Bone marrow-derived macrophages express functional CGRP receptors and respond to CGRP by increasing transcription of c-fos and IL-6 mRNA.

Calcitonin gene-related peptide (CGRP) is a sensory neuropeptide with inflammatory and immunoregulatory properties. CGRP inhibits IL-7 responses by B cell precursors by direct and indirect mechanisms. We recently found that CGRP induces IL-6 and TNF-alpha in long-term bone marrow cultures and that IL-6 and TNF-alpha also inhibit IL-7 responses. Because these are heterogeneous cultures, it was not clear which cells produced IL-6 and TNF-alpha. To determine whether bone marrow-derived macrophages (BMDM) were the source, we did studies to determine whether BMDMs express mRNAs for CGRP receptors and whether CGRP induces c-fos, IL-6, and TNF-alpha mRNA. We found that BMDMs express mRNAs for CRLR and RAMP1, the minimal components for CGRP receptors. CGRP also stimulated dose- and time-dependent increases in c-fos and IL-6. In contrast, CGRP did not induce TNF-alpha in BMDMs. These results suggest that BMDMs are a source of CGRP-induced IL-6 in bone marrow.

The plasmin system is induced by and degrades amyloid-beta aggregates.

Amyloid-beta (Abeta) appears critical to Alzheimer's disease. To clarify possible mechanisms of Abeta action, we have quantified Abeta-induced gene expression in vitro by using Abeta-treated primary cortical neuronal cultures and in vivo by using mice transgenic for the Abeta precursor (AbetaP). Here, we report that aggregated, but not nonaggregated, Abeta increases the level of the mRNAs encoding tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Moreover, tPA and uPA were also upregulated in aged AbetaP overexpressing mice. Because others have reported that Abeta aggregates can substitute for fibrin aggregates in activating tPA post-translationally, the result of tPA induction by Abeta would be cleavage of plasminogen to the active protease plasmin. To gain insights into the possible actions of plasmin, we evaluated the hypotheses that tPA and plasmin may mediate Abeta in vitro toxicity or, alternatively, that plasmin activation may lead to Abeta degradation. In evaluating these conflicting hypotheses, we found that purified plasmin degrades Abeta with physiologically relevant efficiency, i.e., approximately 1/10th the rate of plasmin on fibrin. Mass spectral analyses show that plasmin cleaves Abeta at multiple sites. Electron microscopy confirms indirect assays suggesting that plasmin degrades Abeta fibrils. Moreover, exogenously added plasmin blocks Abeta neurotoxicity. In summation, we interpret these results as consistent with the possibility that the plasmin pathway is induced by aggregated Abeta, which can lead to Abeta degradation and inhibition of Abeta actions.

Calcitonin-gene related peptide (CGRP) inhibits interleukin-7-induced pre-B cell colony formation.

Calcitonin gene-related peptide (CGRP) is a sensory neuropeptide with inflammatory and immunoregulatory activities. Its role in B lymphocyte development was investigated using a pre-B cell colony-forming assay. Physiological concentrations of CGRP inhibited pre-B cell responses to interleukin-7 (IL-7). Inhibition was specific in that it was blocked by the CGRP antagonist CGRP8-37. Adrenomedulin, substance P, and calcitonin had no effect on B cell precursor responses. Similar responses were observed with B220+/IgM- B cell precursors. Inhibition of IL-7 responses in B220+/IgM- cells suggests that CGRP has a direct effect on B cell precursors. Studies with cultured bone marrow-adherent cells found that CGRP also has an indirect effect on IL-7 responses. Cultured bone marrow-adherent cells were treated with CGRP for 24 h, and anti-CGRP was added to the supernatants to neutralize CGRP. Concentrations of CGRP as low as 0.01 nM induced a factor that inhibited colony formation. In contrast, CGRP did not induce an inhibitory factor in cultured bone marrow macrophages, suggesting that CGRP induces an inhibitory factor in some adherent cell other than macrophages. The results show that CGRP has both direct and indirect effects on developing B cells and support a role for CGRP as an inhibitor of early B cell development.

Postmortem elevation in extracellular glutamate in the rat hippocampus when brain temperature is maintained at physiological levels: implications for the use of human brain autopsy tissues.

Postmortem alterations in the neuronal cytoskeleton resemble some aspects of the cytoskeletal disruption associated with neurodegenerative disorders, and are also similar to those observed following ischemia and produced by excitotoxins in vivo and in vitro. This suggests the involvement of excitotoxic mechanisms during the postmortem interval. The purpose of this study was to determine if extracellular levels of glutamate are elevated postmortem. Extracellular levels of GABA and taurine were also monitored using in vivo microdialysis. These three amino acids were analyzed using high-performance liquid chromatography. When postmortem rat brain temperature cooled rapidly to near room temperature, dialysate concentrations of glutamate were not increased in the hippocampal CA1 region during a 2-h postmortem interval, although increased extracellular levels of GABA and taurine were observed. In contrast, maintenance of brain temperature at 37 degrees C resulted in a 12-to-40 fold elevation in extracellular glutamate levels 20-120 min postmortem. In addition, the elevation in dialysate taurine concentration was greater than that observed in rats in which postmortem brain temperature was not maintained. Excitatory amino acid antagonists, NBQX (2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline) and MK-801 (dizocilpine, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cylohepten-5, 10-imine hydrogen maleate blocked the additional elevation in taurine associated with maintaining brain at 37 degrees C, but had less robust effects against glutamate and GABA release. The results indicate that extracellular concentrations of glutamate, taurine and GABA increase in postmortem rat brain when physiologic temperatures are maintained, but that these increases are blunted when brain temperature decreases. After death, the human brain cools much more slowly than does the rat brain. Therefore, extracellular glutamate levels are likely to increase in the postmortem human brain and may contribute to excitotoxic neuronal damage occurring in the interval between death and autopsy.

Thrombin induces surface and intracellular secretion of amyloid precursor protein from human endothelial cells.

Thrombin, a major coagulant and inflammatory mediator, was shown to regulate amyloid precursor protein (APP) secretion. APP is the protein from which the amyloid beta peptide (A(beta)) is derived. A(beta) forms the core of vascular and cerebral plaques in Alzheimer's disease (AD). In this study, human umbilical vein endothelial cells (HUVEC) were used to examine the effects of thrombin on APP expression. Cell supernatants from thrombin-treated HUVEC were immunoblotted to measure secreted APP. Thrombin-induced secretion of APP peaks at approximately 30 min post-treatment. Immunohistochemical analysis found that APP is not colocalized with or secreted through the same pathway as coagulation factor VIII. The secretion of APP is thrombin receptor-mediated, since it is inhibited by the thrombin antagonist N-Acetyl-D-Phe-Pro-1-Amido-4-Guanidino-Butyl-1-Boronic Acid. It also is induced by treatment with a calcium ionophore. Moreover, APP secretion is protein kinase C (PKC)-dependent because it is blocked by the PKC inhibitor bisindolylmaleimide. APP secretion also occurs from the cell surface, possibly through direct cleavage by thrombin. Immunoreactivity on the surface of HUVEC decreased after thrombin treatment but not after treatment with a non-proteolytic thrombin receptor activator. These data suggest that thrombin induces APP secretion through a PKC-dependent mechanism, as well as from the cell surface. Our results are consistent with thrombin playing a role in AD pathology.

Elevated circulating calcitonin gene-related peptide in umbilical cord and infant blood associated with maternal and neonatal sepsis and shock.

The role of the sensory neuropeptide calcitonin gene-related peptide (CGRP) was studied in preterm and term neonates with sepsis and shock. CGRP levels in blood were measured by RIA. The identity of immunoreactive CGRP (irCGRP) in adult and infant human blood was confirmed by reverse phase-HPLC. CGRP levels were analyzed in a total of 189 samples (95 from cord blood and 94 from neonates). The gestational ages ranged from 24 to 43 wk, and the birth weights ranged from 520 to 4445 g. Cord samples were collected immediately after delivery and infant blood samples were collected within 12 h of birth. Samples were coded, and the data were assigned to groups after determination of CGRP levels. There was a weight- and gestation-dependent increase in irCGRP in the newborn population. The direct correlation of circulating CGRP with ascending birth weight and gestation may have significance in the development of the fetus. Infants with and without certain complications were grouped in 500-g intervals. CGRP levels in cord blood were significantly elevated when certain stressful situations existed in the mother. These included culture-positive chorioamnionitis, placental abruption, and severe preeclampsia. There was a similar elevation in CGRP in patient blood in infants with culture-positive sepsis and/or shock with blood pressure <2 SD from the mean for corresponding gestation. CGRP levels did not differ between male and female infants and did not appear to be influenced by type of delivery (vaginal versus cesarean section). There was no significant difference in CGRP level between cord and patient blood in preterm neonates, but at term gestation cord blood levels were slightly higher than those in the patient blood. These results suggest that inflammation and hemodynamic imbalance (e.g. shock) are associated with increased in CGRP levels in the circulation in neonates. Future studies will focus on the biologic effects of elevated CGRP during neonatal complications and will examine the utility of CGRP measurement for diagnosis and treatment of disease in preterm infants.

Catecholamines decrease lymphocyte adhesion to cytokine-activated endothelial cells.

Numerous studies have shown that catecholamines can modulate lymphocyte migration. This effect may be mediated in part by modulation of lymphocyte-endothelial cell interactions, which is dependent on adhesion molecules expressed on both of these cells. Our results show that catecholamines decreased T-cell binding to IL-1 activated endothelial cells in vitro. The decrease in adhesion was not mediated by a change in adhesion molecule expression as LFA-1 and VLA-4 expression on T-cells and ICAM-1 and VCAM-1 expression on endothelial cells were not changed by catecholamine stimulation. T-cells flatten and enlarge the area of surface contact as they adhere to endothelial cells. Image analysis of the number of T-cells bound and the amount of cell spreading over several time points suggests that catecholamines alter the kinetics of T-cell-endothelial cell adhesion. These results support the hypothesis that catecholamines can alter lymphocyte-endothelial interactions in vivo, which in turn would affect lymphocyte migration.

A role for calcitonin gene related peptide (CGRP) in the regulation of early B lymphocyte differentiation.

In previous studies we identified high affinity adenylyl cyclase linked receptors for calcitonin gene related peptide (CGRP) on rat T and B cells, on lymphocyte cell lines including the mouse pre-B cell line 70Z/3, and on cells in mouse bone marrow. The effect of CGRP on early B cell differentiation has been examined using the 70Z/3, and on cells in mouse bone marrow. The effect of CGRP on early B cell differentiation has been examined using the 70Z/3 cell line. CGRP inhibits the lipopolysaccharide (LPS) induction of surface immunoglobulin (sIg) protein expression in 70Z/3 cells, an effect that is associated with a decrease in the steady-state levels of Ig heavy (mu) and light (kappa) chain mRNA. In this report, experiments are described that provide further information on the mechanism by which CGRP inhibits sIg expression. The kinetics of CGRP inhibition of LPS-induced sIg expression was examined in 70Z/3 cells. An optimal window for the inhibitory effect of CGRP on SIg induction occurs at least 24 h after the cells are treated with LPS. To determine whether the inhibitory effects of CGRP on sIg expression are mediated by an inhibition of NF kappa-B translocation to the nucleus, electrophoretic mobility shift assays were performed using nuclear proteins from 70Z/3 cells. There was no difference in NF kappa-B binding activity in cells that had been treated with LPS or LPS + CGRP, suggesting that the inhibitory effect of CGRP is not mediated by an inhibition of NF kappa-B activity. These studies provide further evidence that CGRP plays an inhibitory role in early B cell differentiation. Finally, a model is proposed that describes an integrated role for CGRP in the homeostatic regulation of early B cell differentiation.

Functional characterization of the insulin-like growth factor I receptor on Jurkat T cells.

Insulin-like growth factor I (IGF-I) has been shown to be important in the maintenance, development, and proliferation of various types of leukocytes, particularly T cells. Radio-receptor binding assays demonstrate that Jurkat T cells bind 125I-IGF-I with an affinity of 1.77 nM (Kd) and express approximately 230 receptors/cell. Specificity studies show insulin also binds the IGF-I receptor with an affinity 20-fold lower than that of IGF-I. Interaction of IGF-I with its receptor on Jurkat T cells induces the phosphorylation of tyrosine kinase which is detectable by Western blotting. The 95,000 MW protein detected is equivalent to the molecular weight of the beta chain of the IGF-I receptor described in other types of cells. These studies characterize the binding of IGF-I to its receptor on Jurkat T cells, demonstrate that IGF-I binding induces tyrosine phosphorylation, and support the hypothesis that IGF-I is important in the induction of T cell activation.

Heat shock alters Alzheimer's beta amyloid precursor protein expression in human endothelial cells.

One of the pathological lesions in Alzheimer's disease (AD) is the amyloid or senile plaque. The plaque core is predominantly made up of amyloid beta peptide (A beta), a 42-43 amino acid peptide derived from amyloid precursor protein (APP). APP is a membrane bound glycoprotein which is expressed ubiquitously in many cells. Although normal or pathological functions for APP are not well understood, several observations suggest that APP may play a role in cellular stress and inflammation at the endothelial cell/vascular barrier. APP is found in platelets and endothelial cells, it can inhibit a blood coagulation factor, and secreted APP can be neuroprotective. Changes in expression of APP during cellular stress or inflammation may contribute to pathological deposition of A beta. In the present studies, expression of APP in human endothelial cells was examined following heat shock. In human umbilical vein endothelial cells (HUVECs) exposed to 42 degrees C for 30 min, there was a five- to eight-fold increase in APP mRNA levels which peaked at 4 hr. The increase in APP mRNA was followed by an increase in APP protein immunoreactivity in the cytoplasm in a perinuclear Golgi-like region, and in discrete granular cytoplasmic structures. Immunoblot analysis of APP in the cell media found a transient increase in APP which peaked at 1 hr after heat shock. These results suggest that cellular stress induces the secretion of APP from endothelial cells followed by a subsequent increase in APP mRNA and protein synthesis. The upregulation of APP mRNA and protein supports a cellular stress role for APP.

Characterization of a calcitonin gene-related peptide (CGRP) receptor on mouse bone marrow cells.

Calcitonin gene-related peptide, (CGRP), a vasoactive neuropeptide, is found throughout the peripheral nervous system, and CGRP receptors are present on mature lymphocytes. The current studies describe a CGRP receptor on isolated mouse bone marrow cells. The affinity, distribution and specificity of CGRP receptors were analyzed using radioligand binding assays. [125I]CGRP binding in mouse bone marrow cells was dependent on cell concentration and was stable from 5 to 60 min at room temperature. The average Kd is 3.29 +/- 1.24 nM and the average receptor density is 2796 +/- 365 sites/cell. Competition binding analysis found rat alpha and beta CGRP to be the most inhibitory, (Ki values 0.899 and 0.711 nM, respectively), followed by human alpha CGRP and the antagonist human CGRP8-37. The neuropeptides human and salmon calcitonin did not inhibit [125I]CGRP binding to bone marrow cells. The presence of CGRP receptors on mouse bone marrow cells provides further evidence for a direct role for CGRP in modulating the function and differentiation of cellular components of the immune and inflammatory systems.

Modulation of B lymphocyte differentiation by calcitonin gene-related peptide (CGRP). I. Characterization of high-affinity CGRP receptors on murine 70Z/3 cells.

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide found in both peripheral and central neurons and in certain endocrine tissues. Previous studies have identified specific high-affinity CGRP receptors on normal T and B cells and have described modulatory effects of CGRP on freshly isolated lymphocytes. In these studies several lymphocyte cell lines were screened for 125I-CGRP binding. One cell line, the murine 70Z/3 pre-B cell line, was found to exhibit a high level of 125I-CGRP binding and was used for further characterization of the lymphocyte CGRP receptor. Several protease inhibitors were evaluated for their ability to stabilize 125I-CGRP binding to 70Z/3 cells. Bacitracin, a relatively nonspecific protease inhibitor, and chymostatin, a cysteine protease inhibitor, enhanced 125I-CGRP binding, suggesting that 70Z/3 cells express a protease capable of inactivating CGRP. 125I-CGRP binding had a linear dependence on cell concentration with binding being detectable with as few as 200,000 cells/ml and was rapid, with equilibrium binding being reached by 30 min. Saturation binding studies demonstrated that the 70Z/3 CGRP receptor has both low- and high-affinity binding states, with Kds of approximately 0.2 and 3.3 nM, respectively. The density of the low-affinity binding site was approximately 20,000 sites/cell and was unchanged following LPS treatment. In contrast, LPS treatment for 48 hr induced a fourfold increase in the density of the high-affinity site, from 105 to 401 sites/cell. In competition binding studies, only CGRP and the CGRP antagonist CGRP8-37 inhibited 125I-CGRP binding, whereas the unrelated neuropeptides calcitonin, SP, and CRH had no effect. Inhibition of 125I-CGRP binding by rat CGRP was best described by a two-site model, with Kis of 44.1 pM and 0.6 nM. Inhibition of binding by human CGRP and CGRP8-37 was best described by a one-site model with Kis of 3.92 and 6.50 nM, respectively. The 70Z/3 CGRP receptor protein was biochemically characterized by affinity labeling the receptor protein with 125I-CGRP and the covalent cross-linking reagents DSS or BS. SDS-PAGE analysis revealed a major band of 103,000 MW. In addition a high-molecular-weight complex which did not penetrate the gel was also observed. The presence of CGRP receptors on 70Z/3 cells provides further evidence for the immunomodulatory role for CGRP and provides a model system for studying the cellular mechanisms of action of CGRP in modulating lymphocyte differentiation and function.

Modulation of B lymphocyte differentiation by calcitonin gene-related peptide (CGRP). II. Inhibition of LPS-induced kappa light chain expression by CGRP.

The presence of calcitonin gene-related peptide (CGRP) in nerve endings in lymphoid organs, around blood vessels, and in bone marrow suggests that it might influence the function and differentiation of lymphoid cells. Previous studies identified specific CGRP receptors on mature T and B lymphocytes and on 70Z/3 pre-B cells. In these studies, it was found that CGRP stimulated a rapid, prolonged elevation of cAMP with in 70Z/3 cells with an ED50 of approximately 20 fM. Following CGRP treatment, cAMP levels peaked within 5 min and were still elevated after 60 min. The effect of CGRP on surface immunoglobulin (sIg) expression was examined by treating 70Z/3 cells with CGRP, or combinations of CGRP and LPS, and then measuring sIg expression by FACS. When 70Z/3 cells were treated with LPS, CGRP, or calcitonin for 48 hr, only LPS induced the expression of sIg, increasing the percentage of cells expressing sIg from less than 10% positive in untreated cells to 80-98% positive. Subsequent experiments examined the effect of CGRP on LPS-induced sIg. CGRP inhibited LPS-induced sIg expression at concentrations ranging from 10(-15) to 10(-7) M. The maximal inhibition was observed at CGRP concentrations ranging from 10(-10) to 10(-8) M, and the maximal reduction of sIg expression was about 40%. The inhibitory effect of CGRP was specific in that it could not be mimicked by calcitonin and could be blocked by the CGRP receptor antagonist CGRP8-37. A similar dose-dependent inhibitory effect on LPS induction was observed in 70Z/3 cells treated with LPS and dibutyryl cAMP, suggesting that the inhibitory effect of CGRP on sIg expression is mediated by stimulation of intracellular cAMP. The inhibitory effect of CGRP on LPS induction of sIg appears to be mediated by a reduction in the expression of both mu and kappa mRNA. When mu and kappa expression were examined by Northern blot analysis, it was found that CGRP caused a 50% reduction in the amount of mu and kappa mRNA induced by LPS. The ability of CGRP to inhibit differentiation of 70Z/3 cells and sIg expression provides evidence that CGRP can influence the differentiation of lymphopoietic precursors.

Characterization of functional calcitonin gene-related peptide receptors on rat lymphocytes.

Calcitonin gene-related peptide (CGRP), a vasoactive neuropeptide present in peripheral neurons, is released at local sites of inflammation. In these studies specific high affinity adenylyl cyclase linked CGRP receptors were characterized on rat lymphocytes. The distribution, affinity, and specificity of CGRP receptors was analyzed by radioligand binding. 125I-[His10]CGRP binding to rat lymphocytes was rapid, reaching equilibrium by 20 to 30 min at 22 degrees C, and dependent on cell concentration. The dissociation constants, Kd, for the CGRP receptor on purified T and B lymphocytes are 0.807 +/- 0.168 nM and 0.387 +/- 0.072 nM and the densities are 774 +/- 387 and 747 +/- 244 binding sites/cell, respectively. Competition binding studies determined that rat CGRP inhibits 125I-[His10]CGRP binding to lymphocytes with the highest affinity (Ki = 0.192 +/- 0.073) followed by human CGRP and the CGRP receptor antagonist CGRP8-37. 125I-[His10]CGRP binding to rat lymphocytes was not inhibited by the neuropeptides substance P, calcitonin, or neuropeptide Y. Lymphocyte CGRP receptor proteins were identified by affinity labeling by using disuccinimidyl suberate to covalently cross-link 125I-[His10]CGRP to its receptor. Specifically labeled CGRP binding proteins visualized by SDS-PAGE analysis had molecular masses of 74.5 and 220 kDa. A third high molecular mass protein band which did not penetrate the gel was also observed. In functional studies, CGRP stimulated a rapid, sustained increase in cAMP with an ED50 of approximately 8 pM. In experiments comparing optimal concentrations of isoproterenol, a beta 2-adrenergic agonist, and CGRP, intracellular cAMP elevation after isoproterenol treatment returned to basal levels by 30 min, whereas cAMP was still elevated at 60 min after CGRP treatment. The response to CGRP was specific in that it could be completely blocked by CGRP8-37. The presence of high affinity functional CGRP receptors on T and B lymphocytes provides evidence for a modulatory role for CGRP in regulating lymphocyte function.

Immunomodulation by tachykinin neuropeptides.

Substance P (SP) is an 11-amino-acid neuropeptide found in sensory neurons in the peripheral nervous system. In addition to having well-characterized functions as a peptide neurotransmitter, it also plays a major role in modulating inflammatory and immune responses. SP can alter the proliferative and physiological responses of both lymphocytes and macrophages. These effects are mediated by specific high-affinity SP receptors which have been characterized both kinetically and biochemically. The principle SP binding protein present on human lymphocyte cell membranes is a 58,000-MW hydrophobic glycoprotein. Cellular responses subsequent to the binding of substance P to its receptor that have been identified in various cell populations include phosphatidyl inositol turnover, arachidonic acid metabolism, immunoglobulin synthesis, and enzyme production and secretion. Evidence also suggests that SP modulation of inflammation is a factor in the pathophysiology of certain diseases such as rheumatoid arthritis.

Transient expression of the angiotensin II receptor: a rapid and functional analysis of a calcium-mobilizing seven-transmembrane domain receptor in COS-7 cells.

The mas oncogene/angiotensin II receptor was subcloned into a mammalian expression vector pCDM8 and used to transiently transfect monkey kidney derived COS-7 cells. As a result, the mas transfected COS-7 cells expressed a functional angiotensin II receptor capable of transducing an increase in intracellular Ca2+ following stimulation with angiotensin II. The angiotensin II stimulated changes in Ca2+ could be measured 24 hours after transfection in both a fluorimeter and a fluorescence activated cell sorter. These results describe a rapid method for the functional analysis of the 7-transmembrane domain receptor genes.

Stimulation of rat B-lymphocyte proliferation by corticotropin-releasing factor.

The mitogenic effect of corticotropin-releasing factor (CRF) on rat lymphocytes was investigated. When rat splenocytes were cultured for 48 hr with CFR, a dose-dependent increase in incorporation of 3H-thymidine (3H-Tdr) was observed, with a maximal response at 10 nM CRF. Comparison of the proliferative effect of CRF on enriched populations of B lymphocytes, T lymphocytes, or macrophages revealed that only B lymphocytes responded following treatment with CRF. When lymphocytes derived from different lymphoid tissues were compared, CRF had a greater proliferative effect on lymphocytes derived from gut-associated lymphoid tissue (mesenteric lymph nodes and Peyer's patches) than on lymphocytes from spleen or inguinal lymph nodes; CRF had no effect on thymocytes. Synthetic fragments of CRF were used to determine which portions of the peptide are recognized by lymphocytes. The C-terminal fragments alpha-helical CRF9-41 and CRF21-41 were as potent as native CRF in stimulating B-lymphocyte proliferation, whereas CRF1-20 did not stimulate proliferation. The activity of these peptides suggests that CRF stimulates lymphocyte proliferation by cellular recognition of structural determinants in the C-terminal one-half of the peptide.

Processing of the human IM-9 lymphoblast substance P receptor. Biosynthetic and degradation studies using a monoclonal anti-receptor antibody.

Cellular membrane receptors for the immunostimulatory neuropeptide substance P have been previously identified on the cultured lymphoblast cell line, IM-9. The regulation of this receptor by ligand and the contribution to its molecular weight by N-linked sugars was studied by incubating IM-9 cells for 14 hr in the presence of [35S]met with or without substance P and tunicamycin, respectively. Cells were lysed and the receptor proteins were immunoprecipitated with an anti-receptor monoclonal antibody. SDS-PAGE analysis of untreated cellular lysates revealed specifically precipitated proteins of 38 kD and 33 kD, which were down-regulated by substance P. In tunicamycin-treated cells, whose substance P binding was not affected, the major immunoprecipitated protein had an apparent Mr of 29 kD. The time course of receptor processing was studied by pulse chase analysis. Three proteins of molecular weights 38 kD (mature receptor), 36 kD and 33 kD (receptor precursors) were identified for time periods of 30 min to 4 hr. The half life of the mature receptor and its precursors was approximately 1 hr and 0.5 hr, respectively. Results from the present studies suggest that the lymphocyte substance P receptor is translated as a precursor protein that is glycosylated.

Thymosin fraction 5 (TF5) stimulates secretion of adrenocorticotropic hormone (ACTH) from cultured rat pituitaries.

In vivo administration of a partially purified thymic hormone-containing extract of the thymus gland, TF5, causes an increase in serum glucocorticoids. The lack of a direct effect of TF5 on adrenal corticosterone secretion suggests that it is mediated at the level of the pituitary. Cultured rat pituitary monolayers were used to determine if the effect is mediated by stimulation of ACTH secretion from the pituitary. Two lots of TF5, BPP100 and C114080-01, caused a dose dependent secretion of ACTH from cultured pituitary monolayers. There was a synergistic effect when the cells were treated with both TF5 and corticotropin-releasing factor (CRF). Immunoneutralization studies were done in which the cells were treated with TF5 or CRF and an antibody to CRF. The antibody completely blocked CRF induced ACTH release, but had no effect on TF5 stimulated ACTH release, suggesting that the activity is not due to a CRF-like peptide in TF5. A number of peptides isolated from TF5, and certain other peptides produced by the immune system were evaluated for their ability to stimulate ACTH secretion. These included thymosin (TSN) alpha 1, alpha 11, and beta 4, prothymosin alpha (PT alpha, thymopoeitin 5 (TP5), factuer thymique serique (FTS), interferon alpha (INF alpha), INF gamma, interleukin 1 (IL-1), and interleukin 2 (IL-2). None of these factors had any effect on pituitary ACTH secretion. These results demonstrate that some peptide component of TF5 causes an increase in serum corticosteroids by stimulating pituitary ACTH release.