Chizuko Koseki

Localization of 25-hydroxyvitamin D3-lα-hydroxylase activity in the mammalian kidney

Summary Intra-renal distribution of 25-hydroxyvitamin D 3 (25-OH-D 3 )-lα-hydroxylase activity was studied in single nephron segments prepared from New Zealand White rabbit fetuses (26th to 28th day of gestation). Fetal kidneys were treated with collagenase and the isolated nephrons were micro-dissected into five differrent parts. Each segment of the nephron was incubated with [ 3 H]-25-OH-D 3 . Metabolites of [ 3 H]-25-OH-D 3 were separated by high performance liquid chromatography. The results show that 25-OH-D 3 -1α-hydroxylase activity is localized only in the proximal tubule and that the pars recta of proximal tubule possesses higher activity than the proximal convoluted tubule. The findings indicate that the major and probably exclusive site of 1α,25-(OH) 2 -D 3 synthesis is the proximal tubule not only in birds, as reported by Brunette et al. (8) , but also in mammals.

Binding sites for endothelin-1 in rat tissues: An autoradiographic study

Summary By tissue autoradiography in the rat, we demonstrated that receptors for endothelin-1 (ET-1) were distributed not only in the cardiovascular system but also in the noncardiovascular organs including the brain, lung, intestine, etc. In the brain, the receptors were mainly found in the basal ganglia and brainstem, in which nuclei are known to be cardiovascular regulatory sites. In addition to its direct vasoconstricting action, ET-1 may exert neural cardiovascular control as a neuropeptide.

Autoradiographic localization of [125I]-endothelin-1 binding sites in rat brain

Autoradiograms of [125I]-endothelin (ET) binding in the rat brain demonstrated that the receptors for endothelin are localized mainly in the brainstem, basal ganglia, and cerebellum. Among the many other nuclei in these regions, there also appeared nuclei which are considered to play important roles in the central nervous regulation of the cardiovascular system: they include the nuclei of the anteroventral third ventricle area, the supraoptic nucleus, and the subfornical organ, for example. From these findings, we suggest that ET-1 or its analogous peptide(s) may act as a neuropeptide regulating central nervous functions, including cardiovascular functions.

Functional heterogeneity in the hamster medullary thick ascending limb of Henle's loop

Cellular heterogeneity was examined in the hamster medullary thick ascending limb (MAL) perfused in vitro by electrophysiological measurements with an intracellular microelectrode. Random measurements of fractional resistance of basolateral membrane (RfB) revealed two cell populations, high basolateral conductance (HBC) cells havingRfB of 0.05±0.01 (n=24) and low basolateral conductance (LBC) cells havingRfB of 0.80±0.03 (n=32). Basolateral membrane potentials (VB) were not different between HBC cells and LBC cells (−72.6±1.2,n=43 vs. −70.0±1.2,n=35). Addition of 2 mmol/l Ba2+ to the bath depolarized the basolateral membrane in the HBC cells from −70.4±3.2 to −20.9±5.9 mV (n=8) but not in the LBC cells (from −74.4±1.9 to −72.0±2.1 mV). Increasing K+ or decreasing Cl− in the bathing solution caused marked positive deflection ofVB in the HBC cells but little or no change inVB in the LBC cells. Elimination of Cl− from the lumen or addition of furosemide to the lumen enhanced the potential response of the HBC cells to basolateral application of Ba2+. Accordingly, with Ba2+ present in the bath, the potential response of the HBC cells to a decrease in bath Cl− concentration was enhanced. These observations suggest that a K+ conductance exists in the basolateral membrane of HBC cells in paralled with a Cl− conductance. The basolateral cell membrane of LBC cells also contains a Cl− conductance. In these cells, but not in HBC cells, the potential response to decreasing bath Cl− concentration increased when bath pH was decreased from 7.4 to 6.0 Apparent K+ transference numbers of the luminal membrane were higher in LBC cells (0.74±0.05,n=7) than in HBC cells (0.20±0.02,n=5). From these data, we conclude: (1) there are two distinct cell types in the hamster medullary thick ascending limb; (2) there is a low Cl− conductance in basolateral membrane of LBC cells which is stimulated by low pH.

Nephron heterogeneity: gluconeogenesis from pyruvate in rabbit nephron

In mammals, gluconeogenesis is not a function of the liver only, but also of the kidney cortex. Although the liver plays the main role in blood glucose homeostasis, the kidney haa a ~~~c~t role in certain conditions such as starvation, acidosis, and liver damage [ 1,2]. In the normal physiological condition, the contribution of rat kidney to blood glucose is 25% and fo~ow~g st~ation, -45% [ 1 J. Extensive studies were done to observe the site of gluconeogenesis using various parts of rat nephron [3,4]. These studies were based on distribution of gluconeogenic key enzymes [phosphoenolpyruvate carboxy~m~e (PEP-carboxykinase), glucose-6-phosphatase and fructose-l ,6biphosphatase] along the nephron. Very few data on localization of gluconeogenesis by direct analysis of glucose synthesized by various nephron segments during incubation with precursor are available [S]. Here, we measured glucose produced by various microdissected nephron segments from pyruvate, as a substrate, in order to determine gluconeogenic sites in rabbit nephron. Gluconeogenic activity was found to be the highest in the straight portion of the proximal tubule.

Mechanisms of intracellular pH regulation in the hamster inner medullary collecting duct perfused in vitro

To examine the mechanisms of H+ transport in the mid-inner medullary collecting duct of hamsters, we measured the intracellular pH (pHi) in the in vitro perfused tubules by microscopic fluorometry using 2′,7′-bis(carboxyethyl)-carboxyfluorescein (BCECF) as a fluorescent probe. In the basal condition, pHi was 6.74±0.04 (n=45) in HCO3−-free modified Ringer solution. Either elimination of Na+ from the bath or addition of amiloride (1 mM) to the bath produced a reversible fall in pHi After acid loading with 25 mM NH4Cl, pHi spontaneously recovered with an initial recovery rate of 0.096±0.012 (n=23) pH unit/min. In the absence of ambient Na+, after removal of NH4+, the pHi remained low (5.95±0.10, n=8) and showed no signs of recovery. Subsequent restoration of Na+ only in the lumen had no effect on pHi. However, when Na+ in the bath was returned to the control level, pHi recovered completely. Amiloride (1 mM) in the bath completely inhibited the Na+-dependent pHi recovery. Furthermore, elimination of Na+ from the bath, but not from the lumen, decreased pHi from 6.97±0.07 to 6.44±0.05 (n=12) in the HCO3−/Ringer solution or 6.70±0.03 to 6.02±0.05 (n=8) in the HCO3−free solution. pHi spontaneously returned to 6.76±0.08 with a recovery rate of 0.017±0.5 pH unit/min in the presence of CO2/HCO3−, whereas it did not recover in the absence of CO2/HCO3−. Although elimination of ambient Na+ depolarized the basolateral membrane voltage (VB) from −78±1.2 to −72 ±0.6 mV (n=5, P<0.01), the level of VB was not sufficient to explain the pHi recovery solely by HCO3−entry driven by the voltage. These results indicate that (a) pHi of the inner medullary collecting duct is regulated mainly by a Na+/H+ exchanger in the basolateral membranes, (b) no apparent Na+-dependent H+ transport system exists in the luminal membranes and (c) Na+-independent H+ transport may also operate in the presence of CO2/HCO3−

Difference in molecular size of receptors for α-rat atrial natriuretic polypeptide among the kidney, aorta, and adrenal gland as identified by direct UV-photoaffinity labeling

In order to identify the molecular size of receptors for alpha-rat atrial natriuretic polypeptide (alpha-rANP), we utilized the direct UV irradiation method for photoaffinity labeling with the biologically active [125I] alpha-rANP. In the preparation of isolated glomerulus and the inner medullary collecting duct (IMCT)-rich fraction, the autoradiograms of the electrophoresed sodium dodecyl sulfate (SDS)-polyacrylamide gels showed a single radioactive band which is displaceable with unlabeled alpha-rANP. The dose-dependent displacement fit very well with a binding-inhibition curve representing the binding affinity of 6.5 X 10(-10) M. The molecular size of the ligand-receptor complex was about 65,000 daltons for both glomerulus and IMCT-rich fraction. In contrast, in homogenate of the aorta and adrenal gland, the ligand-receptor complex was 140,000 daltons.

Dual effect of N-ethylmaleimide on Cl− transport across the thin ascending limb of Henle's loop

Effects of SH reagents on Cl− transport were studied in the isolated hamster thin ascending limb of Henles loop (TAL) perfused in vitro. Parachloromercuribenzene sulfonate (PCMBS) at 10−4 M in the bath decreased the relative permeability for Cl−/Na+ (PCl/PNa), as determined by the transmural diffusion voltage (VT) generated under a NaCl concentration gradient, from 2.71±0.16 to 1.11±0.09 (P<0.001). The effect of PCMBS was prevented by the pretreatment with 10−3 M dithiothreitol (DTT). N-Ethylmaleimide (NEM) at 10−3 M in the bath exhibited a dual action on Cl− permeability of the TAL: It inhibited the Cl− permeability in fresh preparations, whereas it stimulated the Cl− permeability in the preparations pretreated with SH reagents including NEM, maleimide and PCMBS. The inhibitory effect was irreversible but the stimulatory effect was reversible. Both responses were prevented by DTT. Since dextranmaleimide did not show any inhibitory effect onPCl/PNa, the SH site responsible for the inhibition may be located inside of the cell. The stimulatory effect of NEM onPCl/PNa was markedly reduced when bath pH was reduced to 5.8. On the other hand, when the bathing fluid was made nominally Ca2+ free, the stimulatory effect of NEM was unaffected, although the basal level ofPCl/PNa was reduced. These observations suggest that the conductive Cl− pathway in the TAL is either stimulated or inhibited by modifying two distinct SH sites. The site of modulation by proton binding may exist distally to these SH sites. The regulatory mechanism involving Ca2+ may be independent of the SH regulatory sites.

Proton secretion in the upper part of the descending limb of long-looped nephron from hamsters.

Abstract The proton transport processes in the upper part of the descending limb of the long-looped nephron (LDLu) from hamsters were studied using a fluorescent dye, 2′,7′-bis(carboxyethyl)carboxyfluorescein (BCECF) in microperfused single nephron preparations. Intracellular pH (pHi), as assessed by the measurement of the fluorescence of BCECF trapped in the cytoplasm, was 7.23 ± 0.05 (n = 18) under nominally HCO3–-free conditions. Ouabain, when added to the bath, decreased pHi by 0.22 units. After an NH4Cl prepulse, the initial proton extrusion rate was 1.23 ± 0.26 (n = 9) pH units/min, and was retarded in the presence of 1 mM amiloride either in the bath or in the lumen. pHi failed to recover when Na+ was eliminated from ambient solutions. These observations suggest that Na+/H+ antiporters exist both in the apical and basolateral cell membranes. By measuring tubular fluid pH (pHt) under stopped flow conditions, we examined whether the hamster LDLu has the capacity to generate and maintain a transmural H+ gradient. After the tubular outflow was obstructed, the luminal fluid was rapidly acidified, reaching a steady-state pH of 6.84 ± 0.09 (n = 7). The steady-state pH was influenced by bath pH. Tubular fluid acidification was not observed in the absence of Na+ and was prevented by ouabain. We conclude that the hamster LDLu has the capability to generate and maintain a transmural proton gradient by proton secretion via a luminal Na+/H+ antiporter which is secondarily driven by the Na+-K+ ATPase in the basolateral membrane.

Basolateral Na/H Exchange in the Hamster Inner Medullary Collecting Duct

Segments of mid-IMCD isolated from male golden hamsters were perfused in vitro. The tubules were then loaded with a pH-sensitive fluorescent probe, BCECF, by adding 2 pM of BCECF-AM to the bath for 15 minutes at 37°C. Then, fluorescence of epithelia was measured with a microscopic fluorometer (Olympus, model OSP3) at the excitation wavelength 490 nm (pH sensitive) and 440 nm (pH insensitive) and at the emission wavelength 530 nm. At the end of the experiments, the pHi was calibrated on perfused tubules by the method of Thomas et ~ 1 . ~ using nigericin.