Dr. Long Jun Dai » fluorescence

Intracellular Mg2+ and magnesium depletion in isolated renal thick ascending limb cells

greedy — Thu, 17 Sep 2009 22:36:15 +0000

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.)

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.) Key words:

cortical thick ascending limb * epithelial cells * fluorescencekidney

* Mg2` entry * primary culture

Address reprint requests to Dr. Gary Quamme, Department of Medicine

1,25(OH)2D3 stimulates Mg2+ uptake into MDCT cells: modulation by extracellular Ca2+ and Mg2+

greedy — Thu, 17 Sep 2009 22:14:46 +0000

Ritchie G, Kerstan D, Dai LJ, Kang HS, Canaff L, Hendy GN, and Quamme GA: 1,25(OH)₂D₃stimulates Mg²⁺ uptake into MDCT cells: modulation by extracellular Ca²⁺ and Mg²⁺. American Journal of Physiology 280:F868-F878, 2001.

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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.

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.

1a,25-dihydroxyvitamin D; calcium/magnesium-

ATP inhibits Mg2+ uptake in MDCT cells via P2X purinoceptors

greedy — Tue, 15 Sep 2009 00:09:22 +0000

Dai LJ, Kand HS, Kerstan D, Ritchie G, and Quamme GA: ATP inhibits Mg²⁺ uptake in MDCT cells via P2X purinoceptors. American Journal of Physiology 281:F833-F840, 2001.

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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.

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.

intracellular magnesium, fluorescence; adenosine triphosphate

Adenosine modulates Mg2+ uptake in distal convoluted tubule cells via A1 and A2 purinoceptors

greedy — Tue, 15 Sep 2009 00:06:08 +0000

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.

electrolyte 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.

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.

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

Aldosterone potentiates hormone-stimulated Mg2+ uptake in mouse distal convoluted tubule cells

greedy — Sat, 12 Sep 2009 01:36:30 +0000

Dai LJ, Ritchie G, Bapty BW, and Quamme GA: Aldosterone potentiates hormone-stimulated Mg²⁺ uptake in mouse distal convoluted tubule cells. American Journal of Physiology 274:F336-F341, 1998.

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Abstract The distal convoluted tubule reabsorbs significant amounts of filtered magnesium that is under hormonal control. In this study, we describe the effects of aldosterone on Mg21 uptake in an immortalized mouse distal convoluted tubule (MDCT) cell line. Intracellular free Mg21 concentration ([Mg21]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. To determine Mg21 entry rate into MDCT cells, they were first Mg21 depleted ([Mg21]i, 0.22 6 0.01 mM) by culturing in Mg21-free media for 16 h and then placed in 1.5mMMgCl2. The rate of change in [Mg21]i as measured as a function of time, d([Mg21]i)/dt, was 164 6 5 nM/s in control cells. We have shown that glucagon or arginine vasopressin (AVP) stimulates Mg21 entry by 63% and 15%, respectively. Incubation of MDCT cells with aldosterone for 16 h did not change the rate of Mg21 uptake (172 6 8 nM/s). However, aldosterone potentiated glucagon- and AVP-stimulated Mg21 uptake rate up to 330 6 39 and 224 6 6 nM/s, respectively. Aldosterone also potentiated glucagon- and AVP-induced intracellular cAMP accumulation in a concentration-independent manner. As cAMP stimulates Mg21 entry in MDCT cells, it is inferred that aldosterone may stimulate Mg21 uptake through intracellular signaling pathways involving cAMP. The actions of aldosterone were dependent on de novo protein synthesis, as pretreatment of the cells with cycloheximide inhibited aldosterone potentiation of hormone stimulation of Mg21 uptake and cAMP accumulation. These studies with MDCT cells suggest that aldosterone may modulate the effects of hormones acting within the distal convoluted tubule to control magnesium absorption.

distal convoluted tubule reabsorbs significant amounts of

filtered magnesium that is under hormonal control. In this

study, we describe the effects of aldosterone on Mg21 uptake

in an immortalized mouse distal convoluted tubule (MDCT)

cell line. Intracellular free Mg21 concentration ([Mg21]i) was

determined on single MDCT cells using microfluorescence

with mag-fura 2. To determine Mg21 entry rate into MDCT

cells, they were first Mg21 depleted ([Mg21]i, 0.22 6 0.01 mM)

by culturing in Mg21-free media for 16 h and then placed in

1.5mMMgCl2. The rate of change in [Mg21]i as measured as a

function of time, d([Mg21]i)/dt, was 164 6 5 nM/s in control

cells. We have shown that glucagon or arginine vasopressin

(AVP) stimulates Mg21 entry by 63% and 15%, respectively.

Incubation of MDCT cells with aldosterone for 16 h did not

change the rate of Mg21 uptake (172 6 8 nM/s). However,

aldosterone potentiated glucagon- and AVP-stimulated Mg21

uptake rate up to 330 6 39 and 224 6 6 nM/s, respectively.

Aldosterone also potentiated glucagon- and AVP-induced

intracellular cAMP accumulation in a concentration-independent

manner. As cAMP stimulates Mg21 entry in MDCT cells,

it is inferred that aldosterone may stimulate Mg21 uptake

through intracellular signaling pathways involving cAMP.

The actions of aldosterone were dependent on de novo protein

synthesis, as pretreatment of the cells with cycloheximide

inhibited aldosterone potentiation of hormone stimulation of

Mg21 uptake and cAMP accumulation. These studies with

MDCT cells suggest that aldosterone may modulate the

effects of hormones acting within the distal convoluted tubule

to control magnesium absorption.

intracellular magnesium; fluorescence

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

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.

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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