Activation of Na+-independent Mg2+ efflux by 20-hydroxyeicosatetraenoic acid in rat renal epithelial cells.

Renal epithelial cells may have Mg(2+) transport pathways that regulate intracellular free Mg(2+) concentration ([Mg(2+)](i)) and reabsorption into the body. In mag-fura 2 fluorescent measurement, extracellular Mg(2+) removal induced a Na(+)-independent [Mg(2+)](i) decrease. The [Mg(2+)](i) decrease was suppressed by methyl arachidonyl fluorophosphonate, a cytosolic and Ca(2+)-independent phospholipase A(2) (iPLA(2)) inhibitor, and bromoenol lactone, an iPLA(2) inhibitor, but it was not suppressed by a secretory phospholipase A(2) inhibitor. On the contrary, the [Mg(2+)](i) decrease was enhanced by the addition of exogenous arachidonic acid (AA). Next, we examined the effect of AA metabolite inhibitors on the [Mg(2+)](i) decrease. 17-octadecynoic acid inhibited the [Mg(2+)](i) decrease, but indomethacin and nordihydroguaiaretic acid did not. In the 17-octadecynoic acid-treated cells, 20-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (20-HETE) recovered the [Mg(2+)](i) decrease. Nicardipine inhibited both the basal and the 20-HETE-enhanced [Mg(2+)](i) decrease. These results suggest that 20-HETE is a key mediator in the activation of Na(+)-independent Mg(2+) efflux.

Polyvalent cation-sensing mechanism increased Na(+)-independent Mg(2+) transport in renal epithelial cells.

Extracellular Ca(2+)/polyvalent cation-sensing receptor (CaSR) is capable of monitoring changes in extracellular polyvalent cation concentrations. In the present study, we investigated whether CaSR agonists reinforce the decrease of intracellular free Mg(2+) concentration ([Mg(2+)](i)) induced by extracellular Mg(2+) plus Na(+) removal. Interestingly, exposure of NRK-52E renal epithelial cells to increasing extracellular Mg(2+) concentrations from 0.8 to 15 mM for 1-2 days resulted in a twofold increase in the levels of CaSR mRNA and protein. By fluorophotometer (with mag-fura 2 fluorescent dye) and atomic absorption spectrophotometer, we confirmed that activation of CaSR by neomycin (0.5 mM) or gadolinium (1 mM) reinforced the decrease of [Mg(2+)](i) induced by Mg(2+) removal in the cells cultured in 10 mM Mg(2+)-containing medium. The neomycin-induced [Mg(2+)](i) decrease was inhibited by nicardipine (50 microM), but not by verapamil (50 microM) or amiloride (0.1 mM). These results indicate that CaSR monitors extracellular Mg(2+) concentration, and probably cause activation of Na(+)-independent Mg(2+)-transport system.

Differential regulation of Na(+),K(+)-ATPase and the Na(+)-coupled glucose transporter in hypertensive rat kidney.

Several Na(+) transporters are functionally abnormal in the hypertensive rat. Here, we examined the effects of a high-salt load on renal Na(+),K(+)-ATPase and the sodium-coupled glucose transporter (SGLT1) in Dahl salt-resistant (DR) and salt-sensitive (DS) rats. The protein levels of Na(+),K(+)-ATPase and SGLT1 in the DS rat were the same as those in the DR rat, and were not affected by the high-salt load. In the DS rat, a high-salt load decreased Na(+),K(+)-ATPase activity, and this decrease coincided with a decrease in the apparent Mechaelis constant (K(m)) for ATP, but not with a change of maximum velocity (V(max)). On the contrary, a high-salt load increased SGLT1 activity in the DS rat, which coincided with an increase in the V(max) for alpha-methyl glucopyranoside. The protein level of phosphorylated tyrosine residues in Na(+),K(+)-ATPase was decreased by the high-salt load in the DS rat. The amount of phosphorylated serine was not affected by the high-salt load in DR rats, and could not be detected in DS rats. On the other hand, the amount of phosphorylated serine residues in SGLT1 was increased by the high-salt load. However, the phosphorylated tyrosine was the same for all samples. Therefore, we concluded that the high-salt load changes the protein kinase levels in DS rats, and that the regulation of Na(+),K(+)-ATPase and SGLT1 activity occurs via protein phosphorylation.

Sodium fluoride increases intracellular calcium in rat renal epithelial cell line NRK-52E.

In our previous experiment using rats, fluoride was reported to cause renal calcification, whose mechanism was deduced to be due to an increase in parathyroid hormone (PTH) secretion. However fluoride-induced renal calcification that was independent of PTH has not been understood well in the nephron of fluoride-treated animals. Thus, we examined the effect of sodium fluoride on intracellular calcium mobilization in a normal rat kidney epithelial cell line (NRK-52E cells). The calcium accumulation was found to be remarkably increased by the addition of sodium fluoride (NaF). The elevation of [Ca2+]i was demonstrated to be due to calcium entry through nifedipine-sensitive calcium channels. In addition, fluoride activates phospholipase C, but inositol 1,4,5-triphosphate (IP3) didn't induce Ca2+ release from the endoplasmic reticulum (ER). Moreover, fluoride alone was deduced to enhance the activity of ER-type Ca2+-ATPase. Finally, on the mechanism of fluoride-induced calcium accumulation in NRK-52E cells, fluoride may activate phospholipase C to generate IP3 and diacylglycerol, and these increases can be elucidated to induce calcium entry through dihydropiridine-sensitive calcium channels. Moreover, fluoride was found to stimulate calcium accumulation through ER-type Ca2+-ATPase into the endoplasmic reticulum. The elevation of ER-type Ca2+-ATPase activity by fluoride was elucidated to operate as a regulatory system to protect against abnormally higher increases in cytosolic calcium concentration via an increase of calcium influx into the endoplasmic reticulum.

Role of glucocorticoid on interleukin-6-induced cellular functions in the mouse macrophage cell line (Mm 1).

To discover a role of glucocorticoid on interleukin-6 (IL-6)-induced responses of a macrophage, we investigated the effect of IL-6 and/or dexamethasone (Dex) on cellular functions of a mouse macrophage cell line (Mm1 cells). The results obtained were as follows. (1) Dex decreased the accumulation of tumor necrosis factor-alpha induced by IL-6, whereas nitric oxide production was enhanced by Dex. Moreover, the enhancement of nitric oxide production could be demonstrated to be associated with stimulation of iNOS mRNA expression by the Dex treatment. (2) Cytotoxic activity of Mm1 cells on mouse B16 melanoma cells was much more enhanced by the co-treatment of IL-6 with Dex than IL-6 treatment alone. (3) Dex promoted further the suppression of proliferation induced by IL-6. (4) DNA fragmentation, introduced by the treatment of cells with IL-6, was further enhanced in the presence of Dex.

Characterization of inhibition by chronic treatment with lithium ion on nerve growth factor-induced neuronal differentiation of rat PC12 pheochromocytoma cells.

To understand the mechanism underlying the neurotoxicity of lithium ion, we investigated the inhibition of the nerve growth factor-induced neuronal differentiation of rat PC12 pheochromocytoma cells induced by treatment with LiCl. Incubation with 0.1-3 mM LiCl from 30 min before nerve growth factor (NGF) treatment attenuated neurite outgrowth. Moreover, incubation with 3 mM LiCl from 24 h before strongly reduced the neurite out-growth. The chronic pretreatment inhibited the NGF-caused induction of acetyl-cholinesterase activity known to be elevated by NGF in transcription-dependent processes, and inhibited expression of c-fos proto-oncogene mRNA. This pretreatment also inhibited the NGF-induced formation of inositol phosphates, accompanied by the significant accumulation of inositol monophosphate. These observations, that chronic treatment with LiCl inhibits the NGF-induced neuronal differentiation in a transcription-dependent manner and inhibits phosphoinositide metabolism, suggest a possible causal relationship between these two events.

Lead inhibits nitric oxide production transiently by mRNA level in murine macrophage cell lines.

Lead inhibited the production of nitric oxide (NO) in cytokine-induced macrophage cell lines of RAW264 and Mm1. The decrease was revealed in the concentration of lead (0.1-10 micrograms/ml) which did not decrease the viability of these cells. The inhibition was proved to be due to the suppression of an inducible type of NO synthase (iNOS) mRNA expression in RAW264 cells. This suppression was found at 24 h and then recovered to the control level (stimulated with cytokine alone) following culture. In the RAW264 cell line, the time course pattern of NO production corresponded to the iNOS mRNA expression. From these results, the inhibition of NO production by, at least in part, mRNA level could be implicated in lead-caused abnormalities in immune functions.

Effect of fluoride on the activities of the Na+/glucose cotransporter and Na+/K(+)-ATPase in brush border and basolateral membranes of rat kidney (in vitro and in vivo).

In this study, renal Na+/K(+)-ATPase activity was demonstrated to be strongly suppressed prior to the glucosuria caused by a fluoride dose (NaF 35 mg/kg, i.p.), and the 50% suppression of the enzyme activity was almost at the same dose of NaF, about 30 mg/kg, i.p. to rats. In the rats, renal Na+/glucose cotransporter activity in brush border membranes was not affected by in vivo NaF, whereas the renal Na+/K(+)-ATPase in basolateral membranes showed a dip in activity 3 h after NaF treatment of the whole animal. Moreover, it was suggested from experiments with inhibitors of calphostin C and KT5720 that protein kinase C, but not protein kinase A, may play an important role in the suppression of Na+/K(+)-ATPase following the administration of fluoride to rats. Na+/glucose cotransporter was fairly insensitive to NaF, being competitively inhibited with a Ki of about 100 mM, whereas Na+/K(+)-ATPase was much more sensitive, with a Ki of about 2 mM. From these results, the elevation of urinary glucose excretion after a single dose of fluoride was deduced to be due to suppression of the renal Na+/K(+)-ATPase activity by a direct and/or secondary action of fluoride, rather than of the corresponding Na+/glucose cotransporter activity.

Inhibitory mechanisms of antibody production by nitrogen oxides released from activated macrophages during the immune response: relationship to energy consumption.

We investigated the relationship between the sensitivity of mouse splenocytes in immune response to nitrogen oxides and energy consumption rate of the cells. Macrophage-like cells (Mm1) pretreated with IL-6 served as the source of the nitrogen oxides. The antibody production of both 2,4,6-trinitrophenyl-keyhole limpet haemocyanin-primed splenocytes and B cell hybridomas was markedly reduced; about 20-40% of splenocytes and B cell hybridomas were killed by co-culture with IL-6-treated Mm1. Cell viability and antibody production were completely restored by the addition of NG-monomethyl L-arginine to the culture medium. The cytotoxicity of the nitrogen oxides was correlated with the distance between effector and target cells. Under conditions of low cytotoxicity, antibody production by B cell hybridomas was suppressed by the nitrogen oxides, this suppression not being correlated with the reduction in cell growth. The sensitivity of the target cells differed in co-cultures of antigen-primed splenocytes and B cell hybridomas with IL-6-treated Mm1. The nitric oxide-sensitivity of the cells corresponded to their 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reducing activity and ATP consumption rate. These findings suggest that nitrogen oxides act as regulatory molecules in immune response in three ways: cytostasis, reduction of cell growth and suppression of antibody synthesis.

Nitric oxide blocks the cell cycle of mouse macrophage-like cells in the early G2+M phase.

The effects of nitric oxide produced by macrophage-like cells (Mm1) on the cell cycle were investigated. Mm1 cells lost proliferative activity in the presence of interleukin-6 (IL-6) and a subpopulation accumulated in the G2+M phase. This level increased in proportion to the incubation time. The DNA content of the cells was slightly lower than that of Mm1 cells treated with vinblastine or demecolcine, drugs which block the cell cycle in the M phase. The peak of the early G2+M phase was reduced by treatment with NG-mono-methyl-L-arginine. However, after treatment with exogenous nitric oxide or sodium nitroprusside, the G0/G1 phase increased, but the early-G2+M and the S phase decreased. The flow cytometry pattern in IL-6-treated Mm1 was the same as that of cytochalasin B-treated Mm1. These data suggest that endogenous nitric oxide affects the microfilament system of IL-6-treated Mm1 cells and blocks the cell cycle in the early G2+M phase.

K+ ionophores inhibit nerve growth factor-induced neuronal differentiation in rat adrenal pheochromocytoma PC12 cells.

Incubation with a K+/H+ ionophore nigericin attenuated the nerve growth factor (NGF)-induced neurite outgrowth in rat pheochromocytoma PC12 cells. However, a Na+/H+ ionophore monensin and a Ca2+ ionophore A23187 did not inhibited the neurite outgrowth. Nigericin also inhibited the NGF-caused induction of acetylcholinesterase and suppression of cell proliferation. These changes were dependent on the amount of the ionophore added to the culture. In addition, a distinct K+ ionophore, valinomycin, similarly inhibited the NGF-induced neuronal differentiation. These results suggest the presence of the K+ ionophore-sensitive mechanism in the NGF-induced differentiation system in PC12 cells.

Synergistic enhancement of nitrite on lysophospholipid-mediated cytolysis.

Nitrogen oxide, which is produced by activated macrophages, has been demonstrated to possess anti-tumor activity. We report herein the synergistic effect of sodium nitrite (NO2-) and/or sodium nitroprusside (SNP) on lysophospholipid (LysoPL)-mediated cytolysis. The incubation of 51Cr-labeled mouse melanoma (B16) cells with NO2- alone for 3 h at 37 degrees C did not induce cytolysis. On the other hand, NO2- significantly enhanced the cytolysis of B16 cells in the presence of lysophosphatidylcholine (LysoPC; 2.0 microM). A similar effect of NO2- on B16-cytolysis was also observed in the presence of 1-O-alkyl-sn-glycero-3-phosphocholine (LysoPAF). In addition, SNP (0.05-0.5 mM) synergistically enhanced B16-cytolysis in the presence of LysoPC. However, nitrate had no effect on the cytolysis of B16 cells treated with LysoPC. Furthermore, NO2- synergistically enhanced the hemolysis of sheep erythrocytes in the presence of LysoPC, but not in the presence of an anti-sheep erythrocyte antibody and complement. These findings suggest that NO2- directly affects membrane damage in the presence of LysoPL.

Extracellular ATP-induced regulation of epidermal growth factor signaling in cultured renal LLC-PK1 cells.

We investigated the effect of extracellular ATP on the interaction of epidermal growth factor (EGF) with its receptor in cultured renal epithelial cells, LLC-PK1. Pretreatment with ATP, but not adenosine, inhibited the binding of 125I-labeled EGF. The inhibition demonstrated by ATP resulted from a decrease in the affinity of EGF receptors for its ligand, with no change in the number of EGF receptors. Incubation of phorbol 12-myristate 13-acetate (PMA) for 30 min mimicked the ATP-mediated inhibition. On the other hand, prolonged pretreatment with PMA, which leads to disappearance of protein kinase C activity, reversed the inhibition. In addition, pretreatment with the protein kinase C inhibitor 1-(5-isoquinoline sulfonyl)-2-methylpiperazine prevented the ATP-mediated inhibition. ATP triggered an increase in inositol 1,4,5-trisphosphate levels and translocation of protein kinase C from cytosol to membranes, consist with the stimulation of phospholipase C and the activation of protein kinase C. These results demonstrate that extracellular ATP attenuates the ligand binding affinity of EGF receptor via the stimulation of phospholipase C, leading to the activation of protein kinase C in the LLC-PK1 cells.

Effect of recombinant human interleukin-6 on nitrite production of mouse myeloid leukemia cells.

The effect of recombinant human interleukin-6 (rhIL-6) on induction of nitrite (NO(-2)) production was investigated in a mouse myeloid leukemia cell line (M1) and a subclone (Mm1). NO(-2) was induced by rhIL-6 (greater than 50 U/ml) in these cell lines. Pretreatment with rhIL-6 (100 U/ml) for 1 day synergistically enhanced the production of NO(-2) in Mm1 by LPS. Furthermore, pretreatment with IL-6 (100 U/ml) shortened the lag time of induction. These results indicate that IL-6 is involved in the regulation of the NO(-2) production in macrophage-like cells.

cAMP activates Cl-/HCO-3 exchange for regulation of intracellular pH in renal epithelial cells.

The role of cAMP in regulation of intracellular pH in the confluent LLC-PK1 cells was investigated. DibutyrylcAMP and forskolin induce intracellular acidification. This acidification is inhibited by DIDS and ethacrynic acid, inhibitors of Na(+)-independent Cl-/HCO3- exchange, and by removal of extracellular Cl-. In addition, Bt2 cAMP causes Cl- entry into LLC-PK1 cells. These results suggest that cAMP activates Cl- transport, namely Na(+)-independent Cl-/HCO3- exchange, which participates in pHi regulation.

Alteration of glucose consumption and adenosine triphosphate content in bone tissue of rats with different ages: the stimulatory effect of zinc.

The alteration in bone metabolism at different ages was investigated by estimating glucose consumption and adenosine triphosphate (ATP) content in a culture system of bone tissue from 3- and 30-week-old rats. The femoral-diaphyseal tissue was removed and cultured for periods up to 48 h in Dulbecco's Modified Eagle Medium. Bone tissue was incubated at 37 degrees C in 5% CO2/95% air in a medium containing either vehicle and zinc sulfate (10(-6) - 10(-4) M). The medium glucose consumed by bone tissue clearly increased in a 48 h-culture in 3-week-old rats, while the increase in 30-week-old rats was slight. The presence of zinc sulfate (10(-6) and 10(-5) M) caused a significant increase in bone glucose consumption in 3- and 30-week-old rats. ATP content in cultured bone tissue from 30-week-old rats fairly fell in comparison with that from 3-week-old rats. Bone ATP contents in 3- and 30-week-old rats were significantly increased by the presence of zinc (10(-4) M). The present findings suggest that bone energy metabolism deteriorates with increasing age, and that zinc has a stimulatory effect in elderly rats.

Changes of phospholipase A2 inhibitory activity in the K+-sensitive actin gelation factor during the differentiation of myeloid leukemia cells.

The phospholipase A2 inhibitory activity of a 38 kDa K+-sensitive actin gelation factor in a murine leukemia cell line (M1) was examined. A specific antibody against 38 kDa protein was found to cross-react with 37 kDa protein (lipocortin) in rat peritoneal exudates. Although the native 38 kDa protein from M1 cells did not block phospholipase A2 activity, pretreatment with alkaline phosphatase produced a form that did inhibit this enzyme. However, a purified 38 kDa protein from differentiated M1 cells blocked phospholipase A2 activity without pretreatment with alkaline phosphatase. Phospholipase A2 inhibitory activity of the 38 kDa protein was not altered by addition of actin. These findings suggest that the phospholipase A2 inhibitory of our 38 kDa protein was induced during differentiation. We also proposed that our 38 kDa protein has the same epitope as lipocortin.