The effects of different steroids on the expression of angiotensin AT1 receptors by the human hepatoma cell line, PLC-PRF-5 was studied. Dexamethasone and aldosterone decreased the specific binding of [3H]angiotensin II to intact PLC-PRF-5 cells by 57 +/- 4% and 54 +/- 2%, respectively, compared to control, untreated cells. EC50 values for dexamethasone, cortisol and aldosterone were 1.8 +/- 0.6, 40 +/- 6, and 310 +/- 20 nM, respectively, suggesting that these effects were mediated via a glucocorticoid receptor. Scatchard analysis revealed that dexamethasone decreased the number of angiotensin AT1 receptors expressed (50 +/- 4% relative to control) with no change in receptor affinity. Treating cells with dexamethasone in the presence of either an angiotensin converting enzyme inhibitor or an angiotensin II receptor antagonist did not prevent the reduction in angiotensin AT1 receptor expression, ruling out a mechanism involving a dexamethasone induced increase in endogenous angiotensin II production. A ribonuclease protection assay established that the steady state level of angiotensin AT1 receptor mRNA in dexamethasone treated cells was reduced to 34.7 +/- 8.4% of untreated cells. The decrease in the number of angiotensin AT1 receptors expressed on the cell surface after treatment with dexamethasone therefore seems likely to reflect the decreased steady state level of the mRNA coding for this receptor.

Radioligand binding studies were undertaken to establish the expression of angiotensin II (AII) receptors on the human hepatoma cell line, PLC-PRF-5. Cell membranes were shown to express a large number of AII receptors with high and low affinity binding sites having Bmax values of 1269 +/- 365 and 4190 +/- 1055 fmol/mg protein and affinities (Kd) of 2.0 +/- 0.3 nM and 8.7 +/- 0.4 nM, respectively. In intact cells a single class of AII binding site was seen with an affinity (Kd) of 6.7 +/- 1 nM and a Bmax value of 315 +/- 32 fmol/mg. In both membranes and intact cells AII, AIII and the selective angiotensin AT1 receptor antagonist, DuP 753, all had a high affinity for the receptor (Ki values in the nanomolar range), but the selective angiotensin AT2 ligands, PD 123177 and p-aminophenylalanine6 AII, had low affinity (Ki values in the micromolar range). These results indicate that the PLC-PRF-5 cells express the angiotensin AT1 receptor subtype. This was further supported by the demonstration of the sensitivity of the receptor to dithiothreitol (DTT). Pretreatment of membranes with DTT reduced [3H]AII binding in a concentration-dependent manner with an IC50 of 4.2 +/- 0.9 mM. The coupling of the AT1 receptor to signal transduction pathways was investigated. In intact cells AII (100 nM) evoked an increase in intracellular calcium ([Ca2+]i). This increase in [Ca2+]i was unaffected by PD 123177 (100 microM) but was abolished by DuP 753 (100 microM). Furthermore, AII (100 nM) did not inhibit forskolin (0.1-10 microM) stimulated cyclic AMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)

In this study we have examined a potential role of the sodium/proton exchange system in the regulation of renin secretion. We found that the inhibitors of the Na+/H+ antiport, amiloride (1 mM) and ethylisopropylamiloride (EIPA, 50 microM), led to a 125% increase of renin secretion from cultured mouse juxtaglomerular cells. The stimulatory effect of EIPA on renin secretion was dependent on the extracellular concentrations of sodium and hydrogen ions. While lowering the extracellular pH from 7.3 to 7.0, and lowering [Na+]e from 130 mM to 5 mM had no effect on basal renin release, it markedly attenuated or even blunted the effect of EIPA on renin secretion. The stimulatory effect of forskolin on renin secretion, however, was not altered by decreases of extracellular pH and of sodium. Inhibition of basal renin release was achieved with angiotensin II (1 microM). In the presence of EIPA the inhibitory effect angiotensin II was markedly attenuated. Although effective on renin secretion, neither amiloride nor EIPA exerted a significant effect on the denovo synthesis of renin in cultured mouse JG cells. These findings are compatible with the idea that an amiloride-sensitive transport process, presumably the Na+/H+ exchanger, acts indirectly as an inhibitory signal transduction system for renin secretion from renal juxtaglomerular cells.

The effects of synthetic [Asu1,7]eel calcitonin (CT) on the unidirectional inflow of Ca2+ were investigated in isolated rat liver cells by measuring the initial rate of 45Ca2+ uptake. CT increased Ca2+ inflow, with EC50 values (concentrations giving half-maximal effect) of 10(-10) M. The action of CT was in evidence within 15 s after the addition of 45Ca2+ to the cells. CT-activated Ca2+ inflow was completely blocked by the presence of the Ca2(+)-antagonist verapamil at a concentration of 10(-8) M. Meanwhile, epinephrine (10(-8) to 10(-4) M) or phenylephrine (10(-8) to 10(-4) M) increased Ca2+ inflow within 60 s after the addition of 45Ca2+ to the cells. Those hormonal effects were additively enhanced by the presence of CT (10(-8) M). Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, increased Ca2+ inflow at a concentration of 10(-9) to 10(-5) M. The presence of CT (10(-8) M) synergistically enhanced PMA-increased Ca2+ inflow at concentrations of 10(-7) to 10(-5) M. The present results suggest that CT can stimulate the rate of Ca2+ inflow in rat liver cells.

In this study, the binding properties of angiotensin receptors were examined in the liver, adrenal, brain, and vascular tissue of the brushtail possum, Trichosurus vulpecula. With 125I-Ile5-angiotensin II as the radioligand, the binding affinity (Ka) and receptor number (R0) were estimated for the liver (Ka = 3.60 +/- 0.31 liters/nmol; R0 = 23.8 +/- 1.30 pmol/g tissue; n = 8) and adrenal (Ka = 1.68 +/- 0.29 liters/nmol; R0 = 1.67 +/- 0.23 pmol/g tissue; n = 8). Specific binding was not found in any of seven areas of the possum brain (n = 6), whereas the expected binding was present in similar areas of the rat brain. Using angiotensin III or the antagonist Sar1-Ala8-angiotensin II as radioligands or changing the composition of the incubation buffer did not alter the outcome. Moreover, the intracerebroventricular injection of 1 and 5 nmol of angiotensin II did not elicit an increase in blood pressure which could be attributed to brain angiotensin II (AII) receptors. Ligand affinities of the adrenal and liver receptors were found to be in the following decreasing order: Val5-AII greater than Ile5-AII = Ile5-AIII greater than Sar1-Ala8-AII greater than Sar1-Gly8-AII greater than Sar1-Leu8-AII greater than Ile5-AI greater than hexapeptide greater than Phe3-Tyr8-AII. The cardiovascular AII receptor was investigated by generating dose-response curves of the pressor activity of Ile5-AII and six AII analogs infused intravenously. It was concluded that liver, adrenal, and vascular AII receptors in the marsupial possum have characteristics similar to those in eutherian mammals. However, the failure to find brain AII receptors raises the possibility that those functions mediated by such receptors in the eutherian brain are absent in the possum and perhaps other marsupials.

Specific angiotensin II (AII) binding sites were identified and characterized in membranes from human term placenta. The binding of iodinated [125I](Sar1)AII was time-dependent and saturable; it could be totally reversed on addition of unlabelled (Sar1)AII and GTP + NaCl. Scatchard plot analysis of dose-dependent [125I](Sar1)AII binding indicated the presence of a single class of binding sites with an equilibrium dissociation constant of 0.27 +/- 0.06 nM and a maximum binding capacity of 38.4 +/- 4.3 fmol/mg protein. The affinity of five AII analogues for the placental receptor was determined in competitive binding assays; the order of inhibitory potency was: (Sar1)AII greater than (Sar1, Ile8)AII approximately (Sar1, Ala8)AII approximately AII greater than angiotensin I greater than (Des-Phe8)AII. (Sar1)AII did not cause any significant change in the basal or stimulated adenylate cyclase activity. In order to investigate the subunit molecular structure of the placenta AII receptor, membranes were covalently labelled with the photoaffinity ligand [125I](Sar1, (4N3Phe)8)AII. Sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by autoradiography showed that the labelling was specifically incorporated into a protein of Mr 92,000 in the presence or absence of dithiothreitol. It therefore appears that the AII receptor from human placenta has the same binding and pharmacological properties as other well-known AII receptors; by contrast, it is characterized by a significantly higher molecular weight, pointing out that structural differences in AII receptors may exist between species.

Vasopressin does not induce glycogenolysis in rabbit hepatocytes; glucagon, angiotensin, phenylephrine and ATP are as potent as with rat hepatocytes, whereas isoprenaline is nearly 10000 times more potent in the rabbit. Binding studies of [3H]vasopressin reveal the complete absence of specific vasopressin receptors on rabbit liver plasma membranes. We verified that vasopressin acts as an antidiuretic and vasopressor agent in the rabbit. We conclude that there is a selective lack of V1 vasopressin receptors in rabbit liver.

We have used the technique of short-term infusion with digitonin to obtain hepatocytes originating either from the periportal or the perivenous zone of the liver acinus [(1985) Biochem. J. 229, 221-226]. Total glycogen phosphorylase content and sensitivity to cyclic AMP-dependent and calcium-mediated glycogenolytic agonists were very similar for both cell sub-populations and did not differ from the values obtained for control cells. We conclude therefore that there is an apparent absence of metabolic zonation as far as receptor-mediated glycogenolysis and glycogenolytic potency is concerned.

The present work delineates the basis for chemical modifications which can be introduced on the angiotensin II (AII) molecule to design probes suitable for indirect affinity techniques, especially for receptor purification. Using the solid-phase synthesis strategy, biotin or dinitrophenyl moieties have been added at the N-terminus of AII, with aminohexanoic acid as spacer arm. The resulting probes, (6-biotinylamido)hexanoyl-AII (Bio-Ahx-AII) and dinitrophenylaminohexanoyl-AII (Dnp-Ahx-AII), were prepared in their monoiodinated and highly labelled radioiodinated forms, with possible sulphoxidation of biotin. In addition to their ability to interact with streptavidin and anti-Dnp antibodies respectively, the two ligands displayed almost unchanged affinities for hepatic AII receptors as compared with AII. Bio-Ahx-AII and Dnp-Ahx-AII behaved as agonists on several AII-sensitive systems. The potential applications of these probes, receptor purification, cell labelling and sorting and histochemical receptor visualization, are discussed.

The design of angiotensin II (A II)-derived probes suitable for indirect affinity techniques is presented. Biotin or dinitrophenyl moieties have been added at the N-terminus of A II, through aminohexanoic acid as spacer arm, to generate (6-biotinylamido)-hexanoyl-AII (Bio-Ahx-AII) and dinitrophenyl- aminohexanoyl-AII (Dnp-Ahx-AII). Monoiodinated and highly labeled radioiodinated forms of these probes have been prepared. The two bifunctional ligands displayed high affinities for rat liver A II receptors (Kd values in the nanomolar range) and their secondary acceptors: streptavidin and monoclonal anti-Dnp antibodies respectively. Bio-Ahx-AII and Dnp-Ahx-AII behaved as agonists on several AII-sensitive systems. Based on these structural assessments, the parent photoactivable azido probe: Bio-Ahx-(Ala1,Phe(4N3)8)A II. A II was synthesized and proved to possess similar biological properties than the non-azido compound. The hepatic A II receptor could be covalently labeled by the radioiodinated probe, with a particularly high yield (15-20%); SDS-polyacrylamide gel electrophoresis of solubilized complexes revealed specific labeling of a 65 Kdaltons binding unit, in agreement with previous data obtained with other azido AII-derived compounds. The potential applications of these probes are: i) receptor purification by combination of its photoaffinity labeling and adsorption of biotin-tagged solubilized hormone-receptor complexes on avidin gels. ii) cell labeling and sorting. iii) histochemical receptor visualization.

The binding of vasopressin, angiotensin II and prazosin (alpha 1-adrenergic antagonist) to purified heavy (GH) and (intermediate + light) (GI + L) rat liver Golgi fractions was studied. The three types of ligands showed a saturable and specific binding in Golgi fractions; the maximal specific binding of [3H]vasopressin, [3H]prazosin and [125I]Sar-N3-Phe-angiotensin II was respectively 5-10%, 20-30% and 30-40% of that detected in purified plasma membranes. The apparent binding affinities of the three ligands were the same whether determined in Golgi fractions or plasma membranes. The presence of vasopressin, alpha 1-adrenergic and angiotensin receptors in very different proportions, as compared to the amount of receptor detected in plasma membranes, in GH and GI + L Golgi fractions was not compatible with the idea that a plasma membrane impurity accounted for the detection of receptor in the purified intracellular particulate fractions. In vivo injection of [125I]Sar-N3-Phe-angiotensin II resulted in a receptor-mediated endocytosis of the iodo-angiotensin analog into the GH and GI + L Golgi fractions. The apparent molecular weight of the irreversible complex, [125I]angiotensin-receptor, was estimated in subcellular fractions using SDS-PAGE electrophoresis. This value was identical after either in vivo or in vitro labelling (MW = 63,000) and was indistinguishable from the molecular weight of the irreversible hormone receptor complex present in the plasma membranes.

The effects of propylthiouracil (PTU) treatment on vasopressin, angiotensin II, glucagon and alpha 1-adrenergic receptors in both developing and adult rats were studied in liver membrane preparations by measuring the binding of the following ligands: [3H][8-lysine]vasopressin, [3H]Sar-angiotensin II, [125I]glucagon and [3H]prazosin, and in the case of glucagon, by measuring adenylate cyclase activation. Whatever the ligand used, in young as well as in adult animals, PTU treatment led to a similar reduction (about 50%) in the maximal number of binding sites (Bmax), without significant changes in the apparent dissociation constant (KD) of labeled hormone for its specific receptor. In normal adult animals, thyroxine treatment, i.e. hyperthyroidism, had an opposite effect on the Bmax (25-50% increase), without changes in the KD. In developing PTU-treated rats, the abnormalities completely disappeared after therapy with increasing physiological doses of thyroxine; consequently they were directly related to thyroid deficiency and not to toxic effects of PTU. Moreover, the abnormalities resulting from induced hypothyroidism were reversible. In developing and adult hypothyroid rats, neither basal, NaF-, nor Gpp(NH)p-stimulated adenylate cyclase activities were significantly affected. Glucagon-sensitive adenylate cyclase activity seemed to be slightly increased (by about 15%), without changes in the apparent activation constant (Kact). These results are considered in parallel with findings on plasmatic glucagon and vasopressin levels, compared with similar previous reports related to renal vasopressin receptors, and discussed with respect to unpublished observations concerning hepatic responsiveness to glycogenolytic hormones in young and adult rats with induced hypothyroidism.

The effects of the alpha 1-adrenergic agonist phenylephrine and the peptide hormones angiotensin II and arg8-vasopressin on cytoplasmic free calcium concentration were investigated in single rat hepatocytes microinjected with the photoprotein aequorin. Hepatocytes responded to physiological concentrations of the glycogenolytic agonists with a series of repetitive Ca transients. In each transient free Ca rose in 2-3s to above 600 nM from a resting level of 200 nM. Transient duration depended on the agonist and ranged from approximately 7s for phenylephrine to approximately 15s for angiotensin. Transient frequency, but not shape or size, depended on agonist concentration. The period ranged from less than 20s to several minutes. We suggest that the frequency of the Ca transients is the principal determinant of the amplitude of the cellular response to calcium-mobilizing agonists.

Evidence has been presented for the existence in rat liver of P2-purinoceptors which are involved in the control of glycogenolysis. Isolated rat hepatocytes and purified liver plasma membranes have been used to study the binding of the ATP analogue adenosine 5'-[alpha- [35S]thio]triphosphate (ATP alpha [35S]) to these postulated P2-purinoceptors. The nucleotide analogue behaves as a full agonist for the activation of glycogen phosphorylase in isolated hepatocytes, 0.3 microM being required for half-maximal activation. Specific binding of ATP alpha [35S] to hepatocytes and plasma membranes occurs within 1 min and is essentially reversible. The analysis of the dose-dependency at equilibrium indicates the presence of binding sites with Kd of 0.23 microM with hepatocytes and Kd of 0.11 microM with plasma membranes. The relative affinities of 10 nucleotide analogues were deduced from competition experiments for ATP alpha [35S] binding to hepatocytes, and these correlated highly with their biological activity (activation of glycogen phosphorylase in hepatocytes). For all the agonists, binding occurs in the same concentration range as the biological effect. These data clearly suggest that the detected binding sites correspond to the physiological P2-purinoceptors involved in the regulation of liver glycogenolysis. The rank order of potency of some ATP analogues suggests that liver possesses the P2Y-subclass of P2-purinoceptors.

The effects of noradenaline (via alpha 1-adrenoceptors) and of the peptidic hormones vasopressin and angiotensin on the Na-K pump have been studied in rat isolated liver cells. The three hormones increased the cytosolic Ca concentration, stimulated the Na-K pump and decreased the internal Na concentration of the cells. The effects were dose-dependent and were blocked by the corresponding antagonists. The simultaneous addition of maximal doses of noradrenaline and angiotensin or vasopressin were not additive suggesting that the hormones use a common mechanism to stimulate the carrier. Incubating the cells in Ca-free medium for long periods (Ca-depletion) increased the Na-K pump activity and reduced the stimulatory action of vasopressin, angiotensin and noradrenaline. The effect of the Ca indicator quin2, used as an intracellular Ca chelator, was also studied. The cells were loaded with a maximal concentration of [3H]-quin2 acetoxymethyl ester in the presence of external Ca for 6 min. The final cell content was 3.1 nmol quin2 mg-1 cell dry wt. In these cells the cytosolic Ca, as monitored from the fluorescence emission of the indicator, was about 200 nM and Na-K pump activity was normal and the cells remained responsive to the three hormones. Loading the cells with quin2 in the absence of external Ca reduced the [Ca]i from 200 nM to about 40 nM and increased the Na-K pump activity but not as a result of a rise in internal Na concentration. In addition, the rat hepatocytes were no longer sensitive to the hormones. It is proposed that Ca inhibits the Na-K pump by binding the internal sites and that vasopressin, angiotensin and noradrenaline stimulate the carrier by interfering with the inhibitory Ca sites.

The action of alpha 1-adrenergic agonists (noradrenaline in the presence of propranolol), vasopressin and angiotensin on the intracellular free Ca2+ concentration, [Ca2+]i, was determined by using the fluorescent dye quin2 in isolated rat liver cells. In the presence of external Ca2+ (1.8 mM), 1 microM-noradrenaline induced an increase in [Ca2+]i up to about 800 nM without apparent delay, whereas 10 nM-vasopressin and 1 nM-angiotensin increased [Ca2+]i to values higher than 1500 nM with a lag period of about 6s. The successive addition of the hormones and of their specific antagonists indicated that the actions of the three Ca2+-mobilizing hormones occurred without apparent desensitization (over 6 min) and via independent receptors. The relative contributions of internal and external Ca2+ pools to the cell response were determined by studying the hormone-mediated [Ca2+]i increase and glycogen phosphorylase activation in low-Ca2+ media (22 microM). In this medium: (1) [Ca2+]i was lowered and the hormones initiated a transient instead of a sustained increase in [Ca2+]i; subsequent addition (2 min) of a second hormone promoted a lesser increase in [Ca2+]i; in contrast, the subsequent addition (2 min) of Ca2+ (1.8 mM) caused [Ca2+]i to increase to a value close to that initiated by the hormone in control conditions, the amplitude of the latter response being dependent on the concentration of Ca2+ added to the medium; (2) returning to normal Ca2+ (1.8 mM) restored the resting [Ca2+]i and allowed the hormone added 2 min later to promote a large increase in [Ca2+]i whose final amplitude was also dependent on the concentration of Ca2+ added beforehand. Similar results were found when the same protocol was applied to the glycogen phosphorylase activation. It is concluded that Ca2+ influx is required for a maximal and sustained response and to reload the hormone-sensitive stores.

Hepatic plasma membranes of female obese mice C57 BL-6 orl ob/ob (ob/ob mice) completely lack vasopressin (VP) receptors of the V1 type whereas kidney VP receptors are normally expressed and functionally coupled to adenylate cyclase. To discover if these alterations are linked to a genetic defect of the V1 receptor, we have studied the binding of VP on liver and kidney membranes of two other models, female diabetic mice C57 BL-6 orl db/db (db/db mice) and female Zucker rats Fatty/orl fa/fa (fa/fa rats), which exhibit different temporal pattern of obesity, hyperinsulinemia and insulin resistance. In addition, since VP is known to exert its vascular response through stimulation of V1 receptors, we have studied the reactivity of VP of isolated tail artery in the three different models, ob/ob and db/db mice and fa/fa rats, and in their respective controls. In all cases, VP kidney receptors and VP vascular reactivity are normal. db/db mice exhibit a marked decrease in hepatic VP receptors whereas a 50% decrease was observed in 32 week fa/fa rats. Angiotensin II and prazosin binding sites are still present as well as the adenylate cyclase response to glucagon. These results suggest that the specific alteration in liver VP receptors is not related to a defect in V1 receptor genetic expression but is specific for liver and appears to parallel the level of hyperinsulinemia and/or insulin resistance.

The effects of the Ca2+-mobilizing hormones noradrenaline, vasopressin and angiotensin on the unidirectional influx of Ca2+ were investigated in isolated rat liver cells by measuring the initial rate of 45Ca2+ uptake. The three hormones increased Ca2+ influx, with EC50 values (concentrations giving half-maximal effect) of 0.15 microM, 0.44 nM and 0.8 nM for noradrenaline, vasopressin and angiotensin respectively. The actions of noradrenaline and angiotensin were evident within seconds after their addition to the cells, whereas the increase in Ca2+ influx initiated by vasopressin was slightly delayed (by 5-15s). The activation of Ca2+ influx was maintained as long as the receptor was occupied by the hormone. The measurement of the resting and hormone-stimulated Ca2+ influxes at different external Ca2+ concentrations revealed Michaelis-Menten-type kinetics compatible with a saturable channel model. Noradrenaline, vasopressin and angiotensin increased both Km and Vmax. of Ca2+ influx. It is proposed that the hormones increase the rate of translocation of Ca2+ through a common pool of Ca2+ channels without changing the number of available channels or their affinity for Ca2+.

V1 vasopressin, angiotensin, alpha-adrenergic, and glucagon receptors in liver were studied on membrane fractions prepared from two groups of jerboas ( Jaculus orientalis) given dry or water-enriched diets for periods of 4 to 7 weeks, and from rats acutely treated with pharmacological amounts of arginine-vasopressin (AVP) or (1-deamino-8-D-arginine)-vasopressin (dDAVP). Tritiated (8-lysine)-vasopressin ([3H]vasopressin), tritiated (1-asparagine-5-valine)-angiotensin II ([3H]angiotensin II), tritiated dihydroergocryptine ([3H] DHEC ), and iodinated glucagon ([125I]-glucagon) were used as specific labeled ligands of these receptors. The V1 vasopressin, angiotensin, alpha-adrenergic, and glucagon receptors detected in both groups of jerboas were identical to receptors found in rat liver plasma membranes in regard to the apparent dissociation constants for their respective labeled ligands. Furthermore, vasopressin receptors in jerboa liver membranes discriminated as efficiently as rat liver receptors between the natural neurohypophyseal peptides arginine-vasopressin and lysine-vasopressin on the one hand and the structural analogs (1-deamino-8-D-arginine)-vasopressin and (4-valine-8-D-arginine)-vasopressin on the other. The reduction of antidiuretic hormone (ADH) secretion in jerboas fed a water-enriched diet compared to those on a dry diet (75 +/- 25 pM versus 372 +/- 86 pM) was accompanied by an increase in the number of liver vasopressin receptors (2.79 +/- 0.53 versus 1.25 +/- 0.14 pmol [3H]vasopressin bound/mg protein). The modifications observed were specific for vasopressin receptors, as judged by the maximal binding capacities of [3H]angiotensin II, [3H] DHEC , and [125I]-glucagon, which remained unchanged in jerboas whatever the levels of endogenous circulating ADH. Similarly, administration of pharmacological doses of AVP by iv infusion to rats induced, 2 hr later, a loss of about 50% of V1 liver vasopressin receptors, while the numbers and apparent dissociation constants of angiotensin, alpha-adrenergic, and glucagon liver receptors remained unchanged, and V2 kidney vasopressin receptors were almost desensitized. For V1 liver and V2 kidney vasopressin receptors, the desensitization process was strikingly dependent on the antidiuretic/glycogenolytic activity ratio of the peptide used. Thus, im injection to rats of dDAVP (an analog possessing a very high antidiuretic/glycogenolytic activity ratio) induced, 1 hr later, a total loss of V2 kidney receptors without modification of the number and apparent dissociation constant of V1 liver receptors.

Angiotensin receptors from rat liver were labeled using four different ligands: (Sar1-(3H)Tyr4)-Angiotensin ll ((3H)SarAll); (Sar1-(3H)Tyr4-lle8)-Angiotensin ll ((3H)SarlleAll); (Sar1-(125I) Tyr4-(4'-N3) Phe8)-Angiotensin ll (IN3All); (Sar1-(125I)Tyr4-(4'N3D-Phe)8)-Angiotensin ll (IN3DPheAll). (3H)SarAll and IN3All behaved like agonists and (3H)SarlleAll and IN3DPheAll like antagonists. All four ligands labeled the same population of sites. The azido derivatives allowed covalent labeling of receptors with a high yield (about 40%). Membranes were solubilized by Triton X-100 under experimental conditions which ensured complete solubilization of the liganded receptors in a stable form (less than 40% dissociation after 20 h). The apparent size of liganded angiotensin receptors was determined by gel filtration on Ultrogel ACA-34 columns and by SDS gel electrophoresis (in the case of covalent labeling). The apparent Stokes radius of solubilized angiotensin receptors was different wether the receptor was labeled with an agonist (Stokes radius = 6.2 +/- 0.1 nm (6) after labeling with (3H)SarAll) or with an antagonist (Stokes radii of 5.5 +/- 0.1 (7), and 5.6 +/- 0.1 nm (4) after labeling with (3H)SarlleAll and IN3DPheAll respectively). After covalent labeling with IN3All angiotensin receptors were eluted as a mixture of light and heavy forms. SDS gel electrophoresis revealed only one molecular entity of Mr 64,000. It is concluded that binding of an agonist to liver angiotensin receptors triggers or stabilizes an interaction with another membrane component involved in the coupling of the receptor to its primary effector.

The activity of phosphorylase a was measured in isolated hepatocytes from fed lean and ob/ob mice after addition of vasopressin, angiotensin, phenylephrine and glucagon. The binding of these hormones to purified liver plasma membranes was also determined. In hepatocytes of ob/ob mice, no increase in phosphorylase a was measured after addition of vasopressin, whereas the other hormones promoted an increase in the activity of the enzyme. No specific vasopressin receptors could be measured on purified liver plasma membrane of ob/ob mice. A decrease in the number of receptors for angiotensin and glucagon, without modification of the affinity, was also observed. No restoration of the number of vasopressin receptors was observed in liver of ob/ob mice starved for 3 days or in younger (5-6 weeks) animals. Vasopressin receptors and vasopressin-stimulated adenylate cyclase, measured on purified kidney medulla membranes, were similar in both lean and ob/ob mice. The data indicate a selective lack of vasopressin receptors and metabolic response in liver of the ob/ob mouse.

Angiotensin binding and the effects of angiotensin on adenylate cyclase activity were determined on purified membranes from the glomerulosa zone of bovine adrenal cortex. Angiotensin II inhibited adenylate cyclase activity in a dose-dependent manner with an A50-value of 2 nM. The angiotensin effect required the presence of GTP. Angiotensin also inhibited ACTH-stimulated activity. Angiotensin binding was sensitive to the same effectors which influenced angiotensin-induced adenylate cyclase inhibition. In the presence of NaCl 100 mM and magnesium, angiotensin interacted with a single population of binding sites (Kd = 4 nM and maximal binding capacity of 440 fmol/mg protein). These results and published data suggest that the ability to inhibit adenylate cyclase might be a general property of angiotensin receptors.

Rat hepatocytes rapidly incorporate [32P]Pi into phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]; their monoester phosphate groups approach isotopic equilibrium with the cellular precursor pools within 1 h. Upon stimulation of these prelabelled cells with Ca2+-mobilizing stimuli (V1-vasopressin, angiotensin, alpha 1-adrenergic, ATP) there is a rapid fall in the labelling of PtdIns4P and PtdIns(4,5)P2. Pharmacological studies suggest that each of the four stimuli acts at a different population of receptors. Insulin, glucagon and prolactin do not provoke disappearance of labelled PtdIns4P and PtdIns(4,5)P2. The labelling of PtdIns4P and PtdIns(4,5)P2 in cells stimulated with vasopressin or angiotensin initially declines at a rate of 0.5-1.0% per s, reaches a minimum after 1-2 min and then returns towards the initial value. The dose-response curves for the vasopressin- and angiotensin-stimulated responses lie close to the respective receptor occupation curves, rather than at the lower hormone concentrations needed to evoke activation of glycogen phosphorylase. Disappearance of labelled PtdIns4P and PtdIns(4,5)P2 is not observed when cells are incubated with the ionophore A23187. The hormone-stimulated polyphosphoinositide disappearance is reduced, but not abolished, in Ca2+-depleted cells. These hormonal effects are not modified by 8-bromo cyclic GMP, cycloheximide or delta-hexachlorocyclohexane. The absolute rate of polyphosphoinositide breakdown in stimulated cells is similar to the rate previously reported for the disappearance of phosphatidylinositol [Kirk, Michell & Hems (1981) Biochem. J. 194, 155-165]. It seems likely that these changes in polyphosphoinositide labelling are caused by hormonal activation of the breakdown of PtdIns(4,5)P2 (and may be also PtdIns4P) by the action of a polyphosphoinositide phosphodiesterase. We therefore suggest that the initial response to hormones is breakdown of PtdIns(4,5)P2 (and PtdIns4P?), and that the simultaneous disappearance of phosphatidylinositol might be a result of its consumption for the continuing synthesis of polyphosphoinositides.