Specific binding of vasoactive intestinal peptide to human circulating mononuclear cells

1983 ◽  
Vol 61 (7) ◽  
pp. 664-671 ◽  
Author(s):  
C. A. Ottaway ◽  
C. Bernaerts ◽  
B. Chan ◽  
G. R. Greenberg
Keyword(s):  
Vasoactive Intestinal Peptide ◽  
Binding Sites ◽  
Mononuclear Cells ◽  
Specific Binding ◽  
Maximal Effect ◽  
Single Class ◽  
Apparent Dissociation Constant ◽  
Peptide Interactions ◽  
Tracer Dilution ◽  
Dose Dependent

The interaction of the neuropeptide vasoactive intestinal peptide with human circulating mononuclear cells has been studied. Mononuclear cells were able to bind radiolabelled vasoactive intestinal peptide; the binding was rapid, reversible, saturable, and specific for vasoactive intestinal peptide. A fragment of vasoactive intestinal peptide (10–28) was 25-fold less effective than intact peptide as a competitor for the binding of the tracer. Secretin was 100-fold less effective as a competitor and glucagon competed poorly even at concentrations 10 000 times greater than that of the tracer molecule. In tracer dilution studies, the binding suggested a single class of binding sites with an apparent dissociation constant (Kd) of 2.4 × 10−10 M and a capacity of 2 000 sites per cell. In the presence of vasoactive intestinal peptide, there was a dose-dependent augmentation of cyclic AMP in the mononuclear cells. The concentration of vasoactive intestinal peptide which produced a half-maximal effect was the same as the Kd for the peptide binding. We conclude that mononuclear cells have specific high-affinity binding sites for vasoactive intestinal peptide. Interactions between mononuclear cells and vasoactive intestinal peptide may be an important mechanism modulating local immune responses within tissues innervated by vasoactive intestinal peptide containing neurons.

Receptors for vasoactive intestinal peptide and secretin on small intestinal epithelial cells

1980 ◽  
Vol 238 (3) ◽  
pp. G190-G196
Author(s):  
H. J. Binder ◽  
G. F. Lemp ◽  
J. D. Gardner
Keyword(s):  
Small Intestine ◽  
Guinea Pig ◽  
Vasoactive Intestinal Peptide ◽  
Binding Sites ◽  
Prostaglandin E1 ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Maximal Effect ◽  
Single Class

Binding of 125I-labeled vasoactive intestinal peptide (VIP) to dispersed enterocytes prepared from guinea pig small intestine was saturable, temperature dependent, and reversible, and reflected interaction of the labeled peptide with a single class of binding sites. Each enterocyte possessed approximately 60,000 binding sites and binding of the tracer to these sites could be inhibited by VIP [concentration for half-maximal effect (Kd), 12 nM] and by secretin (Kd greater than 1 micro M), but not by glucagon, gastrin, cholecystokinin, calcitonin, bombesin, litorin, physalaemin, substance P, eledoisin, serotonin, carbamylcholine, or histamine. With VIP and secretin, there was a close correlation between the relative potency for inhibition of binding of 125I-VIP and that for increasing cellular cAMP. For a given peptide, however, a 10-fold higher concentration was required for half-maximal inhibition of binding than for half-maximal stimulation of cellular cAMP. In addition to inhibiting binding of 125I-VIP and increasing cellular cAMP in enterocytes, secretin caused an increase in short-circuit current across guinea pig small intestine in vitro. Prostaglandin E1 increased cellular cAMP, but did not alter binding of 125I-VIP and the increase in cAMP caused by prostaglandin E1 plus VIP or secretin was equal to the sum of the increase caused by each agent alone.

FSH and LH up-regulate epidermal growth factor receptors in rat granulosa cells

1994 ◽  
Vol 140 (2) ◽  
pp. 171-177 ◽  
Author(s):  
H Fujinaga ◽  
M Yamoto ◽  
T Shikone ◽  
R Nakano
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Granulosa Cells ◽  
Binding Sites ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Single Class ◽  
Epidermal Growth ◽  
Dose Dependent

Abstract Epidermal growth factor (EGF) modulates ovarian folliculogenesis and steroidogenesis and its binding sites have been demonstrated in the ovary. We investigated the localization of EGF-binding sites in the rat ovary, and the effects of FSH and LH on EGF binding to cultured granulosa cells. Autoradiographic localization of 125I-labelled mouse EGF-binding sites was demonstrated in the granulosa and luteal cells. Displacement study and Scatchard analysis showed that a single class of specific binding sites for 125I-labelled mouse EGF was present in the granulosa cells, obtained from the ovaries of immature rats treated with diethylstilboesterol. The number of binding sites and the apparent dissociation constant were 4336 binding sites/cell and 3·42 pmol/l respectively. The granulosa cells were cultured for 48 h at 37 °C in medium alone or with increasing amounts of ovine FSH (oFSH; 1–1000 μg/l). FSH treatment increased 125I-labelled mouse EGF binding to the granulosa cells in a dose-dependent manner. After culture with oFSH (100 μg/l) for 48 h, the cells were cultured in medium alone or with increasing amounts of ovine LH (oLH; 1–1000 μg/l) for an additional 48 h. LH treatment also increased 125I-labelled mouse EGF binding in a dose-dependent manner, compared with the control. However, neither FSH nor LH altered receptor-binding affinity. Furthermore, after culture with oFSH (FSH-primed) or oFSH followed by oLH (LH-primed), tissue plasminogen activator (tPA) activities in the conditioned media were examined by fibrin autography. FSH-primed or LH-primed granulosa cells were more responsive to EGF action to induce an increase in tPA activity. In conclusion, it is suggested that functional receptors for EGF in rat granulosa cells are up-regulated by FSH and LH. Journal of Endocrinology (1994) 140, 171–177

Direct effect of endothelin-1 on the granulosa cells of the porcine ovary

1992 ◽  
Vol 134 (1) ◽  
pp. 59-66 ◽  
Author(s):  
S. Kamada ◽  
T. Kubota ◽  
Y. Hirata ◽  
M. Taguchi ◽  
S. Eguchi ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Binding Sites ◽  
Inositol Phosphate ◽  
Binding Capacity ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Single Class ◽  
Apparent Dissociation Constant ◽  
Endothelin 1 ◽  
Porcine Granulosa Cells

ABSTRACT Specific binding sites for endothelin-1 (ET-1), a novel potent vasoconstrictor peptide, as well as the effects of ET-1 on cytosolic free Ca2+ concentration ([Ca2+]i), intracellular total inositol phosphate (IP) generation and steroidogenesis were studied in cultured porcine granulosa cells. Scatchard analysis of a binding study using 125I-labelled ET-1 indicated the presence of a single class of high-affinity binding sites with almost equal affinity for ET-1 and ET-3: the apparent dissociation constant was 0·59 nmol/l and the maximal binding capacity was 1·84 pmol/mg protein. Affinitylabelling of 125I-labelled ET-1 to the membranes using disuccinimidyl tartarate as a cross-linker revealed one major and one minor band with the apparent molecular weights of 32 kDa and 49 kDa respectively. ET-1 dose-dependently (1−100 nmol/l) induced rapid and transient increases in [Ca2+]i in fura-2-labelled cells. ET-1 also dose-dependently stimulated total IPs in cells prelabelled with myo-[3H]inositol. ET-1 had a slight stimulatory effect on the secretion of progesterone but not of oestradiol from porcine granulosa cells. The present data clearly demonstrate the presence of a non-selective ET receptor (ETB) in porcine granulosa cells coupled with phosphoinositide hydrolysis and [Ca2+]i mobilization, and suggest that ET-1 may play some role in the production of progesterone by porcine granulosa cells. Journal of Endocrinology (1992) 134, 59–66

Dopamine1 receptors in rat kidneys identified with 125I-Sch 23982

1988 ◽  
Vol 255 (5) ◽  
pp. F970-F976 ◽  
Author(s):  
R. A. Felder ◽  
P. A. Jose
Keyword(s):  
Room Temperature ◽  
Specific Binding ◽  
Rat Kidney ◽  
Sch 23390 ◽  
Receptor Density ◽  
Single Class ◽  
Apparent Dissociation Constant ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Inhibition Constants

Dopamine1 receptors were studied in rat kidney using the selective dopamine1 antagonist 125I-labeled Sch 23982. The specific binding of 125I-Sch 23982 (defined by 5 microM Sch 23390) to renal cortical homogenates incubated at room temperature was rapid, saturable with time and ligand concentration, and reversible. Analysis of Rosenthal plots revealed a single class of receptors with an apparent dissociation constant of 12.2 +/- 1.9 nM and maximum receptor density of 1.03 +/- 0.15 pmol/mg protein (n = 6). However, competition experiments with the dopamine1 antagonist Sch 23390 revealed a low- and high-affinity binding site with inhibition constants of 1 x 10(-6) and 1 x 10(-8) M, respectively. The competition experiments were also indicative of dopamine1 receptors with stereoselectivity noted for dopamine1 but not for dopamine2 antagonists. The inhibition constants for dopamine1 antagonists and agonists were two orders of magnitude greater in renal cortical than striatal homogenates. Different buffers affected striatal but not renal cortical binding. Autoradiographic studies revealed 125I-Sch 23982 binding in renal cortical but not medullary tissue. These studies confirm the presence of dopamine1 receptors in the cortex of the rat kidney.

Cholecystokinin downregulates receptors for vasoactive intestinal peptide and secretin in rat pancreatic acini

1990 ◽  
Vol 258 (3) ◽  
pp. G395-G403
Author(s):  
S. Katsushima ◽  
H. Adachi ◽  
T. Honda ◽  
S. Sato ◽  
T. Kusui ◽  
...  
Keyword(s):  
Vasoactive Intestinal Peptide ◽  
Binding Sites ◽  
Specific Binding ◽  
Amylase Secretion ◽  
Affinity Binding ◽  
Pancreatic Acini ◽  
Cck Receptors ◽  
Cyclic Monophosphate ◽  
Apparent Decrease ◽  
Specific Antagonist

We examined the effect of cholecystokinin (CCK) on the receptors for vasoactive intestinal peptide (VIP) and secretin in rat pancreatic acini. CCK decreased the specific binding of 125I-VIP and 125I-secretin by 42 and 51%, respectively. This CCK-induced inhibition was caused by an apparent decrease in the capacity of high-affinity binding sites of VIP and secretin receptors. CR 1409, a specific antagonist of CCK, abolished CCK-induced binding inhibition, whereas 12-O-tetradecanoylphorbol-13-acetate, A23187, and cycloheximide did not affect the binding of the radioligands. Both N2,O2-dibutyryl guanosine 3',5'-cyclic monophosphate (Bt2cGMP) and nitroprusside inhibited the specific binding of 125I-VIP. This inhibition, however, was because of an apparent decrease in the capacity of low-affinity binding sites on VIP receptors. CCK-induced downregulation of VIP and secretin receptors was associated with the diminished acinar response to VIP or secretin-induced adenosine 3',5'-cyclic monophosphate accumulation and amylase secretion, whereas neither Bt2cGMP nor nitroprusside affected VIP-induced amylase secretion. Data suggest that CCK-induced downregulation is mediated by the initial interaction of CCK with CCK receptors followed by some postreceptor process, which appears unrelated to protein kinase C, calcium mobilization, decrease in protein synthesis, or cellular cGMP increases. This downregulation, at least in part, accounts for CCK-induced restricted stimulation of amylase secretion by VIP and secretin.

Corticosterone's metabolite is an agonist for Na+ transport stimulation in A6 cells

1988 ◽  
Vol 255 (4) ◽  
pp. F736-F748 ◽  
Author(s):  
R. L. Duncan ◽  
W. M. Grogan ◽  
L. B. Kramer ◽  
C. O. Watlington
Keyword(s):  
Binding Sites ◽  
Short Circuit ◽  
Type I ◽  
Type Ii ◽  
Na Transport ◽  
Single Class ◽  
Apparent Dissociation Constant ◽  
Lower Affinity ◽  
A6 Cells ◽  
Nuclear Binding

This study tests the hypothesis, in A6 epithelia, that 1) corticosterone stimulates active Na+ transport (short-circuit current, Isc) by an additional receptor mechanism to the type I (mineralocorticoid) and type II (glucocorticoid) mechanisms shared with aldosterone (Aldo) and 2) that the agonist may be 6 beta-OH-corticosterone made in the effector cell. The dose-response relationship of corticosterone at 24 h resolves into two components, by curve fitting, with a 50% effective concentration (EC50) for 10% of maximum Isc stimulation of 2 X 10(-9) M and an EC50 for the other 90% of 3 X 10(-7) M. The EC50 of the smaller component correlates with the apparent dissociation constant (K'd) of corticosterone for high affinity (type II) nuclear binding sites shared with Aldo. In unlabeled analogue competition studies Aldo and corticosterone displaced nuclear binding equally below 10(-8) M [3H]corticosterone, indicating only shared sites. However, nonshared saturable sites (displaced by corticosterone but not by Aldo) were found at [3H]-corticosterone concentrations above 10(-8) M. Concentration-binding curves performed with [3H]corticosterone, in presence of 1,000 X Aldo to displace shared sites, revealed a single class of binding sites with a half-maximal saturation of 2 X 10(-7) M, which is quite similar to the EC50 of the lower affinity component of Isc stimulation by corticosterone at 24 h. Reversed phase high-pressure liquid chromatography of nuclear extracts indicates that the saturable component of bound [3H] was 6 beta-OH-[3H]corticosterone derived from [3H]corticosterone. Thus, A6 cells metabolize corticosterone to 6 beta-OH-corticosterone, which in turn occupies lower-affinity receptors not shared with Aldo or corticosterone, to mediate most of the active Na+ transport stimulation by corticosterone.

Dissociation of contraction and muscarinic receptor binding to isolated smooth muscle cells

1986 ◽  
Vol 251 (4) ◽  
pp. G546-G552 ◽  
Author(s):  
S. M. Collins ◽  
D. J. Crankshaw
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Binding Sites ◽  
Specific Binding ◽  
Muscle Cells ◽  
Muscarinic Agonists ◽  
Isolated Cells ◽  
Single Class ◽  
Antagonist Binding ◽  
Isolated Smooth Muscle Cells

We examined changes in [3H]QNB binding and cell length induced by muscarinic ligands in a suspension of single smooth muscle cells isolated from the canine stomach. Cells contracted following a brief (30 s) exposure to picomolar concentrations of muscarinic agonists and yielded ED50 values of 1.0 +/- 0.7 pM for oxotremorine, 12.5 +/- 1.8 pM for carbachol, and 16.0 +/- 2.9 pM for metacholine. Contraction was inhibited by atropine with a pA2 value of 10.2 +/- 1.1. The binding of [3H]QNB was rapid and reversible and was stereospecific and pharmacologically appropriate. Specific binding of [3H]QNB was saturable and bound with high affinity (KD 1.04 +/- 0.23 nM) to a single class of sites, of which there were approximately 200,000/cell. In competition experiments antagonist binding was generally homogeneous, whereas that of agonists was heterogeneous and subpopulations of binding sites with different affinities for agonists were identified. The Ki value of 8.1 +/- 1.1 nM for inhibition of QNB binding by atropine was greater than the pA2 of 10.2 +/- 1.1 derived from contraction studies. Furthermore, whereas picomolar concentrations of agonists induced cell contraction, substantially higher concentrations (10 nM to 10 mM) were required to inhibit [3H]QNB binding to the isolated cells.

Evidence for a direct action of GH on haemopoietic cells of a marine fish, the gilthead sea bream (Sparus aurata)

1995 ◽  
Vol 146 (3) ◽  
pp. 459-467 ◽  
Author(s):  
J A Calduch-Giner ◽  
A Sitjà-Bobadilla ◽  
P Álvarez-Pellitero ◽  
J Pérez-Sánchez
Keyword(s):  
Binding Sites ◽  
Sparus Aurata ◽  
Binding Capacity ◽  
Specific Binding ◽  
Head Kidney ◽  
Gilthead Sea Bream ◽  
Sea Bream ◽  
Single Class ◽  
Lower Vertebrate

Abstract Receptors for GH were characterized in the head kidney of gilthead sea bream (Sparus aurata), using radioiodinated and biotinylated ligands. The specific binding of radiolabelled recombinant gilthead sea bream GH (rsbGH) to head kidney membrane preparations was dependent on membrane concentration. Salmon prolactin, salmon gonadotrophin and carp gonadotrophin did not compete for 125I-labelled rsbGH-binding sites. Unlabelled rsbGH competitively displaced 125I-labelled rsbGH bound to head kidney membranes. Scatchard plots were always linear, denoting the presence of a single class of binding sites. The binding affinity (Ka=2·7 × 109 m−1) was equivalent to that found in liver membrane preparations, but the binding capacity (2·5 ±0·30 fmol/mg protein) was 50- to 75-fold lower. To identify the cells which express the GH receptor, head kidney smears were incubated with biotinylated rsbGH, followed by incubation with an avidin–biotin complex conjugated to alkaline phosphatase. The reaction with the new-fuchsin substrate gave a red precipitate, showing a specific and intense labelling in erythroblasts, polychromatophilic erythroblasts and myeloblasts. Noticeable binding was observed in myelocytes and immature granulocytes, tending to disappear at the latter stages of granulocyte maturation. Light but appreciable binding was also observed in monocytes, lymphocytes and acidophilic erythroblasts, whereas it was completely absent in proerythrocytes and erythrocytes. The proliferative action of rsbGH and recombinant human IGF-I on in vitro cultures of head kidney cells was demonstrated by a 5-bromo-2′-deoxy-uridine immunoassay. To our knowledge, this is the first report that provides suitable evidence for a role of GH as a haemopoietic growth and differentiation factor in lower vertebrate species. Journal of Endocrinology (1995) 146, 459–467

Atrial natriuretic peptide in the eel, Anguilla anguilla L.: its cardiac distribution, receptors and actions on isolated branchial cells

1992 ◽  
Vol 9 (2) ◽  
pp. 103-114 ◽  
Author(s):  
C. L. Broadhead ◽  
U. T. O'Sullivan ◽  
C. F. Deacon ◽  
I. W. Henderson
Keyword(s):  
Sodium Chloride ◽  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Binding Sites ◽  
Specific Binding ◽  
Anguilla Anguilla ◽  
Chloride Cells ◽  
Dependent Manner ◽  
Single Class ◽  
Atrial Natriuretic

ABSTRACT The presence of atrial natriuretic peptide (ANP) and the nature of its binding sites were studied in freshwater (FW)- and seawater (SW)-adapted eels using a heterologous analogue, that of the rat (rANP). Rat ANP-like immunoreactivity was demonstrated in the cardiac atria and ventricles of both FW and SW eels, and electron-dense ANP-like granules were observed. The atria and ventricles of FW eels contained significantly more granules than those of SW animals and, in both types, the atria were more granular than the ventricles. Specific binding sites for rANP were demonstrated by displacement and uptake experiments using labelled rANP in dispersed eel branchial cell preparations, enriched in chloride cells. The concentration of rANP required to produce a 50% inhibition of binding in FW cells was significantly lower than that in SW cells. Scatchard analyses revealed the presence of two classes of binding site in SW eel branchial cells but only a single class of receptor in FW cells. The affinity of the FW receptor was not significantly different from that of the SW high affinity site. Rat ANP stimulated the production of cyclic GMP (cGMP) in a dose-dependent manner, and both basal and stimulated levels of cGMP were significantly greater in SW branchial cells. These studies suggest that ANP is involved in the adaptation of the euryhaline eel to differing environmental salinities; the levels of the peptide in the heart alter with changing salinity, and the nature of the receptors in the sodium chloride-transporting epithelium of the gill changes in response to the need either to eliminate or to absorb sodium chloride.

Prostaglandin E2 binding sites in porcine oxyntic mucosa: effects of salicylates

1986 ◽  
Vol 64 (5) ◽  
pp. 515-520 ◽  
Author(s):  
B. L. Tepperman ◽  
B. D. Soper
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Prostaglandin E ◽  
Affinity Binding ◽  
Variable Degree ◽  
Single Class ◽  
Oxyntic Mucosa ◽  
High Affinity Binding ◽  
Mucosal Ulceration ◽  
High Affinity

These studies were designed to examine the changes in the characteristics of prostaglandin E2 (PGE2) binding to porcine oxyntic mucosa in the response to oral ingestion of salicylates. Either acetylsalicylic acid (ASA) or salicylic acid (SA) was administered to conscious pigs (100 mg/kg in 30 mL of an equimolar concentration of NaHCO3) once a day for 1, 3, 10, or 20 days. In control experiments a similar volume of 0.3 M NaHCO3 was administered for similar durations. Mucosal ulceration and the characteristics of the binding of [3H]PGE2 to a 30 000 × g membrane preparation of oxyntic mucosa were examined. Generation of mucosal PGE2 was measured by radioimmunoassay. ASA treatment resulted in an increase in the number and severity of mucosal ulcers and a decrease in PGE2 levels within the first treatment day. By day 20 the degree of ulceration had decreased in spite of a persistent reduction of mucosal PGE2 generation. A variable degree of ulceration was observed in SA-treated animals. In control animals only a single class of binding sites for [3H]PGE2 was evident. After 3 days of ASA treatment a second class of binding sites with a high affinity dissociation constant appeared. There was a decrease in the high affinity binding of [3H]PGE2 after 20 days of ASA ingestion. Low affinity binding was not altered. ASA treatment resulted in a significant increase in specific binding capacities for both families of binding sites. SA treatment did not consistently alter PGE2 binding characteristics from control at any time period studied. These data suggest that SA treatment results in a small degree of mucosal damage in the absence of a significant reduction in tissue generation of PGE2 or changes in PGE2 binding. Damage in response to ASA ingestion was associated with a reduction in both endogenous synthesis of PGE2 and an increase in the concentration of both low and high affinity binding sites for PGE2. The reduction in mucosal ulceration on day 20 in spite of depressed endogenous PGE2 coincides with an increase in PGE2 binding.

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