Koji Yoshitomi

Electrophysiological identification of alpha- and beta-intercalated cells and their distribution along the rabbit distal nephron segments.

By cable analysis and intracellular microelectrode impalement in the in vitro perfused renal tubule, we identified alpha- and beta-intercalated (IC) cells along the rabbit distal nephron segments, including the connecting tubule (CNT), the cortical collecting duct (CCD), and the outer medullary collecting duct in the inner stripe (OMCDi). IC cells were distinguished from collecting duct (CD) cells by a relatively low basolateral membrane potential (VB), a higher fractional apical membrane resistance, and apparent high Cl- conductances of the basolateral membrane. Two functionally different subtypes of IC cells in the CCD were identified based on different responses of VB upon reduction of the perfusate Cl- from 120 to 12 mM: the basolateral membrane of beta-IC cells was hyperpolarized, whereas that of alpha-IC cells was unchanged. This is in accord with the hypothesis that the apical membrane of beta-IC cells contains some Cl(-)-dependent entry processes, possibly a Cl-/HCO3- exchanger. Further characterization of electrical properties of both subtypes of IC cells were performed upon lowering bath or perfusate Cl- from 120 to 12 mM, and raising bath or perfusate K+ from 5 to 50 mM. A 10-fold increase in the perfusate K+ had no effect on VB in both subtypes of IC cells. Upon abrupt changes in Cl- or K+ concentration in the bath, a large or a small depolarization of the basolateral membrane, respectively, was observed in both subtypes of IC cells. The electrical properties of alpha- and beta-IC cells were similar among the distal nephron segments, but their distribution was different: in the CNT, which consists of IC cells and CNT cells, 97.3% (36/37) of IC cells were of the beta type. In the CCD, which consists of IC cells and CD cells, 79.8% (79/99) of IC cells were of the beta-type, whereas in the OMCDi 100% (19/19) were of the alpha type, suggesting that the beta type predominates in the earlier and the alpha type in the later segment.

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.

Regulation of cortical collecting duct function: Effect of endothelin☆

We recently showed that endothelin-1 (ET-1) increases cell Ca2+ in the mouse cortical collecting duct. To clarify the cellular action and target cell of ET-1, electrophysiologic techniques and cell Ca2+ measurement were applied to rabbit cortical collecting ducts perfused in vitro. When 10(-8) mol/L ET-1 was added to the bath, a transient increase followed by a sustained increase in cell Ca2+ was observed. A sustained increase in cell Ca2+ lasted 10 to 20 minutes and was associated with a decrease in lumen-negative transepithelial voltage. To confirm the target cell type of ET-1, confocal laser microscopy was used. An increase in cell Ca2+ was observed in the same cell, which also showed an increase in cell Ca2+ in response to arginine vasopressin (AVP), which indicated that the principal cell has ET-1 receptors in the basolateral membrane. When ET-1 was applied to the bath, total cellular membrane resistance (Ri) decreased initially and then gradually increased because of inhibition of the luminal Na+ channel. An initial decrease in Ri was considered an influx of Ca2+ from the basolateral membrane. To further determine the source of an increase in cell Ca2+, the effect of ET-1 was tested in the absence of external Ca2+ and in the presence of a Ca2+ channel blocker in the bath. Cell Ca2+ did not respond to ET-1 in the absence of external Ca2+, a condition in which an AVP-stimulated increase in cell Ca2+ was preserved.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular heterogeneity of ammonium ion transport across the basolateral membrane of the hamster medullary thick ascending limb of Henle's loop.

The epithelia of the medullary thick ascending limb (MAL) consists of two cell types, high (HBC) and low basolateral conductance (LBC) cell, depending on the K+ conductance of the basolateral membrane. The NH4+ conductance distinct from the K+ conductance has been suggested to exist in the proximal tubule, MAL cell, and Xenopus oocyte. The present study was designed to examine whether there is a conductive NH4+ transport system distinct from K+ conductance in two different cell types of the hamster MAL perfused in vitro. The basolateral membrane voltage (VB) was measured by impaling cells with conventional microelectrodes. Addition of NH4+ to the bath depolarized VB in a dose-dependent manner in both cell types. The response was maintained in the absence of HCO3-. When the VB deflection elicited upon 50 mM KCl or NH4Cl in the bath (delta VBK+ or delta VBNH4+) were compared, delta VBNH4+ was almost the same as delta VBK+ in the HBC cell, whereas the former was greater than the latter in the LBC. In the HBC cell, 10 mM Ba2+ in the bath equally suppressed both delta VBK+ and delta VBNH4+, whereas in the LBC cell it suppressed delta VBK+ with a small effect on delta VBNH4+, indicating that NH4+ is transported via a pathway distinct from Ba(2+)-sensitive K+ conductance. The VB deflection by NH4+ was unaffected by addition of 0.1 mM ouabain or 10 microM 5-nitro-2-(3-phenylpropylamino)-benzoate (a Cl- channel blocker) to the bath, excluding the contribution of the Na+, K+ pump or Cl- channel. An abrupt reduction of Na+ in the bath from 200 to 20 mM did not cause any changes in VB, suggesting that a nonselective cation channel may not account for the NH4+ transport. Amiloride at 10 microM inhibited delta VBNH4+ with a higher efficacy in the LBC cell. We conclude that a rheogenic NH4+ transport system independent from the K+ conductance exists in the basolateral membrane of the LBC cell of the hamster MAL, and may play some roles in the regulation of NH4+ transport.

Effect of parathyroid hormone on the connecting tubule from the rabbit kidney: biphasic response of transmural voltage

The effect of parathyroid hormone (PTH) on ion transport was examined by observing transmural (VT) and basolateral membrane voltage (VB) in the in vitro perfused rabbit connecting tubule. Addition of 10 nmol/l PTH to the bath induced a biphasic response of VT, with hyperpolarization followed by depolarization. Chlorophenylthioadenosine cyclic 3′,5′-monophosphate mimicked the effect of PTH, which did not change the VB in the connecting tubule cell, but mainly caused changes in the apical membrane voltage. The VT of distal convoluted tubule and the cortical collecting duct were not affected by PTH. Elimination of Na+ from the lumen abolished the PTH-induced VT responses in the connecting tubule. In the presence of 10 μmol/l amiloride, PTH caused an initial hyperpolarization but did not induce the late depolarization. The same was seen in the absence of luminal Ca2+. Either addition of 0.1 mmol/l ouabain to the bath or elimination of bath Na+ completely abolished the PTH-induced VT changes. The presence of 5 mmol/l Ba2+ in the lumen did not affect the response to PTH. These findings indicate that the initial hyperpolarization may be caused by an increase in Na+ influx across the luminal membrane through an amiloride-insensitive Na+ conductive pathway and that the late depolarization may be caused by the decrease in Na+ influx through the amiloride-sensitive Na+ conductive pathway. Luminal Ca2+ is necessary for the late depolarization caused by PTH. On the basis of these observations, we suggest that PTH initially increase influxes of both Na+ and Ca2+ across the luminal membrane and that an increase in intracellular Ca2+ in turn suppresses Na+ entry through the luminal amiloride-sensitive Na+ channel.

Mechanisms of calcium transport across the basolateral membrane of the rabbit cortical thick ascending limb of Henle's loop

Although net Ca2+ absorption takes place in the thick ascending limb of Henles loop, detailed mechanisms are unknown. Because it has been reported that the Ca2+ entry step across the luminal membrane is mediated by Ca2+ channels inserted by stimulation with parathyroid hormone, we studied the mechanism of Ca2+ transport across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) perfused in vitro by using microscopic fluorometry of cytosolic Ca2+ ([Ca2+]i) with fura-2. The resting [Ca2+]i in this segment was 49.8±4.5 nmol/l. Neither Na+ removal from the bathing solution nor addition of ouabain (0.1 mmol/l) to the bath increased [Ca2+]i, indicating that a Na+/Ca2+ exchanger in the basolateral membrane may not contribute in any major way to [Ca2+]i of CTAL. To confirm our technical accuracy, similar protocols were conducted in the connecting tubule, where the existence of a Na+/Ca2+ exchanger has been reported. In this segment, Na+ removal from the bath increased cell Ca2+ from 148.6 ±6.4 nmol/l to 647.6±132.0 nmol/l, confirming the documented fact. [Ca2+]i in the CTAL was markedly increased when 1 mmol/l NaCN was added to the bath in the absence of glucose. Calmodulin inhibitors (trifluoperazine or W-7) increased [Ca2+]i. When the bath pH was made alkaline, [Ca2+]i was also increased. This response was abolished when Ca2+ was eliminated from the bath, indicating that the Ca2+ entry across the basolateral membrane is dependent on bath pH. Increase in [Ca2+]i induced by an alkaline bath was inhibited by increased the bath K+ from 5 nmol/l to 50 mmol/l, suggesting that the Ca2+ entry system is voltage-dependent. However, the pH-dependent [Ca2+]i increase was unaffected by 0.1–10 μmol/l nicardipine in the bath. We conclude that Ca2+ transport across the basolateral membrane of CTAL is mediated by a pump-and-leak system of Ca2+ rather than a Na+/Ca2+ exchanger secondarily linked to a Na+, K+ pump.

Effects of prostaglandin E2 on membrane voltage of the connecting tubule and cortical collecting duct from rabbits.

1. Effects of prostaglandin E2 (PGE2) on ion transport were examined by observing the transmural (VT) and basolateral membrane voltage (VB) in the in vitro perfused rabbit connecting tubule (CNT) and the cortical collecting duct (CCD). 2. Addition of 1 microM PGE2 to the bath induced a biphasic response of transmural voltage (VT), with initial negative VT deflection followed by positive deflection in the CNT, but monophasic negative deflection in the CCD. Because PGE2 had no affect on the basolateral membrane voltage (VB), PGE2 mainly causes changes in the apical membrane voltage. 3. Elimination of Na+ from the lumen abolished the PGE2‐induced VT response in the CNT. In the presence of 10 microM luminal amiloride, PGE2 caused only an initial negative deflection without causing later positive deflection. The positive VT deflection induced by PGE2 in the CCD was also blocked by luminal amiloride. 4. Addition of ouabain (0.1 mM) to the bath completely abolished the PGE2‐induced VT changes in the CNT, indicating that an intact Na(+)‐K+ pump is a prerequisite for the VT response to PGE2. 5. Addition of 2 mM Ba2+ to the lumen did not affect biphasic VT response to PGE2, indicating that Ba(2+)‐sensitive K+ conductance is not involved. 6. Basolateral addition of 0.1 mM 8‐(4‐chlorophenylthio)‐cAMP inhibited only the negative VT deflection induced by PGE2. 7. The positive VT deflection was blocked by basolateral addition of 50 microM 8‐(N,N‐diethylamino)octyl 3,4,5‐trimethoxy benzoate hydrochloride (TMB‐8), an inhibitor of intracellular Ca2+ release. But elimination of luminal Ca2+ did not affect the biphasic response to PGE2. 8. These findings suggest that the initial negative VT deflection is caused by an increase in Na+ influx across the luminal membrane through an amiloride‐insensitive Na+ conductive pathway, whereas the later positive deflection is caused by the inhibition of Na+ influx through the amiloride‐sensitive Na+ conductive pathway. The cAMP messenger system may be responsible for the initial negative deflection, whereas an increased intercellular Ca2+ release from the store is necessary for the later positive deflection caused by PGE2. The response in the CCD is comparable to the later response in the CNT.

Inhibition of amiloride-sensitive apical Na+ conductance by acetylcholine in rabbit cortical collecting duct perfused in vitro.

We examined effects of acetylcholine (ACh) on the electrical parameters and intracellular Ca2+ concentration ([Ca2+]i) in the isolated rabbit cortical collecting duct (CCD) perfused in vitro using the conventional microelectrode technique and microscopic fluorescence spectrophotometry. ACh (10(-8) to 10(-5) M) in the bath caused a positive deflection of the transepithelial voltage (VT) and an increase in [Ca2+]i. Carbachol also showed similar but smaller effects. The effects of ACh were antagonized by muscarinic receptor antagonists. ACh at 10(-6) M hyperpolarized the apical membrane voltage and increased the fractional resistance of the apical membrane of the collecting duct cells accompanied by a positive deflection of VT and an increase in transepithelial resistance, whereas it did not affect these parameters in the beta-intercalated cells. In the presence of 10(-5) M amiloride in the lumen, the effects of ACh were almost completely abolished. The ACh-induced increase in [Ca2+]i is accounted for by the release of Ca2+ from intracellular store and Ca2+ entry from the bath. In the absence of Ca2+ in the bath, the ACh-induced changes in electrophysiological parameters were significantly smaller than those observed in the presence of Ca2+. Both phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutylate (PDBu), activators of protein kinase C (PKC), also inhibited the apical Na+ conductance. In the presence of PMA or PDBu in the bath, ACh did not show further inhibitory effect. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine, an inhibitor of PKC, partially attenuated the effect of ACh. These observations indicate that ACh inhibits the apical Na+ conductance partly by both increasing [Ca2+]i and activating PKC. Such an action of ACh may partially explain its natriuretic effect.

Morphological and functional heterogeneity of the thick ascending limb of Henle's loop

Abstract In this mini-review, we summarize the morphological and functional correlation of cell heterogeneity in the thick ascending limb of Henles loop. The epithelial cells in the thick ascending limb are of two morphological types, smooth surface (S) and rough surface (R) cells, classified by the morphology of the apical membrane. S cells predominate in the medullary portion, while R cells predominate in the cortical portion. S cells have low apical K+ conductance and high basolateral K+ conductance, whereas R cells have high apical and low basolateral K+ conductance. The S cell participates in K+ reabsorption, while the R cell participates in K+ secretion. Glucocorticoids act on the S cell to increase K+ reabsorption across the medullary thick ascending limb and to accumulate K+ in the renal medulla by the countercurrent multiplication system. This leads, in turn, to an increase in urinary K+ excretion by reducing K+ reabsorption from the inner medullary collecting duct. The R cell is unique in that it has specific NH4+ conductance in the basolateral membrane. It is proposed that this cell may participate in the secretion of NH4+ in the cortical thick ascending limb.