Reported Binding Affinities Research Articles

Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against α-Bungarotoxin

BackgroundNucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period.Principal FindingsHerein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM.ConclusionWe have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers.

Analysis of factor VIIa binding to relipidated tissue factor by surface plasmon resonance.

Kinetic analysis of the tissue factor (TF)-factor VIIa (FVIIa) binding interaction is helpful in investigating the structure-function relationships of TF-FVIIa. However, a wide variation exists among the reported binding affinities of FVIIa to TF, particularly when comparing KD values obtained from functional activity assays versus ligand binding studies. Surface plasmon resonance (SPR) technique was used frequently to investigate binding kinetics of FVIIa to TF in a lipid-free environment. In the present study we used TF embedded in a phospholipid bilayer for determining binding kinectis using SPR. The data revealed that FVIIa had a much higher binding affinity (>100-fold) for TF embedded in the phospholiid bilayer than TF in a lipid-free environment, approaching the KD values that were noted in the enzymatic activity assays. The present data suggest that SPR binding studies using TF embedded in phospholipids is more appropriate for investigating how FVIIa (or FVIIa mutants/derivatives) may interact with TF in physiological settings.

Structure−Activity Relationship and Comparative Docking Studies for Cycloguanil Analogs as PfDHFR-TS Inhibitors

Drug resistance acquired by Plasmodium falciparum (Pf) is a major problem in the treatment and control of malaria. One of the major examples of drug resistance is that caused by mutations in the active site of dihydrofolate reductase (DHFR) of Pf (PfDHFR-TS). A double mutation, A16V+S108T, is specific for resistance to the marketed drug cycloguanil. In this study, we used 58 cycloguanil (2,4-diamino-1,6-dihydro-1,3,5-triazine) derivatives to explore the relationship between various physicochemical properties and reported binding affinity data on wild-type and mutant-type A16V+S108T. Using the Hansch 2D-quantitative structure-activity relationship method, we obtained a parabolic relationship of hydrophobicity of substituents at the N1-phenyl ring with the wild-type binding affinity data. Hydrophobicity being a key property for wild-type binding affinity data, we found steric factors to be crucial for A16V+S108T mutant resistance. We investigated FlexX, GOLD, Glide and Molegro virtual docking programs and 13 different scoring functions on 10 of the cycloguanil derivatives to evaluate which program was best for reproducing the experimental binding mode and correlating the docking scores with the reported binding affinity data. We identified GOLD, using its GoldScore fitness function, as the most accurate docking program for predicting binding affinity data of cycloguanil derivatives to DHFR and Molegro virtual docker, with its template docking algorithm and MolDock [GRID] scoring function, as most accurate for reproducing the experimental binding mode of a reference ligand that is structurally similar to the cycloguanil derivatives studied. We also report an interaction index which best describes the structure-activity relationships exhibited by these analogs in terms of PfDHFR-TS active site interactions.

Binding mode prediction of conformationally restricted anandamide analogs within the CB<sub>1</sub> receptor

BackgroundCB1 cannabinoid receptors are G-protein coupled receptors for endocannabinoids including anandamide and 2-arachidonoylglycerol. Because these arachidonic acid metabolites possess a 20-carbon polyene chain as the alkyl terminal moiety, they are highly flexible with the potential to adopt multiple biologically relevant conformations, particularly those in a bent form. To better understand the molecular interactions associated with binding and steric trigger mechanisms of receptor activation, a series of conformationally-restricted anandamide analogs having a wide range of affinity and efficacy were evaluated.ResultsA CB1 receptor model was constructed to include the extracellular loops, particularly extracellular loop 2 which possesses an internal disulfide linkage. Using both Glide (Schrödinger) and Affinity (Accelrys) docking programs, binding conformations of six anandamide analogs were identified that conform to rules applicable to the potent, efficacious and stereoselective non-classical cannabinoid CP55244. Calculated binding energies of the optimum structures from both procedures correlated well with the reported binding affinity values. The most potent and efficacious of the ligands adopted conformations characterized by interactions with both the helix-3 lysine and hydrophobic residues that interact with CP55244. The other five compounds formed fewer or less energetically favorable interactions with these critical residues. The flexibility of the tested anandamide analogs, measured by torsion angles around the benzene as well as the stretch between side chain moieties, could contribute to the differences in ability to interact with the CB1 receptor.ConclusionAnalyses of multiple poses of conformationally-restricted anandamide analogs permitted identification of favored amino acid interactions within the CB1 receptor binding pocket. A ligand possessing both high affinity and cannabinoid agonist efficacy was able to interact with both polar and hydrophobic interaction sites utilized by the potent and efficacious non-classical cannabinoid CP55940. In contrast, other analogs characterized by reduced affinity or efficacy exhibited less favorable interactions with those key residues.

Structure-based Analysis of HU–DNA Binding

HU and IHF are prokaryotic proteins that induce very large bends in DNA. They are present in high concentrations in the bacterial nucleoid and aid in chromosomal compaction. They also function as regulatory cofactors in many processes, such as site-specific recombination and the initiation of replication and transcription. HU and IHF have become paradigms for understanding DNA bending and indirect readout of sequence. While IHF shows significant sequence specificity, HU binds preferentially to certain damaged or distorted DNAs. However, none of the structurally diverse HU substrates previously studied in vitro is identical with the distorted substrates in the recently published Anabaena HU(AHU)–DNA cocrystal structures. Here, we report binding affinities for AHU and the DNA in the cocrystal structures. The binding free energies for formation of these AHU–DNA complexes range from ∼10–14.5 kcal/mol, representing K d values in the nanomolar to low picomolar range, and a maximum stabilization of at least ∼6.3 kcal/mol relative to complexes with undistorted, non-specific DNA. We investigated IHF binding and found that appropriate structural distortions can greatly enhance its affinity. On the basis of the coupling of structural and relevant binding data, we estimate the amount of conformational strain in an IHF-mediated DNA kink that is relieved by a nick (at least 0.76 kcal/mol) and pinpoint the location of the strain. We show that AHU has a sequence preference for an A+T-rich region in the center of its DNA-binding site, correlating with an unusually narrow minor groove. This is similar to sequence preferences shown by the eukaryotic nucleosome.

Long-Term Effects of Neonatal Exposure to Hydroxylated Polychlorinated Biphenyls in the BALB/cCrgl Mouse

The neonatal mouse model has been a valuable tool in determining the long-term effects of early exposure to estrogenic agents in mammals. Using this model, we compared the effects of 2′,4′,6′-trichloro-4-biphenylol (OH-PCB-30) and 2′,3′,4′,5′-tetrachloro-4-biphenylol (OH-PCB-61) as prototype estrogenic hydroxylated PCBs (OH-PCBs) because they are reported to exhibit relatively high estrogenic activity both in vivo and in vitro. The purpose of this study was to examine the relationship between estrogenicity and carcinogenicity of OH-PCB congeners. The OH-PCBs were tested individually and in combination to determine whether effects of combined OH-PCBs differed from those of these OH-PCBs alone. We evaluated the long-term effects of neonatal exposure to OH-PCBs with treatment doses that were based on the reported binding affinity of specific OH-PCB congeners to estrogen receptor α. BALB/cCrgl female mice were treated within 16 hr after birth by subcutaneous injections every 24 hr, for 5 days. The mice treated with OH-PCB-30 (200 μg/day) or 17β-estradiol (5 μg/day) showed similar increased incidences of cervicovaginal (CV) tract carcinomas (43% and 47%, respectively). In addition, when mice were treated with OH-PCBs as a mixture, a change in the type of CV tract tumor was observed, shifting from predominantly squamous cell carcinomas to adenosquamous cell carcinoma. From our results, we conclude that the individual OH-PCBs tested were estrogenic and tumorigenic in mice when exposed during development of the reproductive tract. These data support the hypothesis that mixtures may act differently and unexpectedly than do individual compounds.

Decrypting the Biochemical Function of an Essential Gene from Streptococcus pneumoniae Using ThermoFluor® Technology

The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphate and pyridoxamine phosphate as equilibrium binding ligands (K(d) approximately 50 pM, K(d) approximately 2.5 microM, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphate binding were strictly conserved. Screening a collection of generic drugs and natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.

Flavin thermodynamics explain the oxygen insensitivity of enteric nitroreductases.

Bacterial nitroreductases are NAD(P)H-dependent flavoenzymes which catalyze the oxygen-insensitive reduction of nitroaromatics, quinones, and riboflavin derivatives. Despite their broad substrate specificity, their reactivity is very specific for two-electron, not one-electron, chemistry. We now describe the thermodynamic properties of the flavin mononucleotide cofactor of Enterobacter cloacae nitroreductase (NR), determined under a variety of solution conditions. The two-electron redox midpoint potential of NR is -190 mV at pH 7.0, and both the pH dependence of the midpoint potential and the optical spectrum of the reduced enzyme indicate that the transition is from neutral oxidized flavin to anionic flavin hydroquinone. The one-electron-reduced semiquinone states of both the free enzyme and an NR-substrate analogue complex are strongly suppressed based on optical spectroscopy and electron paramagnetic resonance measurements. This can explain the oxygen insensitivity of NR and its homologues, as it makes the execution of one-electron chemistry thermodynamically unfavorable. Therefore, we have established a chemical basis for the recent finding that a nitroreductase is a member of the soxRS oxidative defense regulon in Escherichia coli [Liochev, S. I., Hausladen, A., Fridovich, I. (1999) Proc. Natl. Acad. Sci. U.S.A. 96 (7), 3537-3539]. We also report binding affinities for the FMN cofactor in all three oxidation states either determined fluorometrically or calculated using thermodynamic cycles. Thus, we provide a detailed picture of the thermodynamics underlying the unusual activity of NR.

Pharmacologically distinct binding sites in rat brain for [ [formula omitted]]thyrotropin-releasing hormone (TRH) and [ [formula omitted]][3-methyl-histidine 2]TRH

We have used a directed peptide library, in which the histidyl residue of thyrotropin-releasing hormone (TRH) was systematically replaced by a series of 24 natural and unnatural amino acids, to characterise TRH binding sites in rat brain cortex. This was achieved by measuring the ability of library peptides to compete with [ 3 H ][3-Me-His 2]TRH or [ 3 H ]TRH binding to rat cortical homogenates. [ 3 H ][3-Me-His 2]TRH was observed to bind to a single population of high-affinity, low-capacity sites ( K d : 4.54±0.62 nM, N=5; B max: 4.38±0.21 fmol/mg wet weight tissue, N=5), consistent with them being central TRH receptors. Displacement studies showed TRH to bind to these sites with an apparent K i of 22 nM. K i values for the library peptides at [ 3 H ][3-Me-His 2]TRH-labelled sites varied from 10 −3 to 10 −9 M; the potency order was: [3- Me- His 2]> His> Thi> Leu, Phe, Asn> Gln , Arg, Thr, Ala, HomoPhe. All other replacements had K i values >10 −4 M. [ 3 H ]TRH was observed to label a single population of low-affinity, high-capacity sites ( K d : 7.55±1.23 μM, N=6; B max: 3.40±0.63 pmol/mg wet weight tissue, N=6). The affinities of the synthetic peptides for [ 3 H ]TRH-labelled sites did not correlate with their affinities for [ 3 H ][3-Me-His 2]TRH-labelled sites ( r=0.33, N=18, P>0.1). They did, however, correlate significantly with previously reported binding affinities for TRH-degrading ectoenzyme ( r=0.72, N=12, P<0.01). These results strongly indicate that the identity of the low-affinity, [ 3 H ]TRH-labelled site is the membrane-bound enzyme, TRH-degrading ectoenzyme, not a subpopulation of TRH receptors. They also provide the first comprehensive description of the influence of the histidyl residue in TRH on binding of TRH to brain receptors.

α 1A- and α 1B-adrenoceptors are the major subtypes in human saphenous vein

In this study we analyzed the different α 1-adrenoceptor (AR) subtypes present in human saphenous vein (HSV) using reverse transcription polymerase chain reaction (RT-PCR), DNA-DNA hybridization analysis and functional affinities for α-AR antagonists. DNA-DNA hybridization analysis of RT-PCR amplification products confirmed the presence of α 1A- and α 1B-ARs, and low levels of α 1D-AR in HSV. The functional results showed: (1) prazosin, the selective α 1-AR antagonist, phentolamine, the α 1- and α 2-ARs antagonist, WB 4101 and 5-MU, the selective α 1A-AR subtype antagonists were potent, competitive antagonists of noradrenaline (NA)-induced contraction (pA 2 values of 11.03, 8.06, 9.02 and 8.34, respectively). (2) α 1-AR-induced contraction was sensitive to the alkylating effects of CEC (the α 1B and α 1D-AR subtypes antagonist) and (3) The selective α 1D-AR subtype antagonist BMY displayed low affinity (pA 2 values of 6.44). This indicates that the contractile response of the HSV to α 1-AR-induced is predominantly mediated by both α 1A and α 1B-AR subtypes. This was also supported by the good relationship between pA 2 values from the present study and reported binding affinities (pK i) values of various α 1-AR subtype antagonists with cloned human α 1A- and α 1B-AR subtypes (r=0.89 and r=0.98, respectively), but not the α 1D-AR subtype (r=0.67). Our results indicate that α 1A- and α 1B-ARs are the main functional and expressed receptor subtypes in HSV.

Evaluation and Comparison of Ion Permeation and Agonist Selectivities for N-Methyl-[formula omitted]-Aspartate Receptor Channels with Different Subunit Compositions in Bilayer Lipid Membranes Based on Integrated Single-Channel Currents

To quantify the ion-permeation ability of the recombinant ϵ1–4/ζ1 channel activated by agonists, the magnitude of agonist-induced integrated single-channel currents for the ϵ2–4/ζ1 N-methyl-d-aspartate (NMDA) channels in bilayer lipid membranes (BLMs) was evaluated electrochemically based on the single-channel recordings. The recombinant ϵ2–4/ζ1 channels were purified from Chinese hamster ovary cells expressing each channel and incorporated in BLMs formed by the tip-dip method. Three typical agonists, l-glutamate, NMDA, and (2S, 3R, 4S) isomer of 2-(carboxycyclopropyl)glycine (l-CCG-IV), were investigated at a concentration of 50 μM. The magnitude of l-glutamate-induced integrated current was found to depend on the ϵ-subunit composition and to increase in the order of ϵ2/ζ1 > ϵ1/ζ1 ≈ ϵ4/ζ1 > ϵ3/ζ1, which differs from that of the reported binding affinities (EC50) between l-glutamate and each channel type. On the other hand, the magnitude of the integrated currents induced by NMDA and l-CCG-IV did not vary among the four channel types. The order of agonist selectivity toward the ϵ2–4/ζ1 channels in terms of the magnitude of the integrated current was l-glutamate >l-CCG-IV ≈ NMDA for the ϵ2/ζ1 channel, l-CCG-IV > NMDA >l-glutamate for the ϵ3/ζ1 channel, and l-CCG-IV ≈ l-glutamate > NMDA for the ϵ4/ζ1 channel, suggesting that the agonist selectivity also depends on the ϵ-subunit composition. The present study shows that each ϵ1–4/ζ1 channel has its own ability of ion permeation, i.e., its own signal transduction ability, which is not parallel to its binding ability.

Functional assessment of alpha 1-adrenoceptor subtypes in porcine coronary artery.

1. alpha 1-Adrenoceptors are known to play an important role in vasoconstriction in response to adrenergic stimulation. However, the functional importance of alpha 1-adrenoceptor subtypes at the epicardial coronary artery remains unclear. We examined alpha 1-adrenoceptor subtypes by comparing functional affinities for alpha-adrenoceptor antagonists on noradrenaline (NA)-induced vasoconstriction in porcine denuded right coronary arteries. 2. Noradrenaline induced a dose-dependent vasoconstriction in incubated vessel rings. Prazosin and phentolamine were potent and competitive antagonists for NA-induced contraction (pA2 10.27 and 9.03, respectively). In contrast, the selective alpha 2-adrenoceptor antagonist yohimbine had a low affinity (pA2 6.13). Two selective alpha 1A-adrenoceptor antagonists, WB 4101 and 5-methyl urapidil, were potent and competitive antagonists of alpha 1-adrenoceptor-induced contraction (pA2 10.67 and 8.90, respectively) and the selective alpha 1D-adrenoceptor antagonist BMY 7378 had a low affinity (pA2 6.06). Noradrenaline-induced contraction was insensitive to the alkylating effects of chlorethylclonidine. These observations indicate that the vasoconstriction is predominantly mediated by the alpha 1A-adrenoceptor subtype. This was also supported by a good correlation between pA2 values from the present study and reported binding affinities (pKi) of various alpha-adrenoceptor antagonists with cloned human alpha 1A-adrenoceptors (r = 0.98), but not for alpha 1B- or alpha 1D-adrenoceptor subtypes (r = 0.77 and 0.41, respectively). 3. Our results indicate that the alpha 1A-adrenoceptor is the main functional receptor subtype in porcine denuded coronary arteries.

Iodocalcitonin binds to human calcitonin receptors with higher affinity than calcitonin.

The reported binding affinities of cloned human calcitonin receptors (hCTRs) for salmon calcitonin (sCT) vary over a wide range. Because the greatest differences were between values estimated in association binding versus competition binding experiments, we considered the possibility that this variation was due to differences in the affinities of hCTRs for moniodinated sCT (I-sCT) and sCT. We found that I-sCT competed with 125I-sCT for binding with 9.3 +/- 0.58 times the apparent affinity of sCT in COS-1 and Madin Darby canine kidney (MDCK) cells expressing cloned hCTRs. I-sCT was 25 times more potent than sCT in stimulating cAMP formation in MDCK cells expressing hCTRs. These data demonstrate that monoiodinated sCT interacts more avidly with hCTRs than sCT with a consequent enhanced potency in stimulating second messenger formation. Therefore, as I-sCT (or 125I-sCT) binds with different affinity to hCTRs than sCT, affinity measurements must be performed with I-sCT (or 125I-sCT) alone.

Iodocalcitonin binds to human calcitonin receptors with higher affinity than calcitonin

The reported binding affinities of cloned human calcitonin receptors (hCTRs) for salmon calcitonin (sCT) vary over a wide range. Because the greatest differences were between values estimated in association binding versus competition binding experiments, we considered the possibility that this variation was due to differences in the affinities of hCTRs for moniodinated sCT (I-sCT) and sCT. We found that I-sCT competed with 125I-sCT for binding with 9.3 +/- 0.58 times the apparent affinity of sCT in COS-1 and Madin Darby canine kidney (MDCK) cells expressing cloned hCTRs. I-sCT was 25 times more potent than sCT in stimulating cAMP formation in MDCK cells expressing hCTRs. These data demonstrate that monoiodinated sCT interacts more avidly with hCTRs than sCT with a consequent enhanced potency in stimulating second messenger formation. Therefore, as I-sCT (or 125I-sCT) binds with different affinity to hCTRs than sCT, affinity measurements must be performed with I-sCT (or 125I-sCT) alone.

Solution and Solid State Characterization of a Cadmium Octaazacryptand Complex

The constant for cadmium binding by octaazacryptand L = N(CH2CH2NHCH2CH2NHCH2CH2)3N was determined by potentiometry; log βML =18.3(1). Calculation of pM values (pM = −log [M]) show that L has the highest reported binding affinity for cadmium relative to other [2.2.2] polyoxa−polyaza cryptates. Two cadmium cryptate complexes, [CdL](BF4)2 (1a) and [CdL](OAc)2·2H2O (1b), were synthesized by metalation of the free cryptand. The molecular structures of compounds 1a and 1b were determined by X-ray diffraction. Crystallographic data for 1a: trigonal, P3̄1c, Z = 2, a = 9.581(2) Å, c = 17.003(4) Å, V = 1351.2(5) Å3. Crystallographic data for 1b: monoclinic, C2/c, Z = 4, a = 10.500(2) Å, b = 14.782(3) Å, c = 19.395(4) Å, β = 99.30(2)°, V = 2971(1) Å3. The cadmium ions in both complexes are eight-coordinate and display similar coordination geometries but different ligand conformations, designated as parallel and oblique. The cadmium coordination sphere is best described as a bicapped octahedron. Through 2D NMR experiments we have found that the Cd is in a symmetric environment and the conformation of the ligand in solution is consistent with the parallel conformation observed in one of the solid state structures. The 113Cd NMR shift of this eight-coordinate amine complex is 75 ppm relative to 0.1 M aqueous Cd(ClO4)2, which correlates with shifts observed for other [2.2.2] cadmium polyoxa−polyaza cryptates.

Selection of single-stranded DNA molecules that bind and inhibit human thrombin

Aptamers are double-stranded DNA or single-stranded RNA molecules that bind specific molecular targets. Large randomly generated populations can be enriched in aptamers by in vitro selection and polymerase chain reaction. But so far single-stranded DNA has not been investigated for aptamer properties, nor has a target protein been considered that does not interact physiologically with nucleic acid. Here we describe the isolation of single-stranded DNA aptamers to the protease thrombin of the blood coagulation cascade and report binding affinities in the range 25-200 nM. Sequence data from 32 thrombin aptamers, selected from a pool of DNA containing 60 nucleotides of random sequence, displayed a highly conserved 14-17-base region. Several of these aptamers at nanomolar concentrations inhibited thrombin-catalysed fibrin-clot formation in vitro using either purified fibrinogen or human plasma.

Autocrine role of estrogens in the augmentation of luteinizing hormone receptor formation in cultured rat granulosa cells.

The effects of estrogens on gonadotropin-stimulated luteinizing hormone (LH) receptor formation were examined in primary cultures of rat granulosa cells. Granulosa cells were cultured for 3 days with increasing concentrations of follicle-stimulating hormone (FSH) in the presence or absence of native and synthetic estrogens. Follicle-stimulating hormone stimulated LH receptor formation in a dose-dependent fashion, and estrogens enhanced the FSH-stimulated LH receptor content by decreasing the apparent ED50 of FSH. At 6.25 ng/ml FSH, the enhancement in LH receptor was estrogen dose dependent, with an ED50 value of about 3 X 10(-9) M for 17 beta-estradiol. The increased LH receptor content seen in cells treated with FSH and estrogen was correlated with increased cAMP production by these cells in response to LH stimulation. Time course studies revealed enhancement of FSH-stimulated LH receptor induction at 48 and 72 h of culture. Granulosa cells were also cultured with FSH for 2 days to induce functional LH receptors, then further cultured for 3 days with LH in the presence or absence of estrogens. At 30 ng/ml LH, increasing concentrations of estrogens maintained LH receptor content in a dose-dependent fashion, with their relative estrogenic potencies in keeping with reported binding affinities to estrogen receptors. An autocrine role of estrogens on LH receptor formation was further tested in granulosa cells treated with FSH and an aromatase substrate (androstenedione) to increase estrogen biosynthesis. Cotreatment with semipurified estrogen antibodies partially blocked the FSH stimulation of LH receptors, whereas nonimmune serum was ineffective. Also, inclusion of diethylstilbestrol prevented the inhibitory effect of the estrogen antibodies. Thus, local estrogens in ovarian follicles may play an autocrine role in granulosa cells to enhance LH receptor formation and to increase granulosa cell responsiveness to the LH surge, with subsequent ovulation and adequate corpus luteum formation.

Inhibitory actions of calmodulin antagonists on steroidogenesis in zona glomerulosa cells.

The stimulation of aldosterone (Aldo) production in the adrenal glomerulosa cell by angiotensin II (AII) and ACTH is highly dependent on calcium. To determine the role of calmodulin (CM) as a regulator of intracellular calcium during hormonal activation of steroidogenesis, the actions of a series of CM inhibitors on the stimulation of Aldo biosynthesis by AII, the calcium ionophore A23187, ACTH, and cAMP were examined in isolated adrenal glomerulosa cells. The CM antagonists, pimozide, W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and W5 [N-(6-aminohexyl)-1-napthalenesulfonamide] caused dose-dependent inhibition of hormone-stimulated Aldo production, in proportion to their reported binding affinities for CM. The maximum inhibitory concentration (5 microM) of the most potent CM antagonist, pimozide, had no effect on the conversion of progesterone to Aldo. However, submaximal inhibitory concentrations of W7 and W5 also reduced conversion of progesterone to Aldo, indicating that their inhibitory action is not only due to their CM antagonist properties. The relative sensitivities of the stimuli of Aldo release to inhibition by all three CM inhibitors were A23187 = AII greater than ACTH greater than 8-bromo-cAMP (8-Br-cAMP). Aldo responses to ACTH were more sensitive to inhibition by pimozide than those to 8-Br-cAMP, suggesting that ACTH-induced cAMP formation is also dependent on CM. Pimozide (5 microM) completely inhibited the Aldo responses to AII and A23187, whereas the Aldo responses to ACTH and 8-Br-cAMP were only partially inhibited. At submaximal inhibitory concentrations, pimozide caused a decrease in the maximum response to AII without changing the concentration of the peptide required for half-maximum stimulation of Aldo production. The inhibition of Aldo production by pimozide was accompanied by parallel inhibition of pregnenolone accumulation measured in the presence of cyanoketone. No effect of pimozide (5 microM) was observed on the binding of 125I-AII to adrenal glomerulosa cells, placing the CM-dependent site between the receptor and the side-chain cleavage enzyme. These findings indicate that CM-mediated regulation is essential for the steroidogenic response to calcium ionophore and AII, but is only partially required for the responses to ACTH and 8-Br-cAMP.