The relaxant and spasmogenic effects of some xanthine derivatives acting on guinea-pig isolated trachealis muscle.

1. Caffeine (10 mM)-induced relaxation of guinea-pig isolated trachealis was attenuated and converted to a small spasmogenic response on cooling to 22 degrees C. The relaxant response was restored on rewarming to 37 degrees C and was abolished by indomethacin (2.8 microM). Cooling to 22 degrees C in the presence of indomethacin revealed spasmogenic responses to caffeine which were abolished on rewarming to 37 degrees C. 2. Trachealis treated with indomethacin (2.8 microM) was repeatedly dosed with acetylcholine (ACh, 10 microM). Caffeine (1 or 10 mM), added as each ACh-induced spasm reached equilibrium, transiently augmented but then suppressed the spasm. On cooling from 37 degrees C to 12 degrees C, the increment in spasm evoked by caffeine increased relative to the spasm evoked by ACh. 3. Trachealis treated with indomethacin (2.8 microM) was repeatedly dosed with caffeine (10 mM). At 37 degrees C caffeine had little effect but it caused spasm when the tissue was cooled to 32 degrees C. Spasm amplitude increased as cooling progressed to 12 degrees C. Similar results were obtained with caffeine (1 mM). 4. At 37 degrees C, caffeine, enprofylline, 1,3,7,9-tetramethylxanthinium (TMX), theobromine, theophylline, xanthine and forskolin each caused concentration-dependent suppression of tracheal tone. Among the xanthine derivatives the rank order of potency was enprofylline greater than theophylline greater than caffeine greater than theobromine greater than xanthine greater than TMX. 5. In trachealis treated with indomethacin (2.8 microM) and maintained at 12 degrees C, the xanthines each caused concentration-dependent spasm. The rank order of potency was theobromine greater than or equal to theophylline greater than or equal to caffeine greater than or equal to enprofylline greater than xanthine greater than TMX. Forskolin was devoid of spasmogenic activity. 6. Trachealis treated with indomethacin (2.8 microM) and maintained at 12 degrees C, was repeatedly dosed with either caffeine (10 mM) or potassium chloride (KCl, 40 mM). Caffeine-induced spasm was attenuated in a Ca2+-free medium containing EGTA (2 mM), modestly at first but subsequently more profoundly. KCl did not evoke spasm at 12 degrees C but at 37 degrees C the KCl-induced spasm was virtually abolished at its first trail in the Ca2+-free, EGTA-containing medium. 7. It is concluded that caffeine, other alkylated xanthines and xanthine itself share a spasmogenic action in guinea-pig isolated trachealis which is best observed when the tissue is treated with indomethacin (2.8 microM) and maintained at 12 degrees C. The spasmogenic action represents the release of Ca2+ from intracellular sites of sequestration and may not depend on the intracellular accumulation of cyclic AMP. The rank order of spasmogenic potency of the xanthine derivatives differs markedly from their rank order of potency in suppressing the spontaneous tone of the trachealis observed at 370C. Since, at 12 degrees C, TMX is spasmogenic at concentrations identical to those causing relaxation at 37 degrees C, it is likely that TMX penetrates the cell. The relaxant effects of TMX do not, therefore, indicate that methylxanthine-induced relaxation is mediated by a receptor located on the external surface of the cell.

Effects of dimethyl sulphoxide (DMSO) on aggregation of human blood platelets.

The effects were examined of the universal solvent dimethyl sulphoxide (DMSO) on human platelet aggregatory activity, in-vitro, of the endogenous mediators ADP, adrenaline, arachidonic acid, collagen and PAF-acether which are believed to play important roles in cardiovascular diseases in man. DMSO inhibited aggregation induced by all of the mediators in the order ADP greater than adrenaline = arachidonic acid = PAF-acether greater than collagen. Since DMSO is widely used as a solvent for drug substances, an awareness of its intrinsic activity in any such evaluations is essential.

A modified transmembrane migration method for evaluating the spermicidal potency of some nonoxynol compounds.

The transmembrane migration technique, a simple method in-vitro for quantitatively assessing the effects of a drug on human sperm motility, has been evaluated. The original method has been modified to include a preincubation step, and the incubation time has been reduced to 90 min. In semen samples possessing sperm concentrations of less than 75 x 12(6) spermatozoa mL-1 the volume of the lower reservoir has been reduced to 1 mL. This modified method has been used to compare the spermicidal potency of the widely employed non-ionic surfactant nonoxynol-9, with nonoxynol-5 and nonoxynol-15 (containing, respectively, fewer and more ethylene oxide units per molecule). The rank order of spermicidal potency of the compounds evaluated was nonoxynol-9 = nonoxynol-5 greater than nonoxynol-15.

PIP: A modified transmembrane migration method was used to evaluate the spermicidal potency of nonoxynol-9 and to make comparisons with 2 other water-soluble nonoxynols -- nonoxynol-5 and nonoxynol-15. The proportion of sperm which moves across the membrane from the upper to the lower chamber was assessed and expressed as the transmembrane migration ratio (TMR). Semen samples were collected by masturbation after at least 3 days' sexual abstinence, from a pool of 8 healthy volunteers (18-35 years). The characteristics noted of each sample included spermatozoa concentration and sperm motility, the percentage dead sperm being assessed using ethidium bromide, and fluorescence microscopy. The rank order of potency of the nonoxynols examined was nonoxynol-5=nonoxynol 9, which was greater than nonoxynol-15. The varying potency of the nonoxynols with respect to their IC50 values corresponds to the pertinent lipophilic nature of each compound. An observed increase in the IC50 was related to an increase in the side-chain lengths: 5915. This relationship indicates the importance of lipophilicity in the properties of these surfactants to diffuse into the sperm plasma membrane and disturb its conformation and semipermeable nature, preventing the occurrence of both sperm motility and ovum fertilization.

Comparative effects of (+)-propranolol and nonoxynol-9 on human sperm motility in-vitro.

Using the modified transmembrane migration method to measure sperm motility, it was shown that the surfactant nonoxynol-9 alone, was twice as potent as (+)-propranolol alone as a spermicidal agent. Addition of (+)-propranolol to nonoxynol-9 shifted the dose-response curve to the left of the curves for either component alone, and a surprising synergistic action was evident. These observations may form the basis for the development of a new advantageous topical contraceptive combination product.

PIP: Researchers from the Cambridge Research Institute in England conducted i vitro research on the effects of (+)-propranolol and nonoxynol-9, alone and in combination, on human sperm motility. They used modified transmembrane migration method of Hong et al since it allowed dose- response curves to be done on each semen sample from 7 donors. They also applied the PCONLIN computer program to more detailed data to define the shape of the curve more accurately. Nonoxynol-9 exhibited twice the potency of (+)-propranolol. Their drug concentrations at which 50% maximal inhibition occurred (IC50) were .156 mg mL-1 and .373 mg mL-1 respectively. Moreover, with the addition of 1-2.5 mM (+)- propranolol to nonoxynol-9, the dose-response curve moved to the left of the curves for either (+)-propranolol and nonoxynol-9 alone. Therefore the 2 agents exhibited a synergistic effect. Later they applied data from other experiments to a simple effect model and found a 3-10 fold increase in potency of nonoxynol-9 and 1 mM (+)-propranolol together compared to nonoxynol-9 alone. These results gave further credence to a synergistic action of the 2 drugs. The combination which resulted in the greatest inhibition of sperm motility (99.9%) included 1.6 mM nonoxynol-9 and 2.5 mM (+)-propranolol. Nevertheless they did not see a need to use 1 mM (+)-propranolol because the concentration of nonoxynol-9 causing total inhibition of sperm motility was similar. Therefore effective concentrations of each in a topical contraceptive agent can be .5 mg mL-1 nonoxynol-9 and .3 mg mL-1 (+)-propranolol (about 1 mM). This results in using about 1/10th of the nonoxynol-9 used in current contraceptive formulations which reduces the potential problems of sensitivity.

Beta-adrenoceptor antagonists and human sperm motility.

Several beta-adrenoceptor blocking agents have been evaluated for spermicidal activity using a transmembrane migration method. The rank order of potency of the active compounds was: penbutolol greater than (+)-propranolol greater than bufuralol greater than (-)-alprenolol greater than oxprenolol greater than metoprolol. Atenolol, pindolol, practolol, tolamolol were without activity. The observed potencies of spermicidal activity are believed to be unrelated to beta-blocking activities, and we have shown that whilst they are not predictable from lipid solubility or nonspecific membrane properties of the compound alone, both these aspects appear to play a role in this pharmacological activity.

Polyphloretin phosphate (PPP) antagonists of prostaglandin action also inhibit prostaglandin biosynthesis in-vitro.

Several polyphloretin phosphate (PPP) fractions (low mol. wt LC1259; high mol. wt LC1261; crude mixture, LC101) were confirmed in their established property as antagonists of the pharmacological actions of prostaglandins in a preparation of guinea-pig isolated ileum stimulated by prostaglandin (PG)E2. Further samples of the same material were then compared in-vitro with indomethacin in their ability to inhibit prostaglandin biosynthesis from arachidonic acid by a microsomal enzyme preparation. All three PPP fractions potently inhibited prostaglandin generation, with the rank order of potency LC1259 = LC101 = indomethacin greater than LC1261. The oral LD50 in mice was 25 mg kg-1 for indomethacin and greater than 1 g kg-1 for LC101. PPP fractions (especially LC101) may therefore have therapeutic potential as anti-inflammatory agents.

Seasonal variation in the response of adipose tissue to a serum fat-mobilizing factor may be explained in terms of reduced availability of fat cell receptors.

The lipolytic activity of serum obtained from fasted rats on rat isolated rat cells, exhibits seasonal trends. If it is assumed that the serum lipolytic agonist combines with receptors on fat cell membranes, and that the measured physiological response (glycerol release) is proportional to the number of such combinations, then the observed seasonal variation could be due to changes in serum concentration of lipolytic agonist, or the sensitivity of cells towards the same concentration of agonist (or both). On the basis of the observations described herein, it is proposed that the seasonal fluctuation in fat cell sensitivity is the result of some measure of altered availacbility of receptors for the lipolytic agonist. Possible changes in the concentration of circulating agonist, which were not investigated, may also play a role.

Computerized methods of neuropsychological assessment.

There is an increasing trend towards computerization in medicine, including cognition. Nine systems of assessment are reviewed in this article. A number of initiatives involving these systems are in their infancy, and the development of computerized testing is having an influence on their definition.

Prostaglandins and obesity.

In metabolic obesity energy in triglyceride stores is not readily accessible, and lipolysis to free fatty acid and glycerol seems to be somehow restrained. In the normal situation, there is a balance between a forward reaction via cyclic A.M.P. ending in lipolysis and a negative-feedback mechanism in which prostaglandins participate. In metabolic obesity there may be a biochemical error leading to overproduction of prostaglandins; as a result the forward reaction is overwhelmed and lipolysis does not take place. Since prostaglandin antagonists and inhibitors of prostaglandin synthesis are known, this hypothesis is not without therapeutic interest.

Characterization of the beta-adrenoceptor of the adipose cell of the rat.

The dose-response curves of the beta-adrenergic agonists isoprenaline (mixed beta 1 and beta 2), prenalterol (beta 1-selective), noradrenaline (more beta 1 than beta 2) and salbutamol (beta 2-selective) were studied on adipose cells of the rat, in vitro. The observed lipolytic potencies were in the order: isoprenaline greater than noradrenaline greater than salbutamol greater than prenalterol. The effects of beta-adrenergic antagonists betaxolol (beta 1-selective) propranolol (non-selective) and ICI 118551 (beta 2-selective) on lipolysis stimulated by the various beta-adrenergic agonists showed that in each case propranolol was the most potent blocking agent. These observations are not compatible with the concept that regulation of lipolysis in adipose tissue is mediated exclusively either by adrenergic receptors of the classical beta 1 type, or of the classical beta 2 type. We propose therefore, that this beta-adrenergic receptor, because of its non-compliance with the current classification system, be termed a 'beta-3' or beta-hybrid' adrenoceptor. Thus cardio-selective beta-adrenergic blocking agents, like betaxolol, may offer a hitherto unrecognized clinical advantage in obese patients undergoing anti-hypertensive therapy by offering a reduced impediment to hormone-induced utilization of calorie stores in adipose tissue.

Comparative studies of prostaglandin E2, phenylisopropyladenosine, clonidine and propranolol on lipolysis stimulated by methylisobutylxanthine and isoprenaline in hamster fat cells.

The antilipolytic effects of prostaglandin E2 (PGE2), phenylisopropyladenosine (PIA), clonidine (alpha-2 adrenoceptor agonist) and propranolol (beta- adrenoceptor antagonist) on lipolysis stimulated by methylisobutylxanthine (MIX) (2 x 10(-4)M) and isoprenaline (5 x 10(-7)M) were compared in hamster fat cells. PGE2, PIA and clonidine inhibited markedly lipolysis stimulated by MIX, and slightly that stimulated by isoprenaline. Whilst propranolol inhibited markedly lipolysis stimulated by isoprenaline and slightly that stimulated by MIX. These results indicate that the inhibitory activity of PGE2, adenosine and alpha-2-adrenergic receptor stimulation depends upon the presence of MIX, whereas that of the beta-adrenoceptor blockade does not.

Prostaglandins, alpha-adrenergic receptors and lipolysis in hamster fat cells.

In hamster fat cells, PGE2, PGI1 and clonidine (alpha 2-adrenoceptor agonist) were shown to inhibit markedly lipolysis stimulated by methylisobutylxanthine (MIX) (2 X 10(-4) M) and slightly that provoked by isoprenaline (5 X 10(-7) M). The similar inhibitory actions of the alpha 2-adrenoceptor agonist clonidine and PGE2 on lipolysis stimulated by MIX led us to examine possible reversal of the antilipolytic effect of PGE2 by yohimbine (alpha 2-adrenoceptor antagonist). However, the absence of an effect of yohimbine on PGE2, antilipolytic action indicated that the PGE2 receptor was different from the alpha 2-adrenoceptor of hamster fat cells. In these studies, PGE2 was shown to be a more potent antilipolytic agent that PGI2 (IC50 values 2 X 10(-9) and greater than 10(-4) M respectively).

Qualitative and quantitative effects of fenfluramine and tiflorex on food consumption in trained rats offered dietary choices.

The qualitative and quantitative anorectic effects of intraperitoneally-administered fenfluramine and tiflorex were studied in male rats trained during 10 d to a reversed dark/light cycle ('night' 1100-2300 h), and to a food choice (of either a protein-rich or protein-poor meal, or of a carbohydrate-rich or carbohydrate-poor meal) available only during the first 4 h of the dark cycle. On the first day of drug treatment (2.5 mg/kg or 5.0 mg/kg i.p., administered 0.5 h before the end of the light cycle) both fenfluramine and tiflorex provoked a dose-dependent reduction in the consumption of the protein-rich diet, and a reduction in the consumption of the protein-poor diet. In the carbohydrate study, on the first day of drug treatment, there was a dose-related reduction in the consumption of the carbohydrate-rich regime, and marked reduction by both drugs of consumption of the carbohydrate-poor regime (except in the case of the lower dose of fenfluramine, 2.5 mg/kg). There was generally a rapid development of tolerance to the anorectic effects of the anti-obesity drugs, although this was delayed somewhat in the case of tiflorex. These results suggest that drugs like fenfluramine and tiflorex, which enhance central serotoninergic transmission, reduce caloric intake whether the calories are presented as protein or carbohydrate, compatible with the anti-obesity actions of these agents in omnivorous man.

Regulation of adipose tissue metabolism via adrenergic mechanisms: actions of alpha-adrenoceptor agonists on lipolysis in hamster fat cells.

The effects of two alpha-1-adrenergic agonists (phenylephrine, cirazoline) and two alpha-2-adrenergic agonists (clonidine, guanabenz) were investigated on lipolysis stimulated by noradrenaline, isoprenaline, dibutyryl cyclic AMP (dbc AMP) or methylisobutylxanthine (MIX) in hamster adipose cells, in vitro. Phenylephrine enhanced basal lipolysis and that stimulated by dbc AMP and MIX, whereas cirazoline showed no measurable effects. Clonidine produced a slight but significant decrease of basal lipolysis. Both clonidine and guanabenz (from 10(-7) M) provoked a similar and clear inhibition (50-60 per cent) of MIX-stimulated lipolysis, compatible with the view that the alpha-adrenergic receptor of the hamster white fat cell is of the alpha-2 type. Noradrenaline-stimulated lipolysis was unaffect and isoprenaline-stimulated lipolysis moderately inhibited, by clonidine and guanabenz. Since the beta-adrenergic lipolytic effects of noradrenaline are known to be associated with a simultaneous alpha-adrenergic inhibitory stimulus, and one possible explanation of the but moderate inhibition of isoprenaline-induced lipolysis could be simultaneous beta-adrenergic stimulation (resulting in lipolysis), and alpha-adrenergic stimulation (inhibition of lipolysis), as with noradrenaline, the nature of isoprenaline as a pure beta-adrenergic agonist requires clarification.

Anti-lipolytic action of prazosin in hamster fat cells.

Using isolated white fat cells of the hamster, we studied the possible inhibitory effects on lipolysis of the preferentially selective alpha-1-adrenoceptor blocking and anti-hypertensive agent prazosin. Parallel studies were undertaken with the preferentially alpha-2-adrenoceptor blocking agent yohimbine. Neither of the compounds had any significant effects on basal (non-stimulated) lipolysis. When lipolysis was stimulated by noradrenaline or isoprenaline, however, prazosin showed significant anti-lipolytic effects from a concentration of 10(-6) M. Lipolysis stimulated by methylisobutylxanthine was antagonized by prazosin only at a concentration of 10(-4) M, and dibutyrylcyclic adenosine 3'5' monophosphate-stimulated lipolysis was not inhibited at all. It is suggested that prazosin inhibits lipolysis by preventing adenyl cyclase activation by an indirect mechanism not identifiable with an alpha-2-adrenergic function but associated possibly with the formation of a local inhibitory mediator. These observations are compatible with a reduction of the outflow of free fatty acids from adipose stores in patients undergoing prazosin therapy and thus a breaking of the "vicious circle" leading to elevated plasma lipoproteins and may explain, in part at least, the reduction in plasma lipid levels observed during prazosin treatment in man.

Comparative effects of RX 781094, mianserin, yohimbine, rauwolscine and prazosin in reversing clonidine inhibition of MIX-stimulated lipolysis in hamster isolated white fat cells.

Different alpha-adrenergic receptor antagonists: RX 781094, yohimbine and rauwolscine (selective alpha 2-adrenergic antagonists); mianserin (tetracyclic anti-depressant, antagonist at alpha 2-presynaptic autoreceptors) and prazosin (selective alpha 1-adrenergic antagonist) were used at concentrations ranging from 10(-7) to 10(-4) M, to reverse clonidine inhibition of MIX-stimulated lipolysis in the hamster fat cell. In this adipose tissue (like human but unlike rat) there co-exist prolipolytic beta-adrenoceptors and antilipolytic alpha-adrenoceptors. Although no effects were observed with prazosin, RX 781094 was ten times more potent than yohimbine or its isomer rauwolscine in reversing clonidine inhibition of the MIX-stimulated lipolysis. Mianserin was an effective blocker only from a concentration of 10(-4) M, consistent with its relative lack of specificity for alpha 2-adrenoceptors cited elsewhere. Overall these results confirm the utility of this model for testing compounds presumed to act at alpha 2-adrenoceptors.