Desensitization of thyrotrophin-releasing hormone (TRH)-induced growth hormone secretion in chickens: coincident down-regulation of TRH binding to pituitary membranes

Release of GH is stimulated by TRH in chickens. However, for 60 min following a priming injection of TRH, a second injection of TRH is unable to provoke further GH release. TRH binds to the plasma membranes of the pituitary caudal lobe, in which somatotrophs predominate, although the magnitude of [3H]3-methyl-histidine2-TRH ([3H]Me-TRH) binding was reduced (by 25-50%) 30 min after the i.v. administration of a dose of TRH (5 micrograms/kg) maximally effective in provoking GH release. A significant reduction in [3H]Me-TRH binding to caudal lobe membranes was also observed within 15 min of TRH administration and was maintained for at least 60 min. Control levels of [3H]Me-TRH binding were restored 2 h after TRH injection, coincident with the restoration of GH responsiveness to TRH challenge. The suppression of [3H]Me-TRH binding to pituitary membranes 30 min after in-vivo TRH administration was dose related, whereas the maximal (10 min) GH response to TRH was biphasic. The suppression of [3H]Me-TRH binding to chicken pituitary membranes was due to direct pituitary actions of TRH and could be induced by a 30-min exposure to 100 nM TRH in vitro. These results demonstrate that avian pituitary TRH-binding sites differ greatly from mammalian ones in the timing of the onset and duration of down-regulation. Pituitary TRH-binding sites in birds are rapidly and transiently down-regulated following TRH administration in vivo, coincident with the period of GH refractoriness to TRH challenge.     

Thyroidal inhibition of growth hormone secretion in fowl: tri-iodothyronine-induced down-regulation of thyrotrophin-releasing hormone-binding sites on pituitary membranes

The number, but not affinity, of binding sites for [3H]3-methyl-histidine2-TRH ([3H]Me-TRH) on chicken adenohypophysial plasma membranes was increased in chickens made hypothyroid by goitrogen (methimazole) treatment (50 mg/kg per day for 7 days), which also increased circulating GH concentrations. Daily i.p. injection of thyroxine (T4; 100 micrograms/kg for 7 days) had no effect on [3H]Me-TRH binding to pituitary membranes, although it suppressed endogenous GH secretion. Binding of [3H]Me-TRH to pituitary caudal lobe membranes was, however, suppressed by tri-iodothyronine (T3) injected chronically (100 micrograms/kg per day, i.p., for 7 days) or acutely (100 micrograms/kg, 2 h before being killed). The suppression of [3H]Me-TRH binding and inhibition of GH secretion following T3 administration was dose related. Binding of [3H]Me-TRH to caudal lobe membranes was also suppressed following the incubation of pituitary glands with T3 in vitro, and the response was both dose and time related. These results suggest that T3 inhibits GH secretion in fowl by a down-regulation of pituitary TRH receptors. However, other mechanisms are involved in thyroidal inhibition of GH release in birds, since T4 had no effects on [3H]Me-TRH binding yet suppressed GH secretion in vivo.     

Somatostatin binding to chicken adenohypophysial membranes

[125I-Tyr1]-Somatostatin (SRIF)-binding sites were demonstrated on crude plasma membrane preparations from chicken pituitary glands. These binding sites were saturable and of high affinity (dissociation constant less than 1.0 nM) and low capacity (maximal binding capacity less than 200 fmol/mg protein) and were specific for SRIF moieties. The number and affinity of these binding sites in the caudal lobe of the pituitary, in which somatotrophs predominate, were similar to those in the cephalic lobe, in which lactotrophs and thyrotrophs are confined. Gonadotrophs are present in the caudal lobe, but whereas exogenous SRIF inhibited secretagogue-induced GH release from incubated pituitary glands, it had no effect on basal or secretagogue-induced LH release. The half-maximal binding of SRIF to the caudal lobe membranes (3 nM) was similar to that required for half-maximal suppression of TRH-induced GH release, suggesting a role for these binding sites in the regulation of GH secretion in birds.     

Preparation of 3H-[3-M3-His2]TRH as an improved ligand for TRH receptors

[3H]-3-methyl-His2]thyrotropin releasing hormone ([3H]MeTRH) binds to sites in the sheep anterior pituitary gland which appear to be the same as those occupied by [3H]TRH and which can therefore be identified as TRH receptors. In competition experiments performed in parallel, both ligands gave the same number of binding sites, 15 pmol/g wet weight, and showed the same pharmacology for 19 TRH analogs ranging over more than 5 orders of magnitude in potency. The apparent dissociation constant of binding of [3H]MeTRH was about 3.5 nM compared to 29 nM for [3H]TRH. Kinetic experiments with [3H]MeTRH yielded a rate constant for association of 1.4 x 10(7) M-1 min-1 and rate constants for the biphasic dissociation of 5 x 10(-2) min-1 (fast phase) and 7 x 10(-3) min-1 (slow phase). Detailed methods are described for preparation of [3H]MeTRH by reduction of the dehydroproline precursor and its purification by ion exchange and antibody affinity chromatography. The major advantage of the new ligand is that its higher affinity of binding gives relatively less non-specific binding than is obtained with [3H]TRH, particularly in central nervous tissue.     

3H]-Thyroliberin (TRH) binding to nuclei isolated from a pituitary clonal cell line (GH3).

[3H]Thyroliberin (TRH) has been previously shown to enter its target GH3 cells. Intracellular [3H]-TRH was found chemically unmodified and associated to organites, cytosol and nucleus. We studied the [3H]-TRH binding capacity of a highly purified nuclear fraction isolated by an original procedure from GH3 cells. The nuclei still presented their double nuclear envelope. They are able to bind [H]-TRH to the same extent as nuclei isolated from GH3 cells previously exposed to [3H]-TRH. The equilibrium of binding was reached after 2--5 min incubation at 25 degrees or 35 degrees C. The binding is stable at 4 degrees C and partially (50%) dissociated within 15 min at 25 degrees C. 50% of the binding was inhibited by large excess of unlabelled TRH. Nuclei obtained from a variant GH3 cell which has lost its responsiveness to TRH presented only the noncompetitive binding compartment. The binding was found dose dependent and not saturable. Two apparent dissociation constants were evaluated: 1.5--2.5 x 10--8M and 2.10--6M, respectively, for high and low doses of [3H]-TRH. The first one was identical to that previously found for intact GH3 cells. The present data show the existence of specific nuclear binding sites for TRH, establish their characteristics and suggest a possible nuclear site of action for that peptide hormone.     

TRH receptors in fish

The brains of goldfish and other teleosts contain high-affinity binding sites for [3H][3-methyl-His2]thyrotropin-releasing hormone [( 3H]MeTRH) which closely resemble TRH receptors in mammalian brain and pituitary gland in apparent dissociation constant (KD = 3-4 nM), in pharmacology for eight TRH analogs, and in exhibiting marked regional differences in the density of binding sites, with highest binding in the cerebrum and lowest in the cerebellum. Fish brain differs from mammalian brain in containing a prominent additional class of much lower affinity [3H]MeTRH binding sites (KD about 15 microM), of unknown nature, which also exhibit regional differences. Fish pituitary glands contain high-affinity [3H]MeTRH binding sites, but insufficient tissue has prevented full characterization. Little or no saturable binding of [3H]MeTRH was detected in several goldfish peripheral tissues.     

Short-loop inhibition of thyrotrophin-releasing hormone-induced growth hormone secretion in fowl

The i.c.v. administration of 0.1 or 10 micrograms ovine (o)GH to 12- to 16-week-old hypothyroid chickens of a sex-linked dwarf (SLD) strain suppressed the basal plasma GH concentrations, measured 24 h afterwards. The GH response of the oGH-injected SLDs to TRH was suppressed, in a dose-related way, in comparison with that induced by TRH in birds given control injections (10 micrograms) of bovine serum albumin (BSA). Basal circulating concentrations of GH in euthyroid K strain birds of the same age were even lower than in the SLDs following injection of 10 micrograms oGH, and were not further reduced by oGH administration. The GH response to TRH in the K strain birds injected i.c.v. with 0.1 or 10 micrograms oGH was, nevertheless, suppressed in comparison with the BSA-injected K strain controls. The i.c.v. administration of oGH also suppressed circulating concentrations of LH and the LH response to TRH in the K strain birds. Twenty-four hours after i.c.v. administration of oGH (10 micrograms), the somatostatin (SRIF) content in the medial basal hypothalamus of 8-week-old euthyroid cockerels was greater than that in BSA (10 micrograms)-injected controls. At the same time, the binding of [3H]3-methyl-histidine2-TRH to the pituitary caudal and cephalic lobes of GH-injected birds was less than that in the controls. These results suggest that GH regulation in avian species is partly mediated by an inhibitory short-loop mechanism (mediated by hypothalamic SRIF and a down-regulation of pituitary TRH-binding sites) that suppresses basal and secretagogue-induced GH release.     

[3H]xanthine amine congener of 1,3-dipropyl-8-phenylxanthine: an antagonist radioligand for adenosine receptors.

An amine-functionalized derivative of 1,3-dipropyl-8-phenylxanthine has been prepared in tritiated form as a xanthine amine congener ([3H]XAC) for use as an antagonist radioligand for adenosine receptors. [3H]XAC has higher receptor affinity, higher specific activity, lower nonspecific membrane binding, and more favorable hydrophilicity than 1,3-diethyl-8-[3H]phenylxanthine, the xanthine commonly used for adenosine receptor binding. In rat cerebral cortical membranes, [3H]XAC exhibits saturable, specific binding with a Kd of 1.23 nM and a Bmax of 580 fmol/mg of protein at 37 degrees C. N6-(R-Phenylisopropyl)adenosine is a more potent inhibitor of [3H]XAC binding than is 5'-N-ethylcarboxamidoadenosine, indicating that binding is to an A1-adenosine receptor. In the absence of GTP, the inhibition curves for adenosine agonists versus [3H]XAC binding are biphasic, indicating that [3H]XAC is binding to low- and high-affinity agonist states of the A1 receptor. In the presence of GTP, adenosine analogs exhibit monophasic, low-affinity inhibition of binding of [3H]XAC. Inhibition of [3H]XAC binding by theophylline or by various 8-phenylxanthines is monophasic, and the potencies are commensurate with the potencies of these xanthines as adenosine receptor antagonists. The receptor sites in calf brain membranes exhibit a higher affinity (Kd = 0.17 nM) for [3H]XAC, whereas sites in guinea pig exhibit a slightly lower affinity (Kd = 3.0 nM). Densities of [3H]XAC binding sites are similar in brain membranes from all species.     

Alpha 1 adrenoceptors in the ischaemic and reperfused myocardium

The [3H]-prazosin binding activity of membranes isolated from aerobically perfused, ischaemic and reperfused cat and rat hearts was investigated. Alpha 1 adrenoceptors in membranes from aerobically perfused cat hearts had a KD of 0.363 +/- 0.067 nM and a Bmax of 68.8 +/- 7.0 fmol/mg protein; 30 min normothermic global ischaemia caused an increase (P less than 0.01) in density (Bmax, 111.4 +/- 9.3 fmol/mg protein), without any change in affinity (KD 0.430 +/- 0.069 nM). Post-ischaemic reperfusion (15 min) caused the Bmax to return to control values, with no change in KD. Membranes isolated from aerobically perfused rat hearts contained a single population of high affinity alpha 1 adrenoceptor binding sites, with a KD of 0.092 +/- 0.02 nM and a Bmax of 43.02 +/- 2.49 fmol/mg protein. Neither global nor low-flow (0.1 ml/min) ischaemia for either 30 or 60 min, nor post-ischaemic reperfusion, caused any change in either the affinity or density of these binding sites. In both species, and under all the above experimental conditions, the selectivity of the alpha 1 adrenoceptor binding sites was maintained, with phentolamine much greater than rauwolscine in displacing bound [3H]-prazosin. These results show that the ability of ischaemia to increase alpha 1 adrenoceptor density in cardiac membranes is species specific, and that it can occur as a direct (as opposed to a reflex) response to ischaemia.     

Opiate receptor subtypes in the rat hypothalamus and neurointermediate lobe

The potent opiate radioligands [3H]etorphine, [3H]ethylketocyclazocine (EKC), and [3H]naloxone, bound specifically and saturably to a single class of membrane-binding sites in rat neurointermediate lobe (NIL), with Kd values of 3.7, 24, and 51 nM, respectively. In the hypothalamus (Ht), [3H]etorphine bound to specific and saturable sites with a Kd of 2.9 nM. Binding-inhibition studies with [3H]etorphine and unlabeled etorphine-HCl as well as [3H]EKC and unlabeled EKC, revealed high and low affinity binding sites in rat Ht and NIL as well as in the neural lobe of the bovine pituitary gland. [3H]naloxone also bound specifically to two classes of sites in Ht membranes, but to only a single class of low affinity sites in NIL membranes. Specific binding represented 80-90% of total [3H]etorphine binding, about 75% of total [3H]EKC binding, and 45-55% of total [3H]naloxone binding at 22 C in NIL and Ht, respectively. Relative binding potencies derived from Ki values for binding-inhibition studies of [3H]etorphine with opioid peptides and opiates were: NIL, etorphine-HCl greater than dynorphin A greater than naloxone-HCl greater than dynorphin-(1-9) greater than beta-endorphin much greater than alpha-neoendorphin approximately (Leu5)enkephalin approximately DAGO (Tyr-D-Ala-Gly-NMe-Phe-Gly-ol); Ht, etorphine HCl greater than naloxone-HCl greater than beta-endorphin greater than dynorphin A much greater than DAGO greater than morphiceptin much greater than (Leu5)enkephalin. Specific [3H]etorphine binding was also demonstrable after preincubation of NIL membranes with DAGO and (Leu5)enkephalin and after preincubation of Ht membranes with morphiceptin and (Leu5)enkephalin; such binding could be displaced by nonradioactive dynorphin A. In addition, [3H]etorphine binding to bovine neural lobe was displaceable by naloxone-HCl, with an ED50 of 43 nM. Specific ligands for sigma-opiate receptors, such as (+)SKF 10,047 (N-allylnorcyclazocine), phencyclidine (PCP), and (-)cyclazocine, displaced specifically bound [3H]etorphine and [3H]EKC from NIL membranes only at high (micromolar) concentrations. However, specific [3H]PCP sites were of higher affinity in NIL and Ht membranes, with similar Kd values of 102 and 190 nM respectively, and different concentrations (0.15 and 1.32 pmol/mg protein, respectively). These data have revealed several differences in the opiate-binding properties of rat Ht and NIL membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Homologous and heterologous regulation of somatostatin-binding sites on chicken adenohypophysial membranes

Binding of 125I-labelled [Tyr1]-somatostatin (125I-[Tyr1]-SRIF) to pituitary caudal lobe membranes was suppressed in immature chickens 1 and 2 h after i.v. administration of unlabelled SRIF at concentrations of 1-100 micrograms/kg. In-vitro preincubation of chicken pituitary glands for 0.5-4.0 h with 0.1 mumol SRIF/l similarly reduced the binding of 125I-[Tyr1]-SRIF to caudal lobe membrane preparations. After a 4-h incubation in 0.1 mmol SRIF/l, the withdrawal of SRIF from the incubation media was accompanied 4 h later by a partial recovery in the binding of 125I-[Tyr1]-SRIF to pituitary membranes. Passive immunoneutralization of endogenous SRIF resulted in a prompt (within 1 h) and sustained (for at least 24 h) suppression of 125I-[Tyr1]-SRIF binding to pituitary membranes. The i.m. administration of cysteamine (300 mg/kg) to 12-week-old birds depleted hypothalamic SRIF stores and decreased the density of 125I-[Tyr1]-SRIF-binding sites in the caudal and cephalic lobes of the chicken pituitary gland. The reduction in SRIF content and in SRIF-binding sites occurred within 1 h of cysteamine administration and was maintained for at least 24 h. In 6-week-old birds, cysteamine (300 mg/kg) administration suppressed pituitary binding of 125I-[Tyr1]-SRIF for at least 5 days. Circulating concentrations of GH were markedly decreased 1 and 4 h after cysteamine injection, but not after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxytocin and vasopressin: distinct receptors in myometrium

The binding characteristics of [3H]oxytocin [( 3H]OT) and [3H]lysine vasopressin [( 3H]LVP) to nonpregnant human myometrium were investigated. Binding of both radioligands was saturable, time dependent, and reversible. Whereas [3H]OT was found to bind to a single class of sites with high affinity [Kd, 1.5 +/- 0.4 (+/- SEM) nM] and low capacity [maximum binding (Bmax), 34 +/- 6 fmol/mg protein], [3H]LVP bound to two classes of sites, one with high affinity (Kd, 2.2 +/- 0.1 nM) and low capacity (Bmax, 198 +/- 7 fmol/mg protein) and another with low affinity (Kd, 655 +/- 209 nM) and high capacity (Bmax, 5794 +/- 1616 fmol/mg protein). The binding of the labeled peptides also displayed a marked difference in sensitivity to Mg2+ and guanine nucleotides. These differences in binding characteristics as well as the differences in potency of analogs in competing for [3H]OT and [3H]LVP binding indicate the presence of distinct receptors for OT and vasopressin in human myometrium. Pharmacological characterization of the high affinity binding sites for [3H]LVP indicated that these are of the V1 subtype. Although, as suggested by others, vasopressin and OT can bind to the same sites, the presence of distinct receptors for both peptides provides an explanation for the previously reported difference in myometrial responsiveness to OT and vasopressin.     

Characterization of adrenal medullary opioid receptors. I. Binding of opioids to adrenal medullary opioid receptors

We studied the binding of [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE) and [3H] diprenorphine to crude plasma membrane fraction obtained from the bovine adrenal medulla (bovine adrenal medullary membranes) in order to characterize adrenal medullary opioid receptors. The [3H] diprenorphine binding was the highest in crude plasma membrane-mitochondrial fraction among all subcellular fractions studied. The amount of [3H] diprenorphine bound to bovine adrenal medullary membranes was proportional to the protein concentration. Association kinetics of the [3H] diprenorphine binding to bovine adrenal medullary membranes showed that the maximal binding was achieved following 8 min incubation and that the binding conformed the second-order kinetics. [3H] DADLE and [3H] diprenorphine bound to bovine adrenal medullary membranes with high affinities. The Kd and Bmax for the [3H] DADLE binding were found to be 2.9 nM and 57.5 fmole/mg protein, respectively, while those for the [3H] diprenorphine binding were 0.31 nM and 250 fmole/mg protein, respectively. Displacement studies showed that the [3H] diprenorphine binding was inhibited dose-dependently by levorphanol, dynorphin (1-13), beta-endorphin and DADLE. Levorphanol was at least 1000-fold more potent to inhibit the [3H] diprenorphine binding than dextrorphan, indicating stereospecificity of the [3H] diprenorphine binding. Na+, Li+ and K+ (100 mM) diminished the [3H] DADLE binding and enhanced [3H] diprenorphine binding. Na+ (100 mM) increased the Kd value for the [3H] DADLE binding from 2.9 nM to 14.1 nM. Mn++, Ca++ and Mg++ diminished the [3H] diprenorphine binding. Mn++ (1 mM) increased the Bmax value for the [3H] DADLE binding from 95 fmole/mg protein to 450 fmole/mg protein. These effects of Na+ and Mn++ on the [3H] diprenorphine binding were found to be dose-dependent. [3H] Diprenorphine binding to the digitonin-solubilized opioid receptor was also inhibited dose-dependently by Mn++. These results suggest that bovine adrenal medullary membranes contain high affinity and stereospecific opioid receptors and that the binding of opioids to the bovine adrenal medullary opioid receptors is influenced by cations. Binding study also revealed the presence of opioid receptors in human malignant pheochromocytoma. The Kd and Bmax of the [3H] diprenorphine binding to crude membrane fraction obtained from malignant pheochromocytoma were found to be 0.14 nM and 10.4 fmole/mg protein, respectively.     

Epidermal growth factor decreases the concentration of thyrotropin-releasing hormone (TRH) receptors and TRH responses in pituitary GH4C1 cells.

Treatment of pituitary GH4C1 cells with epidermal growth factor (EGF) caused up to a 60% reduction in the amount of [3H]MeTRH bound to specific TRH receptors. The effects of EGF were first detectable after a 2-h incubation and maximal by 24-72 h. EGF elicited a half-maximal response at 0.03 nM. Equilibrium binding analysis was performed on intact cells that had been incubated with or without 10 nM EGF for 96 h. EGF decreased the apparent number of TRH receptors (maximum binding = 0.36 vs. 0.58 pmol/mg protein for EGF-treated and control cells, respectively) without altering the apparent affinity (dissociation constant = 6.4 vs. 7.4 nM). The effects of EGF on TRH receptors were reversible. When EGF was removed from the medium, TRH receptors returned to control levels within 48 h. To assess whether the reduction of TRH receptors was functionally important, the ability of TRH to stimulate phospholipid turnover was measured in cells with a normal complement of TRH receptors and in cells that had been treated with EGF for 72 h to reduce TRH receptor density. EGF significantly blunted the ability of TRH to stimulate release of inositol phosphates from metabolically labeled cells. TRH increased inositol monophosphate accumulation 6.3-fold in control cultures and 2.0-fold in EGF-treated cells. These data show that EGF regulates the concentration of TRH receptors on pituitary GH4C1 cells and the responsiveness of the cells to TRH.     

Sphingosine interacts directly with the receptor complex to inhibit thyrotropin-releasing hormone binding

Sphingosine inhibition of [3H] [N3-Me-His] TRH (MeTRH) binding, previously shown to be independent of its effects on protein kinase-C, has been further characterized in GH3 cell membranes and in a partially purified, digitonin-solubilized receptor preparation. In membranes, as in intact cells, sphingosine inhibited [3H]MeTRH binding by decreasing receptor affinity, but, in contrast to its effect in intact cells, did not affect the number of available binding sites. The inhibition of binding was linear up to 75 microM sphingosine (in the presence of 100 microM BSA at 0.1 mg membrane protein/ml), yielding an apparent Ki of 51 microM. Since GTP decreases the affinity for MeTRH binding in GH3 cell membranes, we studied interactions between GTP and sphingosine. While the effects of low concentrations of GTP gamma S and sphingosine were additive, sphingosine inhibition of MeTRH binding surpassed and was not affected by the addition of maximally inhibitory concentrations of GTP gamma S. Also, sphingosine (75 microM) did not affect the ability of a maximally effective dose of TRH to stimulate the low Km GTPase (vehicle, +35 +/- 5%; sphingosine, +32 +/- 10%); there was a 25% decrease in total GTPase activity in the presence of sphingosine. MeTRH binding to digitonin-solubilized receptors, which had properties similar to those described previously by others, including no effect of GTP on binding, was inhibited by sphingosine. In solubilized receptors, as in membranes, sphingosine caused a decrease in apparent affinity without changes in the number of binding sites. These data suggest that sphingosine interacts directly with the TRH receptor [or an associated factor(s) in the receptor complex] to decrease affinity by a mechanism that does not involve uncoupling of G-proteins.     

Identification of alpha-adrenergic receptor subtypes in the steer stalk-median eminence

The presence of alpha 1- and alpha 2-receptor subtypes were studied in steer stalk-median eminence (SME) using a selective alpha 1-ligand, [3H]-prazosin, and a selective alpha 2-ligand, [3H]-clonidine. Scatchard analysis of the equilibrium isotherms of specific [3H]-prazosin binding indicated a single class of high affinity (Kd = 0.36 +/- 0.05 nM) and low capacity (Bmax = 111 +/- 19 fmol/mg protein) binding sites, which accounted for approximately 26% of the total sites identified by the non-subtype-selective-alpha-antagonist, dihydroergocryptine ([3H]-DHE). Similarly, computer modeling of the biphasic competition curve of prazosin displacing [3H]-DHE also revealed that there are approximately 26% high affinity sites, presumably alpha 1-, and approximately 74% low affinity sites, presumably alpha 2-receptors in the steer SME. A scatchard plot of specific [3H]-clonidine binding was biphasic and was resolved into a high (Kd = 1.60 +/- 0.34 nM) and low (Kd = 34.5 +/- 7.85 nM) affinity site with the binding capacities of 43 +/- 5 and 200 +/- 37 fmol/mg protein, respectively. The total number of alpha 2-sites identified by [3H]-clonidine accounted for approximately 58% of the total sites labeled by [3H]-DHE, which was in good agreement with that calculated from the competition of clonidine for [3H]-DHE binding (approximately 65% alpha 2 and approximately 35% alpha 1). The rank orders of potency of alpha-adrenergic agents to compete for sites labeled by [3H]-prazosin and [3H]-clonidine were consistent with their labeling alpha 1- and alpha 2-subtypes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding sites for [3H]-melatonin in human platelets

A number of in vitro effects of melatonin on human platelets were revealed in previous studies. In order to examine whether high affinity binding sites for [3H]-melatonin were present in membrane preparations of human platelets, a rapid filtration procedure through Whatman GFB paper was employed. Maximal melatonin binding was attained within 3 hr at 0 degree C. Scatchard analysis indicated a single population of binding sites with a dissociation constant (Kd) = 4.1 +/- 0.5 nM and maximal number of binding sites (Bmax) = 24.2 +/- 1.9 fmol/mg protein (mean +/- SEM of five experiments). When various indole analogs were tested for their ability to inhibit [3H]-melatonin binding, the following Ki (nM) were obtained: 6-chloromelatonin (11.4), 2-iodomelatonin (22.0), melatonin (24.7), 5-methoxytryptophol (49.9), N-acetylserotonin (68.9), 6-hydroxymelatonin (78.2), 5-methoxytryptamine (184). Serotonin was a potent inhibitor of [3H]-melatonin binding with a Ki = 20.6 nM. Except for 2-methylserotonin and alpha-methylserotonin, a number of serotonin agonists and antagonists tested did not affect melatonin binding to platelet membranes. Binding experiments carried out at either 0800 or 2000 did not reveal time-dependent differences in Kd or Bmax. The results suggest that high affinity melatonin acceptors are present in human platelets.     

Solubilization and characterization of high-affinity [3H]serotonin binding sites from bovine cortical membranes.

High-affinity [3H]serotonin binding activity has been solubilized from bovine cerebral cortical membranes by using Triton X-100, Tween-80, and octyl-beta-D-glucopyranoside. This mixture of detergents solubilizes the high-affinity [3H]serotonin binding activity present in crude membrane preparations with retention of 75-90% specific binding. The detergent mixture was chosen because it can easily be removed from the solubilized fraction by dialysis and polystyrene bead adsorption, thus permitting further purification and isolation of the binding sites. Saturation analysis reveals multiple components of high-affinity [3H]serotonin binding. In crude bovine cortical membranes, at least two binding components are present. A higher-affinity binding component, as defined from curvilinear Scatchard plots, has a Kd for [3H]serotonin of 1-3 nM, whereas a lower-affinity component has a Kd of 10-20 nM. In the solubilized preparation, only a single class of binding sites is apparent, with a Kd of 50-100 nM. Removal of detergents by dialysis and polystyrene bead adsorption results in restoration of the curvilinear Scatchard plot with apparent Kds similar to those observed in crude membrane preparations and with increased Bmax values for each component. [3H]Serotonin binding activity in the solubilized preparation is stable to Sephacryl S-300 column chromatography and to glycerol gradient sedimentation. Saturation analysis of the peak binding fractions from both these procedures once again yields curvilinear Scatchard plots, indicating that the multiple high-affinity binding components are preserved and migrate together. The molecular weight, Stokes radius, and frictional coefficient of the binding site(s) have been calculated. After detergent removal the solubilized material shows many of the characteristics usually attributed to S1 receptors, such as high affinity for [3H]serotonin and its analogs and low affinity for serotonin antagonists.     

Guanine nucleotide modulation of high affinity gonadotropin-releasing hormone receptors in rat mammary tumors.

We previously suggested that gonadotropin-releasing-hormone (GnRH) analogues activate the phosphoinositide pathway in rat mammary tumor membranes. In the present study we analyzed the binding of GnRH analogues to these membranes and assessed its modulation by guanine nucleotides. [125I]Buserelin (a GnRH superagonist) binding is specific because it is displaced only by GnRH analogues. Scatchard plot analysis reveals high affinity binding sites (Kd = 2.5 +/- 0.8 nM, Bmax = 250 +/- 120 fmol/mg membrane protein) and low affinity binding sites (Kd 1.1 +/- 0.3 microM, Bmax = 200 +/- 105 pmol/mg membrane protein). Guanine nucleotides increased the ED50 of [125I]buserelin displacement, and almost completely eliminated the high affinity binding. Similar results were obtained with [125I]D-Trp6-GnRH--another GnRH superagonist. The inhibition of buserelin binding by guanine nucleotides was specific for nucleotides that interact with G-binding proteins and was dose-dependent with a maximal effect at 10 microM GTP gamma S. Kinetic analysis of buserelin binding revealed that the dissociation rate increased at least 4-fold in the presence of 10 microM GTP gamma S. These results support the hypothesis that GnRH analogues interact directly with mammary tumors and activate a G-protein-dependent transducing mechanism.     

The specific binding of [3H]EO-122, a radiolabeled class I antiarrhythmic drug to rat heart membranes

[3H]EO-122, a radiolabeled class I antiarrhythmic drug, has been used to characterize a new specific binding system to rat heart membranes. The binding is saturable and competitive with unlabeled EO-122 and other antiarrhythmic drugs. In this system, [3H]EO-122 binds to two sites. Site A with an apparent Kd of 33.5 +/- 1.5 nM, Bmax of 1.05 +/- 0.15 pmol/mg protein and Hill coefficient nH = 4. Site B with an apparent Kd of 233 +/- 25 nM, Bmax equals 5.7 +/- 0.61 pmol/mg proteins and nH = 6. The binding to site B indicates that this site is pharmacologically relevant to known class IA antiarrhythmic drugs such as quinidine and procainamide. Lidocaine (class IB) does not interact with this site. Interpretation of the high Hill coefficient suggests that the binding of an antiarrhythmic drug to its pharmacologically relevant binding site exposes additional binding sites and/or modulates the affinity of adjacent binding sites.