Nonsteroidal antiinflammatory agents.

Since the synthesis of aspirin in 1897, aspirin-like or nonsteroidal antiinflammatory drugs (NSAIDs) have been the mainstay of therapy for rheumatoid arthritis. Although of diverse chemical structure, these drugs not only exhibit the same antipyretic, analgesic and antiinflammatory therapeutic actions, but they also manifest identical toxic actions on the gastric mucosa and the kidney. This indicated that a single pharmacological effect was responsible for the properties of NSAIDs, a theory that was confirmed by the epochal discovery by Vane in 1971, that inhibition of the enzyme-producing prostanoids (cyclooxygenase [COX]) produced both the therapeutic and side effects of aspirin-like drugs. However, at equivalent antiinflammatory doses, different NSAIDs exhibited different degrees of toxicity. The reason for this was resolved by the discovery that prostaglandins at sites of tissue damage were synthesized by an inducible COX (COX-2) formed by a gene distinct from that producing the constitutive enzyme (COX-1), responsible for the formation of prostaglandins that serve an essential physiological function. Modification of the structure of drugs showing a moderately selective effect on COX-2, and the elucidation of the crystal structure of both enzymes, has paved the way for the synthesis of NSAIDs that are highly selective for the inducible enzyme and which are, therefore, antiinflammatory without the typical side effects of the classical NSAIDs. The focus on COX-2 has also expanded our knowledge of the pathophysiological significance of prostanoids and raised the possibility of new uses for selective COX-2 inhibitors, for example, in colon cancer, premature labor and possibly Alzheimer's disease. However, the clinical effects of chronic administration of potent, selective COX-2 inhibitors must await the results of ongoing clinical trials.

Relationship between gender difference in intravascular aggregation of platelets and the fibrinolytic pathway in the rat.

Intravascular aggregation of platelets was evaluated in relation to the fibrinolytic system in order to assess the possibility of a "cause-effect" relationship. The spontaneous fibrinolytic activities of the plasma of male rats and of female rats at the various stages of the oestrous cycle were determined. Male rats had higher euglobulin clot lysis time (54.5 +/- 5.3 vs 29.2 +/- 3.1 min; P less than 0.05), higher fibrinogen levels (330.0 +/- 15.8 vs 231.0 +/- 31.1 mg/dl; P less than 0.025) and higher plasminogen activity (8.1 +/- 1.2 vs 6.1 +/- 1.6 plasmin units/ml; P less than 0.05) than female rats. Female rats had higher fibrinolytic index (8.8 +/- 0.8 vs 6.3 +/- 0.3 mg/dl; P less than 0.05) and plasminogen activator activity (99.1 +/- 6.0 vs 76.5 +/- 7.7 Plough units/ml; P less than 0.05) than male rats. The antiplasmin activities were the same in both sexes. During the oestrous cycle in female rats, euglobulin clot lysis time was not significantly different though it was highest during met-oestrous (34.2 +/- 3.6 min). However, pro-oestrous rats had lower fibrinogen (122.9 +/- 5.3 mg/dl; P less than 0.005), higher fibrinolytic index (10.6 +/- 0.8 mg/dl/min; P less than 0.001) and higher plasminogen activator activity (109.4 +/- 7.8 Plough units/ml; P less than 0.05) than rats from the other stages of the oestrous cycle. There were no significant differences in plasminogen content and antiplasmin activity. Using native rats, aggregatory responses to submaximal doses of adenosine diphosphate (20 micrograms/kg) were determined and correlated with the fibrinolytic data in age- and weight-matched rats (of both sexes). Aggregatory responses in all the groups of rats used correlated positively with fibrinogen levels (r = 0.8316; P less than 0.001) and negatively with plasminogen activator activity (r = -0.7839; P less than 0.05). Streptokinase (250-1000 Plough units/kg/hr) and urokinase (1000-4000 Plough units/kg/hr) produced dose-related reductions in intravascular aggregation induced by adenosine diphosphate. The streptokinase effect (but not urokinase effect) was reversed by epsilon-aminocaproic acid. Following the cessation of infusion of streptokinase and urokinase, there was a recovery of the platelets to aggregate to adenosine diphosphate. These observations suggest fibrinolytic pathway-specific effects. However, on its own, epsilon-amino-caproic acid did not affect the aggregatory responses of platelets from pro-oestrous rats. These results suggest that changes in fibrinolytic mechanisms may account for differences observed in intravascular aggregation of platelets of male and female rats and of female rats during the oestrous cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenaline inhibits adenosine diphosphate induced intravascular aggregation of rat platelets through stimulation of alpha 2 adrenoceptors.

STUDY OBJECTIVE: The aim was to determine the receptors involved in the antiaggregatory effects of adrenaline in the rat. DESIGN: The 111indium oxine technique was used to evaluate ADP induced pulmonary accumulation of 111In labelled platelets in adrenalectomised rats (ie, no endogenous adrenaline) in the absence of anticoagulants. Adrenaline was infused (10, 20 and 40 micrograms.kg-1.h-1) and ADP aggregation measured. Adrenoceptor agonists, isoprenaline, methoxamine, and BHT 933, were given to evaluate whether they mimic the adrenaline effects. Adrenoceptor antagonists, yohimbine, propranolol, prazosin, and WY 26392, were given to assess whether they block the adrenaline effects. MEASUREMENTS AND MAIN RESULTS: In all cases, pulmonary accumulation of platelets was measured in response to ADP. Adrenaline dose dependently inhibited platelet aggregation. BHT 933 (1.5 micrograms.kg-1.min-1) mimicked, while phentolamine (1 mg.kg-1), yohimbine (2.5 mg.kg-1), and WY 26392 (0.1, 1.0, 5.0 mg.kg-1) blocked, the adrenaline effects. Methoxamine (3.3 micrograms.kg-1.min-1) produced a relatively weak non-specific inhibition of ADP aggregation. Isoprenaline (33 ng.kg-1.min-1) did not mimic the effects of adrenaline, neither did propranolol (1 mg.kg-1) affect the adrenaline inhibition of the aggregatory responses to ADP. Indomethacin (3 mg.kg-1) blocked the inhibitory effects of adrenaline. CONCLUSIONS: Adrenaline stimulates alpha receptors on platelets but inhibits platelet aggregation. There is no evidence to suggest that beta receptors participated in the effects produced by adrenaline. There is a role for cyclo-oxygenase products in the inhibitory effects of adrenaline on aggregation.

Pharmacodynamic interaction between oestradiol and adrenaline on intravascular aggregation induced by adenosine diphosphate in the rat.

Physiological levels of oestradiol and adrenaline inhibit intravascular aggregation of platelets, and effect dependent on the presence of an adrenal gland secretion (possibly adrenaline) and oestrogens, respectively. This infers a "mutual dependence" phenomenon. Acute adrenalectomy had a biphasic effect on intravascular aggregation: enhancing platelet aggregation when levels of oestradiol are high, e.g. in pro-oestrous rat, and reducing platelet aggregation when levels of oestradiol are nil or very low, i.e., in ovariectomized or di-oestrous rats, respectively. Ovariectomy also had a biphasic effect on platelet aggregation reducing aggregation in the absence of adrenaline and enhancing aggregation in its presence. Removal of endogenous oestradiol (by ovariectomy) and its replacement fully corroborates the "mutual dependence" hypothesis, whereas the removal of endogenous adrenaline (by acute adrenalectomy) and its replacement only partially supports it. It is concluded that, though both oestradiol and adrenaline have inhibitory effects on intravascular aggregation at physiological doses, each seems to need the full compliment of the other to achieve the effect.

Inhibition of intravascular platelet aggregation in the rat by adrenaline.

Intravascular platelet aggregation to adenosine diphosphate (ADP) was measured in anesthetized rats using 111indium-labelled platelets. Acute adrenalectomy increased the aggregatory effect of ADP in vivo, whereas infusions of low concentrations of adrenaline into adrenalectomized rats suppressed ADP-induced platelet aggregation. Similar antiaggregatory effects were seen with the alpha 2 agonist B-HT 933 and the alpha 1 agonist methoxamine, but not with isoprenaline. The effect of adrenaline was inhibited by phentolamine, yohimbine, the selective alpha 2 adrenoceptor antagonist WY 26392 and by indomethacin, but not by propranolol or prazosin. Adrenaline thus inhibits ADP-induced aggregation in vivo by a mechanism that may involve stimulation of an alpha 2 adrenoreceptor and may be dependent on activation of cyclooxygenase enzyme.

Indium-111 oxine technique of studying platelet aggregation in vivo. Some physiological considerations.

Intravascular aggregation in response to ADP, thrombin, arachidonate and collagen has been studied in the rat with a view to throwing more light on the validity, the reproducibility and physiology of the model. The radioisotopic technique of labelling platelets with indium-111 oxine was employed. The model is minimally invasive and involves collecting blood from donor rats, separating and labelling their platelets with indium-111 oxine and assessing the accumulation of platelets in the lungs of recipient rats following the intravascular administration of aggregating agents. Of the parameters evaluated, percentage peak increase in radio-labelled platelet count and area under the curve are good parameters of expressing aggregatory responses. Of the visceral organs evaluated, the lung is the most important organ for assessing platelet accumulation. Of the vascular routes examined, no aggregatory response occurred in the lungs when ADP was injected via any of the intra-arterial routes, and of the intravenous routes, injection via the tail vein gave the highest response. The results of this investigation provide some more detailed technical information to take note of when studying platelet aggregation in vivo by this model. The results also highlight the physiological phenomenon involved.

The influence of adrenaline on gender difference in adenosine diphosphate-induced aggregation of platelets in the rat.

The role of adrenaline on the inhibitory effects of physiological levels of oestradiol on ADP-induced intravascular aggregation has been studied. Platelets from pro-oestrous female rats aggregated less than those from dioestrous and male rats. Following adrenalectomy, there was no longer any difference(s) in the aggregability of the platelets to ADP in any of the rats. Adrenaline infusion (20 mg kg-1 hr-1) restored platelet aggregation to preadrenalectomy levels in pro-oestrous rate. Measurement of spontaneous fibrinolytic activity of the plasma showed highest value in pro-oestrous rats. Adrenalectomy reduced, while adrenaline infusion increased the fibrinolytic activity. The results suggest that the inhibitory effects of oestradiol on intravascular aggregation are dependent on endogenous adrenaline possibly working through the fibrinolytic pathway.

Cryptolepine inhibits platelet aggregation in vitro and in vivo and stimulates fibrinolysis ex vivo.

1. Cryptolepine--the methylquindolanol alkaloid of Cryptolepsis sanguinolenta was evaluated for its antiplatelet and fibrinolytic effects. 2. It exhibited antiplatelet effects in vitro in human, rabbit and rat PRP with EC50 values ranging between 8.1 x 10(-8) M and 1.7 x 10(-7) M for ADP, AA and thrombin. 3. In the rat, it inhibited ADP-aggregation in vivo with delayed onset and prolonged action. 4. In vitro, cryptolepine disaggregated (dose-dependently) platelets aggregated by ADP, AA and thrombin. 5. In addition, it exhibited an indirect fibrinolytic action in the rat possibly by causing the release of plasminogen activators from the vascular endothelium.

Inhibition of adenosine diphosphate-induced intravascular aggregation of rat platelets in vivo by 6-oxo-prostaglandin E1.

The antiaggregatory effects of 6-oxo-PGE1 were evaluated in vivo in the rat using a minimally invasive technique involving 111-Indium labelling of platelets. The antiaggregatory effects on adenosine diphosphate-induced aggregation were compared with those of prostacyclin (PGI2) and prostaglandin E1 (PGE1) following slow infusions and bolus injections. The rank order of antiaggregatory potency was PGI2 greater than 6-oxo-PGE1 greater than PGE1 while the rank order of duration of antiaggregatory effects was PGE1 greater than 6-oxo-PGE1 greater than PGI2. The kinetics of the antiaggregatory effects of these prostaglandins suggests that such actions are not mediated by direct effects on platelets, but through a secondary mechanism.

A minimally invasive technique for the study of intravascular platelet aggregation in anesthetized rats.

A method for monitoring platelet aggregation in vivo in the rat is described, using platelets labeled with indium3+ oxine and recording the increase in radioactivity count in the lung after injection of adenosine diphosphate and collagen. The effects of adenosine diphosphate and collagen are reproducible between animals. 5-Hydroxytryptamine creatine sulfate, which is inactive on rat platelets in vitro, causes aggregation in vivo, and adrenaline, which is proaggregatory in vitro inhibits adenosine diphosphate-induced aggregation in vivo. Female rats are relatively insensitive to the aggregating agents, particularly during proestrus, although when platelets from proestrus females were injected into male rats, their sensitivity was increased. Platelets from male rats injected into female rats in proestrus have low sensitivity.

The mouse anococcygeus muscle as a preparation for the bioassay of oxytocin.

Oxytocin (0.1-10 nM) caused reproducible, dose-related contractions of the male mouse anococcygeus. Desensitization was not a major problem. The muscle was shown to be a useful oxytocin bioassay preparation, having a good index of precision (0.04).

Neuropeptide-induced contraction and relaxation of the mouse anococcygeus muscle.

Isometric tension responses to neuropeptides were recorded from anococcygeus muscles isolated from male mice. This smooth muscle tissue is innervated by inhibitory nonadrenergic, noncholinergic nerves that resemble, ultrastructurally, the peptidergic neurons of the gastrointestinal tract; the physiological function of the anococcygeus is not known. Slow sustained contractions were produced by oxytocin (0.2-20 nM), [Arg8]vasopressin (0.4-200 nM), and [Arg]-vasotocin (0.4-100 nM); the mouse anococcygeus is, therefore, one of the few examples of nonvascular smooth muscle from male mammals to respond to low concentrations of oxytocin and related peptides. Substance P (0.5-8 microM) caused distinctive, biphasic increases in muscle tone of some, but not all, preparations. Other neuropeptides producing contractions were neurotensin (2-100 microM) and thyrotropin-releasing hormone (2-100 microM); the responses were of similar time course and displayed selective cross-desensitization, suggesting that these two peptides act through a common distinct mechanism. Tetradecapeptide somatostatin (10-80 microM) and its analog urotensin II (0.1-5 microM), a dodecapeptide from the urophysis of the teleost fish Gillichthys mirabilis, produced similar slowly developing relaxations of carbachol-induced tone. Piscine urotensin II, of which there are no reported effects on nonvascular mammalian systems, was 20-50 times more potent than somatostatin, a well-established mammalian hormone. Of the peptides studied, only vasoactive intestinal polypeptide (0.05-1 microM) caused rapid powerful relaxations in low concentrations; this is consistent with its proposed involvement in nonadrenergic, noncholinergic neurotransmission in the mouse anococcygeus.

The effect of modification of sympathetic activity on responses to ligation of a coronary artery in the conscious rat.

1 Ligation of a coronary artery was performed in conscious rats whose sympathetic system activity had been altered by various treatments. 2 beta-Adrenoceptor blockade with acute (0.2 mg kg-1 plus 0.1 micrograms kg-1 min-1) or chronic (50-60 mg kg-1 daily for 12 days) propranolol treatment had little effect on arrhythmias, or other responses to ligation. 3 Abrupt withdrawal of chronic propranolol two days before ligation was also without effect. 4 Reduction of sympathetic activity acutely with labetalol (5 mg kg-1), or chronically with adrenomedullectomy and 6-hydroxydopamine treatment, accentuated the adverse effects of ligation. 5 The results of this study suggest that, while activity of the sympathetic system is not detrimental during ligation in the conscious rat, it may be important for survival.

The influence of sex hormones on rat platelet sensitivity to adenosine-diphosphate.

The sensitivity of rat platelets to the aggregating agent adenosine-diphosphate (ADP) was studied. Platelets form male and female rats demonstrated comparable sensitivity. Neither ovariectomy nor castration altered platelet sensitivity to ADP. However platelets form mock-operated female, but not mock-operated male rats were less sensitive. Administration of physiological doses of 17 beta-oestradiol to ovariectomised rats reduced platelet sensitivity. Administration of 17 beta-oestradiol to mock operated female rats increased platelet sensitivity to ADP. The results suggest that physiological amounts of oestrogen induce a loss of platelet sensitivity following surgery or stress, whereas higher concentrations of oestrogens increase platelet sensitivity.