Dr. Long Jun Dai

Dai LJ, Ritchie G, Kerstan D, Kang HS, Cole DE, and Quamme GA: Magnesium transport in  the renal distal convoluted tubule. Physiological Reviews 81:51-84, 2001.

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Abstract Magnesium Transport in the Renal Distal Convoluted Tubule. Physiol Rev 81: 51–84, 2001.—The distal tubule reabsorbs ;10% of the filtered Mg21, but this is 70–80% of that delivered from the loop of Henle. Because there is little Mg21 reabsorption beyond the distal tubule, this segment plays an important role in determining the final urinary excretion. The distal convoluted segment (DCT) is characterized by a negative luminal voltage and high intercellular resistance so that Mg21 reabsorption is transcellular and active. This review discusses recent evidence for selective and sensitive control of Mg21 transport in the DCT and emphasizes the importance of this control in normal and abnormal renal Mg21 conservation. Normally, Mg21 absorption is load dependent in the distal tubule, whether delivery is altered by increasing luminal Mg21 concentration or increasing the flow rate into the DCT. With the use of microfluorescent studies with an established mouse distal convoluted tubule (MDCT) cell line, it was shown that Mg21 uptake was concentration and voltage dependent. Peptide hormones such asparathyroid hormone, calcitonin, glucagon, and arginine vasopressin enhance Mg21 absorption in the distal tubule and stimulate Mg21 uptake into MDCT cells. Prostaglandin E2 and isoproterenol increase Mg21 entry into MDCT cells. The current evidence indicates that cAMP-dependent protein kinase A, phospholipase C, and protein kinase C signaling pathways are involved in these responses. Steroid hormones have significant effects on distal Mg21 transport. Aldosterone does not alter basal Mg21 uptake but potentiates hormone-stimulated Mg21 entry in MDCT cells by increasing hormone-mediated cAMP formation. 1,25-Dihydroxyvitamin D3, on the other hand, stimulates basal Mg21 uptake. Elevation of plasma Mg21 or Ca21 inhibits hormone-stimulated cAMP accumulation and Mg21 uptake in MDCT cells through activation of extracellular Ca21/Mg21-sensing mechanisms. Mg21 restriction selectively increases Mg21 uptake with no effect on Ca21 absorption. This intrinsic cellular adaptation provides the sensitive and selective control of distal Mg21 transport. The distally acting diuretics amiloride and chlorothiazide stimulate Mg21 uptake in MDCT cells acting through changes in membrane voltage. A number of familial and acquired disorders have been described that emphasize the diversity of cellular controls affecting renal Mg21 balance. Although it is clear that many influences affect Mg21 transport within the DCT, the transport processes have not been identified.

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.