Segmental expression of the bradykinin type 2 receptor in rat efferent ducts and epididymis and its role in the regulation of aquaporin 9.

Water and solute transport in the efferent ducts and epididymis are important for the establishment of the appropriate luminal environment for sperm maturation and storage. Aquaporin 9 (AQP9) is the main water channel in the epididymis, but its regulation is still poorly understood. Components of the kinin-kallikrein system (KKS), leading to the production of bradykinin (BK), are highly expressed in the lumen of the male reproductive tract. We report here that the epididymal luminal fluid contains a significant amount of BK (2 nM). RT-PCR performed on epididymal epithelial cells isolated by laser capture microdissection (LCM) showed abundant BK type 2 receptor (Bdkrb2) mRNA expression but no type 1 receptor (Bdkrb1). Double-immunofluorescence staining for BDKRB2 and the anion exchanger AE2 (a marker of efferent duct ciliated cells) or the V-ATPase E subunit, official symbol ATP6V1E1 (a marker of epididymal clear cells), showed that BDKRB2 is expressed in the apical pole of nonciliated cells (efferent ducts) and principal cells (epididymis). Triple labeling for BDKRB2, AQP9, and ATP6V1E1 showed that BDKRB2 and AQP9 colocalize in the apical stereocilia of principal cells in the cauda epididymidis. While uniform Bdkrb2 mRNA expression was detected in the efferent ducts and along the epididymal tubule, marked variations were detected at the protein level. BDKRB2 was highest in the efferent ducts and cauda epididymidis, intermediate in the distal initial segment, moderate in the corpus, and undetectable in the proximal initial segment and the caput. Functional assays on tubules isolated from the distal initial segments showed that BK significantly increased AQP9-dependent glycerol apical membrane permeability. This effect was inhibited by BAPTA-AM, demonstrating the participation of calcium in this process. This study, therefore, identifies BK as an important regulator of AQP9.

Na+/H+ exchangers in the human eccrine sweat duct.

Using an anti-NHE1 antibody, we demonstrate the presence of a Na+/H+ exchanger of isoform 1 (NHE1) in the human eccrine sweat duct. A strong staining was observed at the basolateral membrane of the outer cell layer (NHE1basal), at the junction between inner and outer cells layers (NHE1inter), and along the lateral membranes (NHE1later) of all cells of the duct. At the luminal membrane, no staining was demonstrated either for NHE1 or NHE3. To investigate Na+/H+ mediated proton transport, straight sweat duct portions were isolated and perfused in vitro under HCO3-free conditions. In the presence of basolateral 5-ethyl-N-isopropyl amiloride (EIPA), an acidification of 0.29 +/- 0.03 pH units was observed, whereas no effect was observed with luminal EIPA. Bath sodium removal generated a stronger acidification (0.41 +/- 0.09 pH units). Removal of luminal sodium (in the absence or presence of basolateral EIPA), or low luminal chloride, led to an alkalinization, presumably due to a decrease in intracellular sodium, strongly suggesting functional activity of NHE1inter. We therefore conclude that in the sweat duct, NHE1 plays a major role in intracellular pH regulation.

V-type H+-ATPase in the human eccrine sweat duct: immunolocalization and functional demonstration.

We investigated for the presence of a vacuolar-type H+-ATPase (V-ATPase) in the human eccrine sweat duct (SD). With the use of immunocytochemistry, an anti-V- ATPase antibody showed a strong staining at the apical membrane and a weaker one in the cytoplasm. Cold preservation followed by rewarming did not alter this staining pattern. With the use of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein on isolated and perfused straight SD under HCO-free conditions and in the absence of Na+, proton extrusion was determined from the recovery rate of intracellular pH (dpH(i)/dt) following an acid load. Oligomycin (25 microM), an inhibitor of F-type ATPases, decreased dpH(i)/dt by 88 +/- 6%, suggesting a role for an ATP-dependent process involved in pH(i) recovery. Moreover, dpH(i)/dt was inhibited at 95 +/- 3% by 100 nM luminal concanamycin A, a specific inhibitor of V-ATPases, whereas 10 microM bafilomycin A1, another specific inhibitor of V-ATPases, was required to decrease dpH(i)/dt by 73%. These results strongly suggest that a V-ATPase is involved in proton secretion in the human eccrine SD.

Na+/H+-exchange activity and immunolocalization of NHE3 in rat epididymis.

An acidic luminal pH in the epididymis and vas deferens (VD) helps maintain mature sperm in an immotile state during storage. We have previously shown that the majority of proton secretion in the VD is due to the activity of the vacuolar H+-ATPase. Acidification is dependent on luminal sodium in more proximal regions of the epididymis, and we examined the distribution of the Na+/H+ exchanger, NHE3, by immunofluorescence and measured Na+/H+ exchange (NHE) activity in isolated epididymal tubules. NHE3 was detected in the apical pole of nonciliated cells of the efferent ducts and principal cells (PC) of the epididymis. No staining was seen in the distal cauda epididymidis and the VD. Isolated tubules from the distal initial segment (DIS) and proximal cauda epididymidis were perfused in vitro and loaded with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6')-carboxyfluorescein. Ethylisopropyl amiloride (EIPA) (50 microM) reduced the initial rate of intracellular pH recovery (dpH(i)/dt), in response to an acute acid load, by 51% and 45% in the DIS and cauda epididymidis, respectively. In the DIS, removal of luminal sodium reduced dpH(i)/dt by 52%. HOE694 (50 microM) inhibited all EIPA-sensitive dpH(i)/dt in the DIS, despite the previously reported absence of NHE2 in this region (Cheng Chew SB, Leung GPH, Leung PY, Tse CM, and Wong PYD, Biol Reprod 62: 755-758, 2000). These data indicate that HOE694- and EIPA-sensitive Na+/H+ exchange may participate, together with the H+-ATPase, in luminal acidification in the male excurrent duct.

Hypertonicity decreases basolateral K+ and Cl- conductances in rabbit proximal convoluted tubule.

Collapsed proximal convoluted tubules (PCT) shrink to reach a volume 20% lower than control and do not exhibit regulatory volume increase when submitted to abrupt 150 mOsm/kg hypertonic shock. The shrinking is accompanied by a rapid depolarization of the basolateral membrane potential (VBL) of 8.4 +/- 0.5 mV, with respect to a control value of -54.5 +/- 1.9 mV (n = 15). After a small and transient hyperpolarization, VBL further depolarizes to reach a steady depolarization of 19.5 +/- 1.5 mV (n = 15) with respect to control. In the post-control period, VBL returns to -55.8 +/- 1.5 mV. The basolateral partial conductance to K+ (tK) which is 0.17 +/- 0.01 (n = 5) in control condition, decreases rapidly to nonmeasurable values during the hypertonic shock and returns to 0.23 +/- 0.03 in the post-control period. The basolateral partial conductance to Cl- (tCl), which is 0.05 +/- 0.02 (n = 5) in control, also decreases in hypertonicity to a nonmeasurable value and returns to 0.03 +/- 0.01 in post control. The partial conductance mediated by the Na-HCO3 cotransporter (tNaHCO3), which is 0.48 +/- 0.06 (n = 5) in control condition, remains the same at 0.44 +/- 0.05 (n = 5) during the hypertonic period. Similarly, the membrane absolute conductance mediated by the Na-HCO3 cotransporter (GNa-HCO3) does not vary appreciably. Concomitant with cell shrinkage, intracellular pH (pHi) decreases from a control value of 7.26 +/- 0.01 to 7.13 +/- 0.02 (n = 12) and then remains constant. Return to control solution brings back pHi to 7.28 +/- 0.03. From these results, we conclude that in collapsed PCT, a sustained decrease in cellular volume leads to cell acidification and to inhibition of K+ and Cl- conductances.

Inhibition of basolateral potassium conductance by taurine in the proximal convoluted tubule.

The effect of taurine on the electrophysiological properties of the basolateral membrane of the rabbit proximal convoluted tubule was examined. Short-duration isosmotic pulses of 40 mM taurine in the bath solution induced basolateral membrane depolarizations (delta Vbl) of 6.44 +/- 0.5 mV, which were reduced by 58% in absence of Na+. In presence of barium and quinine, delta Vbl values were reduced by 55% in Na(+)-containing bath solutions and were completely abolished in Na(+)-free solutions. Continuous addition of taurine into the bath solutions for a period of 5 min induced 1) a decrease in the partial conductance of the basolateral membrane to K+ (tK) from 0.39 to 0.23; 2) an increase in the partial conductance to Cl- (tcl) from 0.055 to 0.172; 3) a rapid and transient increase of the partial conductance mediated by the Na-HCO3 cotransporter (tNaHCO3) from 0.37 to 0.52, followed by a progressive decrease to 0.29; and 4) a depolarization of the basolateral membrane of 16 mV. The absolute membrane conductance mediated by the Na-HCO3 cotransporter was not initially affected by taurine, whereas that to K (GK) initially decreased by a maximal factor of 2 after 1 min, followed by a partial recovery after 5 min, and that to Cl (GCl) increased by a factor of 4. Addition of taurine after a hypotonicity-induced cell swelling produced an inhibition of GK comparable to the one observed under isotonic conditions. These results demonstrate the presence of an electrogenic Na-dependent transport of taurine and indicate that taurine inhibits GK.

Cell volume increases of physiologic amplitude activate basolateral K and CI conductances in the rabbit proximal convoluted tubule.

The effects of increases in cell volume (CV) of physiologic amplitude, induced either hypotonically or isotonically, were studied on the three major basolateral conductances of rabbit isolated proximal convoluted tubules. CV increases were produced by a 40 mosmol/kg H2O hypotonic shock or by the isotonic replacement of mannitol by 40 mM glucose or alanine. The hypotonic shock led to an increase in CV of 17 +/- 3% (N = 8), whereas additions of glucose and alanine led to increases in CV of 22.6 +/- 2.5% (N = 7) and 28.3 +/- 3.5 (N = 5), respectively. Under all of these conditions, the absolute conductance mediated by the NaHCO3 cotransporter did not vary appreciably. This allowed determination of the variations of the absolute conductances to potassium (GK) and chloride (GCl) from their measured partial conductances. All three protocols induced significant increases in GK by factors of 2.37 +/- 0.3, 1.43 +/- 0.16, and 1.69 +/- 0.40, and in GCI by factors of 3.32 +/- 0.57, 3.68 +/- 0.75, and 3.90 +/- 1.0 during the hypotonic, glucose, and alanine protocols, respectively. These increases in GK and GCl occurred with a delay compared with the variations in CV, indicating a more elaborate signaling mechanism than stretch-activation of channels that are known to activate channels within seconds. Intracellular pH increased from 7.19 +/- 0.03 to 7.23 +/- 0.03, 7.17 +/- 0.02 to 7.20 +/- 0.02, and 7.13 +/- 0.01 to 7.16 +/- 0.01 after the hypotonic shock and the glucose and alanine additions, respectively. The study presented here demonstrates that there is a close relationship between CV and GK and GCl, independent of the means used (hypotonically or isotonically) to increase CV in rabbit proximal convoluted tubules. CV activation of GK is proposed to account for part of the increase in GK reported previously during activation of transepithelial transport.

Endogenous K+/H+ exchange activity in the Sf9 insect cell line.

The fluorescent pH indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to investigate changes in the intracellular pH (pHi) of individual Sf9 cells in response to changes in the composition of the external medium. Under standard conditions, the resting pHi was 0.2-0.3 unit lower than the extracellular pH (6.5). The extracellular concentration of K+ had a major influence on pHi. Removal of K+ from the medium resulted in a rapid but reversible acidification of the cells. The buffer capacity of the cells was a U-shaped function of pHi with a minimum at about pHi = 6.2. In the presence of K+, a change in the pH of the medium was followed by an equivalent change in pHi. In its absence, however, changes in the external pH had little effect on the pH of the cells. Following removal of K+ from the medium, the cells realkalinized at an initial rate which increased with increasing concentration of added K+. This cation was about 30 times more effective in promoting realkalinization of the cells than Li+ Na+, Rb+, and Cs+. The apparent Km for K(+)-dependent H+ efflux was about 12 mM and was slightly modulated by extracellular pH. These results strongly suggest that, in Sf9 cells, a K+/H+ antiporter plays a key role in the movement of protons across the cell membrane.

Ionic permeabilities induced by Bacillus thuringiensis in Sf9 cells.

The effect of Bacillus thuringiensis insecticidal toxins on the monovalent cation content and intracellular pH (pHi) of individual Sf9 cells of the lepidopteran species Spodoptera frugiperda (fall armyworm) was monitored with the fluorescent indicators potassium-binding benzofuran isophthalate (PBFI) and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The sequential removal of K+ and Na+ from the medium, in the presence of CryIC, a toxin which is highly active against Sf9 cells, caused sharp shifts in the fluorescence ratio of PBFI, demonstrating a rapid efflux of these ions. In Sf9 cells, pHi depends strongly on the activity of a K+/H+ exchanger. In the absence of toxin, removal of K+ from the external medium resulted in a reversible acidification of the cells. In the presence of CryIC, pHi equilibrated rapidly with that of the bathing solution. This effect was both time- and concentration-dependent. In contrast with CryIC, CryIIIA, a coleopteran-specific toxin, and CryIA(a), CryIA(b) and CryIA(c), toxins which are either inactive or poorly active against Sf9 cells, had no detectable effect on pHi. B. thuringiensis endotoxins thus appear to act specifically by increasing the permeability of the cytoplasmic membrane of susceptible cells to at least H+, K+ and Na+.

Hypotonicity increases basolateral taurine permeability in rabbit proximal convoluted tubule.

The permeabilities of the basolateral membrane of rabbit proximal convoluted tubule (PCT) to taurine (PTau) and glucose (PGlc) were estimated under control and hypotonic conditions using the initial rate of increase in cellular volume (CV) induced on isotonic replacement of 40 mM mannitol by one or the other of these substrates. Under control conditions, addition of taurine led to an increase in CV at an initial rate of 7.1 +/- 1.7%/min, leading to a cell swelling of 30.2 +/- 4.8% after 5 min (n = 6). Addition of glucose led to an increase in CV at an initial rate of 30.0 +/- 3.8%/min, leading to a cell swelling of 25.7 +/- 3.1% after 5 min (n = 7). After a period of recovery of 5 min in the absence of taurine or glucose, a 40 mosmol/kg hypotonic shock induced a cell swelling of 14.2 +/- 1.3 and 16.1 +/- 5.2%, respectively, followed by an almost complete volume regulatory decrease after 5 min. At that time, addition of taurine under continuous hypotonicity induced an increase in CV at an initial rate 2.57 +/- 0.17 times larger than that observed under the isotonic condition (P < 0.005), while addition of glucose induced an initial increase in CV identical to that observed under the isotonic condition. The increases in CV observed on addition of taurine were completely abolished in the absence of sodium under both isotonic and hypotonic conditions. The permeability to K+ was also estimated, in the absence of sodium, using the initial rate of increase in CV induced on isotonic replacement of 40 mM N-methyl-D-glucamine by K+.(ABSTRACT TRUNCATED AT 250 WORDS)

Dibutyryl cyclic adenosine monophosphate stimulates the sodium pump in rabbit renal cortical tubules.

The elevation in oxygen consumption (QO2) observed following addition of the sodium ionophore nystatin in suspensions of rabbit renal proximal tubules was significantly increased by 1 mM dibutyryl cyclic adenosine monophosphate (db-cAMP). The QO2 after subsequent addition of strophanthidin to block the sodium pump was unaffected by db-cAMP. However, 10 microM forskolin in the presence of 100 microM IBMX had no significant effect on the QO2 observed following addition of either nystatin or strophanthidin. Nevertheless, we can conclude that db-cAMP does stimulate the sodium pump activity independently of sodium transport mechanisms in the rabbit renal proximal tubule.

Isolation of single mammalian proximal tubule cells: effects of hypotonic shocks on cell yield and function.

Nord et al. [Am J Physiol 1986; 250:F539-F550] proposed a method to give a high yield of proximal tubule cells by exposing a suspension of rabbit cortical tubules to a hypotonic shock in calcium-free media. The present study describes the effects of both amplitude and duration of the hypotonic treatment on some transport-related characteristics of individual cells as compared to the starting tubule suspension. The averaged cell yield increased by an order of magnitude when the osmolality of the hypotonic solution was varied in four steps from 200 (C200 cells) to 70 mosm/kg H2O (C70 cells) while the proportion of trypan blue-positive cells progressively decreased from 33% for C200 cells to 9.5% for C70 cells. An increase in duration of the hypotonic shock from 0.5 to 6 min did not change the cell yield of C200 cells while it significantly increased that of C70 cells by 61%. Basal and ouabain-sensitive oxygen consumption (QO2) increased by 57 and 155%, respectively, from C70 to C200 cells but was approximately one order of magnitude smaller than the QO2 measured for tubule suspension. Intracellular ATP content averaged 5.5 +/- 0.8 nmol/mg for the starting tubule suspension, 4.6 +/- 0.8 nmol/mg for C70 cells but only 1.3 +/- 0.1 nmol/mg for C200 cells. The maximal velocity for phloridzin-sensitive alpha-methyl glucose transport averaged 13.7 +/- 1.7 nmol min-1 mg-1 for C70 cells and only 6.3 +/- 1.3 nmol min-1 mg-1 for C200 cells which is approximately one order of magnitude smaller than what can be expected from a tubule presenting a good access to luminal membrane. We conclude from these results that, in the process of isolating individual cells from a polarized epithelium, membrane transport rates have decreased by one order of magnitude and this reduction is intensified by a large hypotonic shock. In comparison with C200 cells, the cells obtained with a large hypotonic shock give a high yield, a larger proportion of trypan blue-negative cells and their lower overall transport rate allows the cells to maintain a better electrochemical gradient for Na and a higher intracellular ATP level.

Real importance of alanine in renal metabolism: in vitro studies in rat and dog.

In vitro studies were performed on cortical renal tubules to clarify possible differences between dog and rat with regard to alanine production and to define more precisely the role of alanine on ammonia and glucose production by the kidney. It was established that glutamate-pyruvate transaminase has an activity that is seven times lower in the rat than in the dog kidney. At the same time, alanine production from lactate, pyruvate, and glutamate is three times lower in the rat than in the dog kidney. The enzymatic reaction could be completely inhibited in a competitive fashion with aminooxyacetate. O2 consumption and CO2 production by the renal tubules were lower than that observed with glutamine. CO2 production in the rat was lowest. Production of ammonia and glucose by the kidney from alanine during acidosis averaged less than 20% of that produced with L-glutamine. Furthermore, during metabolic acidosis the production of ammonia and glucose from alanine was not augmented and failed to be influenced by increasing the concentration of alanine in the incubation medium.