Shu Uwatoko

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.

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.

C1q solid-phase radioimmunoassay: Binding properties of solid-phase C1q and evidence that C1q-binding IgG complexes in systemic lupus erythematosus are not bound to endogenous C1q

The binding properties of C1q solid-phase radioimmunoassay (C1q SPRIA) were examined, using heat-aggregated IgG (HAG) as the model of immune complexes (IC). The free, liquid-phase C1q, which was added to the C1q-coated tubes prior to the addition of HAG, had little inhibitory effect on binding of HAG to the solid-phase C1q, suggesting that the solid-phase C1q has a higher affinity for HAG than the liquid-phase C1q. On the other hand, more than 60% inhibition was seen when HAG was preincubated with the liquid-phase C1q. These binding properties of HAG to the solid-phase C1q in the presence of the liquid-phase C1q were not essentially altered by the heat inactivation or the addition of EDTA, suggesting that these pretreatments are not essential in C1q SPRIA. Next, in similar kinds of experiments, the binding properties of C1q-binding IgG complexes in SLE sera were investigated. In contrast to HAG, the binding capacity of IgG complexes in SLE sera to the solid-phase C1q was not inhibited by the preincubation with excess liquid-phase C1q. These findings suggest that C1q-binding IgG complexes in SLE sera detected by C1q SPRIA may not be bound to endogenous C1q in the circulation.

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.

Monocyte-mediated suppression of rheumatoid factor production in normal subjects

The regulatory role of normal monocytes in the production of rheumatoid factor (RF) was investigated. Monocyte depletion from normal mononuclear cells (MNC) resulted in an elaboration of IgM RF in 18 of 20 subjects. The increased RF production was inhibited by the addition to the cultures of adherent cells or their culture supernatants. These observations demonstrate the suppressive role of normal monocytes in the production of RF. Supernatants obtained from normal monocytes cultured with indomethacin could suppress the RF production, suggesting that prostaglandin E2 may not be involved in the regulation of IgM RF production.

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

Chronic neutropenia associated with C2 and C9 deficiency.

A Japanese male with a deficiency of the second and ninth components of complement associated with chronic idiopathic neutropenia is presented. In this case the second component of complement is totally deficient while the ninth component is approximately half that of normal control. Neutrophil granulocytes are constantly few, but this case shows no evidence of susceptibility to either viral or bacterial infections. His HLA type is different from that of Caucasians, suggesting that the genetic abnormality responsible for the complement deficiency of this Japanese case is different from that seen in Caucasian patients.