Dr. Long Jun Dai » intracellular adenosine 38 58-cyclic monophosphate

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.

Glucagon and arginine vasopressin stimulate Mg2+ uptake in mouse distal convoluted tubule cells

greedy — Sat, 12 Sep 2009 01:40:44 +0000

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.

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

esters 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.

intracellular magnesium; fluorescence; channel blockers

Insulin stimulates Mg2+ uptake in mouse distal convoluted tubule cells

greedy — Sat, 12 Sep 2009 01:09:13 +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.

Download paper

Abstract Prostaglandins have diverse effects on renal electrolyte reabsorption, inhibiting NaCl absorption in the thick ascending limb and modulating sodium and calcium transport in cortical collecting cells. It is unclear what effect, if any, prostaglandins have on tubular magnesium handling. The effects of prostaglandin E2 (PGE2) were studied on 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 on single MDCT cells 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 173 6 8 nM/s. Indomethacin, 5 μM, diminished basal Mg21 uptake, suggesting that endogenous prostaglandins may stimulate Mg21 entry in control cells. PGE2 stimulated Mg21 entry in a concentration-dependent manner with maximal response of 311 6 12 nM/s, at a concentration of 1027 M, which represented an 80 6 3% increase in uptake rate above control values. This was associated with a sixfold increase in intracellular cAMP generation. PGE2-stimulated Mg21 uptake was completely inhibited with the Rp diastereoisomer of adenosine 38,58-cyclic monophosphothionate (RpcAMPS), a protein kinaseAinhibitor, and U-73122, a phospholipase C inhibitor, and partially by chelerythrine, a protein kinase C inhibitor. Accordingly, PGE2-mediated Mg21 entry rates involve multiple intracellular signaling pathways. These studies demonstrate that PGE2 stimulates Mg21 uptake in a cell line of MDCT.

Prostaglandins have diverse

effects on renal electrolyte reabsorption, inhibiting

NaCl absorption in the thick ascending limb and modulating

sodium and calcium transport in cortical collecting cells. It is

unclear what effect, if any, prostaglandins have on tubular

magnesium handling. The effects of prostaglandin E2 (PGE2)

were studied on 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 on single MDCT cells 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 173 6 8 nM/s. Indomethacin, 5 μM, diminished

basal Mg21 uptake, suggesting that endogenous prostaglandins

may stimulate Mg21 entry in control cells. PGE2

stimulated Mg21 entry in a concentration-dependent manner

with maximal response of 311 6 12 nM/s, at a concentration

of 1027 M, which represented an 80 6 3% increase in uptake

rate above control values. This was associated with a sixfold

increase in intracellular cAMP generation. PGE2-stimulated

Mg21 uptake was completely inhibited with the Rp diastereoisomer

of adenosine 38,58-cyclic monophosphothionate (RpcAMPS),

a protein kinaseAinhibitor, and U-73122, a phospholipase

C inhibitor, and partially by chelerythrine, a protein

kinase C inhibitor. Accordingly, PGE2-mediated Mg21 entry

rates involve multiple intracellular signaling pathways. These

studies demonstrate that PGE2 stimulates Mg21 uptake in a

cell line of MDCT.

intracellular magnesium; fluorescence