Korean J Physiol Pharmacol.  2009 Feb;13(1):9-14. 10.4196/kjpp.2009.13.1.9.

P2 Receptor-mediated Inhibition of Vasopressin-stimulated Fluid Transport and cAMP Responses in AQP2-transfected MDCK Cells

• Kim YH
• Choi YJ
• Bae HR
• Woo JS

• KMID: 2285365
• DOI: http://doi.org/10.4196/kjpp.2009.13.1.9

Abstract

We cultured canine kidney (MDCK) cells stably expressing aquaporin-2 (AQP2) on collagen-coated permeable membrane filters and examined the effect of extracellular ATP on arginine vasopressin (AVP)-stimulated fluid transport and cAMP production. Exposure of cell monolayers to basolateral AVP resulted in stimulation of apical to basolateral net fluid transport driven by osmotic gradient which was formed by addition of 500 mM mannitol to basolateral bathing solution. Pre-exposure of the basolateral surface of cell monolayers to ATP (100 ?M) for 30 min significantly inhibited the AVP-stimulated net fluid transport. In these cells, AVP-stimulated cAMP production was suppressed as well. Profile of the effects of different nucleotides suggested that the P2Y2 receptor is involved in the action of ATP. ATP inhibited the effect of isoproterenol as well, but not that of forskolin to stimulate cAMP production. The inhibitory effect of ATP on AVP-stimulated fluid movement was attenuated by a protein kinase C inhibitor, calphostin C or pertussis toxin. These results suggest that prolonged activation of the P2 receptors inhibits AVP-stimulated fluid transport and cAMP responses in AQP2 transfected MDCK cells. Depressed responsiveness of the adenylyl cyclase by PKC-mediated modification of the pertussis-toxin sensitive Gi protein seems to be the underlyihng mechanism.

Keyword

P2 receptor; ATP; Vasopressin; Adenylyl cyclase; Cyclic AMP

MeSH Terms

Adenosine Triphosphate
Adenylyl Cyclases
Aquaporin 2
Arginine Vasopressin
Baths
Cyclic AMP
Forskolin
Isoproterenol
Kidney
Madin Darby Canine Kidney Cells
Mannitol
Membranes
Naphthalenes
Nucleotides
Pertussis Toxin
Protein Kinase C
Vasopressins
Adenosine Triphosphate
Aquaporin 2
Arginine Vasopressin
Cyclic AMP
Forskolin
Isoproterenol
Mannitol
Naphthalenes
Nucleotides
Pertussis Toxin
Protein Kinase C
Vasopressins

Figure

• 

  Fig. 1. (A) Western blot analysis of AQP2 expression in control (left lane) and AQP2 transfected (right lane) MDCK cells. Cells were transfected with full length cDNA of AQP2. (B, C) Changes in [14C]inulin activity in the apical bathing medium were monitored as an index of net apical to basolateral fluid movement in the presence (closed circle) and absence (open circle) of 10−6 M AVP in control (B) and AQP2 transfected (C) cells. Each point represents mean of 5 experiments.

• 

  Fig. 2. Effect of AVP on net apical to basolateral fluid movement in control and AQP2 transfected MDCK cells. Net apical to basolateral fluid movement was calculated from the changes in [14C]inulin activity in the apical bathing medium. (A) In control cells, in the presence (clsed circle) and absence (open circle) of 10−6 M AVP. (B) In AQP2 transfected cells, in the presence (closed circle) and absence (open circle) of 10−6 M AVP. Each point represents mean±S.E. of 5 experiments.

• 

  Fig. 3. Effect of ATP on AVP-stimulated net apical to basolateral fluid movement. Net apical to basolateral fluid movement was calculated from the changes in [14C]inulin activity in the apical bathing medium in control (A) and AQP2 transfected (B) cells. ATP (10−4 M) was added to basolateral bathing medium 30 min prior to the stimulation with AVP. Data are mean±S.E. of 6 experiments. ∗p<0.01 vs. without ATP.

• 

  Fig. 4. Effect of ATP on cAMP production in AQP2 transfected cells stimulated with AVP, isoproterenol and forskolin. The magnitude of stimulation in cellular cAMP production by exposure for 5 min to 10−6 M each of AVP, isoproterenol (ISP) and froskolin was measured in the presence and absence of ATP (10−4 M). Data are mean±S.E. of 4 experiments. ∗p<0.01 vs. without ATP.

• 

  Fig. 5. Effects of different P2 nucleotide receptor agonists on AVP-stimulated responses. The magnitude of inhibition of AVP-stimulated responses by each nucleotide (10−4 M) was measured via 3-hr fluid movement and 5-min cAMP production. Data are mean±S.E. of 5 experiments. ATP-γS, 5'-O-3-thiotriphosphate; UTP, uridine 5'-triphosphate; MeSATP, 2-methylthio-ATP; MeATP, β,γ-methylene-ATP.

• 

  Fig. 6. Effects of purinergic antagonists on the ATP-induced inhibition of AVP-stimulated responses. The magnitude of ATP-induced inhibition of AVP-stimulated responses of 3-hr fluid movement and 5-min cAMP production was measured in the presence and absence of the P2 receptor antagonist pyridoxalphosphate-24-disulphonic acid (PPADS, 10−5 M) and the P1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 10−5 M). The data represent mean±S.E. of 4 experiments. ∗p<0.01 compared to the respective control.

• 

  Fig. 7. Effects of calphostin C and pertussis toxin on ATP-induced inhibition of AVP-stimulated fluid movement. AVP-stimulated fluid movement was measured in the presence and absence of ATP (10−4 M) in cells pretreated with calphostin C (20 μM) or with pertussis toxin (PTX, 1 μg/ml) for 2 hr. Data are mean±S.E. of 5 experiments. ∗p<0.01 vs. the respective values without ATP.

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