Rat Liver Plasma Membranes Research Articles

NADH oxidase activity of rat liver plasma membrane activated by guanine nucleotides.

The activity of a hormone- and growth-factor-stimulated NADH oxidase of the rat liver plasma membrane responds to guanine nucleotides, but in a manner that differs from that of the classic trimeric and low-molecular-mass monomeric G-proteins. In the absence of added bivalent ions, both GTP and GDP as well as guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) but not guanosine 5'[beta-thio]diphosphate (GDP[beta-S]) stimulate the activity over the range 1 microM to 100 microM. Other di- and tri-nucleotides also stimulate, but only at concentrations of 100 microM or higher. Added bivalent ions are not required either for NADH oxidation or guanine nucleotide stimulation. Bivalent ions (Mg2+ > Mn2+ > or = Ca2+) alone stimulate only slightly at low concentrations and then inhibit at high concentrations. The inhibitions are augmented by GDP or GTP [gamma-S] but not by GTP. Although the activity is the same, or less, in the presence of 0.5 mM MgCl2, GTP at 1-100 nM and other nucleotides at 0.1 mM or 1 mM still stimulate in its presence. The NADH oxidase is activated by mastoparan but aluminum fluoride is weakly inhibitory. Cholera and pertussis toxins elicit only marginal responses. Both the Mg2+ and the GDP and GTP[gamma-S] inhibitions (but not the GTP stimulations) shift to higher concentrations when the membrane preparations are first solubilized with Triton X-100. The results suggest a role for guanine nucleotides in the regulation of plasma membrane NADH oxidase, but with properties that differ from those of either trimeric or the low-molecular-mass G proteins thus far described.

Characterisation of the ATP‐dependent taurocholate‐carrier protein (gp110) of the hepatocyte canalicular membrane

The canalicular domain-specific glycoprotein gp110, which recently has been shown to function as an ATP-dependent taurocholate transporter, has been purified 1800-fold from rat liver plasma membranes. gp110 has been characterised as an integral plasma membrane protein with M(r) of 100,000-115,000 and pI of 2.5-3.5 and possesses a highly glycosylated and negatively charged extra-cellular domain. The broad range of M(r) and pI values results from the existence of numerous glycoforms composed of sialylated N-glycans. After deglycosylation, the polypeptide has M(r) 48,000 and pI 5.0. In primary cultures of rat hepatocytes, gp110 is synthesised with M(r) 110,000, while in the presence of tunicamycin the non-glycosylated form has M(r) 48,000. In the presence of 1-deoxymannojirimycin, two forms of M(r) 83,000 and M(r) 91,000 were found, which were converted by endo-beta-N-acetylglucosaminidase H into a single 52,000-M(r) band, indicating the existence of two basic glycoforms at the oligomannosyl stage of biosynthesis. gp110 was phosphorylated at serine residues in primary cultures of hepatocytes. The sequences of ten internal peptides of gp110 were identical to the sequence of the high-M(r) form of ecto-ATPase, but ecto-ATPase activity from plasma-membrane extracts was not depleted by anti-(gp110) serum. In contrast, Fab fragments of these antibodies inhibit the aggregation of freshly isolated hepatocytes.

Inhibition by cyclosporin A of Adenosine triphosphate-dependent transport from the hepatocyte into bile

Background: Immunosuppressive treatment with cyclosporin A may be associated with impaired hepatobiliary elimination of bile salts and with cholestasis. Inhibition by cyclosporin A of the primary-active adenosine triphosphate (ATP)-dependent transport systems responsible for excretion of bile salts and cysteinyl leukotrienes across the hepatocyte canalicular membrane into bile may explain the cholestatic side effect. Methods: ATP-dependent transport of bile salt and of cysteinyl leukotrienes was studied in human liver plasma membrane vesicles and additionally in rat liver plasma membrane vesicles enriched in canalicular membranes. Results: Inhibition of ATP-dependent taurocholate transport in human liver by 50% was measured at 3 μmol/L cyclosporin A and at 4 μmol/L fujimycin. Kinetic analyses in rat liver indicated non-competitive inhibition by cyclosporin A with respect to ATP and competitive inhibition with respect to taurocholate with inhibition constant (Ki) values of 1.0 and 0.3 μmol/L, respectively. Conclusions: The ATP-dependent export carriers for bile salts and cysteinyl leukotrienes in the hepatocyte canalicular membrane are novel targets for inhibitory side effects of cyclosporin A. Inhibition of ATP-dependent bile salt transport may induce cholestasis.

Plasma membrane p180, which insulin receptor phosphorylates in vivo, is not a tyrosine kinase

The earliest substrates to the transmembrane insulin receptor tyrosine kinase, that would function in insulin signalling, are likely to be associated with the plasma membrane. Rat liver plasma membrane 180, 000 M r protein (p180) is a substrate to the insulin receptor in vitro [Goren et al. (1990) Cellular Signalling 2, 537–555]. The question as to whether p180 is a substrate in vivo was addressed. Half ml 0.9% NaCl or 500 μg insulin was injected into rat livers. Purified plasma membrane glycoproteins from the livers were assayed for in vitro phosphorylation reaction products and endogenous tyrosine-phosphorylated proteins. Membranes from insulin-injected rat livers contained phosphorylated p180 and phosphyrylated insulin receptor β-subunit, whereas saline-injected rat liver membranes contained neither. These data suggested that p180 is an in vivo substrate to the insulin receptor. In vitro p180 is tyrosine-phosphorylated in the absence of insulin. p180, therefore, may be the epidermal growth factor (EGF) receptor or another tyrosine kinase that could be part of a phosphorylation cascade initiated by insulin. Two different experiments suggested that p180 is not the EGF receptor: (i) two-dimensional gel electrophoresis (first dimension—non-equilibrium pH-gradient gel electrophoresis) indicated that p180 is a more basic glycoprotein than EGF receptor; and (ii) based on reverse-phase high pressure liquid chromatography, the tryptic-phosphopeptides of carboxymethyl-Sepharose-purified phosphorylated-p180 were different from those of A431 cell phosphorylated-EGF receptor. Similarly, two different experiments demonstrated that p180 is not a tyrosine kinase: (i) gel-permeation chromatogrpahy separated the insulin receptor from p180 and only insuling receptor was autophosphorylated in vitro; and (ii) membrane proteins not bound to immobilized ATP contained p180. Thus, p180 can associate with the insulin receptor and be phosphorylated in vitro and in vivo; however, p180 does not function in an insulin receptor-mediated phosphorylation cascade.

Identification of a hepatic plasma membrane glutathione [formula omitted] activated by [formula omitted

Rat liver plasma membranes exhibit membrane-bound glutathione S- transferase activity. The specific activity in isolated canalicular membranes was 83 ± 8 mU/ mg protein and 50 ± 3 mU/ mg protein in the sinusoidal membranes. Whereas microsomal and outer mitochondrial glutathione S- transferases were stimulated seven and four-fold with N- ethylmaleimide , respectively, the plasma membrane activity was activated two-fold. Western blot analysis, using an antibody against the microsomal glutathione S- transferase , shows the presence of a 17 kDa protein in canalicular and sinusoidal membrane fractions. The antibody reaction was about three-fold higher in the canalicular compared to the sinusoidal membrane fraction. These data support the conclusion that the plasma membrane glutathione S- transferase is closely related to the microsomal and outer mitochondrial membrane enzyme.

Cardiac alpha 1-adrenoceptors stimulate a high-affinity GTPase activity in sarcolemmal membranes from rabbit atrial and ventricular myocytes.

The interaction between cardiac alpha 1-adrenoceptors and GTP-binding regulatory proteins was characterized in isolated rabbit cardiac myocytes (thereby avoiding interference by other cell types present in the myocardium) by examining the alpha 1-adrenergic stimulation of GTPase activity in sarcolemma-enriched membrane fractions. Stimulation of membrane-associated GTPase activity in both atrial and ventricular myocyte preparations by the alpha 1-adrenergic agonists 1-noradrenaline and methoxamine (in the presence of propranolol) was observed to be both linear with time and saturable. alpha 1-adrenergic stimulation did not change the Km for GTP (0.14-0.21 microM), but increased the Vmax by 39% and 72% above basal levels in atrial and ventricular membranes, respectively. Stimulation of GTPase activity by alpha 1-agonists occurred in a concentration-dependent fashion and was blocked in the presence of the alpha-adrenoceptor antagonists phentolamine and prazosin, but not yohimbine. Prior treatment of myocytes with pertussis toxin had no effect on the alpha 1-adrenergic stimulation of GTPase activity, but inhibited stimulation by muscarinic-receptor activation with carbachol. Finally, photoaffinity labelling of an approximately 75-kDa membrane-bound protein with [alpha-32P]GTP was enhanced in the presence of the alpha 1-agonist methoxamine and abolished by addition of excess non-labelled GTP, suggesting that this GTP-binding protein may interact with cardiac alpha 1-adrenoceptors; a similar GTP-binding protein which may be coupled to alpha 1-adrenoceptors has been reported in rat liver plasma membranes (Im, M. J. & Graham, R. M. (1990) J. Biol. Chem. 265, 18944-18951).

Membrane heterogeneity in isolated rat hepatocytes and liver plasma membrane subfractions: a comparative study using DPH and its cationic derivative TMA-DPH

The fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) and of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) has been studied in hepatocytes isolated from rat liver and in isolated plasma membrane subfractions (cLPM, canalicular membranes and bLPM, basolateral membranes) using frequency domain fluorometry. The decay has been analyzed either by using a model of discrete exponential components or a model that assumes a continous distribution of lifetime values in order to study different aspects of membrane heterogeneity. The results obtained by the two analyses are practically superimposable but the distributional approach allows an evaluation of membrane heterogeneity through the width of the distribution that has shown particularly significant differences when freshly hepatocytes are compared with in vitro aged hepatocytes. Moreover, the comparison of the distributional analysis of the two probes has shown in cLPM a tendency to higher values of the main lifetime component and a narrower distribution width with respect to bLPM. These results indicate changes of membrane domain organization that have been discussed in relation with the specific lipid composition that characterizes the two membrane subfractions. Our results indicate that frequency domain fluorometry may be used to study membrane heterogeneity in intact cells and isolated membranes.

Thiol Groups Are Involved in NADH-Ascorbate Free Radical Reductase Activity of Rat Liver Plasma Membrane

Plasma membranes purified by two-phase partition from rat liver showed an NADH-ascorbate free radical reductase activity of about 14 nmoles NADH oxidized/min/mg protein. This activity was inhibited by N-ethyl maleimide, iodoacetate and iodoacetamide, reagents that covalently block thiol groups. NADH-ascorbate free radical reductase was also inhibited by reduced glutathione and the inhibitions observed with blocking reagents and reduced compounds were additive. These results support the involvement of sulphydryl groups in NADH-AFR reductase and point out the idea that a balance between reduced sulphydryls and oxidized disulfides is required for the optimal function of this activity, considered as part of the transplasma membrane electron transport system.

A protein histidine kinase induced m rat liver by peroxisome proliferators: In vitro activation by Ras protein and guanine nucleotides

A novel protein kinase is induced in rat liver plasma membrane by the administration of peroxisome proliferators. A 36 kDa protein (P36) on the membrane was rapidly phosphorylated in vitro by the kinase and the phosphorylated amino acid was identified as phosphohistidine. Histidine phosphorylation of P36 was activated in vitro by recombinant Ras protein and GTP; both decreased Michaelis constant ( K m) for ATP from 1.25 to 0.25 μM. The novel histidine kinase, products of which have been overlooked due to their acid lability, may participate in cellular signaling and peroxisome proliferators may perturb the pathway.

Monoclonal antibodies to human apolipoprotein AI: probing the putative receptor binding domain of apolipoprotein AI.

We have used four monoclonal antibodies (MAbs) specific for human apolipoprotein (apo) AI, designated AI-1, AI-3, AI-4.1 and AI-4.2, to study the interaction between high-density lipoprotein HDL3 and rat liver plasma membranes. MAbs AI-1 and AI-3 recognize epitopes within residues 28-47 and 140-147 respectively of apoA-I [Allan, Tetaz and Fidge (1991) J. Lipid Res. 32, 595-601]. Two previously unreported MAbs, AI-4.1 and AI-4.2, were raised against purified CNBr fragment 4 (CF4) of apoAI, the C-terminal region. Using e.l.i.s.a. and immunoblotting techniques, we have demonstrated that all four MAbs recognize distinct epitopes within apoAI. Epitope mapping studies using endoproteinase cleavage peptides of CF4 showed that AI-4.1 binds to an epitope within residues 223-233, which is poorly exposed on apoAI molecules associated with lipid. Fab fragments derived from MAb AI-4.2 inhibited the binding of 125I-labelled HDL3 to rat liver plasma membranes, whereas Fab fragments from AI-4.1, AI-3 and AI-1 had little or no effect. In ligand blotting studies with purified CNBr fragments of apoAI and using apoAI-specific antibodies for detection, CF4 showed the highest capacity to recognize two HDL-binding proteins previously identified in rat liver plasma membranes. We propose that the specific interaction between HDL and liver plasma membranes is largely mediated through a binding domain in the C-terminus of apoAI, which is consistent with the involvement of specific receptors for the apolipoprotein moiety of HDL.

Characterization of the effects of adenosine 5′‐[β‐thio]‐diphosphate in rat liver

1. In rat liver cells micromolar concentrations of adenosine 5'-[beta-thio]diphosphate (ADP beta S), activate glycogen phosphorylase by an adenosine 3':5'-cyclic monophosphate (cyclic AMP)- independent mechanism. 2. As with adenosine 5'-triphosphate (ATP), ADP beta S also inhibits the rise in cyclic AMP after glucagon. 3. Cytosolic Ca2+ measured in single cells is rapidly increased with a pattern similar for ADP beta S and for ATP. 4. At variance with ATP, ADP beta S hardly increases inositol 1,4,5-trisphosphate (IP3) levels. 5. Phorbol myristic acetate, which inhibits only slightly the glycogenolytic effect of ATP, almost completely abolishes this effect of ADP beta S. 6. With adenosine 5'-[beta-[35S]thio]diphosphate (ADP beta[35S]) as radioligand, we detected specific purinoceptors on rat liver plasma membranes. Binding consists of a major binding component with KD = 0.7 microM and Bmax = 51 pmol mg-1 of protein, probably mediating the activation of glycogen phosphorylase, and a minor high affinity, low capacity binding component with no obvious function. 7. It is concluded that the differences in biological effects between ATP and ADP beta S may involve different receptors and/or different transduction mechanisms and that ADP beta[35S] can be used to detect the specific binding sites for ADP beta S.

Binding and signal transduction of the cloned vascular angiotensin II (AT1a) receptor cDNA stably expressed in Chinese hamster ovary cells.

The vascular angiotensin (A) II receptor cDNA (AT1a) was transfected into Chinese hamster ovary (CHO) cells to generate the stable cell line CHO-AT1a. This cell line was used to investigate the binding and signal transduction properties of the cloned vascular AT1 receptor. Specific binding of sarcosine1(-)[125I]tyrosine4-isoleucine8-AII ([125I]SI-AII) to CHO-AT1a membranes reached equilibrium after 1 h at 25 degrees C and was consistently greater than 95% of total binding. Saturation binding analyses demonstrated [125I]SI-AII bound to a saturable population of sites on membranes with an equilibrium dissociation constant (KD) of 0.7 nM and a binding site maximum of 1.2 pmol/mg protein. [125I]SI-AII binding to CHO cells was inhibited by the following compounds with a rank order of potency of SI-AII > AII > losartan > AI >> PD 123,177. AII (1 microM) treatment of CHO-AT1a cells caused an increase in inositol phosphates and intracellular calcium relative to basal levels. These responses were blocked by losartan but not by PD 123,177. AII (1 microM) did not effect adenylate cyclase activity in CHO-AT1a cells, whereas the agonist inhibited adenylate cyclase activity in rat liver cell membranes. These effects were blocked by 10 microM losartan. These results indicate that CHO-AT1a cells express functional AT1a receptors which stimulate phospholipase C activity but not adenylate cyclase activity. CHO-AT1a cells should provide a useful model for studies of AT1a receptor domains which are critical to signaling pathways.

Regucalcin modulates hormonal effect on (Ca(2+)-Mg2+)-ATPase activity in rat liver plasma membranes.

The interaction of various hormones and regucalcin on (Ca(2+)-Mg2+)-ATPase activity in rat liver plasma membranes was investigated. The presence of epinephrine (10(-6)-10(-4) M), phenylephrine (10(-6)-10(-4) M), and insulin (10(-8)-10(-7) M) in the reaction mixture produced a significant increase in (Ca(2+)-Mg7+)-ATPase activity, while the enzyme activity was decreased significantly by calcitonin (3 x 10(-8)-3 x 10(-6) M). These hormonal effects, except for calcitonin, were clearly inhibited by the presence of vanadate (10(-4) M) which can inhibit the Ca(2+)-dependent phosphorylation of enzyme. Meanwhile, regucalcin (0.25 and 0.50 microM), isolated from rat liver cytosol, elevated significantly (Ca(2+)-Mg2+)-ATPase activity in the plasma membranes, although this elevation was not inhibited by vanadate (10(-4) M). The epinephrine (10(-5) M) or phenylephrine (10(-4) M)-induced increase in (Ca(2+)-Mg2+)-ATPase activity was disappeared in the presence of regucalcin; in this case the effect of regucalcin was also weakened. However, the inhibitory effect of calcitonin (3 x 10(-6) M) was not weakened by the presence of regucalcin (0.5 microM). Moreover, GTP (10(-5) and 10(-4) M)-induced increase in (Ca(2+)-Mg2+)-ATPase activity was not seen in the presence of regucalcin (0.25 microM). The present finding suggests that the activating mechanism of regucalcin on (Ca(2+)-Mg2+)-ATPase is not involved on GTP-binding protein which modulates the receptor-mediated hormonal effect in rat liver plasma membranes.

Alteration of 5'-nucleotidase properties in rat adipose and liver plasma membranes after 1 Gy whole-body gamma-irradiation.

5'-Nucleotidase activities in rat adipose and liver plasma membranes (PM) were examined at various times after 1 Gy whole-body gamma-irradiation. Changes in the enzyme kinetic parameters were recorded 6 and 14 weeks after exposure: Km decreased while Vmax increased. The enzyme in control PM showed temperature-dependent Arrhenius plots with a lower activation energy at temperatures above a break point at approximately 28 degrees C, whereas 5'-nucleotidase in irradiated PM showed a constant activation energy between 10 and 40 degrees C which was reasonably close to the value observed controls at temperatures below the break point. Furthermore, the interaction of [3H]-AMP with the 5'-nucleotidase catalytic site was studied on adipose PM using a rapid filtration technique. gamma-Irradiation was associated with an increased number of specific AMP-binding sites and decreased ligand affinity. Comparison of the presented data with previous work (Yegutkin et al. 1993a,b) indicate significant functional and structural modification of rat adipose and liver PM following 1 Gy gamma-irradiation and reveals interrelation of these changes.

Modulation of Guanine Triphosphate Nucleotide Binding to p21ras Immunoprecipitates of Rat Liver Plasma Membranes by Agents Affecting Redox State

GTP-γ-[35S] and GTP-γ-[32P] or GTP-α-[32P] bound to plasma membranes of rat liver was immunoprecipitated using anti p21V-H-ras. Binding was enhanced approximately 2-fold by incubation with an exogenous electron acceptor, potassium ferricyanide (but not with potassium ferrocyanide), or oxidized ubiquinone10 and was inhibited or unaffected by incubation with reduced pyridine nucleotides (NADH or NADPH) or reduced ubiquinone10. The results suggest a mechanism of guanine nucleotide exchange that is responsive to oxidation-reduction.

Iodination and binding characteristics of 125I-d(CH2)5 [Tyr(Me)2, Tyr(NH2)9] AVP

With iodinated vasopressin analogue, d(CH2)5[Tyr(Me)2, Tyr(NH2)9] AVP, at position 9, followed by purification by HPLC (specific activity 473-543Ci/mmol), a specific binding was observed in the rat liver plasma membrane fraction. Scatchard analysis indicated a single class of high-affinity binding sites with a Kd of 0.23nM and Bmax of 142fmol/mg protein. V2-agonist, DDAVP, did not displace 125I-vasopressin analogue. These results suggest that 125I-d(CH2)5[Tyr(Me)2, Tyr(NH2)9] AVP with a high specific activity is a useful tool to investigate V1-receptors.

Oleate stimulation of diacylglycerol formation from phosphatidylcholine through effects on phospholipase D and phosphatidate phosphohydrolase.

Hydrolysis of exogenous phosphatidylcholine (PtdCho) to 1,2-diacylglycerol by rat liver plasma membranes was stimulated by oleate concentrations as low as 0.1 mM. In the presence of 75 mM ethanol, the fatty acid also enhanced phosphatidylethanol (PtdEtOH) formation from PtdCho. These effects were also observed with linoleate and arachidonate, but not with saturated fatty acids or detergents, and were minimal in microsomes or mitochondria. Release of [3H]choline from exogenous Ptd[3H]Cho was stimulated by oleate, whereas phosphoryl[3H]choline formation was inhibited. Oleate and other unsaturated, but not saturated, fatty acids also stimulated the conversion of exogenous [14C]phosphatidic acid to [14C]diacylglycerol. These data are consistent with stimulatory effects of these fatty acids on both phospholipase D and phosphatidate phosphohydrolase in liver plasma membranes. The stimulatory effect of guanosine 5'-O-[3-thio]triphosphate) (20 microM) on PtdEtOH and diacylglycerol formation from PtdCho was enhanced by low concentrations of oleate. Phospholipase A2 also stimulated PtdEtOH and diacylglycerol formation from exogenous PtdCho. It is proposed that unsaturated fatty acids may play a physiological role in the regulation of diacylglycerol production through activation of phospholipase D and phosphatidate phosphohydrolase.

Identification of the classical androgen receptor in male rat liver and prostate cell plasma membranes

1. 1. Androgen-specific protein have been identified in cell plasma envelopes: gel filtration chromatography, Scatchard and steroid specificity spectrum analyses show the identity of the protein both from liver and ventral prostate plasma membranes (mol. wt 180,000) and nonidentity of the respective cytosol protein receptors. 2. 2. The protein works only at physiological androgen concentration (0.14–14.0 nM of testosterone). 3. 3. The protein possesses 2-orders higher androgen affinity than rat serum testosterone bincling globulin (TeBG) and 1-order higher affinity compared to cytosol androgen receptor.

Effect of a sunflower oil-supplemented diet on protein kinase activities of rat liver plasma membranes

1. 1. The effect of a sunflower oil-enriched diet on plasma membrane-bound protein kinase C, protein kinase A, casein and tyrosine kinase activities was studied. 2. 2. The diet induced an increase in the content of linoleic acid and a decrease in the content of palmitic acid. The anisotropy parameter ( r s) of the fluorescence probe DPH and S DPH decreased strongly in the experimental group. 3. 3. Protein kinase C was stimulated more than two times. Tyrosine kinase, protein kinase A and casein kinase activities were increased by 65, 57 and 40%, respectively. 4. 4. We suggest that a more fluid lipid environment favours higher plasma membrane-bound protein kinase activities.

Pharmacological characterization of a β 3-receptor agonist (BRL 37,344) and a partial agonist (CGP 12,177A) in neonatal rat liver plasma membranes

The pharmacological properties of BRL 37,344 (sodium-4{2′-[2-hydroxy-2(3-chlorophenyl)ethylamino]-propyl}phenoxyacetatesesquihydrate), a β 3-selective agonist, and CGP 12,177A (-)-4-(3- t-butyl amino-2-hydroxypropoxy) benzimidazole-2-one], a non-selective β-agonists, recently characterized as a partial β 3-agonist in rat adipose tissue, were studied in comparison with isoproterenol, a non-selective β-agonist, in plasma membranes prepared from the livers of newborn rats. Competition binding curves obtained with [ 125I]iodocyanopindolol ([ 125I]CYP) as ligand and isoproterenol or BRL 37,344 as competitor were characterized by the presence of a high and low infinity binding site; the high affinity binding site was no longer detectable when guanidyliidobisphosphate (GppNHp) was present in the incubation mixture. Competition curves with CGP 12,177A were monophasic and independent of GppNHp. In the presence of 10 -7 M of the β 2-selective antagonists ICI 118,551 [erythro-(±)-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol], a concentration which blocks most of the β 2-receptors, ligand binding was reduced to 32% of its maximum. Under these conditions, isoproterenol further displacedthe ligand, and competition curves still displayed the high and the low affinity binding sites; BRL 37,344, however, caused no further displacement of the ligand, except at the highest concentrations. This suggests the BRL 37,344 occupies only the ICI 118,551-sensitive binding sites, i.e. β 2-receptors. Isoproterenol and BRL 37,344 both stimulated adenylate cyclase (EC 4.6.1.1) activity concentration dependently, although the stimulating effect of BRL 37,344 was about half of what was found for isoproterenol. Furthermore, BRL 37,344 inhibited concentration dependently the isoproterenol-induced stimulation of adenylate cyclase, and the inhibition was dependent on the concentration of isoproterenol. The stimulating effect of isoproterenol and BRL 37,344 on adenylate cyclase was blocked by ICI 118,551, whereas the β 1-selective antagonist CGP 20,712A {(±)-(2-(3-carbamoyl-4-hydroxyphenoxy)-ethylamino)-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)-phenoxyl]-2-propanolmethane sulphonate} was ineffective. CGP 12,177A failed to stimulate adenylate cyclase activity. From the results we suggests that BRL 37,344 acts as a β 2-partial agonists in rat liver. The results obtained with CGP 12,177A are typical for a non-selective β-antagonist. We therefore conclude that there is no pharmacological evidence for the presence of β 3-receptors in livers from newborn rats.