Inhibition Of Radioligand Binding Research Articles

Receptor-Mediated Actions of Growth Hormone Releasing Factor on Granulosa Cell Differentiation

GRF promotes follicular maturation and ovulation when administered with FSH in the treatment of infertility. Such actions could be mediated by stimulation of GH secretion and insulin-like growth factor I production, but the known actions of the structurally related hormone, vasoactive intestinal peptide (VIP), on granulosa cell function suggested that GRF may also act directly on the ovary to stimulate follicular development. Radioligand binding and activation studies, performed in granulosa cells from immature estrogen-treated rats, revealed a common receptor for VIP and rat (r) GRF in the ovary. Specific binding of [125I]VIP to granulosa cells was saturable and dependent on time and temperature. The relative potencies of VIP-related peptides for inhibition of radioligand binding were: VIP greater than rGRF greater than peptide histidine isoleucinamide greater than [His1,Nle27] human GRF(1-32)NH2 greater than secretin. In binding studies with the potent GRF agonist, [125I] [His1,Nle27]GRF(1-32)NH2, relative potencies were: rGRF(1-43)OH greater than [His1,Nle27]human GRF(1-32)NH2 greater than VIP greater than peptide histidine isoleucinamide greater than secretin. Glucagon and gastric inhibitory peptide, other peptides of the glucagon superfamily, and unrelated peptides including CRF and beta-endorphin, did not inhibit binding of either radioligand to ovarian receptors. In cultured granulosa cells, rGRF and VIP stimulated cAMP formation, consistent with coupling of their receptors to the adenylate cyclase system, and potentiated FSH-induced cAMP production. Both peptides also amplified FSH-induced progesterone biosynthesis, aromatase activity, and LH receptor formation. These observations demonstrate that rGRF is a potent cAMP-mediated agonist in the rat ovary and acts on a common VIP/GRF receptor in maturing granulosa cells. It is likely that the potentiating effect of administered GRF on gonadotropin-stimulated follicular development in vivo is in part mediated by direct actions of the peptide on the VIP/GRF receptor. Also, since GRF is present in the gonads, it is possible that the locally-produced peptide promotes follicular maturation by paracrine modulation of the stimulatory action of FSH on granulosa cell function.

Properties of amino-terminal parathyroid hormone-related peptides modified at positions 11–13

Biological properties of amino-terminal PTHrP analogues modified in the region 11–13 were examined using ROS 17/2.8 cells. [Leu 11,D-Trp 12,Arg 13,Tyr 36]PTHrP(1–36)amide had a 17-fold lower binding affinity for the receptor (apparent K d: 5 × 10 −8 M ) than [Tyr 36]PTHrP(1–36)amide or [Arg 11,13,Tyr 36]PTHrP(1–36)amide (apparent K d for both: 2 × 10 −9 M ). Moreover, it is only a weak partial agonist despite completely inhibiting radioligand binding. [Leu 11,D-Trp 12,Arg 13,Tyr 36,Cys 38]PTHrP(7–38) and PTHrP(7–34)amide had similar receptor affinities (apparent K ds: 5 × 10 −8 M and 8 × 10 −8 M ), while that of [Nle 8,18,Tyr 34]bPTH(7–34)amide was more than 10-fold lower (apparent K d: 2 × 10 −6 M ). These changes in biological properties suggest that high affinity receptor binding requires both amino- and carboxyl-terminal domains of the PTHrP(1–36) sequence and/or intramolecular interactions which are impaired by the D-Trp substitution for Gly 12.

Brain concentrations of benzodiazepines are elevated in an animal model of hepatic encephalopathy.

Brain extracts from rats with hepatic encephalopathy due to thioacetamide-induced fulminant hepatic failure contained 4- to 6-fold higher concentrations of substances that inhibit radioligand binding to benzodiazepine receptors than corresponding control rat extracts. Both isocratic and gradient-elution HPLC indicated that this inhibitory activity was localized in 3-8 peaks with retention times corresponding to deschlorodiazepam, deschlorolorazepam, lorazepam, oxazepam, diazepam, and N-desmethyldiazepam. The presence of diazepam and N-desmethyldiazepam was confirmed by mass spectroscopy. Both mass spectroscopic and radiometric techniques indicated that the concentrations of N-desmethyldiazepam and diazepam in brain extracts from encephalopathic rats were 2-9 and 5-7 times higher, respectively, than in control brain extracts. While benzodiazepines have been identified previously in mammalian and plant tissues, this report demonstrates that concentrations of these substances are increased in a pathophysiological condition. These findings provide a rational basis for the use of benzodiazepine receptor antagonists in the management of hepatic encephalopathy in humans.

In vitro and in vivo binding of ( E)- and ( Z)-N-(iodoallyl)spiperone to dopamine D 2 and serotonin 5-HT 2 neuroreceptors

Apparent affinities (K i) of ( E)- and ( Z)-N-(iodoallyl)spiperone (( E)- and ( Z)-NIASP) for dopamine D 2 and serotonin 5-HT 2 receptors were determined in competition binding assays. ( Z)-NIASP (K i 0.35 nM, D 2; K i 1.75 nM, 5-HT 2) proved slightly more potent and selective for D 2 sites in vitro than ( E)-NIASP (K i 0.72 nM, D 2; K i 1.14 nM, 5-HT 2). In vivo, radioiodinated ( E)- and ( Z)-[ 125I]-NIASP showed regional distributions in mouse brain which are consonant with prolonged binding to dopamine D 2 receptors accompanied by a minor serotonergic component of shorter duration. Stereoselective, dose-dependent blockade of ( E)-[ 125I]-NIASP uptake was found for drugs binding to dopamine D 2 sites, while drugs selective for serotonin 5-HT 2, α1- adrenergic and dopamine D 1 receptors did not inhibit radioligand binding 2 hr postinjection. Specific binding in striatal tissue was essentially irreversible over the time course of the study, and ( E)-[ 125I]-NIASP gave a striatal to cerebellar tissue radioactivity concentration of 16.9 to 1 at 6 hr postinjection. Thus, ( E)-[ 125I]-NIASP binds with high selectivity and specificity to dopamine D 2 sites in vivo.

Leukotriene D4 binding and signal transduction in rat glomerular mesangial cells

We examined the characteristics of [3H]leukotriene D4 (LTD4) binding to mesangial cells in culture. Binding is stereoselective, specific, saturable, and rapidly reversible. Two binding sites are recognized with dissociation constants and binding site densities at equilibrium of 2.2 and 16.8 nM and 1.1 x 10(4) and 3 x 10(4) binding sites per cell. LTD4, LTE4, (5R,6S)LTD4, LTB4, and the LTD4-receptor antagonist, SKF 104353, competitively inhibit radioligand binding in the following rank order of potency: LTD4 greater than LTE4 = SKF 104353 greater than (5R,6S)LTD4 greater than LTB4. LTD4 also induces time- and concentration-dependent phosphoinositide hydrolysis in mesangial cells. Formation of inositol 1,4,5-trisphosphate (IP3) is maximal at 5 s, followed by a time-dependent increase in inositol monophosphate generation, and inhibited by 100-fold excess concentration of SKF 104353. Addition of LTD4 to mesangial cells is associated with an increase in intracellular pH and dose-dependent stimulation of [3H]thymidine incorporation and mitogenesis. Thus rat mesangial cells possess specific binding sites for LTD4, the activation of which stimulates IP3 formation and induces cellular alkalinization and mitogenic responses. These studies provide insight into the cellular basis for LTD4-mesangial cell interactions, which are of potential pathophysiological relevance during acute glomerular inflammatory injury.

Different Affinities of α2-Agonists for Imidazoline and α2-Adrenergic Receptors

It has recently been shown that imidazoline alpha 2-adrenergic agonists, such as clonidine and UK 14,304, selectively bind to both alpha 2- and imidazoline receptors in basolateral membranes from rabbit renal proximal tubule. In order to define the relative affinity of three antihypertensive alpha 2-agonists for the two classes of receptors, we performed competition studies of imidazoline alpha 2-antagonist 3H-RX 781094 and nonimidazoline antagonist 3H-rauwolscine binding to basolateral membranes from rabbit proximal tubule. The order of potency for inhibition of radioligand binding to basolateral membranes was rilmenidine greater than clonidine greater than guanfacine and clonidine greater than guanfacine greater than rilmenidine for 3H-RX 781094 and 3H-rauwolscine binding, respectively. These data show that not only clonidine, but also rilmenidine and guanfacine, drugs usually used as specific alpha 2-agonists, bind to both alpha 2- and imidazoline receptors. The higher affinity of these molecules for one or the other class of receptors could explain their different capacity to induce hypotension and side effects.

HA-966 antagonizes N-methyl-D-aspartate receptors through a selective interaction with the glycine modulatory site

3-Amino-1-hydroxypyrrolid-2-one (HA-966) has been known for several years as an excitatory amino acid antagonist, acting principally at the N-methyl-D-aspartate (NMDA) receptor subtype. We report here that HA-966 blocks NMDA responses through a selective interaction with the glycine modulatory site present within the receptor complex. In radioligand binding experiments, HA-966 inhibited strychnine-insensitive 3H-glycine binding to rat cerebral cortex synaptic plasma membranes with an IC50 of 17.5 microM. At concentrations up to 1 mM, HA-966 caused minimal inhibition of radioligand binding to the transmitter recognition sites of the NMDA, quisqualate, or kainate receptor subtypes and was similarly inactive against the binding of 3H-strychnine to rat spinal cord/brain stem membranes. In electrophysiological experiments, HA-966 produced a selective block of NMDA responses in a rat cortical slice preparation. The degree of antagonism caused by HA-966 was maximal at 250 microM and was not increased further by raising the HA-966 concentration. Both glycine (1 mM) and D-serine (100 microM) reversed the antagonism of NMDA responses caused by HA-966. In patch-clamp experiments using rat cortical neurons in culture, HA-966 blocked the potentiation of NMDA responses by glycine but had little effect on basal NMDA responses themselves. This profile of antagonism differs from that observed with 7-chlorokynurenate, another recently discovered antagonist of the glycine site on the NMDA receptor (Kemp et al., 1988) and may indicate that glycine antagonists of differing efficacies can exist. Previous experiments with HA-966 may now be interpreted to suggest that activation of the glycine site on the NMDA receptor occurs in vivo and is important for the participation of NMDA receptors in synaptic transmission.

The effects of sulfhydryl-modifying reagents on cholecystokinin-receptor interactions in guinea pig gastric glands.

The effects of a number of different sulfhydryl agents on cholecystokinin (CCK)-binding sites in isolated fundic gastric glands and gastric mucosal membranes from guinea pigs were evaluated. [125I]CCK octapeptide binding was significantly reduced when gastric glands were pretreated with iodoacetamide (IA; 10 mM), p-chloromercuribenzoate (PCMB; 0.1 mM), or N-ethylmaleimide (NEM; 0.1 mM) at 24 C, but not by dithiothreitol (DTT; 1.0 mM) or dinitrothiobenzoic acid (DTNB; 10 mM). In contrast, inclusion of DTT or IA during binding led to significant increases in the amount of radioligand bound, while in the presence of DTNB binding was significantly reduced. Glycine-HCl wash of gastric glands pretreated with these agents indicated that NEM and IA reduced radioligand internalization by about 80% (P less than 0.001) and increased surface binding by 20% (P less than 0.01) and 76% (P less than 0.001), respectively. PCMB inhibited radioligand binding to surface sites by more than 90% and completely inhibited radioligand internalization. Gastric mucosal membranes bound significantly less radioligand after PCMB (80%) and NEM (26%) treatment compared to controls, and significantly more after IA treatment (162%). Scatchard analysis of the CCK-receptor interaction indicated two binding sites with dissociation constants of 0.71 and 11.7 nM, and binding capacities of 3.6 and 15.8 nmol/mg DNA, respectively. NEM and IA significantly increased the Kd of the high affinity site without affecting the binding parameters of the low affinity site; the low affinity site was eliminated by PCMB treatment, and the affinity and capacity of the high affinity site were significantly reduced. In the presence of DTT, [125I]CCK octapeptide binding to gastric glands was enhanced with a significant increase in the amount of radioligand in the internalized pool (39%; P less than 0.001) and no increase in binding to gastric mucosal membranes. These results suggest that sulfhydryl groups play an important role in CCK-receptor interactions.

Amiloride inhibits 22Na uptake and [ 3H]QNB binding in rat submandibular cells

Dispersed acini isolated by collagenase digestion of the rat submandibular gland were used to compare the effects of amiloride and furosemide on the uptake of the isotopic tracer 22Na and on the binding of [ 3H]quinuclidinyl benzylate ([ 3H]QNB). In mM concentrations, both inhibitors reduced 22Na uptake in resting cells 34 and 25–29%, respectively. Acetylcholine (1 μM) enhanced uptake 23% and this effect was reduced 45% by amiloride and 26% by furosemide. Amiloride inhibited the binding of [ 3H]QNB to crude membranes prepared from fresh submanidubular glands in a dose-dependent fashion (IC 50=8×10 −6 M). Furosemide (3×10 −8 to 10 −3 M) did not inhibit radioligand binding. Na influx into resting salivary acini thus appears to occur by both amiloride-sensitive and furosemide-sensitive transport systems. The similar inhibition by furosemide of unstimulated and stimulated uptake of 22Na suggests that acetylcholine does not significantly activate the cotransport system within the time frame (i.e., 2 min) of the experiments. Acetylcholine appears to activate an amiloride-sensitive Na/H antiport, but amiloride blocks cholinergic receptors and may thus affect Na transport by receptor blockade. Other actions of amiloride, such as its ability to penetrate into cells and to act as a weak base which alters intracellular pH, may also contribute to the inhibition of Na entry into salivary cells.

Clonidine-specific antisera recognize an endogenous clonidine-displacing substance in brain.

An endogenous substance in brain, clonidine-displacing substance, binds to the same receptor populations as clonidine and is biologically active. Since receptor binding sites can be modeled by using specific antiligand antibodies, we tested the hypothesis that polyclonal antibodies raised in rat and rabbit against the clonidine analog p-aminoclonidine coupled to hemocyanin would recognize compounds structurally related to clonidine, including clonidine-displacing substance. Binding to anti-p-aminoclonidine antibodies was examined by using a competitive radioimmunoassay with tritiated p-aminoclonidine as the radioligand. Central vasodepressor agents that, like clonidine, are known to bind with high affinity to both imidazole sites and alpha 2-adrenergic receptors in brain inhibited radioligand binding to anti-p-aminoclonidine antibodies. All of these agents contain imidazol(in)e and phenyl ring moieties as part of their chemical structures (e.g., oxymetazoline); a number of other compounds without one or both of these rings failed to cross-react with the antisera. Clonidine-displacing substance, partially purified from bovine brain, also inhibited specific radioligand binding to anti-p-aminoclonidine antibodies. The inhibition was dose dependent and high affinity (IC50, 4 Units). The endogenous substance had no effect on the apparent affinity of the antibodies for the radioligand, but blocked a specific number of binding sites. Immunoprecipitation experiments showed that authentic clonidine-displacing substance, that which displaces tritiated p-aminoclonidine binding to membrane receptors, is recognized by anti-p-aminoclonidine antibodies. We conclude that a unique subset of structural determinants required for ligand interaction with both imidazole and alpha 2-adrenergic receptors is critical for binding to anti-p-aminoclonidine antibodies, and that since clonidine-displacing substance is recognized by highly clonidine-specific antisera, it may also contain these determinants within its structure, namely the imidazol(in)e and phenyl ring systems.

Conus venom interaction with α 2-adrenergic receptors in calf retina membranes

α 2-Adrenergic receptors were identified in calf retina membranes by the specific binding of the radiolabelled antagonist [ 3H]RX 781094. Crude venoms from various Conus species did not interact with the radioligand but were able to inhibit radioligand binding to the α 2-receptors with the following order of potency: C. planorbis (IC 50 = 2.1 μg protein/ml) ≈ C. tessulatus (IC 50 = 2.7) > C. eburneus (IC 50 = 19) > C. textile (IC 50 = 54) > C. geographus (IC 50 = 130). Venom from 17 other species was less or not active at all. Venom competition binding curves were steep and not affected by GTP. In contrast, the ( − )-epinephrine competition binding curve was shallow and underwent a rightward shift and steepening in the presence of GTP. The venom-α 2-receptor interaction was completely inhibited by the calcium chelating reagent EGTA. These data suggest that the venom of certain Conus species contains peptide toxins which are capable of shielding the binding site of α 2-receptors in an antagonistic manner.

A linear analog of atrial natriuretic peptide (ANP) discriminates guanylate cyclase-coupled ANP receptors from non-coupled receptors.

Atrial natriuretic peptide (ANP) contains a disulfide which is generally considered to be required for biological activity. A truncated linear ANP analog, des-Cys105,Cys121-ANP-(104-126) (referred to as analog I), that lacks the 2 cysteine residues of the parent peptide was synthesized. In competition binding studies using rabbit lung membranes, ANP-(103-126) and analog I displaced bound 125I-ANP-(103-126) from specific ANP binding sites 100 and 73%, respectively. The concentrations of ANP-(103-126) and analog I that produced 50% inhibition of radioligand binding to the membranes were 0.26 +/- 0.07 and 0.31 +/- 0.09 nM, respectively. Radioiodinated ANP-(103-126) and analog I were chemically cross-linked to binding sites on rabbit lung membranes, and the labeled membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. 125I-Analog I specifically labeled a 65,000-dalton protein and a 135,000-dalton protein which, under reducing conditions, dissociated into 65,000-dalton subunits. In contrast, 125I-ANP-(103-126) labeled specifically a nonreducible 135,000-dalton protein, in addition to the 65,000-dalton species and the reducible 135,000-dalton species. ANP-(103-126) (100 nM) stimulated rabbit lung particulate guanylate cyclase activity, whereas analog I, at the same concentration, had no effect on cyclic GMP production and did not antagonize the effect of ANP-(103-126). From these observations, we conclude that analog I is a selective ligand which binds to approximately 73% of the total ANP binding sites present in rabbit lung membranes. Unlike ANP-(103-126), analog I does not bind to the remaining 27% of the binding sites and does not activate guanylate cyclase. Binding to the cyclase-linked ANP receptor correlates with the specific labeling by 125I-ANP-(103-126) of the nonreducible 135,000-dalton membrane protein.

Ontogenesis and properties of the convulsant recognition site(s) of the γ-aminobutyric acid (GABA) receptor complex in chicken embryo

Ontogenesis of the convulsant (picrotoxinin or t-butylbicyclophosphorothionate (TBPS) site(s) was defined in chicken embryonic brain with the radioligands [ 35S]TBPS and [ 3H] t-butylbicycloorthobenzoate ([ 3H]TBOB). Binding of the radioligands is detectable from day 6 of incubation. The increase in binding after day 14 of incubation is due to an increase in the number of binding sites. The pharmacological properties of the embryonic recognition site(s) do not undergo significant changes during hatching based on the affinity of embryonic and chick brain membranes for [ 3H]TBOB; the rate of association and dissociation for TBOB; non-competitive inhibition of [ 3H]TBOB binding and modulation of 1R, αS-cis-cypermethrin interaction with the recognition site(s) by GABA; and inhibition of radioligand binding by endrin, picrotoxinin and TBPS. Early development and in vitro susceptibility of the recognition site to convulsive toxicants makes the embryo a possible target for a variety of drugs and environmental toxicants acting at this site.

Purification and characterisation of prolactin from sheep and buffalo pituitaries

A study of the problem of structural variants of proteins and their relative contribution to the expressed immunological and biological activity has been initiated using sheep and buffalo prolactins as models. The feasibility of obtaining immunologically and biologically active prolactin in high yields from the discarded ’acid pellet’ of sheep and buffalo pituitaries has been demonstrated. This permits use of the same batch of glands for purifying lutropin, follitropin and prolactin as side fractions. The major component in preparations of buffalo prolactin has a molecular size of 24 kDa. The preparations were active in a radioligand binding inhibition assay and in a rat liver based radioreceptor assay. Charge and size isomers of sheep prolactin and buffalo prolactin have been observed. The reference sheep prolactin did not, in preliminary work, give any indication of being glycosylated. However radioactive sulphate was found to be incorporated into prolactin-rich fractions of sheep and buffalo pituitariesin vitro. By physico-chemical and immunochemical criteria the [35S]-labelled material was similar to standard reference prolactin. The structural implications of sulphation have been probed.

Inhibition of radioligand binding to receptors: a competitive business

Competitive inhibition of radioligand binding is most simply described as the mutually exclusive binding of two compounds to a common site on a receptor. George Tomlinson argues that apparent competitive inhibition is to be expected for all inhibitory systems, including those involving allosteric (negatively cooperative) interactions, provided that true equilibrium conditions prevail. While thermodynamic considerations may render such behaviour indistinguishable from simple competition, binding studies covering a wide range of radioligand and inhibitor concentrations can sometimes provide insight into the complexity of the system. Noncompetitive inhibition most probably is either artefactual or arises from irreversible or quasiirreversible processes.

Molecular properties of the papaverine binding sites identified in human colonic membranes with 6,7-dimethoxy-4-(4′-amino,3′-[ 125I]iodobenzyl)isoquinoline as probe

The binding sites for papaverine in human intestine were studied with the radioiodinated papaverine derivative, 6,7-dimethoxy-4-(4′-amino,3′-[ 125I]iodobenzyl)isoquinoline ([ 125I]DMABI) as a probe. This drug was shown to bind to two specific binding sites in human colon membranes; one with a low capacity (K d = 0.02 ± 0.01 μM, B max = 0.77 ± 0.15 fmol/mg and another with a high capacity (K d = 12 ± 1.5 μM, B max = 167 ± 20 fmol/mg). The ability of various 4-benzyl isoquinolines derivatives, to inhibit radioligand binding was not influenced by 4′-substitutions, but was influenced by 6,7-substitutions, e.g. 6-hydroxy > 7-hydroxy > 6,7-dihydroxy; and by other substitutions, e.g. 1-CH 3 > N-oxyde. The papaverine probe was further used to examine structural aspects of human colon papaverine binding sites. For this purpose, [ 125I]DMABI-labeled membranes were irradiated with U.V. for 15 min at 4°C. Subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes revealed a major, specifically labeled protein of Mr = 36 000 Da. This photoaffinity labeling was protected by unlabeled DMABI in a dose-dependent manner (IC 50: 0.5 μM).

Characterization of cholecystokinin receptors in toad retina

The binding characteristics, structure, and pharmacologic properties of a cholecystokinin binding protein in toad retinal membranes have been studied. In competition binding studies using 125I-CCK-8, toad retinal membranes exhibited a high affinity binding site having a Ki 50 of 1.5 nM using CCK-8 as competitive ligand. The relative potencies of CCK-related peptides in inhibiting radioligand binding were caerulein > gastrin II ≈ CCK-8 ≈ CCK-33 > CCK-8-DS ≈ gastrin I. L-364,718, a potent inhibitor of peripheral CCK receptors, was ineffective at competition binding at concentrations up to 1 μM; dibutyryl cyclic GMP was modestly effective at competing (K D ∼ 10 mM). Covalent binding of 125I-CCK-33 to toad retinal membranes using chemical cross-linkers or UV irradiation resulted in the labeling of a major M r 62,000 protein and the intermittent labeling of minor components of M r 105,000 and M r 40,000 as determined by SDS-PAGE and autoradiography. The binding of 125I-CCK-33 to retinal membranes and the concomitant labeling of the M r 62,000 component was specifically inhibited by CCK-8 (K D ∼ 1.5 nM). Reduction of membranes with DTT abolished specific binding of 125I-CCK. SDS-PAGE analysis of affinity cross-linked membranes under non-reducing conditions revealed that the M r 62,000 protein migrated with an apparently lower molecular weight. These results suggest that the M r 62,000 CCK binding protein in the toad retina contains an intramolecular disulfide bond(s). The M r 62,000 protein was retained on a wheat germ agglutinin-agarose column and eluted with N-acetyl D-glucosamine, suggesting the glycoprotein nature of this protein. Digestion of the M r 62,000 protein with neuraminidase together with O-glycanase resulted in a discrete product of M r∼60,000. These results indicate that the M r 62,000 protein is a glycoprotein with O-linked oligosaccharide chains. Taken together, these data indicate that the CCK receptor in toad retina has a distinct structure compared to that described in rat pancreas or brain. It will be important to establish whether this difference is reflected in differences in signal transduction mechanisms.

Identification of a receptor binding region on the beta subunit of human follicle-stimulating hormone.

Mouse epidermal growth factor (mEGF) and the beta subunit of follicle-stimulating hormone (hFSH) (hFSH-beta) have been shown to inhibit binding of intact hFSH to its testes membrane receptor in vitro. Both hFSH-beta and mEGF contain the tetrapeptide sequence Thr-Arg-Asp-Leu (TRDL). Previous results demonstrated that synthetic TRDL inhibited binding of intact hFSH to receptor. We therefore investigated the possibility that TRDL was located on an exposed region of FSH-beta using a polyclonal antiserum to hFSH [NHPP anti-hFSH batch 4 (AB4)] which recognized determinants on intact hFSH and its beta subunit, but not the alpha subunit. Pituitary FSH preparations from several mammalian species produced parallel inhibition curves in a heterologous [AB4 and 125I-labeled ovine FSH (125I-oFSH)] radioimmunoassay with relative potencies similar to those observed for the same preparations assayed by radioligand receptor assay. This antiserum also competitively inhibited 125I-FSH binding to receptor. Thus, AB4 appeared to recognize antigenic determinants that are highly conserved and located at or near regions involved with hormone recognition of receptor for FSH. Synthetic TRDL inhibited 50% of 125I-hFSH binding to antiserum at a concentration of 1.36 mg/tube (9 x 10(-3) M). Other tetrapeptides (Thr-Pro-Arg-Lys and Lys-Thr-Cys-Thr) had no inhibitory activity at comparable concentrations. A mixture of the free amino acids T, R, D, and L inhibited radioligand binding only at significantly higher concentrations than TRDL.(ABSTRACT TRUNCATED AT 250 WORDS)

Penbutolol: pharmacokinetics, effect on exercise tachycardia, and in vitro inhibition of radioligand binding.

The pharmacokinetics of penbutolol 40 mg, its reduction in exercise-induced tachycardia, and the in vitro inhibition of radioligand binding to beta-adrenoceptors by plasma have been investigated in 7 healthy volunteers. The peak penbutolol concentration of 285 ng/ml was observed 1.2 h after administration, and the maximum of 4'-OH-penbutolol of 4.76 ng/ml was found after 1.64 h. Penbutolol was detected for up to 48 h, and 4'-OH-penbutolol dropped below the limit of detection after about 10 h. The terminal plasma concentration of penbutolol declined with an average half-life of 19 h. The maximum reduction in exercise-induced tachycardia was 33 beats/min 2.6 h after taking penbutolol. There was still a significant reduction of about 7 beats/min after 48 h. This effect could be adequately explained by the concentration-time course of penbutolol in combination with Clark's model of the concentration-effect relationship. Antagonist activity in plasma caused 91% inhibition of radioligand binding in vitro to beta 2-adrenoceptors on rat reticulocyte membranes 1.6 h after intake of penbutolol. By 48 h after intake, radioligand binding was still significantly inhibited (23%). The in vitro inhibition of radioligand binding by plasma showed a linear correlation with the reduction in exercise-induced tachycardia for all phases of the workload. The time course of the reduction in heart rate was completely explained by the in vitro inhibition of radioligand binding. However, it was not possible to explain the in vitro inhibition of radioligand binding by the concentration-time course of penbutolol using a simple competition model, although both variables were based on the same sampling site. When the in vitro inhibition of radioligand binding was plotted against the penbutolol concentration at the same sampling times (with both variables transformed to multiples of the apparent inhibition constant) the discrepancy became even more apparent as time-related counterclockwise hysteresis. None of the known metabolites of penbutolol can explain the discrepancy between the penbutolol concentration and the inhibition of radioligand binding in vitro. It appears that an other active metabolite is formed, which contributes to the effect in vitro and in vivo and so can explain the observed discrepancy.

Apparent competitive inhibition of radioligand binding to receptors: experimental and theoretical considerations in the analysis of equilibrium binding data.

Radioligand binding and displacement experiments are often interpreted in terms of simple competition between two ligands for occupancy of a single binding site on a receptor. Given our current understanding of the complexities of receptor structure and function, it is probable that more complex interactions occur in many cases. By analysis of a hypothetical two-site receptor model, we show that apparent competitive inhibition can arise in several ways, depending on the specificities of the two sites and the interactions between them. We show that binding experiments can in some cases be used to rule out certain models from among a group of apparently plausible ones, provided that experimental criteria are met which permit a meaningful statistical comparison of models to be made. Ideally, these should include: i) an independent study of ligand and inhibitor binding in the absence of each other; ii) carrying out saturation binding and displacement experiments over as wide a range of ligand and inhibitor concentrations as possible; iii) computerized curve-fitting and statistical analysis as a tool for model-testing. While practical limitations may restrict the attainment of such goals, a thorough study of the equilibrium binding properties of a particular receptor system provides the foundation for the design of more definitive experiments at the molecular level, upon which the proof of any binding model ultimately must rest.