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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.

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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.

]]> http://jamesdai.com/longjundai/2009/09/extracellular-mg2-and-ca2-sensing-in-mouse-distal-convoluted-tubule-cells/feed/ 0 http://jamesdai.com/longjundai/2009/09/intracellular-mg2-and-magnesium-depletion-in-isolated-renal-thick-ascending-limb-cells/ http://jamesdai.com/longjundai/2009/09/intracellular-mg2-and-magnesium-depletion-in-isolated-renal-thick-ascending-limb-cells/#comments Thu, 17 Sep 2009 22:36:15 +0000 greedy http://jamesdai.com/longjundai/?p=118 Dai LJ, and Quamme GA: Intracellular Mg²⁺ and magnesium depletion in isolated renal  thick ascending limb cells. Journal of Clinical Investigation 88:1255-1264, 1991.

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Abstract Magnesium reabsorption and regulation within the kidney occur principally within the cortical thick ascending limb (cTAL) cells of the loop of Henle. Fluorometry with the dye, mag-fura- 2, was used to characterize intracellular Mg2″ concentration (IMg2j11) in single cTAL cells. Primary cell cultures were prepared from porcine kidneys using a double antibody technique (goat anti-human Tamm-Horsfall and rabbit anti-goat IgG antibodies). Basal [Ig2+"] was 0.52±0.02 mM, which was – 2% of the total cellular Mg. Cells cultured (16 h) in high magnesium media (5 mM) maintained basal [Mg2j1i, 0.48±0.02, in the normal range. However, cells cultured in nominally magnesium- free media possessed [Mg2J1j, 0.27±0.01 mM, which was associated with a significant increase in net Mg transport, (control, 0.19±0.03 and low Mg, 0.35±0.01 nmol. mg-' protein min-') as assessed by 'Mg uptake. Mg2'-depleted cells were subsequently placed in high Mg solution (5 mM) and the Mg2" refill rate was assessed by fluorescence. [Mg2"1 returned to normal basal levels, 0.53±0.03 mM, with a refill rate of 257±37 nM/s. Mg2" entry was not changed by 5.0 mM Ca2" or 2 mM Sr2+, Cd2+, Co2+, nor Ba2+ but was inhibited by Mn2+ = La3+ .,Gd3+ Ni2+ , Zn2+ Be2+ at 2 mM. Intracellular Ca2` and "Ca uptake was not altered by Mg depletion or Mg2+ refill, indicating that the entry is relatively specific to Mg2e. Mg2+ uptake was inhibited by nifedipine (117±20 nM/s), verapamil (165±34 nM/s), and diltiazem (194±19 nM/s) but enhanced by the dihydropyridine analogue, Bay K 8644 (366±71 nM/s). These antagonists and agonists were reversible with removal and IMg2+Jj subsequently returned to normal basal levels. Mg2+ entry rate was concentration and voltage dependent and maximally stimulated after 4 h in magnesium-free media. Cellular magnesium depletion results in increases in a Mg2+ refill rate which is dependent, in part, on de novo protein synthesis. These data provide evidence for novel Mg2+ entry pathways in cTAL cells which are specific for Mg2` and highly regulated. These entry pathways are likely involved with renal Mg2` homeostasis. (J. Clin. Invest. 1991. 88:1255-1264.)

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Abstract A local multiorgan donor pancreas procurement program can provide a source for optimized isolation of purified viable islets for transplantation into patients with type 1 diabetes mellitus receiving best medical therapy.

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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.

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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.

]]> http://jamesdai.com/longjundai/2009/09/atp-inhibits-mg2-uptake-in-mdct-cells-via-p2x-purinoceptors/feed/ 0 http://jamesdai.com/longjundai/2009/09/adenosine-modulates-mg2-uptake-in-distal-convoluted-tubule-cells-via-a1-and-a2-purinoceptors/ http://jamesdai.com/longjundai/2009/09/adenosine-modulates-mg2-uptake-in-distal-convoluted-tubule-cells-via-a1-and-a2-purinoceptors/#comments Tue, 15 Sep 2009 00:06:08 +0000 greedy http://jamesdai.com/longjundai/?p=95 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.

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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.

]]> http://jamesdai.com/longjundai/2009/09/beta-adrenergic-agonists-stimulate-mg2-uptake-in-mouse-distal-convoluted-tubule-cells/feed/ 0 http://jamesdai.com/longjundai/2009/09/insulin-stimulates-mg2-uptake-in-mouse-distal-convoluted-tubule-cells-2/ http://jamesdai.com/longjundai/2009/09/insulin-stimulates-mg2-uptake-in-mouse-distal-convoluted-tubule-cells-2/#comments Mon, 14 Sep 2009 23:52:59 +0000 greedy http://jamesdai.com/longjundai/?p=90 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.

]]> http://jamesdai.com/longjundai/2009/09/insulin-stimulates-mg2-uptake-in-mouse-distal-convoluted-tubule-cells-2/feed/ 0 http://jamesdai.com/longjundai/2009/09/glucagon-and-arginine-vasopressin-stimulate-mg2-uptake-in-mouse-distal-convoluted-tubule-cells/ http://jamesdai.com/longjundai/2009/09/glucagon-and-arginine-vasopressin-stimulate-mg2-uptake-in-mouse-distal-convoluted-tubule-cells/#comments Sat, 12 Sep 2009 01:40:44 +0000 greedy http://jamesdai.com/longjundai/?p=87 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.