Sites In Membrane Preparations Research Articles

The affinity and efficacy of naturally occurring catecholamines at beta-adrenoceptor subtypes.

The contribution of affinity and efficacy to the agonistic actions of the naturally occurring catecholamines, (-)-noradrenaline and (-)-adrenaline at beta-adrenoceptor sites were assessed in guinea-pig driven left atrial (beta 1) and K+-depolarized uterine (beta 2) preparations. The dissociation constants of each agonist, required in these calculations, were calculated using radioligand binding techniques. [125I]Iodocyanopindolol bound to sites in membrane preparations of each tissue which have been shown to represent beta 1-(atria) and beta 2-(uterus) adrenoceptors. It was found that (-)-noradrenaline was approximately 10-fold more selective for the beta 1- as opposed to the beta 2-adrenoceptor in the pharmacological studies. Affinity/efficacy calculations indicated that this selectivity was entirely due to a selective affinity for the beta 1-adrenoceptor subtype. (-)-Noradrenaline, (-)-adrenaline and the reference compound (-)-isoprenaline all had approximately the same efficacy at either beta-adrenoceptor subtype.

Increased density of M1 receptors in the hippocampus of juvenile rats chronically deprived of NGF

Binding studies were used to assess the changes in affinity and/or number of M1 muscarinic receptors in hippocampi from juvenile rats chronically deprived of NGF. NGF deprivation was obtained by implanting into right ventricle at postnatal day 2 (P2) hybrydoma cells secreting high levels of monoclonal antibodies against NGF (αD11). Parenteral myeloma cells (P3U) were used as controls. Competition experiments were used to characterise the [ 3 H ]-PNZ binding sites in membrane preparations of hippocampi from rats sacrificed at P15. [ 3 H ]-PNZ bound M1 receptors both in P3U and αD11 group as shown by displacing potency order of antagonists: TLZ=4-DAMP>PNZ> p-F-HHSiD>MTC. The deprivation of NGF for two weeks significantly increased the number of M1 receptors without changing the K i values of antagonists with exception of methoctramine which showed an increase in affinity in αD11 group. Similar changes in binding parameters were already observed after the first week of anti-NGF treatment. In contrast, a treatment for a week with implant at postnatal day 15 failed to produce any changes in M1 binding parameters. These results provide further physiological evidence for developmentally regulated modulatory role of NGF in the cholinergic function in the hippocampus.

Whitefly (Hemiptera: Aleyrodidae) binding site for imidacloprid and related insecticides: a putative nicotinic acetylcholine receptor.

Imidacloprid is used extensively to control sweetpotato whiteflies, Bemisia argentifolii Bellows & Perring [also known as B. tabaci (Gennadius) biotype B]. As a radioligand, [3H]imidacloprid binds rapidly to a single class of high-affinity sites in membrane preparations from whole adult whiteflies with an apparent dissociation constant of 2 nM and maximal binding capacity of 101 fmol/mg protein. Three related compounds (the nitromethylene analog of imidacloprid, acetamiprid, and nitenpyram) inhibit [3H]imidacloprid binding by 50% at 0.40, 2.9, and 57 nM, respectively. The pharmacological profile of the binding site (examined with imidacloprid and the analogs listed above, and nicotine, alpha-bungarotoxin, carbachol, acetylcholine [with paraoxon], and atropine) is consistent with that anticipated for a nicotinic acetylcholine receptor and correlates well with binding results for house fly, Musca domestica L., head membranes under the same conditions. Thus, [3H]imidacloprid is a suitable radioligand to investigate the putative nicotinic acetylcholine receptor of Bemisia and the possible modifications of this target site associated with selection of resistant strains.

CHARACTERIZATION OF A SEROTONIN RECEPTOR IN THE CNIDARIAN RENILLA KOELLIKERI: A RADIOBINDING ANALYSIS

Serotonin (5-HT) binding sites in membrane preparations from the sea pansy Renilla koellikeri were identified using [3H]5-HT as radioligand and unlabelled 5-HT as displacer of specific binding. Saturation and kinetic studies revealed a mixed population of [3H]5-HT binding sites which as a whole displayed slow association kinetics, saturability, negligible dissociation and low affinity. The dopaminergic antagonist trifluoperazine specifically displaced the non-dissociated binding sites, allowing the characterization of a homogenous population of saturable sites exhibiting faster association and full dissociation. Scatchard analysis of this population revealed a KD of 23–34 nM and binding site density (Bmax) of 8.5–20.6 pmol/mg protein depending on body region. The pharmacological profile of the dissociable [3H]5-HT binding sites could not be categorized with that of any of the existing vertebrate and invertebrate 5-HT receptor subtypes in the current nomenclature. Especially noteworthy was the absolute inability of LSD, a ligand of choice for all hitherto identified invertebrate 5-HT receptors, to compete for the binding sites in sea pansy membrane preparations. It is proposed that these binding sites represent an evolutionary forerunner of the primordial 5-HT receptor that diversified as multiple subtypes in higher invertebrates and vertebrates. © 1997 Elsevier Science Ltd

The developmental and circadian variation of melatonin receptors in the chicken spinal cord.

The developmental variation of 2-[125I]iodomelatonin binding sites in membrane preparations of the chicken spinal cords was investigated. In 19-day-old embryos, the maximum number of binding sites (Bmax) was the highest during the development. With the increase in age posthatch, the Bmax decreased gradually and significantly from 1-day-old to 4-month-old chickens. However, there were no significant changes in equilibrium dissociation constant (Kd) among all age-groups studied. To study the circadian variation, the 2-[125I]iodomelatonin binding sites in the spinal cords of 3-week-old chickens were determined at 4-hour intervals throughout a 24-hour period under a 12-hour light/12-hour dark cycle. There was a diurnal rhythm in both Bmax and Kd of [125I]iodomelatonin binding sites in which Bmax decreased and Kd increased in the dark period. The characteristics of developmental and circadian variation of melatonin receptors in the chicken spinal cord suggest that melatonin may exert a direct action on spinal cord functions and that it plays a more important role in the young animal.

Quantitative autoradiography of peripheral opioid binding sites in rat lung

Previous studies in our laboratory have characterized non-conventional opioid binding sites in membrane preparations from both rat and human lung. The studies described in this paper utilized autoradiography to investigate the regional distribution of these [ 3H]morphine binding sites within rat lungs. Specific binding of [ 3H]morphine was saturable and Rosenthal analysis of tissue section wipes revealed the presence of both high-affinity and low-affinity opioid binding sites. The mean ± S.E.M. binding affinity and the mean ± S.E.M. density values for the low-affinity binding site ( K d = 217 ± 160 nM, B max = 12 ± 8 pmol/mg protein) were similar to the values obtained in our previous whole-rat lung membrane binding assays ( K d = 187 ± 36 nM, B max = 13.5 ± 2 pmol/mg protein) (Cabot, P.J., P.R. Dodd, T. Cramond and M.T. Smith, 1994, Eur. J. Pharmacol. 268, 247). Quantitative autoradiography showed that the highest density of opioid binding sites appeared to be present within the alveolar wall (13.2 ± 0.8 pmol/mg protein). A significantly lower ( P < 0.05) density of binding was also observed in the smooth muscle of the trachea and main bronchi (5.5 ± 2.1 pmol/mg protein). However, no morphine binding sites were evident in the smooth muscle surrounding the smaller airways and pulmonary vasculature within the lobes of the rat lung. It remains to be investigated whether the opioid binding sites located within the trachea and main bronchi of the rat airways are the prejunctional opioid receptors on C-afferent nerve fibres which modulate the release of potent inflammatory neuropeptides.

Myometrial steroid concentration and oxytocin receptor density in parturient women at term

The aim of this study was to measure oxytocin receptor concentration in myometrial tissue from term pregnant women with normal and dysfunctional labor and to relate this concentration to the progress of labor and to the levels of estradiol and progesterone in the same myometrium. Myometrial biopsies were obtained from 50 term pregnant women undergoing cesarean section. The patients were categorized as follows: not in labor, normal labor, successful oxytocin-augmented labor, and oxytocin-resistant labor. Specific binding of [ 3H]oxytocin to high-affinity sites in membrane preparations from myometrial tissues was determined. Estradiol and progesterone were assayed using tritiated steroids with a sensitive radioimmunoassay technique. Oxytocin receptor density was significantly lower in oxytocin-resistant labor compared to successful oxytocin-augmentated labor ( P < 0.04) and to spontaneously active normal labor ( P < 0.02). Oxytocin receptor concentration was also significantly lower in non-labor patients compared to normal spontaneous labor ( P < 0.01), and successful oxytocinaugmented labor ( P < 0.02). There was a positive relationship between the progress of cervical dilatation (cm/h) and oxytocin receptor density in the myometrium ( r = 0.408, P < 0.025). The concentration of progesterone and estradiol in the pregnant myometrium did not differ in patients with different types of labor or with the state of uterine contractile activity. Our results suggest that individual myometrial sensitivity is an important determinant of the response to administered oxytocin in humans. Furthermore, myometrial oxytocin receptor expression in vivo seems not be related to ovarian steroid concentration in the myometrium. The low oxytocin receptor density in oxytocin-resistant dystocia needs further investigation.

Characterisation of Melatonin Binding Sites in the Eye of the Japanese Quail (Coturnix japonica)

Melatonin binding sites were examined in the quail eye using 2-[125I]iodomelatonin. Radioreceptor assays indicated similar binding sites in membrane preparations of neural retina (NR) and choroidretinal pigment epithelium (C-RPE) eye components. In both tissues binding of the radioligand was specific, saturale and of high affinity [Kd values NR 50.8 ± 19.5 pM, C-RPE 98.2 ± 35.4 pM, mean ± SEM (n = 4)] and low capacity (Bmax values NR 12.4 ± 2.7 fmol/mg protein. C-RPE 21.5 ± 3.2 fmol/mg protein). Kinetic studies demonstrated that association of 2-[125I]iodomelatonin was rapid and further that this binding was reversible upon the addition of 1 μM melatonin. The order of pharmacological potencies of various indoles tested in 2-[125I]iodomelatonin displacement studies was melatonin > 6-chloromelatonin > 64-hydroxymelatonin >N-acetylscrotonin ⪢ 5-methoxytryptophol > 5-hydroxytryptamine > 5-methoxytryptamine (5-hydroxytryptamine > 5-methoxytryptophol for C-RPE). Studies with guanine nucleotides indicated that the signal transduction mechanism of the binding site may involve a G-protein linkage. This melatonin binding site displays several pharmacological similarities with those investigated in the retina of other species and with those previously characterised in the quail brain.

Further evidence that the classical α1A- and cloned α1c-adrenoceptors are the same subtype

We compared the inactivation of [ 3H]prazosin binding sites in membrane preparations of cell-lines expressing the cloned α 1b, α 1c and α 1d-adrenoceptors after pretreatment with the alkylating agents, 10 μM chlorethylclonidine or 10 nM SZL-49 (1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-(2-bicyclo[2.2.2]octa-2,5-diene-Z-carbonyl)-piperazine). The cloned α 1b-adrenoceptor exhibited a similar inactivation profile to that of the classical α 1B-adrenoceptor of rat liver in that chlorethylclonidine in contrast to SZL-49 produced a marked degree of inactivation. A similarity between the cloned α 1c-adrenoceptor and the classical α 1A-adrenoceptor of rat submaxillary gland was also noted in that both subtypes were highly sensitive to SZL-49 but relatively insensitive to chlorethylclonidine. The cloned α 1d-adrenoceptor displayed a unique profile in that both chlorethylclonidine and SZL-49 produced a marked inactivation of this subtype. The similarity of the alkylation-inactivation profiles between the cloned α 1c and classical α 1A-adrenoceptors support the recent proposal that these two α 1-adrenoceptors in fact correspond to the same subtype.

Increased affinity of histamine H 1 binding to membranes of human myometrium at the end of pregnancy

1. 1. The characterization of H 1 binding sites in membrane preparations of human myometrium obtained from pregnant and non-pregnant women was performed by using 3H-mepyramine as the radioactive ligand. 2. 2. Saturation curve analysis revealed that 3H-mepyramine is bound to a single class of binding sites. Changes in the H 1 site binding parameters were observed at the end of pregnancy, resulting in an increased affinity relative to non-pregnant tissue ( K d : 131.0 ± 8.8 (non-pregnant) and 72.5 ± 7.5 (pregnant) nM, n = 6, P < 0.01). 3. 3. A reduction in receptor concentration at the end of pregnancy was also observed, [ B max : 565.2 ± 43.7 (non-pregnant) and 309.6 ± 25.9 (pregnant) fmol/mg prot, n = 6, P < 0.01]. It is possible that this reduction in B max could be attributed to a dilution factor due to the increase in membraneous proteins that occurs during gestation.

Dopamine D2 receptor gene expression and binding sites in adrenal medulla and pheochromocytoma.

To determine whether dopamine D2 receptors are present in normal and neoplastic chromaffin tissues, 10 pheochromocytomas and 5 human adrenal glands were studied. Dopamine D2 receptor messenger ribonucleic acid corresponding to a single band of approximately 2.5 kilobases was detected in both pheochromocytoma and human adrenal gland by Northern blot analysis. D2 receptor messenger ribonucleic acid levels determined by dot blot analysis were 3.1-fold lower in human adrenal medullas than in pheochromocytomas (P < 0.001). Simultaneous Scatchard analysis of [3H]spiperone binding experiments demonstrated the presence of two different binding sites in membrane preparations from bovine adrenal medullas [R1: Kd = 0.14 nmol/L; binding capacity (Bmax) = 6.2 fmol/mg protein: R2: Kd = 16 nmol/L; Bmax = 223 fmol/mg protein]. Similarly, two binding sites were present in membrane preparations from pheochromocytomas (R1: Kd = 0.39 nmol/L; R2: Kd = 61 nmol/L]. Binding capacities were greatly variable among pheochromocytomas (R1: Bmax = 12.0-372.5 fmol/mg protein; R2: Bmax = 1,000-11,586 fmol/mg protein). The relative potencies of different compounds to displace [3H]spiperone were spiperone > domperidone > (+)-butaclamol > quinpirole > SCH 23390 in bovine adrenal medulla, and spiperone >> domperidone > quinpirole > (+)-butaclamol > SCH 23390 in pheochromocytoma. We conclude that dopamine D2 receptors are synthesized in human adrenal medulla and pheochromocytoma tissues.

Characterization of the inhibitory mechanism of 1-methyl-4-phenylpyridinium and 4-phenylpyridine analogs in inner membrane preparations.

We have investigated the mechanism of the inhibition of membrane-bound NADH dehydrogenase by 1-methyl-4-phenylpyridinium (MPP+) and a series of its 4'-alkyl-substituted analogs of increasing hydrophobicity, as well as their neutral, desmethyl congeners. Comparison of hydrophobicity, as measured by partition coefficients, with the IC50 for the inhibition of NADH oxidase activity in mitochondrial inner membrane preparations shows a negative correlation, but the cationic inhibitors are more effective than the neutral analogs with similar hydrophobicity. The presence of 10 microM tetraphenylboron (TPB-) potentiates the inhibitory power of positively charged analogs up to 4'-pentyl-MPP+, while the neutral inhibitors are unaffected by TPB-. Without TPB-, the more hydrophilic analogs give incomplete inhibition, but the inclusion of TPB- permits the attainment of complete inhibition, accompanied by the appearance of sigmoidal titration curves. These data support the hypothesis that MPP+ analogs, like rotenone, are bound at two sites on the enzyme and occupancy of both is required for complete inhibition. TPB-, by forming ion pairs with the cationic analogs, facilitates their equilibration to both sites in membrane preparations. When present in molar excess over the MPP+ analog, TPB- partially reverses the inhibition by decreasing its concentration in the more hydrophilic binding site. The effect of temperature and of pH on the IC50 values for inhibition support the concept of dual binding sites, and the pH dependence of the inhibition reveals the participation of two ionized protein groups in the binding, one of which may be a thiol group.

Comparison of [ 3H]resiniferatoxin binding by the vanilloid (capsaicin) receptor in dorsal root ganglia, spinal cord, dorsal vagal complex, sciatic and vagal nerve and urinary bladder of the rat

In the present report we compared the properties of [ 3H]resiniferatoxin (RTX) binding by the vanilloid receptors present at different parts of the primary afferent neurons of the rat. We found no major differences in either the affinity or the cooperativity of [ 3H]RTX binding by vanilloid receptors on the cell body, central terminals, peripheral terminals or axons. Specific binding of [ 3H]RTX to dorsal root ganglia, whole spinal cord, dorsal vagal complex, urinary bladder, and sciatic and vagal nerves all followed sigmoidal saturation kinetics indicating positive cooperativity among the binding sites. The cooperativity indexes determined by fitting the data to the Hill equation were 1.82 ± 0.11, 2.21 ± 0.04, 2.55 ± 0.01, 1.91 ± 0.11, 2.03 ± 0.09 and 2.27 ± 0.04, respectively. The dissociation constants in dorsal root ganglia, spinal cord, dorsal vagal complex, urinary bladder, and sciatic and vagal nerve membranes were 46.5 ± 2.7, 29.3 ± 5.1, 28.2 ± 1.2, 60.8 ± 4.4, 59.9 ± 1.9 and 45.2 ± 0.7 pM; the receptor densities were 219 ± 14, 48 ± 5, 67 ± 1, 32 ± 7, 61 ± 9, and 100 ± 20 fmol/mg protein, respectively. We could not show any major differences in the affinities of capsaicin and capsazepine in inhibition of [ 3H]RTX binding by the different membrane preparations either. In all cases the initial enhancement of [ 3H]RTX binding by nonradioactive RTX, capsaicin, and capsazepine confirmed the existence of positive cooperativity among the binding sites. We were unable to detect specific [ 3H]RTX binding sites in membrane preparations of the preoptic area, locus ceruleus, substantia nigra, striatum and paraventricular nuclei of the rat brain under our present conditions. Our results suggest the uniformity of the vanilloid receptors present at different parts of the primary afferent neuron.

125I]iodomelatonin-binding sites in the bursa of Fabricius of birds: binding characteristics, subcellular distribution, diurnal variations and age studies.

The melatonin-binding sites in membrane preparations of the bursa of Fabricius of birds were studied using [125I]iodomelatonin as a radioligand. The binding sites were stable, saturable, reversible and of high affinity. Scatchard analysis of specific binding revealed equilibrium binding constants (Kd) of (means +/- S.E.M.) 43.1 +/- 5.9, 73.3 +/- 7.6 and 35.3 +/- 4.8 pmol/l respectively at the mid-point of the light period (mid-light) in chickens, pigeons and quail, with a total number of binding sites (Bmax) of 1.23 +/- 0.15, 1.33 +/- 0.18 and 0.94 +/- 0.07 fmol/mg protein. The diurnal variation in [125I]iodomelatonin binding showed that the Bmax was 45, 115 and 70% higher at mid-light than at mid-dark in the bursae of chickens, pigeons and quail respectively. The Kd value determined from kinetic analysis was 49.0 +/- 6.4 pmol/l at mid-light in the chicken bursa. The [125I]iodomelatonin-binding sites of chicken bursal membranes had the following order of pharmacological affinities: 2-iodomelatonin > melatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetyl-serotonin > 5-hydroxytryptamine, tryptamine, 5-methoxytryptophol, 1-acetylindole-3-carboxaldehyde, 3-acetylindole, L-tryptophan, 5-hydroxyindole-3-acetic acid, 5-hydroxytryptophan suggesting that the [125I]iodomelatonin-binding sites were highly specific for melatonin. The subcellular distribution of binding sites in the chicken bursa was in the following descending order: nuclear > mitochondrial > microsomal > cytosolic fraction. There was an age-related decrease in [125I]iodomelatonin-binding in chicken bursal membranes, with higher densities in the neonate.(ABSTRACT TRUNCATED AT 250 WORDS)

α 2A Adrenoceptors and non-adrenergic idazoxan binding sites in calf brain and retina are distinct from those in human brain

α 2 Adrenoceptors in membrane preparations of human and calf frontal cortex and of calf retina can be labelled by the antagonists [ 3H]idazoxan, [ 3H]rauwolscine and [ 3H]RX 821002. Present and previous data indicate that [ 3H]idazoxan possesses the highest affinity for the α 2 adrenoceptors in the calf tissues, whereas [ 3H]rauwolscine displays the highest affinity for those in the human frontal cortex. Competition binding experiments with adrenergic and serotonergic drugs further support the notion that the α 2 adrenoceptors in calf frontal cortex and retina are similar, but distinct from the receptors in human frontal cortex. The α 2 adrenoceptors in the three investigated tissues display low affinity for the antagonist prazosin, which suggests that they all belong to the α 2A subclass. The competition binding curves of the α 2A adrenoceptor subtype-selective agonist oxymetazoline are shallow, but undergo a rightward shift and steepening in the presence of GTP. The shallow curves can therefore be attributed to the coupling of the α 2 adrenoceptors to G proteins. The different binding characteristics of the α 2A adrenoceptors from the investigated human and bovine tissues are likely to reflect species-related differences in protein structure. [ 3H]Idazoxan binds also to non-adrenergic sites in membrane preparations from the three tissues. However, the affinity of [ 3H]idazoxan for these sites in calf cortex and retina is appreciably lower than for those in human cortex. The species-related differences of the non-adrenergic idazoxan binding sites may be due to differences in protein structure or even to differences in gene-product.

Characteristics of 2-[125I]iodomelatonin binding sites in the pigeon spleen and modulation of binding by guanine nucleotides.

2-[125]Iodomelatonin binding sites in membrane preparations of pigeon spleen have been characterized. The binding was stable, saturable, reversible, and of high affinity. Rosenthal and Hill analyses showed that the radioligand-receptor interaction involved a single class of binding sites. Analysis of the binding results of spleens collected during mid-light revealed an equilibrium dissociation constant (Kd) of 36.6 +/- 4.8 pmol/l (mean +/- sem, n = 10) and a maximum density (Bmax) of 2.3 +/- 0.2 fmol/mg protein. There was no significant difference in the Kd (46.9 +/- 5.0 pmol/l) or the Bmax values (2.4 +/- 0.3 fmol/mg protein) for spleens collected during mid-dark (n = 9), although the mid-dark serum and pineal melatonin levels were significantly higher (P < 0.05) than the corresponding mid-light values. Kinetic analysis showed a Kd of 8.6 +/- 2.0 pmol/l (n +/- 4), in agreement with that derived from the saturation studies. Except for inhibition by 2-iodomelatonin, melatonin, 6-chloromelatonin, 6-hydroxymelatonin and N-acetylserotonin, the other indoles or neurotransmitters tested have little inhibition on the binding. In addition, guanosine 5'-O-(3-thiophosphate) (GTP gamma S), a nonhydrolysable analog of GTP, was found to inhibit the binding in a dose-dependent manner. Saturation studies revealed that this is due to a decrease in both the affinity and density of the binding sites. These data suggest that a single type of melatonin receptor is found in the pigeon spleen and that the site is coupled to a guinine nucleotide binding protein (G-protein). Our findings support a direct pineal melatonin action on the immune system.

Characterization of serotonin binding sites in insect ( Locusta migratoria) brain

Radioligand binding studies using [ 3H]serotonin, [ 3H]ketanserin and [ 3H]mianserin were used to characterize 5HT receptor sites in membrane preparations from the brain of the locust, Locusta migratoria migratorioides. The [ 3H]serotonin binds reversibly to brain membrane preparations with high affinity and the receptor has 5HT 1 characteristics. However, studies with specific antagonists suggest that the receptor differs pharmacologically from the 5HT 1 receptor subtypes which have been identified in vertebrate brain. Scatchard analysis indicates that the binding sites have a single component with a dissociation constant of 2.98±0.19nM and B max value of 14.45±1.12 pmol/g tissue. Serotonin, bufotenin, N, N-dimethyltryptamine, d-LSD, BOL are equipotent displacers of [ 3H]serotonin binding. Several other potential agonists and antagonists were tested and shown to effect varying degrees of inhibition. Ca and Mg ions have no significant effect on the [ 3H]serotonin binding; however, guanine nucleotides modulate the binding suggesting that G-protein is involved in the serotonin action. No specific, saturable ketanserin binding was found, thus indicating lack of a vertebrate-like 5HT 2 receptor. [ 3H]Mianserin binds to the brain membrane; however, the pharmacology of mianserin binding suggests that mianserin binds to an octapamine-rather than a serotonin-receptor.

Chloroethylclonidine Discriminates between α1A- and α1B-Adrenoceptors in the Presence of Guanosine 5’-Triphosphate in Rabbit Thoracic Aorta

Studies on the displacement of [3H]prazosin binding by the alpha 1-agonist phenylephrine revealed the presence of at least high- and low-affinity binding sites in membrane preparations prepared from rabbit thoracic aorta. Although the low-affinity site was reduced by the pretreatment of tissues with chloroethylclonidine, this site was unaffected by the same pretreatment of membrane preparations that did not contain the GTP analog. However, in membrane preparations with the metabolically stable GTP analog GTP gamma-S (10(-5) M) and single cell preparations, the low-affinity site was completely eliminated by the chloroethylclonidine pretreatment. Displacement studies with the alpha 1-antagonist WB4101 also revealed high- and low-affinity binding sites labeled by [3H]prazosin. Displacement curves of WB4101 obtained from membrane preparations in the presence of GTP gamma-S (10(-5) M) did not differ from those in the absence of GTP gamma-S. These results suggest that the low affinity phenylephrine binding site labeled by [3H]prazosin was selectively bound by the chloroethylclonidine used to pretreat the tissues, membrane preparation containing GTP gamma-S and single cells, and that chloroethylclonidine is able to recognize these two distinct subtypes of alpha 1-adrenoceptors only when GTP gamma-S is present.

The identification and characterization of 125I-labelled iodomelatonin-binding sites in the duck kidney

The melatonin-binding sites in membrane preparations of duck kidney were demonstrated by utilizing 125I-labelled iodomelatonin as a radioligand. Binding at these sites was found to be reversible, saturable, specific and of high affinity. Scatchard analysis of the specific binding revealed an equilibrium binding constant (Kd) of 44.6 +/- 4.4 pmol/l (n = 6) and a total number of binding sites (Bmax) of 6.43 +/- 0.60 fmol/mg protein (n = 6) at the mid-point of the light period (mid-light). The Hill coefficient approached 1.0, suggesting a single class of 125I-labelled iodomelatonin-binding site in the duck kidney. Diurnal variation in 125I-labelled iodomelatonin binding showed that the Bmax was 53.4% higher at mid-light than at mid-point of the dark period (P < 0.05), with no significant variation in Kd. The Kd value determined from kinetic analysis was 22.5 pmol/l in birds at mid-light, which was comparable with values determined from equilibrium studies. The order of pharmacological affinity for 125I-labelled iodomelatonin-binding sites in the duck kidney membrane preparations was: 2-iodomelatonin > melatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin >> 5-methoxytryptamine, 5-methoxytryptophol, 5-hydroxytryptamine, tryptamine, 1-acetylindole-3-carboxaldehyde, 5-hydroxyindole-3-acetic acid, L-tryptophan, 5-methoxyindole-3-acetic acid, 3-acetylindole, acetylcholine, epinephrine, norepinephrine and harmaline. The pharmacological characteristics indicated that 125I-labelled iodomelatonin-binding sites are highly specific for melatonin. Our finding of 125I-labelled iodomelatonin-binding sites in the kidney suggests that melatonin may regulate kidney function.(ABSTRACT TRUNCATED AT 250 WORDS)

Saxitoxin binding to neural membranes from pyrethroid susceptible and resistant tobacco budworm moths Heliothis virescens

1. [ 3H]Saxitoxin, a sodium channel probe, was found to bind reversibly with high affinity to a single class of noninteracting sites in membrane preparations from whole head homogenates of tobacco budworm, Heliothis virescens (F.), moths from a pyrethroid susceptible strain and from a pyrethroid resistant strain with nerve insensitivity and metabolic resistance mechanisms. 2. No significant interstrain differences were detected in saturation ( K D, B max) and kinetic ( k +1, k −1) experiments. 3. Also, five pyrethroids, two formamidines, and DDT had no significant effect on [ 3H]saxitoxin binding when tested at concentrations of 1 × 10 −4 and 1 × 10 −8 M. 4. It appeared that a decreased density in sodium channels in resistant strain moths as compared with that in susceptible strain moths was not a factor in pyrethroid-induced resistance in this particular strain of tobacco budworm.