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Recent findings indicate that platelet-derived growth factor (PDGF) plays a role in the generation of reactive oxygen species (ROS) as second messengers in smooth muscle cells (SMC). To identify the source and signal transduction pathway of ROS formation in SMC, we investigated PDGF-induced ROS formation. Stimulation of SMC with PDGF resulted in a rapid increase of ROS production. Using an inactivating antibody, we identified the increase to be dependent on p22phox, a NAD(P)H-oxidase subunit. ROS release was completely inhibited by the Gi protein inhibitor PTX as well as an antibody against Galphai1,2, however, not by antibodies against Galphai3/0, Gas, and Gbeta1beta2. The effect of PDGF on ROS production in SMC membranes could likewise be mimicked by the use of a recombinant Galphai2 subunit but not by Galphai3, Galphai0, Gas, and Gbetagamma subunits. Immunoaffinity chromatography demonstrated coupling of Galphai1,2 to the PDGF a-receptor, which, after preincubation of the SMC membranes with PDGF, was increased in the absence of GTPgammaS but decreased in the presence of GTPgammaS and prevented by PTX treatment. These data define a novel G protein-dependent mechanism by which PDGF signaling is transduced through direct coupling of the Gai1,2 subunit of the trimeric G proteins to the PDGF tyrosine kinase receptor.

Background: The crucial function of hepatic lipase (HL) in lipid metabolism has been well established, but the relationship between HL activity and coronary artery disease (CAD) is disputed., Methods and Results: We measured HL activity in the postheparin plasma of 200 consecutive men undergoing elective coronary angiography and determined the degree of CAD with the extent score, which has been shown to be better correlated with known risk factors than other measures of CAD extent. We found a significant inverse correlation between HL activity and the extent of CAD (r=-0.19, P<0.01). This association was mainly due to patients with HDL levels >0.96 mmol/L (n=94, r=-0.30, P<0.005). HL activity was lower in 173 patients with CAD than in 40 controls with normal angiograms (286+/-106 versus 338+/-108 nmol. mL(-1). min(-1), P<0.01). To correct for potential confounding factors, we performed multivariate analyses that confirmed the independent association of HL activity with CAD extent. In addition, the presence of the T allele at position -514 in the HL promoter, which leads to a reduced HL promoter activity, was associated with lower HL activity (r=0.30, P<0.001) and higher CAD extent (42.2+/-20.8 versus 35.3+/-23.6 [extent score], P<0.05). In patients with heterozygous familial hypercholesterolemia, calcified lesions in ECG-gated spiral computed tomography were higher in patients with low HL activity (6.3+/-6.8 versus 1.5+/-3.1, P=0.01)., Conclusions: Our data show that low HL activity is associated with CAD. Therefore, HL might be useful for CAD risk estimation and might be a target for pharmacological intervention.

Basic fibroblast growth factor (bFGF), a ligand of receptor protein-tyrosine kinases, promoted the dissociation of G(s) and had antagonistic stimulatory and inhibitory effects on adenylyl cyclase and NADPH oxidase in human fat cell plasma membranes. The bFGF-induced activation of adenylyl cyclase was blocked by COOH-terminal anti-Galpha(s), indicating that it was mediated by Galpha(s). The inhibitory action of bFGF was mimicked by exogenously supplied Gbetagamma-subunits and was reversed by anti-Gbeta(1/2), or betaARK-CT, a COOH-terminal beta-adrenergic receptor kinase fragment that specifically binds free Gbetagamma, indicating that it was transduced by Gbetagamma complexes. The bFGF-induced inhibition of NADPH-dependent H(2)O(2) generation was also reversed by peptide 100-119, an inhibitor of G(s) activation by ligand-occupied beta-adrenergic receptors, indicating that the Gbetagamma complexes mediating the inhibitory action of the growth factor are derived from G(s). The findings suggest a direct, non-kinase-dependent, coupling of bFGF receptor(s) to G(s) and provide the first example of a ligand of receptor protein-tyrosine kinases that is capable of utilizing both types of component subunits of a single heterotrimeric G protein for dual signaling in a single cell type.

Previous studies revealed that human fat cell plasma membranes contain a multireceptor-linked H(2)O(2)-generating system that is under antagonistic control by hormones and cytokines and is stimulated by insulin via Galpha(i2). In this report, it is shown that the inhibitory action of the beta-adrenergic agonist isoproterenol is mediated by G protein betagamma-subunits, based on observations that its action was specifically reversed by anti-Gbeta antibodies or a C-terminal beta-adrenergic receptor kinase-1 fragment containing the Gbetagamma-binding site of the enzyme, and was mimicked by exogenously supplied G protein betagamma-subunits. Isoproterenol signals through a prototypical G(s)-coupled receptor. Consistent with these results, direct activation of G(s) by cholera toxin or by an anti-Galpha(s) antibody exhibiting beta-adrenergic receptor-mimetic properties (K-20) resulted in an isoproterenol-like inhibition of NADPH-dependent H(2)O(2) generation. In addition, a peptide corresponding to the target sequence of K-20 blocked the action of the catecholamine, apparently by competition between the peptide and G(s) for activated beta-adrenergic receptors, indicating that the G protein betagamma-subunits mediating the inhibitory effects of the catecholamine were in fact derived from G(s).

A polyclonal antiserum that recognizes residues 100-119 within the alpha-helical domain of Galpha(s) (K-20) caused a dissociation of G(s) into its component subunits and activated a cholera toxin-sensitive high affinity GTPase. Consistently, the antibody mimicked the stimulatory effects of the beta-adrenergic agonist, isoproterenol, on adenylyl cyclase, which is mediated by Galpha(s), and its inhibitory action on NADPH-dependent H(2)O(2) generation, a Gbetagamma-mediated response. A peptide corresponding to the target sequence of K-20 not only neutralized the receptor-mimetic effects of the antibody but inhibited the whole spectrum of isoproterenol action as well, including its antagonistic effects on adenylyl cyclase and NADPH-dependent H(2)O(2) generation. By contrast, COOH-terminal anti-Galpha(s) selectively inhibited the stimulatory effect of isoproterenol on cAMP formation without affecting its inhibitory effect on NADPH-dependent H(2)O(2) generation. The data are consistent with the concept that beta-adrenergic receptors interact with multiple sites on Galpha(s) each playing a distinct role, and strongly suggest that antibody K-20 defines a novel contact site for beta-adrenergic receptors that localizes to the alpha-helical domain and is essential for eliciting the complete spectrum of beta-adrenergic responses.

Human fat cells possess a multireceptor-linked H2O2-generating system that is activated by insulin. Previous studies revealed that manganese was the sole cofactor required for a hormonal regulation of NADPH-dependent H2O2 generation in vitro. In this report it is shown that the synergistic activation of NADPH-dependent H2O2 generation by Mn2+ and insulin was blocked by GDPbetaS (guanosine 5'-O-(2-thiodiphosphate)), pertussis toxin and COOH-terminal anti-Galphai1-2 or the corresponding peptide. Consistently, manganese could be replaced by micromolar concentrations of GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), which increased NADPH-dependent H2O2 generation by 20-40%. Insulin shifted the dose response curve for GTPgammaS to the left (>10-fold) and increased the maximal response. In the presence of 10 microM GTPgammaS, the hormone was active at picomolar concentrations, indicating that insulin acted via its cognate receptor. The insulin receptor and Gi were co-adsorbed on anti-Galphai and anti-insulin receptor beta-subunit (anti-IRbeta) affinity columns. Partially purified insulin receptor preparations contained Galphas, Galphai2, and Gbetagamma (but no Galphai1 or Galphai3). The functional nature of the insulin receptor-Gi2 complex was made evident by insulin's ability to modulate labeling of Gi by bacterial toxins. Insulin action was mimicked by activated Galphai, but not by Galphao or Gbetagamma, indicating that insulin's signal was transduced via Galphai2. Thus, NADPH oxidase is the first example of an effector system that is coupled to the insulin receptor via a heterotrimeric G protein.

3T3 L1-cells, which undergo adipose conversion in vitro, possess a stimulus-sensitive H2O2-generating system in their plasma membrane, and its properties are virtually identical with those of the insulin-sensitive human fat-cell oxidase [Krieger-Brauer and Kather (1992) J. Clin. Invest. 89, 1006-1013]. Insulin and insulin-like growth factor I were found to be active stimulators of NADPH-dependent H2O2 generation. Surprisingly, the acidic (a) and basic (b) isoforms of fibroblast growth factor (FGF) as well as the AA and BB homodimers of platelet-derived growth factor (PDGF) had antagonistic effects on NADPH-dependent H2O2 generation in plasma membranes which were parallelled by corresponding changes in H2O2 accumulation in intact cells. bFGF and PDGF BB (which inhibit NADPH-dependent H2O2 generation) prevented the adipose conversion of 3T3 L1-preadipocytes, and this effect could be reversed by exogenously supplied H2O2. Conversely, aFGF and PDGF AA, which stimulated H2O2 generation, accelerated adipocyte conversion in the presence of insulin and were adipogenic in themselves. Consistently, expression of the adipocyte phenotype induced by insulin, dexamethasone and isobutylmethylxanthine was enhanced in the presence of exogenous hypoxanthine/xanthine oxidase, whereas antioxidants, such as N-acetylcysteine or ascorbate, suppressed the process of differentiation. It is concluded that the H2O2 produced in response to hormones and cytokines may contribute to the development and maintenance of the differentiated state.

Previous work demonstrated that human fat-cells possess a plasma-membrane-bound H2O2-generating system that is activated by insulin. Here we show that this system is under antagonistic control by various hormones and cytokines that typically act through several distinct receptor families. Similarly to insulin, oxytocin and tumour necrosis factor alpha acted as stimulators of NADPH-dependent H2O2 generation, whereas isoprenaline, a beta-adrenergic agonist, had inhibitory effects. Surprisingly, the acidic and basic isoforms of fibroblast growth factor as well as homodimeric platelet-derived growth factor AA and BB had antagonistic stimulatory and inhibitory effects on NADPH-dependent H2O2 generation. The agents tested acted at discrete ligand-specific receptors and their mechanisms of action were membrane-delimited and occurred in the absence of ATP. These findings implied that established pathways of signal transduction, including receptor kinases or second-messenger-dependent protein kinases A and C, were not involved and placed the stimulus-sensitive H2O2-generating system in a position comparable with adenylate cyclase. It was concluded that the stimulus-sensitive H2O2-generating system of human fat-cells meets all criteria of a universal signal-transducing system for hormones and cytokines that may link ligand binding to cell-surface receptors to changes in the intracellular redox equilibrium.

Insulin caused a transient increase in H2O2 accumulation in human fat cell suspensions that was observed only in the presence of an inhibitor of catalase and heme-containing peroxidases, such as azide, and reached peak levels of 30 microM within 5 min. The cells contained a plasma membrane-bound NADPH oxidase, producing 1 mol H2O2/mol of NADPH oxidation, that was activated on exposure of intact cells to insulin at contrations that are physiologically relevant (0.1-10 nM). The hormone effect was rapid and was due to a selective increase in substrate affinity. The enzyme was magnesium dependent, required a flavine nucleotide for optimal activity, and was most active at pH 5.0-6.5. In contrast to all other hormone- or cytokine-sensitive NADPH oxidases that have been characterized in sufficient detail, the human fat cell oxidase retained its hormone responsiveness after cell disruption, and only Mn2+, but no ATP, was required for a ligand-induced activation in crude plasma membranes. The results demonstrate that insulin utilizes tyrosine kinase-independent pathways for receptor signaling and strongly support the view that H2O2 contributes to the intracellular propagation of the insulin signal.

Previous results demonstrated that the adenosine that accumulates in human fat cell suspensions is derived from extracellular sources (Kather, H. (1988) J. Biol. Chem. 263, 8803-8809). To get insight into the mechanisms responsible for the lack of adenosine release, extracellular adenine nucleotide catabolism was minimized by 10 mmol/liter beta-glycerophosphate and 10 mumol/liter alpha,beta-methyleneadenosine 5'-diphosphate. Intracellular adenine nucleotide catabolism resulted in a release of inosine and hypoxanthine under these conditions that was increased markedly by isoproterenol. Experiments with inhibitors of adenosine deaminase and adenosine kinase indicated that the production of inosine and hypoxanthine proceeded via AMP deamination. Consistently, IMP levels were increased transiently in the presence of isoproterenol. In addition, the cells possessed a nucleotide phosphomonoesterase that was resistant to the inhibitory actions of ATP and alpha,beta-methyleneadenosine 5'-diphosphate and showed preference for IMP over AMP. Adenosine (approximately 1 nmol/10(6) cells/h) was also produced inside the cells. However, adenosine production was unrelated to ATP turnover via adenylate cyclase, and any adenosine formed was immediately reconverted to adenine nucleotides in the absence and presence of isoproterenol. It was concluded that adenosine is not released by intact human adipocytes, because the alternative routes of intracellular AMP catabolism are compartmentalized (at least in functional terms), and adenosine kinase is not saturated with substrate in the absence and presence of isoproterenol.

The effects of beta-adrenergic agonists on ATP utilization and adenine nucleotide breakdown in human adipocytes were examined. The catecholamine-induced increase in cAMP was associated with an enhancement of adenine nucleotide catabolism resulting in an increase in release of inosine and hypoxanthine which can not be reutilized for adenine nucleotide synthesis. Therefore, one-third of total cellular adenine nucleotides were irreversibly lost in the presence of 1 mumol/liter isoproterenol. The catecholamine-induced increase in purine release could be blocked by phosphodiesterase inhibitors, suggesting that cAMP is the main precursor of purines in the presence of beta-adrenergic agonists. However, epinephrine (in the simultaneous presence of the alpha 2-adrenergic blocking agent, yohimbine) and isoproterenol were 10 times more potent in stimulating purine release than in elevating cAMP. In addition, purine release ceased when cAMP was still markedly increased, suggesting a compartmentation of the cyclic nucleotide and/or involvement of the hormone-sensitive, low Km cAMP phosphodiesterase. The results document that white fat cells have an enormous potential for dissipating energy, and demonstrate that the pathway involving cAMP formation and hydrolysis constitutes the principle route of adenine nucleotide catabolism in the presence of beta-adrenergic agonists.

The effects of prazosin on adrenergic-stimulated fat-cell lipolysis and adenylate cyclase activity were investigated. The results revealed that the antilipolytic alpha-adrenergic catecholamine effects on human fat-cell metabolism are mediated via alpha 2-receptor sites displaying extremely low affinity to prazosin, which is alpha 1-site selective. The data provide an explantation for the lack of effect of prazosin on lipid mobilization in vivo. The molecular sites mediating the lipid-lowering action of the drug, however, remain to be elucidated.

Adenosine is known to modulate adenylate cyclase activity in a variety of tissues. We have tested the effects of adenosine on basal activity and the catecholamine- or prostaglandin E1-stimulated activity of the human fat cell adenylate cyclase. Adenosine caused a dose-dependent inhibition of basal enzyme activity as well as of adrenaline-and prostaglandin E1-stimulated rates of 3',5'-cyclic AMP accumulation. The adenosine-induced inhibition was specific since other nucleosides or their respective nitrogenous parent bases, with the exception of adenine, failed to mimic the action of adenosine. In addition, the adenosine-induced inhibition could be reversed by inclusion of adenosine deaminase. The results are compatible with the concept of adenosine acting as an inhibitor of lipolysis at the level of the membrane-bound adenylate cyclase. They show that this nucleoside can also inhibit the prostaglandin E1-induced stimulation of the human fat cell adenylate cyclase thereby suggesting that the effects of adenosine and prostaglandins might be antagonistic under conditions where prostaglandins act as stimulators of lipolysis.

The effects of a single bout of exercise at 40% of maximum aerobic capacity with regard to alimentary lipemia and postprandial lipoproteins was studied in a cross-over design in 12 young healthy male volunteers. In addition to lipids and lipoproteins, lipoprotein lipase, free glycerol, free fatty acids, plasma insulin, and C-peptide concentrations were quantitated. Postprandial exercise reduced alimentary lipemia by 34% while lipoprotein lipase activity rose by 42%. The postprandial fall of high-density lipoprotein (HDL)3 was abolished and the rise of HDL2 accentuated. Free glycerol and free fatty acid concentrations were higher following the meal plus exercise regimen compared to the meal alone. It is concluded that at least part of the chronic effect of exercise may come from additive effects such as observed from individual bouts of muscular activity.

Multiple symmetric lipomatosis has been proposed to be associated with impaired catecholamine-responsiveness of hypertrophic adipose tissue at the level of beta-adrenergic receptors or adenylate cyclase respectively. We have studied the regulation of the adenylate cyclase by guanine nucleotides and adrenaline in 5 subjects suffering from multiple encapsulated lipomata. In the presence of GTP (0.1 mmol/l) basal adenylate cyclase activity averaged 0.5 +/- 0.3 nmol cAMP/mg protein/10 minutes in normal adipose tissue and 1.0 +/- 0.4 nmol cAMP/mg protein/10 minutes in hypertrophic adipose tissue respectively. The synthetic GTP-analogue GMP(PNP) (0.1 mmol/l) increased non-stimulated activity by about 100% in both tissues. Adrenaline (1 mumol/l-1 mmol/l) caused a dose-dependent increase of enzymic activity in both tissues which had a maximum of 130% above basal levels in the presence of GTP and of 300% in the presence of GMP(PNP) respectively. In one of the six subjects suffering from gluteal lipomata normal adipose tissue was obtained from the gluteal as well as the abdominal region on two occasions. Maximally effective concentrations of adrenaline (1 mmol/l) induced a 3-fold increase of enzymic activity in abdominal membranes compared with about a 1.7- and 1.75-fold increase in normal and lipomatous tissue from the gluteal region. The results show that encapsulated lipomata contain a normally reactive adenylate cyclase system.

Human adipocytes are of limited viability (7 +/- 2% release of lactate dehydrogenase/h) and contain active ectophosphatases which are capable of sequentially degrading ATP to adenosine. At densities of 30,000-40,000 cells/ml, human fat cell suspensions accumulated adenosine, inosine, and hypoxanthine, and their concentrations were 38 +/- 8, 120 +/- 10, and 31 +/- 7 nmol/liter after 3 h of incubation. Dipyridamole (10 mumol/liter), an inhibitor of nucleoside transport, caused a 5-7-fold increase in adenosine accumulation which was reduced by 85% on inhibition of ectophosphatases by beta-glycerophosphate and antibodies against ecto-5'-nucleotidase or alpha, beta-methylene 5'-adenosine diphosphate (10 mumol/liter), respectively, indicating that most of the adenosine is produced in the extracellular compartment. Accordingly, the spontaneous accumulation of adenosine was reduced beyond 5 nmol/liter on inhibition of ectophosphatase activities or removal of extracellular AMP by AMP deaminase (4 units/ml). Added adenosine (30 nmol/liter) disappeared until its concentration approached 5 nmol/liter. Isoproterenol (1 mumol/liter) had no effect on adenosine accumulation regardless whether purine production from extracellular sources was minimized or not. In contrast to adenosine, the concentrations of inosine and hypoxanthine displayed only a modest decrease (30-50%) on inhibition of ectophosphatase activities. In addition, isoproterenol caused a 2-3-fold increase in inosine and hypoxanthine production which was concentration-dependent and could be inhibited by propranolol. It is concluded that the adenosine that accumulates in human adipocyte suspensions is almost exclusively derived from adenine nucleotides which are released by leaking cells. By contrast, inosine and hypoxanthine are produced inside the cells, and the release of these latter purines appears to be linked to ATP turnover via adenylate cyclase.

Some of the effects of native bovine parathyroid hormone and of the synthetic aminoterminal 1-34 fragment on the adenylate cyclase activity of human fat cell ghosts were studied. Saturating concentrations of both hormone preparations caused a significant increase of enzyme activity by about 200-300%. Guanosine 5'-triphosphate (0.1 mM) inhibited basal enzyme activity but had no substantial effect on parathyroid hormone-stimulated enzyme activity. The guanosine 5'-triphosphate analogue, 5'-guanylyl-imidodiphosphate, produced about a threefold enhancement of basal and parathyroid hormone-stimulated enzyme activities under standard conditions (5 mM Mg+2, 1mM ATP, pH 8.0, 30 degrees C). Activation by parathyroid hormone was not influenced by beta-adrenergic blockade in contrast to stimulation by epinephrine. The sensitivity of the enzyme system to the native and the synthetic parathyroid hormone was, however, abolished after pretreatment of the fat cells with trypsin (1 mg/ml). The stimulatory effects of epinephrine and NaF were not affected by pretreatment with trypsin. The results suggest that human fat cells, like rat adipocytes, contain a multireceptor-coupled adenylate cyclase.

It has been shown that human gastric mucosa contains a prostaglandin-sensitive adenylate cyclase system. Of the prostaglandins tested the E-type induced an about 3--3.5-fold increase of enzyme activity. The prostaglandin A2 by causing an about 2.5-fold enhancement of cAMP formation and the F-prostaglandins (1.4--2.0-fold stimulation) were less effective in activating the enzyme system. Maximally effective concentrations of histamine and prostaglandin E2 were additive with respect to enzyme activity indicating that both hormones act via individual adenylate cyclases.

The antisecretory effects of a new histamine H2-receptor antagonist, ranitidine hydrochloride, have been investigated on basal and pentagastrin-stimulated acid secretion in healthy volunteers 5 and 10 h after oral administration of 150 mg. In addition, the 24-h intragastric pH-profiles have been measured in patients undergoing parenteral nutrition after three doses of 150 mg ranitidine per day. A 40% inhibition of basal acid output has been noted even 10 h after drug intake. The intragastric pH-values were raised above 5 for at least 24 h. The new H2-antagonist ranitidine has been proven to be a potent and long-acting antisecretory compound.

Adenylate cyclase of human fat cell ghosts shows a biphasic response towards prostaglandin E2 with inhibition occurring at nanomolar concentrations of the hormone and stimulation at concentrations beyond 10(-6) mol/liter. The expression of the inhibitory effect is critically dependent on GTP. Under the conditions employed (1 mmol/liter ATP, 5 mmol/liter Mg2+, 30 degrees C) the inhibitory component of prostaglandin E2 became apparent at GTP concentrations exceeding 10(-6) mol/liter. The prostaglandin E2-induced inhibition displayed characteristic features of prostaglandin action in intact fat cells with respect to the effective concentrations and degree of inhibition. It is concluded that prostaglandin E2 is capable of inducing antagonistic effects upon lipolysis via interaction with the membrane-bound adenylate cyclase.

Several prostaglandins prevent ulcer formation (called cytoprotection) by a mechanism other than inhibition of gastric acid secretion. One suggestion is that they increase cyclic AMP in non-parietal cells. A variety of prostaglandins with potent cytoprotective properties were tested for their capacity to modulate adenylate cyclase activity in homogenates of human gastric mucosa. Prostaglandin E2, prostacyclin (PGI2) and 15(S)-methyl-PGE2 stimulated the cyclase in human gastric mucosal biopsy specimens in a dose-dependent manner. Cytoprotective prostaglandins without antisecretory properties such as PGF2 beta were also able to activate the enzyme system dose-dependently. In contrast, cytoprotective prostaglandins such as PGD2, the PGE1-analogue, SC-29333, and the prostaglandin-like compound C83 did not stimulate human gastric adenylate cyclase. Whereas PGD2 did not modulate enzyme activity at all, SC-29333 and C83, at concentrations greater than 10 mumol/l, inhibited basal and PGE2-stimulated enzyme activities. These studies suggest that cyclic AMP is not directly related to the cytoprotective effect of prostaglandins, at least in human gastric mucosa.

Alimentary lipemia was studied in 12 healthy young men with and without exercise. Three sets of experiments were performed. While continuous exercise of 90 min duration significantly reduced postprandial triglycerides by 26% (study I), this effect could not be observed when exercise was interrupted for 5 min after each 25 min (study II). Plasma free fatty acid concentrations, in the latter experiment, were significantly higher (by 311%) than during rest. When, in a third experiment continuous exercise was compared with intermittent physical activity, the latter condition significantly increased postprandial triglyceridemia, most probably due to precipitous rises of free fatty acids on each interruption of ergometry. It is concluded that in the third experiment the balance between triglyceride removal and triglyceride synthesis was shifted toward the latter. Whether exercise lowers, leaves unaltered, or raises plasma triglyceride levels may depend on subtle changes of experimental design.

Some of the effects of GTP on human fat cell adenylate cyclase activity were studied. This nucleotide caused a dose-dependent inhibition of basal activity without affecting the hormone-activated rate of cAMP formation. Maximal effects were observed at GTP-concentrations of about 1 x 10(-4) M. The relative extent of hormonal stimulation was about 1.5-fold increased in the presence of 0.1 mM GTP.

The effects of various beta-blocking agents upon the catecholamine-activated adenylate cyclase of human fat cell ghosts were studied. Both non-selective and cardio-selective beta-blocking drugs are capable in inhibiting the human enzyme system. The concentrations of the non-selective beta-blockers (bupranolol, alprenolol, propranolol, 4-hydroxypropranolol, prindolol and oxyprenolol) required to produce half maximal inhibition of isoproterenol-stimulated rates of cAMP-formation were found to be in a narrow range (5X10(-7) M-3X10(-6) M).The cardioselective beta-blocking agents, however, were only 1/100 (methypranol) to 1/1000 (atenolol, practolol) as potent as the non-selective drugs.

The effects of guanine nucleotides on basal and parathyroid hormone-stimulated adenylate cyclase of human fat cell ghosts were studied. GTP (10(-7)-10(-3) M) caused a dose-dependent inhibition of basal enzyme activity, but it had no significant effect on PTH-stimulated rates of cAMP-formation. The guanine nucleotide analogue 5'-guanylyl-imidodiphosphate GMP (PNP) when applied in the same concentration range, stimulated basal as well as PTH-activated adenylate cyclase activity up to 300%. GMP (PNP) activation was non-linear with time. PTH-activated the human fat cell adenylate cyclase via an individual receptor distinct from beta-adrenergic receptor sites.

The orientation of SH-groups within the fat cell membrane involved in adrenaline (and NaF) action was studied by comparing the effects of uncoupled p-CMB with those of a large derivative of this reagent -- p-CMB-dextran --. Preincubation of intact adipocytes with uncoupled p-CMB caused a dose-dependent inhibition of adrenaline (and NaF) stimulated adenylate cyclase activity as determined in ghosts prepared subsequently. Preincubation with p-CMB-dextran, however, influenced neither the lipolytic response to adrenaline nor the catecholamine (and NaF) activated adenylate cyclase activity. When p-CMB-dextran was present during ghost preparation, a dose-dependent inhibition of adrenaline (and NaF) stimulated adenylate cyclase activity was observed. These results suggest that the binding sites of adrenaline as well as SH-groups essential for activity of adenylate cyclase are not localized near enough to the exterior surface to be accessible for p-CMB-dextran. The polarity in the sensitivity of fat cell adenylate cyclase could not be observed when p-CMB-dextran was added directly to intact or fragmented ghosts. The abbreviations used are: Cyclic AMP, cyclic adenosine 3',5'-monophosphate; p-CMB, p-chloromercuribenzoate.