Evidence that UTP and ATP regulate phospholipase C through a common extracellular 5'-nucleotide receptor in human airway epithelial cells

Extracellular ATP and UTP produced a rapid accumulation of inositol phosphates in human airway epithelial cells (CF/T43). The order of agonist potencies for a series of nucleotide analogues differed markedly from that of the classically described P2x- or P2y-purinergic receptors. UTP was the most potent agonist and was fully efficacious; ATP and adenosine-5'-O-(3-thiotriphosphate) were also full agonists. In contrast, 2-methylthio-ATP, adenosine-5'-O-(2-thiodiphosphate) and alpha,beta-methylene-ATP were without effect. ADP and UDP had little or no effect at concentrations as high as 100 microM, and deoxyribose and dideoxyribose compounds were inactive. The effects of ATP and UTP were not additive, whereas bradykinin- or histamine-stimulated inositol phosphate production was additive with the effects of ATP or UTP. Preincubation of cells with either UTP or ATP resulted in a parallel loss of responsiveness to both agonists. Desensitization was specific for the response to nucleotides, because no ATP- or UTP-induced effect on the response to histamine or bradykinin was observed. Pertussis toxin treatment of CF/T43 cells produced a 30-40% decrease in the response to ATP or UTP, which correlated with the ADP-ribosylation of 41- and 43-kDa proteins. Bradykinin and histamine responses were not modified by pertussis toxin. Guanine nucleotides had little effect on the inositol phosphate response in intact CF/T43 cells at concentrations below 100 microM. However, in streptolysin-O-permeabilized cells GTP-gamma S produced a concentration-dependence activation of inositol phosphate formation. UTP or ATP had little effect in permeabilized cells in the absence of guanine nucleotides but markedly increased inositol phosphate formation in the presence of guanine nucleotides. Taken together, these results suggest that UTP and ATP activate a 5'-nucleotide receptor on CF/T43 cells that is distinct from the classically defined P2x- and P2y-purinergic receptors. Activation of phospholipase C by this receptor involves, at least in part, a guanine nucleotide-binding regulatory protein.     

Evidence for a nucleotide receptor on adrenal medullary endothelial cells linked to phospholipase C and phospholipase D.

1. We have investigated whether the 'atypical' P2-purinoceptor previously described on adrenal microvasculature endothelial cells is a nucleotide receptor (responds to pyrimidines and purines) and is linked to phospholipase D as well as phospholipase C. 2. Cultured bovine adrenal medullary endothelial (BAME) cells responded to the pyrimidine UTP, as well as the purines. The total [3H]-inositol phosphate responses were with a rank order of UTP > ATP- = adenosine 5'-O-(3-thio-triphosphate) (ATP gamma S) >> 2MeSATP. The selective P2x agonist beta, gamma-methylene ATP was inactive. 3. Construction of dose-response curves to ATP, ATP gamma S and UTP in the presence and absence of additional agonists showed that responses to ATP gamma S and UTP were not additive, nor were those to UTP and ATP. This suggests that purines and pyrmidines acted via a common nucleotide receptor. 4. 32P-labelled BAME cells, in the presence of butanol, produced [32P]-phosphatidylbutanol (PBut) when stimulated with ATP gamma S or the protein kinase C activator, tetradecanoyl phorbol acetate (TPA). 5. Cells labelled with [3H]-palmitate and stimulated in the presence of butanol generated [3H]-PBut with the same order of agonist potencies seen for inositol phosphate responses. 6. The protein kinase C inhibitor, Ro 31-8220, abolished TPA and agonist stimulation of [3H]-PBut production. 7. These observations, and our related studies on bovine aortic endothelial cells, provide the first demonstration of a phospholipase C linked nucleotide receptor on vascular endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a vasoconstriction-mediating receptor for UTP,distinct from the P2 purinoceptor,in rabbit ear artery

Summary 1. The mechanism of uridine 5-triphosphate-(UTP-)induced vasoconstriction was studied in the rabbit ear artery. The arteries were incubated and perfused at a constant rate of flow. Vasoconstriction was measured as an increase in perfusion pressure. 2. Noradrenaline, adenosine 5'-triphosphate (ATP) and UTP caused concentration-dependent vasoconstriction. ATP and UTP were approximately equipotent. 3. The vasoconstrictor effect of UTP 300 mol/l was enhanced by a mixture of atropine, diphenhydramine and methysergide (1 mol/l each) and not affected by indometacin 10 mol/l. 4. Prazosin (0.01 –1 mol/l) and phentolamine (1–10 mol/l) reduced the vasoconstrictor effect of UTP 300 mol/l by up to 34%. Prazosin 1 mol/l failed to diminish the vasoconstrictor effect of UTP 300 mol/l after the sympathetic nerves had been destroyed with 6-hydroxydopamine. 5. , -Methylene-ATP (10–50 ol/l) elicited transient vasoconstriction. Subsequently, vasoconstrictor responses to ATP 100 or 300 pmol/1 were reduced by 88%, whereas responses to UTP 100 gmol/1 were enhanced, responses to UTP 300 mol/l decreased by only 32% and responses to UTP 1000 gmol/1 reduced by 74%. After in vitro-denervation with 6-hydroxydopamine or in the presence of phentolamine 1 mol/l throughout, a, -methylene-ATP (10–50 mol/l) reduced the vasoconstrictor effect of UTP 300 mol/l by 44% and 43%, respectively. 6. We suggest that, in the rabbit ear artery, the non-adrenergic and , -methylene-ATP-resistant vasoconstrictor response to UTP is mediated by a separate receptor mechanism, distinct from the P₂ purinoceptor. Send offprint requests to K. Starke     

Stimulation by the nucleotides, ATP and UTP of mitogen-activated protein kinase in EAhy 926 endothelial cells.

1. We have investigated the characteristics of activation of the 42kDa isoform of mitogen-activated protein (MAP) kinase in response to various nucleotides in the endothelial cell line EAhy 926. 2. Adenosine 5'-triphosphate (ATP) in the concentration range 0.1-100 microM stimulated the rapid and transient tyrosine phosphorylation and activation of the 42 kDa isoform of MAP kinase in EAhy 926 endothelial cells which peaked at 2 min and returned to basal values by 60 min. ATP also stimulated a similar response in primary cultured bovine aortic endothelial cells. 3. Uridine 5' triphosphate (UTP) also stimulated the 42 kDa isoform of MAP kinase with similar potency to ATP (EC50 values 5.1 +/- 0.2 microM for UTP; 2.9 +/- 0.8 microM for ATP), whilst the selective P2Y-purinoceptor agonist, 2-methylthioATP (2-meSATP) was without effect up to concentrations of 100 microM. In bovine aortic endothelial cells however, UTP and 2-meSATP both stimulated MAP kinase. 4. Pretreatment of cells for 24 h with 12-O tetradecanoyl phorbol 13-acetate resulted in the loss of the alpha and epsilon isoforms of protein kinase C (PKC) and virtual abolition of nucleotide-stimulated MAP kinase activity (> 90% inhibition). 5. Preincubation for 30 min with the PKC inhibitor, Ro-31 8220 (10 microM) reduced MAP-kinase activation at 2 min but potentiated the response at 60 min. 6. Removal of extracellular calcium in the presence of EGTA reduced the MAP kinase activation in response to UTP by approximately 30-50%. 7. Pretreatment with pertussis toxin (18 h, 50 ng ml-1) did not significantly affect the UTP-mediated activation of pp42 MAP kinase. 8. These results show that in the EAhy 926 endothelial cell line, nucleotides stimulate activation of MAP kinase in a protein kinase C-dependent manner through interaction with a P2U-purinoceptor.     

Evidence from desensitization studies for distinct receptors for ATP and UTP on the rat superior cervical ganglion.

Prolonged contact of the rat superior cervical ganglia (SCG) with the purine alpha,beta-methylene-ATP (alpha,beta-Me-ATP) selectively depressed responses to ATP and alpha,beta-Me-ATP but not responses to uridine 5'-triphosphate (UTP), potassium or adenosine. Prolonged contact with the pyrimidine UTP selectively depressed responses to UTP but not responses evoked by alpha,beta-Me-ATP, potassium or adenosine. These results are consistent with the presence of P2-purinoceptors and pyrimidinoceptors on the rat SCG and the hypothesis that pyrimidinoceptors exist within the nervous system.     

Evidence for a new G protein-coupled cannabinoid receptor in mouse brain.

The purpose of these studies was to support the hypothesis that an undiscovered cannabinoid receptor exists in brain. [(35)S]GTP gamma S binding was stimulated by anandamide and WIN55212-2 in brain membranes from both CB(1)(+/+) and CB(1)(-/-) mice. In contrast, a wide variety of other compounds that are known to activate CB(1) receptors, including CP55940, HU-210, and Delta(9)-tetrahydrocannabinol, failed to stimulate [(35)S]GTP gamma S binding in CB(1)(-/-) membranes. In CB(1)(-/-) membranes, SR141716A affected both basal and anandamide- or WIN55212-2-induced stimulation of [(35)S]GTP gamma S binding only at concentrations greater than 1 microM. In CB(1)(+/+) membranes, SR141716A inhibited only 84% of anandamide and 67% of WIN55212-2 stimulated [(35)S]GTP gamma S binding with an affinity appropriate for mediation by CB(1) receptors (K(B) approximately 0.5 nM). The remaining stimulation seemed to be inhibited with lower potency (IC(50) approximately 5 microM) similar to that seen in CB(1)(-/-) membranes or in the absence of agonist. Further experiments determined that the effects of anandamide and WIN55212-2 were not additive, but that the effect of mu opioid, adenosine A1, and cannabinoid ligands were additive. Finally, assays of different central nervous system (CNS) regions demonstrated significant activity of cannabinoids in CB(1)(-/-) membranes from brain stem, cortex, hippocampus, diencephalon, midbrain, and spinal cord, but not basal ganglia or cerebellum. Moreover, some of these same CNS regions also showed significant binding of [(3)H]WIN55212-2, but not [(3)H]CP55940. Thus anandamide and WIN55212-2 seemed to be active in CB(1)(-/-) mouse brain membranes via a common G protein-coupled receptor with a distinct CNS distribution, implying the existence of an unknown cannabinoid receptor subtype in brain.     

Evidence for a peripheral mechanism in cardiac opioid withdrawal

Studies involving heart catecholaminergic systems in morphine-dependent rats have not established whether the adaptive changes observed in the heart during morphine withdrawal are mediated peripherally or centrally. In this study, naloxone (Nx), naloxone methiodide (NxM) and N-methyl levallorphan (NML), quaternary derivatives of Nx and levallorphan, respectively, that do not cross the blood-brain barrier, were administered to morphine-dependent rats and catecholamines and their metabolites determined in the right ventricle. Rats were made dependent on morphine by implantation of morphine pellets for 7 days. On day 8 animals received s.c. injections of saline, Nx (1 mg/kg), NxM (5 mg/kg) or NML (5 mg/kg) and were decapitated 30 min later. Noradrenaline (NA) and its metabolites normetanephrine (NMN) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) and dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were determined by high-performance liquid chromatography with electrochemical detection. After NxM or NML administration to morphine-dependent rats there was a pronounced increase in NMN and DOPAC levels, as well as in NA and DA turnovers (as estimated by NMN/NA and DOPAC/DA ratios, respectively) in the right ventricle. Similarly, giving Nx to morphine-dependent rats increased NMN and DOPAC levels and NA and DA turnovers. In addition, in the paraventricular nucleus of the hypothalamus (PVN) NA and DA turnover, measured as the MHPG/NA or DOPAC/DA ratios, increased after Nx administration but not after NxM or NML These results suggest that the changes in cardiac sympathetic activity observed during morphine withdrawal are due to intrinsic mechanisms outside the central nervous system. These data may be important for understanding the adaptive changes induced in the heart in subjects dependent on opioids.     

Evidence for muscarinic receptors in endothelial cells from combined functional and binding studies

The aim of this study was to characterize muscarinic receptors of the bovine coronary artery by means of a combination of mechanical relaxation and contraction responses and radioligand binding data. Fresh helical strips of bovine coronary artery with intact endothelium relaxed in response to low concentrations (0.03-1 microM) of acetylcholine (ACh) and contracted at higher concentrations while endothelium-denuded strips only contracted. The ED50 for relaxation was 0.13 microM and that for contraction 1.8 microM (without endothelium); in the presence of endothelium, contraction dose-response curves were shifted to the right and the maximum contraction was reduced. In order to determine the location of the receptors mediating vasorelaxation, apparent affinity constants (KA) of ACh for relaxant and contractile effects were determined by irreversible blockade of a fraction of receptors with propyl benzilylcholine mustard (PBCM). The affinity constants (KA) were 0.22 microM for relaxation and 13 microM (with endothelium) and 20 microM (without endothelium) for contraction. In competition binding experiments against the muscarinic antagonist, [3H]N-methylscopolamine ([3H]NMS), the apparent affinity (KI) of ACh for binding sites in homogenates of endothelium-free coronary artery was 16 microM which was not different from the affinity constant determined in functional contraction experiments. Thus, the affinity constant of ACh determined for relaxation responses with endothelium-preserved vessels had no correlate in the binding affinity as determined with endothelium-free arteries. These findings indicate that bovine coronary arteries are relaxed by ACh through muscarinic receptors located on the endothelium whereas contractions are mediated by receptors on smooth muscle cells.     

Evidence for an A2-subtype adenosine receptor on pancreatic glucagon secreting cells.

The effects of a 5'-substituted analogue of adenosine, 5'-N-ethylcarboxamidoadenosine (NECA) have been studied on glucagon secretion in vitro, using the isolated pancreas of the rat perfused in the presence of glucose (2.8 mM). NECA provoked a peak of glucagon secretion, the kinetics of which were comparable to those previously obtained with adenosine. The effect was concentration-dependent and appeared at nanomolar concentrations. The EC50 was approximately 4 X 10(-8) M. A comparison of relative potency between adenosine and NECA showed that NECA was about 800 fold more potent than adenosine in inducing glucagon secretion. Theophylline (50 microM) considerably decreased the peak of glucagon secretion induced by 1.65 microM NECA and totally suppressed the effect of 16.5 nM NECA. These results indicate the involvement of an adenosine receptor. These and other previous results (low stereoselectivity of N6-phenylisopropyladenosine) provide evidence for an adenosine receptor of the A2-subtype being involved in glucagon secretion.     

Evidence for an intracellular action of platelet-activating factor in bovine cultured aortic endothelial cells

Bovine culture endothelial cells (BAECs) generate platelet-activating factor (Paf) following activation by bradykinin (Bk 0.1 nM), the ionophore, A23187 (3 microM), and ATP (10 microM), but Paf is not released from the cells. These stimuli also elicit generation of prostacyclin (PGI2). The specific and competitive Paf receptor antagonists, WEB 2086 (0.1-1.0 microM) and CV 6209 (0.01-0.1 microM), inhibited Bk-, A23187- and, to a lesser extent, ATP-induced PGI2 generation but had no effect on basal PGI2 generation. These data suggest a role for intracellular Paf in signal transduction.     

Human malignant glioma cells expressing functional formylpeptide receptor recruit endothelial progenitor cells for neovascularization

Endothelial progenitor cells (EPCs) are involved in tumor neovascularization with undefined mechanisms. In this study, we explored the role of formylpeptide receptor, a G protein-coupled receptor, expressed by human malignant glioma cells in neovascularization of malignant glioma. EPCs were isolated from human umbilical cord blood and their migratory capability and tubulogenesis induced by the supernatant of U87 glioblastoma (GBM) cell line were examined. We also assessed the recruitment and incorporation of EPCs into orthotopic intracranial tumors formed by implanted U87 GBM cells. The supernatant of control U87 cells induced high levels of migration and tubule-formation in vitro by EPCs. In contrast, the chemotactic and tubule-stimulating activities on EPCs in the supernatant of U87 cells with FPR knocking down by small interference (si) RNA were significantly attenuated. In addition, the number of EPCs recruited and incorporated into intracranial glioma xenografts was significantly higher in tumors formed by control U87 cells than tumors formed by U87 cells containing FPR-siRNA. Our results suggest that expression of functional FPR in glioma cells plays an important role in regulating vasculogenesis by EPCs, which constitute a novel target for anti-angiogenic therapy in gliomas.     

Evidence for a pulmonary B3 bradykinin receptor

We have examined pulmonary effects of bradykinin (Bk) in vivo and in vitro in guinea pigs and their potential inhibition by antagonists of Bk B1 and B2 receptors. Bk was a potent bronchoconstrictor in vivo and caused contractions of isolated, epithelium-denuded trachealis. D-Arg[Hyp3,D-Phe7]-Bk (NPC567) and D-arg[Hyp3,Thi5,8,D-Phe7]-Bk (NPC349), B2 receptor antagonists, were weak inhibitors of Bk-induced bronchoconstriction in vivo and were virtually inactive as antagonists of Bk-induced airway smooth muscle contraction. Several other B2 antagonists as well as B1 antagonist, des-Arg9-[Leu8]-Bk, did not inhibit Bk-induced tracheal contraction. The B1 receptor agonist des-Arg9-Bk was without effect on tracheal tone. Tracheal responses to Bk were unaffected by antagonists of muscarinic, histamine, serotonin, and catecholamine receptors. The inability of the antagonists to inhibit Bk is unlikely to be due to their degradation, because NPC567 was only weakly active in the presence of inhibitors of kininase I (EC 3.4.11.2), kininase II (EC 3.4.15.1), and neutral endopeptidase (EC 3.4.24.11). These studies were corroborated by ligand binding experiments in guinea pig and ovine airways. In [3H]Bk binding, the Bk antagonists had no effect in guinea pig trachea, slightly displaced [3H]Bk in ovine trachea, and inhibited approximately 60% of total specific binding in lung. des-Arg9-[Leu8]-Bk and several other agents, including atropine, neurokinin A, substance P, and vasoactive intestinal peptide, had no effect on lung Bk binding. Bk and its analogs were not degraded during the binding assay. These data suggest that pulmonary tissue, particularly in the large airways, contains a novel Bk binding site, a B3 receptor, which may be involved in Bk-induced bronchoconstriction.     

Evidence for a P2-purinoceptor mediating vasoconstriction by UTP,ATP and related nucleotides in the isolated pulmonary vascular bed of the rat.

1. The vasoconstrictor effects of uridine 5''-triphosphate (UTP), uridine 5''-diphosphate (UDP), uridine 5''-monophosphate (UMP) and uridine were tested in the isolated pulmonary vascular bed of the rat. Comparison was made with the effects of adenine nucleotides, adenosine 5''-triphosphate (ATP), adenosine 5''-diphosphate (ADP), adenosine 5''-monophosphate (AMP) and with adenosine. The effect of P2x-purinoceptor desensitization and blockade was compared on the vascular responses to uracil and adenine nucleotides. 2. At doses ranging from 10(-8) to 10(-5) mol, UTP elicited dose-dependent vasoconstriction. UDP was equiactive to UTP, while UMP and uridine did not show vasomotor activity. Similarly, ATP showed dose-related vasoconstrictor activity. ADP was less potent than ATP in eliciting vasoconstriction, while AMP was active only at the higher doses tested and adenosine was ineffective. 3. Vasoconstriction was produced by ATP analogues with the following order of potency: alpha, beta-methylene ATP > ATP gamma S > beta, gamma-methylene ATP > 2-methylthio ATP > or = ATP. 4. Desensitization of P2x-purinoceptors by the selective agonist alpha, beta-methylene ATP did not modify the vasoconstrictor activity of UTP and UDP and only partially reduced vasoconstrictor responses to ATP, while it abolished vascular responses to alpha, beta-methylene ATP itself. 5. The antagonists of P2-purinoceptors, suramin and pyridoxalphosphate-6-azophenyl-2'', 4''-disulphonic acid (PPADS), did not affect vascular responses to UTP and UDP, but reduced vasoconstriction evoked by beta, gamma-methylene ATP and ATP by about 70 and 30%, respectively. 6. This study demonstrates that uracil nucleotides, UTP and UDP, elicit vasoconstriction in the rat pulmonary vascular bed. In addition to confirming the presence of classical P2x-purinoceptors, these results also suggest the presence of a distinct purinoceptor subtype which mediates UTP- and ATP- evoked vasoconstriction in the rat pulmonary circulation.     

Quantitation of extracellular UTP using a sensitive enzymatic assay.

The wide distribution of the uridine nucleotide-activated P2Y2, P2Y4 and P2Y6 receptors suggests a role for UTP as an important extracellular signalling molecule. However, direct evidence for UTP release and extracellular accumulation has been addressed only recently due to the lack of a sensitive assay for UTP mass. In the present study, we describe a method that is based on the uridinylation of [14C]-glucose-1P by the enzyme UDP-glucose pyrophosphorylase which allows quantification of UTP in the sub-nanomolar concentration range. The UTP-dependent conversion of [14C]-glucose-1P to [14C]-UDP-glucose was made irreversible by including the pyrophosphate scavenger inorganic pyrophosphatase in the reaction medium and [14C]-glucose-1P and [14C]-UDP-glucose were separated and quantified by HPLC. Formation of [14C]-UDP-glucose was linearly observed between 1 and 300 nM UTP. The reaction was highly specific for UTP and was unaffected by a 1000 fold molar excess of ATP over UTP. Release of UTP was measured with a variety of cells including platelets and leukocytes, primary airway epithelial cells, rat astrocytes and several cell lines. In most resting attached cultures, extracellular UTP concentrations were found in the low nanomolar range (1-10 nM in 0.5 ml medium bathing 2.5 cm2 dish). Up to a 20 fold increase in extracellular UTP levels was observed in cells subjected to a medium change. Extracellular UTP levels were 10-30% of the ATP levels in both resting and mechanically-stimulated cultured cells. In unstirred platelets, a 1:100 ratio UTP/ ATP was observed. Extracellular UTP and ATP increased 10 fold in thrombin-stimulated platelets. Detection of UTP in nanomolar concentrations in the medium bathing resting cultures suggests that constitutive release of UTP may provide a mechanism of regulation of the basal activity of uridine nucleotide sensitive receptors.     

Characterization of histamine receptor sub-types regulating prostacyclin release from human endothelial cells.

1. The histamine receptor sub-types that are involved in the initiation and maintenance of prostacyclin (PGI2) release from human endothelial cells have been investigated. 2. Endothelial cells cultured from umbilical vein (HUVEC) were incubated with either histamine, the selective H1-receptor agonists, 2-methyl histamine (2-MeHA) or thiazolylethylamine (ThEA), the H1-agonist/H3-antagonist, beta-histidine (beta-His), the selective H2-agonist, dimaprit, the H2-agonist/H3-antagonist, impromidine, the selective H3-agonist, (R)alpha-methylhistamine ((R)alpha-MeHA) and the H3-antagonist, thioperamide. 3. The H1-agonists and the H3-agonist (R)alpha-MeHA induced a concentration (100 nM-1 mM) and time-dependent release of PGI2 as determined by radioimmunoassay for 6-keto-PGF1 alpha, but were less potent than histamine itself. The rank order of potency was the same following 30 min and 24 h incubation, i.e. histamine > ThEA > 2-MeHA > beta-His > (R)alpha-MeHA. 4. Histamine and 2-MeHA (1 microM-1 mM), ThHEA (10 microM-1 mM) and (R)alpha-MeHA (1 mM), but not beta-His, induced a significantly greater increase in PGI2 release after 24 h incubation than after 30 min incubation (P < 0.05). 5. Neither the selective H2-agonist, dimaprit, nor the H2-agonist/H3-antagonist, impromidine alone induced release of PGI2. 6. The H1-antagonist, mepyramine (10 microM), abolished release of PGI2 induced by histamine, the H1-agonists and (R)alpha-MeHA but the H2-antagonist cimetidine (10 microM) and the H2/H3-antagonist, burimamide (10 microM) did not significantly modulate PGI2 release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the thromboxane receptor mediating prostacyclin release from cultured endothelial cells.

The thromboxane A2 (TXA2) mimetic, 9,11-dideoxy-11,9-epoxymethano-prostaglandin F 2 alpha (U46619), mobilized calcium in the bovine aortic endothelial cell line AG4762 and stimulated release of prostacyclin from these cells. The U46619-stimulated release of prostacyclin could be inhibited by TXA2 antagonists with the order of potency [Is-[1 less than a, 2 less than b(5z), 3 less than b, 4 less than a]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo- [2.2.1]hept-2-yl]-5- heptenoic acid (SQ29548) greater than 4-[2-(4-chlorobenzene-sulphonamido) ethyl]phenylacetic acid (BM13505) greater than 4-[2-(phenylsulphonamido)-ethyl]phenoxyacetic acid (BM13177), which was consistent with release being mediated by a TXA2 (TP) receptor. The TP receptor ligands, [3H]SQ29548 and 9,11-dimethylmethano-16(3-[125I]iodo-4-hydroxyphenyl)-13,14-dih ydr o-13-aza- 15-omega-o-tetranor-thromboxane ([125I]-PTA-OH), both appeared to bind to a homogenous population of sites in AG4762 cell membranes. The affinities of [3H]SQ29548 and [125I]PTA-OH were approximately 10 nM and approximately 0.3 nM, respectively, and the density of sites labelled by either ligand was approximately 25 fmol/mg protein. Under conditions where equilibrium was approached, the specific binding of [3H] SQ29548 or [125I]PTA-OH was displaced by SQ29548, BM13505 and BM13177 with the same order of potency and similar apparent affinities as in the functional assay, suggesting that these binding sites represent bona fide TP receptors.     

Isolated cardiac cells for electropharmacological studies.

Single cardiac myocytes provide a model widely used to characterize the electrophysiological properties of drugs and to identify new therapeutic targets. This review focuses on isolation procedures to obtain single cardiac myocytes from several mammal species, including humans, and on patch-clamp technique as a useful method to investigate the molecular mechanism of druy actions.     

A contraction-mediating receptor for UTP, presumably P2Y2, in rat vas deferens

The possible existence of a contraction-mediating P2-receptor for uracil nucleotides was investigated in the rat vas deferens. In order to minimize breakdown of nucleotides, Evans blue was used as an inhibitor of ectonucleotidases. UTP was degraded by rat vas deferens tissue, and the degradation was inhibited by Evans blue (100 microM). In the absence of other drugs, UTP and UDP elicited marginal contractions. Evans blue (100 microM) greatly enhanced contractions elicited by the uracil nucleotides. When the medium contained alpha,beta-MeATP (100 microM) in addition to Evans blue in order to desensitize contraction-mediating P2X1-receptors, responses to UTP and UDP were not changed; in contrast, responses to alpha,beta-MeATP were virtually abolished and contractions elicited by ATP and ADP were greatly reduced; EC50 values were 122 microM for UTP and 58 microM for ATP under these conditions. The P2-receptor antagonist suramin attenuated contractions elicited by UTP (320 microM) and alpha,beta-MeATP (32 microM) in the presence of Evans blue; pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS) also reduced responses to alpha,beta-MeATP but, at up to 100 microM, did not alter contractions elicited by UTP. Incubation of vasa deferentia in nominally calcium-free medium almost abolished the response to alpha,beta-MeATP (32 microM), while a major part of the contraction elicited by UTP (320 microM) was preserved. In the presence of Evans blue and alpha,beta-MeATP, prior addition of UDP (3200 microM) or ATP (320 microM), without washout, markedly reduced the response to UTP (320 microM); UTP and ATP also reduced the response to UDP; in contrast, prior addition of UTP or UDP did not alter the contraction to ATP. The results demonstrate the existence of a contraction-mediating, uracil nucleotide-sensitive P2Y-receptor in rat vas deferens, distinct from the P2X1-receptor. Pharmacological analysis indicates that it is P2Y2.     

Cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Evidence for a homologous nature among various mammalian species

The presence and properties of the Ah receptor were examined in the guinea pig, rat, hamster, monkey, and three different strains of mice. These species and strains have demonstrated differences in sensitivity and variability of response to 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. All species examined, with the exception of DBA/2J mice, possess similar amounts of binding protein with high affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin in hepatic tissue. Numerous dibenzo-p-dioxin congeners and polycyclic aromatic hydrocarbons demonstrated a similar rank order ability to bind to receptor molecules from these species. When analyzed by gel-exclusion high-performance liquid chromatography, hepatic cytosolic receptors from all species eluted at volumes corresponding to a similar molecular weight range. Association of the hepatic Ah receptor with the nuclear fraction was observed in all cases following the i.p. treatment of guinea pig, rat, C57BL/6J mouse, or hamster with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin. In all species and tissues examined, with the exception of hamster duodenum and thymus, the highest concentrations of receptor were localized in the liver, lung, thymus, intestine, and kidney. Exceptionally high concentrations of receptors were also observed in guinea pig testes. These findings indicate that, despite species and tissue specific differences in the biochemical and toxicological responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds, a number of different mammalian species possess Ah receptors with similar properties. Thus, the correlative differences between certain strains of mice in terms of altered specific binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and sensitivity to this compound may be unique and not necessarily applicable to other species. Although all data indicate that the receptor mediates these responses, it appears that species- and tissue-specific differences may be determined by a number of additional factors. These results also suggest the conservation of some, as yet unknown, functional role of the receptor molecule.