2. When, at constant pHe, pHi was lowered with the NH4Cl prepulse technique, or by a transient increase in CO2 tension, [Ca2+]i invariably increased, the magnitude of the rise being proportional to delta pHi. Since similar results were obtained in Ca(2+)-free solutions, the results suggest that the rise in [Ca2+]i was due to calcium release from intracellular stores. The initial alkaline transient during NH4Cl exposure was associated with a rise in [Ca2+]i. However, this rise seemed to reflect influx of Ca2+ from the external solution. Thus, in Ca(2+)-free solution NH4Cl exposure led to a decrease in [Ca2+]i. This result and others suggest that, at constant pHe, intracellular alkalosis reduces [Ca2+]i, probably by enhancing sequestration of calcium. When cells were exposed to a CO2 transient at reduced pHe, Ca2+ rose initially but then fell, often below basal values. Similar results were obtained when extracellular HCO3- concentration was reduced at constant CO2 tension. Unexpectedly, such results were obtained only in Ca(2+)-containing solutions. In Ca(2+)-free solutions, acidosis always raised [Ca2+]i. It is suggested that a lowering of pHe stimulates extrusion of Ca2+ by ATP-driven Ca2+/2H+ antiport.

The regulation of intracellular pH is strongly dependent on extracellular pH in cultured rat astrocytes and neurons.

We studied the mechanisms regulating intracellular pH (pHi) in cultured rat astrocytes and neurons, with particular reference to the influence of extracellular pH (pHe) on these mechanisms, using microspectrofluorometric monitoring from single cells, loaded with the pH-sensitive fluorophore BCECF. The pH regulatory mechanisms differ between neurons and astrocytes. The experimental data suggest the presence of a Na+/H+ and a Na(+)-independent HCO3-/Cl- exchanger in both types of cells, while astrocytes, in addition, utilise a Na(+)-dependent HCO3-/Cl- exchanger for regulating acid transients. In both cell types the pH regulatory mechanisms are strongly dependent on pHe. Thus, at pHe 6.85 or below, there was no recovery of pHi. Steady state pHi was also strongly dependent on pHe, in both astrocytes and neurons. The pHi recovery following normalisation of pHe was very rapid, (indicating that a prolonged exposure to a low pH stimulates pH regulating mechanisms), and was inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and amiloride, or in the absence of Na+. The results challenge the concept of a H(+)-regulatory site solely at the internal side of the exchanger regulating pHi to a constant value.

Buffer capacity of rat cortical tissue as well as of cultured neurons and astrocytes.

The primary objective of this work was to assess the intrinsic nonbicarbonate buffer capacity (beta i) of cultured neurons and astrocytes and to compare the beta i values obtained to those of neocortical tissue. A second objective was to determine the pH dependence of beta i. Titration of homogenates of whole-brain cortical tissue and cultured neurons with NaOH and HCl gave beta i values of 25-30 mmol.l-1 x pH-1. The buffer capacity was essentially constant in the pH range of 6-7. Astrocytes showed a higher buffer capacity and a clear relationship between beta i and pH. However, beta i decreased when pH was reduced from 7 to 6. The beta i values derived from microspectrofluorometric studies on neurons and astrocytes were surprisingly variable, ranging from 10 to 50 mmol.l-1 x pH-1. The ammonia "step method" suggested that beta i increased dramatically when pH was lowered from 7 to 6 but the propionic "step method" failed to reveal such a pH dependence. Some techniques obviously give erroneous values for beta i, presumably because changes in buffer base concentration (due to transmembrane fluxes of H+, HCO3-, NH4+ or anions of weak acids) violate the principles upon which the calculations are based. From the results obtained by direct titration and with the propionate technique, we tentatively conclude that beta i in neurons and astrocytes are approximately 20 and 30 mmol.l-1 x pH-1, respectively. We further suggest that the term "intrinsic buffer capacity", as commonly used, is redefined.

Intracellular pH regulation in cultured rat astrocytes in CO2/HCO3(-)-containing media.

We studied the regulation of intracellular pH (pHi) and the mechanisms of pHi regulation in cultured rat astrocytes using microspectrofluorometry and the pH-sensitive fluorophore 2',7'-bis(carboxyethyl-)-5,6-carboxyfluorescein. Control pHi was 7.00 +/- 0.02 in HCO3(-)-containing solutions at an extracellular pH of 7.35. Addition of 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) or amiloride decreased pHi, as did removal of extracellular Na+, while removal of extracellular Cl- was followed by an increase in pHi. Following exposure to an acid transient induced by increasing the CO2 content from 5 to 15%, pHi rapidly returned to base line, with an average initial rate of recovery of 0.10 pH units min-1 (corresponding to a mean acid extrusion rate of 6.3 +/- 0.36 mmolo l-1 min-1). Regulation of pHi was impaired when either amiloride or DIDS was added or Cl- was removed. This inhibition was enhanced when both DIDS and amiloride were present, and pHi regulation was completely blocked in the absence of extracellular Na+. The rapid regulation of pHi normally seen following a transient alkalinisation was not inhibited by amiloride or removal of Na+, but was partially inhibited by DIDS and by the absence of extracellular Cl-. The results are compatible with the presence of at least three different pHi-regulating mechanisms: a Na+/H+ antiporter, a Na(+)-dependent HCO3-/Cl- exchanger (both regulating pHi during a transient acidification), and a passive Cl-/HCO3- exchanger (regulating pHi during transient alkalinisation). The results fail to provide firm evidence of the presence of an electrogenic Na+/HCO3- symporter.