1. The effects of three analogues of NG-nitro-L-arginine (L-NOARG) and NG-monomethyl-L-arginine (L-NMMA), inhibitors of nitric oxide (NO) synthase, on hydrogen peroxide (H2O2)-induced endothelial cell injury were studied. 2. Endothelial cell injury was assessed by measuring the release of intracellular lactate dehydrogenase (LDH) and 51Cr. 3. Addition of H2O2 (250-1,000 microM) to endothelial cells induced the release of LDH dose-dependently. The release of LDH was reduced by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME, 10(-4)-4 x 10(-3) M), L-NOARG (10(-4)-4 x 10(-3) M) and NG-nitro-L-arginine benzyl ester (L-NABE, 10(-4)-4 x 10(-3) M), inhibitors of NO synthase. 4. L-NOARG analogues also reduced H2O2-induced 51Cr release from endothelial cells, while L-NMMA had no effect. 5. The protective effect of L-NAME was not reversed by addition of L-arginine (L-Arg, 1-10 mM). 6. Both L-NAME and L-NMMA completely inhibited L-Arg metabolism to L-citrulline coupled with NO synthesis. 7. These findings suggest that L-NOARG analogues but not L-NMMA reduced H2O2-induced endothelial cell injury, and that these effects may not be related to inhibition of NO production.
1. Coronary arteries from bovines (BCA) and pigs (PCA) were used for measuring endothelium-dependent relaxation in the presence of L-NG nitroarginine and indomethacin. As some compounds tested have been found to have an inhibitory effect on autacoid-activated endothelial Ca2+ signalling, endothelium-dependent relaxation was initiated with the Ca2+ ionophore A23187. 2. The common compounds for modulating arachidonic acid release/pathway, mepacrine and econazole only inhibited L-NG nitroarginine-resistant relaxation in BCA not in PCA. In contrast, proadifen (SKF 525A) diminished relaxation in BCA and PCA. Mepacrine and proadifen inhibited Hoe-234-initiated relaxation in BCA and PCA, while econazole only inhibited Hoe 234-induced relaxation in PCA. Due to the multiple effects of these compounds, caution is necessary in the interpretation of results obtained with these compounds. 3. The inhibitor of Ca(2+)-activated K+ channels, apamin, strongly attenuated A23187-induced L-NG nitroarginine-resistant relaxation in BCA while apamin did not affect L-NG nitroarginine-resistant relaxation in PCA. 4. Pertussis toxin blunted L-NG nitroarginine-resistant relaxation in BCA, while relaxation of PCA was not affected by pertussis toxin. 5. Thiopentone sodium inhibited endothelial cytochrome P450 epoxygenase (EPO) in PCA but not in BCA, while L-NG nitroarginine-resistant relaxation of BCA and PCA were unchanged. Protoporphyrine IX inhibited EPO in BCA and PCA and abolished L-NG nitroarginine-resistant relaxation of BCA not PCA. 6. An EPO-derived compound, 11,12-epoxy-eicosatrienoic acid (11,12-EET) yielded significant relaxation in BCA and PCA in three out of six experiments. 7. These findings suggest that L-NG nitroarginine-resistant relaxation in BCA and PCA constitutes two distinct pathways. In BCA, activation of Ca(2+)-activated K+ channels via a pertussis-toxin-sensitive G protein and EPO-derived compounds might be involved. In PCA, no selective inhibition of L-NG nitroarginine-resistant relaxation was found.
Some postganglionic sympathetic axons possess P 2Y ‐like P 2 ‐purinoceptors which, when activated, decrease the release of noradrenaline. We examined the question of whether such receptors also occur at the noradrenergic axons in the rat brain cortex. Slices of the brain cortex were preincubated with [ ³ H]‐noradrenaline, then superfused with medium containing desipramine (1 μ m ) and stimulated electrically, in most experiments by trains of 4 pulses/100 Hz.
The selective adenosine A 1 ‐receptor agonist, N ⁶ ‐cyclopentyl‐adenosine (CPA; 0.03‐3 μ m ) as well as the non‐subtype‐selective agonist 5′‐N‐ethylcarboxamido‐adenosine (NECA; 0.3‐3 μ m ) reduced the evoked overflow of tritium, whereas the adenosine A 2a ‐receptor agonist, 2‐ p ‐(2‐carbonylethyl)‐phenethylamino‐5′‐N‐ethylcarboxamido‐adenosine (CGS‐21680; 0.003–30 μ m ) and the adenosine A 3 ‐receptor agonist N ⁶ ‐2‐(4‐aminophenyl)ethyl‐adenosine (APNEA; 0.03‐3 μ m ) caused no change. Of the nucleotides tested, ATP (30–300 μ m ), adenosine‐5′‐0‐(3‐thiotriphosphate) (ATP7S; 30–300 μ m ), adenosine‐5′‐0‐(2‐thiodiphosphate) (ADPγS; 30–300 μ m ), P 1 , P 4 ‐di(adenosine‐5 ⁻ )‐tetraphosphate (Ap 4 A; 30–300 μ m ) and the preferential P 2Y ‐purinoceptor agonist, 2‐methylthio‐ATP (300 μ m ) decreased the evoked overflow of tritium. The P 2X ‐purinoceptor agonist, α,β‐methylene‐ATP (3–300 μ m ) caused no change.
The A 1 ‐selective antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX; 10 n m ) attenuated the effects of the nucleosides CPA (apparent p K B value 9.8) and NECA as well as of the nucleotides ATP (apparent p K B 9.3), ATP7S (apparent p K B 9.2) and ADPβS (apparent p K B 8.7). CGS‐21680 and APNEA were ineffective also in the presence of DPCPX. The A 2 ‐selective antagonist 1,3‐dipropyl‐8‐(3,4‐dimethoxystyryl)‐7‐methylxanthine (KF‐17837) reduced the effects of CPA, NECA and ATPγS only when given at a concentration of 300 n m but not at 10 n m .
The P 2 ‐purinoceptor antagonists, suramin (300 μ m ), reactive blue 2 (30 μ m ) and cibacron blue 3GA (30 μ m ) did not change the effect of CPA. Suramin and cibacron blue 3GA shifted the concentration‐response curve of ATPγS to the right (apparent p K B values 3.7 and 5.0, respectively). Reactive blue 2 also attenuated the effect of ATPγS, and cibacron blue 3GA attenuated the effect of ATP, but in these cases the agonist concentration‐response curves were not shifted to the right. There was no antagonistic effect of suramin against ATP and ADPβS.
The results indicate that rat cerebrocortical noradrenergic axons possess, in addition to the known adenosine A 1 ‐receptor, a separate purinoceptor for nucleotides (P 2 ) which, in contrast to the A 1 ‐receptor, is blocked by suramin, reactive blue 2 and cibacron blue 3GA. Nucleotides such as ATP and ATPγS activate both receptors. Inconsistencies in antagonist effects against nucleotides are probably due to this activation of two receptors. The presynaptic P 2 ‐purinoceptor is P 2Y ‐like, as it is in the peripheral sympathetic nervous system.
1. The effects of the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), on mean circulatory filling pressure (MCFP), total peripheral resistance (TPR), cardiac output (CO) and resistance to venous return (Rv) were studied in rats. 2. In conscious, unrestrained rats, L-NAME (0.5-16 mg kg-1) dose-dependently increased mean arterial pressure (MAP) but not MCFP, an inverse index of venous compliance, either in the absence or presence of the ganglionic blocker mecamylamine (10 mg kg-1). 3. In pentobarbitone-anaesthetized rats, L-NAME (2, 4, 8 mg kg-1) increased MAP and reduced CO in a dose-related manner but did not change MCFP, TPR (+84, +140 and +192%) as well as Rv (+62, +72, +110%) were dose-dependently increased by L-NAME. 4. Our results show that L-NAME reduces CO by increasing arterial as well as venous resistances. L-NAME does not affect MCFP.
1. The cardiovascular and vasorelaxant effects of (+)-glaucine and of a semisynthetic derivative (N-carbethoxysecoglaucine) were studied in rats. 2. N-carbethoxysecoglaucine did not modify either systolic arterial pressure or heart rate values in conscious (25 mg kg-1, p.o.) and anaesthetized normotensive rats (5 mg kg-1, i.v.). Furthermore, this compound showed no activity in the experiments carried out on rat isolated aorta [contractility and 45Ca2+ influx assays (5 microM)] and did not modify the rate and force of contraction in rat isolated atria (5 microM). 3. In conscious normotensive rats, oral administration of (+)-glaucine (25 mg kg-1) did not modify either systolic arterial pressure or heart rate. 4. In anaesthetized normotensive rats, (+)-glaucine (5 mg kg-1, i.v.) produced a remarkable fall in mean arterial pressure (MAP) accompanied by a significant decrease in heart rate. In the same preparation, (+)-glaucine (5 mg kg-1, i.v.) did not modify the cardiovascular effects induced by noradrenaline (NA) (5 micrograms kg-1) and 5-hydroxytryptamine (5-HT) (300 micrograms kg-1) but markedly inhibited those induced by nicotine (200 micrograms kg-1). 5. In isolated intact aorta of rat, (+)-glaucine (0.15-5 microM) competitively inhibited the contractions induced by NA (with a pA2 value of 7.14) and non-competitively those induced by 5-HT (in normal Krebs solution) and Ca2+ (in depolarizing Ca(2+)-free high-K+ 50 mM solution), with depression of the maximal response and with pD2 values of 5.56 and 5.26, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
The efflux of 86Rb from rat isolated pancreatic islets preloaded with the isotope and perifused in vitro, has been used to monitor the effects of sulphonylureas on the potassium permeability, Pk, of pancreatic beta-cells. Tolbutamide (5 microM to 5 mM) had a dual effect, causing initially a decrease in 86Rb efflux (the 'on' response) which was rapidly superseded on drug removal by a large phasic increase in 86Rb efflux (the 'off' response). Each kinetic response had a different dose-dependency: the 'on' response was half-maximal at tolbutamide concentrations of 0.02 mM, maximal at 0.2 mM and decreased by concentrations greater than 0.2 mM whereas the 'off' response was half-maximal at 0.07 mM, maximal at 0.7 mM, with further increases in concentration (up to 5 mM) causing no further change in magnitude. Analysis of the time- and concentration-dependency of tolbutamide action, by presenting increasing concentrations (0 to 1.4 mM) of tolbutamide as a ramp or step function, established a critical dependence of the kinetics of 86Rb efflux during and after exposure to tolbutamide upon the initial rate of increase of the tolbutamide concentration rather than its final steady state. In the presence of quinine (10 microM), D600 (50 microM), or tetraethylammonium (20 mM), the secondary increase in 86Rb following tolbutamide (0.7 mM) removal was totally inhibited. Co2+ (2.56 mM) not only blocked the secondary 'off' response but also potentiated the initial 'on' response of tolbutamide. Glibenclamide produced a rapid decrease in 86Rb efflux but at a much lower concentration (10 microM) than tolbutamide and with no 'off' response apparent over a wide range of concentration (1 to 100 microM); moreover the decrease in 86Rb efflux was sustained and only slowly reversible. It is concluded that tolbutamide has two opposing actions on islet beta-cell 86Rb efflux, and therefore PK: (i) a tendency to increase a calcium-sensitive PK by stimulating calcium entry into the cell and (ii) a decrease in PK that may be due to a direct effect on the calcium-sensitive PK itself. The more sustained pharmacological action of glibenclamide is explained by the longer-lasting decrease in PK that it produces.
1. The influence of the nitric oxide (NO) biosynthesis inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) on the gastric relaxation induced by peripheral vagal stimulation was investigated in the anaesthetized rat.
2. Peripheral vagal stimulation (10 Hz, 10 V, 1 ms for 20 s) induced a reproducible biphasic response: a short-lasting increase followed by a more pronounced decrease in intragastric pressure. This response also occurred in reserpinized animals (5 mg kg-1, i.p., 24 h before the experiment) while atropine (1 mg kg-1, i.v.) abolished the initial increase in intragastric pressure.
3. L-NAME (1-30 mg kg-1, i.v.) induced an increase in arterial blood pressure. L-NAME (1 mg kg-1, i.v.) had no influence on the vagally induced gastric response while L-NAME (10 and 30 mg kg-1 i.v.) significantly changed it: the initial increase in intragastric pressure was enhanced while the decrease in intragastric pressure was reduced or abolished. N(G)-nitro-L-arginine (L-NNA, 10 mg kg-1, i.v.) had the same effect.
4. An i.v. infusion of phenylephrine (10-mu-g kg-1 min-1) inducing a pressor response similar to that produced by L-NAME (30 mg kg-1, i.v.) did not influence the vagal gastric response. Infusion of L-arginine (300 mg kg-1 bolus, then 100 mg kg-1 h-1) starting 30 min beforehand, reduced the pressor effect and prevented the influence of L-NAME (10 mg kg-1, i.v.) on the vagal gastric response. After injection of both atropine (1 mg kg-1, i.v.) and L-NAME (30 mg kg-1, i.v.), the vagally induced decrease in intragastric pressure was similar to that obtained under control conditions.
5. These results are consistent with NO being released and inducing gastric relaxation during peripheral vagal stimulation. In addition to NO, another inhibitory non-adrenergic non-cholinergic neurotransmitter is released.
The effect of forskolin and several H 2 ‐agonists was investigated on the activity of adenylate cyclase in homogenates of guinea‐pig lung parenchyma.
Histamine, 0.1 μM to 1 mM, dimaprit, 1 μM to 10 mM, 4‐methyl histamine, 0.1 μM to 10 mM, impromidine, 10 nM to 10μM and forskolin, 1 nM to 100 μM, all produced a dose‐dependent stimulation of adenylate cyclase activity above the basal level.
The histamine H 1 ‐receptor antagonist mepyramine, 10 μM, and β‐adrenoceptor antagonist propranolol, 10 μM, had no effect on the stimulation by histamine of adenylate cyclase.
The dose‐response curve for stimulation by histamine of adenylate cyclase was shifted to the right in a dose‐dependent manner by increasing concentrations of several H 2 ‐antagonists. Schild plots constructed for each H 2 ‐antagonist produced straight lines with slopes not significantly different from unity. The equilibrium dissociation constants obtained for the H 2 ‐antagonists in this study were similar to those previously reported for inhibition of dimaprit‐induced relaxation of the pre‐contracted lung strip, inhibition of [ ³ H]‐tiotidine binding to homogenates of guinea‐pig lung parenchyma and inhibition of histamine‐stimulated adenylate cyclase in guinea‐pig gastric mucosa.
1. The effects of noradrenaline, ATP, adenylyl-imidodiphosphate (AMP-PNP), adenosine, alpha,beta-methylene-ATP and the P2-purinoceptor antagonist, suramin on N'-acetyl-5-hydroxytryptamine production were studied in cultured denervated rat pineal glands. 2. Noradrenaline (3 nM-1 microM) increased N'-acetyl-5-hydroxytryptamine production as measured both in the gland and the culture medium. 3. In noradrenaline (10 nM)-stimulated pineal glands, ATP (0.03 nM-1 mM) or AMP-PNP (0.1 microM-1 mM) increased N'-acetyl-5-hydroxytryptamine production in a concentration-dependent manner. 4. Alpha,beta-Methylene-ATP at the concentration of 0.1 mM, but not 3 microM, attenuated the enhancement by ATP (0.1 mM) of noradrenaline (10 nM)-induced N'-acetyl-5-hydroxytryptamine production. 5. Suramin (0.1 mM) blocked the potentiating effect of ATP (0.1 mM), but not the potentiating effect of adenosine (0.1 mM) in glands incubated with noradrenaline (10 nM). 6. These findings suggest that the rat pineal gland possesses P2-purinoceptors which when stimulated potentiate the effect of noradrenaline but do not, by themselves, induce an increase in N'-acetyl-5-hydroxytryptamine production.
1. Conscious, Long Evans rats, chronically instrumented for the measurement of regional haemodynamics, were used to assess responses to 3 min infusions of the potassium channel opener, BRL 38227 (1 and 10 micrograms kg-1 min-1) or adrenaline (0.05 and 0.5 microgram kg-1 min-1) in the absence and in the presence of NG-nitro-L-arginine methyl ester (L-NAME; 3 mg kg-1 h-1), an inhibitor of nitric oxide biosynthesis. 2. In the absence of L-NAME, the low dose of BRL 38227 caused slight hypotension and tachycardia, accompanied by small increases in mesenteric and hindquarters blood flow only. However, there were increases in renal, mesenteric and hindquarters vascular conductances. L-NAME had no effect on any of these responses. 3. The high dose of BRL 38227 caused substantial hypotension and tachycardia. Renal and hindquarters flows did not change significantly, but there was a marked increase in mesenteric flow. There were only modest increases in renal and hindquarters vascular conductances but a substantial mesenteric vasodilatation. In the presence of L-NAME, there was a slight reduction of the latter but no other changes in the responses to BRL 38227. 4. In the absence of L-NAME, the low dose of adrenaline caused slight hypotension but a marked tachycardia. There were no changes in renal or mesenteric blood flow but a clear-cut increase in hindquarters flow. Renal and mesenteric vascular conductances showed only small rises, in contrast to the substantial hindquarters vasodilatation. In the presence of L-NAME, there was significant attenuation of the tachycardia and of the increases in hindquarters flow and vascular conductance in response to adrenaline.5. The high dose of adrenaline caused marked hypotension and tachycardia. Renal flow did not change, but there was a fall in mesenteric and a marked rise in hindquarters flow. Renal vascular conductance showed a slight increase but mesenteric vascular conductance did not change significantly, whereas there was a substantial hindquarters vasodilatation. In the presence of L-NAME, adrenaline caused an increase in blood pressure but no significant change in heart rate; the renal vasodilatation was abolished, there was a mesenteric vasoconstriction, and the hindquarters vasodilatation was markedly reduced. L-NAME also attenuated the tachycardia induced by adrenaline in animals with no cardiac baroreflexes.6. The present results indicate that L-NAME-sensitive mechanisms are involved in the vasodilator and tachycardic effects of adrenaline. The relative lack of effect of L-NAME on responses to BRL 38227 indicates that the changes in the responses to adrenaline were not non-specific or due to changes in haemodynamic status caused by L-NAME. The results raise the possibility that the 'hypertensinogenic' properties of endogenous adrenaline could be amplified when nitric oxide biosynthesis is impaired.
The effects of two inhibitors of nitric oxide synthase, N G ‐monomethyl l ‐arginine ( l ‐NMMA) and N G ‐nitro l ‐arginine ( l ‐NOARG), were examined on non‐adrenergic non‐cholinergic (NANC) inhibitory transmission in the rat anococcygeus, bovine retractor penis (BRP) and bovine penile artery.
In the rat anococcygeus, l ‐NMMA (10–1000 μ m ) produced a concentration‐dependent augmentation of guanethidine (30 μ m )‐induced tone and inhibited NANC relaxation at all frequencies tested (0.1–20 Hz): the maximum inhibition obtained was 56 ± 6% ( n = 6). l ‐NOARG (0.3–30 μ m ) also augmented tone and inhibited NANC relaxation in a concentration‐dependent manner, but unlike l ‐NMMA the maximum inhibition was 100%.
In the BRP, l ‐NMMA (10–100 μ m ) had no effect on tone or NANC‐induced relaxation, but at 1000 μ m tone was increased and NANC relaxation inhibited by 25 ± 7% ( n = 6). l ‐NOARG (0.3–30 μ m ) produced a concentration‐dependent increase in tone and inhibition of NANC relaxation. As in the rat anococcygeus, inhibition of NANC relaxation was complete.
The effects of l ‐NMMA and l ‐NOARG were stereospecific since d ‐NMMA (10–1000 μ m ) and d ‐NOARG (1–1000 μ m ) had no effect on tone or NANC relaxation of the rat anococcygeus or BRP.
l ‐Arginine (10–300 μ m ) had no effect by itself on NANC‐induced relaxation of the rat anococcygeus or BRP. It did, however, reverse the ability of l ‐NMMA (10–1000 μ m ) to augment tone and inhibit NANC relaxation in the rat anococcygeus and BRP. The actions of low concentrations l ‐NOARG (0.3–10 μ m ) were also reversed by l ‐arginine (300 μ m ), but those of higher concentrations were not. d ‐Arginine (1000 μ m ) had no effect on the ability of l ‐NMMA or l ‐NOARG to augment tone and inhibit NANC relaxation in the anococcygeus and BRP.
On the bovine penile artery, both l ‐NMMA (100μ m ) and l ‐NOARG (30 μ m ) augmented the tone induced by guanethidine (30 μ m ) and 5‐hydroxytryptamine (0.2 μ m ) in an endothelium‐dependent manner. l ‐NMMA had no effect on NANC‐induced relaxation, but inhibited acetylcholine‐induced endothelium‐dependent relaxation. l ‐NOARG abolished NANC relaxation at all frequencies tested and inhibited acetylcholine‐induced relaxation. d ‐NOARG (30 μ m ) had no effect on NANC or acetylcholine‐induced relaxation.
The ability of l ‐NOARG to abolish NANC‐induced relaxation in the rat anococcygeus, BRP and bovine penile artery suggests that the l ‐arginine‐nitric oxide pathway mediates neurotransmission in all three tissues. The effectiveness of l ‐NMMA in blocking NANC relaxation in the rat anococcygeus but not the BRP and bovine penile artery suggests a species difference in the neuronal nitric oxide synthase. The neuronal and endothelial nitric oxide synthases in the penile artery also appear to differ.
We have studied both the electrophysiological and contractile effects of the purine nucleotide, adenosine‐5′‐triphosphate (ATP), as well as a number of its structural analogues as agonists at P 2X purinoceptors in the rat vas deferens in vitro .
Electrophysiological effects were investigated by a whole cell voltage clamp technique (holding potential − 70 mV) with fast flow concentration‐clamp applications of agonists in single isolated smooth muscle cells. ATP, 2‐methylthio adenosine‐5′‐triphosphate (2‐MeSATP) and α, β methylene adenosine‐5′‐triphosphate (αβ‐meATP) all evoked inward currents over a similar concentration range (0.3–10 μ M ), being approximately equipotent with similar concentrations for threshold effects (0.3 μ M ). ADP (10 μ M ) also evoked a rapid current of similar peak amplitude to that seen with ATP (10 μ M ).
α,β‐meATP was the most potent agonist in producing contractions of the rat vas deferens whole tissue preparation, with a threshold concentration equal to that in the electrophysiological studies (0.3 UM). However, ATP and 2‐MeSATP were at least ten times less potent in studies measuring contraction than in the electrophysiological studies. Furthermore, their concentration‐effect curves were shallow with smaller maximal responses than could be achieved with α,β‐meATP. ADP, AMP and adenosine were inactive at concentrations up to 1 mM. The rank order of agonist potencies observed for contraction was α,β‐meATP≫ATP = 2‐MeSATP.
Measurement of inorganic phosphate (iP), as a marker of purine nucleotide metabolism in the vas deferens whole tissue preparation, indicated that ATP and 2‐MeSATP were rapidly metabolized, whereas α,β‐meATP was stable for up to 2 h. Removal of divalent cations prevented breakdown of ATP and 2‐MeSATP, suggesting that metabolism involved a Ca ²⁺ /Mg ²⁺ ‐dependent enzyme.
It appears that in isolated preparations of rat vas deferens, the low potency of ATP and 2‐MeSATP can be explained by rapid agonist breakdown by ectonucleotidases. However, this is not the case in the single cell studies where the use of rapid concentration‐clamp applications revealed the true potency of the agonists. Under such conditions the three agonists were all equal in potency indicating that the rank order of agonist potencies of α,β‐meATP≫ATP = 2‐MeSATP is not in fact characteristic of smooth muscle P 2X ‐purinoceptors as commonly believed.
1. A comparative study was carried out between the adenosine receptor mediating a stimulation of cyclic AMP formation in guinea-pig cerebral cortical slices with the adenosine receptor mediating relaxation of phenylephrine precontracted guinea-pig aortic rings. 2. [3H]-cyclic AMP accumulation in [3H]-adenine-prelabelled guinea-pig cerebral cortical slices was stimulated by adenosine and its analogues with the following EC50 values (microM): 5'-N-ethylcarboxamidoadenosine (3.1 +/- 0.3) > 2-chloroadenosine (10 +/- 2) > adenosine (109 +/- 15). 3. 2-Chloroadenosine and adenosine elicited maximal responses for [3H]-cyclic AMP accumulation that were 100 +/- 7 and 71 +/- 6% of the maximal response to 5'-N-ethylcarboxamidoadenosine, respectively. CGS 21680 (100 microM) and DPMA (100 microM) elicited -2 +/- 2 and 12 +/- 3% of the response to 100 microM 5'-N-ethylcarboxamidoadenosine. 4. Estimation of antagonist potencies at the A2 adenosine receptor of cerebral cortex showed a rank order of potency (K1, nM): xanthine amino congener (35 +/- 3) > 8-cyclopentyl-1,3-dipropylxanthine (130 +/- 22) > PD 115,199 (407 +/- 82) > 3,7-dimethyl-1-propargylxanthine (13 +/- 2 microM). 5. Adenosine analogues produced long-lasting relaxation of phenylephrine-precontracted aortic rings with the following rank order of potency (EC50 values, microM): 5'-N-ethylcarboxamidoadenosine (0.68 +/- 0.06) > 2-chloroadenosine (4.3 +/- 0.6) > adenosine (104 +/- 13). Maximal relaxations elicited by these agents were 71 +/- 3, 98 +/- 1, and 100 +/- 1%, respectively. CGS 21680 and DPMA at 100 microM elicited smaller relaxations of the precontracted tissues (12 +/- 2 and 43 +/- 15%, respectively). 6. Antagonism by xanthine derivatives of the 5'-N-ethylcarboxamidoadenosine-induced relaxation of aortic rings showed the following rank order of potency (Ki, nM): xanthine amino congener (17 +/- 4) > 8-cyclopentyl-1,3-dipropylxanthine (171 +/- 36) > PD 115,199 (341 +/- 64) > 3,7-dimethyl-1-propargylxanthine (5520 +/- 820). 7. We conclude that the A2 adenosine receptor mediating relaxation of phenylephrine-contracted aortic rings is an A2b adenosine receptor which exhibits certain minor differences from the A2b receptor which stimulates cyclic AMP accumulation in cerebral cortical slices.
1. The aim of this investigation was to study the relationship between contractile responsiveness, activation of the L-arginine pathway and tissue levels of guanosine 3':5'cyclic monophosphate (cylic GMP) in aortic rings removed from rats 4 h after intraperitoneal administration of bacterial endotoxin (E. coli. lipopolysaccharide, LPS, 20 mg kg-1). 2. LPS-treatment resulted in a reduction of the sensitivity and maximal contractile response to noradrenaline (NA). 3. Depression of the maximal contractile response was restored to control by 6-anilo-5,8-quinolinedione (LY 83583, 10 microM), which prevents activation of soluble guanylate cyclase. 4. Cyclic GMP levels in tissue from LPS-treated rats were 2 fold greater than cyclic GMP levels detected in tissue from control (saline-treated) rats. The LPS-induced increase in cyclic GMP content was observed both in the presence and absence of functional endothelium. 5. Addition of L-arginine 1 mM) to maximally contracted aortic rings produced significantly relaxation of rings from LPS-treated rats but not rings from control animals. In the LPS-treated group, addition of L-arginine was also associated with a significant increase in cyclic GMP content. L-Arginine had no effect on the cyclic GMP content of control rings. D-Arginine (1 mM) was without effect. 6. In rings from LPS-treated rats, NG-nitro-L-arginine methyl ester (L-NAME, 300 microM), an inhibitor of nitric oxide (NO) production, increased the contractile response to NA and prevented the LPS-induced increase in cyclic GMP content. In control rings, L-NAME increased the NA sensitivity only when the endothelium remained intact and reduced the cyclic GMP content of these rings to that of control endothelium-denuded rings. 7. These results demonstrate that LPS-induced hyporeactivity to NA occurs secondarily to activation of the L-arginine pathway and subsequent activation of soluble guanylate cyclase in vascular tissue. In addition they suggest that LPS induces the production of an NO-like relaxing factor in non-endothelial cells.
It has been hypothesized that in patients with benign prostatic hyperplasia, selective antagonism of the α1A-adrenoceptor-mediated contraction of lower urinary tract tissues may, via a selective relief of outlet obstruction, lead to an improvement in symptoms.
The present study describes the α1-adrenoceptor (α1-AR) subtype selectivities of two novel α1-AR antagonists, Ro 70-0004 (aka RS-100975) and a structurally-related compound RS-100329, and compares them with those of prazosin and tamsulosin. Radioligand binding and second-messenger studies in intact CHO-K1 cells expressing human cloned α1A-, α1B- and α1D-AR showed nanomolar affinity and significant α1A-AR subtype selectivity for both Ro 70-0004 (pKi 8.9: 60 and 50 fold selectivity) and RS-100329 (pKi 9.6: 126 and 50 fold selectivity) over the α1B- and α1D-AR subtypes respectively. In contrast, prazosin and tamsulosin showed little subtype selectivity.
Noradrenaline-induced contractions of human lower urinary tract (LUT) tissues or rabbit bladder neck were competitively antagonized by Ro 70-0004 (pA2 8.8 and 8.9), RS-100329 (pA2 9.2 and 9.2), tamsulosin (pA2 10.4 and 9.8) and prazosin (pA2 8.7 and 8.3 respectively). Affinity estimates for tamsulosin and prazosin in antagonizing α1-AR-mediated contractions of human renal artery (HRA) and rat aorta (RA) were similar to those observed in LUT tissues, whereas Ro 70-0004 and RS-100329 were approximately 100 fold less potent (pA2 values of 6.8/6.8 and 7.3/7.9 in HRA/RA respectively).
The α1A-AR subtype selectivity of Ro 70-0004 and RS-100329, demonstrated in both cloned and native systems, should allow for an evaluation of the clinical utility of a ‘uroselective’ agent for the treatment of symptoms associated with benign prostatic hyperplasia.
British Journal of Pharmacology (1999) 127, 252–258; doi:10.1038/sj.bjp.0702541
Hydroxamate derivatives have been attracted considerable attention, due to their broad pharmacological properties. Recent studies reported their potential use in the treatment of cardiovascular diseases, arthritis or infectious diseases. However, the inhibitory mechanisms of hydroxamate derivatives in inflammation remain to be elucidated. In an effort to develop a novel pharmacological agent that could suppress abnormally activated macrophages, we investigated a novel aliphatic hydroxamate derivative, WMJ-S-001, and explored its anti-inflammatory mechanisms.
RAW264.7 macrophages were exposed to lipopolysaccharide (LPS) in the absence or presence of WMJ-S-001. COX-2 expression and signaling molecules activated by LPS were assessed.
The LPS-induced COX-2 expression was suppressed by WMJ-S-001. WMJ-S-001 inhibited p38MAPK, NF-κB subunit p65 and C/EBPβ phosphorylation in cells exposed to LPS. Treatment of cells with a p38MAPK inhibitor (p38MAPK inhibitor III) markedly inhibited LPS-induced p65 and C/EBPβ phosphorylation and COX-2 expression. LPS-increased p65 and C/EBPβ binding to the COX-2 promoter region was suppressed in the presence of WMJ-S-001. In addition, WMJ-S-001 suppression of p38MAPK, p65 and C/EBPβ phosphorylation, and subsequent COX-2 expression were restored in cells transfected with mitogen-activated protein kinase phosphatase-1 (MKP-1) dominant negative (DN) mutant. WMJ-S-001 also caused an increase in MKP-1 phosphatase activity in RAW264.7 macrophages.
WMJ-S-001 may cause MKP-1 activation to dephosphorylate p38MAPK, resulting in the decrease in p65 and C/EBPβ binding to the COX-2 promoter region and COX-2 down-regulation in LPS-stimulated RAW264.7 macrophages. The present study suggests that WMJ-S-001 may be a potential drug candidate in alleviating LPS-associated inflammatory diseases.
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The effects of imidazopyrazine derivative, SCA40, on the activity of single large conductance, Ca ²⁺ ‐activated K ⁺ (BK Ca ) channels in inside‐out and outside‐out patches from bovine tracheal smooth muscle (BTSM) cells in culture have been compared with those of two established BK Ca channel openers, NS 004 and NS 1619.
The presence of BK Ca channels on inside‐out patches of BTSM membranes was confirmed by the single channel conductance (240 pS), selectivity for K ⁺ , dependence of channel activity on [Ca ²⁺ ], and sensitivity to the selective BK ca channel blocker, iberio toxin.
NS 004 and ND 1619 (3‐30 μ M ) induced concentration‐related increases in open state probability of BK ca channels when applied to either inside‐out or outside‐out BTSM patches, thus confirming that these compounds are activators of the BK Ca channel in this preparation.
SCA40 (0.1‐10 μ M ) had no effect on the activity of BK Ca channels when applied to either inside‐out or outside‐out patches which subsequently responded to the application of NS 004 (10‐20 μ M ).
It is concluded that SCA40 does not have a direct effect on BK ca channel activity in BTSM patches and that the previously reported relaxant action of SCA40 on tracheal smooth muscle is unlikely to be mediated by this mechanism.
The adhesion molecule mucosal addressin cell adhesion molecule (MAdCAM) plays an essential role in the recruitment of lymphocytes to specialized high endothelial venules of the gastrointestinal tract and in their excessive tissue extravasation observed in inflammatory conditions, such as Crohn's disease. We have characterized the in vitro pharmacological properties of two monoclonal antibodies blocking MAdCAM, MECA-367 and PF-00547659, and determined their pharmacokinetic/pharmacodynamic profiles in vivo.
Functional adhesion assays and surface plasmon resonance were used to characterize, in vitro, the pharmacological properties of MECA-367 and PF-00547659. The in vivo effects of MECA-367 and PF-00547659 on restriction of beta(7) (+) memory T lymphocytes were determined in mice and macaques, respectively, over the pharmacological dose range to confirm pharmacokinetic/pharmacodynamic relationships.
MECA-367 and PF-00547659 bound with high affinity to mouse and human MAdCAM with K(d) values of 5.1 and 16.1 pmol.L(-1) respectively and blocked the adhesion of alpha(4)beta(7) (+) leukocytes to MAdCAM with similar potency. MECA-367 and PF-00547659 induced a similar, dose-dependent two- to threefold increase in circulating populations of beta(7) (+) memory T-cells in the mouse and macaque; without affecting the beta(7) (-) populations.
PF-00547659 has potential utility in the treatment of inflammatory conditions by blocking tissue homing of activated alpha(4)beta(7) (+) leukocytes. The characterization of a rodent cross-reacting antibody as a surrogate for PF-00547659 in the search for potential pharmacological biomarkers and the determination of efficacious doses was effective in addressing the restricted orthologous cross-reactivity of PF-00547659 and the challenges this poses with respect to efficacy and safety testing.
20-Hydroxyeicosatetraenoic acid is a potent vasoconstrictor that contributes to cerebral ischaemia. An inhibitor of 20-Hydroxyeicosatetraenoic acid synthesis, TS-011, reduces infarct volume and improves neurological deficits in animal stroke models. However, little is known about how TS-011 affects the microvessels in ischaemic brain. Here, we investigated the effect of TS-011 on microvessels after cerebral ischaemia.
TS-011 (0.3 mg·kg(-1) ) or a vehicle was infused intravenously for 1 h every 6 h in a mouse model of stroke, induced by transient occlusion of the middle cerebral artery occlusion following photothrombosis. The cerebral blood flow velocity and the vascular perfusion area of the peri-infarct microvessels were measured using in vivo two-photon imaging.
The cerebral blood flow velocities in the peri-infarct microvessels decreased at 1 and 7 h after reperfusion, followed by an increase at 24 h after reperfusion in the vehicle-treated mice. We found that TS-011 significantly inhibited both the decrease and the increase in the blood flow velocities in the peri-infarct microvessels seen in the vehicle-treated mice after reperfusion. In addition, TS-011 significantly inhibited the reduction in the microvascular perfusion area after reperfusion, compared with the vehicle-treated group. Moreover, TS-011 significantly reduced the infarct volume by 40% at 72 h after middle cerebral artery occlusion.
These findings demonstrated that infusion of TS-011 improved defects in the autoregulation of peri-infarct microcirculation and reduced the infarct volume. Our results could be relevant to the treatment of cerebral ischaemia.
Intrathecal (i.t.) administration of prostaglandin E 2 (PGE 2 ) to conscious mice was reported to induce allodynia, a state of discomfort and pain evoked by innocuous tactile stimuli through prostaglandin E receptor subtype EP 1 and hyperalgesia through prostaglandin E receptor subtypes EP 2 and/or EP 3 . In the present study, we investigated the effects of an EP 1 antagonist on these sensory disorders by use of ONO‐NT‐012 or AH6809.
ONO‐NT‐012 dose‐dependently antagonized the PGE 2 ‐induced allodynia but had no effect on the PGE 2 ‐induced hyperalgesia by the hot plate test. On the other hand, AH6809 blocked the PGE 2 ‐induced hyperalgesia at the highest dose examined (50 μ kg ⁻¹ ) but had no effect on the PGE 2 ‐induced allodynia. The i.t . injection of AH6809 or ONO‐NT‐012 alone did not have any effect on the response to noxious or innocuous stimuli.
Increasing doses (5 pg kg ⁻¹ ‐500 ng kg ⁻¹ ) of ONO‐NT‐012 produced parallel shifts to the right of the dose‐response curves to PGE 2 . The Schild plot regression line was linear and the slope was close to unity. The pA 2 value against PGE 2 was calculated to be 9.96.
The present study demonstrates that i.t . administration of PGE 2 exerts allodynia through EP 1 in the mouse spinal cord and that ONO‐NT‐012 is a highly potent, simple competitive antagonist for the PGE 2 ‐induced allodynia.
The aim of this study was to determine whether a synthetic inhibitor of the interleukin‐1β converting enzyme (ICE) displays oral activity in models of inflammation.
To this end, the ICE inhibitor, SDZ 224‐015, was examined in rat paw oedema, pyrexia and nociception tests.
SDZ 224‐015 (0.3–300 μg kg ⁻¹ ) potently reduced carrageenin‐induced paw oedema, with an oral ED 50 of approximately 25 μg kg ⁻¹ . This effect was independent of endogenous glucocorticoid, as shown by retention of activity upon adrenalectomy.
Pyrexia induced by lipopolysaccharide (0.1 mg kg ⁻¹ s.c.) or by interleukin‐1β (100 ng i.v.) was also reduced, over a similar dose‐range to oedema (oral ED 50 S 11 μg kg ⁻¹ and 4 μg kg ⁻¹ respectively).
SDZ 224‐015 (0.2–5 mg kg ⁻¹ , p.o.) displayed analgesic activity in the Randall‐Selitto yeast‐inflamed paw pressure test, significant at a dose of 1 mg kg ⁻¹ , p.o. 6 Thus, SDZ 224‐015 has potent oral activity in several acute models for inflammation, suggesting that ICE inhibitors may constitute a novel type of anti‐inflammatory agent.
Peroxynitrite is a strong oxidant that results from reaction between NO and superoxide. It has been recently proposed that peroxynitrite plays a pathogenetic role in inflammatory processes. Here we have investigated the therapeutic efficacy of raxofelast, a new hydrophilic vitamin E-like antioxidant agent, in rats subjected to carrageenan-induced pleurisy.
In vivo treatment with raxofelast (5, 10, 20 mg kg−1 intraperitoneally 5 min before carrageenan) prevented in a dose dependent manner carrageenan-induced pleural exudation and polymorphonuclear migration in rats subjected to carrageenan-induced pleurisy. Lung myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels, as well as histological organ injury were significantly reduced by raxofelast.
Immunohistochemical analysis for nitrotyrosine, a footprint of peroxynitrite, revealed a positive staining in lungs from carrageenan-treated rats. No positive nitrotyrosine staining was found in the lungs of the carrageenan-treated rats, which received raxofelast (20 mg kg−1) treatment.
Furthermore, in vivo raxofelast (5, 10, 20 mg kg−1) treatment significantly reduced peroxynitrite formation as measured by the oxidation of the fluorescent dihydrorhodamine 123, prevented the appearance of DNA damage, the decrease in mitochondrial respiration and partially restored the cellular level of NAD+ in ex vivo macrophages harvested from the pleural cavity of rats subjected to carrageenan-induced pleurisy.
In conclusion, our study demonstrates that raxofelast, a new hydrophilic vitamin E-like antioxidant agent, exerts multiple protective effects in carrageenan-induced acute inflammation.
British Journal of Pharmacology (1999) 126, 407–414; doi:10.1038/sj.bjp.0702275
Sodium p ‐benzyl‐4‐[1‐oxo‐2‐(4‐chlorobenzyl)‐3‐phenyl propyl]phenyl phosphonate (N‐0164) selectively inhibited the formation of thromboxane‐A 2 from prostaglandin endoperoxides by human platelet microsomes in a dose‐dependent manner (IC 50 2.2 × 10 ⁻⁵ m or 11.6 μg/ml).
N‐0164 was approximately 15 to 20 times as potent as indomethacin as an inhibitor of thromboxane‐A 2 formation. In contrast, indomethacin was 20 times as potent as N‐0164 as an inhibitor of prostaglandin endoperoxide formation from arachidonic acid (IC 50 2.6 × 10 ⁻⁵ m or 9.4 μg/ml).
Spiral strips of dog coronary arteries relaxed in the presence of prostaglandin endoperoxides and were contracted by prostaglandin E 2 and thromboxane‐A 2 and were therefore used to distinguish between prostaglandins and their intermediate precursors, the endoperoxides.
Neither indomethacin nor N‐0164 (both 50 μg/ml) significantly inhibited the formation of prostaglandin‐like activity from the endoperoxides following incubation with indomethacin‐pretreated rabbit kidney medulla microsomes.
It is not known whether this action of N‐0164 is related to its ability to antagonize certain actions of prostaglandins (and related compounds) or whether N‐0164 can penetrate the cell membrane to inhibit thromboxane formation in the intact cell.
Selective inhibition of thromboxane formation by drugs such as N‐0164 may be useful both clinically and as a pharmacological tool to elucidate the patho‐physiological roles of the thromboxanes.
The ability of sodium p ‐benzyl‐4‐[1‐oxo‐2‐(4‐chlorobenzyl)‐3‐phenylpropyl]phenyl phosphonate (N‐0164) to antagonize contractions produced by prostaglandins E 2 and F 2α on isolated preparations of gerbil, rat and guinea‐pig gastrointestinal muscle has been studied.
N‐0164 was found to be a potent, partially selective prostaglandin antagonist in these isolated smooth muscle preparations. The blockade produced by N‐0164 in the isolated stomach strip of the rat had some, but not all, the characteristics of a competitive antagonism.
N‐0164 produced a dose‐dependent decrease in tone in the rat stomach strip that was abolished by pretreatment of the preparation with indomethacin.
N‐0164 prevented diarrhoea induced by prostaglandin E 2 in mice when given by intraperitoneal injection but was less effective when given orally.
N‐0164 inhibited oedema induced with croton‐oil and pyridine‐ether in the mouse ear.
N‐0164 delayed the onset of erythema following ultraviolet irradiation of guinea‐pig skin only when an equimolar amount of pralidoxime chloride was added to the vehicle.
It is concluded that N‐0164 is a potent, partially selective prostaglandin antagonist on several isolated smooth muscle preparations. N‐0164 exhibits activity in vivo particularly following local application when problems associated with penetration and distribution are minimized.