beta-Adrenergic agonists stimulate Mg2+ uptake in mouse distal convoluted tubule cells

greedy — Mon, 14 Sep 2009 23:57:34 +0000

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.

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.

Mg2+/Ca2+ sensing inhibits hormone-stimulated Mg2+ uptake in mouse distal convoluted tubule cells

greedy — Sat, 12 Sep 2009 01:33:23 +0000

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.

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.

intracellular magnesium; magnesium

Dr. Long Jun Dai » extracellular calcium

beta-Adrenergic agonists stimulate Mg2+ uptake in mouse distal convoluted tubule cells

Mg2+/Ca2+ sensing inhibits hormone-stimulated Mg2+ uptake in mouse distal convoluted tubule cells