Dr. Long Jun Dai

Bapty BW, Dai LJ, Ritchie G, Jirik F, Canaff L, Hendy GN, and Quamme GA: Extracellular  Mg²⁺-and Ca²⁺-sensing in mouse distal convoluted tubule cells. Kidney International 53:583-592,     1998.

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Abstract An immortalized cell line (designated MDCT) has been extensively used to investigate the cellular mechanisms of electrolyte transport within the mouse distal convoluted tubule. Mouse distal convoluted tubule cells possess many of the functional characteristics of the in vivo distal convoluted tubule. In the present study, we show that MDCT cells also possess a polyvalent cation-sensing mechanism that is responsive to extracellular magnesium and calcium. Southern hybridization of reverse transcribed-polymerase chain reaction (RT-PCR) products, sequence determination and Western analysis indicated that the calcium-sensing receptor (Casr) is expressed in MDCT cells. Using microfluorescence of single MDCT cells to determine cytosolic Ca21 signaling, it was shown that the polyvalent cation-sensing mechanism is sensitive to extracellular magnesium concentration ([Mg21]o) and extracellular calcium concentration ([Ca21]o) in concentration ranges normally observed in the plasma. Moreover, both [Mg21]o and [Ca21]o were effective in generating intracellular Ca21 transients in the presence of large concentrations of [Ca21]o and [Mg21]o, respectively. These responses are unlike those observed for the Casr in the parathyroid gland. Finally, activation of the polycationsensitive mechanism with either [Mg21]o or [Ca21]o inhibited parathyroid hormone-, calcitonin-, glucagon- and arginine vasopressin-stimulated cAMP release in MDCT cells. These studies indicate that immortalized MDCT cells possess a polyvalent cation-sensing mechanism and emphasize the important role this mechanism plays in modulating intracellular signals in response to changes in [Mg21]o as well as in [Ca21]o.

Dai LJ, Bapty BW, Ritchie G, and Quamme GA: Glucagon and arginine vasopressin stimulate Mg²⁺ uptake in mouse distal convoluted tubule cells. American Journal of Physiology 274:F328-F335, 1998.

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Abstract Glucagon and arginine vasopressin (AVP) enhance renal magnesium conservation through actions within the loop of Henle and the distal tubule. Studies were performed on an immortalized mouse distal convoluted tubule (MDCT) cell line to characterize the cellular actions of these hormones on Mg21 transport in this segment of the distal tubule. Glucagon and AVP increased cellular cAMP concentrations by about fivefold above basal levels in normal and Mg21-depleted cells. Intracellular free Mg21 concentration ([Mg21]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. To assess Mg21 uptake, MDCT cells were first Mg21 depleted (0.22 6 0.01 mM) by culturing in Mg21-free media for 16 h and then placed in 1.5 mM MgCl2, and the [Mg21]i was determined. [Mg21]i returned to basal levels, 0.53 6 0.02 mM, with a mean refill rate, d([Mg21]i)/dt, of 164 6 5 nM/s. Both glucagon and AVP stimulated Mg21 uptake into MDCT cells, 196 6 11 and 189 6 6 nM/s, respectively, at concentrations of 3 3 1027 M and 1027 M, respectively. Enhanced Mg21 uptake for each of the hormones was concentration dependent and inhibited by the channel blocker, nifedipine. Hormone stimulation of Mg21 entry was not dependent on protein synthesis. 8-Bromo-cAMP, 1024 M, enhanced Mg21 uptake (225 6 13 nM/s), whereas phorbol testers were without effect. Finally, protein kinaseAinhibition prevented glucagon and AVP stimulation of Mg21 uptake, supporting the notion that the cAMP pathway is important as expected in the hormone action. These studies demonstrate that glucagon and AVP stimulate Mg21 uptake in MDCT cells and suggest that these hormones act to control magnesium conservation in the convoluted segment of the distal tubule.

Dai LJ, Ritchie G, Bapty BW, and Quamme GA: Aldosterone potentiates hormone-stimulated  Mg²⁺ uptake in mouse distal convoluted tubule cells. American Journal of Physiology 274:F336-F341, 1998.

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Abstract The distal convoluted tubule reabsorbs significant amounts of filtered magnesium that is under hormonal control. In this study, we describe the effects of aldosterone on Mg21 uptake in an immortalized mouse distal convoluted tubule (MDCT) cell line. Intracellular free Mg21 concentration ([Mg21]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. To determine Mg21 entry rate into MDCT cells, they were first Mg21 depleted ([Mg21]i, 0.22 6 0.01 mM) by culturing in Mg21-free media for 16 h and then placed in 1.5mMMgCl2. The rate of change in [Mg21]i as measured as a function of time, d([Mg21]i)/dt, was 164 6 5 nM/s in control cells. We have shown that glucagon or arginine vasopressin (AVP) stimulates Mg21 entry by 63% and 15%, respectively. Incubation of MDCT cells with aldosterone for 16 h did not change the rate of Mg21 uptake (172 6 8 nM/s). However, aldosterone potentiated glucagon- and AVP-stimulated Mg21 uptake rate up to 330 6 39 and 224 6 6 nM/s, respectively. Aldosterone also potentiated glucagon- and AVP-induced intracellular cAMP accumulation in a concentration-independent manner. As cAMP stimulates Mg21 entry in MDCT cells, it is inferred that aldosterone may stimulate Mg21 uptake through intracellular signaling pathways involving cAMP. The actions of aldosterone were dependent on de novo protein synthesis, as pretreatment of the cells with cycloheximide inhibited aldosterone potentiation of hormone stimulation of Mg21 uptake and cAMP accumulation. These studies with MDCT cells suggest that aldosterone may modulate the effects of hormones acting within the distal convoluted tubule to control magnesium absorption.

Bapty BW, Dai LJ, Ritchie G, Jirik F, Canaff L, Hendy GN, and Quamme GA: Mg²⁺/Ca²⁺ sensing inhibits hormone-stimulated Mg²⁺ uptake in mouse distal convoluted tubule cells. American Journal of Physiology 275:F353-F360, 1998.

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Abstract The distal convoluted tubule plays a significant role in renal magnesium conservation. An immortalized mouse distal convoluted tubule (MDCT) cell line has been extensively used to study the cellular mechanisms of magnesium transport in this nephron segment. MDCT cells possess an extracellular polyvalent cation-sensing mechanism responsive to Mg21, Ca21, and neomycin. The present studies determined the effect of Mg21/ Ca21 sensing on hormone-mediated cAMP formation and Mg21 uptake in MDCT cells. MDCT cells were Mg21 depleted by culturing in Mg21-free media for 16 h, and Mg21 uptake was measured by microfluorescence after placing the depleted cells in 1.5 mM MgCl2. The mean rate of Mg21 uptake was 164 6 5 nM/s in control MDCT cells. Activation of Mg21/Ca21 sensing with neomycin did not affect basal Mg21 uptake (155 6 5 nM/s). We have previously reported that treatment of MDCT cells with either glucagon or arginine vasopressin (AVP) stimulated Mg21 entry. In the present studies, the addition of extracellular Mg21 or Ca21 inhibited glucagon- and AVP-stimulated cAMP formation and Mg21 uptake in concentration-dependent manner with half-maximal concentrations of ,1.5 and 3.0 mM, respectively. Exogenous cAMP or forskolin stimulated Mg21 uptake in the presence of Mg21/Ca21 sensing activation.We infer from these studies that Mg21/Ca21-sensing mechanisms located in the distal convoluted tubule may play a role in control of distal magnesium absorption.

Dai LJ, Bapty BW, Ritchie G, Kerstan D, and Quamme GA: Insulin stimulates Mg²⁺ uptake in  mouse distal convoluted tubule cells. American Journal of Physiology 277:F907-F913, 1999.

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Abstract Prostaglandins have diverse effects on renal electrolyte reabsorption, inhibiting NaCl absorption in the thick ascending limb and modulating sodium and calcium transport in cortical collecting cells. It is unclear what effect, if any, prostaglandins have on tubular magnesium handling. The effects of prostaglandin E2 (PGE2) were studied on immortalized mouse distal convoluted tubule (MDCT) cells by measuring cellular cAMP formation with radioimmunoassays and Mg21 uptake with fluorescence techniques. Intracellular free Mg21 concentration ([Mg21]i) was measured on single MDCT cells using microfluorescence with mag-fura 2. To assess Mg21 uptake, MDCT cells were first Mg21 depleted to 0.22 6 0.01 mM by culturing in Mg21-free media for 16 h and then placed in 1.5 mM MgCl2, and the changes in [Mg21]i were determined. [Mg21]i returned to basal levels, 0.53 6 0.02 mM, with a mean refill rate, d([Mg21]i)/dt, of 173 6 8 nM/s. Indomethacin, 5 μM, diminished basal Mg21 uptake, suggesting that endogenous prostaglandins may stimulate Mg21 entry in control cells. PGE2 stimulated Mg21 entry in a concentration-dependent manner with maximal response of 311 6 12 nM/s, at a concentration of 1027 M, which represented an 80 6 3% increase in uptake rate above control values. This was associated with a sixfold increase in intracellular cAMP generation. PGE2-stimulated Mg21 uptake was completely inhibited with the Rp diastereoisomer of adenosine 38,58-cyclic monophosphothionate (RpcAMPS), a protein kinaseAinhibitor, and U-73122, a phospholipase C inhibitor, and partially by chelerythrine, a protein kinase C inhibitor. Accordingly, PGE2-mediated Mg21 entry rates involve multiple intracellular signaling pathways. These studies demonstrate that PGE2 stimulates Mg21 uptake in a cell line of MDCT.