Adrenal Cortical Membranes Research Articles

Identification of Five Genes as a Potential Biomarker for Predicting Progress and Prognosis in Adrenocortical Carcinoma.

Background: Adrenocortical carcinoma (ACC) is a limited endocrine fatality with a minor diagnosis and rare remedial options. The progressive and predictive meaning of message RNA (mRNA) expression oddity in ACC has been studied extensively in recent years. However, differences in measurement platforms and lab protocols as well as small sample sizes can render gene expression levels incomparable.Methods: An extensive study of GEO datasets was conducted to define potential mRNA biomarkers for ACC. The study compared the mRNA expression profiles of ACC tissues and neighboring noncancerous adrenal tissues in the pair. The study covered a sum of 165 tumors and 36 benign control samples. Hub genes were identified through a protein-protein interaction (PPI) network and Robust Rank Aggregation method. Then the Cancer Genome Atlas (TCGA) and Oncomine database were used to perform the validation of hub genes. 4 ACC tissues and 4 normal tissues were collected and then Polymerase Chain Reaction (PCR), Western-blot and immunofluorescence were conducted to validate the expression of five hub genes.Results: We identified five statistically significant genes (TOP2A, NDC80, CEP55, CDKN3, CDK1) corrected with clinical features. The expression of five hub genes in TCGA and Oncomine database were significantly overexpressed in ACC compared with normal ones. Among all the TCGA ACC cases, the strong expression of TOP2A (logrank p=1.4e-04, HR=4.7), NDC80 (logrank p=8.8e-05, HR=4.9), CEP55 (logrank p=5.2e-07, HR=8.6), CDKN3 (log rank p=2.3e-06, HR=7.6) and CDK1 (logrank p=7e-08, HR=11) were correlated with low comprehensive survival, disease free survival (logrank p < 0.001), pathology stage and pathology T stage (FDR < 0.001). PCR results showed that the transcriptional levels of these five genes were significantly higher in ACC tissues than in normal tissues. The western blotting results also showed that the translational level of TOP2A was significantly higher in tumor tissues than in normal tissues. The results of immunofluorescence showed that TOP2A was abundantly observed in the adrenal cortical cell membrane and nucleus and its expression in ACC tissues was significantly higher than that in normal tissues.Conclusions: The distinguished five genes may be utilized to form a board of progressive and predictive biomarkers for ACC for clinical purpose.

Novel Ouabain Receptors in Bovine Adrenal Cortex: Evidence for Ouabain Transport

Novel high affinity binding sites for ouabain (NOR) are present in bovine adrenal cortex (Ward SC, Hypertension 2002; 39[pt. 2]:536). To distinguish NOR from classical sodium‐potassium pumps (Na pumps), we used fresh adrenal cortical membranes. Scatchard analysis showed that K+ (20mM) reduced the apparent Kd for ouabain binding to Na pumps from 4.6 ± 0.2 to 240 ± 7.2 nM with no change in Bmax (10.1 ± 0.1 vs. 10.3 ± 0.2 pmoles/mg). K+ increased the Kd for ouabain binding to NOR and increased the Bmax (0.15 ± 0.02 vs. 0.26 ± 0.06 pmoles/mg). Moreover, the effect of Na+ was more dramatic; Na+ inhibited ouabain binding to Na pumps with no change in Bmax, while Na+ increased the apparent Kd for ouabain binding to NOR and increased Bmax &gt; 25‐fold. Ouabain binding to the Na pump in stored membranes remained constant for up to 50 days whereas NOR related binding declined with a t1/2 of 6.8 days. The association of ouabain with membranes was 0.538 pmoles/mg when [Na+]o » [Na+]i, and was 0.152 pmoles/mg when [Na+]o « [Na+]i, and was sensitive to permeable anions. NOR binding was sensitive to osmolarity. Photoactive analogs of ouabain labeled a 35 kDa polypeptide. We conclude that NOR mediates the secondary active sodium‐dependent transport of 3H‐ouabain into sealed membrane vesicles and that this process is electrogenic. NOR may mediate the secretion of cardiac glycosides by the adrenal cortex in vivo. Supported by HL078870 and HL075584.

Differential stereoselectivity of d- and l- myo-inositol 1,2,4,5-tetrakisphosphate binding to the inositol 1,4,5-trisphosphate receptor and 3-kinase

d- and l- myo-inositol 1,2,4,5-tetrakisphosphate (Ins(1,2,4,5)P 4) were investigated for their ability to bind to the d- myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3) receptor in a bovine adrenal cortical membrane fraction, to mobilize intracellular Ca 2+ stores in Xenopus oocytes, and to bind to the rat brain Ins(1,4,5)P 3 3-kinase overexpressed and purified in E. coli. In competitive binding experiments with the Ins(1,4,5)P 3 receptor, d-Ins(1,2,4,5)P 4 effectively displaced [ 3H]Ins(1,4,5)P 3 in a concentration-dependent manner with a potency comparable to that of d-Ins(1,4,5)P 3, while l-Ins(1,2,4,5)P 4 was ∼50-fold less effective than d-Ins(1,4,5)P 3 and d-Ins(1,2,4,5)P 4. The dl-Ins(1,2,4,5)P 4 racemate bound to the Ins(1,4,5)P 3 receptor with an apparent intermediate efficiency. Injection of d-Ins(1,2,4,5)P 4 into oocytes evoked a chloride current dependent on intracellular Ca 2+ mobilization in which the agonists ranked in a similar order of potency as in the Ins(1,4,5)P 3 receptor binding. On the other hand, d-Ins(1,2,4,5)P 4 only inhibited the binding of [ 3H]Ins(1,4,5)P 3 to 3-kinase very weakly with a markedly reduced potency compared to d-Ins(1,4,5)P 3, indicating that d-Ins(1,2,4,5)P 4 is not an effective competitor in the phosphorylation of [ 3H]-Ins(1,4,5)P 3 by 3-kinase. The results, therefore, clearly indicate that d-Ins(1,2,4,5)P 4 is as effective as d-Ins(1,4,5)P 3 in the binding to the receptor but not 3-kinase, and access of Ins(1,2,4,5)P 4 over the Ins(1,4,5)P 3 receptor calls for stringent stereospecificity with d-Ins(1,2,4,5)P 4 being the active form in dl-Ins(1,2,4,5)P 4-mediated Ca 2+ mobilization.

Binding and activity of the nine possible regioisomers of myo-inositol tetrakisphosphate at the inositol 1,4,5-trisphosphate receptor

All 9 racemic regiosomers (15 enantiomerically) of myo-inositol tetrakisphosphates (IP 4s): DL-Ins(1,2,4,5)P 4 [ A], DL-Ins(1,2,4,6)P 4 [ B], Ins(1,2,3,5)P 4 [ C], Ins(1,3,4,6)P 4 [ D], Ins(2,4,5,6)P 4 [ E], DL-Ins(1,3,4,5)P 4 [ F], DL-Ins(1,2,5,6)P 4 [ G], DL-Ins(1,2,3,4)P 4 [ H] and DL-Ins(1,4,5,6)P 4 [ I] [Chang S-K., Chang YT. Synthesis of all possible regioisomers of myo-inositol tetrakisphosphate. J Chem Soc Chem Commun 1995; 11–13] were investigated for their ability to bind to the D- myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3] receptor in bovine adrenal cortical membranes, and for their ability to mobilize 45Ca 2+ from Ins(1,4,5)P 3-sensitive Ca 2+ stores in permeabilized Chinese hamster ovary (CHO) cells. DL-Ins(1,2,4,5)P 4 (K i = 11 nM) bound to Ins(1,4,5)P 3 receptors with an affinity only 2-fold lower than Ins(1,4,5)P 3 (K i = 6 nM), Ins(1,2,3,5)P 4 Ins(1,3,4,6)P 4, Ins(2,4,5,6)P 4, DL-Ins(1,3,4,5)P 4, DL-Ins(1,2,3,4)P 4 and DL-Ins(1,4,5,6)P 4 bound with affinities of between 0.4–0.7 μM. DL-Ins(1,2,4,6)P 4 and DL-Ins(1,2,5,6)P 4 bound to the Ins(1,4,5)P 3 receptor with low affinity (approximately 2–3 μM). All but one of the IP 4s mediated release of 45Ca 2+ from stores of permeabilized CHO cells with a similar rank order of potency as that for Ins(1,4,5)P 3 receptor binding, being between 16-fold and 50-fold less potent at releasing 45Ca 2+ compared with their apparent binding affinities to the Ins(1,4,5)P 3 receptor. The notable exception was Ins(1,2,3,5)P 4, which showed an approximately 200-fold lower potency compared with its affinity for the Ins(1,4,5)P 3 receptor. Ins(1,2,3,5)P 4 may be a useful lead compound for the rational design of novel synthetic Ins(1,4,5)P 3 analogues possessing structure-activity profiles with relatively high binding affinity, but low intrinsic efficacy, and hence partial agonists and antagonists at the Ins(1,4,5)P 3 receptor.

The pharmacologic profile of 606A, a novel angiotensin II receptor antagonist.

The pharmacologic profile of a novel angiotensin I (AT1) receptor antagonist 606A was studied in various in vitro and in vivo preparations. The 606A showed a high affinity at AT1 receptors [inhibition constant (Ki), 12.8 +/- 0.4 nM] in rabbit adrenal cortical membrane and a low affinity to AT2 receptors (Ki, > 1 mM) in bovine cerebellar membrane, indicating potent and selective AT1 properties. In the guinea pig aorta, 606A reduced the maximal angiotensin II-induced contraction (pD'2, 9.06 +/- 0.04), whereas EXP3174 showed suppression of the maximum response and a shift to the right of the concentration-response curve at lower and higher concentrations, respectively (conventionally calculated pd'2, 8.61 +/- 0.23). The 606A had no effects on KC1-, norepinephrine-, serotonin-, and endothelin-1-induced contractions or any agonist activities. In anesthetized dogs, 606A inhibited the angiotensin II-induced pressor response 35 times more potently than losartan. In renal hypertensive rats and spontaneously hypertensive rats (SHRs), 606A decreased systolic blood pressure 10 and 3 times more potently than losartan, respectively, without any chronotropic effects. By repeated administration of 606A to SHRs for 2 weeks, an augmentation of the hypotensive effect was observed No rebound phenomena occurred after discontinuation. These results indicate the 606A is a potent AT1-selective insurmountable angiotensin II receptor antagonist having more potent angiotensin II receptor antagonistic and hypotensive effects than losartan in in vivo models. 606A is suggested to be a useful agent for the treatment of patients with hypertension.

Candesartan (CV-11974) dissociates slowly from the angiotensin AT 1 receptor

The mechanisms of the insurmountable antagonism of 2-ethoxy-1-[[2′-(1 H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1 H-benzimidazole-7-carboxylic acid, candesartan (CV-11974), an angiotensin AT 1 receptor antagonist, on angiotensin II-induced rabbit aortic contraction were examined in contraction and binding studies. Preincubation of the rabbit aorta with CV-11974 (0.1 nM) for 30 min reduced the maximal contractile response to angiotensin II by approximately 50%. This insurmountable antagonism of CV-11974 was reversed in the presence of losartan (1 μM), a surmountable angiotensin AT 1 receptor antagonist. The inhibitory effect of CV-11974 on angiotensin II-induced contraction persisted longer after washing than did that of losartan but was not irreversible. Scatchard analysis of [ 3H]CV-11974 binding in bovine adrenal cortical membranes indicated the existence of a single class of binding sites ( K d = 7.4 nM). Competition binding studies using angiotensin II receptor agonists and antagonists have demonstrated that [ 3H]CV-11974 binding sites may be identical to angiotensin AT 1 receptors. The dissociation rate of [ 3H]CV-11974 binding ( t 1 2 = 66 min ) was 5 times slower than that of [ 125I]angiotensin II binding ( t 1 2 = 12 min ). These results suggest that the insurmountable antagonism by CV-11974 is due to its slow dissociation from angiotensin AT 1 receptors.

N-3-substituted pyrimidinones as potent, orally active, AT1 selective angiotensin II receptor antagonists.

A novel series of nonpeptide angiotensin II (A II) antagonists containing a pyrimidinone ring which carries a C-linked biphenyltetrazole moiety and a carboxyheteroaryl group on the 3-position have been prepared. Their affinity for the AT1 receptor was determined in a binding assay on rat adrenal cortical membranes. The in vivo antihypertensive properties were tested by evaluating the inhibition of the pressor response to A II followed by iv and id administration. Extensive molecular modeling studies, including comparison of molecular electrostatic potential distributions, conformational analysis, and overlays on a computational pharmacophore model of A II, were used to evaluate structural parameters of the new compounds, in comparison to other known A II antagonists (e.g., DUP-753 and SK&F 108566). According to the modeling studies, the introduction of a (carboxyheteroaryl)methyl moiety at the 3-position of the pyrimidinone ring led to derivatives with increased potency. Methyl 2-[[4-butyl-2-methyl-6-oxo-5-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl ]- 4-yl]methyl]-1-(6H)-pyrimidinyl]methyl]-3-thiophenecarboxylate (3k, LR-B/081), one of the most potent compounds in the series (Ki = 1.4 nM), exhibited a marked antihypertensive activity on oral administration to conscious renal hypertensive rats, with long duration of action. It was selected for clinical evaluation in the treatment of hypertension in man.

Pharmacology of CS-866, a novel nonpeptide angiotensin II receptor antagonist

CS-866, (5-methyl-2-oxo-1,3-dioxole-4-yl)methoxy-4-(1-hydroxy-1-methylethyl)-2-propyl-10{4-[2-(tetrazol-5-yl)-phenyl]phenyl}methylimidazol-5-carboxyl a prodrug type angiotensin receptor antagonist, is deesterified to the active acid, RNH-6270. RNH-6270 inhibited [ 125I]angiotensin II binding to bovine adrenal cortical membranes (angiotensin AT 1 receptors) with an IC 50 value of 7.7 nM, but not [ 125I]angiotensin II binding to bovine cerebellar membranes (angiotensin AT 2 receptors), indicating the selectivity of the compound for angiotensin AT 1 receptors. In guinea pig aortas, RNH-6270 reduced the maximal response of the concentration-contractile curve for angiotensin II (pD' 2 = 9.9), but had no effect on the contractile response induced by phenylephrine or KCl. In conscious rats, intravenously injected RNH-6270 inhibited angiotensin II-induced pressor responses in a dose-dependent manner, and orally administered CS-866 produced a long-lasting inhibition of angiotensin II pressor responses. SK & F-525A, a P-450 inhibitor, suppressed the angiotensin II inhibitory effect of losartan, but not that of CS-866. These results demonstrate that RNH-6270 is a potent and AT 1-selective angiotensin receptor antagonist and that, after oral administration, CS-866 has a long-lasting angiotensin II inhibitory action which is not affected by drug metabolizing enzymes in the liver.

Dithiothreitol, sodium chloride, and ethylenediaminetetraacetic acid increase the binding affinity of [ 125I]angiotensin IV to AT 4 receptors in bovine adrenal cortex

The present studies demonstrate that the sulfhydryl reducing agent, dithiothreitol (DTT), increases the specific binding of [ 125I]angiotnesin IV ([ 125I]AIV) to AT 4 receptors in bovine adrenal cortical membranes. Both the degree of stimulation and the pharmacological selectivity of [ 125I]AIV binding in the presence of DTT were quantitatively different depending on the contents of the assay buffer. Similar effects were also observed using a different sulfhydryl reducing agent, 2-mercaptoethanol (2-MCE). These sulfhydryl reducing agents (100 m M) produced a 200% increase in specific [ 125I]AIV binding in an assay buffer that has been used to characterize the novel AT 4 receptor subtype. A much larger stimulation (700%) of specific [ 125I]AIV binding was found when the assay was conducted in a buffer that has been used to characterize ligand binding to the AT 1 receptor. Ligand association studies indicated that 0.3 n M [ 125I]AIV displayed similar equilibrium kinetics and stability in both the AT 4 and AT 1 buffers. Ligand saturation studies indicated that [ 125I]AIV bound with high affinity ( K d = 6 nM ) in the AT 4 buffer system, but bound with lower affinity ( K d = 32 nM ) in the AT 1 buffer system. Removal of NaCl and EDTA from the AT 4 buffer also resulted in low-affinity [ 125I]AIV binding ( K d = 33 nM ). The subsequent inclusion of NaCl, EDTA, or DTT resulted in higher-affinity [ 125I]AIV binding ( K d s = 3–14 nM ). No significant effects on the apparent density ( B max ) of AT 4 receptors were observed. These results show that DTT specifically increases the specific binding of [ 125I]AIV under conditions of both high and low affinity for the AT 4 receptor, which can be regulated by NaCl and/or EDTA. Further, the effects of DTT shown here on the AT 4 receptor are similar to its reported actions on the AT 2 receptor and indicate that putative receptor disulfide bridges may be important in the regulation of receptor affinity.

Nonpeptide angiotensin II receptor antagonists. Synthesis and biological activity of benzimidazolecarboxylic acids.

A series of 2-substituted-1-[(biphenyl-4-yl)methyl]-1H-benzimidazole-7- carboxylic acids was prepared from the key intermediate 3-amino-2-[[(biphenyl-4- yl)methyl]amino]benzoate (6a-c) in order to clarify the structure-activity relationships of various analogues of 2-butyl-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-ben zimidazole- 7-carboxylic acid (CV-11194), a potent and long acting angiotensin II (AII) receptor antagonist. The AII antagonistic activity of the benzimidazoles was investigated by in vitro assays, which included an AII receptor binding assay and AII-induced vasocontraction assay, as well as by in vivo assays such as an AII-induced pressor response in rats. Most of the benzimidazoles showed high affinity for the AII receptor (IC50 value, 10(-6)-10(-7) M) and inhibited the AII-induced pressor response at 1 or 3 mg/kg po, and the effects were more potent than those of CV-11194 and DuP 753. The structure-activity relationship studies on the binding affinity and the inhibition of AII-induced pressor response suggested that straight chains of a certain length (e.g., ethoxy groups, ethyl groups) were the best as substituents at the 2-position and that their steric factors, lipophilicity, and electronic effects affected the potency of the AII antagonistic action. Both a carboxyl group at the 7-position and a tetrazole ring at the 2'-position were particularly important for potent and orally active AII antagonistic activity and a long-acting hypotensive effect. The representative compound, 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H- benzimidazole-7-carboxylic acid (26b, CV-11974), inhibited the specific binding of [125I]AII to bovine adrenal cortical membrane with an IC50 value of 1.1 x 10(-7) M. The AII-induced contraction of rabbit aortic strips was antagonized by CV-11974 (IC50 value, 3.0 x 10(-10) M). Oral administration of CV-11974 to conscious normotensive rats at 1 mg/kg resulted in long-lasting inhibition of the AII-induced pressor response. CV-11974 at 0.1-1 mg/kg iv reduced blood pressure dose-dependently in spontaneously hypertensive rats.

Comparison of Ins(1,4,5)P 3 receptors from rat cerebellum and bovine adrenal cortex

Ins(1,4,5)P 3 receptors in adrenal cortical and cerebellar membranes can be distinguished by their affinities for Ins(1,4,5)P 3 as well as the potencies with which heparin and Mg 2+ inhibit binding. We have found that the differences in Ins(1,4,5)P 3 affinity and heparin inhibition are maintained upon receptor solubilization and purification. In contrast to this, heparin-agarose affinity purification of solubilized cerebellar receptors reduces the potency of Mg 2+ inhibition to that in adrenal cortex. These results suggest that Ins(1,4,5)P 3 receptors in adrenal cortex are structurally distinct from those in cerebellum. Monoclonal antibodies raised against C- and N-terminal regions of mouse cerebellar Ins(1,4,5)P 3 receptors recognize 250–300-kDa proteins in both rat cerebellum and bovine adrenal cortex.

The angiotensin hexapeptide 3–8 fragment potently inhibits [ 125I]angiotensin II binding to non-AT 1 or -AT 2 recognition sites in bovine adrenal cortex

In the present studies, ligand competition experiments were conducted to examine the ability of angiostensin II peptide agonists and nonpeptide AT 1- and AT 2-selective receptor antagonists to inhibit the binding of [ 125I]angiostensin II to bovine adrenal cortical membranes. Angiostensin II, angiostensin III, the All-(3–8) hexapeptide fragment of angiotensin II, and the AT 1-selective receptor antagonist L-158,809, inhibited [ 125I]angiostensin II binding in a biphasic fashion indicative of a ligand interaction at more than one recognition site. Approximately 20% of low affinity [ 125I]angiostensin II binding was inhibited only by high micromolar concentrations of L-158,809. RG 13647 (1(-1,4-benzodioxan-2-methyl)-5-diphenylacetyl-4,5,6,7-tetrahydro-1H-imidazo-[4,5,c]- pyridine-6-carboxylic acid) represents a potent and AT 2-selective analog of PD 123177 and showed weak activity in competing for [ 125I]angiostensin II binding with an IC 50 value of 100 μM. When subsequent competition studies were conducted in the presence of 1 μM L-158,809 to block [ 125I]angiotensin II to the AT 1 receptor subtype, the angiotensin II agonists produced monophasic inhibition curves with AII-(3–8) showing the greatest activity (IC 50 = 6 nM) followed by angiotensin III (IC 50 = 15 nM) ⪢ angiotensin II (IC 50 = 110 nM). RG 13647 was not found to significantly inhibit this portion of [ 125I]angiotensin II binding. These data demonstrate that bovine adrenal cortex contains both the AT 1 receptor subtype, as well as, a novel class of [ 125I]angiotensin II recognition sites which may be analogous to the recently described angiotensin IV (AT 4) receptor.

Angiotensin II Receptor Heterogeneity

The possibility of receptor heterogeneity in the angiotensin II (AII) system has been suggested previously, based on differences in Kd values or sensitivity to thiol reagents. One of our earliest indications was the frequent observation of incomplete inhibition of the binding of AII to adrenal cortical membranes. Autoradiographic studies demonstrated that all of the labeling of the rat adrenal was blocked by unlabeled AII or saralasin, but not by DuP 753. The predominant receptor in the rat adrenal cortex (80%) is sensitive to dithiothreitol (DTT) and DuP 753, and is designated AII-1. The residual sites in the adrenal cortex and almost all of the sites in the rat adrenal medulla are insensitive to both DTT and DuP 753, but were blocked by EXP655. These sites have been confirmed by ligand binding studies and are designated AII-2. The rabbit adrenal cortex is unique in yielding a nonuniform distribution of AII-2 sites around the outer layer of glomerulosa cells. In the rabbit kidney, the sites on the glomeruli are AII-1, but the sites on the kidney capsule are AII-2. Angiotensin III appears to have a higher affinity for AII-2 sites since it inhibits the binding to the rabbit kidney capsule but not the glomeruli. Elucidation of the distribution and function of these diverse sites should permit the development of more selective and specific therapeutic strategies.

Characteristics of inositol 1,4,5-trisphosphate binding to rat cerebellar and bovine adrenal cortical membranes: evidence for the heterogeneity of binding sites

The equilibrium and kinetic binding characteristics of D-inositol 1,4,5-trisphosphate (Ins(1.4.5)P 3) have been studied in membrane preparations of rat cerebellum and bovine adrenal cortex. Saturation analysis of isotopic dilution binding data demonstrated apparent K D values for Ins(1,4,5)P 3 binding of 23 ± 5 nM and 3.0 ± 1.3 nM for cerebellar and adrenal cortical preparations, respectively, with approximately 20-fold greater receptor density present in the cerebellar preparation (B max: 10.2 ± 2.5 pmol/mg protein). Kinetic analysis confirmed the equilibrium binding-derived K D value for cerebellum (K D: 39.9 nM), but revealed a second, very high affinity site (K D: 0.06 nM) to be present in adrenal cortex. The affinity differences between the investigated preparations was also observed with respect to the IC 50 values obtained for inhibition of specific [ 3H]Ins(1,4,5)P 3 binding by a number of inositol polyphosphate analogues including D-inositol 2,4,5-trisphosphate, DL-inositol 1,4,5-triphosphorothioate and L-Ins(1,4,5)P 3. In contrast, the Ins(1,4,5)P 3-receptor antagonist heparin displayed greater potency for the cerebellar (IC 50: 16.5 ± 6.2 μg · ml −1) compared to the adrenal cortical preparation (IC 50: 51.0 ± 6.1 μg · ml −1). The apparent differences between the Ins(1,4,5)P 3 receptors characterized in the two tissue preparations are discussed.

Ring-deleted analogs of atrial natriuretic factor inhibit adenylate cyclase/cAMP system. Possible coupling of clearance atrial natriuretic factor receptors to adenylate cyclase/cAMP signal transduction system.

We have recently shown that atrial natriuretic factor (ANF) inhibits adenylate cyclase activity in rat platelets where only one population of ANF receptors (ANF-R2) is present, indicating that ANF-R2 receptors may be coupled to the adenylate cyclase/cAMP system. In the present studies, we have used ring-deleted peptides which have been reported to interact with ANF-R2 receptors also called clearance receptors (C-ANF) without affecting the guanylate cyclase/cGMP system, to examine if these peptides can also inhibit the adenylate cyclase/cAMP system. Ring-deleted analog C-ANF4-23 like ANF99-126 inhibited the adenylate cyclase activity in a concentration-dependent manner in rat aorta, brain striatum, anterior pituitary, and adrenal cortical membranes. The maximal inhibition was about 50-60% with an apparent Ki between 0.1 and 1 nM. In addition, C-ANF4-23 also decreased the cAMP levels in vascular smooth muscle cells in a concentration-dependent manner without affecting the cGMP levels. The maximal decrease observed was about 60% with an apparent Ki of about 1 nM. Furthermore, C-ANF4-23 was also able to inhibit cAMP levels and progesterone secretion stimulated by luteinizing hormone in MA-10 cell line. Other smaller fragments of ANF with ring deletions were also able to inhibit the adenylate cyclase activity as well as cAMP levels. Furthermore, the stimulatory effects of various agonists such as 5'-(N-ethyl)carboxamidoadenosine, dopamine, and forskolin on adenylate cyclase activity and cAMP levels were also significantly inhibited by C-ANF4-23. The inhibitory effect of C-ANF4-23 on adenylate cyclase was dependent on the presence of GTP and was attenuated by pertussis toxin treatment. These results indicate that ANF-R2 receptors or so-called C-ANF receptors are coupled to the adenylate cyclase/cAMP signal transduction system through inhibitory guanine nucleotide regulatory protein.

Heterogeneity of [3H]inositol 1,4,5-trisphosphate binding sites in adrenal-cortical membranes. Characterization and validation of a radioreceptor assay

1. The characterization of a radioreceptor assay for determining Ins(1,4,5)P3 concentration in tissue extracts is described which utilizes the binding of [3H]Ins(1,4,5)P3 to an adrenal-cortex membrane fraction. 2. Analysis of [3H]Ins(1,4,5)P3 binding by isotope dilution demonstrated an apparent single population of binding sites (KD 3.65 +/- 0.18 nM, Bmax. 872 +/- 70 fmol/mg of protein). Specific binding of [3H]Ins(1,4,5)P3 was enhanced at alkaline pH values (maximum at pH 8.5), with complete loss of specific binding at pH less than 6. These binding sites displayed strict stereo- and positional specificity for Ins(1,4,5)P3, with L-Ins(1,4,5)P3, Ins(1,3,4)P3 and DL-Ins(1,3,4,5)P4 causing 50% displacement of specific [3H]Ins(1,4,5)P3 binding (IC50 values) at concentrations of 14 +/- 3 microM, 3.0 +/- 0.3 microM and 0.53 +/- 0.03 microM respectively. 3. Kinetic analysis of binding data, however, revealed a high-affinity [3H]Ins(1,4,5)P3 binding site (KD 0.052 nM) in addition to the lower-affinity site (KD 2.53 nM) already demonstrated in displacement studies. 4. It is shown that the presence of the high-affinity site can be exploited to increase the sensitivity of the [3H]Ins(1,4,5)P3 radioreceptor assay, allowing accurate detection of 20 fmol of Ins(1,4,5)P3 in 300 microliters of tissue extract. 5. Further validation of the specificity of the above assay for Ins(1,4,5)P3 was provided by incubating tissue extracts with either a 5-phosphatase or 3-kinase preparation. It was shown that identical loss occurred of both Ins(1,4,5)P3 mass and [3H]Ins(1,4,5)P3, added to parallel incubations. 6. The ability of the assay to measure basal and agonist-stimulated increases in Ins(1,4,5)P3 concentration has been demonstrated with rat cerebral cortex and bovine tracheal smooth-muscle slices and a range of cultured and isolated cell preparations.

Nonpeptide Angiotensin II Receptor Antagonists: A Novel Class of Antihypertensive Agents

The most direct approach to block the function of the renin-angiotensin system would be to antagonize angiotensin II (AII) at the level of its receptor. However, the AII receptor antagonists currently available, such as saralasin, are peptides which still retain agonistic activity and lack oral bioavailability. We have identified the N-benzylimidazoles, S-8307 and S-8308, as weak, but selective nonpeptide AII receptor antagonists. These initial leads were subsequently converted into more potent compounds, such as EXP6155, EXP6803 and EXP7711, while maintaining the selectivity. The compounds displace 3H-AII from its specific binding sites in adrenal cortical membranes and smooth muscle cells. They competitively inhibit the vasoconstrictor response to AII in various in vivo and in vitro preparations, but do not influence those to KCl, norepinephrine, and vasopressin. Converting enzyme and renin are not affected by these agents. In renal hypertensive rats and sodium-depleted dogs our compounds cause a sustained decrease in arterial pressure following intravenous and oral (EXP7711) administration, and are devoid of agonistic properties. Taken together, these nonpeptide structures are true competitive AII receptor antagonists and represent a new class of effective antihypertensive agents.

Angiotensin II differentially affects cyclic AMP formation in intact adrenal glomerulosa cells and in purified membrane preparations

In the present study we have investigated the cyclic AMP (cAMP) responses to angiotensin II (AII) in isolated rat adrenal glomerulosa cells and in purified membrane preparations. When cells were incubated with 10 nM AII cAMP cellular content increased 2-fold at 5 min and 3-fold at 10 min, then rapidly declined. The effect of AII was dose-dependent with EC 50 of 4 nM and was mediated by AII receptors as shown by the pharmacological characterization with AII analogs and AII receptor antagonists. Since AII inhibited cAMP formation in purified adrenal cortical membrane preparations, the stimulatory effect observed in intact cells could be indirect and mediated by other intracellular events.

New signal transduction mechanisms of atrial natriuretic factor: Inhibition of phosphorylation of protein kinase C and a 240 kDa protein in adrenal cortical plasma membrane by cGMP dependent and independent mechanisms

The effects of synthetic atrial natriuretic factor (ANF) on the state of protein phosphorylation in plasma membranes of bovine adrenal cortex have been studied in vitro . ANF (1×10 −8M – 1×10 −7M) specifically inhibited the phosphorylation of two distinct proteins of 78 kDa and 240 kDa. Immunoblotting with specific antiserum to protein kinase C produced evidence that 78 kDa protein is most likely the protein kinase C whose phosphorylation is inhibited by both ANF and cGMP. However, cGMP did not affect the phosphorylation of 240 kDa protein, indicating a new cGMP-independent mechanism of ANF action in the adrenal, which is compatible with the lack of action of cGMP and its analogs in ANF-induced inhibition of aldosterone secretion from adrenal cortex. The inhibition of phosphorylation of putative protein kinase C by ANF or cGMP indicates a hitherto unknown signal transduction mechanism of ANF.

Inhibitory effect of atrial natriuretic factor on adenylate cyclase activity in adrenal cortical membranes

The effect of rat synthetic atrial natriuretic factor (ANF) on adenylate cyclase activity was studied in adrenal cortical membranes. Synthetic ANF (Arg 101-Tyr 126) inhibited adenylate cyclase activity in a concentration-dependent manner. The maximum inhibition observed was about 25% with an apparent K i of 5 x 10 −11 to 10 −10 M. Various hormones such as isoproterenol, dopamine, prostaglandin (PGE 1) and ACTH-stimulated adenylate cyclase to various degrees and ANF inhibited the stimulatory effect of all these hormones to some degree but never abolished it. In addition, ANF was also able to inhibit the stimulation exerted by forskolin which activates adenylate cyclase by receptor-independent mechanism. This is the first study demonstrating the inhibitory effect of ANF on adrenal cortical adenylate cyclase. From these results it can be suggested that the inhibition of adenylate cyclase may be one of the mechanisms through which ANF exerts the inhibitory effect on steroidogenesis stimulated by various hormones and agents.