adenosine-5--o-(3-thiotriphosphate) and 8-bromoadenosine-3--5--cyclic-monophosphorothioate

Although in LLC-PK cells ATP depletion has been shown to result in alterations of cytoskeleton actin and an inhibition of Na+/H+ exchanger activity, there is little information concerning the regulation of this exchanger in the distal luminal membrane by ATP and actin filaments. The present study examined the direct effect of ATP and cytochalasin B on the Na+/H+ exchanger activity in the proximal and distal tubule luminal membranes. The presence of 100 microM ATP in the luminal membrane vesicles from rabbit proximal tubules did not influence the Ethyl Isopropyl Amiloride sensitive Na+ uptake by these membranes. In contrast, the same treatment of luminal membranes from distal tubules significantly enhanced the exchanger activity from 0.22 +/- 0.04 to 0.39 +/- 0.08 pM/microg/10 sec (P < 0.02). When ATP was replaced by its nonhydrolysable form, ATPgammas, the effect on the distal luminal membrane was strongly diminished suggesting that the action of the nucleotide implicates a phosphorylation step. Confirming this hypothesis, addition of 300-microM-Rp cAMP, a protein kinase A inhibitor, completely abolished the effect of ATP. In view of the fact that a tight relationship has been described between ATP, the cytoskeleton complex and the exchanger activity, we studied the effect of cytochalasin B on this activity. The presence of 20 microM cytochalasin B in the distal luminal membrane vesicles induced, as observed with ATP, a significant increase in the Na+ uptake. However, the actions of ATP and cytochalasin B were not additive. These results suggest that firstly, ATP and short actin filaments of the cytoskeleton regulate the distal luminal isoform through an intramembranous mechanism and secondly, a phosphorylation mechanism is, at least partially, implicated in the action of ATP. In contrast, the proximal tubule exchanger is regulated through different mechanisms.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine Triphosphate; Amiloride; Animals; Cytochalasin B; Cytoskeleton; Cytosol; Enzyme Inhibitors; Intracellular Membranes; Ion Transport; Kidney Tubules, Distal; Kidney Tubules, Proximal; Kinetics; Nephrons; Phosphorylation; Protein Isoforms; Rabbits; Sodium; Sodium-Hydrogen Exchangers; Thionucleotides

Extracellular ATP and ATP gamma S (1-1000 microM) stimulated cyclic AMP (cAMP) production in undifferentiated HL-60 cells. The potency order for adenine nucleotides and adenosine was ATP gamma S > ATP > > ADP > 3 AMP = Adenosine. Indomethacin (50 microM) had no effect on ATP-induced cAMP production. ATP and ATP gamma S also suppressed cell growth and induced differentiation as revealed by fMLP-stimulated beta-glucuronidase release 48 h after exposure. The potency order for the induction of fMLP-stimulated beta-glucuronidase release by adenine nucleotides and adenosine was ATP gamma S > 3 ATP > ADP > AMP = Adenosine approximately 0. The protein kinase A inhibitor Rp-8-Br-cAMPS (10-200 mM) suppressed ATP-induced differentiation but had no effect on ATP-dependent growth suppression. UTP which, like ATP, activates P2U receptors on HL-60 cells, had no effect on cAMP production, cell growth, or differentiation. The data suggest the existence of a novel receptor for ATP on undifferentiated HL-60 cells that is coupled to the activation of adenylate cyclase and cAMP-dependent differentiation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenine Nucleotides; Adenosine; Adenosine Triphosphate; Cell Differentiation; Cell Division; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; HL-60 Cells; Humans; Kinetics; N-Formylmethionine Leucyl-Phenylalanine; Receptors, Purinergic P2; Thionucleotides; Uridine Triphosphate