DuP 532: a second generation of nonpeptide angiotensin II receptor antagonists

DuP 532 is a novel nonpeptide angiotensin II (AII) receptor antagonist under development for the treatment of hypertension. DuP 532 is a more potent antihypertensive agent in renal hypertensive rats (ED30 = 0.042 mg/kg, i.v.) and displays a similar or longer duration of action than the previously described AII antagonist, DuP 753. DuP 532, in contrast to DuP 753, is a noncompetitive antagonist of AII-induced contractions of rabbit aortic strips (KB = 1.1 x 10(-10) M). However, the inhibition of AII binding by DuP 532 in rat adrenal cortex does not correlate with either the aortic contractile response or with the hypotensive response. Assay conditions were evaluated and the presence or absence of BSA was shown to markedly affect the apparent binding affinity of DuP 532 and other 5-carboxylic acid derivatives. DuP 753 and other compounds were much less affected. The IC50 for DuP 532 was 4.7 x 10(-6) M with and 3 x 10(-9) M without BSA. The IC50s for DuP 753 were 1.7 x 10(-8) M with and 5 x -9 M without BSA. Both compounds with or without BSA did not completely inhibit AII binding which is characteristic of AT1 selectivity. BSA also reduced the effect of DuP 532 on the AII-induced contractions of rat main pulmonary artery preparations and the AII-induced Ca2+ mobilization in rat aortic smooth muscle cells. DuP 532 was very specific for AT1 receptors and did not interfere with receptors associated with neurotensin, prazosin, bradykinin, nitrendipine, or vasopressin. It is concluded that DuP 532 represents a new class of specific, but noncompetitive. AII receptor antagonists whose binding characteristics may provide new insight into AII receptor function.     

Identification of angiotensin II receptor subtypes

We have demonstrated the existence of two distinct subtypes of the angiotensin II receptor in the rat adrenal gland using radioligand binding and tissue section autoradiography. The identification of the subtypes was made possible by the discovery of two structurally dissimilar, nonpeptide compounds, DuP 753 and EXP655, that show reciprocal selectivity for the two subtypes. In the rat adrenal cortex, DuP 753 inhibited 80% of the total AII binding with an IC50 value on the sensitive sites of 2 x 10(-8) M, while EXP655 displaced only 20%. In the rat adrenal medulla, EXP655 gave 90% inhibition of AII binding with an IC50 value of 3.0 x 10(-8) M, while DuP 753 was essentially inactive. The combination of the two compounds completely inhibited AII binding in both tissues.     

DuP 753, the selective angiotensin II receptor blocker, is a competitive antagonist to human platelet thromboxane A2/prostaglandin H2 (TP) receptors.

DuP 753 is a potent, selective angiotensin II type 1 (AT1) receptor antagonist. The possibility was investigated that DuP 753 may crossreact with thromboxane A2/prostaglandin H2 (TP) receptors. DuP 753 inhibited the specific binding of the TP receptor antagonist [3H]SQ 29,548 (5 nM) in human platelets with kd/slope factor values of 9.6 +/- 1.4 microM/1.1 +/- 0.02. The AT2-selective angiotensin receptor ligand, PD 123,177 was a very weak inhibitor of specific [3H]SQ 29,548 binding in platelets (Kd/slope factor:200 microM/0.86). [3H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (Kd: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (Bmax: 3597, 4597, 3109 fmol/mg protein, respectively). Platelet aggregation induced by the TP receptor agonist U 46,619 was concentration-dependently inhibited by DuP 753 (IC50 = 46 microM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors.     

Molecular characterization of angiotensin II type II receptors in rat pheochromocytoma cells.

The binding sites and biochemical effects of angiotensin (A) II were investigated in rat pheochromocytoma (PC12W) cells. Sarcosine1, [125I]-tyrosine4, isoleucine8-AII ([125I]-SI-AII) bound to a saturable population of sites on membranes with an equilibrium dissociation constant (Kd) of 0.4 nM and a binding site maximum of 254 fmol/mg protein. Competitive displacement of [125I]-SI-AII by agonists and antagonists elucidated a rank order of potency of AIII greater than or equal to AII greater than PD 123177 greater than AI greater than [des-Phe]AII [AII(1-7)] much greater than DuP 753. The stable guanine nucleotide analog 5'-guanylyl imidodiphosphate did not alter the binding affinity or slope of the inhibition curves for AI, AII, AIII, or AII(1-7). Treatment of PC12W cells with AII or AIII did not affect the free intracellular calcium concentration, phosphoinositide metabolism, arachidonate release, cyclic GMP, or cyclic AMP concentrations. [125I]-AII binding sites remained on the cell surface and were not internalized after 2 h at 37 degrees C. Angiotensin II did not stimulate tyrosine, serine, or threonine phosphorylation. Northern analysis of PC12W mRNA with an AT1 receptor gene probe failed to produce an RNA:DNA hybrid at low stringency. These data indicate that PC12W cells express a homogeneous population of AT2 binding sites which differ significantly from AT1 receptors in signal transduction and molecular structure. AT2 sites may act via potentially novel, biochemical pathways or, alternatively, be vestigial receptors.     

Discrimination of angiotensin II receptor subtype distribution in the rat brain using non-peptidic receptor antagonists

The non-peptidic angiotensin II receptor subtype selective antagonists, DuP 753 and PD123177, were used to characterize angiotensin II receptor binding sites in the rat brain. Competitive receptor autoradiography with 125I-Sar1-Ile8 angiotensin II defined a regional distribution of binding sites that were sensitive to either DuP 753 (designated AII alpha subtype) or PD123177 (designated AII beta subtype). Whereas most brain nuclei could be assigned to a category containing a predominant subtype, a multiple receptor subtype analysis indicated that some regions are homogeneous, while others contain a mixture of both AII alpha and AII beta subtypes.     

Specific, high-affinity binding sites for angiotensin II on Mycoplasma hyorhinis.

Mycoplasmataceae are known to express various proteins that are similar to those present in mammals. We report a strain of Mycoplasma hyorhinis isolated from opossum kidney cells with specific, high-affinity binding sites for human angiotensin II (Kd = 5.1 +/- 1.9 nM). In contrast, two strains of M. hominis revealed no specific binding. These binding sites resembled mammalian angiotensin II receptors by their high affinity and by their sensitivity to dithiothreitol. However, they are different from mammalian angiotensin II receptors in that they bind angiotensin I with high affinity (Kd = 1.6 +/- 0.29 nM) but not angiotensin III (Kd approximately 330,000 nM). [125I]-angiotensin II binding was not inhibited by angiotensin receptor subtype antagonists DuP 753 and CGP 42112A but it was sensitive to bacitracin and aprotinin. Positions Asp1, Ile5, His6 and Pro7 were essential for binding to M. hyorhinis as deletion of these residues led to a more than 10,000-fold decrease in affinity.     

Angiotensin II stimulates phosphoinositide turnover and phosphorylase through AII-1 receptors in isolated rat hepatocytes

Angiotensin II stimulated the activity of phosphorylase a (EC50 approximately 3 nM). The effect of two receptor subtype-selective nonpeptide antagonists, DuP 753 (AII-1 selective) and PD123177 (AII-2 selective), was studied. It was observed that DuP 753 inhibited the effect of angiotensin II (IC50 100 nM) but in contrast, PD123177 was without effect on this action of the peptide hormone. Angiotensin II stimulated the labeling of phosphatidylinositol (resynthesis) and the release of inositol phosphates (breakdown). These effects of angiotensin II were blocked by DuP 753 but not by PD123177. The antagonists were without effect by themselves on these parameters. The results clearly indicate that angiotensin II receptors of the AII-1 subtype are coupled to phosphoinositide turnover and mediate phosphorylase activation in isolated rat hepatocytes.     

DuP 753, the selective angiotensin II receptor blocker, is a competitive antagonist to human platelet thromboxane A2/prostaglandin H2 (TP) receptors

DuP 753 is a potent, selective angiotensin II type 1 (AT1) receptor antagonist. The possibility was investigated that DuP 753 may crossreact with thromboxane A₂/prostaglandin H₂ (TP) receptors. DuP 753 inhibited the specific binding of the TP receptor antagonist [³H]SQ 29,548 (5 nM) in human platelets with k_d/slope factor values of 9.6±1.4 μM/1.1±0.02. The AT2-selective angiotensin receptor ligand, PD 123,177 was a very weak inhibitor of specific [³H]SQ 29,548 binding in platelets (K_d/slope factor:200 μM/0.86). [³H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (K_d: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (B_max: 3597, 4597, 3109 fmol/mg protein, respectively). Platelet aggregation induced by the TP receptor agonist U 46,619 was concentration-dependently inhibited by DuP 753 (IC₅₀=46 μM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors.     

Pharmacological characterization of angiotensin II binding sites in the canine pancreas.

High affinity 125I-angiotensin II (Ang II) binding sites were characterized in the canine pancreas. Total binding increased with protein concentration and equilibrium was reached within 60-90 min at 22 degrees C. Specific binding was saturable and averaged 70% of total. Scatchard analysis of binding yielded a KD of 0.48 +/- 0.18 nM with a Bmax of 32.8 +/- 6.5 fmol/mg protein (mean +/- SEM, n = 6). The addition of the reducing agent dithiothreitol increased specific binding two-fold. The rank order of displacement of 125I-Ang II binding by native angiotensin peptides was Ang II greater than or equal to Ang III greater than AngI greater than Ang(1-7) much greater than Ang(1-6). The use of the specific Ang II antagonists CGP 42112A, PD 123177, and DuP 753 revealed that the pancreas expresses two receptor subtypes. The majority of Ang II binding sites in the pancreas could be classified as type 2 (AT2), although type 1 (AT1) sites were also detected. In vitro autoradiography revealed binding sites localized over islet cells, acinar and duct cells, as well as the pancreatic vasculature. In addition, the autoradiographic studies confirmed the predominance of the AT2 receptor subtype throughout the pancreas.     

Effect of DuP 753, a nonpeptide angiotensin II receptor antagonist, on the drinking responses to acutely administered dipsogenic agents in rats.

The present studies examine the effect of the nonpeptide angiotensin II (AII) type 1 receptor antagonist, DuP 753, on water intake in rats treated with dipsogenic stimuli, which are thought to induce drinking via release of renin and subsequent formation of AII. Subcutaneous administration of DuP 753 in doses that are known to inhibit drinking induced by AII failed to inhibit the water intake of rats following subcutaneous administration of the beta-adrenoceptor agonist isoproterenol. The peptide antagonist1 Sar, 8Ileu-AII, which blocks both AII type 1 and AII type 2 receptors, also failed to inhibit isoproterenol-induced drinking, suggesting that neither subtype is involved in this drinking response. Additional studies verified previous reports that acute subcutaneous administration of both the beta-adrenoceptor antagonist propranolol and the angiotensin I-converting enzyme inhibitor captopril could block the drinking response to subcutaneous administration of isoproterenol. Subcutaneous administration of DuP 753 also failed to inhibit the drinking responses to subcutaneous administration of serotonin, 5-hydroxytryptophan, hypertonic saline, and polyethylene glycol. However, central intraventricular administration of DuP 753 inhibited the drinking response to subcutaneous administration of isoproterenol. The results are discussed in terms of the importance of AII in mediating isoproterenol-, serotonin-, and 5-hydroxytryptophan-induced water intake and suggest a need to readdress this mechanism.     

Physicochemical characterization of photoaffinity-labeled angiotensin II receptors

Specific receptor sites for angiotensin II (AII) were analyzed in the adrenal cortex and other target tissues including liver, anterior pituitary gland, and smooth muscle, after covalent labeling with the radioactive photoaffinity analog 125I-[Sar1,(4-N3)Phe8]-AII. The photoreactive AII derivative retained high affinity for adrenal receptors and full steroidogenic activity in adrenal glomerulosa cells. In bovine adrenal cortex, covalent labeling of AII receptors by the photoreactive analog was specifically inhibited by [Sar1]AII with an IC50 of about 5 nM. The Mr of the covalent AII-receptor complex during polyacrylamide gel electrophoresis of the labeled protein under reducing conditions was 58,000. Under non-reducing conditions, a minor band with Mr of 105,000 was also observed. Two labeled species were also found during gel permeation chromatography of the detergent-solubilized complex, with Mrs of 64,000 and 86,000. The pl of the solubilized photolabeled complex was absorbed to wheat germ lectin Sepharose 6MB and could be eluted by N-acetylglucosamine. The Mrs of specific AII-binding sites in several target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed significant differences within and between species. The most striking differences were between rat adrenal cortex (79,000) and both rat liver (60,000) and bovine adrenal cortex (58,000). After enzymatic deglycosylation, the Mr of the major component present in the bovine and rat adrenal cortex decreased by 40% and 55% to 35,000 and 34,000, respectively, suggesting that variations in carbohydrate content contribute to the physical heterogeneity of AII receptors in individual target tissues.     

Localization of angiotensin II receptor subtypes in the rabbit adrenal and kidney

The localization of subtypes of the angiotensin II receptor has been determined by autoradiographic techniques using iodinated angiotensin II and two nonpeptide antagonists that exhibit selective affinities. DuP 753 specifically displaces type 1 sites (AII-1) and PD123177 inhibits only type 2 sites (AII-2). The rabbit adrenal cortex contains predominately AII-1 sites and the few AII-2 sites that are present are nonuniformly distributed. In the rabbit kidney, the fibrous outer sheath contains exclusively AII-2 sites whereas the glomeruli of the renal cortex and the renal medulla exhibit only AII-1 sites.     

AT1 Angiotensin Receptors Mobilize Intracellular Calcium in a Subclone of NG108–15 Neuroblastoma Cells

The effects of angiotensin II (AII) and related peptides on the mobilization of internal Ca2+ were studied in a subclone of NG 108-15 cells. The subclone, C1, was prepared by fluorescence-activated cell cloning using a rapid response kinetics and a large response magnitude following stimulation by AII as the selection criteria. Angiotensin I, AII, and angiotensin III (AIII) stimulated Ca2+ mobilization in the C1 cells in a concentration-dependent manner (1 nM-100 microM), yielding EC50 values of 437 +/- 80 nM (n = 4; slope = 1.6 +/- 0.3), 57 +/- 8 nM (n = 12; slope = 1.5 +/- 0.3), and 36 +/- 5 nM (n = 7; slope = 1.4 +/- 0.3), respectively. AIII was significantly more potent than AII (p less than 0.05). In contrast, Des-Phe8-AII, AII-hexapeptide (AII 3-8), and p-NH2-Phe6-AII (1-10 microM) were inactive as agonists. Although the effects of AII and AIII in C1 and parent NG108-15 cells were totally inhibited by the AT1 receptor-selective nonpeptide antagonist, DUP-753 (0.3-1 microM), the AT2-selective antagonists, EXP-655 and CGP42112A (1-10 microM), failed to block the effects of AII. DUP-753 (0.3-100 nM) produced dextral shifts of the AII-induced concentration-response curves and yielded an estimated affinity constant (pA2) of 8.5 +/- 0.2 (n = 16) using single-point analysis involving different concentrations of DUP-753. These data compared well with those obtained for the inhibition of AII-induced aortic contractions by DUP-753 (pA2 = 8.5) reported previously by others.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of angiotensin II receptor subtypes in rat liver

Radioligand binding studies identified two classes of 125I-angiotensin II-binding sites in rat liver membranes. High affinity binding sites (Kd = 0.35 +/- 0.13 nM, N = 372 +/- 69 fmol/mg of protein) were inactivated by dithiothreitol (0.1-10 mM) without any apparent change in low affinity binding sites (Kd = 3.1 +/- 0.8 nM, N = 658 +/- 112 fmol/mg of protein). Dithiothreitol inactivation was readily reversible but could be made permanent by alkylation of membrane proteins with iodoacetamide. Angiotensin II stimulation of glycogen phosphorylase in isolated rat hepatocytes (maximal stimulation 780%, EC50 = 0.4 nM) was completely inhibited by 10 mM dithiothreitol, a concentration which also abolished high affinity site binding; phosphorylase stimulation by glucagon and norepinephrine under these conditions was unaltered. Angiotensin II inhibition of glucagon-stimulated adenylate cyclase activity in hepatocytes required higher angiotensin II concentrations (EC50 = 3 nM) than phosphorylase stimulation and was not affected by dithiothreitol. Fractional occupancy of high affinity binding sites by 125I-angiotensin II correlated closely with angiotensin II-mediated phosphorylase stimulation, whereas occupancy of low affinity sites paralleled inhibition of adenylate cyclase activity. These data indicate that the physiologic effects of angiotensin II in rat liver are mediated by two distinct receptors, apparently not interconvertible, and provide the first evidence for angiotensin II receptor subtypes with differing biochemical features and mechanisms of action.     

The conformation and activity relationship of benzofuran type of angiotensin II receptor antagonists

As a continuing effort to establish the structure and activity relationship in a benzofuran type of angiotensin II antagonist, we synthesized various regioisomers and performed a series of QSAR analyses. The conformational analyses of target isomers were carried out using molecular mechanics and fine-tuned using the information from the NMR NOE experiment. The conformations of compounds with a good binding activity are quite similar to that of DuP753, a prototype of AII antagonist, suggesting that these compounds also bind to the same site of AII receptor. We then studied the compounds with a varied length of the hydroxyl group bearing side chain to find out the optimum distance between the hydroxyl group and the imidazole ring. The CoMFA with these compounds gave acceptable statistical measures (cross-validated r2 and conventional r2 to be 0.881 and 0.974, respectively) and the map was well consistent with the previously proposed pharmacophore.     

Molecular cloning of a ferret angiotensin II AT(1) receptor reveals the importance of position 163 for Losartan binding

A complementary DNA for the angiotensin II (AngII) type 1 (AT(1)) receptor from Mustela putorius furo (ferret) was isolated from a ferret atria cDNA library. The cDNA encodes a protein (fAT(1)) of 359 amino acids having high homologies (93-99%) to other mammalian AT(1) receptor counterparts. When fAT(1) was expressed in COS-7 cells and photoaffinity labeled with the photoactive analogue (125)I-?Sar(1), Bpa(8)AngII, a protein of 100 kDa was detected by autoradiography. The formation of this complex was specific since it was abolished in the presence of the AT(1) non-peptidic antagonist L-158,809. Functional analysis indicated that the fAT(1) receptor efficiently coupled to phospholipase C as demonstrated by an increase in inositol phosphate production following stimulation with AngII. Binding studies revealed that the fAT(1) receptor had a high affinity for the peptide antagonist ?Sar(1), Ile(8)AngII (K(d) of 5. 8+/-1.4 nM) but a low affinity for the AT(1) selective non-peptidic antagonist DuP 753 (K(d) of 91+/-15.6 nM). Interestingly, when we substituted Thr(163) with an Ala residue, which occupies this position in many mammalian AT(1) receptors, we restored the high affinity of this receptor for Dup 753 (11.7+/-5.13 nM). These results suggest that position 163 of the AT(1) receptor does not contribute to the overall binding of peptidic ligands but that certain non-peptidic antagonists such as Dup 753 are clearly dependent on this position for efficient binding.     

The influence of glucose concentration on angiotensin II-induced hypertrophy of proximal tubular cells in culture.

Incubation of cultured murine proximal tubular cells in serum-free media containing 450 mg/dl of glucose resulted in cellular hypertrophy as defined by an increase in cell size, total protein content, and synthesis after 72 h. 10 nM angiotensin II further increased this hypertrophy, but failed to have any effect on cells grown in 100 mg/dl glucose. This enhancement by angiotensin II was blocked by treatment with 1 microM of the angiotensin-receptor antagonist DuP 753. Although cells incubated in either glucose media exhibited similar high-affinity angiotensin II-receptors, the receptor density was elevated only in cells grown in the presence of high glucose. Stimulation of cells in high glucose for 60 min with 10 nM angiotensin II also reduced significantly intracellular cAMP concentrations. This was not the case for proximal tubular cells cultured in normal glucose. Our results indicate that high glucose and angiotensin II have additive effects on the induction of hypertrophy in renal tubular cells.     

Relevant role of Leu265 in helix VI of the angiotensin AT1 receptor in agonist binding and activity

The finding of critical residues for angiotensin II (AII) binding and receptor signalling in helices V and VI led us to assess if, in this region of the receptor, aliphatic side chains might play a role in the agonist-mediated mechanism. Two mutations of the angiotensin AT1 receptor were designed to explore a possible role of a leucine at two positions, Leu265 and Leu268. Thus two mutants, L265D and L268D, were prepared through single substitutions of Leu265, located in the C-terminal region of transmembrane VI (TM-VI), and Leu268, in the adjoining region of the third extracellular loop (EC-3), for an aspartyl residue, and were stably transfected into Chinese hamster ovary (CHO) cells. Ligand-binding experiments and the functional assays determining inositol phosphate (IP) production were performed in these cells expressing these mutants. No significant changes were found in the binding affinity for the ligands, AII, DuP753, and [Sar1Leu8]AII in the mutant L268D. Moreover, the relative potency and the maximum effect on IP production of this mutant were similar to those of the wild-type receptor. In contrast, L265D mutant AT1 receptor, located within the transmembrane domain, markedly decreased binding affinity and ability to stimulate phosphatidylinositol turnover. Our results suggest that the hydrophobic side chain of Leu265, at the C-terminal portion of the AT1's TM-VI, but not Leu268, which belongs to the EC-3 loop, might interact with the AII molecule. On the other side, the aliphatic side chain of Leu265 may be involved in the formation of the ligand binding sites through allosteric effects, thus helping to stabilize the receptor structure around the agonist binding site for full activity.     

Intracisternal galanin/angiotensin II interactions in central cardiovascular control

The aim of this work was to investigate the interactions between angiotensin II (Ang II) and galanin(1-29) [GAL(1-29)] or its N-terminal fragment galanin(1-15) [GAL(1-15)] on central cardiovascular control. The involvement of angiotensin type1 (AT1) receptor subtype was analyzed by the AT1 antagonist, DuP 753. Anesthesized male Sprague-Dawley rats received intracisternal microinjections of Ang II (3 nmol) with GAL(1-29) (3 nmol) or GAL(1-15) (0.1 nmol) alone or in combination. The changes in mean arterial pressure (MAP) and heart rate (HR) recorded from the femoral artery were analyzed. The injection of Ang II and GAL(1-15) alone did not produce any change in MAP. However, coinjections of both Ang II and GAL(1-15) elicited a significant vasopressor response. This response was blocked by DuP 753. Ang II and GAL(1-15) alone produced an increase in HR. The coinjections of Ang II with GAL(1-15) induced an increase in HR not significantly different from the tachycardia produced by each peptide. The presence of DuP 753 counteracted this response. GAL(1-29) alone elicited a transient vasopressor response that disappeared in the presence of Ang II. The coinjections of Ang II with GAL(1-29) and with DuP 753 restored the transient vasopressor effect produced by GAL(1-29). GAL(1-29) produced a slight but significant tachycardic effect that was not modified in the presence of Ang II. The presence of DuP 753 did not modify the tachycardic response produced by Ang II and GAL(1-29). These results give indications for the existence of a differential modulatory effect of Ang II with GAL(1-15) and GAL(1-29) on central blood pressure response that might be dependent on the activity of the angiotensin AT1 receptor subtype.     

Two distinct pathways in the down-regulation of type-1 angiotension II receptor gene in rat glomerular mesangial cells.

The mRNA level of the type-1 angiotensin II receptor (AT1) was down-regulated by angiotensin II in cultured rat glomerular mesangial cells. The effect was maximum with 1 microM AII at 6 h, sensitive to cycloheximide, and specific to AT1 since this phenomenon was blocked by DuP753, an AT1 antagonist, but not by type-2 antagonist PD123319. Dibutyryl cAMP, forskolin, and cholera toxin also caused AT1 down-regulation. These effects were not altered by either the protein kinase A inhibitor H-8 or cycloheximide. Calcium ionophore A23187, pertussis toxin, protein kinase C inhibitor staurosporine, or prolonged incubation with phorbol ester were without effect. These results suggest that there are at least two pathways to down-regulate AT1 mRNA; one way is an angiotensin II-induced, protein kinase C-independent, and cycloheximide-sensitive pathway and the other is an angiotensin II-independent, cAMP-induced, and cycloheximide-insensitive pathway.