James B. Polson

Multiple high-affinity cAMP-phosphodiesterases in human T-lymphocytes

Cyclic nucleotide phosphodiesterases (PDEs) are the only enzymes that inactivate intracellular cyclic AMP (cAMP). Because the functions of T-lymphocytes are modulated by cAMP levels, the isozymes of PDE in these cells are potential targets for new drugs designed to modify the bodys immunity through selective alteration of T-lymphocyte PDE activity. Cyclic GMP and 3(2H)-pyridazinone-4,5- dihydro-6-[4-(1H-imidazol-1-yl)phenyl]-5-methyl-monohydrochloride (CI-930) selectively inhibit the catalytic activity of one of the two high affinity cAMP-PDE isozyme families known to occur in mammals, whereas d,l-1,4-[3-butoxy-4-methoxybenzyl]-2-imidazolidinone (Ro 20-1724) selectively inhibits the other. The objectives of this investigation were: (1) to determine whether human T-lymphocytes contain one or both of these pharmacologically distinguishable high-affinity cAMP-PDEs, and (2) to determine the effects of selective inhibitors of these PDEs on lymphocyte blastogenesis. High-affinity cAMP-PDE was found in both the soluble and particulate fractions of T-lymphocyte sonicates. Cyclic GMP and CI-930 inhibited PDE in the particulate fraction better than in the soluble fraction, but the converse was found for Ro 20-1724. CI-930 or Ro 20-1724, used alone, attenuated T-lymphocyte blastogenesis, but neither suppressed it completely. In combination, the same PDE inhibitors caused greater suppression of blastogenesis than either produced alone. The results indicate that human T-lymphocytes contain both CI-930- and Ro 20-1724-inhibitable isozymes. Either of the isozymes can modulate human T-lymphocyte blastogenesis, but inhibition of both isozymes produces synergistic antiblastogenic effects.

Characteristics of histamine tachyphylaxis in canine tracheal smooth muscle

SummaryIn isolated canine tracheal smooth muscle, repeated administrations of histamine result in a rapid reduction in contractile response to about 15% of the initial contraction (tachyphylaxis). Development of this tachyphylaxis is specific inasmuch as: 1) it does not develop to acetylcholine (10−6 M or 10−4 M), or serotonin (10−5 M); and 2) maximally developed histamine tachyphylaxis is not associated with a parallel reduction in response to acetylcholine. Pretreatment with propranolol (10−5 M) or phentolamine (10−4 M) does not prevent tachyphylaxis: however, pretreatment with atropine (10−4 M) does prevent tachyphylaxis in about 50% of the animals tested.Tachyphylaxis to histamine can be reversed in a dose- and time-dependent fashion with prostaglandin synthesis inhibiting agents. The order of potency obtained with such compounds (indomethacin > mefenamic acid > oxyphenbutazone > acetylsalicylic acid) is consistent with potencies for inhibition of prostaglandin synthesis found in the literature. Also, in indomethacin pretreated strips in which tachyphylaxis to histamine was prevented, exogenous addition of PGE2 (1.42×10−10 M to 2.84×10−9 M) and PGA2 in a high concentration (2.9×10−9 M) are capable of selectively reducing the response to histamine without an effect on acetylcholine-induced contractions. These data suggest that the mechanism of histamine tachyphylaxis in the canine tracheal smooth muscle preparation involves prostaglandin synthesis.

High pressure liquid chromatography of cyclic nucleotide phosphodiesterase from purified human T-lymphocytes

The cyclic nucleotide phosphodiesterase (EC 3.1.4.17) in extracts of purified human peripheral blood T-lymphocytes was examined by ion exchange high pressure liquid chromatography. Four peaks of activity were isolated. The first peak of activity selectively hydrolyzed cyclic GMP. The following 3 peaks of activity (Ia, IIa and IIIa) were selective for cyclic AMP. The selective low Km cyclic AMP-phosphodiesterase inhibitor, Ro 20-1724 (d,1-1,4-[3-butoxy-4-methoxybenzyl]-2-imidazolidinone), did not inhibit the activity in Ia whereas it did inhibit the activity in IIa and IIIa (IC50 = 17 microM). The authors conclude that ion exchange high pressure liquid chromatography described in this communication is a useful method for the isolation of different forms of cyclic nucleotide phosphodiesterase activity from human T-lymphocytes.

Pharmacological and biochemical activities of some monomethylxanthine and methyluric acid derivatives of theophylline and caffeine

Abstract Some monomethylxanthine and methyluric acid derivatives of theophylline and caffeine have been studied to explore whether they possess pharmacological and biochemical activities similar to those of their parent compounds. Both 3-methylxanthine and 1-methylxanthine, but not 1,3-dimethyluric acid or 3-methyluric acid, produced the same maximal relaxation of guinea pig tracheal muscle as did theophylline. The EC 50 values for theophylline and 3-methylxanthine were not significantly different, whereas those for 1-methylxanthine, 1,3-dimethyluric acid and 3-methyluric acid were significantly higher than that if theophylline. In the Langendorff guinea pig heart, theophylline and 3-methylxanthine caused essentially identical increases in cardiac contractile force. Although less effective than theophylline, 1-methylxanthine and caffeine produced equivalent increases in cardiac contractility. At concentrations higher than those effective for the methylxanthines, 1,3-dimethyluric acid markedly increased contractile force. 3-Methylxanthine inhibited cyclic AMP phosphodiesterase to a lesser extent than did theophylline at both 1.4 and 400 μM cyclic nucleotide concentrations. However, at the higher substrate concentration, cyclic GMP phosphodiesterase activity was inhibited by 3-methylxanthine more than by theophylline. Thus, it appears that the monomethylxanthine and methyluric acid derivatives of theophylline and caffeine possess a spectrum of pharmacological activity similar to that of their parent compounds, a finding which raises important questions about various aspects of the current therapeutic use of methylxanthines.

Analysis of the relationship between pharmacological inhibition of cyclic nucleotide phosphodiesterase and relaxation of canine tracheal smooth muscle

Abstract Dose-response curves for muscular relaxation produced by five phosphodiesterase inhibitors were compared to curves for inhibition of phosphodiesterase-catalyzed breakdown of cyclic AMP or cyclic GMP. The rank-order of potency of the agents as muscular relaxants was similar to their order of potency as phosphodiesterase inhibitors. When enzyme activity was measured with 1.5. 38 or 400 μM cyclic AMP or 1.5 μM cyclic GMP as substrate, it was found that two agents, caffeine and SQ 20,009 [1-ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b) -pyridine-5-car☐ylic acid, ethyl ester, HCl], inhibited activity in concentrations equal to or slightly less than were required for muscular relaxation. The remaining three agents, theophylline, MIX (1-methyl-3-isobutylxanthine), and ICI 58,301 (3-acetamido-6-methyl-8- n -propyl-s-triazolo[4,3-a]pyrazine), were required in 2.4 to 10.3-fold higher concentrations for enzyme inhibition than for muscular relaxation. It was concluded that, although these findings are generally consistent with the hypothesis that phosphodiesterase inhibition is important in the mechanism of action of the drugs tested, it may be necessary to take into account additional, presently unknown factors to explain fully the relaxant effects of these drugs on respiratory smooth muscle.

Correlation between inhibition of a cyclic GMP phosphodiesterase and relaxation of canine tracheal smooth muscle.

Inhibition of partially purified cyclic nucleotide phosphodiesterase activity as well as pharmacologically induced relaxation of respiratory airways smooth muscle was examined to determine whether any correlation between these two effects could be found. The phosphodiesterase in extracts of canine tracheal smooth muscle was chromatographed on a DEAE Bio-Gel A column and eluted with a sodium chloride gradient. The peak I activity hydrolyzed cGMP at a higher rate than cAMP although the apparent Km values for these two cyclic nucleotides were relatively close. Comparison of the Ki values for alkylxanthine inhibitors of peak I activity correlated remarkably well with the EC50 values of the same compounds as relaxants of canine tracheal smooth muscle strips. It is concluded that inhibition of the peak I enzyme may cause accumulation of an intracellular pool of cyclic nucleotide and thus produce or contribute to the muscle relaxant effects that were observed.

The site of action of Ptychodiscus brevis toxin within the parasympathetic axonal sodium channel h gate in airway smooth muscle

The red tide toxin produced by Ptychodiscus brevis ( PBTX ) may cause cough, sneezing, and asthma. Previous in vitro studies with isolated canine tracheal smooth muscle demonstrated that PBTX stimulates sodium channels of parasympathetic nerve endings and thus causes a contractile response. The present study investigated the mechanism of the PBTX effect on canine tracheal smooth muscle. Repeated exposure of the muscle strip to PBTX (final concentration 46 micrograms/ml) followed by washout of the toxin resulted in reestablishment of baseline tension but a failure of contraction on further addition of PBTX . However, veratridine and scorpion toxin (SCT), which are voltage-sensitive sodium channel activators, still induced contraction. Furthermore, the contraction caused by veratridine was enhanced by a high dose of PBTX , whereas contraction caused by SCT was not. Responses to veratridine and SCT as well as PBTX (previously reported) were blocked by tetrodotoxin (a sodium channel blocker), while acetylcholine responsiveness remained intact. These results indicate that PBTX receptors in parasympathetic nerves influence Na+ flux at the h gate, that these receptors differ from the veratridine and SCT receptors, and that the conformational change in the receptors induced by PBTX affects the tissue response to veratridine.

Pulmonary patterns of adenosine-3′, 5′-cyclic monophosphate accumulations in response to adrenergic or histamine stimulation in Bordetella pertussis-sensitized mice

Abstract Susceptible strains of mice when injected with Bordetella pertussis vaccine develop a marked hypersensitivity to histamine and other agents of pharmacologic interest. The possibility was raised that this hypersensitivity is due to a reduced responsiveness of adenylate cyclase to adrenergic stimulation in various tissues of the pertussis-sensitized organism. This paper describes experiments showing that, in contrast to some other tissues, the resting level of adenosine-3′,5′-cyclic monophosphate (cAMP) in lungs, as well as the capacity of the pulmonary cAMP system to respond to adrenergic activation, as effected by naturally occurring or synthetic catecholamines, is unaltered by the pertussis sensitization of the animals. Another finding here-in described is the pulmonary cAMP rise in both normal and pertussis-sensitized mice after histamine administration. Using beta-adrenergically blocked and adrenalectomized mice, evidence was obtained indicating that the increased amounts of cAMP after histamine may have resulted from an adrenergically mediated effect.

In vitro red tide toxin effects on human bronchial smooth muscle

Airborne Ptychodiscus brevis toxin (PBTX), produced by Ptychodiscus brevis (Florida red tide), induces cough, rhinorrhea, watery eyes, and sneezing in normal individuals and wheezing in subjects with asthma. The mechanism of PBTX-induced contractile response has been investigated by the authors in vitro in dog and rat tissue. PBTX stimulates neuronal sodium channels, resulting in activation of autonomic cholinergic and adrenergic nerve endings in canine upper and lower airway smooth muscle and in rat vas deferens, respectively. This article concerns the investigation of the effect and mechanism of action of PBTX on human airways in order to determine the unique role of the toxin in the pathogenesis of asthma. PBTX elicited contractions of isolated human airway smooth muscle with a threshold concentration of 0.1 micrograms/ml, very similar to values obtained in canine lower airways. Pharmacologic analysis demonstrated that atropine (10(-6) mol/L) blocked the response to both PBTX and acetylcholine; tetrodotoxin (10(-7) mol/L) blocked PBTX but not acetylcholine; and verapamil (10(-5) mol/L) attenuated but neostigmine (10(-8) mol/L) potentiated the response to PBTX. Other selected blockers did not affect the PBTX response. These data indicate that PBTX produces contraction of human lower airway smooth muscle via stimulation of cholinergic nerve fiber sodium channels. The concept that PBTX triggers asthma through this mechanism is strengthened by these results.

The effect of prostaglandin E2 on histamine-stimulated calcium mobilization as a possible explanation for histamine tachyphylaxis in canine tracheal smooth muscle

SummaryIsolated strips of canine tracheal smooth muscle rapidly lost their responsiveness to histamine when placed in a zero calcium Krebs buffer. Responsiveness to acetylcholine, however, was not rapidly lost, and following 120 min of incubation in zero calcium buffer with frequent washes, 10% of the contractile response still remained. The kinetics of each loss of response suggest that primarily a loosely bound source of calcium is mobilized by histamine and a more tightly bound source is mobilized by acetylcholine. Consistent with these data were the effects of the calcium antagonist verapamil. In normal calcium Krebs solution, dose-response curves to histamine were markedly reduced by verapamil while acetylcholine responses were relatively unaffected.In calcium depleted tracheal strips, indomethacin potentiated the calcium dose-response curve, determined by incremental readdition of calcium in the presence of histamine (10−4 M), with comparatively little effect on the calcium doseresponse curve in the presence of acetylcholine (10−6 M). Also, in indomethacin pretreated tracheal strips, a reduction in the histamine-calcium dose-response curve could be produced by exogenous addition of 2.8×10−9 M and 2.8×10−8 M PGE2. In the acetylcholine-calcium responses there was a significant reduction only at 2.8×10−8 M PGE2. These data suggest that histamine mobilizes primarily a loosely bound, possibly extracellular source of calcium necessary for contraction, and this histamine-stimulated calcium mobilization is sensitive to the effects of PGE2.