| 1. |
|
|
| 2. |
- Hou, Mingyan, et al.
(författare)
-
Increase in cardiac P2X1-and P2Y2-receptor mRNA levels in congestive heart failure
- 1999
-
Ingår i: Life Sciences. - 1879-0631. ; 65:11, s. 1195-1206
-
Tidskriftsartikel (refereegranskat)abstract
- We wanted to study the expression of P2-receptors at the mRNA-level in the heart and if it is affected by congestive heart failure (CHF). To quantify the P2 receptor mRNA-expression we used a competitive RT-PCR protocol which is based on an internal RNA standard. The P2 receptor mRNA-expression was quantified in hearts from CHF rats and compared to sham-operated rats. Furthermore, the presence of receptor mRNA was studied in the myocardium from patients with heart failure. In the sham operated rats the G-protein coupled P2Y-receptors were expressed at a higher level than the ligand gated ion-channel receptor (P2X1). Among the P2Y-receptors the P2Y6-receptor was most abundantly expressed (P2Y6 > P2Y1 > P2Y2 = P2Y4 > P2X1). A prominent change was seen for the P2X1- and P2Y2-receptor mRNA levels which were increased 2.7-fold and 4.7-fold respectively in the myocardium from the left ventricle of CHF-rats. In contrast, the P2Y1-, P2Y4- and P2Y6-receptor mRNA levels were not significantly altered in CHF rats. In human myocard the P2X1-, P2Y1-, P2Y2-, P2Y6- and P2Y11-receptors were detected by RT-PCR in both right and left atria and ventricles, while the P2Y4-receptor band was weak or absent. In conclusion, most of the studied P2-receptors were expressed in both rat and human hearts. Furthermore, the P2X1- and P2Y2-receptor mRNA were upregulated in CHF, suggesting a pathophysiological role for these receptors in the development of heart failure.
|
|
| 3. |
- Kim, HS, et al.
(författare)
-
Methanocarba modification of uracil and adenine nucleotides: High potency of northern ring conformation at P2Y(1), P2Y(2), P2Y(4), and P2Y(11) but not P2Y(6) receptors
- 2002
-
Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 1520-4804 .- 0022-2623. ; 45:1, s. 208-218
-
Tidskriftsartikel (refereegranskat)abstract
- The potency of nucleotide antagonists at P2Y(1) receptors was enhanced by replacing the ribose moiety with a constrained carbocyclic ring (Nandanan, et al. J. Med. Chem. 2000, 43, 829842). We have now synthesized ring-constrained methanocarba analogues (in which a fused cyclopropane moiety constrains the pseudosugar ring) of adenine and uracil nucleotides, the endogenous activators of P2Y receptors. Methanocarba-adenosine 5'-triphosphate (ATP) was fixed in either a Northern (N) or a Southern (S) conformation, as defined in the pseudorotational cycle. (N)-Methanocarba-uridine was prepared from the 1-amino-pseudosugar ring by treatment with beta-ethoxyacryloyl cyanate and cyclization to form the uracil ring. Phosphorylation was carried out at the 5'-hydroxyl group through a multistep process: Reaction with phosphoramidite followed by oxidation provided the 5'-monophosphates, which then were treated with 1,1'-carbonyldiimidazole for condensation with additional phosphate groups, The ability of the analogues to stimulate phospholipase C through activation of turkey P2Y(1) or human P2Y(1), P2Y(2), P2Y(4), P2Y(6), and P2Y(11) receptors stably expressed in astrocytoma cells was measured. At recombinant human P2Y(1) and P2Y(2) receptors, (N)-methanocarba-ATP was 138- and 41-fold, respectively, more potent than racemic (S)-methanocarba-ATP as an agonist. (N)methanocarba-ATP activated P2Y(11) receptors with a potency similar to ATP. (N)-Methanocarba-uridine 5'-triphosphate (UTP) was equipotent to UTP as an agonist at human P2Y2 receptors and also activated P2Y(4) receptors with an EC50 of 85 nM. (N)-Methanocarba-uridine 5'-diphosphate (UDP) was inactive at the hP2Y(6) receptor. The vascular effects of (N)-methanocarba-UTP and (N)-methanocarba-UDP were studied in a model of the rat mesenteric artery, The triphosphate was more potent than UTP in inducing a dilatory P2Y(4) response (pEC(50) = 6.1 +/- 0.2), while the diphosphate was inactive as either an agonist or antagonist in a P2Y(6) receptor-mediated contractile response. Our results suggest that new nucleotide agonists may be designed on the basis of the (N) conformation that favors selectivity for P2Y(1), P2Y(2), P2Y(4), and P2Y(11) receptors.
|
|
| 4. |
- Malmsjö, Malin, et al.
(författare)
-
Characterization of contractile P2 receptors in human coronary arteries by use of the stable pyrimidines uridine 5'-O-thiodiphosphate and uridine 5'-O-3-thiotriphosphate
- 2000
-
Ingår i: Journal of Pharmacology and Experimental Therapeutics. - 1521-0103. ; 293:3, s. 755-760
-
Tidskriftsartikel (refereegranskat)abstract
- The present study was designed to evaluate the relative contribution of the different contractile P2 receptors in endothelium-denuded human coronary arteries by use of extracellular nucleotides, including the stable pyrimidines uridine 5'-O-3-thiotriphosphate (UTPgammaS) and uridine 5'-O-thiodiphosphate (UDPbetaS). The isometric tension of isolated vessel segments was recorded in vitro, and P2 receptor mRNA expression was examined by reverse transcription-polymerase chain reaction. alphabeta-Methylene-adenosine triphosphate (alphabeta-MeATP) elicited contractions at a low concentration (pEC(50) = 5.2), indicating the presence of contractile P2X receptors. The P2Y responses were analyzed after P2X receptor desensitization with 10 microM alphabeta-MeATP. The stable nucleotides UTPgammaS and adenosine 5'-O-3-thiotriphosphate (ATPgammaS), which are agonists of P2Y(2) or P2Y(4) receptors, were approximately 2 log units more potent than the endogenous UTP and ATP (pEC(50) = 4.6 and 3.8 for UTPgammaS and ATPgammaS). The efficacy of these responses were approximately double that of the P2X agonist alphabeta-MeATP (E(max) = 125% for UTPgammaS, 126% for ATPgammaS, and 68% for alphabeta-MeATP), suggesting a primary role for contractile P2Y(2/4) receptors. The P2Y(2) receptor agonist diadenosine tetraphosphate also stimulated contraction, whereas the selective P2Y(1) agonist adenosine 5'-O-thiodiphosphate and the selective P2Y(6) agonist UDPbetaS had no effect. Reverse transcription-polymerase chain reaction analysis of mRNA from endothelium-denuded human coronary arteries demonstrated strong bands for P2Y(2) and P2X(1), although bands for P2Y(1), P2Y(4), and P2Y(6) receptor mRNA could also be detected. In conclusion, the stable pyrimidines UDPbetaS and UTPgammaS are important tools for P2 receptor subtype characterization in intact tissues with ectonucleotidase activity. Extracellular nucleotides elicit contraction of human coronary arteries primarily by activation of P2Y(2) and P2X receptors, whereas a role for P2Y(1) and P2Y(6) receptors can be excluded. Antagonists of P2Y(2) and P2X receptors may be useful in the treatment of coronary vasospastic disorders.
|
|
| 5. |
- Malmsjö, Malin, et al.
(författare)
-
Congestive heart failure induces downregulation of P2X1-receptors in resistance arteries
- 1999
-
Ingår i: Cardiovascular Research. - 1755-3245. ; 43:1, s. 219-227
-
Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Congestive heart failure (CHF) is accompanied by enhanced peripheral sympathetic nerve activity, increased vascular resistance and impaired peripheral blood flow. Besides noradrenaline and neuropeptide Y, the sympathetic nervous system also releases ATP, which has contractile effects mediated by different subtypes of P2-receptors on the vascular smooth muscle cells. The present study was designed to examine postsynaptic changes of the contractile responses to ATP and other extracellular nucleotides in CHF. METHODS: CHF was induced by left coronary artery ligation resulting in a reproducible myocardial infarction in Sprague-Dawley rats. Contractile responses were examined in cylindrical segments of aorta and the mesenteric artery after endothelium removal. To determine if an altered response was regulated on the transcriptional level, competitive reverse transcription polymerase chain reaction (RT-PCR) was used to estimate the amount of P2X1-receptor mRNA. RESULTS: ATP, which is both a P2X1- and a P2Y-receptor agonist, induced a weaker contraction in the mesenteric artery from CHF as compared to sham operated rats. A decrease in both potency and maximum contraction was shown for the selective P2X1-receptor agonist, alpha beta-MeATP, in the mesenteric artery (pEC50 = 6.04 vs. 5.76, Cmax = 57% vs. 33%, sham vs. CHF operated rats), but not in the aorta. Competitive RT-PCR also revealed decreased P2X1-receptor mRNA levels in CHF operated rats in the mesenteric artery (9106 x 10(3) vs. 714 x 10(3) molecules/microgram, sham vs. CHF operated rats), while it remained unaltered in the aorta. To study the P2Y-receptor induced contractile effects, the P2X1-receptors were first desensitised with alpha beta-MeATP (10(-5) M for 8 min). After P2X1-receptors desensitisation, UTP and UDP induced strong contractions in both the mesenteric artery and in the aorta, while ATP and ADP were much less effective. These contractions were not altered by CHF, indicating that vascular contraction mediated by P2Y-receptors are unaffected by CHF. CONCLUSION: CHF induces downregulation of P2X1-receptor stimulated contraction in the mesenteric artery depending on decreased mRNA synthesis for the receptor, while the P2Y-receptor activity remains unchanged. Downregulation of P2X1-receptors appears to be specific for peripheral resistance arteries. This may represent a compensatory response to enhanced peripheral sympathetic nerve activity and increased vascular resistance in CHF.
|
|
| 6. |
- Malmsjö, Malin, et al.
(författare)
-
Endothelial P2Y receptors induce hyperpolarisation of vascular smooth muscle by release of endothelium-derived hyperpolarising factor
- 1999
-
Ingår i: European Journal of Pharmacology. - 1879-0712. ; 364:2-3, s. 169-173
-
Tidskriftsartikel (refereegranskat)abstract
- The effects of P2Y receptor agonists on smooth muscle membrane potential in isolated ring segments of rat mesenteric artery were examined by intracellular microelectrodes. In the presence of inhibitors of nitric oxide-synthase and cyclo-oxygenase, the selective P2Y1 receptor agonist adenosine 5'-O-thiodiphosphate (ADPbetaS) induced endothelium-dependent membrane hyperpolarisations, which were abolished by a combination of the K+ channel inhibitors charybdotoxin and apamin, providing direct evidence that ADPbetaS releases endothelium-derived hyperpolarising factor (EDHF). 2-MethylthioATP and ATP, each which stimulates both endothelial P2Y receptors and P2X receptors on the smooth muscle cells, also elicited hyperpolarisation, but only after desensitisation of P2X receptors with alphabeta-methylATP indicating that simultaneous activation of P2X receptors may counteract the action of EDHF. In conclusion, activation of endothelial P2Y receptors induce release of EDHF.
|
|
| 7. |
- Malmsjö, Malin, et al.
(författare)
-
Enhanced acetylcholine and P2Y-receptor stimulated vascular EDHF-dilatation in congestive heart failure
- 1999
-
Ingår i: Cardiovascular Research. - 1755-3245. ; 43:1, s. 200-209
-
Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Congestive heart failure (CHF) is accompanied by impaired peripheral blood flow and endothelial dysfunction with decreased release of nitric oxide (NO). Strong evidence supports the existence of another vasodilatory substance, endothelium derived hyperpolarising factor (EDHF), which has not previously been studied in CHF. METHOD: CHF was induced by left coronary artery ligation resulting in a reproducible myocardial infarction in Sprague Dawley rats. Vasodilatory responses to acetylcholine and extracellular nucleotides (ATP, ADP beta S, ADP and UTP) were examined in cylindrical segments of the mesenteric artery, precontracted with noradrenaline. The combined NO- and EDHF-dilatation (after inhibition of cyclo-oxygenase pathways) was called "total dilatation", as indomethacin had only minor effects in this system. NO-dilatation was studied in segments pretreated with indomethacin and the potassium channel inhibitors charybdotoxin (10(-7.5) M) and apamin (10(-6) M), while EDHF-dilatations were studied in the presence of indomethacin (10(-5) M) and L-NOARG (10(-3.5) M). RESULTS: EDHF-dilatations in CHF were strongly up-regulated for ACh (36% vs. 73%; sham vs. CHF operated rats), ADP beta S (10% vs. 42%), ADP (0% vs. 21%) and UTP (3% vs. 35%). These dilatations were abolished by a combination of charybdotoxin and apamin, confirming that they were mediated by EDHF. The NO-dilatations on the other hand were down-regulated in CHF as compared to sham operated rats for ACh (93% vs. 76%; sham vs. CHF operated rats), ADP beta S (61% vs. 37%). ADP (60% vs. 30%), ATP (49% vs. 34%) and UTP (65% vs. 47%), while a minor decrease was seen in the total dilatation for ACh (87% vs. 75%; sham vs. CHF operated rats), ADP beta S (47% vs. 42%), ADP (59% vs. 39%), ATP (52% vs. 39%) and UTP (59% vs. 44%). CONCLUSION: In this model of non-atherosclerotic CHF there was a minor decrease in the total dilatation and a marked down-regulation of the NO-mediated dilatation, while the EDHF-dilatation was up-regulated. Increased EDHF-activity in CHF may represent a compensatory response to decreased NO-activity to preserve endothelial function and tissue perfusion.
|
|
| 8. |
- Malmsjö, Malin, et al.
(författare)
-
P2U-receptor mediated endothelium-dependent but nitric oxide-independent vascular relaxation
- 1998
-
Ingår i: British Journal of Pharmacology. - : Wiley. - 1476-5381 .- 0007-1188. ; 123:4, s. 719-729
-
Tidskriftsartikel (refereegranskat)abstract
- 1. The dilator effect of extracellular adenosine triphosphate (ATP) has mainly been characterized as a direct effect on smooth muscle or as an endothelium-dependent effect mediated by nitric oxide (NO) or prostaglandins. We tested the hypothesis that endothelium-derived hyperpolarizing factor (EDHF) may also be involved. Dilator effects were studied in vitro by continuous recording of isomeric tension in cylindrical segments of rat blood vessels precontracted by noradrenaline (NA), in the presence of indomethacin (10 microM). 2. By screening different blood vessels in the rat we found that both acetylcholine (ACh) and ATP dilate mesenteric arteries with a resting tone of 1 mN by an endothelium-dependent non-NO mechanism. With an increased resting tone (4 mN) the dilatation was mediated by NO. Thus by varying the resting tension the different dilator mechanisms could be examined. However, in the carotid artery the dilatation was solely mediated by an endothelium-dependent NO mechanism, even at different resting tones (1 and 4 mN). 3. The N-nitro-L-arginine methyl ester (L-NAME)-resistant dilatation to ACh and ATP was further inhibited by the NO-scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), indicating L-NAME insensitive NO-synthesis. 4. In carotid arteries and mesenteric arteries at high resting tones (4 mN) the ATP-dilatation was totally inhibited by endothelium removal or L-NAME (10(-3) M). In mesenteric arteries at low resting tone (1 mN) the ATP, UTP (uridine-triphosphate) and 2-MeSATP (2methylthioATP)-dilatation was totally inhibited by endothelium removal. However, L-NAME in combination with indomethacin attenuated only 5% of the UTP dilatation, 70% of the ATP dilatation but all of the 2-MeSATP-dilatation. The inhibitors of Ca2+-activated K+ channels charybdotoxin (0.5 x 10(-7) M) together with apamin (10(-6) M), and the cytochrome P450 inhibitor, SKF 525A (10(-4) M), each in combination with indomethacin. L-NAME and PTIO (0.5 x 10(-3) M) totally abolished the remaining ATP and UTP-dilatation. This indicates a dilatation mediated by an endothelium-dependent non-NO factor, probably EDHF. 5. Agonist potency (UTP>ATP>>2-MeSATP), indicates that the EDHF-mediated dilatation was stimulated by a P2U-receptor, possibly by a selective pyrimidine-receptor. In contrast, a P2Y-receptor stimulated NO-mediated dilatation (2-MeSATP=ATP>UTP). 6. In conclusion, the dilator effects of ATP and especially UTP can be mediated by an endothelium-dependent non-NO-mediated mechanism, probably EDHF, mediated by a P2U-receptor, possibly a selective pyrimidine-receptor, while NO-mediated dilatation is stimulated mainly by a P2Y1-receptor. Furthermore, the EDHF-dilatation is dependent on the resting tone of the blood vessel.
|
|
| 9. |
- Malmsjö, Malin, et al.
(författare)
-
P2X receptors counteract the vasodilatory effects of endothelium derived hyperpolarising factor
- 2000
-
Ingår i: European Journal of Pharmacology. - 1879-0712. ; 390:1-2, s. 173-180
-
Tidskriftsartikel (refereegranskat)abstract
- Dilatory responses of extracellular nucleotides were examined in the precontracted isolated rat mesenteric artery. Dilatation mediated by endothelium-derived hyperpolarising factor (EDHF) was studied in the presence of Nomega-nitro-L-arginine (L-NOARG) and indomethacin, and was most potently induced by the selective P2Y(1) receptor agonist adenosine 5'-O-thiodiphosphate (ADPbetaS), while 2-methylthioadenosine triphosphate (2-MeSATP) and adenosine triphosphate (ATP) were almost inactive. However, after P2X receptor desensitisation (with alphabeta-methylene-adenosine triphosphate, alphabeta-MeATP), 2-MeSATP and ATP potently stimulated EDHF-mediated dilatation. This can be explained by simultaneous activation of endothelial P2Y receptors that release EDHF, and depolarising P2X receptors on smooth muscle cells. Uridine triphosphate (UTP) also induced potent dilatation, suggesting EDHF release via P2Y(2)/P2Y(4) receptors. Uridine diphosphate (UDP) had only minor dilatory effects, and when pretreated with hexokinase it was almost inactive, suggesting a minor role for P2Y(6) receptors. The nitric oxide (NO) mediated dilatation was studied in the presence of charybdotoxin, apamin and indomethacin. ADPbetaS, 2-MeSATP, ATP and UTP were all potent relaxant agonists suggesting NO release via P2Y(1) and P2Y(2)/P2Y(4) receptors, while UDP was much less potent and efficacious. P2X receptor desensitisation had only minor effect on the NO-mediated dilatations. In conclusion, both EDHF and NO-mediated dilatation can be induced by activation of P2Y(1) and P2Y(2)/P2Y(4) receptors. P2X receptor stimulation of smooth muscle cells selectively counteracts the dilatory effect of EDHF.
|
|
| 10. |
- Malmsjö, Malin, et al.
(författare)
-
P2Y receptor-induced EDHF vasodilatation is of primary importance for the regulation of perfusion pressure in the peripheral circulation of the rat.
- 2002
-
Ingår i: Acta Physiologica Scandinavica. - : Wiley. - 0001-6772. ; 174:4, s. 301-309
-
Tidskriftsartikel (refereegranskat)abstract
- Extracellular nucleotides have been shown to induce vasodilatation of conductance arteries by release of the endothelium-derived hyperpolarizing factor (EDHF). As small resistance arteries are of greater importance for blood pressure regulation, a whole rat mesenteric arterial bed preparation was used in the present study when evaluating the physiological relevance for EDHF in mediating nucleotide dilatation. Dilatory responses were examined after pre-contraction with noradrenaline in the presence of 10 mM indomethacin. Adenosine-5'-O-thiodiphosphate (ADPbetaS), adenosine triphosphate (ATP) and uridine triphosphate (UTP) induced vasodilatation (pEC50=6.5-7 and E(max)=40-70%), while uridine diphosphate (UDP) was ineffective. Endothelium-derived hyperpolarizing factor was studied in the presence of 0.5 mM Nvarpi-nitro-L-arginine (L-NOARG). ADPbetaS and UTP induced strong and potent EDHF-dilatations, while ATP only had a weak effect (E(max)=25%). After P2X1 receptor desensitization with 10 microM alphabeta-methylene-adenosine triphosphate, the ATP response was significantly increased (E(max)=65%). Putatively, this could be because of simultaneous activation of both endothelial P2Y receptors and P2X1 receptors on smooth muscle cells, which resulted in the release of EDHF and subsequent hyperpolarization, and depolarization, respectively. Nitric oxide (NO) was studied in the presence of 60 mM K+. ADPbetaS, ATP and UTP induced weak NO dilatations, suggesting a minor role for NO as compared with EDHF. In conclusion, extracellular nucleotides stimulate dilatation by activating P2Y(1) and P2Y(2)/P2Y(4) receptors, but not P2Y(6) receptors. The dilatory responses are mediated primarily by EDHF in the peripheral vascular bed.
|
|
