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Kang, Hyung Sub, Dirk Kerstan, Long-Jun Dai, Gordon Ritchie, and Gary A. Quamme. Adenosine modulates [Mg.sup.2+] uptake in distal convoluted tubule cells via [A.sub.1] and [A.sub.2] purinoceptors. Am J Physiol Renal Physiol 281: F1141-F1147, 2001.--tk;1Adenosine plays a role in the control of water and electrolyte reabsorption in the distal tubule. As the distal convoluted tubule is important in the regulation of renal [Mg.sup.2+] balance, we determined the effects of adenosine on cellular [Mg.sup.2+] 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 [Mg.sup.2+] uptake was measured with fluorescence techniques using mag-fura 2. To assess [Mg.sup.2+] uptake, MDCT cells were first [Mg.sup.2+] depleted to 0.22 [+ or -] 0.01 mM by being cultured in [Mg.sup.2+]-free media for 16 h and then placed in 1.5 mM Mg[Cl.sub.2]; next, changes in intracellular [Mg.sup.2+] concentration ([[Mg.sup.2+]][.sub.i]) were determined. [[Mg.sup.2+]][.sub.i] returned to basal levels, 0.53 [+ or -] 0.02 mM, with a mean refill rate, d([[Mg.sup.2+]][.sub.i])/dt, of 137 [+ or -] 16 nM/s. Adenosine stimulates basal [Mg.sup.2+] uptake by 41 [+ or -] 10%. The selective [A.sub.1] purinoceptor agonist [N.sup.6]-cyclopentyladenosine (CPA) increased intracellular [Ca.sup.2+] and decreased parathyroid hormone (PTH)-stimulated cAMP formation and PTH-mediated [Mg.sup.2+] uptake. On the other hand, the selective [A.sub.2] receptor agonist 2- [p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethyl-carboxamidoadenosine (CGS) stimulated [Mg.sup.2+] entry in a concentration-dependent fashion. CGS increased cAMP formation and the protein kinase A inhibitor RpcAMPS inhibited CGS-stimulated [Mg.sup.2+] 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 [A.sub.2] agonist-mediated [Mg.sup.2+] entry. Aldosterone potentiated CGS-mediated [Mg.sup.2+] entry, and elevation of extracellular [Ca.sup.2+] diminished CGS-responsive cAMP formation and [Mg.sup.2+] uptake. Accordingly, MDCT cells possess both [A.sub.1] and [A.sub.2] purinoceptor subtypes with intracellular signaling typical of these respective receptors. We conclude that adenosine has dual effects on [Mg.sup.2+] uptake in MDCT cells through separate [A.sub.1] and [A.sub.2] purinoceptor pathways. intracellular magnesium; fluorescence; intracellular calcium transients; intracellular adenosine 3'5-cyclic monophosphate; immortalized mouse distal convoluted tubule cells Received 16 March 2001; accepted in final form 25 July 2001

The physiological effect of the prostaglandin E2 (PGE2) on Mg2+ intake into immortalized mouse distal convoluted tubule (MDCT) cells was investigated. It was established that PGE2 mediates renal hemodynamics, inhibits the absorption of sodium chloride within the thick ascending limb and regulates sodium and water transport in the cortical collecting duct. It was discovered that PGE2 activated the reabsorption of Mg2+ within the MDCT cells through the increased levels of cellular cyclic adenosine monophosphate.

Arginine vasopressin (AVP) and glucagon modulates magnesium uptake in the distal convoluted tubules and the loop of Henle. An immortalized line of mouse distal convoluted cells was used in the investigation of the mechanism of modulation. The calcium channel blocker nifedipine inhibited magnesium uptake as did the inhibition of protein kinase A. The latter finding indicate that cyclic adenosine monophosphate is important in signal transduction in the modulation of magnesium uptake by glucagon and vasopressin.

An immortalized cell line from the distal convoluted tubule of mice was used in the investigation of the effect of aldosterone on magnesium reabsorption at the distal convoluted tubule. By itself, aldosterone does not have any effect on magnesium reabsorption. However, it potentiates the magnesium reabsorption promoting effects of glucagon and arginine vasopressin. Cyclic adenosine monophosphate and protein synthesis appear to play a role in the aldosterone-modulated increase in magnesium reabsorption.

The cellular effects of acid-base changes on Mg2+ uptake in immortalized mouse distal convoluted tubule (MDCT) cells which exhibited the functional properties of intact distal convoluted tubules. Analysis of Mg2+ uptake in rodent MDCT cells indicated the role of the transmembrane electrical potential in modulating Mg2+ uptake in MDCT cells. Furthermore, elevations in the transmembrane voltage activated Ca2+ uptake and increased the driving force for Ca2+ movement into the cell.

Immortalized mouse distal convoluted tubules (MDCT) were analyzed to determine the properties and cellular mechanisms that mediate renal Mg2+ uptake. Analysis of magnesium transport in immortalized murine MDCT indicated the role of transmembrane voltage in affecting Mg2+ uptake. Furthermore, administration of amiloride enhanced Mg2+ renal tubular transport in a dose-dependent manner.

Phosphorylation events regulate the sodium/calcium ion (Na+/Ca2+) exchange in cells of the porcine cortical thick ascending limb (CTAL) of the loop of Henle. Phosphorylation changes either the exchanger protein or other proteins that alter the exchange activity. The cytoplasmic region of the protein is necessary for the regulation. Treatment of CTAL cells with phosphatase suppressors increases Ca2+ exchange even at normal Na+ levels. Calmodulin suppressors inhibit the exchange at normal Na+ levels. This indicates that phosphatases and calmodulin-dependent kinases regulate the exchange.

Sodium-calcium exchange in the cortical segment of the thick ascending limb (CTAL) of Henle's loop was investigated using microfluorometry techniques in isolated porcine CTAL cells. The addition of ouabain elevated intracellular sodium concentration while removal of extracellular sodium or voltage depolarization with potassium chloride resulted in the immediate increase in calcium concentration, suggesting the presence of the Na+/Ca2+ exchanger. This result was confirmed using hybridization and DNA sequencing techniques.

1,25[(OH).sub.2][D.sub.3] stimulates [Mg.sup.2+] uptake into mouse MDCT cells: modulation by extracellular [Ca.sup.2+] and [Mg.sup.2+]. Am J Physiol Renal Physiol 280: F868-F878, 2001.--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 1[Alpha],25-dihydroxyvitamin D [1,25[(OH).sub.2][D.sub.3]] on magnesium transport. In this study, we examined the effect of 1,25[(OH).sub.2][D.sub.3] on distal cellular magnesium uptake and the modulation of this response by extracellular [Ca.sup.2+] and [Mg.sup.2+] in an immortalized mouse distal convoluted tubule (MDCT) cell line. MDCT cells possess the divalent cation-sensing receptor (CaSR) that responds to elevation of extracellular [Ca.sup.2+] and [Mg.sup.2+] concentrations to diminish peptide hormone-stimulated [Mg.sup.2+] uptake. [Mg.sup.2+] uptake rates were determined by microfluorescence in [Mg.sup.2+]-depleted MDCT cells. Treatment of MDCT cells with 1,25[(OH).sub.2][D.sub.3] for 16-24 h stimulated basal [Mg.sup.2+] uptake in a concentration-dependent manner from basal levels of 164 [+ or -] 5 to 210 [+ or -] 11 nM/s, representing a 28 [+ or -] 3% change. Pretreatment with actinomycin D or cycloheximide abolished 1,25[(OH).sub.2][D.sub.3]-stimulated [Mg.sup.2+] uptake (154 [+ or -] 18 nM/s), suggesting that 1,25[(OH).sub.2][D.sub.3] stimulates [Mg.sup.2+] uptake through gene activation and protein synthesis. Elevation of extracellular [Ca.sup.2+] inhibited 1,25[(OH).sub.2][D.sub.3]-stimulated [Mg.sup.2+] uptake (143 [+ or -] 5 nM/s). Preincubation of the cells with an antibody to the CaSR prevented the inhibition by elevated extracellular [Ca.sup.2+] of 1,25[(OH).sub.2][D.sub.3]-stimulated [Mg.sup.2+] uptake (202 [+ or -] 8 nM/s). Treatment with an anti-sense CaSR mRNA oligodeoxynucleotide also abolished the effects of extracellular [Ca.sup.2+] on 1,25[(OH).sub.2][D.sub.3]-responsive [Mg.sup.2+] entry. This showed that elevated extracellular calcium modulates 1,25[(OH).sub.2]D-mediated responses through the CaSR. In summary, 1,25[(OH).sub.2][D.sub.3] stimulated [Mg.sup.2+] uptake in MDCT cells, and this is dependent on de novo protein synthesis. Elevation of extracellular [Ca.sup.2+], acting via the CaSR, inhibited 1,25[(OH).sub.2][D.sub.3]-stimulated [Mg.sup.2+] entry. These data indicate that 1,25[(OH).sub.2][D.sub.3] has important effects on the control of magnesium entry in MDCT cells and these responses can be modulated by extracellular divalent cations. 1[Alpha],25-dihydroxyvitamin D; calcium/magnesium-sensing receptor; adenosine 3',5'-cyclic monophosphate measurements; intracellular magnesium determinations; magnesium uptake; fluorescence

I. INTRODUCTION: IMPORTANCE OF THE DISTAL CONVOLUTED TUBULE IN RENAL MAGNESIUM BALANCE Control of total body magnesium homeostasis principally resides within the nephron segments of the kidney. The proximal tubule [...]

Kang, Hyung Sub, Dirk Kerstan, Long-Jun Dai, Gordon Ritchie, and Gary A. Quamme. [Beta]-Adrenergic agonists stimulate [Mg.sup.2+] uptake in mouse distal convoluted tubule cells. Am J Physiol Renal Physiol 279: F1116-F1123, 2000.--[Beta]-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, [Beta]-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 [Mg.sup.2+] uptake with fluorescence techniques. Intracellular free [Mg.sup.2+] concentration ([[[Mg.sup.2+]].sub.i]) was measured in single MDCT cells by using microfluorescence with mag-fura-2. To assess [Mg.sup.2+] uptake, MDCT cells were first [Mg.sup.2+] depleted to 0.22 [+ or -] 0.01 mM by culturing in [Mg.sup.2+] -free media for 16 h and then placed in 1.5 mM Mg[Cl.sub.2], and the changes in [[[Mg.sup.2+]].sub.i] were determined. [[[Mg.sup.2+]].sub.i] returned to basal levels, 0.53 [+ or -] 0.02 mM, with a mean refill rate, d([[[Mg.sup.2+]].sub.i])/dt, of 168 [+ or -] 11 nM/s. Isoproterenol stimulated [Mg.sup.2+] entry in a concentration-dependent manner, with a maximal response of 252 [+ or -] 11 nM/s, at a concentration of [10.sup.-7] M, that represented a 50 [+ or -] 7% increase in uptake rate above control values. This was associated with a sixfold increase in intracellular cAMP generation. Isoproterenol-stimulated [Mg.sup.2+] 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, isoproterenol-mediated [Mg.sup.2+] entry rates involve multiple intracellular signaling pathways. Aldosterone potentiated isoproterenol-stimulated [Mg.sup.2+] uptake (326 [+ or -] 31 nM/s), whereas elevation of extracellular [Ca.sup.2+] inhibited isoproterenol-mediated cAMP accumulation and [Mg.sup.2+] uptake, 117 [+ or -] 37 nM/s. These studies demonstrate that isoproterenol stimulates [Mg.sup.2+] uptake in a cell line of mouse distal convoluted tubules that is modulated by hormonal and extracellular influences. isoproterenol; propranolol; intracellular magnesium; fluorescence; intracellular 3',5'-cyclic adenosine monophosphate; protein kinase A; phospholipase C; protein kinase C; phorbol ester; aldosterone; extracellular calcium

Dai, Long-Jun, Gordon Ritchie, Brian W. Bapty, Dirk Kerstan, and Gary A. Quamme. Insulin stimulates [Mg.sup.2+] uptake in mouse distal convoluted tubule cells. Am. J. Physiol. 277 (Renal Physiol. 46): F907-F913, 1999.--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 [Mg.sup.2+] uptake with fluorescence techniques using mag-fura 2. To assess [Mg.sup.2+] uptake, MDCT cells were first [Mg.sup.2+] depleted to 0.22 [+ or -] 0.01 mM by culturing in [Mg.sup.2+]-free media for 16 h and then placed in 1.5 mM Mg[Cl.sub.2], and the changes in intracellular [Mg.sup.2+] concentration ([[[Mg.sup.2+]].sub.i]) were measured with microfluorescence. [[[Mg.sup.2+]].sub.i] returned to basal levels, 0.53 [+ or -] 0.02 mM, with a mean refill rate, d([[[Mg.sup.2+]].sub.i])/dt, of 164 [+ or -] 5 nM/s. Insulin stimulated [Mg.sup.2+] entry in a concentration-dependent manner with maximal response of 214 [+ or -] 12 nM/s, which represented a 30 [+ or -] 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 [+ or -] 3 pmol [multiplied by] mg [protein.sup.-1] [multiplied by] 5 [min.sup.-1]). Genistein, a tyrosine kinase inhibitor, diminished insulin-stimulated [Mg.sup.2+] uptake (169 [+ or -] 11 nM/s), but did not change insulin-mediated cAMP formation (47 [+ or -] 5 pmol [multiplied by] mg [protein.sup.-1] [multiplied by] 5 [min.sup.-1]). PTH stimulates [Mg.sup.2+] entry, in part, through increases in cAMP formation. Insulin and PTH increase [Mg.sup.2+] uptake in an additive fashion. In conclusion, insulin mediates [Mg.sup.2+] entry, in part, by a genistein-sensitive mechanism and by modifying hormone-responsive transport. These studies demonstrate that insulin stimulates [Mg.sup.2+] 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. intracellular magnesium; fluorescence; insulin; genistein; tyrosine kinase; intracellular adenosine 3',5'-cyclic monophosphate; parathyroid hormone; aldosterone; extracellular calcium sensing; neomycin