[Quantitative parameters of drug-receptor interactions and receptor reserves. I. Models I-3-R and I-3-S]. / Uvahy o kvantitativních parametrech farmakon-receptorové interakce a o receptorové rezerve. I: Modely I-3-R a I-3-S.

On a simple model of a reaction-chain from the pharmacon-receptor complex (RA) formation the formation of a ternal complex with substrate (S) and its activation to a product the authors investigate the meaning of the term "affinity" and "internal activity" and the behaviour of the model with substrate and velocity limits at various sites. If K1 = dissociation constant of the complex RA, K2 = dissociation constant of the complex RAS, then at S1 much greater than R1 (i.e. without receptor reserve) the obtained value ED50 described usually as KA = K1K2: (K2 + [St]) and if K1 is constant it is variable in relation to the substrate concentration. In this way continuous differences of apparent affinity of the same substance to the same receptor under different biological conditions are possible. At R1 much greater than St (i.e. with receptor reserve) apparent KA = K1K2: (K2 + [Rt]) and thus K1 can be assessed by blocking receptors with an irreversible antagonist. The authors describe the calculation and very simple graphical method for assessment of basic parameters using an irreversible antagonist. After linear plotting of ED50 (= KA) on the chi-axis and maximal effects on the gamma-axis and after extrapolation of the connecting line of the two points (results without antagonist and results with antagonist) the intersecting point with the chi-axis gives value K1. This makes it possible, among others, to assess also the "intrinsic efficacy" of the substance, the ratio of receptors eliminated by the antagonist, prediction of the behaviour of typical curves during gradual elimination of receptors by a blocker and to assess possibly also the half-life of binding of the irreversible blocker with the receptor.

[2 different populations of adrenergic alpha receptors in the splenic capsule in rats]. / Dve rozdílné populace adrenergních alpha-receptoru v capsula linenalis krysy.

Using the contraction of the smooth muscle of rat spleen capsula, studies of the character of the alpha-adrenergic receptors involved in this reaction where performed in vitro. By computer analysis, the flat dose-response curve of noradrenaline shows the presence of two different subpopulations of alpha-receptors, each being responsible for approximately 50% of the maximal overall contraction. The two subpopulations differ roughly one hundred times by their apparent affinity for noradrenaline (pD2 cca 8 and pD2 cca 6). Solely the high-affinity component is competitively antagonized by the alpha 1-blocker prazosin, needs necessarily extracellular Ca2+ for evoking the contraction and is desensitized by previous contact with a high concentration of noradrenaline. Neither of the two components is influenced in its action by calcium-channel blockers nifedipine and verapamil. The needed exogenous calcium thus enters the cell by different routes than by those affected by the mentioned blockers.

[Closer characterization of alpha-receptor subpopulations in the smooth muscle of the splenic capsule in rats]. / Pokus o blizsí charakteristiku alpha-receptorových supopulací v hladkém svalu krysí capsula lienalis.

Two different subpopulations of adrenergic alpha-receptors -- "high-affinity" (pD2 less than 8) and "low-affinity" (pD2 less than 6) ones -- are involved in alpha-adrenergic contractions of the rat spleen muscle evoked by noradrenaline in vitro. Only the "high-affinity" subpopulation is agonistically influenced by the alpha 1-mimetic phenylephrine (pD2 cca 6, pKA cca 4.8), antagonistically influenced by prazosin (pA2 cca 9); it has a substantial receptor reserve for noradrenaline and a somewhat lower reserve for phenylephrine, it can be desensitized by these mimetics and it requires the presence of exogenous Ca2+ in the medium for evoking the contraction. -- Only the "low-affinity" subpopulation is agonistically influenced by the alpha 2-mimetic UK-14304. Both subpopulations are activated by noradrenaline and competitively blocked by yohimbine. The effect of neither subpopulation is influenced by nifedipine. According to the new classification (11) the "high-affinity" receptors can be described as alpha 1A, the "low-affinity" ones as alpha 2A. Contrarily to current data, the peculiar feature of this second subpopulation is a low affinity for yohimbine (pA2 less than 7) and a certain extent of blockability by phenoxybenzamine.

Morphine and dynorphins in lipid mobilization in rats in vitro.

In vitro lipid-mobilizing activities of morphine (MO) and dynorphin-(1-10) amide (DYA) were compared using adipocytes of young adult rats (body weight 170-200 g, age 50-60 days, diameter of adipocytes 54 +/- 1.12 microns) and of 20-month-old rats (body weight 500-690 g, diameter of adipocytes 99.5 +/- 3.0 microns). The adipokinetic activities of adenosine deaminase (ADA) and dynorphin-(1-13) (DY) were also tested. In the experiments 0.16 units of ADA did not influence basal or stimulated lipolysis, whereas DY exerted a slight but statistically significant adipokinetic effect. In three experimental series the EC50 of MO ranged between 0.89 and 14.60 mumol l-1, the EC50 of DYA in young rats was estimated as 0.8 mumol l-1 and in old animals, 1.3 mumol l-1. Statistically significant differences in the lipid-mobilizing potency between DYA and MO could be observed only in one experimental series in young rats. Expressed in percent of maximum lipolysis induced by isoprenaline, the maximum lipolytic response to DYA in young animals was significantly lower (Emax 26.1 +/- 1.9) when compared to the maximum effect of MO (Emax 76.3 +/- 8.5 and 68.9 +/- 3.5, respectively). Adipocytes of old rats seemed to be more sensitive not only against MO but also against DYA. When studying lipolysis no signs of competitive dualism could be observed in the interaction between MO and DYA. The possibility of two or more independent lipid-mobilizing mechanisms in the effect of the two opioids compared cannot be excluded.

Graphical orientation procedure for the estimation of Kd and Bmax values of ligand binding to two receptor subpopulations.

Within a few minutes, the procedure described here provides to the investigator, performing binding studies, information about Kd and Bmax values of two possibly present receptor subpopulations. The procedure is based on the comparison of the experimental curve with values of ligand binding to only one receptor population at 4 defined levels of radioligand binding. The binding parameters are derived from comparison of 4 horizontal deviations at these levels with tabulated deviations. Kd and Bmax values can be further confirmed by comparison of the experimental data with the simulated binding curve obtained by entering the binding parameters found in the formula for ligand binding to two receptor subpopulations. The practical use of this procedure was demonstrated both for simulated and for experimental data; it was confirmed by comparison with binding parameters obtained by computer programs used for the evaluation of receptor heterogeneity.

A simple and rapid orientation procedure for the estimation of affinities (1/Kd) and maximum binding capacities (Bmax) of ligand binding to two receptor subpopulations.

This paper describes a simple and rapid procedure for the estimation of specific parameters (dissociation constants, Kd and maximum binding capacities, Bmax) of ligand binding to two receptor subpopulations. This procedure provides, in a few minutes, the investigator, performing the actual binding studies, the necessary information about receptor heterogeneity, enabling the investigator to plan further experiments. The procedure is based on the graphical comparison of experimental binding data (ligand binding to one or two receptor subpopulations) with the theoretical values of ligand binding to one receptor population at four levels). The values of Kd and Bmax for high- and low-affinity receptors are derived from 4 horizontal deviations of experimental data from a theoretical data plot at these levels by their comparison with tabulated deviations. The correctness of the estimated parameters can be confirmed by the comparison of experimental data with those simulated on the basis of applying the values of Kd and Bmax found in the formula for ligand binding to two receptor subpopulations. The practical applicability of this procedure was demonstrated both on simulated and experimental data, and confirmed by the well known computer programs for evaluating receptor heterogeneity, namely "LIGAND" and "Affinity spectra".

Inhibitory effects of pentacaine and some related local anaesthetics on rat hepatic adenylate cyclase.

In the present study effects of a new local anaesthetics, pentacaine (trans-2-pyrolidinocyclohexylester of 3-pentyloxyphenylcarbamic acid), and of some chemically related compounds on rat hepatic adenylate cyclase activity were studied under various experimental conditions. As compared with tetracaine, the local anaesthetics tested showed stronger inhibitory effects, regardless of the type of stimulating agents used to activate adenylate cyclase. The most potent effect was observed with pentacaine. Its inhibitory effects on glucagon, guanylylimidodiphosphate (Gpp/NH/p), sodium fluoride or forskolin stimulated activity suggest that it may directly act on the catalytic unit of adenylate cyclase. The same conclusion can be drawn based on its inhibitory effects on adenylate cyclase, regardless ATP concentrations used as the enzyme substrate, and on octylpyranoside solubilized enzyme activated by preincubation of the enzyme preparation with Gpp/NH/p. Structure-activity studies have suggested that the pentacaine molecule as a whole and none of its parts alone or its analogs are responsible for the inhibitory effect. However, the inhibitory effects of these compounds on the rat adenylate cyclase activity do not correlate with their local anaesthetic properties. The possibility of using adenylate cyclase inhibitors to decrease cyclic AMP production under pathological conditions, like in cholera, known to be due to a high adenylate cyclase activity, is discussed.

Effect of oestradiol on catecholamine-stimulated lipolysis.

The authors studied the effect of a single in vivo dose of oestradiol (OE) on adrenergic lipolysis in the epididymal adipose tissue of adult and juvenile male rats, and the effect of OE on plasma free fatty acids (FFA), cholesterol and beta-lipoprotein levels at various intervals after its administration. It was found that OE injected 24 h beforehand in vivo (s.c.), in doses of 100 and 200 micrograms X kg-1 body weight, significantly potentiated the lipid-mobilizing action of the catecholamines noradrenaline (NOR) and isoprenaline (ISO) in adult rats (the action of ISO was potentiated more intensively); in addition, the adipose tissue became more sensitive to the action of NOR, but not of ISO. Raising the dose of OE to 400 micrograms X kg-1 did not enhance the potentiation of the lipolytic action of the catecholamines any further; on the contrary, the lipid mobilizing effect of the catecholamines was potentiated less than after half this dose. Following the s.c. injection of an oily OE solution, the lipolytic effect was potentiated after more than 7 h; the potentiation was strongest after 12 h, but only as far as the maximum attainable degree of lipolysis was concerned. Potentiation of adrenergic lipolysis was found only in adult male rats. In male rats weighing 130-150 g the lipolytic effect of catecholamines (in mumol/g adipose tissue) was significantly greater than in adult animals and the pre-administration of OE did not potentiate adrenergic lipolysis any further. Determination of plasma FFA, cholesterol and beta-lipoprotein levels 1, 2, 4 and 6 hours after the s.c. injection of OE showed only nonsignificant changes (an increase in FFA and a decrease in cholesterol). The authors consider it important to distinguish between the effect of OE on catecholamine-stimulated lipolysis in depot adipose tissue and its effect on lipid metabolism. In their opinion, the dose-dependent effect of OE on muscular and metabolic adrenergic reactions could be one of the factors co-reversible for certain side reactions to steroid contraceptives.

Relationship between morphine and isoprenaline-induced lipolysis. Quantitative aspects.

In order to estimate the relationship of the adipokinetic effect of morphine (MO) to adrenergic-mediated lipolysis the interaction between MO and isoprenaline (ISO) was studied in epididymal adipose tissue of rats and perirenal adipose tissue of rabbits. Two preparations of adipose tissue (AT)-trimmed tissue and fat cells (FC)-were used. Increment of non-esterified fatty acids (NEFA) release into the medium during 90 min of incubation was the measure of lipolysis. In rats, especially in FC, MO stimulated NEFA release up to 900% of basic values and also in rabbits the lipid-mobilizing action of MO was found as high as 320% of the non-stimulated level. The net increase due to MO expressed as % of maximum lipolysis induced by ISO was fluctuating between 30-50% except rat FC, where MO reached almost the level of ISO maximum effect. The calculated pD2 values of MO were in FC of both species 5.8, in trimmed AT 5.1 (rats) and 5.3 (rabbits). Different concentrations of MO (1-100 mumol/l) did not significantly alter the concentration-response adipokinetic curve of ISO. Naloxone in concentrations of 1 and 10 mumol/l did not exert any antagonistic action in MO-induced lipolysis. The MO effect is most probably not mediated via adrenergic receptors; the possibility of opiate receptor involvement has not been excluded. Pronounced species differences between rats and rabbits in sensitivity to the lipolytic morphine effect were observed.

Adenylate cyclase activity in crude plasma membranes of subcutaneous adipocytes during ontogenetic development of rats.

In order to explain the differences in the hormone stimulated lipolysis during ontogenic development of rats, the activity of adenylate cyclase was determined in crude plasma membranes of subcutaneous adipocytes of 5, 14, 21 and 45 to 55-day-old animals. Stimulatory effects of nonhormonal and hormonal agents were expressed as the increment in percentage of basal values which were not significantly changed in the age groups studied. The highest stimulatory effect was observed after sodium fluoride in 14 and 21-day-old rats. Guanylylimidodiphosphate and GTP revealed the lowest stimulatory effects in adult animals (greater than 45-day-old). The beta-adrenergic agent isoproterenol revealed the highest stimulatory effect in the 5 and 45-day-old group while in the preparation from 14-day-old rats the adenylate cyclase activity was significantly lower. On the other hand, tetracosactide (beta 1-24-corticotropin) revealed the smallest stimulatory effect on the preparation from 5-day-old rats; its stimulatory effect steadily increased and reached the highest value in adenylate cyclase preparations from adult animals. It can be concluded that the adenylate cyclase system in subcutaneous adipocytes is already basically mature at early ontogenic stages of development in rats. Nevertheless, the explanation for the small variations of the enzyme activity in different age groups requires further study.

Some adrenergic beta-blocking agents affecting lipolysis in human adipose tissue in vitro.

Antagonistic actions of beta-blocking drugs on isoproterenol-induced lipolysis were studied in human omental adipose tissue. Competitive interaciton characterized by the following pA2 values was found: propranolol 8.7; trimepranol 8.7; practolol 7.1; H 35/25 6.0. The plot of pA2 values of these drugs for human versus rat adipose tissue is linear with slope 2.0 indicating a higher differentiation of beta-antagonist actions in human than in rat adipose tissue.

Some adrenomimetic drugs affecting lipolysis in human adipose tissue in vitro.

In human adipose tissue in vitro, dose-response curves were followed for different adrenomimetics releasing free fatty acids and glycerol into an albumin-containing medium. Phenylephrine, salbutamol and t-butyl-norsynephrine produced lipolytic actions whose maximum did not exceed 20% of the maximal isoproterenol lipolysis. Phentolamine (1 x 10(-5) M) did not potentiate the effect of any of these drugs. When using salbutamol or t-butyl-norsynephrine for antagonizing isoproterenol actions, typical antagonism was found. The parallel shift of the isoproterenol curves showed pA2 values 5.8 for salbutamol and 5.7 for t-butyl-norsynephrine. Comparison of human and rat adipose tissue reactivity shows identical affinities of the drugs studied in both tissues, but related to isoproterenol, diminished intrinsic activities of salbutamol and t-butyl-norsynephrine in human adipose tissue.

7-oxa-13-prostynoic acid and polyphloretin phosphate as non-specific antagonists of the stimulatory effects of different agents on adenylate cyclase from various tissues.

7-oxa-13-prostynoic acid (OPA) and polyphloretin phosphate (PPP) are believed to act as specific antagonists of prostaglandin action. In order to estimate their specificity, the inhibitory effects of these drugs were tested on the activity of adenylate cyclase from several tissues which were stimulated by prostaglandins and several other compounds. In adenylate cyclase preparation from L-fibroblasts both OPA (0.15-1.5 MM) and PPP (0.01-1.0 MG/ML) antagonized not only the stimulatory effects of PGE but also the stimulatory effects of sodium fluoride and increased enzyme activity due to the previous treatment of cell cultures by cholera toxin. Both OPA and PPP produced a dose dependent depression of adenylate cyclase activity to zero values both under basal conditions and after stimulation by sodium fluoride and various hormones in all preparations studied, including rat liver, heart, brain, epididymal adipose tissue, small intestine, renal cortex and renal medulla. The present results indicate that both prostaglandin antagonists may, in higher concentrations, act as nonspecific inhibitors of the catalytic unit of adenylate cyclase rather than specific antagonists of the prostaglandin effects on adenylate cyclase.

The absence of stimulation of lipolysis by papaverine, a strong inhibitor of phosphodiesterase.

Quantitative studies of the action of theophylline and papaverine were performed in rat epididymal fat pads, both on the lipolytic effect and on the activity of phosphodiesterase, adenylate cyclase and protein kinase. Papaverine, a stronger inhibitor of phosphodiesterase than theophylline, did not produce lipolysis. The maximum lipolytic effect (glycerol release) of theophylline was much higher than that of epinephrine and nearly approached the effect exerted by dibutyryl cyclic AMP. While theophylline potentiated or was without any effect on lipolysis produced by epinephrine and dibutyryl cyclic AMP, papaverine at concentration 10- minus 3 M reduced the effect of both drugs as well as of theophylline by 90 per cent. These concentrations of papaverine also strongly inhibited the activity of adenylate cyclase. Neither papaverine nor theophylline prevented the activation of protein kinase by cyclic AMP. The data suggest that the lack of a lipolytic effect of papaverine migth be caused by a combination of its inhibitory effect on adenylate cyclase and direct inhibition of activation of triglyceride lipase.

Adrenergic lipolysis in human adipose tissue in vitro.

In human adipose tissue in vitro, dose-response curves of lipolytic agents in releasing free fatty acids and glycerol into an albumine-containing medium were followed. Norepinephrine and adrenaline produced about 20% of the maximal effect of isoproterenol, isopropylnorsynephrine about 40%, theophylline more than 100%. Found pD2 values were approximately 7.4 for isoproterenol and norepinephrine, approximately 5.9 for isopropylnorsynephrine. Phentolamine (1 times 10-5 M) elevated the maximal norepinephrine effects up to the isoproterenol maxima. Phenoxybenzamine (1 times 10-5 M) had significantly lower potentiating effects. Phenylephrine depressed lipolytic actions of isoproterenol (1 times 10-6 M) showing the same pI2 value 3.0 as well in human as in rat adipose tissue and exerted per se weak lipolytic effects also. It is concluded neither potentiating actions of alpha-blockers nor depressing effects of phenylephrine in human adipose tissue seem related to affecting adrenergic alpha-receptors. The existence of antilipolytic actions of adrenergic alpha-receptors is questioned.