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.

Dr. Long Jun Dai » immortalized mouse distal convoluted tubule cells

ATP inhibits Mg2+ uptake in MDCT cells via P2X purinoceptors

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