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The effect non-steroidal anti-inflammatory drugs on formation and release of glycosaminoglycans (GAG) and cyclic 3',5'-AMP levels was studied in embryonic mouse fibroblasts. The results were compared and correlated with the action of these drugs on cyclic 3',5'-AMP-dependent as well as independent protein kinase obtained from bovine diaphragm. 1. Phenylbutazone dose-dependently decreased cyclic 3',5'-AMP levels and GAG secretion both in unstimulated and PGE1 stimulated cells. 2. Indometacin decreased cyclic 3',5'-AMP levels and GAG secretion only in cells with elevated cyclic 3',5'-AMP levels after stimulation by PGE1. 3. Sodium salicylate decreased cyclic 3',5'-AMP levels in the presence and absence of PGE1. However, GAG secretion was reduced only in cells with elevated cyclic 3',5'-AMP levels, since the drug activated cyclic 3',5'-AMP-independent protein kinase activity, thus presumably precluding changes in GAG formation at low levels of cyclic 3',5'-AMP. 4. Mefenamic acid decreased cyclic 3',5'-AMP levels in cells stimulated by PGE1, whereas GAG secretion was increased both in the absence and presence of PGE1. This increase in GAG secretion was closely correlated to an enhanced cyclic 3',5'-AMP-dependent and independent protein kinase activity. The results indicate that non-steroidal anti-inflammatory drugs may exert their effects on GAG formation by interfering with cyclic 3',-5'-AMP formation or function.

Benzydamine dose-dependently increased cyclic 3',5'-AMP levels in isolated fat cells and competitively inhibited phosphodiesterase activity (Ki = 1.1 mM). ATP levels of isolated cells and cyclic 3',5'-AMP-dependent protein kinase activity were not affected. However, benzydamine caused a dose-dependent inhibition of lipolysis in isolated fat cells stimulated by norepinephrine or dibutyryl cyclic 3',5'-AMP. The enhancement of cyclic 3',5'-AMP formation may be due to the local anaesthetic properties of benzydamine. The antilipolytic effect appears to result from the direct inhibition of hormone sensitive triglyceride lipase.

In experiments with isolated epididymal fat cells, paraoxone caused a dose-dependent inhibition of lipolysis in the LD50 dose range (10−7 - 10−5 M). The inhibition occurred in unstimulated as well as stimulated lipolysis elicited by 10−6 M norepinephrine or 3 × 10−3 M dibutyryl-A-3′, 5′-MP. The type of inhibition showed a noncompetitive pattern at an apparent Ki = 1.1 × 10−6 M. Obidoxime, HS 3 and HS 6 did not abolish the 50% inhibition of maximum lipolysis elicited by 10−6 M paraoxone. Neither a reactivation nor a direct interaction between paraoxone and oximes was observed in this test system.

Cyclic 3′,5′-AMP-dependent protein kinase activity is inhibited by phenylbutazone both in the presence and absence of the cyclic nucleotide. Mefenamic acid stimulates the activity of the enzyme up to 10−4 M, being less effective at higher concentrations. These findings closely correlate with the influence of both drugs on lipolysis. Other antilipolytic drugs such as benzydamine, adrenergic blocking agents, insulin, prostaglandin E1 or nicotinic acid do not affect the activity of cyclic 3′,5′-AMP-dependent protein kinase. The results indicate that only in a few cases the effect on protein kinase activity may be causal for the antilipolytic action of drugs.

Sodium salicylate and to a lesser extent acetylsalicylic acid both inhibit the activity of cyclic 3′,5′-AMP-dependent protein kinase measured by the phosphorylation of histone. The competitive type of inhibition is only seen in the presence of cyclic 3′,5′-AMP. In the absence of cyclic 3′,5′-AMP the unstimulated activity of protein kinase is increased. The impairment of function of protein kinase correlates with the previously reported inhibition of binding of cyclic 3′,5′-AMP to cyclic 3′, 5′-AMP-dependent protein kinase and indicates that the action of sodium salicylate may involve the “regulatory site” of the protein kinase.

Dopaminergic stimulants (amantadine, amphetamine, apomorphine, nomifensine and L-dopa plus benserazide) increased cyclic GMP levels in the medial forebrain and cerebellum of mice. Cyclic AMP levels were not significantly altered under these conditions. Drug-induced stereotyped behaviour correlated in intensity and duration to the changes in cyclic GMP levels in the medial forebrain. Amantadine, apomorphine and nomifensine showed a linear dose response relationship, but differed as to the extent and time course of the increase in cyclic GMP. Amantadine and apomorphine were were more effective in elevating cyclic GMP in the medial forebrain than in the cerebellum. Amphetamine produced an exponential dose-related elevation of cyclic GMP in both parts of the brain, being more effective in the cerebellum than in the medial forebrain at high doses, thus indicating a complex mechanism of action. L-Dopa (50 mg/kg) and benserazide (40 mg/kg) alone did neither significantly increase cyclic GMP levels nor induce stereotyped behaviour. However, in animals pretreated with benserazide (15 min prior to L-odopa) L-dopa produced a significant elevation of cyclic GMP and stereotyped behaviour.

Glucocorticoids (GCs) reduced cyclic AMP levels and inhibited glycosaminoglycan (GAG) synthesis in secondary embryonic mouse fibroblast cultures, when cells were incubated for short periods (30 min). The order of potency was dexamethasone greater than prednisolone greater than hydrocortisone. The effect was more marked, when cyclic AMP levels and GAG synthesis were increased by addition of PGE1. Glucocorticoids exerted no longer an inhibitory effect on cyclic AMP and GAG synthesis in cultures pretreated for 48 h with the steroids. Addition of PGE1 caused a stronger rise in cyclic AMP and GAG synthesis than in controls without GC-preincubation. This enhancement was even more pronounced, when PGE1 was added together with the GCs. The reversal of the inhibitory effect of the GCs into a potentiating effect following preincubation correlated to a reducation of endogenous PGE formation in the cultures. Short-term treatment with GCs did not reduce endogenous PGE levels, but prolonged incubation markedly decreased PGE levels. PGE formation recovered following addition of fresh medium after the 48 h incubation with the steroids, but the amount of PGE formed remained significantly lower than in untreated cultures. Non-glucocorticoid steroid hormones did not decrease PGE levels. The results indicate that the apparent loss of inhibitory activity of GCs on cyclic AMP and GAG synthesis observed after prolonged incubation may result from a reduction of endogenous PGE formation which renders the cells more sensitive to the stimulatory effect of exogenous PGE1.

Paraoxon exerts its antilipolytic effect on the lipolytic system of fat cells by a direct interaction with the triglyceride lipase. It does not affect other components of the lipolytic system such as adenyl cyclase, phosphodiesterase, ATP-levels or binding of cyclic 3′,5′-AMP-dependent protein kinase. Phosphorylation of protein mediated by cyclic 3′,5′-AMP-dependent protein kinase is not impaired by paraoxon.