Serum KL-6 as a novel disease marker in adolescent and adult cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) is a chronic progressive disease leading to obstructive pulmonary impairment, fibrosis and shortened life expectancy. Serum levels of KL-6, high molecular weight human MUC1 mucin, are increased in the majority of patients with various interstitial lung disorders. Whether they are also elevated in CF has not been investigated before. OBJECTIVE: To evaluate whether serum KL-6 levels are elevated and correlate with pulmonary function variables in CF. DESIGN: Serum KL-6, lactate dehydrogenase (LDH) and C-reactive protein (CRP) levels were measured in 72 consecutive CF and 80 age- and sex-matched healthy control subjects. The relationship between serum KL-6 levels and pulmonary function variables was analyzed. RESULTS: Serum KL-6 levels in CF patients were significantly increased compared to healthy subjects. Receiver operating characteristic curve analysis revealed that the diagnostic accuracy of KL-6 was better than that of LDH and CRP. Serum KL-6 levels showed an inverse relationship with vital capacity (VC) % predicted and forced expiratory volume in one second (FEV1) % predicted. CONCLUSIONS: Serum KL-6 levels are elevated and appear to be correlated with pulmonary function variables in CF. These results suggest that KL-6 may be a useful noninvasive marker to monitor disease severity.

Glutamine and alanyl-glutamine dipeptide reduce mesenteric plasma extravasation, leukocyte adhesion and tumor necrosis factor-α (TNF-α) release during experimental endotoxemia.

Glutamine (GLN) appears to be an essential nutrient during organism development and critical illness. The aim of our study was to evaluate the effects of GLN and its generic preparation alanyl-glutamine-dipeptide (DIP) on the microcirculation in endotoxemia in rats and its effects on tonus or aortal rings in vitro. Male Lewis rats (n=40) were separated in 4 groups. Group 1 (CON) served as healthy control group while the other groups received an endotoxin bolus i.v. (5 mg/kg lipopolysaccharide, LPS i.v.). In group 3 (LPS+GLN) 0.75 g/kg-1 GLN i.v. before LPS challenge was administered. In group 4 (LPS+DIP) DIP containing 0.75 g/kg GLN was given. Leukocyte-endothelial interactions and mesenteric plasma extravasation were determined at 0, 1 and 2 hours during the experiment by intravital fluorescence microscopy (IVM). Cytokine release (TNF-alpha, IL-1 beta, IL-6, IL-10) was measured by ELISA. GLN treatment reduced leukocyte adherence (-49.7% vs. LPS group, p<0.05) and plasma extravasation (-12.3% vs. LPS group, p<0.05) significantly during endotoxemia compared to untreated LPS animals. In group 4 (DIP+LPS), a decrease of leukocyte adherence (-56.0%) and mesenteric plasma extravasation (-18.8% vs. LPS group, p<0.05) was also found. TNF-alpha levels were reduced in both GLN and DIP (p<0.05). In vitro experiments demonstrated that glutamine agents could attenuate the response to contracting agents in presence of the vascular endothelium, implying nitric oxide pathway. In vivo, GLN as well as DIP pre-treatment diminish the detrimental impact of endotoxemia on the mesenteric microcirculation and the TNF-alpha release, the effects whose clinical importance should be further examined.

Pharmacokinetics of inhaled colistin in patients with cystic fibrosis.

OBJECTIVES: Inhaled colistin is commonly used in patients with cystic fibrosis (CF), but only limited data are available to define its pharmacokinetic profile. PATIENTS AND METHODS: We performed a multicentre study in 30 CF patients to assess sputum, serum and urine concentrations after a single dose of 2 million units of colistin administered by inhalation. In a subgroup of patients we also compared the efficacy of two different nebulizers for administration of inhaled colistin. RESULTS: Serum concentrations of colistin reached their maximum 1.5 h after inhalation and decreased thereafter. Serum concentrations were well below those previously reported for systemic application in all patients. A mean 4.3+/-1.3% of the inhaled dose was detected in urine. Elimination characteristics did not differ significantly from those previously reported for systemic application. A positive correlation was found between forced expiratory volume in 1 s (FEV1) in per cent predicted and both AUC and maximal colistin concentrations in serum (Cmax). Maximum sputum concentrations were at least 10 times higher than the MIC breakpoint for Pseudomonas aeruginosa proposed by the British Society for Antimicrobial Chemotherapy. Although sputum drug concentrations decreased after a peak at 1 h, the mean colistin concentrations were still above 4 mg/L after 12 h. No differences were seen in polymyxin E sputum concentrations, for CF patients between the two nebulizer systems. CONCLUSIONS: The low systemic and high local concentrations of colistin support the use of inhaled colistin in CF patients infected with P. aeruginosa.

Nucleotide-evoked relaxation of rat vas deferens: possible mechanisms.

ATP causes relaxation of the K(+)-contracted rat vas deferens. Possible sites of action were investigated. ATP and adenosine relaxed the vas deferens precontracted with 80 mM K(+); EC(50) values and maximal relaxations averaged, respectively, 760 microM and 56% for ATP and 74 microM and 30% for adenosine. The adenosine P1 receptor antagonist 8-(para-sulfophenyl)theophylline (8-SPT) reduced relaxations caused by adenosine and low concentrations of ATP, as did the Rp-diastereomer of adenosine 3',5'-cyclic phosphorothioate (Rp-cAMPS), an inhibitor of protein kinase A. The phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) augmented responses to adenosine and low concentrations of ATP. alpha,beta-Methylene ADP, an inhibitor of 5'-nucleotidase, reduced relaxations caused by ATP to a similar extent as did 8-SPT. In the presence of an almost saturating concentration of adenosine, ATP caused further relaxation. Conversely, in the presence of ATP, adenosine had little effect. Like ATP, UTP and other nucleoside triphosphates relaxed the vas deferens. The P2 receptor antagonists reactive blue 2, acid blue 25 and 4,4'-diisothiocyanotostilbene-2,2'-disulphonate (DIDS) attenuated the relaxation caused by ATP; suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulphonate (PPADS), Evans blue, trypan blue, reactive red 2 and brilliant blue G had no effect. Three non-selective inhibitors of protein kinases, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), staurosporine and (8R*,9S*,11S*)-(-)-9-hydroxy-9-carboxy-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinden-1-one (K-252b), markedly reduced the relaxation caused by ATP. The results indicate that adenosine, derived from enzymatic dephosphorylation, contributes to the relaxant effect of ATP, presumably by activation of a smooth muscle adenosine receptor linked to the accumulation of cAMP and activation of protein kinase A. Yet, the main part of the response to ATP is mediated by a site distinct from the adenosine receptor. The pharmacological properties of this site differ from known P2 receptor subtypes. Possibly, the nucleotide-evoked relaxation is due to a phosphoryl transfer catalyzed by an ecto-protein kinase.

Nucleotide-evoked relaxation of rat vas deferens--a possible role for endogenous ATP released upon alpha(1)-adrenoceptor stimulation.

The possibility was tested that endogenous ATP released upon alpha(1)-adrenoceptor activation causes relaxation of the rat vas deferens smooth muscle. ATP, 2-methylthio ATP and adenosine relaxed the vas deferens precontracted with 80 mM K(+). The metabolically stable P2 receptor agonists alpha,beta-methylene ATP (alpha,beta-MeATP) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) had little or no effect. The adenosine P1 receptor antagonist 8-(para-sulfophenyl)theophylline did not significantly affect the response to ATP. The P2 receptor antagonist reactive blue 2 markedly reduced the relaxation (by up to 73%); suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and acid blue 129 caused no change. ATP, but not alpha,beta-MeATP, also attenuated contractions elicited by noradrenaline at resting tension; reactive blue 2 blocked the inhibitory effect of ATP. Reactive blue 2, by itself, enhanced the response to noradrenaline (by up to 36%); suramin, PPADS and acid blue 129 caused no change. In the presence of the ATP-degrading enzymes apyrase and nucleotide pyrophosphatase, the facilitatory effect of reactive blue 2 was lost. Apyrase, by itself, enhanced the response to noradrenaline (by 13%). The results indicate that endogenous ATP, released from rat vas deferens smooth muscle upon alpha(1)-adrenoceptor stimulation, causes relaxation. The site of action of ATP is not a typical smooth muscle P2Y receptor.

Stimulation of mouse cultured sympathetic neurons by uracil but not adenine nucleotides.

Cultured neurons from the paravertebral sympathetic chain of rats possess excitatory P2X as well as excitatory uracil nucleotide-sensitive P2Y receptors. Preliminary observations had indicated that the analogous neurons of mice lacked P2X receptors. This difference was now investigated. Thoracolumbar sympathetic neurons from one- to three-day-old mice were cultured for seven days. When the neurons were preincubated with [3H]noradrenaline and then superfused, ATP failed to cause any change in tritium outflow. UTP (3-300 microM) and UDP (30-100 microM), in contrast, caused marked increases, and so did nicotine (3-100 microM). The effect of UTP was not changed by suramin but abolished by tetrodotoxin and in the absence of calcium. The effect of nicotine was antagonized by hexamethonium and also abolished by tetrodotoxin and in the absence of calcium. Pre-exposure to UDP prevented the effect of UTP. In neurons studied by means of whole-cell patch-clamp techniques under current clamp, ATP lacked any effect. UTP (100 microM), UDP (100 microM) and nicotine (10 microM) caused depolarization accompanied by action potentials. Pre-exposure to UDP prevented the effect of UTP. In neurons studied under voltage clamp, ATP, UTP and UDP failed to cause any detectable current. Nicotine (10 microM), in contrast, elicited inward currents. Neither UTP nor UDP reduced the M-type potassium outward current. These results demonstrate a pronounced difference between cultured sympathetic neurons from the mouse and the rat paravertebral chain. Neurons from both species possess the nicotinic acetylcholine receptor. Neurons from both species also possess uracil nucleotide-sensitive P2Y receptors which, when activated, mediate depolarization, action potential firing and noradrenaline release; these effects are not due to inhibition of M-type potassium channels. Only the rat but not the mouse neurons, however, possess P2X receptors which, when activated, mediate cation entry, depolarization, action potential generation and transmitter release. The absence of functional P2X receptors makes the mouse neurons suitable for further study of the uracil nucleotide-sensitive P2Y receptors.

M-type K+ currents in rat cultured thoracolumbar sympathetic neurones and their role in uracil nucleotide-evoked noradrenaline release.

Cultured sympathetic neurones are depolarized and release noradrenaline in response to extracellular ATP, UDP and UTP. We examined the possibility that, in neurones cultured from rat thoracolumbar sympathetic ganglia, inhibition of the M-type potassium current might underlie the effects of UDP and UTP. Reverse transcriptase-polymerase chain reaction indicated that the cultured cells contained mRNA for P2Y(2)-, P2Y(4)- and P2Y(6)-receptors as well as for the KCNQ2- and KCNQ3-subunits which have been suggested to assemble into M-channels. In cultures of neurones taken from newborn as well as from 10 day-old rats, oxotremorine, the M-channel blocker Ba(2+) and UDP all released previously stored [(3)H]-noradrenaline. The neurones possessed M-currents, the kinetic properties of which were similar in neurones from newborn and 9 - 12 day-old rats. UDP, UTP and ATP had no effect on M-currents in neurones prepared from newborn rats. Oxotremorine and Ba(2+) substantially inhibited the current. ATP also had no effect on the M-current in neurones prepared from 9 - 12 day-old rats. Oxotremorine and Ba(2+) again caused marked inhibition. In contrast to cultures from newborn animals, UDP and UTP attenuated the M-current in neurones from 9 - 12 day-old rats; however, the maximal inhibition was less than 30%. The results indicate that inhibition of the M-current is not involved in uracil nucleotide-induced transmitter release from rat cultured sympathetic neurones during early development. M-current inhibition may contribute to release at later stages, but only to a minor extent. The mechanism leading to noradrenaline release by UDP and UTP remains unknown.

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.

Role of action potentials and calcium influx in ATP- and UDP-induced noradrenaline release from rat cultured sympathetic neurones.

Adenine and uracil nucleotides release noradrenaline from rat postganglionic sympathetic neurones by activation of P2X-receptors and distinct receptors for uracil nucleotides, respectively. The present study on cultured neurones of rat thoracolumbal paravertebral ganglia has analysed the involvement of action potentials and calcium influx in the nucleotide-induced transmitter release. ATP and UDP (100 microM each) caused a marked release of previously incorporated [3H]noradrenaline. The P2-receptor antagonists suramin (300 microM) and cibacron blue 3GA (3 microM) decreased the ATP-induced but not the UDP-induced release. The response to ATP was decreased by the sodium channel blocker tetrodotoxin (0.5 microM; by 47%), by the N-type calcium channel blocker omega-conotoxin GVIA (100 nM; by 35%), and by the alpha2-adrenoceptor agonist UK-14,304 (1 microM; by 45%); it was not changed by the potassium channel blocker tetraethylammonium (10 mM). The response to UDP was abolished by tetrodotoxin, greatly decreased by omega-conotoxin (by 78%), also abolished by UK-14,304, and increased by tetraethylammonium (by 410%). ATP (100 microM) caused a marked increase in intra-axonal free calcium as measured by fura-2 microfluorimetry. Withdrawal of extracellular calcium diminished the calcium response to ATP by 85%, and tetrodotoxin and omega-conotoxin diminished it by about 40%. As studied with the amphotericin B-perforated patch method, ATP (100 microM) induced a large depolarisation and action potential firing. UDP (100 microM) induced only a small depolarisation but it also elicited action potentials. UDP increased the excitability of the neurones. The results indicate that the ATP-induced release of noradrenaline depends on influx of calcium from the extracellular space. Calcium passes through two structures: voltage-gated channels and - probably - the P2X-receptor itself. Only that component of ATP-induced transmitter release which is triggered by opening of voltage-gated calcium channels is sensitive to modulation by alpha2-adrenoceptors. In contrast to ATP, the UDP-induced release of noradrenaline is entirely due to generation of action potentials followed by calcium influx through voltage-gated channels. All of the UDP-induced release is therefore sensitive to alpha2-adrenoceptor modulation.

Noradrenaline release from cultured mouse postganglionic sympathetic neurons: autoreceptor-mediated modulation.

The possible existence of alpha2-autoreceptors, P2-autoreceptors, and adenosine A1- or A2A-receptors was studied in cultured thoracolumbar postganglionic sympathetic neurons from mice. The cells were preincubated with [3H]noradrenaline and then superfused. The selective alpha2-adrenoceptor agonist UK 14,304 reduced the electrically evoked overflow of tritium. When the cultures were stimulated by trains of increasing pulse number, ranging from a single pulse to 72 pulses at 3 Hz, the concentration-inhibition curve of UK 14,304 was shifted progressively to the right and the maximal inhibition obtainable became progressively smaller. Six alpha-adrenoceptor antagonists shifted the concentration-inhibition curve of UK 14,304 in a parallel manner to the right. Neither ATP (3-300 microM), adenosine (0.01-100 microM), the selective A1-receptor agonist cyclopentyladenosine (1-1,000 nM), nor the selective A2A-receptor agonist CGS-21680 (1-10,000 nM) changed the basal or the electrically evoked overflow of tritium. It is concluded that the cultured neurons possess presynaptic, release-inhibiting alpha2-autoreceptors. As in intact tissues, the effectiveness of presynaptic alpha2-adrenergic inhibition depends on the "strength" of the releasing stimulus. The pK(D) values of the six antagonists against UK 14,304 indicate that the autoreceptors belong to the pharmacological alpha2D and hence the genetic alpha(2A/D) subtype of alpha2-adrenoceptor. Neither P2-autoreceptors nor receptors for adenosine, the degradation product of ATP, were detected.

A study of the mechanism of the release of ATP from rat cortical astroglial cells evoked by activation of glutamate receptors.

Glutamate and the selective agonists at ionotropic glutamate receptors N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and kainate release ATP from superfused primary cultures of rat cortical astrocytes. The mechanism of this release was investigated. The release of ATP elicited by N-methyl-D-aspartate and kainate was abolished or greatly reduced in the absence of external calcium as well as in the presence of cadmium (1 mM) and nicardipine (10 microM). The release of ATP elicited by AMPA, in contrast, was not changed by these interventions. The calcium ionophore ionomycin (5 microM) released ATP in the presence but not in the absence of external calcium. No release was obtained with alpha-latrotoxin. Of several compounds tested as potential blockers of ATP transporters or channels only glibenclamide (100 microM) and diphenylamine-2-carboxylate (500 microM), which block the cystic fibrosis transmembrane conductance regulator, caused any change: both reduced the effect of AMPA without changing the effects of N-methyl-D-aspartate and (only glibenclamide tested) kainate. Lithium (1 mM) abolished the release of ATP evoked by glutamate and AMPA and significantly reduced the release evoked by N-methyl-D-aspartate and kainate. The three glutamate receptor agonists did not increase the release of lactate dehydrogenase. The results confirm the previous observation that activation of N-methyl-D-aspartate, AMPA and kainate receptors induces release of ATP from astrocytes in culture. Two different mechanisms seem to be involved. The N-methyl-D-aspartate- and kainate-induced release of ATP requires an influx of calcium, is not due to neuron-like exocytosis, is not mediated by cystic fibrosis transmembrane conductance regulator or a mechanism regulated by cystic fibrosis transmembrane conductance regulator, and is reduced (by an unknown mechanism) but not abolished by lithium. The AMPA-induced release does not require extracellular calcium, may be mediated by cystic fibrosis transmembrane conductance regulator or a mechanism regulated by cystic fibrosis transmembrane conductance regulator, and is abolished (by an unknown mechanism) by lithium. The ability of astrocytes to both release ATP and respond to ATP suggests that ATP may act as an autocrine or paracrine messenger between these glial cells.

Concomitant blockade of P2X-receptors and ecto-nucleotidases by P2-receptor antagonists: functional consequences in rat vas deferens.

In order to assess the consequences of a concomitant blockade of P2X-receptors and ecto-nucleotidases, effects of 13 P2-receptor antagonists were investigated on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alpha,beta-MeATP) and ATP and on the removal of ATP from the incubation medium by vas deferens tissue. Increasing concentrations of all antagonists reduced and finally abolished contractions elicited by alpha,beta-MeATP (3 microM), with IC50-values ranging from 1.1 to 100 microM. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonate (PPADS), 6-azophenyl-4-amino-5-hydroxy-naphthalene-1,3-disulphonate (NH02), 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and uniblue A also progressively reduced and finally abolished contractions elicited by ATP (1 mM). 8,8'-[Carbonylbis(imino-3, 1-phenylenecarbonyl-imino)]-bis-(1,3,5-naphthalenetrisulphonate ) (NF023), suramin, pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS), trypan blue and reactive blue 19, in contrast, caused only partial blockade, by 34-43% maximally; reactive blue 2 and reactive red 2 had no effect; and 6,6'-(1,1'-biphenyl-4,4'-diylbisazo)-bis-4-amino-5-hydroxy-naphtha lene-1,3-disulphonate (NH01) and Evans blue even enhanced the response to ATP. For antagonists causing full or partial inhibition, the IC50-values against ATP were close to those against alpha,beta-MeATP. All antagonists attenuated the removal of ATP, with IC25%-values ranging from 0.8 microM to >320 microM. The results confirm the frequent combination, in one antagonist molecule, of P2-receptor blockade and blockade of ecto-nucleotidases. This dual action underlies the effect of such compounds on contractions of the vas deferens elicited by ATP which, for certain substances (e.g., suramin, reactive blue 2), can be explained by a simple model in which the antagonist simultaneously blocks the degradation of ATP and a single contraction-mediating receptor (P2X1). Several observations, however, do not conform with this model, and the existence of multiple contraction-mediating receptors for ATP or multiple, pharmacologically distinct ecto-nucleotidases has to be considered.

On the suitability of adenosine 3'-phosphate 5'-phosphosulphate as a selective P2Y receptor antagonist in intact tissues.

Agonist and antagonist effects of the putative P2Y1 receptor antagonist adenosine 3'-phosphate 5'-phosphosulphate (PAPS) were studied in intact tissues. In the carbachol-precontracted guinea-pig taenia coli, PAPS caused prominent relaxation (EC50 3.3 microM). The response was attenuated by the P2 receptor antagonists 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and reactive red 2 with apparent Kd values (0.27 and 0.29 microM) indicating that PAPS acts through the non-P2Y receptor, which is the site of action of alpha,beta-methylene ATP (alpha,beta-MeATP) in the taenia coli. Incubation with PAPS (10-100 microM) attenuated the P2Y receptor-mediated relaxation caused by adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS); PAPS (100 microM) also attenuated the relaxation caused by alpha,beta-MeATP, as well as the alpha1-adrenoceptor-mediated response to noradrenaline. In the noradrenaline-precontracted rat aorta, PAPS caused minor relaxation (EC50 24.7 microM), which was reduced by the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS; 1 microM), indicating that PAPS activates endothelial P2Y receptors. Incubation with PAPS (10 and 100 microM) attenuated the P2Y receptor-mediated relaxation caused by ADPbetaS; PAPS (100 microM) also attenuated the P2U receptor-mediated relaxation caused by UTP and the muscarine receptor-mediated response to acetylcholine. In rat vas deferens, PAPS (100 microM) attenuated the P2X receptor-mediated contraction elicited by alpha,beta-MeATP but did not alter the alpha1-adrenoceptor-mediated response to noradrenaline. The results indicate that PAPS attenuates P2Y receptor-mediated relaxation in intact tissues. However, due to its limited subtype selectivity and non-P2 receptor effects, the nucleotide is not a suitable antagonist for this subtype.

On the suitability of adenosine 3'-phosphate 5'-phosphosulphate as a selective P2Y receptor antagonist in intact tissues.

Agonist and antagonist effects of the putative P2Y1 receptor antagonist adenosine 3'-phosphate 5'-phosphosulphate (PAPS) were studied in intact tissues. In the carbachol-precontracted guinea-pig taenia coli, PAPS caused prominent relaxation (EC50 3.3 microM). The response was attenuated by the P2 receptor antagonists 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and reactive red 2 with apparent Kd values (0.27 and 0.29 microM) indicating that PAPS acts through the non-P2Y receptor, which is the site of action of alpha,beta-methylene ATP (alpha,beta-MeATP) in the taenia coli. Incubation with PAPS (10-100 microM) attenuated the P2Y receptor-mediated relaxation caused by 5'-O-(2-thiodiphosphate) (ADPbetaS); PAPS (100 microM) also attenuated the relaxation caused by alpha,beta-MeATP, as well as the alpha1-adrenoceptor-mediated response to noradrenaline. In the noradrenaline-precontracted rat aorta, PAPS caused minor relaxation (EC50 24.7 microM), which was reduced by the P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS; 1 microM), indicating that PAPS activates endothelial P2Y receptors. Incubation with PAPS (10 and 100 microM) attenuated the P2Y receptor-mediated relaxation caused by ADPbetaS; PAPS (100 microM) also attenuated the P2U receptor-mediated relaxation caused by UTP and the muscarine receptor-mediated response to acetylcholine. In rat vas deferens, PAPS (100 microM) attenuated the P2X receptor-mediated contraction elicited by alpha,beta-MeATP but did not alter the alpha1-adrenoceptor-mediated response to noradrenaline. The results indicate that PAPS attenuates P2Y receptor-mediated relaxation in intact tissues. However, due to its limited subtype selectivity and non-P2 receptor effects, the nucleotide is not a suitable antagonist for this subtype.

P2-receptor-mediated inhibition of noradrenaline release in the rat pancreas.

The aim of the study was to find out whether, and if so through which receptors, nucleotides modulate the release of noradrenaline in the rat pancreas. Segments of the pancreas were preincubated with [3H]-noradrenaline, superfused with medium containing desipramine (1 microM) and yohimbine (1 microM), and stimulated electrically, in most experiments by 60 pulses/l Hz. The adenosine A1-receptor agonist N6-cyclopentyl-adenosine (CPA; EC50 32 nM), the non-subtype-selective adenosine receptor agonists adenosine (EC50 15 microM) and 5'-N-ethylcarboxamidoadenosine (NECA; EC50 135 nM), and the nucleotides ATP (EC50 13 microM), adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS; EC50 19 microM) and adenosine-5'-O-(2-thiodiphosphate) (ADPbetaS; EC50 16 microM) decreased the evoked overflow of tritium. The adenosine A2A-agonist 2-p-(2-carboxyethyl)-phenethylamino-5 '-N-ethylcarboxamido-adenosine (CGS 21680) caused no change. The concentration-response curve of CPA was shifted to the right by the A -antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 10 nM; pKd 9.1) but, like the concentration-response curve of adenosine, hardly affected by the P2-receptor antagonist cibacron blue 3GA (30 microM). Combined administration of a high concentration of DPCPX (1 microM) and 8-phenyltheophylline (10 microM) abolished the effects of CPA and NECA. The concentration-response curves of ATP and ADPbetaS were shifted to the right by both DPCPX (10 nM; pKd 8.7 and 8.9, respectively) and cibacron blue 3GA (30 microM; pKd 5.0 and 5.2, respectively). The antagonist effects of DPCPX (10 nM) and cibacron blue 3GA (30 microM) against ATP were additive in a manner compatible with the blockade of two separate receptors for ATP. In the presence of the high concentration of DPCPX (1 microM) and 8-phenyltheophylline (10 microM), ATP and ADPbetaS still decreased evoked tritium overflow, and this decrease was attenuated by additional administration of cibacron blue 3GA (30 microM). The P2-antagonists cibacron blue 3GA, reactive blue 2, reactive red 2, and to a limited extent also suramin and 8-(3,5-dinitro-phenylenecarbonylimino)- 1,3,5-naphthalenetrisulphonate (XAMR0721), increased the evoked overflow of tritium by up to 114%. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonate (PPADS) caused no change. The results indicate that the postganglionic sympathetic axons of the rat pancreas possess A1-adenosine and P2-receptors. Both receptors mediate an inhibition of noradrenaline release. The presynaptic P2-receptors are activated by an endogenous ligand, presumably ATP, during appropriate trains of action potentials. This is the first demonstration of presynaptic P2-receptors at postganglionic sympathetic neurons that are located in prevertebral ganglia.

P2-receptor antagonists: IV. Blockade of P2-receptor subtypes and ecto-nucleotidases by compounds related to reactive blue 2.

Effects of reactive blue 2 and twelve structurally related compounds were studied on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alpha,beta-MeATP; mediated by P2X-receptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; mediated by P2Y-receptors), and the degradation of ATP by rat vas deferens tissue. All compounds, except acid blue 41 and acid blue 129 (at up to 100 microM), shifted the concentration-response curve of alpha,beta-MeATP in the rat vas deferens to the right. Most increased, but uniblue A greatly decreased, the maximum of the curve. In the case of cibacron blue 3GA and reactive blue 19, of which three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from unity. The apparent Kd values of the effective substances ranged between 0.7 and 111 microM. Most compounds increased the contraction of the rat vas deferens elicited by high K+. In the guinea-pig taenia coli, all compounds, except uniblue A and reactive blue 19 (at up to 100 microM), shifted the concentration-response curve of ADPbetaS to the right in a parallel manner. In the case of acid blue 129 and acid blue 80, of which three concentrations were tested, the slope of the Arunlakshana-Schild regression did not differ from unity. The apparent Kd values of the effective substances were between 0.7 and 69 microM. Most compounds also reduced the relaxation of the guinea-pig taenia coli elicited by noradrenaline. The removal of ATP from the medium by vas deferens tissue was decreased only by reactive blue 2, cibacron blue 3GA, uniblue A and reactive blue 19, with IC25% values between 17 and 62 microM. The structure-activity relationships for P2X- and P2Y-receptor blockade in this series are strikingly dissimilar. In reactive blue 2 and its isomers, for example, both the 1-amino-anthraquinone-2-sulphonate core and the 'side-chain' of the molecule are involved in P2X-receptor binding; P2Y-receptor affinity, in contrast, resides largely or totally in the anthraquinone core. The most promising antagonists are uniblue A which is P2X- versus P2Y-selective and acid blue 129 which is P2Y- versus P2X-selective, both with few, if any, non-P2-receptor effects at concentrations blocking the respective P2-subtype.

Characterization by antagonists of P2-receptors mediating endothelium-dependent relaxation in the rat aorta.

The receptors through which 2-methylthio ATP (MeSATP), adenosine 5'-O-(2-thiodiphosphate) (ADP beta S), UTP and ATP elicit endothelium-dependent relaxation of noradrenaline-precontracted rings of the rat aorta were characterized by means of a series of antagonists. The acetylcholine-induced relaxation and the degradation of MeSATP, UTP and ATP were also studied. The potency of the nucleotides at producing relaxation decreased in the order MeSATP (EC50 0.24 microM) > ADP beta S (0.43 microM) > UTP (1.09 microM) > ATP (3.53 microM). MeSATP, ADP beta S and UTP did not cause relaxation when the endothelium had been destroyed; high concentrations of ATP still caused some relaxation. The relaxation by MeSATP, ADP beta S and UTP became very small after treatment of the rings with NG-nitro-L-arginine methyl ester; the relaxation by ATP was less affected. Pre-exposure to MeSATP (100 microM) abolished or almost abolished the relaxation normally elicited by MeSATP and ADP beta S, did not change that elicited by UTP and slightly enhanced the relaxation elicited by ATP. Of nine compounds examined as antagonists, six attenuated selectively the effect of some or all of the nucleotides (as compared to acetylcholine): suramin, reactive blue 2, pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS), pyridoxalphosphate-6-azophenyl-2',4'-disulphonate (PPADS), reactive red 2 and 5,5'-(1,1'-biphenyl-4,4'-diylbisazo)-bis-7-amino -6-hydroxy-naphthalene-1,4-disulphonate (NH05). Decreases of maximal relaxations and slopes different from unity in Schild plots often indicated non-competitive kinetics of the antagonism. For each of the six 'selective' antagonist, the apparent Kd values against MeSATP and against ADP beta S were similar: none of the six differentiated between MeSATP and ADP beta S. Also, for each of four 'selective' antagonists, the apparent Kd values against UTP and against ATP were similar: none of the four differentiated between these two nucleotides (two antagonists did not act against UTP and ATP in the 'selective' concentration range). On the other hand, for five of the six 'selective' antagonists (the exception being NH05), the apparent Kd values against MeSATP and ADP beta S were considerably lower than those against UTP and ATP. At the highest concentrations tested against agonist-evoked relaxations, the antagonists did not alter the removal from the incubation medium, by pieces of rat aorta, of MeSATP, UTP and ATP. It is concluded that nucleotides cause endothelium-dependent relaxation of the rat aorta through two sites: a P2Y-receptor and a P2U-receptor. The receptors may be pharmacologically similar to a bovine endothelial P2Y (P2Y1) and a cloned rat P2U (P2Y2) receptor, respectively. ATP acts mainly through the P2U-receptor. Suramin, reactive blue 2, iso-PPADS, PPADS and reactive red 2 are more potent at the P2Y- than the P2U-receptor. NH05 does not discriminate between the two receptors but is the most potent P2U antagonist so far described.

P2-receptor-mediated inhibition of serotonin release in the rat brain cortex.

The possibility of a P2-receptor-mediated modulation of the release of serotonin in the rat brain cortex was investigated in occipito-parietal slices preincubated with [3H]serotonin and then superfused and stimulated electrically (10 pulses, 1 Hz). Adenosine receptor agonists decreased the stimulation-evoked overflow of tritium at best slightly; the selective A1 agonist N6-cyclopentyl-adenosine caused no change. Several nucleotides had more marked effects: ATP (3-1000 microM), adenosine-5'-O-(3-thiotriphosphate) (3-300 microM) and P1,P5-di(adenosine-5')-pentaphosphate (3-300 microM) decreased the evoked overflow by up to ca 35%. AMP, alpha,beta-methylene-ATP and UTP produced smaller decreases and 2-methylthio-ATP and UMP caused no change. The inhibition by ATP was attenuated both by the P1-receptor antagonist 8-(p-sulphophenyl)-theophylline (100 microM) and by the P2-receptor antagonist suramin (300 microM) but was not changed by indomethacin (10 microM) and NG-nitro-L-arginine (10 microM). We conclude that the release of serotonin in the rat brain cortex is inhibited through presynaptic P1-receptors (which are not A1) as well as P2-receptors. Inhibition of release via P2-receptors has been previously shown for noradrenaline (brain cortex and hippocampus) and dopamine (neostriatum) and, hence, may be widespread. Differences between transmitter systems exist, however, in the degree of their sensitivity to presynaptic P2-receptor-mediated modulation.