Effects of mitoxantrone on action potential and membrane currents in isolated cardiac myocytes.
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1. The effects of mitoxantrone (MTO), an anticancer drug, on the membrane electrical properties of cardiac myocytes were investigated using the whole-cell clamp technique. 2. In isolated guinea-pig ventricular myocytes, 30 microM MTO induced a time-dependent prolongation of action potential duration (APD) which was occasionally accompanied by early afterdepolarizations. APD prolongation was preserved in the presence of 10 microM tetrodotoxin and showed reverse rate-dependence. 3. Both the inward rectifier K+ current (I(KI)) and the delayed rectifier K+ current (I(K)) of guinea-pig ventricular myocytes were significantly depressed by 30 microM MTO. The rapidly activating component of I(k) (I(Kr)) seemed to be preferentially blocked by MTO. The transient outward current was not affected by MTO in rat ventricular myocytes. 4. Thirty microM MTO had no direct effect on the L-type Ca2+ current (I(Ca(L))), but reversed the inhibitory effect of 1 microM carbamylcholine but not the A1-adenosine receptor agonist (-)-N6-phenylisopropyladenosine (1 microM) on I(Ca(L)) enhanced by 50 nM isoprenaline in guinea-pig ventricular myocytes. In guinea-pig atrial mycotyes, 30 microM MTO inhibited by 93% the muscarinic receptor gated K+ current (I(K,ACh)) evoked by 1 microM carbamylcholine, whereas I(K,ACh) elicited by 100 microM GTPgammaS, a nonhydrolysable GTP analogue, was only decreased by 12%. 5. The specific binding of [3H]QNB, a muscarinic receptor ligand, to human atrial membranes was concentration-dependently displaced by MTO (1-1000 microM). 6. In conclusion, MTO blocks cardiac muscarinic receptors and prolongs APD by inhibition of I(KI) and I(Kr). The occasionally observed early afterdepolarizations may signify a potential cardiac hazard of the drug. (+info)
Recruitment of a heparan sulfate subunit to the interleukin-1 receptor complex. Regulation by fibronectin attachment.
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In this study, we identified an adhesion-regulated subunit of the interleukin-1 (IL-1) receptor complex. Transfection of fibroblasts with an IL-1 receptor-EGFP construct showed that the fusion protein was located at focal adhesions in cells attaching to fibronectin. Fibronectin attachment caused enhancement in endogenous IL-1 type I receptor levels from on average 2500 to 4300 receptors/cell. In addition, matrix attachment resulted in a decrease in binding affinity (Ka) from 1.0 x 10(9) (M-1) to 5.6 x 10(8) (M-1), due to a 2-fold reduction in association rate constant. The adhesion-mediated effects were reversed by soluble heparin. Cross-linking experiments showed that in cells attached to fibronectin, 50-70% of the radiolabeled IL-1 was associated with a heparinase sensitive, high molecular mass component of about 300 kDa, with a core protein of 80-90 kDa. Formation of the complex was dependent on cell interaction with the heparin binding region in fibronectin and required IL-1/type I IL-1 receptor binding. This report demonstrates the recruitment of a heparan sulfate to the IL-1 receptor complex, following attachment to fibronectin, which correlates with alterations in receptor function. The data suggest that the heparan sulfate constitutes an attachment regulated component of the IL-1 receptor complex with the role of mediating matrix regulation of IL-1 responses. (+info)
Functionalized congeners of 1,4-dihydropyridines as antagonist molecular probes for A3 adenosine receptors.
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4-Phenylethynyl-6-phenyl-1,4-dihydropyridine derivatives are selective antagonists at human A3 adenosine receptors, with Ki values in a radioligand binding assay vs [125I]AB-MECA [N(6)-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyl-adenosine] in the submicromolar range. In this study, functionalized congeners of 1,4-dihydropyridines were designed as chemically reactive adenosine A3 antagonists, for the purpose of synthesizing molecular probes for this receptor subtype. Selectivity of the new analogues for cloned human A3 adenosine receptors was determined in radioligand binding in comparison to binding at rat brain A1 and A2A receptors. Benzyl ester groups at the 3- and/or 5-positions and phenyl groups at the 2- and/or 6-positions were introduced as potential sites for chain attachment. Structure-activity analysis at A3 adenosine receptors indicated that 3,5-dibenzyl esters, but not 2,6-diphenyl groups, are tolerated in binding. Ring substitution of the 5-benzyl ester with a 4-fluorosulfonyl group provided enhanced A3 receptor affinity resulting in a Ki value of 2.42 nM; however, a long-chain derivative containing terminal amine functionalization at the 4-position of the 5-benzyl ester showed only moderate affinity. This sulfonyl fluoride derivative appeared to bind irreversibly to the human A3 receptor (1 h incubation at 100 nM resulting in the loss of 56% of the specific radioligand binding sites), while the binding of other potent dihydropyridines and other antagonists was generally reversible. At the 3-position of the dihydropyridine ring, an amine-functionalized chain attached at the 4-position of a benzyl ester provided higher A3 receptor affinity than the corresponding 5-position isomer. This amine congener was also used as an intermediate in the synthesis of a biotin conjugate, which bound to A3 receptors with a Ki value of 0.60 microM. (+info)
Inhibition of beta(2)-adrenergic and muscarinic cholinergic receptor endocytosis after depletion of phosphatidylinositol bisphosphate.
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Recent evidence supporting a role for phosphoinositides in the endocytosis of phospholipase C-coupled receptors has prompted an investigation of whether there exists a similar requirement for the internalization of adenylyl cyclase-linked receptors. When 1321N1 astrocytoma cells, which possess both muscarinic cholinergic receptors (mAChRs) that couple to phospholipase C and beta-adrenergic receptors (beta(2)-ARs) linked to adenylyl cyclase, were pretreated with wortmannin (WT) at a concentration known to inhibit phosphatidylinositol 4-kinase activity, the labeling of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP(2)) was reduced. Stimulation of phosphoinositide breakdown by activation of mAChRs in WT-pretreated cells led to a further depletion of PIP(2). As previously demonstrated for SH-SY5Y neuroblastoma, inclusion of WT inhibited the endocytosis of mAChRs in 1321N1 cells by >85%. In contrast, the internalization of beta(2)-ARs was only partially ( approximately 30%) prevented. However, when the concentration of PIP(2) was further reduced by exposure of WT-pretreated 1321N1 cells to a muscarinic agonist, the endocytosis of beta(2)-ARs was substantially inhibited (>70%). Lower concentrations of WT (100 nM) that were sufficient to fully inhibit phosphatidylinositol 3-kinase activity had no effect on either phosphoinositide synthesis or receptor endocytosis. The results indicate that the agonist-induced endocytosis of an adenylyl cyclase-linked receptor such as the beta(2)-AR, like that of the phospholipase C-coupled mAChR, is dependent on the synthesis of phosphoinositides and, in particular, that of PIP(2). (+info)
Mechanism-based pharmacokinetic-pharmacodynamic modeling of antilipolytic effects of adenosine A(1) receptor agonists in rats: prediction of tissue-dependent efficacy in vivo.
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In this study, we analyzed the antilipolytic effects of six N(6)-cyclopentyladenosine analogs in rats and developed a mechanistic pharmacokinetic-pharmacodynamic model to quantify and predict the tissue-selective action of adenosine A(1) receptor agonists in vivo. Freely moving rats received an i.v. infusion of vehicle or compound over 15 min. Arterial blood samples were taken at regular time intervals for the determination of concentrations of drugs using HPLC analysis and of nonesterified fatty acids (NEFAs). All N(6)-cyclopentyladenosine analogs that were investigated produced a significant decrease in the NEFA plasma concentration after i.v. infusion. The pharmacokinetic behavior of each ligand was described by a standard two-compartment model. The pharmacokinetic parameter estimates then were used to simultaneously fit the individual (n = 6-8) time-NEFA concentration profiles for each agonist to a physiological indirect response model in combination with the Hill equation to obtain estimates of the NEFA elimination rate constant (k(e)) and upper asymptote (fractional inhibition), midpoint location, and midpoint slope parameter (alpha, pEC(50), and n(H), respectively) of the concentration-effect relationship. Subsequently, the data were analyzed with the operational model of agonism to obtain estimates of in vivo affinity and efficacy. It was estimated that the in vivo density and/or coupling of adenosine A(1) receptors mediating antilipolytic effects is approximately 38 times higher compared with the receptors mediating bradycardia. The model predicts that it is possible to design ligands that produce significant inhibition of lipolysis and are completely devoid of cardiovascular effects in vivo. (+info)
Proteinase-activated receptor 2 (PAR(2)): development of a ligand-binding assay correlating with activation of PAR(2) by PAR(1)- and PAR(2)-derived peptide ligands.
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A cloned rat proteinase-activated receptor (PAR)(2)-expressing cell line (KNRK-rPAR(2)) was used to study the structure-activity relationships (elevated intracellular Ca(2+)) for a series of: 1) PAR(1)-derived receptor-activating ligands (PAR(1)-APs) [SFLLR (P5), SFLLR-NH(2) (P5-NH(2)), SFLLRNP (P7), SFLLRNP-NH(2) (P7-NH(2)), and TFLLR-NH(2) (TF-NH(2))] and 2) PAR(2)-derived-activating-peptides (PAR(2)-APs) [SLIGRL-NH(2) (SL-NH(2)), SLIGR-NH(2) (GR-NH(2)), and SLIGKV-NH(2) (KV-NH(2))]. The activities of the PAR-APs were compared with the PAR(2)-AP analog trans-cinnamoyl-Leu-Ile-Gly-Arg-Leu-Orn-NH(2) tc-NH(2)), which as a [(3)H]propionyl derivative ([(3)H]propionyl-tc-NH(2)) was used to develop a radioligand-binding assay for PAR(2). The relative potencies of the PAR-APs in the Ca(2+)-signaling assay were tc-NH(2) = SL-NH(2) > KV-NH(2) congruent with P5-NH(2) > GR-NH(2) > P7-NH(2) > P7 > P5 > TF-NH(2). The reverse sequence PAR-APs, LSIGRL-NH(2) (LS-NH(2)), LRGILS-NH(2) (LR-NH(2)), FSLLRY-NH(2) (FSY-NH(2)), and FSLLR-NH(2) (FS-NH(2)), as well as the Xenopus PAR(1)-AP TFRIFD-NH(2), were inactive. The relative biological potencies of the peptides were in accord with their ability to compete for the binding of [(3)H]propionyl-tc-NH(2) (tc-NH(2) = SL-NH(2) > GR-NH(2) congruent with P5-NH(2) > P5) to KNRK-rPAR(2) cells, whereas inactive peptides (FS-NH(2); LR-NH(2)) showed no appreciable binding competition. Our data therefore validate a ligand-binding assay for the use in studies of PAR(2) and indicate that the relative biological potencies of the PAR(1)-APs for activating rat PAR(2) parallel their ability to activate human PAR(1). The relative receptor-binding activities of the PAR-APs, although in general agreement with their relative biological activities, point to differences in the intrinsic receptor-activating activities between the several PAR-APs. The binding assay we have developed should prove of use for the further study of PAR(2)-ligand interactions. (+info)
Characterization of prejunctional and postjunctional muscarinic receptors of the ascending reflex contraction in rat ileum.
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The ascending reflex contraction of the small intestine involves predominantly cholinergic neurotransmission. The orally projecting neural excitatory pathway of the myenteric reflex was studied in an in vitro model of rat ileal segments. The contractile response elicited by aboral field stimulation was significantly inhibited by a range of muscarinic receptor antagonists. Methoctramine and tripitramine (both M(2) selective, pIC(50) = 9.3 and 8.8, respectively), darifenacin and hexahydrosiladifenidol (both M(3) selective, pIC(50) = 7.3 and 7.7, respectively), and pirenzepine (M(1) selective, pIC(50) = 7.0). In radioligand binding experiments on synaptosomal and smooth muscle plasma membrane fractions, we examined whether prejunctional or postjunctional muscarinic receptors exist that could potentially contribute to the reflex contraction. In the synaptosomal fraction, the muscarinic ligand [(3)H]N-methylscopolamine labeled a homogeneous population of receptors (Hill coefficient = 1) with a K(d) value of 0.31 +/- 0.09 nM and a B(max) value of 185 +/- 16.6 fmol/mg protein. The ratio of potency of subtype-selective muscarinic receptor antagonists in competition studies was tripitramine (pK(i) = 8.7 +/- 0.3) > 1/6 x methoctramine (pK(i) = 7.9 +/- 0.02) > 1/25 x darifenacin (pK(i) = 7. 3 +/- 0.2) > 1/316 x hexahydrosiladifenidol (pK(i) = 6.2 +/- 0.1) > 1/2511 x pirenzepine (pK(i) = 5.3 +/- 0.1). In the smooth muscle plasma membrane fraction, the K(d) value was 0.29 +/- 0.05 nM and the B(max) value was 770 +/- 29 fmol/mg. The competition studies revealed a similar ratio of potency of the respective antagonists. These data suggest that muscarinic M(2) receptors, located at prejunctional and postjunctional sites, are predominantly involved in the ascending reflex contraction. (+info)
Comparison of the ligand binding and signaling properties of human dopamine D(2) and D(3) receptors in Chinese hamster ovary cells.
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Human dopamine D(2) (hD(2)) and D(3) (hD(3)) receptors were expressed at similar, high expression levels in Chinese hamster ovary (CHO) cells, and their coupling to G proteins and further signal transduction pathways were compared. In competition radioligand-binding experiments, guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) treatment of hD(2S)- or hD(3)-CHO cell membranes induced a rightward shift and steeping of the dopamine inhibition curve. This effect was pronounced for hD(2) receptors and small for hD(3) receptors. Activation of G proteins was investigated in [(35)S]GTPgammaS-binding assays. Dopamine stimulated [(35)S]GTPgammaS binding 330 and 70% over basal levels on hD(2)-CHO and hD(3)-CHO cell membranes, respectively. (+)-7-(Dipropylamino)-5, 6,7,8-tetrahydro-2-naphthalenol and PD128907 were partial agonists for both receptors. Haloperidol, risperidone, raclopride, and nemonapride inhibited dopamine-stimulated [(35)S]GTPgammaS binding with potencies comparable to their binding affinities for hD(2) and hD(3) receptors in CHO cell membranes; inverse agonism could not be detected with this assay. Receptor stimulation by dopamine inhibited forskolin-induced cyclic AMP formation in hD(2)-CHO and hD(3)-CHO cells by 70%. Furthermore, the extracellular acidification rate increased when hD(2)-CHO and hD(3)-CHO cells were stimulated by dopamine; this effect was abolished by pertussis toxin pretreatment. In this study, we could demonstrate clear functional effects at different levels of the signaling cascade of hD(2) and hD(3) receptors in CHO cells when expressed at high levels. High-affinity agonist binding to hD(2) and hD(3) receptors was still present, but effects of receptor-G protein uncoupling at hD(3) receptors were small, indicating that hD(3) receptors maintain relatively high-affinity agonist binding in the absence of G proteins. (+info)