ABSTRACT
The recently cloned canine P2Y11 receptor (cP2Y11) and its human homolog (hP2Y11) were stably expressed in Chinese hamster ovary cells (CHO-K1) and 1321N1 human astrocytoma cells, and their agonist selectivities and coupling efficiencies to phospholipase C and adenylyl cyclase were assessed. Adenosine triphosphate nucleotides were much more potent and efficacious at the hP2Y11 receptor than their corresponding diphosphates in promoting both inositol phosphate and cyclic AMP accumulation. In contrast, adenosine diphosphate nucleotides were considerably more potent at the cP2Y11 receptor than their corresponding triphosphate analogs. The tri- versus diphosphate specificity of the two receptors was further confirmed in studies using Ca(2+) mobilization as a measure of receptor activation under conditions that minimized nucleotide degradation. Moreover, 2-methylthioadenosine-5'-triphosphate and 2-methylthioadenosine-5'-diphosphate were 58- and 75-fold more potent than ATP and ADP, respectively, at the cP2Y11 receptor compared with only 2- to 3-fold more potent at the hP2Y11 receptor. Mutational analysis revealed that the change of Arg-265, which is located at the juxtaposition of transmembrane domain 6 and the third extracellular loop in the hP2Y11 receptor, to glutamine in the cP2Y11 receptor is at least partly responsible for the diphosphate selectivity but not the increased sensitivity to 2-thioether-substituted adenine nucleotides at the canine receptor. These results imply a key role for a positively charged arginine residue in contributing to the recognition of extracellular nucleotides by the P2Y11 receptor and perhaps other P2Y receptors.
Subject(s)
Adenine Nucleotides/metabolism , Membrane Proteins/metabolism , Receptors, Purinergic P2/metabolism , Amino Acid Substitution , Animals , Arginine/genetics , Arginine/metabolism , Cyclic AMP/metabolism , Dogs , Glutamine/genetics , Glutamine/metabolism , Humans , Hydrolysis , Inositol/metabolism , Lipid Metabolism , Membrane Proteins/genetics , Protein Conformation , Protein Structure, Tertiary , Receptors, Purinergic P2/genetics , Uridine Diphosphate/metabolism , Uridine Triphosphate/metabolismABSTRACT
1. The human P2Y(11) (hP2Y(11)) receptor was stably expressed in two cell lines, 1321N1 human astrocytoma cells (1321N1-hP2Y(11)) and Chinese hamster ovary cells (CHO-hP2Y(11)), and its coupling to phospholipase C and adenylyl cyclase was assessed. 2. In 1321N1-hP2Y(11) cells, ATP promoted inositol phosphate (IP) accumulation with low microM potency (EC(50)=8.5+/-0.1 microM), whereas it was 15 fold less potent (130+/-10 microM) in evoking cyclic AMP production. 3. In CHO-hP2Y(11) cells, ATP promoted IP accumulation with slightly higher potency (EC(50)=3.6+/-1.3 microM) than in 1321N1-hP2Y(11) cells, but it was still 15 fold less potent in promoting cyclic AMP accumulation (EC(50)=62.4+/-15.6 microM) than for IP accumulation. Comparable differences in potencies for promoting the two second messenger responses were observed with other adenosine nucleotide analogues. 4. In 1321N1-hP2Y(11) and CHO-hP2Y(11) cells, down regulation of PKC by chronic treatment with phorbol ester decreased ATP-promoted cyclic AMP accumulation by 60--80% (P<0.001) with no change in its potency. Likewise, chelation of intracellular Ca(2+) decreased ATP-promoted cyclic AMP accumulation by approximately 45% in 1321N1-hP2Y(11) cells, whereas chelation had no effect on either the efficacy or potency of ATP in CHO-hP2Y(11) cells. 5. We conclude that coupling of hP2Y(11) receptors to adenylyl cyclase in these cell lines is much weaker than coupling to phospholipase C, and that activation of PKC and intracellular Ca(2+) mobilization as consequences of inositol lipid hydrolysis potentiates the capacity of ATP to increase cyclic AMP accumulation in both 1321N1-hP2Y(11) and CHO-hP2Y(11) cells.
Subject(s)
Adenylyl Cyclases/metabolism , Receptors, Purinergic P2/metabolism , Type C Phospholipases/metabolism , Adenine Nucleotides/pharmacology , Adenosine Triphosphate/pharmacology , Adenylyl Cyclases/physiology , Animals , CHO Cells , Calcium/metabolism , Cricetinae , Cyclic AMP/biosynthesis , Enzyme Activation , Humans , Inositol Phosphates/pharmacology , Phosphodiesterase Inhibitors/pharmacology , Polymerase Chain Reaction , Protein Kinase C/metabolism , Receptors, Dopamine D1/biosynthesis , Receptors, Purinergic P2/physiology , Second Messenger Systems/physiology , Tumor Cells, Cultured , Type C Phospholipases/physiologyABSTRACT
The nucleotide selectivities of the human P2Y(4) (hP2Y(4)) and rat P2Y(4) (rP2Y(4)) receptor stably expressed in 1321N1 human astrocytoma cells were determined by measuring increases in intracellular [Ca(2+)] under conditions that minimized metabolism, bioconversion, and endogenous nucleotide release. In cells expressing the hP2Y(4) receptor, UTP, GTP, and ITP all increased intracellular [Ca(2+)] with a rank order of potency of UTP (0.55) > GTP (6.59) = ITP (7.38), (EC(50), microM). ATP, CTP, xanthine 5'-triphosphate (XTP), and diadenosine 5',5"'-P(1), P(4)-tetraphosphate (Ap(4)A), all at 100 microM, were inactive at the hP2Y(4) receptor. In cells expressing the rP2Y(4) receptor, all seven nucleotides increased intracellular [Ca(2+)] with similar maximal effects and a rank order of potency of UTP (0.20) > ATP (0. 51) > Ap(4)A (1.24) approximately ITP (1.82) approximately GTP (2. 28) > CTP (7.24) > XTP (22.9). Because ATP is inactive at the hP2Y(4) receptor, we assessed whether ATP displayed antagonist activity. When coapplied, ATP shifted the concentration-response curve to UTP rightward in a concentration-dependent manner, with no change in the maximal response. A Schild plot derived from these data gave a pA(2) value of 6.15 (K(B) = 708 nM) and a slope near unity. Additionally, CTP and Ap(4)A (each at 100 microM) inhibited the response to an EC(50) concentration of UTP by approximately 40 and approximately 50%, respectively, whereas XTP had no effect. The inhibitory effects of ATP, CTP, and Ap(4)A were reversible on washout. Thus, ATP is a potent agonist at the rP2Y(4) receptor but is a competitive antagonist with moderate potency at the hP2Y(4) receptor.
Subject(s)
Adenosine Triphosphate/pharmacology , Purinergic P2 Receptor Agonists , Purinergic P2 Receptor Antagonists , Adenosine Diphosphate/pharmacology , Animals , Dinucleoside Phosphates/pharmacology , Humans , Rats , Receptors, Purinergic P2/metabolism , Species Specificity , Tumor Cells, Cultured , Uridine Diphosphate/pharmacologyABSTRACT
It is generally accepted that ATP is costored and coreleased with noradrenaline from the sympathetic nerve terminals. The pacemaker region of the mammalian heart is highly innervated with the sympathetic nervous system. It is possible, therefore, that ATP released from nerve terminals can act on pacemaker cells and modulate heart beat activity. However, the physiological role(s) of extracellular ATP in mammalian heart beat has been little evaluated, even though the effect of extracellular ATP observed in in vivo studies has been attributed to the formation of adenosine, the catabolic product of ATP, which is a cardiodepressant. The present study investigated the effect of extracellular ATP on L-type calcium channel currents of guinea-pig single sinoatrial nodal cells, by using the whole cell patch clamp technique. Application of extracellular ATP caused a concentration-dependent and reversible inhibition of calcium channel currents with a 50% inhibitory concentration of 100 microM. The presence of the P2-purinoceptor antagonist suramin (1, 10 and 100 microM), reactive blue 2 (1 and 10 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 50 and 100 microM) or the adenosine receptor antagonists 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX, 0.1 microM) and 8-phenyltheophylline (10 microM) failed to affect the inhibitory action of extracellular ATP on calcium channel currents. The relative rank order of potency of different nucleotides and nucleosides, at a concentration of 100 microM, on the inhibition of calcium channel currents was as follows: ATP = alpha, beta-methylene-ATP >> 2-methylthioATP > or = adenosine 5'-O-(3-thiotriphosphate) >> UTP = ADP > AMP > or = adenosine. Dialysis, by way of the patch pipette, of the cell interior with specific protein kinase C inhibitor staurosporine (70 nM) or calphostin C (500 nM) abolished extracellular ATP-induced inhibition of calcium channel currents. Therefore, extracellular ATP-mediated inhibition of calcium channel currents in guinea-pig single sinoatrial nodal cells involves activation of protein kinase C.
Subject(s)
Adenosine Triphosphate/pharmacology , Calcium Channels/drug effects , Protein Kinase C/pharmacology , Sinoatrial Node/cytology , Child, Preschool , Extracellular Space , Heart/physiology , Humans , Sinoatrial Node/physiologyABSTRACT
1. The effects of extracellular adenosine 5'-triphosphate ([ATP]zero) on the L-type Ca2+ channel currents in guinea-pig single sinoatrial nodal (SAN) cells, isolated by enzymatic dissociation, were investigated by use of whole-cell patch-clamp techniques. 2. The application of [ATP]zero (2 microM-1 mM) produced an inhibitory effect on the L-type Ca2+ channel current peak amplitude (10 mM Ba2+ as charge carrier) in a concentration-dependent and reversible manner with an IC50 of 100 microM and a Hill coefficient of 1.83. 3. The presence of the adenosine receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 microM) and 8-phenyltheophylline (10 microM) did not affect the [ATP]zero-induced inhibition of the Ca2+ channel currents. Adenosine (100 microM) had little effect on the basal Ca2+ channel currents. Adenosine 500 microM, caused 23% inhibition of the Ca2+ channel current, which was abolished by 0.1 microM DPCPX. 4. The presence of the P2-purinoceptor antagonists, suramin (1, 10 and 100 microM), reactive blue 2 (1 and 10 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 50 and 100 microM) failed to affect the inhibitory action of [ATP]zero on Ca2+ channel currents. 5. The relative rank order of potency of different nucleotides and nucleosides, at a concentration of 100 microM, on the inhibition of the Ca2+ channel currents is as follows: adenosine 5'-triphosphate (ATP) = alpha,beta-methylene-ATP (alpha,beta MeATP) > > 2-methylthioATP (2-MeSATP) > or = adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) > > uridine 5'-triphosphate (UTP) = adenosine 5'-diphosphate (ADP) > adenosine 5'-monophosphate (AMP) > or = adenosine. 6. These results suggest that [ATP]zero may play an important role in the heart beat by inhibiting the L-type Ca2+ channel currents in single SAN cells. This inhibitory effect is not due to the formation of adenosine resulting from the enzymatic degradation of [ATP]zero. Based on the relative order of inhibitory potency of different nucleotides and nucleosides on the L-type Ca2+ channel currents and the ineffectiveness of the purinoceptor antagonists tested, a novel type of purinoceptor may be involved.
Subject(s)
Adenosine Triphosphate/metabolism , Calcium Channels/metabolism , Sinoatrial Node/metabolism , Adenosine Triphosphate/pharmacology , Animals , Calcium Channel Blockers/pharmacology , Calcium Channels/drug effects , Electrophysiology , Extracellular Space/metabolism , Female , Guinea Pigs , In Vitro Techniques , Male , Nucleosides/pharmacology , Nucleotides/pharmacology , Patch-Clamp Techniques , Platelet Aggregation Inhibitors/pharmacology , Purinergic Antagonists , Purinergic P1 Receptor Antagonists , Pyridoxal Phosphate/analogs & derivatives , Pyridoxal Phosphate/pharmacology , Sinoatrial Node/cytology , Theophylline/analogs & derivatives , Theophylline/pharmacology , Xanthines/pharmacologyABSTRACT
The purpose of the investigation was to determine the effects of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) on the isolated rabbit aortic strips. The results showed that (1) the rabbit aortic strips with intact endothelium contracted in a dose-dependent manner to exogenous AA between the concentrations of 2.5 x 10(-6) mol/L and 4 x 10(-5) mol/L. EPA did not affect the tension of the relaxing strips, but inhibited the contractile response to AA (2 x 10(-5) mol/L), IC50 = 9.94 x 10(-6) mol/L. DHA showed no significant effect on the contractile response to AA. (2) In the endothelium-damaged rabbit aorta, the contraction of strips to AA was drastically diminished. The inhibitory effect of EPA on the contraction to AA was almost vanished. (3) The contraction of rabbit aortic strips to AA was shown to be abolished by indomethacin (1.5 x 10(-5) mol/L), a cyclooxygenase inhibitor. (4) Radioimmunoassay showed that exogenous AA increased the tissue levels of 6-keto-PGF1 alpha, TXB2 and their ratio. Lower dose of EPA (1.5 x 10(-6) or 1.5 x 10(-5) mol/L) did not affect the tissue levels of 6-keto-PGF1 alpha and TXB2. However, EPA at higher concentrations decreased the metabolites of AA significantly. These findings suggest that exogenous AA induces the contraction of rabbit aortic strips in vitro, which is related to the endothelial cells and mediated by the metabolites(s) of cyclooxygenase, likely endothelium-derived contracting factor (EDCF).(ABSTRACT TRUNCATED AT 250 WORDS)