Neurotensin receptors in canine intestinal smooth muscle: preparation of plasma membranes and characterization of (Tyr3-125I)-labelled neurotensin binding

To study the binding of (Tyr3-125I)-labelled neurotensin to intestinal muscle, plasma membranes have been purified from dog intestinal circular smooth muscle. Purification was done by differential centrifugation followed by separation on a sucrose gradient. Electron microscopic study revealed that the dissected circular muscles used as the source of membranes were free of myenteric plexus and that the plasma membrane fraction obtained was free of any mitochondria or synaptosomes. The fraction used was obtained at the interface of 14%-33% sucrose density on the gradient and was 25-times enriched in the plasma membrane marker enzyme 5'-nucleotidase activity as compared to post-nuclear supernatant. This fraction contained negligible activity of mitochondrial membrane marker enzyme cytochrome c oxidase and low activity of a putative endoplasmic reticulum marker enzyme NADPH-cytochrome-c reductase. This membrane fraction contained a high density of neurotensin binding sites. This binding was studied by kinetic and by saturation approaches. Analysis of data from saturation binding studies by the computer programs (EBDA and LIGAND) suggested the presence of a two-site model (Kd1 = 0.118 nM, Kd2 = 3.18 nM, Bmax1 = 9.73 fmol/mg and Bmax2 = 129.8 fmol/mg). A part of specifically bound neurotensin was rapidly dissociated. No cooperativity between the two receptor types could be detected. A kinetic analysis of binding gave the Kd value equal to 0.107 nM. Carboxy terminal amino acid residues 8-13 were found to be essential for the binding activity and replacement of Tyr11 by tryptophan reduced the affinity of the peptide by 10 times in displacement studies. Binding was modulated by sodium ions and a guanine nucleotide Gpp[NH]p. MgCl2, CaCl2 and KCl were also found to reduce the specific binding. Evidence was found of a high specific binding to another membrane fraction poor in plasma membranes and rich in synaptosomes. We concluded that plasma membrane of canine intestinal circular muscle contains neurotensin receptors with recognition properties distinct from those obtained in previous studies of neurotensin binding sites in murine tissues. Another neurotensin binding site may be present on neuronal membranes.     

Covalent labeling of neurotensin receptors in rat gastric fundus plasma membranes

Neurotensin receptors from plasma membranes of rat gastric fundus smooth muscle were specifically and covalently labeled either by using the photoreactive analogue ¹²⁵I-labeled azidobenzoyl (Trp¹¹)-neurotensin or by cross-linking (monoiodo-Tyr³)neurotensin to the membrane preparation by means of disuccinimidyl suberate. Analysis of plasma membranes by sodium dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the same protein band with an apparent molecular weight of 110,000 was specifically labeled by both methods. This band consisted of a single chain protein since its apparent size was found to be the same with or without reduction of membrane samples before electrophoresis. Only neurotensin and its biologically active analogues were able to protect plasma membranes against specific labeling of the protein band of molecular weight 110,000. Comparison of these results with those obtained from rat brain synaptic membranes shows that although rat central and peripheral neurotensin receptors exhibit similar specificities towards a series of neurotensin analogues, their subunit structures are different.     

Purification and characterization of neurotensin receptor from rat brain with special reference to comparison between newborn and adult age rats.

Scatchard analysis of saturation curves was performed to compared newborn and adult rat neurotensin receptor using [3H] neurotensin as a tracer. The membrane fraction of newborn rat cerebral cortex has a single population of neurotensin receptor (Kd = 0.13 nM, Bmax = 710 fmol/mg protein), whereas adults have two distinct neurotensin binding sites (high affinity site, Kd1 = 0.13 nM; low affinity site, Kd2 = 20 nM). High affinity neurotensin receptor, solubilized with digitonin, was purified from newborn rat cortex by affinity chromatography. An overall purification of 14,000-fold was achieved. The binding of [3H] neurotensin to the purified receptor is saturable and specific, with a Kd of 0.45 nM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 2-mercaptoethanol revealed purified material of a single major band of Mr = 55,000.     

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 kinin binding sites: identity of B2 receptors in the epithelium and the smooth muscle of the guinea pig ileum.

Binding of [125I-Tyr8]bradykinin (BK) was measured in homogenates of epithelial and smooth muscle layers of the guinea pig ileum. Binding assays were performed at 4 degrees C for 40 min (smooth muscle) or 90 min (epithelium) in 25 mM PIPES buffer at pH 6.8 in the presence of 1 mM 1,10-phenanthroline, 140 micrograms/mL bacitracin, 1 mM captopril, 1 mM dithiothreitol, and 0.1% bovine serum albumin. Specific binding of [125I-Tyr8]BK (0.32 nM) to epithelial and smooth muscle cell membranes was linearly related to protein concentration between 0.05 and 0.5 mg/mL. Equilibrium experiments showed that specific binding of [125I-Tyr8]BK was saturable and Scatchard analysis indicated the presence of a high affinity site with a Kd value of 1.6 nM and a Bmax of 156 fmol/mg of protein in the epithelial cell membranes. In smooth muscle membranes, Kd was 1.8 nM and the maximum number of binding sites was 58 fmol/mg of protein. Unlabelled peptides, namely bradykinin, [Tyr8]BK, [Hyp3]BK, D-Arg[Hyp3]BK, [Hyp3,Tyr(Me8)]BK, and kallidin displaced [125I-Tyr8]BK binding while other peptides, angiotensin II and substance P, had no effect. A series of B2-receptor antagonists displaced [125I-Tyr8]BK from specific binding sites with IC50 values ranging from 16 to 152 nM on epithelial cell membranes; similar values were obtained from smooth muscle cell membranes. These findings suggest that the binding sites in both preparations are of the B2 type. B1-receptor agonists and antagonists were found to be inactive at concentrations up to 10(-4) M. Results obtained in the two preparations were compared and a positive highly significant correlation was demonstrated between the two sets of data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.     

Pharmacology and molecular identification of vasoactive intestinal peptide (VIP) receptors in normal and cancerous gastric mucosa in man

In human antral membranes, VIP and its natural analogs inhibited the binding of HPLC-purified 125I-VIP, according to the following order of potency: VIP greater than rh GRF greater than helodermin greater than r PHI greater than PHM greater than p PHI greater than hp GRF greater than h, p secretin. No specific binding was detected in plasma membranes purified from the human fundus. In human antral membranes, Scatchard plots were compatible with the existence of two classes of VIP receptors, the first class with high affinity and low binding capacity (Kd = 0.1 nM, Bmax = 10 fmol/mg protein) and another class with a low affinity and higher binding capacity (Kd = 12) nM, Bmax = 1,000 fmol/mg protein). The structure of the VIP receptor in purified plasma membranes prepared from human antral glands and from the HGT-1 human gastric cancer cells was subsequently probed using the cross-linking reagent DSP and 125I-VIP. In agreement with the pharmacological study and the Scatchard analysis of the binding data, SDS gel electrophoresis of the solubilized receptor identified two radiolabeled peptides Mr 67,000 and 34,000 containing disulfide bonds. According to its sensitivity to low doses of VIP and to GTP, the Mr 67,000 binding site represents the membrane domains involved in the physiologial regulation of adenylate cyclase by VIP in normal and transformed human gastric epithelia.     

Neurotensin receptors in canine intestinal smooth muscle: preparation of plasma membranes and characterization of (Tyr3−125I)-labelled neurotensin binding

To study the binding of (Tyr³−¹²⁵I)-labelled neurotensin to intestinal muscle, plasma membranes have been purified from dog intestinal circular smooth muscle. Purification was done by differential centrifugation followed by separation on a sucrose gradient. Electron microscopic study revealed that the dissected circular muscles used as the source of membranes were free of myenteric plexus and that the plasma membrane fraction obtained was free of any mitochondria or synaptosomes. The fraction used was obtained at the interface of 14%–33% sucrose density on the gradient and was 25-times enriched in the plasma membrane marker enzyme 5′-nucleotidase activity as compared to post-nuclear supernatant. This fraction contained negligible activity of mitochondrial membrane marker enzyme cytochrome c oxidase and low activity of a putative endoplasmic reticulum marker enzyme NADPH-cytochrome-c reductase. This membrane fraction contained a high density of neurotensin binding sites. This binding was studied by kinetic and by saturation approaches. Analysis of data from saturation binding studies by the computer programs (EBDA and LIGAND) suggested the presence of a two-site model (K_d1 = 0.118 nM, K_d2 = 3.18 nM, B_max1 = 9.73 fmol/mg and B_max2 = 129.8 fmol/mg). A part of specifically bound neurotensin was rapidly dissociated. No cooperativity between the two receptor types could be detected. A kinetic analysis of binding gave the K_d value equal to 0.107 nM. Carboxy terminal amino acid residues 8–13 were found to be essential for the binding activity and replacement of Tyr¹¹ by tryptophan reduced the affinity of the peptide by 10 times in displacement studies. Binding was modulated by sodium ions and a guanine nucleotide Gpp[NH]p. MgCl₂, CaCl₂ and KCl were also found to reduce the specific binding. Evidence was found of a high specific binding to another membrane fraction poor in plasma membranes and rich in synaptosomes. We concluded that plasma membrane of canine intestinal circular muscle contains neurotensin receptors with recognition properties distinct from those obtained in previous studies of neurotensin binding sites in murine tissues. Another neurotensin binding site may be present on neuronal membranes.     

Receptors for neurotensin in canine small intestine.

We have previously characterized the neurotensin receptors on the circular smooth muscle (CM) of the canine small intestine (1). In the present studies, using radioligand binding technique, neurotensin receptors were localized on the membranes from deep muscular (DMP) and the submucous plexus while no binding was observed on either the longitudinal smooth muscle or myenteric plexus membranes. The high affinity binding sites (Kd 0.1-0.2 nM) on DMP membranes were similar to those on CM; the low affinity component was of much lower affinity (Kd approximately 40 nM). DMP had 4-6 times higher density of binding sites than the CM. The recognition properties of DMP receptors were similar to those on the CM and reduced sulfhydryl groups were required for the binding activity. The action of neurotensin on the contractility of the canine small intestine, therefore, appears to be through a direct action on the circular smooth muscle and through the prejunctional action on the DMP neurons through distinct receptors. Thiol groups in the neurotensin receptors may be important for the receptor function.     

Neurotensin receptors on the rat liver plasma membranes

Neurotensin (NT) is now classified as a brain-gut peptide in the central nervous system and gastrointestinal tract. In the present study, we characterized the NT receptors on the rat liver plasma membranes. The specific binding of [3H]NT was time dependent, reversible, and saturable. Scatchard analysis of the specific binding data yielded two classes of binding sites, a high affinity site and a low affinity site. The average maximum number of binding sites (Bmax) amounted to 13.3 +/- 1.1 fmol/mg protein at high affinity site and 122.3 +/- 21.5 fmol/mg protein at low affinity site, respectively. The dissociation constant (Kd) had values of 0.39 +/- 0.01 nM at high affinity site and 8.1 +/- 1.1 nM at low affinity site, respectively. The amount of specifically bound [3H]NT was significantly reduced in the presence of mono and divalent cations, EDTA, EGTA and a peptidase inhibitor bacitracin, NT1-13 competed with [3H]NT for its binding site with an IC50 of 0.19 nM at high affinity site (0.2 nM concentration of [3H]NT) and 0.7 nM at low affinity site (4.0 nM concentration of [3H]NT). Xenopsin, a NT analogue separated from the skin of Xenopus laevis, was equipotent (IC50 0.75 nM) with NT1-13 at 4.0 nM concentration of [3H]NT. C-terminal sequence of NT contains the structure necessary for interaction with NT binding sites whereas N-terminal sequence had no binding activity. Since NT has a hyperglysemic and a hypercholesterolemic effects in rats, these NT receptors on the rat liver plasma membranes may be involved in the hyperglycemia and/or hypercholesteroremia induced by NT.     

Galanin receptor and its ligands in the rat hippocampus

Receptors for the 29-amino-acid peptide, galanin, in membranes from the rat ventral hippocampus were examined using chloramine-T-iodinated porcine galanin as ligand. The equilibrium binding of 125I-galanin showed the presence of a high-affinity binding site (Kd = 1.91 +/- 0.40 nM). The concentration of the high-affinity-binding sites was 107 +/- 15 fmol/mg membrane protein. The on rate constant was estimated to be 2.6 +/- 0.1 M-1 min-1 at 37 degrees C. The affinity of rat galanin (differing in three amino acid residues from the porcine protein) was equal to that of porcine galanin. The 125I--galanin-binding site is a trypsin-sensitive membrane protein, which is heat-denaturated at 60 degrees C within 5 min. The effect of GTP and its analogs and of pertussis-toxin-catalyzed ADP-ribosylation on the binding of 125I-galanin suggest that the galanin receptor is coupled to an inhibitory G protein (Gi protein). 127I-galanin was shown to be a ligand with affinity equal to that of galanin in displacing 125I-galanin. The 125I-galanin-binding site in the ventral hippocampus recognizes as a ligand the tryptic fragments 1-20 and 21-29 of rat galanin and the synthetic fragments 12-29, 18-29 and 21-29 of porcine galanin. None of these afforded full inhibition of the binding of fragment 1-29 of 125I-galanin at a concentration of 1 microM.     

Galanin binding sites in rat gastric and jejunal smooth muscle membrane preparations.

Receptors for galanin in membranes from the rat gastric and jejunal smooth muscle were studied using [125I] radioiodinated synthetic porcine galanin. Specific binding was time and temperature dependent. At 32 degrees C radioligand was degraded in the presence of smooth muscle membranes in a time-dependent manner. At optimal experimental conditions, the equilibrium binding analyses showed the presence of a single population of high affinity binding sites in both the rat stomach and jejunum (Kd value of 2.77 +/- 0.78 nM and 4.93 +/- 1.74 nM for stomach and jejunal smooth muscle membranes, respectively). The concentration of the high affinity binding sites was 58.19 +/- 11.04 and 32.36 +/- 5.68 fmol/mg protein, for gastric and jejunal preparations, respectively. Specific binding was completely inhibited by 10(-6) M of nonradioactive galanin; was 75% blocked by 1 microM of galanin(9-29); it was 10% blocked by 1 microM of galanin(15-29). Galanin(1-15) at a concentration of 1 microM was ineffective for inhibiting [125I]galanin binding. Deletion of four C-terminal amino acid residues from galanin(9-29) to give galanin(9-25) also resulted in almost complete loss of affinity. Radioiodinated galanin and N-terminally deleted fragments had receptor binding potency in the following order: galanin(1-29) greater than galanin(9-29) greater than galanin(15-29). We conclude that the C-terminal part of the galanin chain is important for the rat gastric and jejunal smooth muscle membrane receptor recognition and binding and that N-terminal amino acid sequences are probably not so important, since galanin(1-15) was not active but galanin(9-29) retained most of the receptor binding activity.     

[TRP11]-Neurotensin and xenopsin discriminate between rat and guinea-pig neurotensin receptors

The binding and biological activities of neurotensin and two analogues, [Trp¹¹]-neurotensin and xenopsin, in which a tryptophan replaces the neurotensin residue Tyr¹¹, were compared in rat and guinea-pig. The binding activity of the three peptides was measured as their ability to inhibit the binding of [³H]neurotensin to rat and guinea-pig brain synaptic membranes. Their biological activities were measured as their effects on the contractility of rat and guinea-pig ileal smooth muscle preparations. In binding as well as biological assays, it was found that [Trp¹¹]-neurotensin and xenopsin were as potent as neurotensin in the rat. In contrast, the two analogues were about 10 times less potent than neurotensin in the guinea-pig. These findings reveal differences between rat and guinea-pig neurotensin receptors. Such species-related differences in neurotensin receptors should be considered when comparing the activity of neurotensin analogues in assays using tissue preparations from various animal species.     

Characterization and visualization of neurotensin binding to receptor sites in human brain

The binding of monoiodo [125I-Tyr3]-neurotensin to human brain was characterized and visualized using radioreceptorassay and autoradiographic techniques. Specific binding to homogenates of human substantia nigra at 25 degrees C was maximal at 20 min, reversible and saturable. Scatchard analysis of equilibrium data indicated the existence of two populations of binding sites with Kd values of 0.26 nM and 4.3 nM. Corresponding binding capacities were 26 and 89 fmol/mg of protein. Neurotensin analogs inhibited the binding of iodinated neurotensin with relative potencies that demonstrated the crucial role of the C-terminal hexapeptide portion of neurotensin for binding to its receptors. Autoradiography of human substantia nigra sections incubated with iodinated neurotensin revealed high levels of specific binding in the nucleus paranigralis and substantia nigra, pars compacta, and low levels in the substantia nigra, pars reticulata.     

The Na+ channel in mammalian cardiac cells. Two kinds of tetrodotoxin receptors in rat heart membranes

The properties of interaction of both tetrodotoxin (TTX) and tritiated ethylenediamine tetrodotoxin [3H] en-TTX) were studied in rat heart membranes at different stages of development and in cultured cells. Studies by electrophysiology and by 22Na+ flux measurements on cardiac cultured cells indicate that the functional form of the Na+ channel is of low affinity for TTX (250-700 nM). Binding experiments (bioassay and [3H]en-TTX binding) on cultured cardiac cells from newborn rats indicate the presence of both high and low affinity binding sites for TTX with dissociation constants (Kd) of 1.6 and 135 nM, respectively. On homogenates of hearts taken just after birth, [3H]en-TTX binding reveals no high affinity binding site for TTX but the presence of a low affinity binding site with a Kd of 125 nM. This result was confirmed by kinetic studies and competition experiments. Conversely, binding studies on homogenates and extensively purified membranes from adult ventricles reveal the presence of both high and low affinity binding sites for TTX with Kd values of 1.5 and 170 nM, respectively. The maximum binding capacity for the low affinity binding sites is 45 times higher than that of the high affinity binding sites. High affinity sites do not exist at the fetal stage or at birth, but after 5 days their number gradually increases to reach a maximum level around 45 days after birth. Conversely, the number of low affinity binding sites is essentially invariant between birth and adulthood. Monolayers of cardiac cells from hearts at 2 days after birth which have no high affinity TTX-binding sites in vivo develop both high and low affinity binding sites for TTX in vitro. The results presented here are the first direct demonstration of the coexistence in rat heart plasma membrane of two families of binding sites for TTX.     

High affinity angiotensin II receptors in myocardial sarcolemmal membranes. Characterization of receptors and covalent linkage of 125I-angiotensin II to a membrane component of 116,000 daltons

High affinity receptors for angiotensin II have been identified on purified cardiac sarcolemmal membranes. Equilibrium binding studies were performed with 125I-labeled angiotensin II and purified sarcolemmal vesicles from calf ventricle. The curvilinear Scatchard plots were evaluated by nonlinear regression analysis using a two-site model which identified a high affinity site Kd1 = 1.08 +/- 0.3 nM and N1 = 52 +/- 10 fmol/mg of protein and a low affinity site Kd2 = 52 +/- 16 nM and N2 = 988 +/- 170 fmol/mg of protein. Monovalent and divalent cations inhibited the binding of 125I-angiotensin II by 50%. The affinity of angiotensin II analogs for the receptor was determined using competitive binding assays; sarcosine, leucine-angiotensin II (Sar,Leu-angiotensin II), Kd = 0.53 nM; angiotensin II, Kd = 2.5 nM; des-aspartic acid-angiotensin II, Kd = 4.81 nM; angiotensin I, Kd = 77.6 nM. There is a positive correlation between potency in inducing positive inotropic response in myocardial preparations reported by others and potency for the hormone receptor observed in the binding assays. Pseudo-Hill plots of the binding data showed that agonists display biphasic binding with Hill numbers around 0.65 while antagonists recognized a single class of high affinity receptors with Hill numbers close to unity. These data were confirmed using 125I-Sar,Leu-angiotensin II in equilibrium binding studies which showed that this antagonist bound to a single class of receptor sites; Kd = 0.42 +/- 0.04 nM and N = 1050 +/- 110 fmol/mg of protein. Competition-binding experiments with this 125I-peptide yielded monophasic curves with Hill numbers close to unity for both agonists and antagonists. Membrane-bound 125I-angiotensin II was covalently linked to its receptor by the use of bifunctional cross-linking reagents such as dithiobis(succinimidyl propionate) and bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone. Analysis of the membranes showed the labeling of a component with an apparent Mr = 116,000. The affinity labeled species showed characteristics expected of a functional component of the high affinity receptor. The affinity labeling of this membrane component was inhibited by nanomolar angiotensin II or Sar,Leu-angiotensin II. Together these data indicate that high affinity receptors exist for angiotensin II that most likely mediate the positive inotropic effects of this hormone on myocardial cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of angiotensin II receptor subtypes in rat liver

Radioligand binding studies identified two classes of 125I-angiotensin II-binding sites in rat liver membranes. High affinity binding sites (Kd = 0.35 +/- 0.13 nM, N = 372 +/- 69 fmol/mg of protein) were inactivated by dithiothreitol (0.1-10 mM) without any apparent change in low affinity binding sites (Kd = 3.1 +/- 0.8 nM, N = 658 +/- 112 fmol/mg of protein). Dithiothreitol inactivation was readily reversible but could be made permanent by alkylation of membrane proteins with iodoacetamide. Angiotensin II stimulation of glycogen phosphorylase in isolated rat hepatocytes (maximal stimulation 780%, EC50 = 0.4 nM) was completely inhibited by 10 mM dithiothreitol, a concentration which also abolished high affinity site binding; phosphorylase stimulation by glucagon and norepinephrine under these conditions was unaltered. Angiotensin II inhibition of glucagon-stimulated adenylate cyclase activity in hepatocytes required higher angiotensin II concentrations (EC50 = 3 nM) than phosphorylase stimulation and was not affected by dithiothreitol. Fractional occupancy of high affinity binding sites by 125I-angiotensin II correlated closely with angiotensin II-mediated phosphorylase stimulation, whereas occupancy of low affinity sites paralleled inhibition of adenylate cyclase activity. These data indicate that the physiologic effects of angiotensin II in rat liver are mediated by two distinct receptors, apparently not interconvertible, and provide the first evidence for angiotensin II receptor subtypes with differing biochemical features and mechanisms of action.     

Characterization of hepatic epidermal growth factor receptors in the developing rat

Binding of 125I-labeled epidermal growth factor (EGF) was characterized in basolateral plasma membranes prepared from the livers of 21-day gestation fetuses, 14-day-old sucklings and adult Sprague-Dawley rats using a self-generating Percoll gradient method. The membrane preparations employed have been previously assayed in terms of plasma membrane protein yield, enrichment of various marker enzymes and sodium-dependent bile acid and amino acid transport. 125I-EGF binding was saturable and time and temperature dependent. Equilibrium analyses showed that the suckling period is characterized by a marked decrease in overall hepatic EGF binding capacity (460 +/- 50 fmol/mg protein) compared to either the fetal period (1290 +/- 160 fmol/mg) or to adults of either sex (males = 1540 +/- 230, females 1010 +/- 130 fmol/mg). Treatment of the suckling rat with parenteral EGF resulted in a 78% reduction in the observed binding capacity when assessed 2 h after growth factor administration. Comparison of binding affinities revealed no significant difference between the suckling and adult preparations (Kd = 0.40 +/- 0.03 vs. 0.39 +/- 0.02 nM, respectively); however, both preparations differed significantly from the fetal group which exhibited a decreased affinity of binding with a higher overall dissociation constant (Kd = 0.68 +/- 0.06 nM). Thus, it appears that major ontogenetic changes occur in the rat hepatic ligand/receptor system for epidermal growth factor. These changes are discussed in the context of transitional events in mammalian development such as birth and weaning.     

Neurotensin receptors on circular smooth muscle of canine small intestine: role of disulfide bridges

We have studied the effect of sulfhydryl agents on the binding of 125I-tyr3-neurotensin to the purified plasma membranes from circular smooth muscle and on the in vitro response of circular muscle strips of canine small intestine to neurotensin. Dithiothreitol (DTT) enhanced the binding by about 80%. Cysteine (a reductant) also enhanced the binding while cystine (an oxidant) reduced the binding to the similar extent. DTT stimulated the tissue in the organ bath and abolished the stimulatory response to low concentrations of neurotensin. The stimulatory response to acetylcholine was not altered by DTT. The implications of the role of disulfide bridges in the neurotensin response is discussed.     

Characterization of neuropeptide Y binding sites in rat cardiac ventricular membranes

Neuropeptide Y (NPY) binding sites in rat cardiac ventricular membranes have been characterized in detail. 125I-NPY bound to the membranes with high affinity. Binding was saturable, reversible and specific, and depended on time, pH and temperature. Analysis of the binding data obtained under optimal conditions, 2 hr, 18 degrees C and at pH 7.5, revealed the presence of low and high affinity binding sites. The high affinity binding sites had an apparent dissociation constant (Kd) of 0.38 nM and a binding capacity (Bmax) of 7.13 fmol/mg protein. The apparent Kd and Bmax for low affinity binding sites were 22.34 nM and 261.25 fmol/mg protein, respectively. Peptides unrelated to NPY did not compete with 125I-NPY for the binding sites even at 1 microM concentrations, whereas homologous peptides, peptide YY (PYY) and pancreatic polypeptide (PP), and NPY(13-36) inhibited 125I-NPY binding but with lower potency compared to NPY. 125I-NPY binding was sensitive to the nonhydrolyzable GTP analog, Gpp(NH)p, suggesting that the NPY receptor is coupled to the adenylate cyclase system. The ventricular membrane receptor characterized in this study may play an important role in mediating the physiological effects of NPY in the heart.