Evidence for a single class of somatostatin receptors in ground squirrel cerebral cortex

In the present study we characterized high-affinity somatostatin (SRIF) binding sites (Kd = 2.06 +/- 0.32 nM and Bmax = 295 +/- 28 fmol/mg protein) in cerebral cortex membrane preparations of European ground squirrel using 125I-[Tyr0-D-Trp8]-SRIF14 as a radioligand. The inhibition of radioligand specific binding by SRIF14, as well as by its agonists (SRIF28, Tyr0-D-Trp8-SRIF14, SMS 201 995) was complete and monophasic, thus revealing a single population of somatostatinergic binding sites. Radioautographic analysis of 125I-[Tyr0-D-Trp8]-SRIF14 labeled brain sections confirmed the results of our biochemical study. The homogeneity of SRIF binding sites in the ground squirrel neocortex was not dependent on the animal's life-cycle phase.     

Effects of monovalent and divalent cations and of guanine nucleotides on binding of vasopressin to the rat mesenteric vasculature

The rat mesenteric vasculature contains high affinity binding sites specific for [3H]Arg8-vasopressin which mediate its vasoconstrictor action. We have investigated the in vitro effect of monovalent and divalent cations and guanine nucleotides on the interactions between [3H]Arg8-vasopressin and its receptor in this preparation. Binding was increased by divalent cations from fourfold in the presence of Mg2+ at 5 mM to ninefold in the presence of Mn2+ at 5 mM. The potency order of divalent cations to increase binding was Mn2+ greater than Co2+ greater than Ni2+ greater than Mg2+ greater than Ca2+ approximately equal to control without cations. Addition of Na2+ or other monovalent cations (K+, Li+, and NH4+) in the presence or absence of divalent cations reduced binding significantly. Analysis of saturation binding curves showed a single high affinity site. In the presence of 5 mM Mn2+, binding capacity (Bmax) increased to 139 +/- 23 fmol/mg protein. Receptor affinity was enhanced (KD decreased to 0.33 +/- 0.07 nM). In presence of 5 mM Mg2+ or 150 mM Na+, Bmax and affinity were reduced. The addition of 100 microM GTP or its nonhydrolyzable analogue, Gpp(NH)p, reduced receptor affinity in the presence of Mn2+ + Na+, Mg2+, and Mg2+ + Na+, but not in the presence of Mn2+ alone. Computer modeling of competition binding curves demonstrated that in contrast with saturation studies, the data were best explained by a two-site model with high affinity, low capacity sites and low affinity, high capacity sites. Mn2+ or Mn2+ + Na+ with or without guanine nucleotides resulted in a predominance of high affinity sites. GTP or Gpp(NH)p in the presence of Mg2+ or Mg2+ + Na+ induced a reduction of affinity of the high affinity binding sites and the number of these sites. In the presence of Mg2+ + Na+ and guanine nucleotides, high affinity sites were maximally decreased. An association kinetic study indicated that the association rate constant (K+1) was increased by divalent cations and reduced by guanine nucleotides, without change in the dissociation rate constant (K-1). The equilibrium dissociation constant (KD) calculated with these rate constants (K-1/K+1) was similar to that obtained in saturation experiments at steady state. Dissociation kinetics were biphasic, indicating the presence of two receptor states, one of high and one of low affinity, associated with a slow and a rapid dissociation rate. Cations and guanine nucleotides interact with one or more sites closely associated with vasopressin receptors, including possibly with a GTP-sensitive regulatory protein, to modulate receptor affinity for vasopressin.     

Characterization, Regional Distribution, and Subcellular Distribution of 125I-Tyr1-Somatostatin Binding Sites in Rat Brain

Abstract: ¹²⁵I-Tyr₁-somatostatin binds reversibly, in a saturable manner, and with high affinity to membranes from rat brain. Kinetic and saturation data measured at equilibrium lead to K_Dvalues of 0.4 nM for cortical membranes. The binding is not affected significantly by seven neuropeptides and drugs unrelated structurally to somatostatin (SRIF) while native SRIF, Tyr₁-SRIF, and D-Trp⁸-D-Cys¹⁴-SRIF displace ¹²⁵I-Tyr₁-SRIF in a dose-dependent manner, with K_i of 0.23 nM, 0.90 nM, and 0.11 nM, respectively. Binding sites for ¹²⁵I-Tyr₁-SRIF were found in 9 out of 11 central structures; there was a significant correlation between binding capacity and endogenous SRIF levels measured by radioimmunoassay. In each of the two structures containing the most binding sites, the cortex and the preoptic area, Scatchard analysis suggests a single population of sites with apparent affinities of 0.8 nM and 1.4 nM, respectively. Subcellular fractionation of these two regions reveals that more than 60% of ¹²⁵I-Tyr₁-SRIF specific binding of the homogenate is found in the crude mitochondrial pellet (P₂), which contains synaptosomes. When P₂ is further fractionated on a discontinuous sucrose gradient, most of the initial P₂ binding is recovered from membrane fractions. Each of nine SRIF analogs, with a single alanine substitution, displaces ¹²⁵I-Tyr₁-SRIF binding on cortical membranes in the same order of potency as on adenohypophyseal membranes (r= 0.84). The data demonstrate the presence of SRIF binding sites in the rat brain, with kinetic characteristics comparable to those found in the adenohypophysis, and they provide a biochemical basis for the multiple functions of SRIF in brain.     

Analogues of Somatostatin Bind Selectively to Brain Somatostatin Receptor Subtypes

Somatostatin (SRIF) is a neurotransmitter that produces its multiple effects in the CNS through interactions with membrane-bound receptors. Subtypes of SRIF receptors are found in the CNS that are distinguished by their sensitivities to the cyclic hexapeptide MK-678, such that SRIF1 receptors are sensitive to MK-678 and SRIF2 receptors are insensitive to MK-678. In the present study, we further examined the selectivities of a series of structurally diverse SRIF analogues for SRIF receptor subtypes. SRIF receptors were labeled by 125I-Tyr11-SRIF, which has indistinguishable affinities for SRIF receptor subtypes. The inhibition by MK-678 was incomplete, indicating this peptide is highly selective for a subtype of SRIF receptor that we have termed the SRIF1 receptor. The binding of 125I-MK-678 to SRIF1 receptors was monophasically inhibited by SRIF, the octapeptides (such as SMS-201-995), and the hexapeptides (such as MK-678), consistent with the highly selective labeling of a subtype of SRIF receptor. In contrast, the smaller CGP-23996-like analogues did not inhibit 125I-MK-678 binding to SRIF1 receptors. The binding of 125I-CGP-23996 to SRIF receptors was inhibited by SRIF and the octapeptides with Hill coefficients of less than 1, indicating that 125I-CGP-23996 labels multiple SRIF receptor subtypes. The hexapeptides and CGP-23996-like compounds produced only partial inhibitions of 125I-CGP-23996 binding, which were additive, indicating selective interactions of these compounds with the different receptor subpopulations labeled by 125I-CGP-23996. 125I-Tyr11-SRIF binding and 125I-CGP-23996 binding to SRIF receptors were likewise only partially affected by 100 microM guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a concentration that completely abolishes specific 125I-MK-678 binding to SRIF1 receptors. The component of 125I-CGP-23996 labeling that was sensitive to GTP gamma S was also MK-678 sensitive. Thus, two subpopulations of SRIF receptors exist in the CNS. The SRIF1 receptor is sensitive to cyclic hexapeptides such as MK-678 and to GTP gamma S but insensitive to smaller CGP-23996-like compounds. The SRIF2 receptor is sensitive to the CGP-23996-like compounds and can be selectively labeled by 125I-CGP-23996 in the presence of high concentrations of the hexapeptides or GTP gamma S because, unlike the SRIF1 receptor, the SRIF2 receptor is insensitive to these agents. The SRIF receptor subtype-selective peptide analogues will be useful in the future characterization of the functions mediated by SRIF receptor subtypes in the CNS.     

Modulation of the affinity of the vasopressin receptor by magnesium ions.

The binding of [3H]vasopressin (AVP) and the 125I-labelled vasopressin antagonist (VP-AT) d(CH2)5[Tyr2(Me),Tyr9(NH2)]AVP to rat liver membranes was examined with or without the addition of milimolar concentrations of divalent cations. The binding of vasopressin was enhanced by Mg2+ and Co2+ and markedly decreased by EGTA. The addition of EGTA and Mg2+ together restored the binding to a value similar to that of Mg2+ alone. On the contrary, the addition of Mg2+, Co2+, EGTA, and the combination of EGTA and Mg2+ decreased the binding of VP-AT to rat liver membranes. Kinetic analyses showed that Mg2+ increased the Kd twofold for VP-AT; that is from 0.13 nM to 0.28 nM. Moreover, it showed that the receptor with or without the addition of Mg2+ consists of a single population of binding sites, indicating that the receptor is switched from a high affinity to a low affinity state for VP-AT in the presence of 10 mM Mg2+. GTP gamma S was unable to block the effect of Mg2+ on the binding of VP-AT. These results suggest that this divalent cation interacts with receptor itself producing a conformational changes which thus modulates the affinity of the receptor.     

Characterization of 17-beta-estradiol-dependent and -independent somatostatin receptor subtypes in rat anterior pituitary

Previous studies from this laboratory showed that treatment with 17-beta-estradiol (E2) caused an acquisition of inhibitory effect of somatostatin (SRIF) on prolactin release with an increased number of SRIF-binding sites in the rat anterior pituitary. The aim of this study was to characterize the E2-dependent SRIF receptor in comparison with the E2-independent one, which was expressed in ovariectomized rats. The following observations were obtained: 1) both of the E2-dependent and E2-independent SRIF receptors, measured with 125I-Tyr11-SRIF as a radiolabeled ligand, were enriched in the plasma membrane fraction of the cells, displaying a single class of binding site (E2-dependent: Kd, 32 pM, Bmax, 2.3 pmol/mg protein; E2-independent: Kd, 83 pM, Bmax, 0.26 pmol/mg protein). The ligand binding to both receptors was sensitive to monovalent and divalent cations, and GTP. 2) Among the SRIF analogs tested, the relative potencies of SRIF28 and its analog and cyclosomatostatin compared with SRIF were lower in the E2-dependent receptor than in the E2-independent one. 3) A cross-linking study with N-hydroxysuccinimidyl-4-azido-benzoate revealed that the molecular weight of the cross-linked E2-dependent receptor was approximately 94,000, whereas that of the E2-independent one was 82,000, irrespective of the presence of a reducing reagent. The molecular weight of SRIF receptor from normal male or female rat pituitary was similar to the E2-independent type. 4) Both types of the cross-linked SRIF receptors were solubilized by sucrose monolaurate, adsorbed to a wheat germ agglutinin-agarose column, and eluted with N-acetyl-glucosamine. 5) SRIF inhibited the forskolin-stimulated adenylate cyclase activity in the pituitary membranes from E2-treated rats, but it did not in the E2-depleted membranes. These results demonstrate that there are at least two subtypes of SRIF receptor in the rat anterior pituitary, one of which is exclusively expressed by the treatment with E2, and that these subtypes are distinct with respect to ligand binding specificity, molecular weight, and coupling to adenylate cyclase inhibition.     

Regulation by divalent cations of 3H-baclofen binding to GABAB sites in rat cerebellar membranes

When investigating the effects of divalent cations (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+ and Ni2+) on 3H-baclofen binding to rat cerebellar synaptic membranes, we found that the specific binding of 3H-baclofen was not only dependent on divalent cations, but was increased dose-dependently in the presence of these cations. The effects were in the following order of potency: Mn2+ congruent to Ni2+ greater than Mg2+ greater than Ca2+ greater than Sr2+ greater than Ba2+. Scatchard analysis of the binding data revealed a single component of the binding sites in the presence of 2.5 mM MgCl2, 2.5 mM CaCl2 or 0.3 mM MnCl2 whereas two components appeared in the presence of 2.5 mM MnCl2 or 1 mM NiCl2. In the former, divalent cations altered the apparent affinity (Kd) without affecting density of the binding sites (Bmax). In the latter, the high-affinity sites showed a higher affinity and lower density of the binding sites than did the single component of the former. As the maximal effects of four cations (Mg2+, Ca2+, Mn2+ and Ni2+) were not additive, there are probably common sites of action of these divalent cations. Among the ligands for GABAB sites, the affinity for (-), (+) and (+/-) baclofen, GABA and beta-phenyl GABA increased 2-6 fold in the presence of 2.5 mM MnCl2, in comparison with that in HEPES-buffered Krebs solution (containing 2.5 mM CaCl2 and 1.2 mM MgSO4), whereas that for muscimol was decreased to one-fifth. Thus, the affinity of GABAB sites for its ligands is probably regulated by divalent cations, through common sites of action.     

Cationic modulation of follicle-stimulating hormone binding to granulosa cell receptor

Magnesium (Mg2+) increases binding of follicle-stimulating hormone (FSH) to membrane-bound receptors and increases adenylyl cyclase activity. We examined the effects of divalent and monovalent cations on FSH binding to receptors in granulosa cells from immature porcine follicles. Divalent and monovalent cations increased binding of [125I]iodo-porcine FSH (125I-pFSH). The divalent cations Mg2+, calcium (Ca2+) and manganese, (Mn2+) increased specific binding a maximum of 4- to 5-fold at added concentrations of 10 mM. Mg2+ caused a half-maximal enhancement of binding at 0.6 mM, whereas Ca2+ and Mn2+ had half-maximal effects at 0.7 mM and 0.8 mM, respectively. The monovalent cation potassium (K+) increased binding a maximum of 1.5-fold at an added concentration of 50 mM, whereas the monovalent cation (Na+) did not increase binding at any concentration tested. The difference between K+ and Na+ suggested that either enhancement of binding was not a simple ionic effect or Na+ has a negative effect that suppresses its positive effect. Ethylenediamine tetraacetic acid, a chelator of Mg2+, prevented binding of 125I-pFSH only in the presence of Mg2+, whereas pregnant mare's serum gonadotropin, a competitor with FSH for the receptor, prevented binding in both the absence and the presence of Mg2+. Guanyl-5-ylimidodiphosphate (Gpp[NH]p) inhibited binding of 125I-pFSH in the absence or presence of Mg2+, but only at Gpp(NH)p concentrations greater than 1 mM. We used Mg2+ to determine if divalent cations enhanced FSH binding by increasing receptor affinity or by increasing the apparent number of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionic and GTP regulation of binding of platelet-activating factor to receptors and platelet-activating factor-induced activation of GTPase in rabbit platelet membranes

Specific binding of 3H-labeled platelet-activating factor (PAF) to rabbit platelet membranes was found to be regulated by monovalent and divalent cations and GTP. At 0 degrees C, inhibition of [3H]PAF binding by sodium is specific, with an ED50 of 6 mM, while Li+ is 25-fold less effective. On the contrary, K+, Cs+, and Rb+ enhance the binding. The divalent cations, Mg2+, Ca2+, and Mn2+ enhance the specific binding 8-10-fold. From both Scatchard and Klotz analyses, the inhibitory effect of Na+ is apparently due to an increase in the equilibrium dissociation constant (KD) of PAF binding to its receptors. However, the Mg2+-induced enhancement of the PAF specific binding may be attributed to an increased affinity of the receptor and an increased availability of the receptor sites. In the presence of Na+, PAF receptor affinity decreased with increasing temperature with a 100-fold sharp discontinuous decrease in receptor affinity at 24 degrees C. In contrast, the Mg2+-induced increase is independent of temperature suggesting that the Mg2+ regulatory site is different from Na+ regulatory site. [3H]PAF binding is also specifically inhibited by GTP; other nucleotides have little effect. PAF also stimulates hydrolysis of [gamma-32P]GTP with an ED50 of 0.7 nM, whereas 3-O-hexadecyl-2-O-acetyl-sn-glyceryl-1-phosphorylcholine showed no activity even at 10 microM. Moreover, such stimulatory effect of PAF is dependent on Na+ and can be abolished by the PAF-specific receptor antagonist, kadsurenone, but not by an inactive analog, kadsurin B. These results suggest that the PAF receptor may be coupled with the adenylate cyclase system via an inhibitory guanine nucleotide regulatory protein.     

High affinity divalent cation binding to actin. Effect of low affinity salt binding

Monomeric actin labeled with the fluorescent probe N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS-actin) displays a fast fluorescence intensity increase immediately upon addition of salt and then a slow fluorescence intensity change concurrent with Ca2+/Mg2+ exchange at the high affinity divalent cation binding site on actin. The fast change appears to reflect competitive binding of K+ at low affinity (nonspecific) sites and of Mg2+ or Ca2+ at low and intermediate affinity sites. Binding of cation at the low affinity sites (but apparently not at the intermediate affinity sites) results in an increase in k-Ca and k-Mg and thus a decrease in affinity for divalent cations at the high affinity site. The effect of Mg2+ on k-Ca is twice that of K+ for equal fractional saturations of the low affinity binding, and the effect of K+ and Mg2+ together on k-Ca reflects competitive binding at the low affinity sites. Thus the affinity and kinetics of divalent cation binding at the high affinity site of actin are significantly affected by concurrent cation binding at low affinity sites.     

Thermogenic and lipogenic activities in brown adipose tissue of I-strain mice.

Specific binding of 125I-labelled human somatotropin was demonstrated in isolated hepatocytes from male mice. In the presence of divalent cations (Ca2+ and Mg2+) the binding of 125I-labelled human somatotropin was competitive with ovine prolactin. Scatchard analysis of competition data indicated a KD of 1.4 +/- 0.2 nM and a binding capacity of 13 000 +/- 2000 sites/cell. In the absence of divalent cations and in the presence of EDTA, human and bovine somatotropins were found to be equally effective to displace bound 125I-labelled human somatotropin, while ovine prolactin showed a weak competition. In this case, the binding capacity was 8400 +/- 1500 sites/cell and the KD was 1.1 +/- 0.1 nM.     

Guanyl nucleotide and divalent cation regulation of cortical S2 serotonin receptors

Computer-assisted quantitative analysis of radioligand binding to rat cortical S2 serotonin receptors indicates the existence of two affinity states of the same receptor population. Monophasic antagonist competition curves for [3H]ketanserin-labelled sites suggest a uniform population of receptors with one affinity state for antagonists. Biphasic competition curves of agonists suggest that agonists discriminate high- and low-agonist-affinity forms of the S2 receptors. The affinities of agonists for the high- and low-affinity states, and the apparent percentages of high agonist-affinity forms varies with different agonists. The guanine nucleotides GTP and guanyl-5'-imido-diphosphate [Gpp(NH)p], as well as divalent cations, modulate the proportion of the sites with high affinity for agonists as evidenced by their ability to shift the agonist competition curves for [3H]ketanserin-labelled S2 receptors. GTP and Gpp(NH)p effects appear to be agonist-specific, as they do not affect antagonist competition for [3H]ketanserin-labelled S2 receptors, or [3H]ketanserin binding to S2 receptors. ATP and ADP have little or no effect on the binding properties of S2 serotonin receptors, whereas GDP is less potent than GTP. The presence of these specific nucleotide effects are the first evidence suggesting involvement of a guanine nucleotide-binding protein in the mechanism of agonist interaction with the S2 serotonin receptor. In general, the binding properties of [3H]ketanserin-labelled S2 serotonin receptors strongly resemble those of adenylate-cyclase coupled receptors such as the beta-adrenergic, the alpha 2-receptor, and the D-2 dopamine receptor. This may indicate the S2 serotonin receptor is coupled to adenylate cyclase activity, through a GTP binding protein.     

Different ionic requirements for somatostatin receptor subpopulations in the brain

Two populations of brain somatostatin (SS) receptors, one with high affinity for the somatostatin octapeptide analogue SMS 201-995 (SS1 type) and one poorly sensitive to this analogue (SS2 type) have been characterised in regard to their ionic requirements using two radioligands, the iodinated Tyr3 derivative of the octapeptide SS analog SMS 201-995 and the iodinated [Tyr11]-SS. Specific binding of 125I-[Tyr11]-SS to rat cortex membrane homogenates can be increased by approximately 180% in presence of 5 mM Mg2+. The increase in number of binding sites seen by Mg2+ is not accompanied by a marked increase in affinity for SS but for SMS 201-995: the low affinity binding for SMS 201-995 seen in absence of Mg2+ is replaced in part by higher affinity binding in presence of these ions. SMS 201-995 sensitive SS1 receptor subpopulation measured with 125I-204-090, a specific ligand for SS1 subpopulation, is massively increased in presence of Mg2+. However, SMS 201-995 insensitive SS2 receptor population measured with 125I-[Tyr11]-SS in presence of excess SMS 201-995 is unchanged in presence of Mg2+. The Mg2+-dependency can also be observed with autoradiography for extra cortical, i.e. hippocampal, brain SS receptors. 120 mM Na+ does not affect the total brain SS receptor population, but reduces the specific binding of SS1 receptors and increases that of SS2 receptors. Therefore, the rat brain, in particular the cortex, possesses a SMS 201-995-sensitive, Mg2+-dependent SS receptor subpopulation (SS1) as well as a SMS 201-995-insensitive, Mg2+-independent SS population (SS2).     

The hepatic angiotensin II receptor. II. Effect of guanine nucleotides and interaction with cyclic AMP production

Guanine nucleotides were observed to modify the binding of 125I-angiotensin II to rat hepatic plasma membrane receptors. GTP and its nonhydrolyzable analogues greatly increased the dissociation rate of bound 125I-angiotensin II and altered hormone binding to the receptor under equilibrium conditions. In the absence of GTP, 125I-angiotensin II labeled both high affinity sites (Kd1 = 0.46 nM, N1 = 650 fmol/mg) and low affinity sites (Kd2 = 4.1 nM, N2 = 1740 fmol/mg). In the presence of guanine nucleotides, the affinities of the two sites were unchanged, but the number of high affinity sites decreased markedly to 52 fmol/mg. In analogous experiments using the angiotensin II antagonist, 125I-sarcosine1,Ala8-angiotensin II (125I-saralasin), guanine nucleotides minimally affected the interaction of 125I-saralasin with its receptor, increasing the dissociation rate 1.9-fold and the Kd 1.4-fold. The guanine nucleotide inhibition of agonist binding required a cation such as Na+ or Mg2+, with a maximal effect occurring at about 1 mM Mg2+. In liver plasma membranes prepared in EDTA, angiotensin II inhibited basal and glucagon-stimulated adenylate cyclase activities by 30% and 10%, respectively. Angiotensin II also caused a 40% inhibition of glucagon-stimulated cyclic AMP accumulation in intact hepatocytes, with a half-maximal effect occurring at 1 nM. The inhibition by angiotensin II of adenylate cyclase in membranes and of cAMP levels in intact cells could be reversed by the antagonist sarcosine1,Ile8-angiotensin II. Vasopressin caused a smaller 26% inhibition of glucagon-stimulated cyclic AMP accumulation. The ability of angiotensin II to inhibit cyclic AMP synthesis may provide an explanation for the observed effects of guanine nucleotides on 125I-angiotensin II binding to plasma membranes.     

Characterization of the putative anxiolytic SM-3997 recognition sites in rat brain

In order to clarify the mechanism of action of the putative nonbenzodiazepine anxiolytic SM-3997 [3a alpha,4 beta,7 beta,7a alpha)-Hexahydro-2-(4-(4-(2-pyrimidinyl)-1- piperazinyl)-butyl)-4,7-methano-1H-isoindole-1,3 (2H)-dione dihydrogen citrate), in vitro binding studies with radiolabeled compound were performed. 3H-SM-3997 bound rapidly, reversibly and in a saturable manner with high affinity to rat brain hippocampal membranes (Kd = 9.4 nM, Bmax = 213 fmol/mg protein). This specific binding was displaced by 5-hydroxytryptamine (5-HT) and related compounds. Especially, 8-OH-DPAT, a 5-HT-1A selective agonist, bound with the highest affinity to these binding sites. 3H-SM-3997 binding, however, was not displaced by a variety of other neurotransmitters, neuropeptides and some other drugs. EDTA and physiological concentration of Na+ inhibited this specific binding, but several divalent cations, Mn2+, Ca2+ and Mg2+, enhanced this binding. GTP decreased the affinity of these binding sites for 3H-SM-3997 without changing the number of binding sites, but GMP and ATP did not influence 3H-SM-3997 binding. Furthermore, 3H-SM-3997 bound with marked regional selectivity to hippocampal membranes. These characteristics and the regional distribution of 3H-SM-3997 binding sites were very similar to those of 3H-8-OH-DPAT binding sites (5-HT-1A receptors). Therefore, these results indicate that SM-3997 binds selectively and with high affinity to 5-HT-1A receptors in rat brain and may be an agonist.     

Identification of the angiotensin II receptor in rat mesenteric artery.

Specific binding sites of high affinity and low capacity for 125I-angiotensin II have been identified in a membrane fraction derived from arterial arcades of the rat mesentery. Heterogeneity of binding sites and extensive tracer degradation necessitated the use of nonlinear regression methods for the analysis of radioligand binding data. Forward and reverse rate constants for the high affinity sites obtained by three experimental approaches were in good agreement and gave a dissociation equilibrium constant (Kd) of 19-74 pM (95% confidence interval). Affinities for a number of angiotensin-related peptides calculated from competitive binding curves were in the order 125I-angiotensin II = angiotensin II greater than angiotensin III greater than [Sar1,Ile8]angiotensin II greater than [Sar1,Gly8]angiotensin II. Angiotensin I and biochemically unrelated peptides had virtually no effect on binding of tracer angiotensin II. The divalent cations Mn2+, Mg2+ and Ca2+ stimulated 125I-angiotensin II binding at concentrations of 2-10 mM, as did Na+ at 50-100 mM. In the presence of Na+ or Li+, K+ had a biphasic effect. The chelating agents EDTA and EGTA were inhibitory, as were the thiol reagents dithiothreitol and cysteine. This study defined angiotensin II binding sites in a vascular target tissue of sufficiently high affinity to interact rapidly with plasma angiotensin II at physiological concentrations.     

Alteration of somatostatin but not growth hormone-releasing factor pituitary binding sites in obese Zucker rats.

The present study was designed to determine whether the diminution of growth hormone (GH) secretion that occurs in obese Zucker rats is related to alterations of GH-releasing factor (GRF) or somatostatin (SRIF) pituitary binding sites. Cold saturation studies were performed in pituitary homogenates of 4-month-old lean and obese rats, using [125I-Tyr10]hGRF(1-44)NH2 as radioligand and [127I-Tyr10]hGRF-(1-44)NH2 as competitor, and in pituitary membrane preparations, using [125I-Tyr0, D-Trp8]SRIF14 as radioligand and [127I-Tyr0, D-Trp8]SRIF14 as competitor. In lean rats, analysis of the curves by the Ligand program revealed the presence of two distinct classes of GRF binding sites, the first being of high affinity (0.74 +/- 0.11 nM) and low capacity (118 +/- 31 fmol/mg protein), the second being of lower affinity (880 +/- 240 nM) and higher capacity (140 +/- 35 pmol/mg protein), and of a single class of SRIF binding sites (affinity: 0.40 +/- 0.12 nM; capacity: 24 +/- 6 fmol/mg protein). In obese rats, no difference was observed in GRF binding parameters for both classes of sites, but the concentration of somatostatin binding sites was reduced by 67% when compared to their lean littermates. These findings suggest that the SRIF pituitary receptors are down-regulated in obese Zucker rats and indicate that no alteration of GRF pituitary binding sites contribute to the blunted GH secretion observed in this model of obesity.     

A Study of Somatostatin Receptors in Amygdaloid-Kindled Rat Brain

Abstract: Kindling is an animal model of epilepsy. Previously somatostatin (SRIF) was implicated in seizure activity in the brain. Recently we reported a significant increase in brain SRIF content in the temporal cortices and cortices of kindled rats. Since the interaction between the neurotransmitter and the receptor eventually is responsible for the biological response, the present study was undertaken to examine evidence for the participation of SRIF receptor in the kindled state. In this study we present a procedure for detection of SRIF receptors using radiolabeled (D-Tyr⁸)-SRIF as a tracer. The present study indicates that in kindled rats there are no differences in the total number or affinity of the binding sites in the temporal cortex and a slight increase in the total number of binding sites in the cortex when compared with controls. These results, in view of our other observations, suggest that in kindled rat brain there may be an increased release of SRIF but no down-regulation of SRIF receptors in temporal cortex and cortex. There appears to be a significant decrease in the number of SRIF receptors in kindled hippocampus. The mechanism by which this occurs remains unclear.     

3H-dopamine binding to rat striatal D-2 and D-3 sites: Enhancement by magnesium and inhibition by guanine nucleotides and sodium

Previous studies have demonstrated high affinity ³H-dopamine binding sites on mammalian striatal membranes. These putative dopamine receptors of unknown physiological significance have been termed D-3 sites. Such studies have failed, however, to demonstrate high affinity ³H-dopamine binding to D-2 sites, which can be labeled by ³H-butyrophenones, and which represent the putative dopamine receptors most stronly implicated in the behavioral correlates of dopaminergic CNS activity. We now report that preincubation of membrane homogenates with Mg⁺⁺ and inclusion of Mg⁺⁺ (1–10mM) or other divalent metal cations during binding allows high affinity D-2 specific ³H-dopamine binding in rat striatal membranes, and that these ions also increase the Bmax of D-3 specific ³H-dopamine binding. GTP, GDP, and GppNHp can completely abolish all D-2 specific ³H-dopamine binding, while only a magnesium-dependent portion of D-3 sites appears to be GTP sensitive. These data are consistent with the hypothesis that the striatal D-2 receptor exists in two agonist affinity states whose interconversion is effected by guanine nucleotides and divalent metal cations. The GTP sensitive/magnesium dependent nature of ³H-dopamine binding to so-called D-3 sites suggests that some such sites may in fact represent a high agonist-affinity state of the D-1 adenylate cyclase stimulating dopamine receptor also found in this tissue.     

Regulation of follicle-stimulating hormone binding to receptors on bovine calf testis membranes by cholera toxin-sensitive guanine nucleotide binding protein

In a previous study we reported that FSH receptors in bovine testes membranes are physically and functionally associated with a guanine nucleotide-binding protein (N protein). In this study we examined the mechanism whereby GTP binding to N protein regulates FSH binding to its receptors. Binding of FSH to receptors decreased in the presence of GTP in a dose-dependent and noncompetitive manner. This effect did not require the presence of Mg+2 and is in contrast to the reported requirement for Mg+2 for GTP effects on human CG binding to ovarian receptors. Equilibrium binding experiments indicated that decreased hormone binding in the presence of GTP was not due to a decrease in the number of FSH receptors per se; rather, the altered binding isotherm was the result of a decrease in affinity of receptors for FSH. Moreover, the dissociation of [125I]human FSH from preformed FSH-receptor complex was rapid in onset and significantly accelerated in the presence of GTP. In a series of nucleotides, GTP was most effective in causing this effect. Evidently, occupancy of GTP binding sites on the N protein, including low affinity and high capacity sites, is necessary for GTP regulation of FSH binding to receptors. The fact that pretreatment of bovine testis membranes with cholera toxin plus NAD, but not pertussis toxin plus NAD, eliminates the GTP effect on FSH binding to its receptors suggests that the GTP regulatory binding protein mediating the GTP regulation of FSH binding is probably Ns and not Ni. Further characterization of FSH receptor sensitivity to GTP, however, indicated that the N protein involved does not exhibit all of the characteristics reported for Ns. For example, the affinity of GTP for N protein is relatively low even under conditions where GTP hydrolysis has a minimal effect in reducing the total concentration of GTP. Also, the absence of a requirement for Mg+2 in high affinity FSH receptor-N protein coupling is different from the requirement for Mg+2 seen with the beta-adrenergic receptor and Ns. Moreover, the N protein which mediates GTP regulation of FSH-receptor binding appears to be relatively insensitive to N-ethylmaleimide, unlike the N-ethylmaleimide sensitivity of the turkey erythrocyte Ns. These results suggest that differences may exist in the structure-function features of GTP regulatory binding protein associated with different types of hormone ligands and receptors.