The effect of acetylsalicylate on aggregation and arachidonate metabolism by human platelets suspended in plasma or buffer.

Acetylsalicylate inhibits prostaglandin and thromboxane production by human platelets suspended in plasma or buffer. Acetylsalicylate inhibits arachidonate-induced aggregation of human platelets suspended in plasma, but the effect of acetylsalicylate on arachidonate-induced aggregation of human washed platelets in buffer has not been reported. We have therefore studied this in relation to arachidonate metabolism in human platelets suspended in plasma or buffer. Platelets suspended in plasma and in buffer were both prepared from each donor, who had not taken acetylsalicylate or like-acting drugs for at least 7 days. Acetylsalicylate was 1500 times less potent in inhibiting arachidonate-induced aggregation in buffer (IC50 = 27.3 +/- 7.5 (s.e.m.)mM) than it was in plasma (IC50 = 18.3 +/- 6.0 microM); whereas it was only 4 times less potent in inhibiting thromboxane production in buffer (IC50 = 110 +/- 51.0 microM) than in plasma (IC50 = 25.3 +/- 8.9 microM). The acetylsalicylate concentration required to inhibit aggregation in buffer was sufficient to inhibit 12-hydroxyeicosatetraenoic acid production whereas the concentration that inhibited thromboxane production in buffer was not. These results indicate that arachidonate-induced aggregation of platelets in buffer may depend on product(s) of lipoxygenase rather than of cyclooxygenase, and is hence insensitive to inhibition by acetylsalicylate compared with arachidonate-induced aggregation of platelets in plasma.

Inhibition by adenosine analogs of opiate withdrawal effects.

The stable derivatives of adenosine, 2-chloroadenosine and N6-cyclohexyladenosine, with high affinity for the A1 (Ri) adenosine receptor, suppress the naloxone-precipitated withdrawal contracture of the opiate-dependent guinea-pig ileum in vitro. These adenosine derivatives also inhibit naloxone-precipitated jumping, diarrhea and weight-loss in morphine-dependent mice. This effect was not due to sedation, since (i) 2-chloroadenosine was effective at a non-sedative dose and (ii) sedative doses of chlordiazepoxide were ineffective.

Sites and mechanisms of dependence in the myenteric plexus of guinea pig ileum.

The isolated guinea pig ileum provides a model in which drug dependence can be induced in normal neurons. The characteristics of opiate dependence in the ileum closely resemble those of dependence in whole animals. Convergent dependence on normorphine, clonidine, and adenosine can be separately induced in the ileum in vitro. Use of selective antagonists indicates that both acetylcholine and substance P participate in the withdrawal response associated with all three of these dependencies. The demonstration that adenosine derivatives suppress opiate withdrawal in the guinea pig ileum and in mice raises the possibility that they might act similarly in man. The point at which the dependencies on normorphine, clonidine, and adenosine converge is probably below their separate recognition sites and is possibly at the level of adenylate cyclase regulation.

Novel form of drug-dependence--on adenosine in guinea pig ileum.

There is evidence that dependence on opiates occurs in neurones bearing their specific receptors, whose activation inhibits the neurone. Thus, incubation of guinea pig ileum with an opiate induces in the final cholinergic motoneurone of the myenteric plexus a dependence that closely resembles in basic characteristics opiate dependence in whole animals. A comparable, but distinct dependence can be induced by incubating the ileum with clonidine. Since adenosine also inhibits the final cholinergic motoneurone, by activating a specific purine receptor, we have tested whether it, too, can induce a distinct dependence in this neurone. To demonstrate dependence, we challenged the ileum by removing drug or by adding the selective purine receptor antagonist, 8-phenyltheophylline, which does not substantially inhibit phosphodiesterase, or caffeine. We found that incubation of the ileum with adenosine, or with the more potent derivative, 2-chloroadenosine, induced a novel form of drug dependence, made manifest by withdrawal of inducing drug, but not by antagonists of opiates or clonidine.

Endogenous peptides that inhibit brain cyclic AMP phosphodiesterase.

Peptide extracts of rat brain powerfully inhibited the cyclic AMP phosphodiesterase activity of rat brain homogenate. Similar extracts of ox brain showed comparable although less potent activity. Preliminary investigation of the physicochemical properties of brain extracts indicated that the rat brain extract contained an active peptide of low molecular weight (about 1400), whereas ox brain contained two such peptides (about 1400 and 900). These studies indicate that endogenous oligopeptides that inhibit cyclic AMP phosphodiesterase are present in brain. Experiments on several pure peptides known to be present in brain. Experiments on several pure peptides known to be present in the CNS showed that the majority were inactive against brain phosphodiesterase, but ACTH(1-24), somatostatin, substance P and Lys8-vasopressin, in descending order of potency, were active. To help distinguish the peptides found in rat and ox brain extracts from known peptides, preliminary analyses of amino acid composition were performed. These suggested that the peptides found in brain extracts were distinct from known peptides having the ability to inhibit cyclic AMP phosphodiesterase.

Model of opiate dependence in the guinea-pig isolated ileum.

1 Segments of ileum, incubated for 2-24 h at 22 degrees C with normorphine (0.01 - 1.0 muM), in the presence of hexamethonium, contracted when challenged with naloxone (0.03 muM). No response to this dose of naloxone was induced either by incubation in control solution without opiate for 2-24 h or by exposure of the preparation to opiate for 30 min at 37 degrees C.2 When segments were incubated for 24 h, the size of the response to naloxone was directly related both to the normorphine concentration in the incubation fluid (0.01 to 0.1 muM), and to the concentration of naloxone applied (0.03 to 0.1 muM).3 A spontaneous withdrawal contracture was elicited in ilea that had been incubated with normorphine (1.0 muM), when the normorphine-containing bathing fluid was exchanged for one without opiate.4 Normorphine restored to resting level the tension of the withdrawal contracture, whether it had been elicited spontaneously or by naloxone challenge.5 Addition of naloxone (1.0 muM) to normorphine (1.0 muM) in the incubation fluid abolished the withdrawal contracture to subsequent challenge with naloxone.6 Naloxone elicited a contracture from segments incubated for 24 h at 22 degrees C with levorphanol (0.1 muM) but not from those incubated with dextrorphan.7 Application of (+)-naloxone (0.03 muM) to segments previously incubated with normorphine (0.1 muM) did not elicit a contracture.8 The contracture elicited by naloxone in preparations incubated with morphine (10 muM) was associated with a reduction in sensitivity to the acute inhibitory effect of morphine on the electrically-evoked response.9 Addition of hyoscine (0.5 muM) immediately after challenge with naloxone restored the tension of the withdrawal contracture to resting level.10 Tetrodotoxin (3.0 muM) given before challenge, prevented naloxone from eliciting a withdrawal contracture.11 The inclusion of 5-hydroxytryptamine (10 muM) with morphine (10 muM) inhibited the induction of tolerance to morphine.12 These experiments, together with those described earlier, indicate that incubation with opiate induces a dependence in the final cholinergic motor neurones of the myenteric plexus, manifested as a contracture of the longitudinal muscle on removal of opiate or administration of an antagonist. This dependence is associated with tolerance, expressed as a decrease in sensitivity to inhibition by morphine of the electrically-evoked contracture. Tolerance and dependence are induced and withdrawal precipitated through specific and stereospecific opiate receptors.

Clonidine dependence in the guinea-pig isolated ileum.

1 Compared with the response of preparations incubated in solutions without clonidine, a three to four fold increase in the magnitude of the contracture of the longitudinal muscle to challenge with phentolamine (1.0 mum) was induced by incubating the guinea-pig isolated ileum at 22 degrees C for 24 h with clonidine (1.0 mum) in Krebs solution containing hexamethonium (70 mum). Incubation of the ileum with clondine (1.0 mum) for 0.5 h at 37 degrees C did not increase responsiveness to phentolamine.2 The increase in responsiveness to phentolamine was directly related to the clonidine concentration in the incubation fluid over the range 0.01 to 1.0 mum.3 The magnitude of the contracture to phentolamine of ilea incubated with clonidine (1.0 mum) (withdrawal contracture) was directly related to the challenge dose of phentolamine over the range 0.3 to 1.0 mum.4 Yohimbine (1.0 mum) or piperoxane (1.0 mum) elicited a response comparable to that elicited by phentolamine but propranolol (1.0 mum) was inactive.5 Addition of phentolamine (1.0 mum) to clonidine (1.0 mum) in the incubation fluid abolished the increased response of the preparation to subsequent challenge with phentolamine.6 Addition of hyoscine (0.5 mum) immediately after challenge with phentolamine restored the tension of the withdrawal contracture to its resting level.7 Tetrodotoxin (3.0 mum) given before challenge, prevented phentolamine from eliciting a withdrawal contracture.8 Ileal segments incubated with clonidine (1.0 mum) were unresponsive to challenge with naloxone (100 nm); and segments incubated with normorphine (1.0 mum) were unresponsive to phentolamine (1.0 mum), although responsive to naloxone.9 Normorphine (1.0 mum) restored to resting level the tension of the clonidine withdrawal contracture; and clonidine (0.1 mum) restored to resting level the tension of the contracture to naloxone in ileal segments incubated with normorphine.10 These experiments indicate that incubation with clonidine induces, in the final cholinergic motor neurones of the myenteric plexus of the isolated ileum, a dependence the withdrawal from which is expressed as a contracture in response to alpha-adrenoceptor antagonists.11 Although opiate receptors are not involved in clonidine dependence nor alpha-adrenoceptors in opiate dependence, the findings that normorphine suppresses the clonidine withdrawal-contracture and that clonidine suppresses the contracture of opiate-dependent ileum to naloxone, suggest that the withdrawal effect studied in both clonidine and normorphine dependence in this preparation is mediated by release of acetylcholine from the final motor neurone.

Comparisons between the abilities of various human and ovine plasmas to inhibit prostaglandin synthesis.

The ability of various human and ovine blood plasmas to inhibit prostaglandin synthesis in vitro has been tested. Human plasmas were significantly more potent in their ability to inhibit prostaglandin synthesis than their counterpart ovine plasmas. In general, female plasma had greater inhibitory activities than male plasmas and adult plasmas were more active than fetal plasmas. There was no simple correlation between the activity of plasmas as inhibitors of prostaglandin synthesis and their respective albumin or haptoglobin contents.

Character and meaning of quasi-morphine withdrawal phenomena elicited by methylxanthines.

A quasi-morphine withdrawal syndrome (QMWS) is a pattern of behavior closely resembling the true withdrawal syndrome in the opiate-dependent animal, which can be elicited acutely by a nonopiate drug in an opiate-naive animal. The main criteria proposed for the QMWS, in addition to its resembling the true withdrawal syndrome, are that the effects of opiates and of opiate antagonists on the QMWS should parallel those on true opiate withdrawal. Drugs that wholly or largely fulfill these criteria are 3-isobutyl-1-methylxanthine (IBMX), theophylline, caffeine, ICI 63197, and RO 201724. From the evidence given, it is concluded that these drugs act by inhibiting brain cyclic AMP phosphodiesterase, thus raising the level of cyclic AMP in appropriate neurons. These findings are consistent with the view that the molecular mechanisms of opiate dependence is the hypertrophy of a neuronal cyclic AMP system in compensation for the inhibition by opiate of an adenylate cyclase. Our studies and those of others suggest that: a) very rapid tests for opiate activity and for addictive liability can be devised by use of IBMX; b) opiates may be used clinically to counter poisoning by caffeine or theophylline; and c) a relationship may exist between caffeine consumption and opiate addiction.

Prostaglandin synthesis inhibition by maternal and fetal sheep plasma and its relation to haptoglobin and albumin levels.

Mammalian plasmas and sera have been reported to contain endogenous inhibitors of prostaglandin synthesis (EIPS), but the identity and role of these suggested inhibitors is as yet undetermined. Albumin and haptoglobin have been proposed as possible inhibitors, and it has been suggested that EPIS may have a part to play in the control of PG production during pregnancy and in the neonatal period. As part of a series of studies aimed at elucidating the identity and role of EIPS, maternal and fetal blood samples were collected from chronically catheterized pregnant ewes, and plasma levels of albumin, haptoglobin and EIPS activity determined. Pregnant ewe plasma possessed high EIPS activity and fetal lamb plasma little or no EIPS activity. Levels of albumin and/or haptoglobin did not consistently parallel that of EIPS activity. A post-operative rise (4 sheep studied) and a pre-parturition nadir (1 sheep studied) in maternal plasma EIPS activity were also noted. The possible physiological significance of these results is discussed.

Specific receptors for prostaglandins in airways.

The relative bronchomotor activities of prostaglandins (PG) E1, E2, F2 alpha, F2 beta and I2 and of three synthetic E prostaglandin analogues (TR4161, TR4367 and TR4752) were determined on a large number of isolated preparations of guinea-pig trachea and human bronchial muscle. Each prostaglandin was capable of eliciting both contraction and relaxation, the relative incidence of these responses partly depending on concentration. TR4161 was a virtually pure relaxant; TR4367 was virtually devoid of bronchomotor activity; and TR4752 was a potent relaxant, devoid of contractant activity. The results also provided distinct rank orders of approximate potency for contraction and relaxation. Tachyphylaxis to the relaxant activities of PGE1 and TR4752 confirmed the underlying contractant activity of the two natural E prostaglandins. Antagonism with a high dose of indomethacin of the contractant actions of PGE1, PGE2 and PGF2 alpha confirmed the presence of relaxant activities in each. Inhaled aerosols of the same natural and synthetic prostaglandins were evaluated for irritant activity on the airways, using the cough response of the restrained conscious cat. All of them, except TR4161, elicited severe coughing. The rank order of potencies for irritancy differed from those for tracheobronchial contractant and relaxant activities. These findings suggest that the three responses studied arise from the activation of three distinct PG receptors in the airways. We propose the terms chi (contractant), psi (relaxant) and omega (irritant) for these putative receptors for prostaglandins or possibly other prostanoids.