Na+/H+ exchanger (NHE) has a critical role in regulation of intracellular pH (pHi) in the renal proximal tubular cells. It has recently been shown that dopamine inhibits NHE in the renal proximal tubules. Nevertheless, there is a dearth of information on the effects of long-term (chronic) dopamine treatment on NHE activities. This study was performed to elucidate the pHi regulatory mechanisms during the chronic dopamine treatments in renal proximal tubular OK cells. The resting pHi was greatly decreased by chronic dopamine treatments. The initial rate and the amplitude of intracellular acidification by isosmotical Na+ removal from the bath medium in chronically dopamine-treated cells were much smaller than those in control. Although it seemed to be attenuated in Na+-dependent pH regulation system, Na+-dependent pHi recovery by NHE after intracelluar acid loading in the dopamine-treated groups was not significantly different from the control. The result is interpreted to be due to the balance between the stimulation effects of lower pHi on the NHE activity and counterbalance by dopamine. Our data strongly suggested that chronic dopamine treatment increased intrinsic intracellular buffer capacity, since higher buffer capacity was induced by lower resting pHi and this effect could attenuate pHi changes under extracellular Na+-free conditions in chronically dopamine-treated cells. Our study also demonstrated that intracellular acidification induced by chronic dopamine treatments was not mediated by changes in NHE activity.
Baths ; Dopamine* ; Hydrogen-Ion Concentration* ; Kidney* ; Opossums*

OBJECTIVETo study, at the cytological level, the basic concept of Chinese medicine that "the Kidney (Shen) controls the bone".

METHODSKaempferol was isolated form Rhizoma Drynariae (Gu Sui Bu, GSB) and at several concentrations was incubated with opossum kidney (OK) cells, osteoblasts (MC3T3 E1) and human fibroblasts (HF) at cell concentrations of 2×10(4)/mL. Opossum kidney cell-conditioned culture media with kaempferol at 70 nmol/L (70kaeOKM) and without kaempferol (0OKM) were used to stimulate MC3T3 E1 and HF proliferation. The bone morphological protein receptors I and II (BMPR I and II) in OK cells were identified by immune-fluorescence staining and Western blot analysis.

RESULTSKaempferol was found to increase OK cell growth (P<0.05), but alone did not promote MC3T3 E1 or HF cell proliferation. However, although OKM by itself increased MC3T3 E1 growth by 198% (P<0.01), the 70kaeOKM further increased the growth of these cells by an additional 127% (P<0.01). It indicates that the kidney cell generates a previously unknown osteoblast growth factor (OGF) and kaempferol increases kidney cell secretion of OGF. Neither of these media had any significant effect on HF growth. Kaempferol also was found to increase the level of the BMPR II in OK cells.

CONCLUSIONSThis lends strong support to the original idea that the Kidney has a significant influence over bone-formation, as suggested by some long-standing Chinese medical beliefs, kaempferol may also serve to stimulate kidney repair and indirectly stimulate bone formation.

3T3 Cells ; Animals ; Cell Line ; Culture Media, Conditioned ; Intercellular Signaling Peptides and Proteins ; secretion ; Kaempferols ; pharmacology ; Kidney Tubules ; physiology ; secretion ; Medicine, Chinese Traditional ; Mice ; Opossums ; Osteoblasts ; chemistry

This study was undertaken to examine the effect of ethanol on Na+-dependent phosphate (Na+-Pi) uptake in opossum kidney (OK) cells, an established renal proximal tubular cell line. Ethanol inhibited Na+-dependent component of phosphate uptake in a dose-dependent manner with I50 of 8.4%, but it did not affect Na+-independent component. Similarly, ethanol inhibited Na+-dependent uptakes of glucose and amino acids (AIB, glycine, alanine, and leucine). Microsomal Na+-K+-ATPase activity was not significantly altered when cells were treated with 8% ethanol. Kinetic analysis showed that ethanol increased Km without a change in Vmax of Na+-Pi uptake. Inhibitory effect of n-alcohols on Na+-Pi uptake was dependent on the length of the hydrocarbon chain, and it resulted from the binding of one molecule of alcohol, as indicated by the Hill coefficient (n) of 0.8-1.04. Catalase significantly prevented the inhibition, but superoxide dismutase and hydroxyl radical scavengers did not alter the ethanol effect. A potent antioxidant DPPD and iron chelators did not prevent the inhibition. Pyrazole, an inhibitor of alcohol dehydrogenase, did not attenuate ethanol-induced inhibition of Na+-Pi uptake, but it prevented ethanol-induced cell death. These results suggest that ethanol may inhibit Na+-Pi uptake through a direct action on the carrier protein, although the transport system is affected by alterations in the lipid environment of the membrane.
Alanine ; Alcohol Dehydrogenase ; Amino Acids ; Antioxidants ; Catalase ; Cell Death ; Cell Line ; Chelating Agents ; Ethanol* ; Glucose ; Glycine ; Hydroxyl Radical ; Iron ; Kidney* ; Membranes* ; Opossums* ; Reactive Oxygen Species* ; Superoxide Dismutase

Cisplatin treatment increases the excretion of inorganic phosphate in vivo. However, the mechanism by which cisplatin reduces phosphate uptake through renal proximal tubular cells has not yet been elucidated. We examined the effect of cisplatin on Na+-dependent phosphate uptake in opossum kidney (OK) cells, an established proximal tubular cell line. Cells were exposed to cisplatin for an appropriate time period and phosphate uptake was measured using [32P]-phosphate. Changes in the number of phosphate transporter in membranes were evaluated by kinetic analysis, [14C]phosphonoformic acid binding, and Western blot analysis. Cisplatin inhibited phosphate uptake in a time- and dose-dependent manner, and also the Na+-dependent uptake without altering Na+-independent uptake. The cisplatin inhibition was not affected by the hydrogen peroxide scavenger catalase, but completely prevented by the hydroxyl radical scavenger dimethylthiourea. Antioxidants were ineffective in preventing the cisplatin-induced inhibition of phosphate uptake. Kinetic analysis indicated that cisplatin decreased Vmax of Na+-dependent phosphate uptake without any change in the Km value. Na+-dependent phosphonoformic acid binding was decreased by cisplatin treatment. Western blot analysis showed that cisplatin caused degradation of Na+-dependent phosphate transporter protein. Taken together, these data suggest that cisplatin inhibits phosphate transport in renal proximal tubular cells through the reduction in the number of functional phosphate transport units. Such effects of cisplatin are mediated by production of hydroxyl radicals.
Antioxidants ; Blotting, Western ; Catalase ; Cell Line ; Cisplatin ; Epithelial Cells* ; Foscarnet ; Hydrogen Peroxide ; Hydroxyl Radical ; Kidney ; Kinetics ; Membranes ; Opossums ; Phosphate Transport Proteins

The characteristics of Na+/-dependent cycloleucine uptake was investigated in OK cells with regard to substrate specificity and regulation by protein kinase C (PKC). Inhibition studies with different synthetic and natural amino acids showed a broad spectrum affinity to neutral amino acids regardless of their different side chains including branched or aromatic, indicating that the Na+/-dependent cycloleucine uptake in OK cells is mediated by System B-o or System B degree -like transporter rather than the classical System A or ASC. Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate, but not 4 alpha-PMA elicited a time-dependent biphasic stimulation of Na+/-dependent cycloleucine uptake, which produced early transient peak at 30 min and late sustained peak at 180 min. Both the early and late stimulations by PMA were due to an increase in Vmax and not due to a change in Km. PKC inhibitors blocked both the early and late stimulation by PMA, while protein synthesis inhibitors blocked the late stimulation only. These results suggest the existence and regulation by PKC of System B degree or System B degree -like broad spectrum transport system for neutral amino acids in OK cells.
Amino Acid Transport Systems* ; Amino Acids ; Amino Acids, Neutral ; Cycloleucine ; Kidney* ; Opossums* ; Phorbol 12,13-Dibutyrate ; Protein Kinase C ; Protein Synthesis Inhibitors ; Substrate Specificity

Lipopolysaccharide (LPS), given in vivo, modulates opossum esophageal motor functions by inducing the inducible nitric oxide synthase (iNOS), which increases nitric oxide (NO) production. Superoxide, a NO scavenger, is generated during this endotoxemia. Superoxide is cleared by superoxide dismutase (SOD) and catalase (CAT) to protect the physiological function of NO. This study examined whether lower esophageal sphincter (LES) motility, NO release, and iNOS and nitrotyrosine accumulation in the LES are affected by LPS in vitro. Muscle strips from the opossum LES were placed in tissue baths containing oxygenated Krebs buffer. NO release was measured with a chemiluminescence NOx analyzer, and Western blots were performed to analyze iNOS and nitrotyrosine production. The percent change in resting LES tone after a 6-hour exposure to LPS was significantly increased compared to pretreatment values. The percent LES relaxation upon electrical stimulation was significantly decreased in the control group at 6 hours, indicating that the LPS treatment had an effect. The NO concentration in the tissue bath of LPS-treated muscle without nerve stimulation was significantly less than that of LPS treatment combined with SOD/CAT or SOD/CAT alone. iNOS and nitrotyrosine were detectable and increased over time in the LES muscle of both the control and LPS-treated groups. Antioxidant enzymes may play a role in regulating NO-mediated neuromuscular functions in the LES.
Tyrosine/analogs & derivatives/chemistry ; Time Factors ; Superoxide Dismutase/metabolism ; Opossums ; Nitric Oxide Synthase Type II/metabolism ; Nitric Oxide/*chemistry/metabolism ; Muscles/metabolism ; Male ; Luminescence ; Lipopolysaccharides/chemistry/metabolism ; Female ; Esophageal Sphincter, Upper/*anatomy & histology/metabolism ; Esophageal Sphincter, Lower/*anatomy & histology/metabolism ; Catalase/metabolism ; Blotting, Western ; Antioxidants/chemistry/metabolism ; Animals