Gastrin-CCK-B type receptors on human T lymphoblastoid Jurkat cells

The presence of specific receptors for gastrointestinal hormones on T cells and their involvement in the immune response are still matters of debate. We reported the effects of gastrin-cholecystokinin (CCK)-related peptides on J.RT3-T3.5 Jurkat cells. A single class of high-affinity binding sites (dissociation constant approximately 0.1 nM) for gastrin and CCK-8 was evident on these cells. These peptides dose-dependently induced a transient increase in intracellular Ca2+ concentration ([Ca2+]i), which was independent of extracellular Ca2-. L-365,260 was 150- to 300-fold more potent than L-364,718 to inhibit radiolabeled ligand binding or peptide-stimulated [Ca2+]i increase, confirming the gastrin-CCK-B nature of the receptor. Gastrin caused a rise in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] level within 5 s of stimulation. Finally, gastrin increased interleukin (IL)-2 secretion in J.RT3-T3.5 cells. We conclude that 1) J.RT3-T3.5 cells possess "gastrin-CCK-B type" receptors coupled to phospholipase C activation, Ins(1,4,5)P3 formation, and Ca2+ release from intracellular Ca2+ pools, and 2) these receptors could be involved in the regulation of IL-2 production.

Download Full-text

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

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y86-085 ◽

1986 ◽

Vol 64 (5) ◽

pp. 515-520 ◽

Cited By ~ 3

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.

Download Full-text

PGE2 binding, synthesis, and distribution in hen oviduct

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1985.248.1.e80 ◽

1985 ◽

Vol 248 (1) ◽

pp. E80-E88

Author(s):

G. Asboth ◽

H. Todd ◽

M. Toth ◽

F. Hertelendy

Keyword(s):

Binding Sites ◽

Scatchard Analysis ◽

Affinity Binding ◽

Shell Gland ◽

Temperature Dependent ◽

Single Class ◽

High Affinity Binding ◽

High Affinity ◽

Prostaglandin F2 Alpha ◽

20 Nm

Prostaglandin E2 (PGE2) bound specifically to particulate fractions prepared from the vagina and uterus (shell gland) portions of the hen oviduct in a time and temperature dependent fashion. Scatchard analysis indicated a single class of high-affinity binding sites in the vagina (Kd congruent to 1 nM), whereas the myometrium exhibited two kinds of binding site populations (Kd1 congruent to 1 nM, Kd2 congruent to 20 nM). It is suggested that these binding sites represent specific PGE2 receptors mediating the effects of PGE2 in oviductal smooth muscle. Vaginal particulate fractions produced approximately four times more prostanoids from [3H]-arachidonate than did uterine preparations. In the presence of epinephrine both tissues synthesized mainly thromboxane (TxB2), PGE2, and significantly less prostaglandin F2 alpha (PGF2 alpha). Addition of glutathione (GSH) or cytosol prepared from the oviduct markedly increased the yield of PGE2 at the expense of TxB2. Of the five morphologically discrete regions of the oviduct the vagina, infundibulum, and uterus contained the highest amounts of PGE and PGF, whereas the magnum and isthmus portions contain the least. TxB2 and 6-keto PGF1 alpha could not be detected in significant quantities in either region. These studies support the notion that PGE2 play a key role in the physiology of oviposition.

Download Full-text

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648890 ◽

1994 ◽

Vol 72 (03) ◽

pp. 465-474 ◽

Cited By ~ 20

Author(s):

Neelesh Bangalore ◽

William N Drohan ◽

Carolyn L Orthner

Keyword(s):

Binding Sites ◽

Protein C ◽

Umbilical Vein ◽

Activated Protein C ◽

Affinity Binding ◽

Cell Binding ◽

High Affinity Binding ◽

High Affinity ◽

Gla Domain ◽

Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

Download Full-text