Characterization of vasoactive intestinal peptide (VIP) receptors in mammalian lung

125I-VIP bound specifically to sites on human, rat, guinea pig, and rabbit lung membranes with a dissociation constant (KD) of 60-200 pM and binding site maxima of 200-800 fmol/mg of protein. The presence of a second lower affinity site was detected but not investigated further. High affinity 125I-VIP binding was reversible and displaced by structurally related peptides with an order of potency: VIP greater than rGRF greater than PHI greater than hGRF greater than secretin = Ac Tyr1 D Phe2 GRF. 125I-VIP has been covalently incorporated into lung membranes using disuccinimidyl suberate. Sodium dodecyl sulfate-polyacrilamide gel electrophoresis of labeled human, rat, and rabbit lung membranes revealed major 125I-VIP-receptor complexes of: Mr = 65,000, 56,000, and 64,000 daltons, respectively. Guinea pig lung membranes exhibited two 125I-VIP-receptor complexes of Mr = 66,000 and 60,000 daltons. This labeling pattern probably reflects the presence of differentially glycosylated forms of the same receptor since treatment with neuroaminidase resulted in a single homogeneous band (Mr = 57,000 daltons). Soluble covalently labeled VIP receptors from guinea pig and human lung bound to and were specifically eluted from agarose-linked wheat germ agglutinin columns. Our studies indicate that mammalian lung VIP receptors are glycoproteins containing terminal sialic acid residues.     

Solubilization of active and stable receptors for vasoactive intestinal peptide from rat liver

Vasoactive intestinal peptide (VIP) receptors were solubilized from rat liver using the zwitterionic detergent CHAPS. Optimal conditions of solubilization were obtained with 5 mM CHAPS and 2.5 mg protein/ml. The binding of 125I-VIP to CHAPS extracts was time- and pH-dependent, saturable and reversible. The following order of potency of unlabeled VIP-related peptides for inhibiting 125I-VIP binding was observed: VIP greater than helodermin greater than peptide histidine isoleucine amide (PHI) greater than rat growth hormone releasing factor (rGRF) greater than secretin. This peptide specificity is identical to that of rat liver membrane-bound receptors. VIP binding activity in the CHAPS extract was destroyed by trypsin or dithiothreitol in accordance with the known sensitivity of membrane-bound receptors to these agents. VIP receptors in CHAPS extracts were stable for at least 5 days at 4 degrees C. Scatchard analysis of equilibrium binding data indicated the presence in CHAPS extracts of high (H) and low (L) affinity binding sites with the following characteristics: KdH = 0.27 nM and BmH = 34 fmol/mg protein; KdL = 51 nM and BmL = 1078 fmol/mg protein. The guanine nucleotide GTP inhibited 125I-VIP binding to soluble receptors and enhanced the dissociation of soluble VIP-receptor complexes, suggesting that GTP-binding proteins were functionally associated with VIP receptors in solution. Gel filtration of solubilized VIP receptors on Sephacryl S-300 revealed a single binding component with a Stokes radius of 6.1 nm. It is concluded that active VIP receptors can be extracted from liver membranes by CHAPS. The availability of this CHAPS-soluble, stable and functional receptor from a tissue which can be obtained in large amounts represents a major step toward the purification of VIP receptors.     

VIP receptors from porcine liver: high yield solubilization in a GTP-insensitive form.

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membranes using CHAPS. The binding of 125I-VIP to solubilized receptors was reversible, saturable and specific. Scatchard analysis indicated the presence of one binding site with a Kd of 6.5 +/- 0.3 nM and a Bmax of 1.20 +/- 0.15 pmol/mg protein. Solubilized and membrane-bound receptors displayed the same pharmacological profile since VIP and VIP-related peptides inhibited 125I-VIP binding to both receptor preparations with the same rank order of potency e.g. VIP greater than helodermin greater than rat GRF greater than rat PHI greater than secretin greater than human GRF. GTP inhibited 125I-VIP binding to membrane-bound receptors but not to solubilized receptors supporting functional uncoupling of VIP receptor and G protein during solubilization. Affinity labeling of solubilized and membrane-bound VIP receptors with 125I-VIP revealed the presence of a single molecular component with Mr 55,000 in both cases. It is concluded that VIP receptors from porcine liver can be solubilized with a good yield, in a GTP-insentive, G protein-free form. This represents a major advance towards the purification of VIP receptors.     

Solubilization of the active vasoactive intestinal peptide receptor from human colonic adenocarcinoma cells

The non-ionic detergent n-octyl-beta-D-glucopyranoside was used to solubilize the VIP (vasoactive intestinal peptide) receptor from human colonic adenocarcinoma cell line HT29-D4. The binding of monoiodinated 125I-VIP to the solubilized receptor was specific, time-dependent, and reversible. Scatchard analysis of data obtained from competitive displacement of monoiodinated 125I-VIP by native VIP suggested the presence of two classes of VIP binding sites with Kd values of 0.32 and 46.7 nM. The binding capacities of these two classes were 1.7 x 10(10) and 30.2 x 10(10) sites/mg of proteins, respectively. The solubilized receptor retained the specificity of the human VIP receptor towards the peptides of the VIP/secretin/glucagon family. The order of potency in inhibiting monoiodinated 125I-VIP binding was VIP (IC50 = 1.0 x 10(-9) M) much greater than peptide histidine methionine amide (IC50 = 10(-7) M) greater than growth hormone-releasing factor (IC50 = 3 x 10(-7) M) greater than secretin (IC50 greater than 10(-6) M); glucagon had no effect on VIP binding. The reducing agent dithiothreitol inhibited in a dose-dependent manner the binding of 125I-VIP. Covalent cross-linking experiments between the solubilized receptor and 125I-VIP showed that after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography two major and one minor polypeptides of Mr 67,000, 72,000, and 83,000 were specifically labeled. When analyzed by gel filtration, the n-octyl-beta-D-glucopyranoside-solubilized 125I-VIP-receptor complex was resolved into two major peaks with molecular mass in the range of 60-70 and 270-300 kDa. Thus, the soluble form of the VIP receptor was probably a multimeric complex in which disulfide bonds may play an important role to hold the receptor in an active configuration.     

Limited proteolysis of the vasoactive intestinal peptide receptor: comparison of its folded structure in the membrane-bound and detergent-solubilized states

Limited proteolysis was used to probe and compare the conformation of the rat lung vasoactive intestinal peptide (VIP) receptor in membrane-bound and detergent-solubilized states. It had been shown previously that the activity of the detergent-solubilized VIP receptor is sensitive to the nature of the detergent used for extraction (Patthi, S., Simerson S. and Velicelebi, G. (1988) J. Biol. Chem., 263, 19363-19369). Receptors that were extracted from the membrane using digitonin retained the ability to bind 125I-VIP, while those solubilized in Triton X-100 displayed little or no detectable activity. In order to correlate the differences observed in the activity of the receptor with its folded state, membrane-bound and detergent-solubilized receptors were covalently labeled with 125I-VIP and subjected to limited proteolysis using trypsin, chymotrypsin or carboxypeptidase Y. Digitonin-solubilized receptors most closely resembled the membrane-bound protein in terms of protease sensitivity and proteolytic cleavage products. By contrast, receptors solubilized in Triton X-100 displayed increased sensitivity to proteases and produced distinctly different proteolytic patterns. Thus, the differences observed in the activities of receptors solubilized in digitonin and those solubilized in Triton X-100 could be correlated with detectable differences in the conformation of the protein in each respective detergent solution. These results suggest that digitonin provides an environment that is more compatible with the native folded state of the receptor, similar to its conformation in the membrane.     

Characterization of receptors for vasoactive intestinal peptide solubilized from the lung

The zwitterionic detergent CHAPS was used to solubilize functional receptors for vasoactive intestinal peptide (VIP) from guinea pig lung. The solubilized receptors were resolved by high performance gel filtration in 3 mM CHAPS into two active fractions with apparent Stokes radii of 5.9 +/- 0.1 and 2.3 +/- 0.1 nm. The binding of 125I-VIP to the two receptor fractions was time-dependent, reversible, and saturable. Trypsin destroyed the binding activity of the receptor fractions, indicating their proteinic nature. Unlabeled VIP competitively displaced the binding of 125I-VIP to the 5.9-nm fraction (IC50 = 240 pM) and the 2.3-nm fraction (IC50 = 1.2 microM). Scatchard analysis indicated a single class of binding sites in each receptor fraction, with Kd values 300 pM and 0.97 microM for the 5.9- and 2.3-nm Stokes radii fractions, respectively. When the high affinity, 5.9-nm Stokes radius fraction was rechromatographed in 9 nM CHAPS, 46% of the binding activity eluted in the low affinity, 2.3-nm Stokes radius fraction, indicating that the latter is a product of dissociation of the high affinity receptor complex. GTP inhibited the binding of 125I-VIP to the high affinity complex but not the low affinity species. Scatchard plots of VIP binding by the high affinity receptors treated with GTP suggested the presence of two distinct binding sites (Kd 4.4 and 153 nM), compared to a single binding site (Kd = 0.3 nM) obtained in untreated receptors. The nonhydrolyzable GTP analog, guanyl-5'-yl-imidodiphosphate, inhibited VIP binding by the high affinity receptor fraction with potency nearly equivalent to that of GTP. These observations suggest that GTP-binding regulatory proteins are functionally coupled to the VIP-binding subunit in the high affinity receptor complex. The peptide specificity characteristics of the two receptor fractions were different. Peptide histidine isoleucine and growth hormone releasing factor, peptides homologous to VIP, were 87.5- and 22.9-fold less potent than VIP in displacing 125I-VIP binding by the high affinity receptor complex, respectively. On the other hand, growth hormone-releasing factor was more potent (22.7-fold) and peptide histidine isoleucine was less potent (31.3-fold) than VIP in displacing the binding by the low affinity species.     

Functional and immunological evidence for stable association of solubilized vasoactive-intestinal-peptide receptor and stimulatory guanine-nucleotide-binding protein from rat liver

We have reported the solubilization of complexes between vasoactive intestinal peptide (VIP) and its receptor from rat liver in a GTP-sensitive form of Mr 150,000 [Couvineau, A., Amiranoff, B. & Laburthe, M. (1986) J. Biol. Chem. 261, 14482-14489]. In the present study, we demonstrate a stable association of solubilized VIP receptor and stimulatory guanine nucleotide-binding protein (Gs protein), taking advantage of the ability of the glycoproteic VIP receptor (Mr 48,000), and the inability of the Gs protein, to adsorb to wheat germ agglutinin (WGA). 125I-VIP-receptor complexes solubilized in Triton X-100 were adsorbed on WGA-Sepharose, extensively washed and the radioactivity retained was eluted with 1 mM GTP showing that: (a) radioactivity corresponds to free 125I-VIP and (b) alpha s (Mr 42,000) and beta (Mr 35,000) subunits of Gs protein are detectable in the GTP eluate by immunoblotting using antisera against these subunits. Such an effect of GTP implied that a stable ternary complex consisting of VIP, receptor and Gs protein had been adsorbed to WGA-Sepharose. When Triton-solubilized 125I-VIP-receptor complexes were adsorbed on WGA-Sepharose, then retained material was specifically eluted with 0.3 M N-acetylglucosamine, analysis of the sugar eluate showed the following results. (a) GTP induces the dissociation of 125I-VIP-receptor complexes of Mr 150,000 contained in the eluate indicating that 125I-VIP-receptor-G protein complexes had been adsorbed to the WGA column. (b) The Mr-42,000 alpha s subunit can be specifically ADP-ribosylated by cholera toxin. (c) Immunoblotting using antisera against the alpha s and beta subunits of Gs protein, reveals Mr-42,000 and Mr-35,000 components corresponding to alpha s and beta subunits, respectively. (d) Affinity cross-linking using dithiobis(succinimidyl-propionate) of 125-I-VIP-receptor complexes eluted from the WGA column reveals a major band corresponding to Mr 150,000. Immunoblotting using antisera against the beta-subunit shows the presence of the beta subunit (Mr 35,000) in this Mr-150,000 component. In conclusion, these data provide functional and immunochemical evidence for the physical association of solubilized VIP-receptor complexes with alpha s and beta subunits of Gs protein.     

Autoradiographic localization of vasoactive intestinal polypeptide receptors in the rat mesenteric vascular tree

By the use of combined in vitro radioreceptor binding and autoradiographic techniques, we analyzed the pharmacological properties and the anatomical localization of the vasoactive intestinal polypeptide (VIP) receptor in rat superior mesenteric artery and in medium and small mesenteric artery branches. 125I-VIP was bound by sections of rat superior mesenteric artery in a manner consistent with the labeling of specific VIP receptors, with Kd and Bmax values of 0.23 nM and 0.71 pmol/mg protein respectively. Inhibition of 125I-VIP binding with VIP and related peptides gives the following rank order of potency: VIP greater than peptide histidine methionine greater than secretin. Light microscope autoradiography reveals specific VIP binding sites within the medial layer of superior mesenteric artery and its branches. Medium and small sized vessels are richer in 125I-VIP binding sites than the larger ones.     

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.     

Hydrodynamic characterization of vasoactive intestinal peptide receptors extracted from rat lung membranes in Triton X-100 and n-octyl-beta-D-glucopyranoside

Rat lung membrane vasoactive intestinal peptide (VIP) receptors were covalently labeled with 125I-VIP, extracted in Triton X-100 and n-octyl-beta-D-glucopyranoside, and analyzed by gel filtration and sucrose density gradient sedimentation. The fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and the identity of the 125I-VIP.receptor complex was demonstrated by its co-migration with the covalently labeled 55-kDa receptor unit identified previously. Furthermore, the radioactivity in the peak corresponding to the 125I-VIP.receptor complex was displaced in the presence of unlabeled VIP in a dose-dependent manner. The following hydrodynamic properties were determined for VIP receptors in each detergent solution: in Triton X-100, Stokes radius of 6.1 +/- 0.4 nm, sedimentation coefficient (S20,w) of 7.35 +/- 0.45 S, and partial specific volume (v) of 0.809 +/- 0.015 ml/g; in n-octyl-beta-D-glucopyranoside, Stokes radius of 5.6 +/- 0.00 nm, S20,w of 10.87 +/- 0.22 S, and partial specific volume of 0.783 +/- 0.020 ml/g. The apparent molecular weight of the 125I-VIP.receptor.detergent complex was calculated as 270,000 +/- 36,000 in Triton X-100 and 320,000 +/- 32,000 in n-octyl-beta-D-glucopyranoside. The amount of detergent bound to the receptor was estimated by using the two sets of hydrodynamic data and the significantly different partial specific volumes of the two detergents. Thus, the molecular weight of the receptor alone was calculated as 54,600 daltons, indicating that approximately 3.9 g of Triton X-100 and 4.9 g of n-octyl-beta-D-glucopyranoside were bound per g of receptor. This species contained the 55-kDa binding unit and appeared to be glycosylated as evidenced by its specific binding to wheat germ agglutinin-Sepharose. These results indicate that the rat lung VIP receptor is a glycoprotein with a single polypeptide chain of 55 kDa. The large amount of detergent bound suggests that the receptor is extensively embedded in the membrane.     

Molecular identification of receptors for vasoactive intestinal peptide in rat intestinal epithelium by covalent cross-linking. Evidence for two classes of binding sites with different structural and functional properties

The cleavable cross-linking reagent dithiobis (succinimidyl propionate) or DTSP was shown to link 125I-labeled vasoactive intestinal peptide (125I-VIP) covalently to its receptors in rat intestinal epithelial membranes. DTSP treatment of 125I-VIP-labeled membranes inhibited the dissociation of VIP-receptor complexes in a way which was dependent on both time and concentration (ED50 = 200 microM). Polyacrylamide gel electrophoresis of membrane proteins revealed three 125I-VIP-protein complexes of Mr 76 000, 36 000 and 17 000. The labeling of those compounds was not observed when: (a) treatment of membranes by DTSP was omitted; (b) the reagent quench, ammonium acetate, was added together with DTSP; (c) DTSP-treated membranes were incubated with 2-mercaptoethanol which reduces the disulfide bond present within DTSP. Labeling of Mr-76 000 and Mr-36 000 complexes was specific in that it could be abolished by native VIP, while the labeling of the Mr-17 000 was not. Densitometric scanning of autoradiographs indicated that: (a) labeling of the Mr-76 000 complex was abolished by low VIP concentrations (0.03--10 nM), by VIP agonists with the relative potency VIP greater than a peptide having N-terminal histidine and C-terminal isoleucine amide greater than secretin, and by GTP (10(-5)--1 mM) but was unaffected by various other peptide hormones; (b) labeling of the Mr-36 000 complex was inhibited by high VIP concentrations (1--300 nM), by VIP agonists at high concentrations but was not affected by GTP and various peptide hormones. Assuming one molecule of 125I-VIP was bound per molecule of protein, two proteins with Mr-73 000 and 33 000 were identified as VIP binding sites. The Mr-73 000 protein displays many characteristics (affinity, specificity, discriminating power toward agonists, sensitivity to GTP regulation) of the high-affinity VIP receptors mediating adenylate cyclase activation. The Mr-33 000 protein displays the characteristics (affinity, specificity) of a low-affinity VIP binding site. This study thus shows the molecular characteristics of the VIP receptor and further argues for the molecular heterogeneity of VIP binding sites.     

Effects of PHI on vasoactive intestinal peptide receptors and adenylate cyclase activity in lung membranes. A comparison in man, rat, mouse and guinea pig

The presence of receptors, recognized by vasoactive intestinal peptide (VIP) as well as by PHI (a peptide with N-terminal histidine and C-terminal isoleucine amide), was documented in lung membranes from rat, mouse, guinea pig and man by the ability of these receptors, once occupied, to stimulate adenylate cyclase. In lung membranes from rat, mouse and guinea pig, the capacity of VIP, PHI and secretin to stimulate the enzyme and the potency of the same peptides to compete with 125I-VIP for binding to VIP receptors were similar, the affinity decreasing in the order: VIP greater than PHI greater than secretin. In addition, dose-effect curves were compatible with the coexistence of high-affinity and low-affinity VIP receptors, in the four animal species considered. If PHI was able to recognize all VIP receptors it could not, however, discriminate the subclasses of VIP receptors.     

Receptors for Vasoactive Intestinal Polypeptide on Isolated Synaptosomes from Rat Cerebral Cortex. Heterogeneity of Binding and Desensitization of Receptors

Abstract: Vasoactive intestinal polypeptide (VIP) is a neuropeptide that causes neurone excitation in the brain cortex. VIP receptors were studied in subcellular fractions isolated from rat cerebral cortex. The receptor binding of ¹²⁵I-VIP was greatest in the synaptosomal fraction at membrane protein concentrations of 50–100 μg/ml, a temperature of 37°C, and a pH from 7.4 to 7.7. Under these conditions the concomitant proteolytic degradation of ¹²⁵I-VIP was approximately 10% after 60 min of incubation. The binding of 60 pmoI/L ¹²⁵I-VIP reached steady-state after 60 min and was maintained up to 240 min. At steady-state, the receptor-bound 125I-VIP was displaced by unlabelled VIP with half-maximal inhibition (IC₅₀) at a concentration of approximately 3 nmol/L. The binding of ¹²⁵I-VIP in the concentration range of 10 pmol/L to 6 nmol/L was superimposable on the VIP displacement curve. The Scatchard plot was curvilinear with upward concavity, which can be interpreted to represent two classes of receptors with K_D of 2.5 and 125 nmol/L, one class of receptors with negative cooperative interactions, or heterogeneity of the ¹²⁵I- VIP preparation. The total amount of receptors was 9.5 pmol/mg of membrane protein. Secretin displaced receptor-bound 125I-VIP with an IC₅₀ of 0.3 μmol/L, whereas glucagon snowed no inhibition up to 1 μmol/L. The dissociation of receptor-bound ¹²⁵I-VIP was biexponential with rate constants (k₂) of 4.1 – 10^?3 and 0.18 min^?1 corresponding to half-times of approximately 170 and 4 min, respectively. The size of the two components was dependent on the duration of the ¹²⁵I-VIP association period. Initially, both components increased; at steady-state, the rapid component declined, whereas the slow component increased to approximately 70% after 120 min. The association rate constants (k₁) were estimated from the initial velocities as 10⁶ and 4. 10⁶ L. mol^?1. min^?1, and a calculation of the K_D as k₂/k₁ gave values of 4.1 and 45 nmol/L, respectively. In conclusion, the presence of receptors for VIP on synaptosomes from the cerebral cortex supports the role of VIP as a neurotransmitter in the brain. The receptor binding was heterogeneous, suggesting the presence of two classes of receptors. The binding kinetics showed a time-dependent transition of VIP receptors from a low- to a high-affinity state, which may be interpreted as desensitisation of synapses to the action of VIP.     

Vasoactive intestinal peptide receptor on liver plasma membranes: Solubilization and cross-linking

The hepatic receptor for VIP was solubilized from rat liver plasma membranes with 1.4% digitonin and shown to conserve its ability to bind to the ligand. This solubilized receptor demonstrated the high affinity and specificity for VIP (K_D1 nM, binding preference: VIP > PHI > secretin > thymosin ₁) which were observed with the nonsolubilized VIP receptor on intact liver plasma membranes. ¹²⁵I-VIP was next cross-linked to either the solubilized or nonsolubilized receptor using disuccinimido suberate or disuccinimido dithiobis(propionate), and the resulting complexes analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. A broad autoradiographic band which demonstrated a high affinity for VIP was identified at Mr 56,000 (53,000 in the absence of the reducing agent dithiothreitol) for both the solubilized and nonsolubilized receptors. We have thus been able to solubilize from rat liver plasma membranes a receptor with high affinity and specificity for VIP, and confirmed its structural similarity with the native VIP receptor in nonsolubilized membranes using cross-linking techniques.     

Multiplicity of receptors for vasoactive intestinal polypeptide (VIP): differential effects of apamin on binding in brain, uterus and liver

Apamin is a neurotoxic octadecapeptide from bee venom, which has been shown to inhibit the non-adrenergic, non-cholinergic inhibitory innervation of the smooth muscle of the gut. Since vasoactive intestinal polypeptide (VIP) has been proposed as a possible inhibitory neurotransmitter, the effect of apamin on the receptor binding of 125I-VIP was studied using the following assays: (1) isolated synaptosomes from rat cerebral cortex, (2) crude plasma membranes from hog uterine smooth muscle, and (3) purified plasma membranes and isolated hepatocytes from hog liver. Apamin inhibited the receptor-bound 125I-VIP on membranes from brain or myometrium, although the binding affinity was 100-1000 times lower than for VIP. The displacement curves for VIP and apamin were parallel suggesting that apamin interacts with both the low and high affinity VIP receptors. In membranes and cells from liver, apamin was unable to displace receptor-bound 125I-VIP in concentrations up to 50 mumol/l. The findings suggest that the VIP receptors in liver are different from those in the brain cortex and myometrium.     

Insertion and internalization of vasoactive intestinal peptide (VIP) receptors in murine CD4 T lymphocytes.

The specific binding of vasoactive intestinal peptide (VIP) to murine lymphocytes was investigated. CD4 T cells from mesenteric lymph nodes (MLN) bound more 125I-VIP than did unseparated MLN lymphocytes at 37 degrees C, but not at 4 degrees C. The differences between the amount of 125I-VIP bound by the CD4 T cells and unseparated MLN lymphocytes at 37 degrees C depended upon a difference in the amount of the ligand that was internalized by the cells. The rate of insertion of unoccupied VIP receptors from the cytoplasm into the cell membrane (370 receptors/cell/min), the rate constants for internalization of ligand occupied VIP receptors (0.55 min-1) and unoccupied VIP receptors (0.11 min-1), and the rate constant for the elimination of internalized VIP (0.07 min-1) by CD4 T cells were evaluated. These results provide new understanding of the behaviour of VIP receptors on lymphocytes and indicate a mechanism by which CD4 T lymphocytes can homologously regulate their surface expression of VIP receptors in the presence of ambient VIP.     

Autoradiographic visualization of CNS receptors for vasoactive intestinal peptide

Receptors for VIP were characterized in the rat CNS. 125I-VIP bound with high affinity to rat brain slices. Binding was time dependent and specific. Pharmacology studies indicated that specific 125I-VIP binding was inhibited with high affinity by VIP and low affinity by secretin and PHI. Using in vitro autoradiographic techniques high grain densities were present in the dentate gyrus, pineal gland, supraoptic and suprachiasmatic nuclei, superficial gray layer of the superior colliculus and the area postrema. Moderate grain densities were present in the olfactory bulb and tubercle, cerebral cortex, nucleus accumbens, caudate putamen, interstitial nucleus of the stria terminalis, paraventricular thalamic nucleus, medial amygdaloid nucleus, subiculum and the medial geniculate nucleus. Grains were absent in the corpus callosum and controls treated with 1 microM unlabeled VIP. The discrete regional distribution of VIP receptors suggest that it may function as an important modulator of neural activity in the CNS.     

Properties of vasoactive intestinal peptide-receptor interaction in rat liver membranes

The properties of the specific receptors for vasoactive intestinal peptide (VIP) in rat liver plasma membranes have been studied by using 125I-VIP as a tracer. The binding of the peptide was a reversible, saturable and specific process, as well as time and temperature dependent. Peptide inactivation was also dependent on time and temperature and remained relatively low in the standard conditions used, as it happened in the inactivation of the binding sites. The binding data were compatible with the existence of two classes of VIP receptors: a high affinity (Kd = 4.2 x 10(-10) M) and low binding capacity (1.5 pmol VIP/mg protein) class and another one of low affinity (Kd = 1.7 x 10(-7) M) and high binding capacity (38.6 pmol VIP/mg protein). The specificity of the binding sites of VIP was established from the fact that binding of 125I-VIP was inhibited by native VIP and by 60-fold higher concentrations of secretin but not by the parent hormone glucagon, by insulin or somatostatin at concentrations as high as 10(-6) M.     

Solubilization of the liver vasoactive intestinal peptide receptor. Hydrodynamic characterization and evidence for an association with a functional GTP regulatory protein

Vasoactive intestinal peptide (VIP) receptors were solubilized using the nondenaturing detergent Triton X-100 after occupancy of rat liver membrane-bound receptors with 125I-VIP. Gel filtration and ultracentrifugation on sucrose density gradients revealed the existence in the soluble macromolecular fraction of two labeled components: a major (80%) heavy component and a minor (20%) light one. The two components exhibit the following hydrodynamic parameters: Stokes radius, 5.8 nm: s20,w, 5.98 s; Mr, 150,000; frictional ratio, 1.52 for the major; and Stokes radius, 3.0 nm: s20,w, 3.98 s; Mr = 52,000; frictional ratio, 1.12 for the minor component. The labeling of these components was specific in that it dramatically decreased when unlabeled VIP was added together with 125I-VIP. The pharmacological specificity was also assessed by using 10 nM histidylisoleucineamide (a VIP agonist). Many lines of evidence indicate that the light component (Mr = 52,000) is the VIP-receptor complex while the heavy component (Mr = 150,000) is a ternary complex consisting of VIP, the receptor, and a guanine nucleotide regulatory protein, probably Ns. GTP is required to dissociate 125I-VIP from the heavy component whereas it is ineffective on the light component. This effect is nucleotide specific. After cholera toxin-induced [32P]ADP ribosylation of liver membranes, a high peak of 32P radioactivity containing the alpha subunit (Mr = 42,000) of the Ns protein is coeluted with the heavy component on Sephacryl S-300. By mild urea (2 M) treatment, the heavy component is converted into the light without significant dissociation of 125I-VIP. When a Triton extract of membranes prelabeled with 125I-VIP is treated with dithiobis(succinimidyl propionate) subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis reveals a major band corresponding to Mr = 150,000. Alternatively, when prelabeled membranes are directly treated with the cross-linker, a major complex of Mr = 51,000 is observed. This may be related to different accessibility of the cross-linker to the site at which the receptor and the Ns protein interact in the two conditions. In conclusion, these data represent initial reports on the successful solubilization of functional VIP-receptor complexes and provide evidence for an interaction between liver VIP-receptor complexes and a GTP-binding protein.     

Autoradiographic localisation of VIP receptors in human lung

Localisation and pharmacological properties of the VIP receptor in human lung sections are described. The receptor density determined by saturation analysis using 125I-VIP is approx. 100 fmol/mg protein, with a Kd of approx. 600 pM. Inhibition of 125I-VIP binding with VIP and related peptides gives a rank order of potency: VIP greater than peptide histidine methionine greater than secretin. Light microscope autoradiography reveals specific VIP binding sites, with a high density over the pulmonary artery smooth muscle and the alveolar walls and with a lower density over the bronchial epithelium.