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.     

Solubilization and functional reconstitution of human neuropeptide FF2 receptors

Neuropeptide FF (NPFF, FLFQPQRFamide) receptors modulate endogenous opioid functions. Here, we report the solubilization of the human NPFF₂ receptor expressed in Chinese hamster ovary (CHO) cells by the zwitterionic detergent Chaps. Chaps solubilization resulted in the abolishment of specific agonist binding activity, which was restored by a polyethylene glycol (PEG) precipitation method. Reincorporation after the precipitation step into liposomes made of endogenous lipids issued from CHO membranes or exogenous lipids significantly enhanced the specific agonist binding activity and G-protein coupling. This method of solubilization and lipid reconstitution could be useful for studies of NPFF receptors.     

Peptide binding to intestinal epithelium: distinct sites for insulin, EGF and VIP

Several peptides, including insulin, epidermal growth factor and vasoactive intestinal polypeptide bind to intestinal epithelial cells. However, it is unclear whether one binding site binds several peptides or whether separate sites exist for each peptide. These studies were designed to examine the specificity of peptide binding sites on intestinal epithelial cells. Peptide binding was measured directly with [125I]radiolabelled peptides to isolated enterocytes prepared from rabbit ileum. The characteristics of insulin and epidermal growth factor binding were similar. Both insulin and epidermal growth factor specific binding was saturable, directly correlated to cell concentration and temperature and pH dependent. The total number of insulin binding sites per cell was 4500, that for epidermal growth factor was 2280. Scatchard analysis for both peptides produced curvilinear plots. Dissociation of both peptides from the binding site was increased in the presence of their respective unlabelled peptide. However, insulin specific binding was not altered by epidermal growth factor, and epidermal growth factor specific binding was unaffected by insulin. Further, both insulin and epidermal growth factor failed to inhibit the specific binding of vasoactive intestinal polypeptide to ileal enterocytes, and vasoactive intestinal polypeptide did not inhibit insulin or epidermal growth factor specific binding. These studies demonstrate that insulin, epidermal growth factor and vasoactive intestinal polypeptide interact with three distinct membrane binding sites on the enterocyte.     

The binding characteristics of cholinergic sites in rabbit spermatozoa

Binding of neurotrophic ligands to rabbit spermatozoa was studied. Nicotinic cholinergic antagonists, [3H]alpha-bungarotoxin and [3H]dihydro-beta-erythroidine (DE), bound with high affinity to different sites in the tails of rabbit spermatozoa with the former binding to 10,207 sites/cell and the latter to 562 sites/cell. alpha-Bungarotoxin and DE sites resemble nicotinic sites in brain in binding affinity and specificity. [3H]Quinuclidinyl benzilate (QNB), a muscarinic cholinergic antagonist, also bound with high affinity to a single class of sites located in the heads and tails of rabbit spermatozoa. The binding characteristics of the sperm muscarinic site are similar to muscarinic sites in both innervated and noninnervated cells. Rabbit spermatozoa incubated for 16-18 h in a medium which supported motility for an extended period possessed fewer binding sites than nonincubated spermatozoa for [3H] alpha-bungarotoxin and [3H]QNB and the KD for the latter ligand was also lower. Ligands specific for the kappa and delta opiate receptors showed no affinity for rabbit spermatozoa.     

Pivotal role of cAMP in the activation of liver glycogen breakdown in high-fat diet fed mice

Aims

Liver glycogen catabolism was evaluated in male Swiss mice fed a high-fat diet rich in saturated fatty acids (HFD) or normal fat diet (NFD) during one week.

Main methods

Liver glycogenolysis (LG) and liver glucose production (LGP) were measured either under basal or stimulated conditions (infusion of glycogenolytic agents). Thus, isolated perfused livers from HFD and NFD mice were infused with glycogenolytic agents, i.e., glucagon, epinephrine, phenylephrine, isoproterenol, adenosine-3′-5′-cyclic monophosphate (cAMP), N⁶,2′-O-dibutyryl-cAMP (DB-cAMP), 8-bromoadenosine-cAMP (8-Br-cAMP) or N⁶-monobutyryl-cAMP (N6-MB-cAMP). Moreover, glycemia and liver glycogen content were measured.

Key findings

Glycemia, liver glycogen content and basal rate of LGP and LG were not influenced by the HFD. However, LGP and LG were lower (p < 0.05) in HFD mice during the infusions of glucagon (1 nM), epinephrine (20 μM) or phenylephrine (20 μM). In contrast, the activation of LGP and LG during the infusion of isoproterenol (20 μM) was not different (HFD vs. NFD). Because glucagon showed the most prominent response, the effect of cAMP, its intracellular mediator, on LGP and LG was investigated. cAMP (150 μM) showed lower activation of LGP and LG in the HFD group. However, the activation of LGP and LG was not influenced by HFD whether DB-cAMP (3 μM), 8-Br-cAMP (3 μM) or N6-MB-cAMP (3 μM) were used.

Significance

The activation of LGP and LG depends on the intracellular availability of cAMP. It can be concluded that cAMP played a pivotal role on the activation of LG in high-fat diet fed mice.     

Distribution and characterization of motilin receptors in the cat

We demonstrate binding of [¹²⁵I][Nle¹³-po]motilin to homogenates of cat gastric and small intestinal, but not to colonic smooth muscle tissue. The density was (B_max in fmol/mg protein): 0 (fundus); 12 ± 2 (corpus); 22 ± 3 (antrum); 55 ± 12 (duodenum); 44 ± 10 (jejunum); 17 ± 1 (ileum); 0 (colon). A significant (p < 0.05) difference was found between the dissociation constant for motilin in the stomach (pK_d = 8.84 ± 0.06) and in the small intestine (pK_d = 8.58 ± 0.08). The motilides erythromycin-A (EM-A), EM-523, and EM-A N-oxide displaced labeled [Nle¹³-po]motilin bound to cat duodenal receptor with potencies (pK_d) of 5.47 ± 0.23, 7.60 ± 0.24, and < 4.3, respectively. Studies with [Leu¹³-po]motilin fragments showed that the N-terminus of motilin interacts with the receptor. In the tissue bath, duodenal strips mounted in the longitudinal direction responded to motilin, EM-523, and EM-A (pEC₅₀: 8.29 ± 0.08; 7.12 ± 0.12; 5.99 ± 0.15). The compounds had a comparable intrinsic activity (83 ± 3%; 80 ± 5%; 82 ± 5% of the response to ACh), which was unaffected by atropine, TTX, hexamethonium, and zacopride but reduced by verapamil and calcium-free medium. Cat stomach and small intestine possess smooth muscle motilin receptors, which have comparable properties as those found in man and in rabbit.     

Sequence analysis,structure and binding site prediction of Sigma 1 receptor protein by in silico method

Sigma 1 Receptor is a subtype of opioid receptor that participates in membrane remodeling and cellular differentiation in thenervous system. Sigma1 Receptor protein with amino acid length ranging from 229 is widely distributed in the liver andmoderately in the intestine, kidney, white pulp of the spleen, adrenal gland, brain, placenta and the lung. In this study, the threedimensional structure for sigma 1 receptor protein has been developed by in- silico analysis based on evolutionary trace analysis of37 sigma proteins from different sources. The present work focus on identification of functionally important residues and itsinteraction with antipsychotic drugs reported in literature.     

VIP receptors in mesenteric and coronary arteries: a radioligand binding study

Using a biologically active radioligand, [Tyr(125I)10]VIP, we have identified and characterized receptors for vasoactive intestinal peptide (VIP) on membranes prepared from the rat superior mesenteric artery and bovine coronary arteries. Binding was specific, saturable, reversible and dependent on time and temperature. Scatchard analysis suggested the presence of a high and a low affinity binding site in each arterial system with the following binding constants: the rat mesenteric artery, KD = 0.22 +/- 0.02 and 13.6 +/- 7.8 nM (corresponding maximum number of binding sites, RO = 606 +/- 44 fmol/mg protein and 2.1 +/- 0.2 pmol/mg protein); bovine circumflex coronary artery, KD = 0.10 +/- 0.01 and 37.8 +/- 16.1 nM (corresponding RO = 369 +/- 65 fmol/mg protein and 2.0 +/- 0.7 pmol/mg protein); bovine left and right descending coronary arteries, KD = 0.12 +/- 0.03 and 21.3 +/- 6.4 nM (corresponding RO = 472 +/- 7 fmol/mg protein and 2.2 +/- 0.3 pmol/mg protein). The arterial VIP receptors did not recognize secretin, glucagon, apamin or bovine parathyroid hormone, and had reduced affinity for PHI, PHM and growth hormone releasing factors (GRF). These recognition properties were, by and large, similar to those seen in the bovine cerebral arteries although a between-species heterogeneity of recognition function could be deduced from the differences in the competitive binding of rat and bovine vascular VIP receptors with the corresponding species-specific GRFs.     

Endocytosis and degradation of serglycin in liver sinusoidal endothelial cells

We have previously reported that liver sinusoidal endothelial cells (LSECs) are responsible for the clearance of monocyte chondroitin sulfate proteoglycan serglycin from the circulation (øynebråten et al.(2000) J. Leukocyte Biol. 67; 183–188). The aim of the present study was to investigate the kinetics of degradation of endocytosed serglycin in primary cultures of LSECs. The final degradation products of serglycin labelled biosynthetically in the glycosaminoglycan (GAG) chains with [³H] in the acetyl groups of N-acetyl galactosamine residues, [¹⁴C] in the pyranose rings, or [³⁵S] in the sulfate groups were identified as[³H]-acetate, [¹⁴C]-lactate and [³⁵S]-sulfate. Comparison of the rate of release of degradation products from the cells after endocytosis of serglycin labelled chemically with ¹²⁵I in the tyrosine residues, or biosynthetically with [³⁵S] or [³H] in the sulfate or acetyl groups, respectively, showed that ¹²⁵I appeared more rapidly in the medium than [³⁵S]-sulfate and [³H]-acetate. Judging from the speed of appearance of free ¹²⁵I both intracellularly and in the medium, the core protein is degraded considerably more rapidly than the GAG chains.Desulfation of the GAG chains starts after the GAG chains are released from the core protein. Generation of lactate and acetate as the final products from degradation of the carbon skeleton of the GAG chains indicates that catabolism of endocytosed macromolecules in LSECs proceeds anaerobically.     

Postnatal development of somatostatin levels and its binding sites in rabbit gastric mucosa

Using a specific radioimmunoassay technique, we have determined somatostatin-like immunoreactivity (SLI) in acid extracts of gastric (fundic and antral) mucosa as well as the specific binding of ¹²⁵I-Tyr¹¹-somatostatin to cytosol of the stomach of 0 to 150 days postnatal rabbits. The levels of somatostatin in both fundus and antrum decreased from birth up to day 5 followed by a sharp increase from 5 to 10 days, then decreased progressively until day 35. After this age, the somatostatin concentration remained relatively stable. The number of specific somatostatin binding sites of both high- and low-affinity increased gradually (without changes in the affinity values) with the development of rabbits, reaching the adult level by 35 days. However, there was an apparent lack of high-affinity sites immediately after birth (day 0). The somatostatin binding sites had characteristics identical with those found in adult animals with regard to their respective specific ligands.     

A high affinity binding site for cytokinin to a particulate fraction in carrot suspension cells

Summary Carrot suspension cells contain one class of high affinity binding sites for cytokinin in an 80,000 × g particulate fraction. Binding of [8-¹⁴C]-benzylaminopurine (BA) to this fraction assayed by a sedimentation method was found to be optimal at pH 6.0 and thermolabile. Specific binding was proved in competition experiments in which labelled BA was displaced by increasing concentrations of unlabelled BA. Scatchard plots of these results displayed a dissociation constant (K_d) of 33 ± 6 nM. The number of binding sites found was 1,100 ± 120 fmol g^–1 fresh weight which is equivalent to a frequency of 23,000 binding sites per cell. The specificity of the binding sites to cytokinins and their analogues followed the sequence BA with highest affinity, kinetin, zeatin, iP and adenine. The cytokinin ribosides generally had a lower affinity than their cytokinin bases, and the affinity decreased in the order [9 R] BA, [9 R] iP, [9 R] Z, [9 R] A.     

The lysosomal degradation of neuromedin B is dependent on tripeptidyl peptidase-I: evidence for the impairment of neuropeptide degradation in late-infantile neuronal ceroid lipofuscinosis

Late-infantile neuronal ceroid lipofuscinosis (CLN2), previously known as the late-infantile form of Batten disease, is a lysosomal storage disease which results from mutations in the gene that codes for tripeptidyl peptidase-I (TPP-I). This disease is characterised by progressive neurodegeneration in young children although the molecular mechanisms responsible for neuronal cell death are unclear. TPP-I is an exopeptidase which removes N-terminal tripeptides from small peptides, including several peptide hormones. We report that the degradation of the neuropeptide, neuromedin B, by mouse brain cells is restricted to lysosomes and that the pattern of degradation products is consistent with a predominant role for TPP-I. Neuromedin B is degraded by a similar pathway in a mouse neuronal cell line and also in cultured human fibroblasts. A specific inhibitor of TPP-I is able to abolish neuromedin B degradation in a variety of cell types. Fibroblasts from CLN2 patients, which are deficient in TPP-I activity, are unable to degrade neuromedin B. These observations suggest that TPP-I is the predominant proteolytic enzyme responsible for the intracellular degradation of neuromedin B. The inability of cells from CLN2 patients to degrade neuromedin B and other neuropeptides may contribute to the pathogenesis of the disease.     

Autoradiographic localization of specific atrial natriuretic peptide binding sites on immunocytochemically identified cells in cultures from rat and guinea-pig hearts

Summary Dissociated cell culture preparations from rat and guinea-pig atria and interatrial septum, and from rat ventricles were studied using a combined autoradiographic and immunocytochemical approach. Alphaatrial natriuretic peptide (¹²⁵I-ANP_1-128) binding sites were confined to subpopulations of identified non-neuronal cells in each type of culture preparation, and had distinct patterns of labelling. The density of ANP_1-28 binding sites was substantially greater in guinea-pig cultures than in rat cultures and was least in rat ventricular cultures. ANP_1-28-labelled subpopulations of S-100-like immunoreactive glial cells were only seen in guinea-pig cultures. Von Willebrand factor (vWF)-like immunoreactive endothelial cells and vWF-negative endothelioid cells expressed ANP_1-28 binding sites in both the guinea-pig and rat atrial cultures, but were unlabelled in rat ventricular cultures. In contrast, labelled subpopulations of fibronectin-like immunoreactive fibroblasts were present in all of the three types of culture preparation studied. ANP-like immunoreactive myocytes were present in both atrial and ventricular cultures. These cells did not, however, express ANP_1-28 binding sites.     

Atrial and brain natriuretic peptides share binding sites on cultured cells from the rat trachea

Summary We examined the distribution of binding sites for alpha-atrial natriuretic peptide (¹²⁵I-ANP_1–28) and the recently discovered porcine brain natriuretic peptide (¹²⁵I-pBNP) on immunocytochemically identified cells in dissociated culture preparations of the rat trachea. Specific binding sites for both ¹²⁵I-ANP_1–28 and ¹²⁵I-pBNP were evenly distributed over distinet subpopulations of smooth muscle myosin-like immunoreactive muscle cells, fibronectin-like immunoreactive fibroblasts and S-100-like immunoreactive glial cells. Neither keratin-like immunoreactive epithelial cells nor protein gene product 9.5-like immunoreactive paratracheal neurones expressed natriuretic peptide binding sites, although autoradiographically labelled glial cells were seen in close association with both neuronal cell bodies and neurites. The binding of each radiolabelled peptide was abolished by the inclusion of either excess (1 M) unlabelled rat ANP or excess unlabelled porcine BNP, suggesting that ANP and BNP share binding sites in the trachea. Furthermore, the ring-deleted analogue, Des-[Gln¹⁸, Ser¹⁹, Gly²⁰, Leu²¹, Gly²²]-ANF_4–23-NH₂, strongly competed for specific ¹²⁵I-ANP_1–28 and ¹²⁵I-pBNP binding sites in the tracheal cultures; this suggests that virtually all binding sites were of the clearance (ANP-C or ANF-R₂) receptor subtype.     

Specific binding sites for corticosterone in isolated cells and plasma membrane from rat liver

Summary The specific binding of [³H]corticosterone to hepatocytes is a nonsaturable, reversible and temperature-dependent process. The binding to liver purified plasma membrane fraction is also specific, reversible and temperature dependent but it is saturable. Two types of independent and equivalent binding sites have been determined from hepatocytes. One of them has high affinity and low binding capacity (K _D=8.8nm andB _max=1477 fmol/mg protein) and the other one has low affinity and high binding capacity (K _D=91nm andB _max=9015 fmol/mg). In plasma membrane only one type of binding site has been characterized (K _D=11.2nm andB _max=1982 fmol/mg). As it can be deduced from displacement data obtained in hepatocytes and plasma membrane the high affinity binding sites are different from the glucocorticoid, progesterone nuclear receptors and the Na⁺,K⁺-ATPase digitalis receptor. Probably it is of the same nature that the one determinate for [³H]cortisol and [³H]corticosterone in mouse liver plasma membrane. Beta-and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [³H]corticosterone binding to hepatocytes and plasma membranes; therefore, these binding sites are independent of adrenergic receptors. The binding sites in hepatocytes and plasma membranes are not exclusive for corticosterone but other steroids are also bound with very different affinities.     

Intracellular transport and degradation of I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

In vivo covalent binding of aflatoxin B1 and aflatoxin M1 to liver DNA of rat,mouse and pig

[¹⁴C]Aflatoxin B₁ (AFB₁) was isolated from cultures of Aspergillus parasiticus grown on [1-¹⁴C]sodium acetate. Covalent binding of AFB₁ to liver DNA of rat and mouse was determined 6–8 h after oral administration. The effectiveness of covalent binding, expressed as DNA binding per dose in the units of a ‘Covalent Binding Index’ (CBI), (μmol aflatoxin/mol DNA nucleotides)/(mmol aflatoxin/kg animal), was found to be 10 400 for rats and 240 for mice. These CBI partly explain the different susceptibility of the two species for the incidence of hepatic tumors.The corresponding values for pig liver DNA, 24 and 48 h after oral administration, were found to be as high as 19 100 and 13 300. DNA-binding has not so far been reported for this species although it could represent an appropriate animal model for studies where a human-like gastrointestinal tract physiology is desirable.Aflatoxin M₁ (AFM₁) is a metabolite found in the milk of cows that have been fed AFB₁-contaminated diet. [¹⁴C]AFM₁ was also found to be produced by cultures of A. parasiticus giving a yield of about 0.3% of the total aflatoxins. A test for covalent binding to rat liver DNA revealed a CBI of 2100 showing that AFM₁ must also be regarded as a strong hepatocarcinogen. It is concluded that AFB₁ contaminations should be avoided in dairy feed.     

Neuropeptide profiling of the bovine hypothalamus: thermal stabilization is an effective tool in inhibiting post-mortem degradation

The hypothalamus is the central regulatory region of the brain that links the nervous system to the endocrine system via the pituitary gland. It synthesizes and secretes neuropeptide hormones, which in turn act to stimulate or inhibit the secretion of pituitary hormones. We have undertaken a detailed MS investigation of the peptides present in the bovine hypothalamus by adapting a novel heat stabilization methodology, which improved peptide discovery to direct our studies into the molecular mechanisms involved in bovine reproduction. The untreated samples contained large numbers of protein degradation products that interfered with the analysis of the neuropeptides. In the thermally stabilized samples, we were able to identify many more neuropeptides that are known to be expressed in the bovine hypothalamus. Furthermore, we have characterized a range of post-translational modifications that indicate the presence of processed intact mature neuropeptides in the stabilized tissue samples, whereas we detected many trimmed or truncated peptides resulting from post-mortem degradation in the untreated tissue samples. Altogether, using an optimized workflow, we were able to identify 140 candidate neuropeptides. We also nominate six new candidate neuropeptides derived from proSAAS, secretogranin-2 and proTRH.     

Evidence for the role of glycoprotein G of respiratory syncytial virus in binding of Neisseria meningitidis to HEp-2 cells

Abstract Viral glycoproteins G and F are expressed on the surface of cells infected with respiratory syncytial virus (RSV). We investigated the role of these proteins in the previously reported enhanced binding of Neisseria meningitidis to RSV-infected HEp-2 cells. Virus particles attached to bacteria were detected by immunofluorescence with flow cytometry. Binding of FITC-labelled bacteria to RSV-infected cells was significantly inhibited by monoclonal antibody against glycoprotein G. Unlabelled bacteria interfered with binding of the anti-G monoclonal antibody to these cells. These interactions were not found with a monoclonal antibody against glycoprotein F. We propose that glycoprotein G of RSV expressed on the surface of infected cells might act as an additional receptor for meningococci.     

Distribution of mRNA and binding sites of adrenoceptors and muscarinic receptors in the rat heart

Since there exist some obscurities in the expression of mRNAs and their receptors in the heart, we have investigated the gene expression (mRNA levels) of adrenoceptors (alpha1A-, alpha1B-, beta1-, beta2-, beta3-) and muscarinic receptors (M2) and the density of receptor binding sites (alpha1A-, alpha1B-, beta1-, beta2-adrenoceptors, muscarinic receptors). Moreover, the heart regions consist of tissue rich in ganglion cells (that are of importance in heart neural circuits) and those virtually free of them (myocytes). Therefore, we have examined the differences in the distribution of mRNAs/receptor binding sites in the atrial samples of the heart rich in ganglion cells vs. those are virtually free of them. Binding sites and mRNAs of muscarinic receptors and alpha1B-adrenoceptors differ in their distribution in different heart regions. The mRNAs for beta1- and beta2-adrenoceptors were almost equally distributed herein, while the amount of beta-adrenoceptors significantly differs in the heart regions. The alpha1A- and beta3-adrenoceptors mRNAs were also found in all investigated heart regions, but at significantly lower level and have not shown region differences. This is a new finding, especially to beta3-adrenoceptors, as they were not regularly found in each heart regions. alpha1B-adrenoceptors have similar distribution of their mRNAs and binding sites in some heart parts. Thus, we can conclude that there are noticeable differences in the presence of receptors in heart regions that contain ganglion cells in comparison to those are virtually free of them.