Parathyroid hormone increases thiol proteinase activity by activation of protein kinase C in cultured kidney tubule cells (OK).

Chronic exposure (24 h) to parathyroid hormone (PTH) increases the intracellular proteolytic activity in cultured opossum kidney cells 2-fold at physiological PTH concentrations (10(-12) mol/l). This increase can be blocked by E-64, an inhibitor of thiol proteinases. The phorbol ester TPA mimicks the effect of PTH, whereas the calcium ionophore A23187 reduces the intracellular proteinase activity. Forskolin and dibutyrylic cAMP do not elevate proteinase activity. The protein kinase C inhibitor staurosporine is equally effective in blocking the TPA- and PTH-induced proteinase activity increase. These data indicate that PTH increases the intracellular thiol proteinase activity by an activation of protein kinase C and not by the cAMP dependent way.

Basic, not acidic fibroblast growth factor stimulates proliferation of cultured human retinal pigment epithelial cells.

PURPOSE: In this study, we evaluated a possible effect of acidic and basic fibroblast growth factor (aFGF, bFGF) on the proliferation of human retinal pigment epithelial (RPE) cells in culture. As the RPE is the primary source for bFGF in the retina, such an effect would suggest autocrinic actions of FGFs. METHODS: Primary cultures of human and porcine RPE and an established human RPE cell line (D407) were subjected to aFGF and bFGF at different culture conditions. Cell proliferation was determined using the BrdU non-radioactive nucleotide analogue assay, and total protein was measured colorimetrically. The cells were subjected to aFGF and bFGF from 0.1 to 100 ng/ml for 1 to 14 days. RESULTS: In the presence of 100 ng/ml bFGF, cell proliferation doubled from day 2 (143+/-12 units) to day 6 (227+/-17). This effect was neither seen without bFGF nor with aFGF at the same concentration. The stimulating effect of bFGF on cell proliferation was dose-dependent, the ED50 being around 1-10 ng/ml. The bFGF effect was markedly greater at high fetal calf serum concentration (10% vs. 1%). No bFGF effect was seen on cells of the established human RPE cell line D407 nor on primary cultures from porcine RPE. CONCLUSIONS: bFGF, in contrast to its analogue aFGF, stimulates cell proliferation in cultured human RPE cells. It may act as an autocrinic agent (secretion by and action on the same cell) and thus be a specific regulator for cell proliferation in repair and replacement of the RPE cell monolayer.

Magnetometric studies on the reticulo-endothelial system of the liver after administration of different lipid emulsions.

A comparison of the burdening effects on the reticulo-endothelial system (RES) by soybean oil emulsions with two different emulsifiers [soybean lecithin (SOB) and egg lecithin (EGG)] or by a perfluorochemical emulsion (Fluosol-DAR, PFC) was performed using a magnetometric method. This method is based on measuring the relaxation of injected iron oxide (gamma-Fe2O3) above the liver after magnetization in a strong external magnetic field. Three different methods of evaluation of the data were chosen. First, according to the conventional monoexponential function, second, according to a monoexponential function with a constant, and third, using the ratio of the initial dynamic to total magnetic field strength. In contrast to SOB and EGG, the PFC emulsion which was used depressed the RES-capacity to less than 31% of the control values (p less than 0.001). Following the administration of SOB the phagosomal motion was significantly lowered after 6 h (p less than 0.01) and 1-2 days (p less than 0.05); thereafter no significant difference of the relaxation constants remained as compared to the control group. The fatty emulsion with egg lecithin showed no significant lowering of the RES-capacity during the entire observation period (p greater than 0.05). Our results indicate that the RES-capacity is diminished not only by a PFC emulsion, but also temporarily by a soybean lecithin emulsion, though not by an egg lecithin emulsion, when given in the same dosages.

Localization and capacity of the last step of mercapturic acid biosynthesis and the reabsorption and acetylation of cysteine S-conjugates in the rat kidney.

We investigated the capacity and the localization of N-acetylation of the mercapturic acid precursor S-benzyl-L-cysteine (BC), as well as the tubular reabsorption of this compound in the rat kidney in vivo et situ by renal clearance and continuous microinfusion and microperfusion experiments. In renal clearance experiments. 450 mumol BC was infused intravenously for 180 min. During the time of BC infusion and the following 180 min, the two kidneys excreted 400 mumol or 90% of the infused BC dose as the mercapturate N-acetyl-S-benzyl-L-cysteine (AcBC). Comparison of the amounts of BC and AcBC entering the left kidney via the renal artery with those leaving it via the renal vein and the ureter showed that 0.13 +/- 0.04 mumol BC/min (mean +/- SEM) was extracted and 0.24 +/- 0.08 mumol AcBC/min was formed by one kidney. The intrarenal acetylation can account for the formation of 38% of the mercapturate excreted in the final urine. In additional experiments, 50 pmol/min [14C]BC was microinfused into single superficial tubules at three different sites. During microinfusion into early proximal tubules, the final urine contained 16.3 +/- 1.8% of the microinfused radioactivity as AcBC, but no BC. When [14C]BC was microinfused into late proximal tubules, 13.0 +/- 2.3% of the infused label was recovered as BC, 28.1 +/- 2.3% as AcBC. During microinfusion into early distal tubules, the final urine contained no AcBC, but 90.3 +/- 2.1% of the infused [14C]BC was recovered.(ABSTRACT TRUNCATED AT 250 WORDS)

Receptor-mediated endocytosis of albumin in cultured opossum kidney cells: a model for proximal tubular protein reabsorption.

The accumulation of fluorescein (FITC)-labelled bovine albumin was measured against the extracellular-fluid-phase marker FITC-inulin within confluent monolayers of the opossum kidney cell line OK. Fluorescence and electron microscopic pictures show that FITC-albumin is taken up by endocytosis and appears in a vesicular intracellular distribution. The uptake of FITC-albumin was quantified by measuring the cell-adherent fluorescence fluorimetrically. FITC-albumin uptake shows a time- and concentration-dependent saturation kinetics in contrast to the non-saturable FITC-inulin uptake, and exceeds the latter more than tenfold at low concentrations. Half-maximum saturation occurs at 20-30 mg/l. Initial FITC-albumin uptake/mg protein is stimulated by cell maturation, being six-to sevenfold higher in the confluent than in the subconfluent state, while FITC-inulin uptake is unchanged. Both an elevation of ambient osmolality to 600-750 mOsm/kg and disruption of the cytoskeleton by cytochalasin B (0.1 mmol/l) reduce initial FITC-albumin uptake by 50%-60% in a non-additive fashion. Albumin endocytosis is reduced both in acidic (pH 5.4) and alkaline (pH 8.4) medium, but does not depend on extracellular sodium, calcium or chloride. High concentrations of fetal calf serum or unlabelled albumin reduce FITC-albumin endocytosis dose-dependently. The present study is the first to investigate both the protein uptake and the fluid-phase endocytosis in a cultured proximal tubular cell line, using these cells as a model system-for proximal tubular protein reabsorption.

Properties and regulation of pH-dependent cation channels in the apical membrane of cultured proximal tubule cells.

The established opossum kidney (OK) cell line serves as a model system for ion and substrate transport in the renal proximal tubule. Previous experiments on OK cells revealed a channel-mediated Na+ conductance which is regulated by intracellular pH (pHi). In this study we report on patch clamp experiments determining the properties and pHi dependence of a cation channel located in the apical membrane. This channel is selective for sodium over chloride but discriminates poorly between the monovalent cations Na+,K+,Li+ and Cs+. Its open probability (P(o)) rises at depolarising membrane potentials. Under normal conditions the channel is inactive in the cell-attached patch mode and is activated upon excision. However, after excision the channel usually runs down within 30-90 s which cannot be overcome by either altering the Ca(2+)-concentration (10(-3) mol/l, 10(-6) mol/l, Ca(2+)-free) or adding 1 mmol/l Mg-ATP to the bath solution. In the cell-attached patch mode the channel could be activated by decreasing pHi from pH 7.4 to pH 6.5, by either the ammonium prepulse technique or the nigericin K+ method, in more than 50% of the experiments performed. In the renal proximal tubule such a non-selective cation channel would constitute a functional Na+ channel and might therefore support Na+ reabsorption especially during the intracellular acidification due to hormonal inhibition of the Na+/H+ exchanger.

Mercapturic acid formation in cultured opossum kidney cells.

We investigated the last step of mercapturic acid formation, the N-acetylation of cysteine S-conjugates, in the established opossum kidney (OK) cell line which exhibits characteristics of the proximal tubule. S-Benzyl-L-cysteine was used as a model substance for such a cysteine S-conjugate. We succeeded in showing that OK cells absorb S-benzyl-L-cysteine via an active transport system which is inhibitable by phenylalanine. This transport follows Michaelis-Menten kinetics and the two characterizing parameters were determined: the Michaelis-Menten constant Km = 1.8 mmol/l, and the maximum of the difference between the intracellular and the extracellular concentration of S-benzyl-L-cysteine delta Cmax = 19.4 mmol/l. S-Benzyl-L-cysteine is converted to N-acetyl-S-benzyl-L-cysteine at a constant rate, which is independent of the extracellular S-benzyl-L-cysteine concentration. Under the tested experimental conditions this is probably due to saturation of the microsomal N-acetyltransferase catalyzing this reaction. In conclusion, we have shown that OK cells are a suitable model for studying mercapturate formation. They take up S-benzyl-L-cysteine mainly via the same carrier as phenylalanine, which is known to be transported in the rat by the high-capacity, low-affinity neutral amino acid carrier, and convert it to N-acetyl-L-benzyl-S-cysteine.

Expression of fibroblast growth factors 1 and 2 in a human retinal pigment epithelium cell line (K1034).

The primary source of fibroblast growth factors (FGFs) is the retinal pigment epithelium (RPE). Investigations on FGF secretion in RPE primary cultures are hampered by the rapid run-down of cell vitality after a few passages. Therefore, long-term experiments require an alternative to primary cultures. We detected FGF-1 and FGF-2 in the established human K1034 cell line by immunohistochemistry. In addition, mRNA for both FGFs was found by RT-PCR. By immunohistochemistry, the signal was more pronounced with FGF-2 than with FGF-1. K1034 is capable of expressing both FGF-1 and FGF-2. With respect to these features, this cell line can be used as an alternative to primary cultured human RPE cells.

Electrical properties of cultured renal tubular cells (OK) grown in confluent monolayers.

UNLABELLED: OK cells grown to confluent monolayers were investigated by microelectrode techniques and microinjection. Cell membrane potential difference (PDm) in bicarbonate-free solution is -61.8 +/- 0.6 mV (n = 208), cell membrane resistance (Rm) amounts to 1.4 +/- 0.2 k omega. cm2 (n = 8). The apparent transference number for potassium (t'k+) is 71 +/- 3% (n = 28) and can be reduced by 3 mmol/l BaCl2 to 7.5 +/- 4.0%; (n = 8). In the presence of extracellular CO2 and HCO3- (pH 7.4) the cells acidify by 0.34 +/- 0.05 pH units (n = 12). This leads to a depolarization of PDm by 8.4 +/- 1.8 mV (n = 8), an increase in Rm by 49 +/- 10% (n = 10), and a reduction of K+-conductance to 63 +/- 5% (n = 13). Intracellular acidification by the NH4Cl-prepulse technique also inhibits K+-conductance and depolarizes the membrane. Recovery from an intracellular acid load is reflected by cell membrane repolarization. This recovery can be inhibited by amiloride (10(-3) mol/l). Na+- and Cl- -conductances could not be detected. The transepithelial resistance (Rte) of OK cell monolayers 1 day after plating is 41 +/- 6 omega.cm2 and decreases with time after plating. Intercellular communication (electrical or dye coupling) was not observed. CONCLUSIONS: 1. The membrane potential of OK cells is largely determined by a pH-sensitive, barium-blockable K+-conductance. 2. Amiloride-blockable Na+/H+-exchange is reflected by membrane potential changes via this K+-conductance. 3. Monolayers of OK cells are electrically leaky.

pHi-dependent membrane conductance of proximal tubule cells in culture (OK): differential effects on K(+)- and Na(+)-conductive channels.

Confluent monolayers of the established opossum kidney cell line were exposed to NH4Cl pulses (20 mmol/liter) during continuous intracellular measurements of pH, membrane potential (PDm) and membrane resistance (R'm) in bicarbonate-free Ringer. The removal of extracellular NH4Cl leads to an intracellular acidification from a control value of 7.33 +/- 0.08 to 6.47 +/- 0.03 (n = 7). This inhibits the absolute K conductance (gK+), reflected by a decrease of K+ transference number from 71 +/- 3% (n = 28) to 26 +/- 6% (n = 5), a 2.6 +/- 0.2-fold rise of R'm, and a depolarization by 24.2 +/- 1.5 mV (n = 52). In contrast, intracellular acidification during a block of gK+ by 3 mmol/liter BaCl2 enhances the total membrane conductance, being shown by R'm decrease to 68 +/- 7% of control and cell membrane depolarization by 9.8 +/- 2.8 mV (n = 17). Conversely, intracellular alkalinization under barium elevates R'm and hyperpolarizes PDm. The replacement of extracellular sodium by choline in the presence of BaCl2 significantly hyperpolarizes PDm and increases R'm, indicating the presence of a sodium conductance. This conductance is not inhibited by 10(-4) mol/liter amiloride (n = 7). Patch-clamp studies at the apical membrane (excised inside-out configuration) revealed two Na(+)-conductive channels with 18.8 +/- 1.4 pS (n = 10) and 146 pS single-channel conductance. Both channels are inwardly rectifying and highly selective towards Cl-. The low-conductive channel is 4.8 times more permeable for Na+ than for K+. Its open probability rises at depolarizing potentials and is dependent on the pH of the membrane inside (higher at pH 6.5 than at pH 7.8).

Electrogenic transport of neutral and dibasic amino acids in a cultured opossum kidney cell line (OK).

A study has been made of electrogenic cellular uptake of amino acids resulting in the depolarization of cell membrane potential (PDm) in confluent monolayers of an established opossum kidney (OK) cell line using conventional and pH-selective microelectrodes. Apical superfusion of neutral and dibasic amino acids rapidly depolarized the cell membrane, while application of acidic amino acids had no effect on PDm. The depolarization in response to L-phenylalanine and L-arginine was stereoselective, dose-dependent and saturable. 10 mmol/l of L-phenylalanine reduced PDm by 4.8 +/- 0.4 mV (n = 51) in a completely sodium-dependent way and the concentration necessary for half-maximal depolarization (C1/2) was about 1.5 mmol/l. On the other hand, the C1/2 for L-arginine was about 0.02 mmol/l. The maximal depolarization produced by L-arginine (measured at 10 mmol/l) amounted to 6.8 +/- 1.2 mV (n = 10) and this was not affected when extracellular sodium was replaced by choline (6.3 +/- 1.2 mV; n = 10). The depolarizations induced by L-phenylalanine and L-arginine were significantly additive (p less than 0.001). The intracellular pH of OK cells was 7.09 +/- 0.03 (n = 11) and did not change during L-arginine application. We conclude that (1) carrier-mediated uptake of neutral and dibasic amino acids into OK cells is at least partially electrogenic. (2) L-Phenylalanine is transported by a Na+-symport. (3) In contrast, L-arginine depolarizes PDm independently of extracellular sodium. (4) Electrogenic uptake of acidic amino acids is not detectable in OK cells.

Renal tubular transport of glutathione in rat kidney.

Filtered glutathione (gamma-glutamyl-cysteinyl-glycine or GSH) is rapidly hydrolyzed by brush-border enzymes facing the tubular lumen and is reabsorbed in the form of the constituent amino acids. The first step of hydrolysis is catalyzed by gamma-glutamyltransferase (gamma-GT). We investigated localization and capacity of the rat renal glutathione degradation/reabsorption during elevation of the filtered load (intravenous infusion of 12 resp. 18 mumol GSH/min). Fractional excretion went up from about 0.003 to 0.31 +/- 0.02 SEM during infusion of the lower and to 0.49 +/- 0.03 SEM during infusion of the higher glutathione dose. GSH degradation/reabsorption took place along the entire proximal tubule and was partially saturated by a 150-200-fold elevation of the normal filtered load. Net reabsorption of GSH up to the last accessible superficial loop was significantly lower during infusion of 18 mumol GSH/min (0.3 mumol/min) than during infusion of 12 mumol GSH/min (1.6 mumol/min). In further experiments, infusion of 18 mumol GSH/min was preceded by the i.v. administration of acivicin (0.5 mmol/kg body wt.), an inhibitor of gamma-GT. In these experiments, fractional glutathione deliveries to late proximal and early distal tubules did not significantly differ from 1, fractional excretion of GSH at the same time was 1.46 +/- 0.11 SEM, revealing net secretion of GSH with the final urine. Tubular secretion of GSH in the acivicin-treated animals occurred either in distal tubules and/or collecting ducts or in the proximal tubules of deep nephrons which are not accessible to micropuncture.(ABSTRACT TRUNCATED AT 250 WORDS)