Shigeo Taniguchi

Calpain-mediated degradation of p35 to p25 in postmortem human and rat brains

Tau in Alzheimer neurofibrillary tangles has been shown to be hyperphosphorylated and CDK5, GSK3, MAP kinase and SAP kinases are the candidate kinases for the phosphorylation of tau. Recently, it was reported that the conversion of p35, the activator of CDK5, to p25 was upregulated in Alzheimers disease (AD) brains, and that p35 is cleaved to yield p25 by calpain. Here we show that p35 is rapidly cleaved to p25 in rat and human brains within a short postmortem delay and that the conversion of p35 to p25 is partially dependent on calpain activity. Immunoblot analysis of brains prepared from patients with AD or age‐matched control individuals with a short postmortem delay revealed no specific increase in the levels of p25 in AD brains, whereas the levels of active form of calpain were increased in AD brains compared to the those in controls. These observations suggest that the conversion of p35 to p25 is a postmortem degradation event and may not be upregulated in AD brains.

The neuropathology of frontotemporal lobar degeneration with respect to the cytological and biochemical characteristics of tau protein

Pathological examinations, using a panel of tau and other antibodies, were performed on the brains from 55 consecutively acquired cases of frontotemporal lobar degeneration (FTLD). Clinically, these comprised 31 cases of frontotemporal dementia (FTD), 10 cases of motor neurone disease inclusion dementia (MNDID), seven cases of progressive aphasia (PA), four cases of semantic dementia (SD) and three cases of progressive apraxia (PAX). Tau pathology, in the form of neurofibrillary tangles (NFTs) and glial cell tangles, was present in six cases of FTD with parkinsonism linked to chromosome 17, five of these cases resulting from +16 splice‐site mutation and one from +13 mutation in the tau gene. The insoluble tau proteins were comprised mostly of four‐repeat (4‐R) isoforms. Eight other cases of FTD, one of PA and all three cases of PAX showed tau‐positive inclusions (Pick bodies) and swollen cells (Pick cells), characteristic of Picks disease. In these cases, the insoluble tau proteins were present in most instances as three‐repeat (3‐R) tau isoforms, although two cases with a mixture of 3‐R and 4‐R isoforms were seen. One other case of FTD showed an unusual pathology characterized by massive extracellular deposition of tau protein, composed of 4‐R tau isoforms, within white matter without neuronal or glial cell inclusions. However, 33 (60%) of 55 FTLD cases showed no tau pathology in the brain, except for the rare NFTs, composed of a mix of 3‐R and 4‐R isoforms, in some of the more elderly cases. Of these 33 cases, 13 had FTD, 10 had MNDID, six had PA and four had SD. The pathological changes present were those of a superficial cortical laminar microvacuolation with mild subpial and subcortical gliosis; the 10 MNDID cases had ubiquitin‐positive inclusions in the cerebral cortex and hippocampus. These 33 nontau FTLD cases, along with five Alzheimers disease (AD) and six Huntingtons disease (HD) cases with severe pathology, showed a variable loss of soluble tau proteins, broadly comparable with the extent of neuronal loss from the cortex and loss of the intracortical perikaryal marker, NeuN, but unrelated to proteins within afferent projection fibres such as neurofilament and α‐synuclein. Levels of tau mRNA were decreased in parallel in the tau‐negative FTLD cases and in the severe AD and HD cases. Hence, the loss of tau from these 33 nontau FTLD cases is just one aspect of a neurodegenerative process that destroys many components of the nerve cell machinery and does not represent a specific disordering of the cells ability to form tau proteins or incorporate these into microtubules.

Functional and molecular evidence for Na+-HCO3– cotransporter in human corneal endothelial cells

Abstract. Although bicarbonate transport in corneal endothelium has been suggested to be coupled to Na+, the underlying molecular mechanism has not been clarified. In the present study we investigated whether a recently cloned Na+-HCO3– cotransporter (NBC-1) is responsible for this process, and, if so, whether the endothelium expresses a separate isoform or one of the other two isoforms that have recently been identified (kNBC-1 from kidney and pNBC-1 from pancreas). Using primers designed for specific and common regions we demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) that both kNBC-1 and pNBC-1 are expressed in cultured human corneal endothelial cells. In addition functional studies with a pH-sensitive fluorescence probe were performed. In the presence of HCO3–/CO2 a pH regulatory process was demonstrated which depends on the presence of Na+ and membrane potential, but is independent of Cl– and is inhibited by the disulfonic stilbene DIDS. These results support the presence of NBC-1 as the major bicarbonate transport system in corneal endothelium.

Biphasic Regulation of Na+-HCO3− Cotransporter by Angiotensin II Type 1A Receptor

Abstract—Although angiotensin (Ang) II is known to regulate renal proximal transport in a biphasic way, the receptor subtype(s) mediating these Ang II effects remained to be established. To clarify this issue, we compared the effects of Ang II in wild-type mice (WT) and Ang II type 1A receptor–deficient mice (AT1A KO). The Na+-HCO3− cotransporter (NBC) activity, analyzed in isolated nonperfused tubules with a fluorescent probe, was stimulated by 10−10 mol/L Ang II but was inhibited by 10−6 mol/L Ang II in WT. Although valsartan (AT1 antagonist) blocked both stimulation and inhibition by Ang II, PD 123,319 (AT2 antagonist) did not modify these effects of Ang II. In AT1A KO, in contrast, this biphasic regulation was lost, and only stimulation of NBC activity by 10−6 mol/L Ang II was observed. This stimulation was blocked by valsartan but not by PD 123,319. More than 10−8 mol/L Ang II induced a transient increase in cell Ca2+ concentrations in WT, which was again blocked by valsartan but not by PD 123,319. However, up to 10−5 mol/L Ang II did not increase cell Ca2+ concentrations in AT1A KO. Finally, the addition of arachidonic acid inhibited the NBC activity similarly in WT and AT1A KO, suggesting that the inhibitory pathway involving P-450 metabolites is preserved in AT1A KO. These results indicate that AT1A mediates the biphasic regulation of NBC. Although low-level expression of AT1B could be responsible for the stimulation by 10−6 mol/L Ang II in AT1A KO, no evidence was obtained for AT2 involvement.

Positive and negative regulation of cell proliferation through prostaglandin receptors in NIH-3T3 cells.

Among major eicosanoids and their analogs, prostaglandin (PG) F2α > PGD2 > PGE1 ≥ PGE2 > iloprost, a stable agonist of PGI2, dose‐dependently stimualted DNA synthesis in quiescent NIH‐3T3 cells. PGF2α, PGD2, and PGE2, in that order, formed inositol phosphates and elevated intracellular CA2+ ([Ca2+]i) but did not form cAMP nor inhibit forskolin‐induced cAMP formation. Iloprost, PGI2, and PGE1 induced cAMP formation dose‐dependently with an ED50 of around 10−7 M, and PGE2 at more than 10−6 M did it. [3H]PGF2α and [3H]PGD2 bindings to membranes from NIH‐3T3 cells were displaced in the order of PGF2α > PGD2 ≥ PGE2, while [3H]PGE2 binding was displaced by PGE2 > PGD2 ≥ PGF2α. Expression of mRNA encoding EP1 and EP4 (EP2) subtypes could be detected by reverse transcription‐ polymerase chain reaction using primers specific for EP1 and EP4 (EP2) cDNAs, but not that of EP3 subtype mRNA. The dose dependence of cAMP formation on iloprost and PGI2 and that of [Ca2+]i, elevation on PGF2α, D2, and E2 were similar to that of [3H]thymidine incorporation on the corresponding agonists. Fluprostenol (1 μM), a PGF2α receptor agonist> 17‐phenyl‐trinor‐PGE2 (1 μM), an EP1 receptor agonist stimulated [3H]thymidine incorporation, but an EP3 receptor agonist, ONO‐AP‐324, nor an EP4 (EP2) receptor agonist, 11‐deoxy‐PGE1 (1 μM) did not. Iloprost, dibutyryl cAMP, forskolin, or cholera toxin, when applied alone, enhanced [3H]thymidine incorporation, while they inhibited [3H]thymidine incorporation induced by submaximal concentrations of PGF2α or epidermal growth factor (EGF), when applied within 12 hr after agonist stimulation.

Intracellular pH regulatory mechanism in a human renal proximal cell line (HKC-8): evidence for Na+/H+ exchanger, Cl–/HCO3– exchanger and Na+-HCO3– cotransporter

In the present study we investigated whether an immortalized human renal proximal cell line, HKC-8, expresses a recently cloned Na+-HCO3– cotransporter (NBC-1) and, if so, which isoform (kNBC-1 from kidney or pNBC-1 from pancreas) is expressed in this cell line. Cell pH (pHi) measurements using a pH-sensitive fluorescence probe in the absence of HCO3–/CO2 revealed the presence of a Na+/H+ exchanger that required high concentrations of amiloride for full inhibition. In the presence of HCO3–/CO2 another pHi recovery process, dependent on Na+ but independent of Cl–, was identified. This process was electrogenic and was inhibited by 4,4′-diisothiocyanatodihydrostilbene-2,2′-disulphonic acid (DIDS), being consistent with the Na+-HCO3– cotransporter. In addition, the pHi responses to Cl– removal were compatible with the presence of a Na+-independent Cl–/HCO3– exchanger that was also inhibited by DIDS. Reverse transcriptase polymerase chain reaction (RT-PCR) using primers designed for specific and common regions detected mRNAs of both kNBC-1 and pNBC-1 and Western blot analysis confirmed the expression of NBC-1 protein. These results indicate that HKC-8 has transport activities similar to intact proximal tubules and also suggest that both kNBC-1 and pNBC-1 may contribute to the Na+-HCO3– cotransport activity in this cell line.

Effect of parathyroid hormone on acid/base transport in rabbit renal proximal tubule S3 segment

The effect of parathyroid hormone (PTH) on acid/base transport in isolated rabbit renal proximal tubule S3 segment was investigated with double-barreled and conventional microelectrodes. PTH (10 nM) induced a small depolarization and enhanced the initial rates of cell pH (pHi) increase and cell Cl− ([Cl−]i) decrease in response to bath Cl− removal by 28.0±2.1% and 31.0±6.4% respectively. The calculated initial HCO3− influx to bath Cl− removal was also enhanced by 28%. On the other hand, PTH reduced the initial rate of pHi decrease to luminal Na+ removal in the absence of HCO3−/CO2 by 20.4±3.9%. The PTH-induced depolarization was not accompanied with changes in steadystate pHi or [Cl−]i levels, but was greatly attenuated in the presence of ouabain (0.1 mM). Either dibutyrylcAMP (0.1 mM) plus theophylline (1 mM) or forskolin (10 μM) alone could reproduce all the effects of PTH. These results indicate that (a) PTH inhibits the luminal Na+/H+ exchanger but stimulates the basolateral Cl−/HCO3− exchanger in the S3 segment; (b) the PTH-induced depolarization largely results from inhibition of Na+/K+-ATPase and (c) all these effects are at least partly mediated by a cAMP-dependent mechanism.

Activation of the basolateral Cl- conductance by cyclic AMP in rabbit renal proximal tubule S3 segment.

The regulatory mechanism of basolateral Cl− conductance in rabbit renal proximal tubule S3 segments was investigated with conventional and Cl− sensitive microelectrodes. After the basolateral Cl−/HCO3−exchanger was blocked by 4,4′-diisothiocyanatostilbene-2, 2′-disulphonic acid (DIDS) we increased the bath K+ concentration from 5 mmol/l to 20 mmol/l, which depolarized the cells and thereby increased intracellular Cl− activity ([Cl−]i). This [Cl−]i response was enhanced by +63% in the presence of forskolin (20 μmol/l), by +40% in the presence of dibutyryl adenosine 3′,5′-cyclic monophosphate (db-cAMP) (1 mmol/l) and by +44% in the presence of parathyroid hormone (PTH, 10 nmol/l), whereas it was inhibited by a Cl− channel blocker, indanyl-oxyacetic acid (IAA-94, 0.3 mmol/l). In addition, forskolin, PTH and chlorophenylthio-cAMP enhanced the electrogenic response to removal of bath Cl− after the blockade of K+ conductance, and this activation was also sensitive to IAA-94. On the other hand, 2 μmol/l ionomycin and 0.5 μmol/l phorbol myristate failed to activate the [Cl−]i response to elevation of bath K+ concentration and the electrogenic response to Cl− removal, and ionomycin had no effect even in the absence of DIDS. These results indicate that this basolateral Cl− conductance can be activated by cAMP, while neither the increase in cytosolic Ca2+ nor the activation of protein kinase C has direct effects on this conductance.

Distribution of β2-adrenergic receptor mRNA expression along the hamster nephron segments

Distribution of β2‐adrenergic receptor mRNA expression along the microdissected hamster nephron segments was examined by the reverse transcription‐polymerase chain reaction (RT‐PCR) technique. Conventional RT‐PCR using a set of primers on separate exons could not be applied for the detection of β2‐adrenergic receptor mRNA because of its intronless nature. We used the ‘rapid amplification of cDNA ends’ protocol [(1985) Proc. Natl. Acad. Sci. USA 85, 8998‐9002] as a maneuver for RT‐PCR of an intronless gene. Using this method, we successfully located hamster β2‐adrenergic receptor mRNA only in glomeruli and early proximal convoluted tubule along the nephron segments tested.

Stable Measurements of Intracellular Calcium Concentrations with Fura-2 in Isolated Rabbit Renal Proximal Tubules

The experiments were done to determine the optimal conditions for measurement of intracellular Ca2+ concentrations ([Ca2+]i) with a fluorescence dye fura-2 in isolated