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The binding of the alpha 2-agonist [3H]UK 14304 on Wistar rat adipocyte membranes was separated in two distinct components: one was displaceable by adrenaline or other alpha 2-adrenergic agents and possessed the characteristics of alpha 2-adrenoceptors while the other, non-adrenergic in nature, was only recognized by some imidazoline derivatives [3H]idazoxan binding shared the same characteristics. The non-adrenergic sites labeled by both radioligands are similar to those described for [3H]idazoxan on other tissues such as brain cortex, smooth muscle and kidney. Even though they were about 10-fold more numerous than the true alpha 2-adrenoceptors, the non-adrenergic binding sites were not directly involved in the antilipolytic action of UK 14304 since alpha 2-antagonists devoid of interaction with these sites (yohimbine, phentolamine) totally blocked the UK 14304 effect. However, the existence of such a type of site impairs direct quantification of alpha 2-adrenoceptors in rat adipocytes. The use of [3H]RX 821002 (2-(2-methoxy-1,4-benzodioxan-2yl)imidazoline) allowed an accurate quantification of rat adipocyte alpha 2-adrenoceptors (Bmax = 35 +/- 2 fmol/mg protein, Kd = 2.6 +/- 0.6 nM) since it did not interact with non-adrenergic binding sites and exhibited the highest alpha 2-blocking properties among the various alpha 2-antagonists tested. [3H]RX 821002 binding analysis revealed that alpha 2-adrenoceptors are, on rat adipocytes; (i) less numerous than in other species well known for their alpha 2-adrenergic inhibitory regulation of lipolysis (human, hamster, rabbit); (ii) slightly different in nature from the receptors of these species since they had weaker affinity for clonidine and yohimbine; and however (iii) not of the typical alpha 2-B subtype since the affinity of prazosin was lower than that of oxymetazoline in displacing [3H]RX 821002 or [3H]yohimbine binding.

1. The binding of a new alpha 2-adrenoceptor antagonist, [3H]-RX821002 (2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline), was investigated in human platelet membranes and compared with [3H]-yohimbine binding parameters. 2. Analysis of kinetic data revealed association and dissociation time courses consistent with a simple biomolecular reaction. Saturation isotherms showed that [3H]-RX821002 labelled a higher total number of alpha 2-binding sites (224 +/- 31 vs 168 +/- 24 fmol mg-1 protein) than [3H]-yohimbine and with higher affinity (Kd: 0.92 +/- 0.06 vs 1.51 +/- 0.08 nM). Moreover [3H]-RX821002 exhibited a lower percentage of nonspecific binding 3. The difference in total binding is due to a better labelling of the alpha 2-adrenoceptors in the low affinity state by [3H]-RX821002 since the labelled receptors number in high affinity state was identical with the two radioligands. 4. [3H]-RX821002 binding displayed a specificity similar to that obtained with [3H]-yohimbine. The potency of various compounds acting on adrenoceptors was: yohimbine greater than oxymetazoline greater than UK14304 greater than (-)-adrenaline greater than prazosin greater than or equal to (+)-adrenaline greater than isoprenaline. This order of potency is classical for an alpha 2A-adrenoceptor. 5. RX821002 is a more potent alpha 2-adrenoceptor antagonist than yohimbine on adrenaline-induced platelet aggregation. 6. These results indicate that [3H]-RX821002 is a suitable ligand for the identification of human platelet alpha 2-adrenoceptors.

[3H]Idazoxan binding to myometrial membranes was investigated in four groups of ewes under different steroid hormone status: control, estradiol-treated and progesterone plus estradiol-treated ovariectomized ewes and pregnant ewes. [3H]Idazoxan binding to myometrial membrane fractions was saturable, reversible, specific and of high affinity. The affinity did not vary significantly between the four groups of ewes (2.8 less than KD less than 4.7 nM). Maximal binding capacity varied significantly among groups: binding of [3H]idazoxan was lower in control ovariectomized ewes than in either estradiol or progestagen plus estrogen-treated ewes (maximal binding capacity, 73 +/- 11 fmol/mg of protein vs. 108 +/- 16 and 318 +/- 65, respectively). The highest [3H]idazoxan binding was measured in pregnant ewes (maximal binding capacity, 1302 +/- 256 fmol/mg of protein). Based on the saturation studies with accurate nonspecific binding definition (phentolamine vs. epinephrine), and on the relative order of potency for selected adrenergic drugs, it could be stated that the binding sites labeled by [3H]idazoxan in our study exhibited most of the alpha-2 adrenoceptor properties. Nevertheless, these alpha-2 adrenoceptors obviously differed from the standard alpha-2A-subtype based on Ki values obtained with yohimbine and prazosin in competition studies of [3H]idazoxan binding. The increase in the number of alpha-2 adrenoceptors under progesterone domination, and especially during gestation, supported the hypothesis that this adrenoceptor subtype could play a major role in the control of the motility pattern of the ovine pregnant uterus.

It is questioned whether alterations of the sympathetic nervous system can explain the specific increment of the adipocyte alpha 2-adrenoceptor observed during white adipose tissue (WAT) enlargement and fat cell size increment. The impact of a 6-hydroxydopamine-induced sympathectomy, validated by determination of the WAT norepinephrine content, was tested on isolated white fat cell alpha 2-adrenoceptor function and binding [( 3H]RX 821002 and [3H]UK 14304 binding). Chemical sympathectomy of standard adult hamsters, neither alters the alpha 2-adrenergic-dependent antilipolysis nor the adipocyte alpha 2-adrenergic binding. A slight hypersensitivity of the beta 1-adrenergic lipolytic response was observed, without modification in beta-adrenergic binding [( 125I]cyanopindolol binding). Compared with controls, caloric restriction (10 days) of adult hamsters induced a large decrease in adipocyte alpha 2-adrenoceptor (P less than 0.001) with a parallel decrement in the mean fat cell size (P less than 0.001) and alpha 2-adrenergic responsiveness while there was no significant variation of the WAT norepinephrine content (on a per pad basis). 6-Hydroxydopamine treatment of the restricted animals (from day 6 to day 9) induced a higher preservation of adipocyte alpha 2-adrenoceptor density (P less than 0.001), no change in alpha 2-adrenergic responsiveness, and higher preservation of mean fat cell size (P less than 0.05) than in the restricted only animals. No significant modification in the beta-adrenergic binding was observed whatever the conditions. It was concluded that the specific increment in the adipocyte alpha 2-adrenoceptor during fat mass enlargement was not directly dependent on sympathetic nervous system alterations. Nevertheless, the sympathetic-dependent mobilization of the fat stores, after induction of caloric restriction, can indirectly modulate the alpha 2-adrenoceptor density in the adipocyte by promoting changes in white fat cell size.

Previous results indicated that the rabbit could represent a suitable model for investigations on the functional role of alpha 2-adrenoceptors in fat cells, but the characterization of these receptors was not resolved yet. In the present report, imidazoline compounds were used to attempt a better definition of rabbit adipocyte alpha 2-receptivity by means of lipolysis and binding studies. Lipolysis data showed that UK14304 is a full alpha 2-adrenoceptor agonist promoting a strong antilipolysis in rabbit fat cells. Moreover, the methoxy derivative of idazoxan, RX821002, is a more potent antagonist of UK14304-induced antilipolysis than idazoxan or yohimbine. Whereas [3H]yohimbine failed to bind at rabbit adipocyte alpha 2-adrenoceptors, [3H]UK14304 and [3H]RX821002 are valuable tools to study this receptor. Analysis of binding data demonstrated that [3H]UK14304 labels the high-affinity-state receptor while [3H]RX821002 binds to the whole alpha 2-adrenergic population. Inhibition studies of [3H]RX821002 and [3H]UK14304 binding by various compounds confirmed the alpha 2-adrenergic nature of the sites labelled by both radioligands. The other alpha 2-adrenoceptor radioligand, [3H]idazoxan, labelled binding sites which are insensitive to catecholamines. Competition studies of [3H]idazoxan binding with imidazoline derivatives revealed structure-activity relationships different from those of alpha 2-adrenoceptors. The most striking observation is that substitutions in the 2-position of idazoxan markedly reduce the affinity for the non-adrenergic sites, whereas the alpha 2-potency is increased or unchanged.

The pharmacology of the alpha-2 adrenoceptor of the human adipocyte was improved by using some new alpha-2 antagonists from different chemical families (imidazolines, benzazepines and benzofuroquinolizines) in biological and binding assays. Moreover, investigations were also carried out to define the binding properties of a new imidazolinic antagonist, RX821002 [2-(2-methoxy-1,4-benzodioxan-2yl)-2-imidazoline], which could be a potential radioligand. [3H]RX821002 binding was very rapid and reversible. Saturation isotherms indicated that [3H]RX821002 labeled, with high affinity, a homogeneous population of noninteracting binding sites with a mean Kd of 0.98 +/- 0.05 nM (n = 6). The binding of [3H]RX821002 on the human fat cell alpha-2 adrenoceptor displayed a specificity which is strictly similar to that obtained with [3H]rauwolscine and which is classical for an alpha-2 A adrenoceptor. The binding parameters of [3H]RX821002 were compared with those obtained with the classical alpha-2 antagonist [3H]yohimbine. Analysis of the data indicate: 1) that [3H]RX821002 exhibited higher affinity; 2) that the nonspecific binding of [3H]RX821002 was very low; 3) that the total number of sites (maximum binding values) defined with [3H]RX821002 was significantly higher than that defined with [3H]yohimbine. This difference was not due to a specific preferential labeling of one of the two affinity states of the receptor, but suggested that [3H]yohimbine does not label the whole receptor population; 4) that [3H]RX821002 specific binding was less sensitive to magnesium chloride and GTP than [3H]yohimbine binding; and 5) that [3H]RX821002 can be used suitably for identification of alpha-2 adrenoceptors on the intact adipocyte.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of selected alpha-agonists and alpha-antagonists on theophylline-induces lipolysis were investigate in isolated hamster white fat cells, alpha 2-Agonists (tramazoline, clonidine) inhibited theophylline-induced lipolysis while in alpha 1-agonist (methoxamine) was without any effect. The inhibitory effect of alpha 2-agonists was suppressed by yohimbine (alpha 2-antagonist), whereas alpha 1-antagonists were inefficient. This result implies that the alpha-adrenergic receptor of hamster fat cells is of the alpha 2-type, although postsynaptically.

1. The small population of [3H]clonidine binding sites in rat fat cell membranes do not have the characteristics of typical alpha 2-adrenoceptors. 2. Clonidine (an alpha 2-adrenoceptor agonist) has no antilipolytic effect on rat fat cells stimulated by theophylline. 3. In contrast to the rat, [3H]clonidine labels an alpha 2-adrenoceptor in hamster fat cell membranes and clonidine exerts a strong antilipolytic effect on theophylline-stimulated lipolysis.

alpha-Adrenergic receptor which inhibit lipolysis in human and hamster fat cells are known to be of the alpha 2-type. By measuring the lipolytic activity on isolated fat cells and by studying the [3H]-yohimbine binding on the fat cell membranes, we tried to determine the alpha-adrenergic potency of the alpha-adrenolytic drug nicergoline. 1. Clonidine inhibits, in a concentration-dependent manner, the theophylline-stimulated lipolysis. This antilipolytic effect is suppressed by various alpha 2-adrenolytic agents but not by alpha 1-adrenolytic agents. Nicergoline is found to partially and competitively inhibit the antilipolytic effect of clonidine. 2. Specific binding of [3H]-yohimbine on fat cell membranes was a rapid and saturable process and was of high affinity (KD = 3.1 +/- 0.3 nM, Bmax = 615 +/- 90 f.mol./mg protein). 3. At low concentration, nicergoline displaced the specific binding of [3H]-yohimbine (EC50 = 0.440 microM). Compared to other alpha-adrenolytic agents, the affinity decreased in the following order: yohimbine greater than or equal to phentolamine greater than piperoxane greater than nicergoline much greater than prazosin. 4. The results show that the alpha-adrenergic antagonists capacity to suppress the antilipolytic effect of clonidine on isolated adipocytes is directly correlated with the ability to inhibit [3H]-yohimbine binding on fat cell membranes. Moreover we conclude that, on this model, nicergoline exhibits alpha 2-adrenolytic properties.

The dog platelet alpha-adrenergic receptor was characterized using [3H]clonidine and [3H]yohimbine. The binding of both radioligands was rapid and reversible at 25 degrees C; saturation and kinetic experiments revealed a single population of binding sites. The number of [3H]yohimbine sites was 2-3-fold higher than the number of [3H]clonidine sites as reported in other tissues containing alpha2-adrenoceptors. The various alpha-agonists and antagonists displaced [3H]clonidine and [3H]yohimbine with an order of potency indicating alpha2-adrenoceptor specificity. Neither (-)adrenaline nor clonidine infusions (0.5 micrograms/min/kg during 3 hr) modified the number of [3H]yohimbine and [3H]clonidine sites or the affinity of the ligands for the alpha2-sites of the dog platelet. Oral administration of clonidine (3 X 150 micrograms/day) did not alter the binding parameters of either ligand.

It is well known that both the sympathetic nervous system and several hormones control adipose tissue lipolysis. However, the relative importance of these different lipolytic agents may vary according to the animal species and according to the adipose territories in a same species. The action of noradrenaline administration on tibial intraosseous lipolysis was compared with the effects of splanchnic nerve and lumbar sympathetic chain stimulation in chloralose-anaesthetized dogs. The osseous nutrient artery was infused through the homolateral femoral artery in order to check constant blood flow. The levels of free fatty acids (FFA) were compared in femoral artery blood and in the osseous nutrient vein. Noradrenaline was injected in the infusion line. Lumbar sympathetic chain and splanchnic nerve were stimulated at frequencies of 3-10 Hz and 5-10 Hz respectively. Noradrenaline (at 411 p mol/min i.e. a dose three times higher than that required for adrenaline) induced FFA mobilization from tibial adipose bone marrow. Splanchnic nerve stimulation elicited an increase in the FFA levels in the nutrient vein whereas lumbar sympathetic chain stimulation had no remained without any effect. It is concluded that circulating adrenaline seems more important than noradrenaline released by sympathetic nerve endings in the control of activity of tibial bone marrow adipocytes.

Guanine nucleotides and sodium were found to reduce the affinity of adrenaline for the alpha 2-adrenoceptor sites labelled by [3H]yohimbine in human fat cell membranes. Surprisingly, the negative regulation by these agents was far weaker when clonidine, the alpha 2-agonist prototype was used. The results are discussed in relation to the weaker or the lack of adenylate cyclase inhibition promoted by clonidine and indicate that the formation of the lower affinity state of the alpha-receptor is essential to the alpha 2-adrenoceptor-mediated adenylate cyclase inhibition.

Investigations were carried out to demonstrate the function and the possible advantage of the interplay between beta 1 and alpha 2 adrenoceptor sites in the regulation of human subcutaneous fat-cell lipolysis. alpha 2 and beta adrenoceptor binding studies were conducted with antagonist radioligands and revealed that alpha 2-adrenoceptors ([3H]yohimbine and [3H]rauwolscine binding sites) are more numerous than beta 1-adrenoceptors ([3H]dihydroalprenolol and [3H]CGP-12177 binding sites) in human fat-cell membranes. Physiological agonists epinephrine and norepinephrine competed with [3H]-ligand sites with a higher affinity for alpha 2 sites than for beta 1 sites. Epinephrine exhibited a higher affinity than norepinephrine for the alpha 2 sites; the two amines had the same affinity for beta 1 sites. In lipolysis studies conducted in the absence of adenosine deaminase the beta lipolytic action of the biological amines predominated; after alpha 2-adrenoceptor blockade by yohimbine or idazoxan, the amines exhibited an intrinsic activity similar to that of isoproterenol. When adenosine was prevented from accumulating in the incubation medium by inclusion of adenosine deaminase, low concentrations of epinephrine and norepinephrine preferentially exerted an antilipolytic action. We conclude that: he lipolytic response in abdominal human subcutaneous fat cells to physiological amines results from the interplay between beta 1-and alpha 2-adrenoceptor stimulation; alpha 2 adrenoceptors, with their higher number and higher affinity for the physiological amines, and the adrenoceptor population involved at the lowest (i.e. physiological) concentrations of the amines.(ABSTRACT TRUNCATED AT 250 WORDS)

A study was designed to assess whether a period of physical exercise (100 min; 60% of maximal aerobic power), known to promote sympathetic nervous system activation, had a functional impact on human fat cell responsiveness to catecholamines and beta-agonists. The lipolytic activity of isolated sc fat cells was measured before and after a period of exercise in trained and sedentary women with a similar body mass index values [20.4 +/- 0.5 (+/- SE) and 21.3 +/- 0.5 kg/m2] and an equivalent mean fat cell volume (0.216 +/- 0.022 vs. 0.278 pm 0.026 nL). Before exercise, compared with those of sedentary women, the adipocytes of trained women had greater lipolytic responsiveness to catecholamines and beta-agonists; moreover, dibutyryl cAMP had greater lipolytic efficiency on the adipocytes of the trained women. These results suggest that the differences in responsiveness of the adipocytes from sedentary and trained women are due to functional differences at a postreceptor level of the lipolytic cascade. The lipolytic (beta-adrenergic) and antilipolytic (alpha 2-adrenergic) responses of the adipocytes from both groups did not change after the period of physical activity. These results demonstrate, whatever the physical condition of the subjects, that desensitization of beta- and alpha 2-adrenergic responsiveness does not occur after a period of intense physical activity which promotes physiological activation of the sympathetic nervous system.

Lipolytic activity of human isolated fat cells from different fat deposits was studied. The purpose of the present investigations was to determine the epinephrine responsiveness, with regard to alpha- and beta-adrenergic receptor site activity, of omental and subcutaneous adipocytes (abdominal or from the lateral part of the thigh). Adipocytes were obtained from normal subjects or from obese subjects on iso- or hypocaloric diets. The lipolytic effect of epinephrine varied according to the fat deposits, while the beta-lipolytic effect of isoproterenol was more stable (Fig. 1). We explored the possible involvement of adrenergic alpha-receptors, in order to explain these results. The potentiating action of phentolamine on epinephrine-induced lipolysis, and the antilipolytic effect of alpha-agonists on basal or theophylline--induced lipolysis, were found to be a good indication of alpha-adrenergic activity. The alpha-adrenergic antilipolytic effect was most prominent in adipose tissue from the lateral part of the thigh, and less noticeable in omental adipocytes. In conclusion, the inability of epinephrine to induce lipolysis, and the epinephrine-induced inhibition of lipolysis observed when the basal rate of FFA release was spontaneously increased in subcutaneous fat-cells of the thigh, could be explained by an increased alpha adrenergic responsiveness (Fig. 2). Moreover, various alpha-adrenergic agonists (phenylephrine, noradrenaline and adrenaline) showed a clear inhibiting effect on theophylline-stimulated adipocytes from the thigh. The pharmacological study of the antilipolytic effect of epinephrine on theophylline-induced lipolysis showed that the inhibition was linked to a specific stimulation of the alpha-receptors of the subcutaneous adipocytes (Fig. 4). From the different sets of experiments, it is shown that the modifications in the lipolytic effect of epinephrine on adipocytes of different areas could be explained by the occurrence of a variable alpha-adrenergic effect initiated by catecholamine. Furthermore, theophylline stimulation of lipolysis provides an accurate system to investigate the alpha-inhibiting effect of catecholamines. Our study was completed by the investigation of the lipolytic activity of subcutaneous fat cells from obese subjects submitted to a hypocaloric diet (800-1 000 Cal/day). An increased alpha-inhibitory effect of epinephrine was shown on the increased basal lipolytic activity observed in the fat cells of obese subjects on a hypocaloric diet (Fig. 5); a similar effect was observed when these adipocytes were stimulated by theophylline. To conclude, these investigations allow the alpha-adrenergic effect to be considered as a regulator mechanism of the in vitro lipolytic activity in human adipose tissue, since the antilipolytic effect is operative whenever the basal rate of lipolysis is increased (spontaneously, after caloric restriction, or with a lipolytic agent such as theophylline).

The present review summarizes recent data on fat cell adrenoceptors with the aim of clarifying the role played by catecholamines in the regulation of adipocyte metabolism. Part of the review is focused on the possible interest of animal models for the study of catecholamine-mediated effects in human fat cells. It is now clearly demonstrated that human, hamster, dog and rabbit fat cells possess three basic types of adrenoceptor: the beta 1-, alpha 2- and alpha 1-adrenoceptors identified in biological assays or binding studies with selected radioligands. The rat is an exception in the species commonly studied as catecholamines exert an exclusive lipolytic effect through beta-adrenoceptor stimulation, there are no alpha 2-adrenoceptors in rat white fat cells although an alpha 1-adrenoceptor does exist. In human fat cells, physiological amines are lipolytic or antilipolytic. Binding studies have revealed that alpha 2-adrenoceptors are three to four times more numerous than beta 1-adrenoceptors. Moreover physiological amines, in particular epinephrine, have a higher affinity for alpha 2-sites than for beta 1-sites. Dose-response studies of the effect of epinephrine on adenosine-deaminase or isoproterenol-stimulated fat-cells demonstrate an inhibitory effect of epinephrine on lipolysis promoted by stimulation of alpha 2-adrenoceptors which occurs before the commonly described beta 1-adrenergic effect which promotes stimulation of lipolysis. This aspect and its putative physiological interest is described and discussed. Intraspecific variations in adrenergic responses of adipocytes have been briefly analysed. The appearance and disappearance of alpha 2-adrenoceptors according to the extent of adipose tissue and increment of fat cell size are discussed. Variations of adrenergic responsiveness during fasting, calorie restriction or chronic stimulation of the adipocytes by physiological amines are also discussed.

The effects of clomipramine treatment (5 mg/kg s.c. a day during 21 days) on dog platelet alpha 2-adrenoreceptivity were investigated. The radioligand binding studies on platelet membrane preparations showed that the antidepressant treatment promotes a decrease in the number of [3H]-yohimbine binding sites. The measurement of the inhibition of PGE1-induced cyclic AMP accumulation in the whole platelet indicated that the biological event immediately associated with alpha 2-adrenergic stimulation was not modified. The aggregatory response to ADP or ADP plus adrenaline was totally abolished in clomipramine-treated platelets. The diastolic pressor responses to noradrenaline or adrenaline injections (after propranolol and prazosin administration) in chloralose anaesthetized dogs were not affected by clomipramine treatment. This work shows that variations in the binding capacities of alpha 2-adrenergic sites are not always linked to modifications of related physiological events.

The beta-adrenergic receptors of isolated human fat cells were identified using a new hydrophilic beta-adrenergic radioligand (+/-)[3H]CGP-12177. The results were compared with those from [3H]dihydroalprenolol binding to fat cells and membranes. [3H]CGP-12177 binding to isolated fat cells showed lower nonspecific binding (less than 15% of total binding) than the lipophilic [3H]dihydroalprenolol (40-60%) at 3 times the KD. At 37 degrees C, [3H]CGP-12177 binding was rapid, reversible, of high affinity (1.2 +/- 0.3 nM) and saturable. The total number of binding sites per cell in subcutaneous adipocytes was 25,000 +/- 6,000 and was equivalent to that found using membrane fractions. Displacement of [3H]CGP-12177 bound to adipocytes by propranolol was stereoselective, consistent with competition at a single site, and had the same characteristics as in membranes. The displacement curves of the beta 1-selective antagonists (atenolol and betaxolol) were biphasic, the high affinity displacement accounting for 70% of the total binding sites. Beta-adrenergic agonists also competed with [3H]CGP-12177 binding in the order of potency: (-) isoproterenol greater than (-) norepinephrine greater than (-) epinephrine, similar to that found in membranes and in in vitro studies on the lipolytic activity of isolated fat cells. This study demonstrates that the sites specifically labeled by [3H]CGP-12177 are the physiological beta-adrenoceptors and also shows that the ligand is better than [3H]dihydroalprenolol for the accurate identification of these receptors in intact human adipocytes. The methodology, which requires biopsies of less than 1 gram of adipose tissue, can be of potential interest for clinical studies investigating the status of fat cell beta-adrenoceptors in various pathophysiological situations.

Spontaneous and stimulated lipolytic activity of subcutaneous abdominal adipocytes from obese subjects treated by a hypocaloric diet (800-1000 kcal/d) for 13-15 d were studied. A strong increase in basal lipolytic activity of the adipocytes occurred after the restrictive diet treatment. Linked to this phenomenon, an important alpha-adrenergic antilipolytic effect of adrenaline appeared whereas, the beta-stimulating effect of isoproterenol (beta-adrenomimetic drug) was not affected. The increased alpha-inhibitory effect of adrenaline was shown to theophylline-stimulated adipocytes of energy-restricted subjects. It is concluded that after a period of hypocaloric diet, the adrenaline-dependent lipolysis of subcutaneous abdominal fat cells of obese subjects is reversed. The lipolytic response induced by adrenaline, described in the adipocytes of obese controls, develop towards an alpha-adrenergic antilipolytic response after the hypocaloric treatment. Our data suggest that, in states with increased rate of lipolysis, there is an increased readiness for alpha-adrenergic response.

In this review, the results obtained in commercial livestock with certain beta-adrenergic agonists (clenbuterol and cimaterol) having an anabolic potential associated with lipid mobilizing properties are considered. The first chapter summarizes major data concerning the effects of beta-agonists on growth and carcass composition in cattle, sheep and pigs. The effect of clenbuterol and cimaterol on carcass quality is to increase the deposition of protein while reducing fat accretion. Then, we briefly consider the physiology and pharmacology of the sympathoadrenal system with a special attention to the distribution and properties of beta-adrenoceptors of various tissues which are putative targets for the beta-adrenergic agonists. Several mechanisms liable to be responsible for the anabolic action of these compounds are also discussed. This chapter includes the evaluation of the effects of beta-agonist on central nervous system and pancreas. A special attention is devoted to their metabolic impact on adipose tissue and muscle. In isolated fat cells, beta-agonists promote stimulation of lipolysis associated with reduction of lipogenesis and of insulin action. The in vitro effects on adipocytes are consistent with the in vivo effects of the compounds. Beta-agonist impact on protein synthesis and muscle accretion is also discussed with reference: 1) to the vascular effects of the compounds that should modify the nutrient flow into the muscle, 2) to a reduction of proteolysis mainly observed for the moment in in vitro studies, 3) to the possible beta-adrenergic-dependent enhancement of insulin action on the muscle. However, more direct experimental evidence is still needed to clearly assess the nature of the action(s) of such anabolic agents on muscle.

Rabbit white adipose tissue, in vitro, may not respond to epinephrine by an increase in the rate of lipolysis although beta adrenergic receptors may be postulated from our experiments with isoproterenol. When basal rate of lipolysis is high, an antilipolytic effect may be shown for low concentrations of epinephrine (0.01 and 0.1 mug/ml). However, epinephrine and an alpha adrenergic blocker, phentolamine (5 mug/ml), acting together, abolish the antilipolytic effect and better still a strong lipolytic effect of epinephrine is unmasked in such a case. The best evidence of these results is that inhibitory alpha, adrenergic receptors are involved in Rabbit white fat cells. These facts partially explain the absence of lipolytic response with epinephrine previously described.

3H-clonidine bound to membrane sites of human fat cells, which have the characteristics of alpha 2-adrenoceptors. Specific binding was rapid, reversible and saturable. [3H]Clonidine binding was of high affinity with a KD of 3.9 nM and with a maximal occupancy of 348 fmol/mg protein. The correlation between alpha-adrenergic agonist or antagonist affinities for the membrane [3H]clonidine binding site with their physiological potencies demonstrates the usefulness of the human fat cell as a model for investigating postsynaptic alpha 2-adrenoceptor properties and regulation.

1. Lipid mobilization during a hypocaloric diet may be enhanced by a pharmacological approach using beta 2-adrenoceptor agonists or alpha 2-adrenoceptor antagonists. Studies were undertaken in the dog, an animal model presenting fat cell antilipolytic alpha 2- and lipolytic beta-adrenoceptors, in order, firstly, to demonstrate the presence of beta 2 subtype adrenoceptors on adipocytes and, secondly, to compare the effects of procaterol (beta 2-adrenoceptor agonist) and of yohimbine (alpha 2-adrenoceptor antagonist) on metabolic, endocrinological and cardiovascular parameters. 2. Procaterol strongly stimulates lipolysis in dog adipocytes in vitro. The utilisation of selective beta 1- and beta 2-adrenoceptor antagonists (bisoprolol and ICI 118,551) in both lipolysis and binding studies (displacement of [3H]-dihydroalprenolol binding) demonstrated the presence of the two beta-adrenoceptor subtypes in dog fat cells. 3. Infusion of either yohimbine or procaterol (10 and 0.4 nmol min-1 kg-1, respectively for 30 min), provoked an equivalent increase in plasma non-esterified fatty acids (+100%). Procaterol, but not yohimbine, induced hyperglycaemia (+120%). Plasma insulin was weakly enhanced by yohimbine (+120%) as compared to the increase given by procaterol (+500%). 4. Both drugs stimulated sympathetic nervous system activity, as indicated by the increased plasma noradrenaline concentration, but only yohimbine increased the plasma adrenaline level. 5. Cardiovascular measurements indicated that procaterol strongly enhances heart rate and transiently decreases mean blood pressure. Yohimbine exhibits a weaker effect on heart rate and slightly increases mean blood pressure. 6. The present work clearly indicates that lipid mobilization is enhanced during fasting in the dog by selective beta 2-adrenoceptor stimulation or by alpha 2-adrenoceptor blockade. This enhanced lipolytic effect may result either from a direct action of the drugs on the adrenoceptors of fat cells or from an activation of the sympathetic nervous system. Procaterol suffers major limitations since it strongly increases heart rate, immunoreactive insulin and glycaemia. On the other hand, yohimbine induces only minor modifications both in cardiovascular and endocrinological parameters.

The human adenocarcinoma cell-line HT29 was used as a model to investigate the binding properties of a new antagonist radioligand of the imidazoline series, [3H]RX821002. All aspects of [3H]RX821002 binding conclusively prove that this radioligand is a valuable tool for labelling alpha 2A-adrenoceptors. [3H]RX821002 binding was very rapid and reversible. Computer-assisted analysis of kinetic data revealed association and dissociation time courses consistent with a simple bimolecular reaction. Saturation isotherms indicated that [3H]RX821002 labeled with high affinity a single population of non-interacting sites displaying a KD of 1.7 +/- 0.1 nM. Adrenoceptor agonists and antagonists inhibited [3H]RX821002 and [3H]yohimbine binding with a strictly similar rank order of potency which is characteristic of alpha 2A-adrenoceptors. The binding parameters of [3H]RX821002 were compared with those of other commercially available [3H]antagonists, [3H]yohimbine and [3H]idazoxan. Analysis of the saturation isotherms for the three radioligands showed that (1) [3H]RX821002 was the radioligand exhibiting the lower percentage of non-specific binding and the better affinity, (2) the Bmax of [3H]RX821002 was significantly higher than that of [3H]yohimbine. The difference in Bmax was not due to better labelling of one of the two affinity states of the receptor but was greatly reduced in glycylglycine buffer, suggesting that, in Tris-Mg2+ buffer, [3H]yohimbine does not label the entire alpha 2-adrenoceptor population.

Imidazolinic radioligands ([3H]UK 14304, [3H]idazoxan and [3H]RX 821002) were used for the identification of alpha 2-adrenoceptors on hamster fat cell membranes since there are limitations to the use of [3H]yohimbine and [3H]clonidine, which suggest alpha 2-adrenoceptor heterogeneity. Biological assays (lipolysis measurements) were performed on isolated fat cells and binding studies were carried out on fat cell membranes. The imidazolinic derivative, UK 14304, was a full agonist as compared to clonidine. Idazoxan and RX 821002 (2-(2-methoxy-1,4-benzodioxan-2yl)-2-imidazoline), a recently developed alpha 2-antagonist, were more potent alpha 2-antagonists than yohimbine in this fat cell model. [3H]UK 14304 was the most suitable agent for the quantification of the 'high-affinity state' alpha 2-adrenoceptors in binding studies since it did not exhibit the sensitivity to the composition of the buffer shown by [3H]clonidine. Although it is a potent alpha 2-antagonist, [3H]idazoxan had major limitations for use in the identification of alpha 2-adrenoceptors in this cell model since it also bound to 'non-adrenaline displaceable' binding sites which were revealed when imidazolinic derivatives (phentolamine) were used instead of adrenaline to determine the non-specific binding. We demonstrated that [3H]RX 821002 was a more suitable radioligand than [3H]yohimbine for labelling hamster fat cell alpha 2-adrenoceptors (KD = 1.0 +/- 0.1 nM, Bmax = 776 +/- 60 fmol/mg protein). Moreover, since it exhibited low affinity for 'imidazoline-preferring sites', it represents a valuable ligand even in tissues possessing such binding sites. We suggest that [3H]RX 821002 can be used to identify alpha 2-adrenoceptors in various tissues when these sites cannot be labelled with [3H]yohimbine.

The imidazoline ligand, [3H]idazoxan, labels a large population of high-affinity binding sites in rabbit fat cell membranes (Bmax = 1370 +/- 91 fmol/mg protein; KD = 1.6 +/- 0.6 nM) when imidazoline derivatives are used for definition of non-specific binding. [3H]Idazoxan sites are not alpha 2-adrenoceptors as assessed by competition studies which showed that epinephrine, norepinephrine and yohimbine do not inhibit [3H]idazoxan binding. Naphazoline, tramazoline and the Na+/H+ exchange inhibitor, amiloride, completely inhibited [3H]idazoxan binding. The Ki values were 9, 27 and 48 nM, respectively.

Epinephrine cannot stimulate adipocytes' lipolysis of human subcutaneous adipose tissue (lateral part of the thigh) while a clear lipolytic action can be shown on the omental tissue. However, at the same concentrations, isoproterenol (a beta-agonist) exerts a strong adipokinetic effect on adipocytes of the both types of adipose tissue. An alpha-adrenolytic (phentolamine) enhances the lipolytic action of epinephrine on the omental adipose tissue and unmasks a lipolytic action of epinephrine on the subcutaneous. Epinephrine antagonizes the lipolysis induced by theophyline on the subscutaneous adipocytes, this action is increased by propranolol (a beta-adrenergic blocking agent). The unresponsiveness to epinephrine of the subcutaneous adipose tissue studied here could be linked to a strong antilipolytic alpha-adrenergic effect.

The effect of the alpha-adrenergic agonist clonidine on lipolysis was investigated in omental dog fat cells. In presence of theophylline, clonidine markedly enhanced lipolysis. This effect was competitively inhibited by the (H2)-receptor blocking agent cimetidine while the H1-histamine receptor antagonist (mepyramine) was without any significant effect. This result suggests that clonidine stimulates dog adipocyte lipolysis through histamine (H2)-receptor activation. Histamine and clonidine stimulated dog fat cell adenylate cyclase; the stimulation was suppressed by cimetidine.

Investigations were carried out to analyse the interactions of alpha 2-antagonists (yohimbine, idazoxan, SK & F-86,466) with human fat cell alpha 2-adrenoceptors. All the alpha 2-antagonists enhanced the lipolytic potencies of epinephrine with an order of potency: yohimbine greater than idazoxan greater than SK & F-86,466; the same order was also found in 3H-yohimbine competition studies on human fat cell membranes. The most potent agent, yohimbine, was administered orally in humans to define the conditions of appearance and the time-course of a putative lipid-mobilizing action. Oral yohimbine administration (0.2 mg kg-1) elevated plasma glycerol and non-esterified fatty acids in fasting healthy subjects without significant action on heart rate or blood pressure during the time-course of the experiment. The lipid-mobilizing action of yohimbine was reinforced during physical exercise, completely suppressed after a meal and partially blocked by administration of propranolol (0.5 mg kg-1; 60 min before yohimbine). Plasma norepinephrine concentrations were increased (40-50%) after oral yohimbine administration. The rise in plasma catecholamine concentration elicited by yohimbine was not modified by propranolol treatment. The lipid-mobilizing effect of yohimbine could be attributable to: (i) the increase in synaptic norepinephrine with a resultant increment in lipolysis by beta-adrenergic agonism; (ii) a decrease in alpha 2-adrenoceptor stimulation of human fat cell alpha 2-adrenoceptors; (iii) a blockade of presynaptic alpha 2-adrenoceptors. The use of highly selective alpha 2-antagonists will allow investigations into alpha 2-adrenoceptors, which may represent a novel locus for pharmacological intervention in lipid-mobilization strategies.(ABSTRACT TRUNCATED AT 250 WORDS)

[3H]-yohimbine binding sites were quantified in platelets from Parkinsonians with no clinical signs of dysautonomia. Never-treated Parkinsonians had a lower specific binding than control subjects. This alteration was associated with decreased epinephrine-induced platelet aggregation. Treatment with dopaminergic agents induced a significant increment of [3H]-yohimbine binding sites. These results show that Parkinson's disease is associated with a reduced number of peripheral alpha 2 adrenoceptors and that dopaminergic agents induce partial recovery.

Theophylline-stimulated human subcutaneous adipocytes were incubatged in vitro in the presence of selected alpha-adrenergic agents in order to characterize the alpha-adrenoceptor of human fat cells. Inhibition of theophylline-induced lipolysis occurred with the agents tested; clonidine was the most potent agonist while methoxamine had no effect. The relative order of potency of the various agonists was: clonidine > adrenaline > phenylephrine > methoxamine; this order is consistent with the classification of agonists described for the presynaptic alpha 2-receptor. Moreover, selected antagonists were used in order to antagonise clonidine inhibition of theophylline-induced lipolysis. The order of potency of the alpha-antagonists for the human alpha-adrenoceptor of adipocytes was: yohimbine > piperoxane > phenoxybenzamine > prazosin. This order is consistent with an alpha 2 type receptor. In conclusion, the results demonstrate that the alpha-adrenergic receptor which inhibits lypolysis in human fat cells is of the alpha 2 type. it is noteworthy that although localized postsynaptically this alpha-receptor can be classified as alpha 2 like the commonly known presynaptic alpha-adrenergic receptor which inhibits noradrenaline release from sympathetic neurons.

Rat adipose tissue possesses two Bordetella pertussis toxin (PTX) substrates and, in the same 39-41 kDa molecular mass range, positive immunoreactivity has also been reported with antibodies against the alpha subunit of Go, the major brain GTP-binding protein (G-protein). In this study, the presence of the brain Go alpha subunit at 39 kDa in adipocytes was reassessed, since direct correspondence between PTX substrates and Go alpha immunoreactivity has not yet been clearly established. On resolutive SDS/polyacrylamide-gel electrophoresis, the PTX substrates of human adipocytes were compared with the three PTX substrates found in brain. No ADP-ribosylated substrate at the level of the 39 kDa brain Go alpha could be detected in adipocyte membranes. Immunoblotting of human adipocyte membranes stained with our anti-Go alpha antibodies confirmed the presence of a positive immunoreactivity in this tissue, but the apparent molecular mass of the immunoreactive polypeptide in adipocytes was higher than that found in nervous tissues. Taken together, these results indicate that the brain Go alpha subunit is not present in adipose tissue. They also suggest the existence of a G-protein in adipocytes which is immunologically related to Go alpha but having a slightly higher molecular mass.