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.

Insulin stimulates Mg2+ uptake in mouse distal convoluted tubule cells

greedy — Mon, 14 Sep 2009 23:52:59 +0000

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.

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.

Dr. Long Jun Dai » aldosterone

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

Insulin stimulates Mg2+ uptake in mouse distal convoluted tubule cells