Dr. Long Jun Dai

Ritchie G, Kerstan D, Dai LJ, Kang HS, Canaff L, Hendy GN, and Quamme GA:  1,25(OH)₂D₃ stimulates Mg²⁺ uptake into MDCT cells: modulation by extracellular Ca²⁺ and Mg²⁺. American Journal of Physiology 280:F868-F878, 2001.

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Abstract The distal convoluted tubule plays a significant role in renal magnesium conservation. Although the cells of the distal convoluted tubule possess the vitamin D receptor, little is known about the effects of 1a,25-dihydroxyvitamin D [1,25(OH)2D3] on magnesium transport. In this study, we examined the effect of 1,25(OH)2D3 on distal cellular magnesium uptake and the modulation of this response by extracellular Ca21 and Mg21 in an immortalized mouse distal convoluted tubule (MDCT) cell line. MDCTcells possess the divalent cation-sensing receptor (CaSR) that responds to elevation of extracellular Ca21 and Mg21 concentrations to diminish peptide hormone-stimulated Mg21 uptake. Mg21 uptake rates were determined by microfluorescence in Mg21-depleted MDCT cells. Treatment of MDCT cells with 1,25(OH)2D3 for 16–24 h stimulated basal Mg21 uptake in a concentration-dependent manner from basal levels of 164 6 5 to 210611 nM/s, representing a 2863% change. Pretreatment with actinomycin D or cycloheximide abolished 1,25(OH)2D3- stimulated.Mg21 uptake (154 6 18 nM/s), suggesting that 1,25(OH)2D3 stimulates Mg21 uptake through gene activation and protein synthesis. Elevation of extracellular Ca21 inhibited 1,25(OH)2D3-stimulated Mg21 uptake (143 6 5 nM/s). Preincubation of the cells with an antibody to the CaSR prevented the inhibition by elevated extracellular Ca21 of 1,25(OH)2D3-stimulated Mg21 uptake (202 6 8 nM/s). Treatment with an antisense CaSR mRNA oligodeoxynucleotide also abolished the effects of extracellular Ca21 on 1,25(OH)2D3-responsive Mg21 entry. This showed that elevated extracellular calcium modulates 1,25(OH)2D-mediated responses through the CaSR. In summary, 1,25(OH)2D3 stimulated Mg21 uptake in MDCT cells, and this is dependent on de novo protein synthesis. Elevation of extracellular Ca21, acting via the CaSR, inhibited 1,25(OH)2D3-stimulated Mg21 entry. These data indicate that 1,25(OH)2D3 has important effects on the control of magnesium entry in MDCT cells and these responses can be modulated by extracellular divalent cations.

Dai LJ, Kand HS, Kerstan D, Ritchie G, and Quamme GA: ATP inhibits Mg²⁺ uptake in MDCT cells via P2X purinoceptors. American Journal of Physiology 281:F833-F840, 2001.

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Abstract Nucleotides have diverse effects on water and electrolyte reabsorption within the distal tubule of the nephron. As the distal tubule is important in control of renal Mg21 balance, we determined the effects of ATP on cellular Mg21 uptake in this segment. The effects of ATP on immortalized mouse distal convoluted tubule (MDCT) cells were studied by measuring Mg21 uptake with fluorescence techniques. The mean basal Mg21 uptake rate was 165 6 6 nM/s. ATP inhibited basal Mg21 uptake and hormone-stimulated Mg21 entry by 40%. Both P2X (P2X1– P2X5 subtypes) and P2Y2 receptor subtypes were identified in MDCT cells using differential RT-PCR. Activation of both receptor subtypes with selective agonists increased intracellular Ca21 concentration, P2X purinoceptors by ionotropicgated channels, and P2Y receptors via G protein-mediated intracellular Ca21 release. The more relatively selective P2X agonists [b,g-methylene ATP (b,g-Me-ATP) and 29- and -39- O-(4-benzoyl-benzoyl)-ATP] inhibited arginine vasopressin (AVP)- and parathyroid hormone (PTH)-mediated Mg21 uptake whereas agonists more selective for P2Y purinoceptors (UTP, ADP, and 2-methylthio-ATP) were without effect. Removal of extracellular Ca21 diminished b,g-Me-ATP-mediated increase in intracellular Ca21 and inhibition of AVPstimulated Mg21 entry. We conclude from this information that ATP inhibited Mg21 uptake in MDCT cells through P2X purinoceptors expressed in this distal convoluted tubule cell line.

Kang HS, Kerstan D, Dai LJ, Ritchie G, and Quamme GA: Adenosine modulates Mg²⁺ uptake in distal convoluted tubule cells via A₁ and A₂ purinoceptors. American Journal of Physiology 281:F1141-F1147, 2001.

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Abstract Adenosine plays a role in the control of water andelectrolyte reabsorption in the distal tubule. As the distal convoluted tubule is important in the regulation of renal Mg21 balance, we determined the effects of adenosine on cellular Mg21 uptake in this segment. The effect of adenosine was studied on immortalized mouse distal convoluted tubule (MDCT) cells, a model of the intact distal convoluted tubule. The rate of Mg21 uptake was measured with fluorescence techniques using mag-fura 2. To assess Mg21 uptake, MDCT cells were first Mg21 depleted to 0.22 6 0.01 mM by being cultured in Mg21-free media for 16 h and then placed in 1.5 mM MgCl2; next, changes in intracellular Mg21 concentration ([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 137 6 16 nM/s. Adenosine stimulates basal Mg21 uptake by 41 6 10%. The selective A1 purinoceptor agonist N6-cyclopentyladenosine (CPA) increased intracellular Ca21 and decreased parathyroid hormone (PTH)-stimulated cAMP formation and PTH-mediated Mg21 uptake. On the other hand, the selective A2 receptor agonist 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-59-N-ethylcarboxamidoadenosine (CGS) stimulated Mg21 entry in a concentration- dependent fashion. CGS increased cAMP formation and the protein kinase A inhibitor RpcAMPS inhibited CGS-stimulated Mg21 uptake. Selective inhibition of phospholipase C, protein kinase C, or mitogen-activated protein kinase enzyme cascades with U-73122, Ro-31-8220, and PD- 98059, respectively, diminished A2 agonist-mediated Mg21 entry. Aldosterone potentiated CGS-mediated Mg21 entry, and elevation of extracellular Ca21 diminished CGS-responsive cAMP formation and Mg21 uptake. Accordingly, MDCT cells possess both A1 and A2 purinoceptor subtypes with intracellular signaling typical of these respective receptors. We conclude that adenosine has dual effects on Mg21 uptake in MDCT cells through separate A1 and A2 purinoceptor pathways.

Kang HS, Kerstan D, Dai LJ, Ritchie G, and Quamme GA:  beta-Adrenergic agonists stimulate Mg²⁺ uptake in mouse distal convoluted tubule cells. American Journal of Physiology 279:F1116-F1123, 2000.

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Abstract b-Adrenergic agonists influence electrolyte reabsorption in the proximal tubule, loop of Henle, and distal tubule. Although isoproterenol enhances magnesium absorption in the thick ascending limb, it is unclear what effect, if any, b-adrenergic agonists have on tubular magnesium handling. The effects of isoproterenol were studied in 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 in single MDCT cells by 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 168 6 11 nM/s. Isoproterenol stimulated Mg21 entry in a concentration-dependent manner, with a maximal response of 252 6 11 nM/s, at a concentration of 1027 M, that represented a 50 6 7% increase in uptake rate above control values. This was associated with a sixfold increase in intracellular cAMP generation. Isoproterenol-stimulated Mg21 uptake was completely inhibited with RpcAMPS, a protein kinase A inhibitor, and U-73122, a phospholipase C inhibitor, and partially blocked by RO 31– 822, a protein kinase C inhibitor. Accordingly, isoproterenolmediated Mg21 entry rates involve multiple intracellular signaling pathways. Aldosterone potentiated isoproterenolstimulated Mg21 uptake (326 6 31 nM/s), whereas elevation of extracellular Ca21 inhibited isoproterenol-mediated cAMP accumulation and Mg21 uptake, 117 6 37 nM/s. These studies demonstrate that isoproterenol stimulates Mg21 uptake in a cell line of mouse distal convoluted tubules that is modulated by hormonal and extracellular influences.

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 Insulin has been shown to be a magnesium-conserving hormone acting, in part, through stimulation of magnesium absorption within the thick ascending limb. Although the distal convoluted tubule possesses the most insulin receptors, it is unclear what, if any, actions insulin has in the distal tubule. The effects of insulin were studied on immortalized mouse distal convoluted tubule (MDCT) cells by measuring cellular cAMP formation with radioimmunoassays and Mg21 uptake with fluorescence techniques using 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 intracellular Mg21 concentration ([Mg21]i) were measured with microfluorescence. [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. Insulin stimulated Mg21 entry in a concentration-dependent manner with maximal response of 214 6 12 nM/s, which represented a 30 6 5% increase in the mean uptake rate above control values. This was associated with a 2.5-fold increase in insulin-mediated cAMP generation (52 6 3 pmol·mg protein21 ·5 min21). Genistein, a tyrosine kinase inhibitor, diminished insulin-stimulated Mg21 uptake (169 6 11 nM/s), but did not change insulin-mediated cAMP formation (47 6 5 pmol·mg protein21 ·5 min21). PTH stimulates Mg21 entry, in part, through increases in cAMP formation. Insulin and PTH increase Mg21 uptake in an additive fashion. In conclusion, insulin mediates Mg21 entry, in part, by a genistein-sensitive mechanism and by modifying hormone-responsive transport. These studies demonstrate that insulin stimulates Mg21 uptake in MDCT cells and suggest that insulin acts in concert with other peptide and steroid hormones to control magnesium conservation in the distal convoluted tubule.

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.

Dai LJ, Raymond L, Friedman PA, and Quamme GA: Mechanisms of amiloride stimulation of  Mg²⁺ uptake in immortalized mouse distal convoluted tubule cells. American Journal of Physiology 272:F249-F256, 1997.

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Abstract The distal convoluted tubule reabsorbs – 10% of the filtered magnesium, which is -70% of that delivered to it from the loop of Henle. The cellular mechanisms of magnesium transport in the distal convoluted tubule are not known. Amiloride has been reported to promote magnesium conservation. Studies were performed on immortalized mouse distal convoluted tubule (MDCT) cells to characterize distal magnesium transport and the effects of amiloride. Intracellular free Mg”+ concentration ( [Mg2+]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. Basal [Mg”+]i was 0.53 t 0.01 mM, which is -2% of the total cellular magnesium. To assess Mg2+ uptake, MDCT cells were first Mg2+ depleted (0.22 t 0.01 mM) by culturing in Mg2+-free media for 8-16 h and then placed in 5 mM MgC12, and the [Mg2+]i was determined. [Mg2+]i returned to basal levels (0.50 t 0.04 mM) with refill rate, d([Mg2+]i)ldt, of 181 t 33 r&I/s. Mg2+ entry rate was concentration dependent; a concentration of -0.1 mM resulted in half-maximal uptake rates. Mg”+ uptake was inhibited by La3+ (36 t 17 nM/s), Mn2+ (56 t 25 r&I/s), and nitrendipine (52 t 18 nM/s), but not Ca2+ (225 t 70 nnlvs). Mg2+ uptake was influenced by the transmembrane voltage; hyperpolarization either with the addition of valinomycin or the substitution of bath NaCl with NaSCN stimulated Mg2+ influx (205 ? 3 and 561 t 54 r&I/s, respectively). Depolarization with external KC1 diminished Mg2+ uptake (57 t 25 r&I/s). These data provide evidence for novel Mg2+ entry pathways in MDCT cells that are specific for Mg2+ and activated by an increase in transmembrane voltage. Because amiloride leads to a hyperpolarization of the apical membrane, we postulated that amiloride may enhance Mg2+ transport by influencing the membrane voltage. Amiloride (50 pM) increased Mg2+ uptake (235 t 79 nM/s) in a concentration.-dependent manner (half-maximal concentration of 35 uM amiloride). Accordingly, the distal diuretic, amiloride, inhibits Na+ transport, hyperpolarizes the apical membrane, and results in a stimulation of Mg2+ uptake in MDCT cells. These results provide the cellular basis for the clinical use of amiloride to bring about renal magnesium conservation.