Joint ultrasound baseline abnormalities predict a specific long-term clinical outcome in systemic lupus erythematosus patients.

Objective To describe long-term clinical and serological outcome in all systemic lupus erythematosus (SLE) domains in SLE patients with hand arthralgia (HA) and joint ultrasound (JUS) inflammatory abnormalities, and to compare them with asymptomatic SLE patients with normal JUS. Methods SLE patients with HA who presented JUS inflammatory abnormalities ('cases') and SLE patients without HA who did not exhibit JUS abnormalities at baseline ('controls') were included. All SLE clinical and serological domain involvement data were collected. End follow-up clinical activity and damage scores (systemic lupus erythematosus disease activity index (SLEDAI), Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR)) were recorded. JUS inflammatory abnormalities were defined based on the Proceedings of the Seventh International Consensus Conference on Outcome Measures in Rheumatology Clinical Trials (OMERACT-7) definitions. Statistical analyses were carried out to compare 'cases' and 'controls'. Results A total of 35 patients were recruited. The 'cases', n = 18/35, had a higher incidence of musculoskeletal involvement (arthralgia and/or arthritis) through the follow-up period (38.9% vs 0%, p = 0.008) and received more hydroxychloroquine (61.1% vs 25.0%, p = 0.034) and methotrexate (27.8% vs 0%, p = 0.046) compared to 'controls', n = 17/35. Other comparisons did not reveal any statistical differences. Conclusions We found SLE patients with arthralgia who presented JUS inflammatory abnormalities received more hydroxychloroquine and methotrexate, mainly due to persistent musculoskeletal involvement over time. JUS appears to be a useful technique for predicting worse musculoskeletal outcome in SLE patients.

Comparative analysis of two emerging rice seed bacterial pathogens.

Seed sterility and grain discoloration limit rice production in Colombia and several Central American countries. In samples of discolored rice seed grown in Colombian fields, the species Burkholderia glumae and B. gladioli were isolated, and field isolates were compared phenotypically. An artificial inoculation assay was used to determine that, although both bacterial species cause symptoms on rice grains, B. glumae is a more aggressive pathogen, causing yield reduction and higher levels of grain sterility. To identify putative virulence genes differing between B. glumae and B. gladioli, four previously sequenced genomes of Asian and U.S. strains of the two pathogens were compared with each other and with two draft genomes of Colombian B. glumae and B. gladioli isolates generated for this study. Whereas previously characterized Burkholderia virulence factors are highly conserved between the two species, B. glumae and B. gladioli strains are predicted to encode distinct groups of genes encoding type VI secretion systems, transcriptional regulators, and membrane-sensing proteins. This study shows that both B. glumae and B. gladioli can threaten grain quality, although only one species affects yield. Furthermore, genotypic differences between the two strains are identified that could contribute to disease phenotypic differences.

Prevalence and predictors of vitamin D deficiency in non-supplemented women with systemic lupus erythematosus in the Mediterranean region: a cohort study.

OBJECTIVES: Over the past few years researchers have suggested that vitamin D plays a diverse role in autoimmune diseases such as systemic lupus erythematosus (SLE). We sought to determine the prevalence and predictors of vitamin D deficiency in a cohort of non-supplemented female SLE patients from the Mediterranean region. METHODS: We carried out a prospective cohort study on all SLE patients who had visited the Department of Rheumatology at the Parc de Salut MAR (Barcelona, Spain) between June 2007 and December 2008, excluding those who had been taking vitamin D supplements (total: 73 patients, all female). For each patient, demographic information was collected; scores were measured for disease severity [SLE Disease Activity Index (SLEDAI)] and structural damage [Systemic Lupus International Collaborating Clinic/American College of Rheumatology, (SLICC/ACR) Damage Index]; pharmacological treatment was recorded; analytical variables were analysed; and plasma levels of 25-hydroxy vitamin D [25(OH)D] were quantified. RESULTS: Among the patients in our cohort, 68.5% [95% confidence interval (CI) 60.3-79.2] exhibited vitamin D deficiency [plasma level of 25(OH)D < 30 ng/mL]. The predictors for vitamin D deficiency were daily sunscreen use [odds ratio (OR) 1.67, p = 0.02] and high body mass index (BMI) (OR 1.32 when adjusted for seasons and patient age, p = 0.04). We did not find any correlation between vitamin D deficiency and SLEDAI score (p = 0.31), SLICC/ACR score (p = 0.82), or any other of the variables. CONCLUSIONS: Vitamin D insufficiency is highly prevalent among SLE patients, even in southern regions. Sunscreen use and obesity increase the risk. Clinicians should be aware of these factors and supplement SLE patients at risk of vitamin D deficiency accordingly.

Association between fibromyalgia and psychiatric disorders in systemic lupus erythematosus.

OBJECTIVES: Systemic lupus erythematosus (SLE) is an autoimmune disease that may affect many organs, with musculoskeletal symptoms being the most common. Fibromyalgia (FM) is frequent in SLE patients. Psychiatric disorders such as anxiety and depression are also present in many SLE patients. The aim of our study is to determine the relationship between FM and psychiatric symptoms (PS), both anxious (AS) and depressive (DS), and its impact on health status in SLE patients. METHODS: In a total of 84 SLE patients we studied the presence of both FM and PS using specific questionnaires (Hamilton). We also evaluated health status and SLE disease activity by both the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) and serological markers. Patients performed the Short Form 12 (SF-12) questionnaire as a quality of life measure. Qualitative variables were compared using Pearson's chi-square test and Fisher's exact test, and Student's t-test was used for quantitative variables. The Mann-Whitney U-test was applied if a normal distribution was not observed. RESULTS: Thirty patients were diagnosed with FM (35.7%), 16 had clinical signs DS (19%) and 30 had clinical signs AS (35.7%). We found a statistically significant association between FM and AS (p<0.001), and between FM and DS (p<0.001). Higher SF-12 physical component and mental component scores were observed in FM group compared to non-FM group (p<0.001). We have not found any associations between SLE activity and FM and PS. CONCLUSIONS: There is a high prevalence of FM in SLE patients, and a strong association with DS and AS. FM contributes to worsening health status in SLE patients. SLE activity has little or no impact either on psychiatric symptoms or FM.

Organ-specific inhibition of metastatic colon carcinoma by CXCR3 antagonism.

Liver and lung metastases are the predominant cause of colorectal cancer (CRC)-related mortality. Recent research has indicated that CXCR3/chemokines interactions that orchestrate haematopoetic cell movement are implicated in the metastatic process of malignant tumours, including that of CRC cells to lymph nodes. To date, however, the contribution of CXCR3 to liver and lung metastasis in CRC has not been addressed. To determine whether CXCR3 receptors regulate malignancy-related properties of CRC cells, we have used CXCR3-expressing CRC cell lines of human (HT29 cells) and murine (C26 cells) origins that enable the development of liver and lung metastases when injected into immunodeficient and immunocompetent mice, respectively, and assessed the effect of CXCR3 blockade using AMG487, a small molecular weight antagonist. In vitro, activation of CXCR3 on human and mouse CRC cells by its cognate ligands induced migratory and growth responses, both activities being abrogated by AMG487. In vivo, systemic CXCR3 antagonism by preventive or curative treatments with AMG487 markedly inhibited the implantation and the growth of human and mouse CRC cells within lung without affecting that in the liver. In addition, we measured increased levels of CXCR3 and ligands expression within lung nodules compared with liver tumours. Altogether, our findings indicate that activation of CXCR3 receptors by its cognate ligands facilitates the implantation and the progression of CRC cells within lung tissues and that inhibition of this axis decreases pulmonary metastasis of CRC in two murine tumour models.

Bombesin stimulates adhesion, spreading, lamellipodia formation, and proliferation in the human colon carcinoma Isreco1 cell line.

The neuropeptide bombesin and its mammalian homologue, gastrin-releasing peptide (GRP), enhance proliferation in some but not all human tumor cell lines. The pathophysiological relevance of the bombesin/GRP receptor (GRP-R), which is expressed in 30% of human colon tumor cell lines and in 24-40% of native tumors, has not been clearly assessed at this time. We studied the effects of bombesin in the recently characterized human colon carcinoma Isreco1 cell line. Competitive reverse transcription-PCR showed a high GRP-R mRNA level in Isreco1 cells, and binding studies confirmed the expression of bombesin/GRP-subtype receptors (Kd = 0.42 nM; Bmax = 18,000 sites/cell). Exposure to bombesin resulted in an increase of intracellular calcium concentrations. Bombesin (1 nM) induced cell spreading at 24 h (21.7+/-1.6% versus 6.4+/-0.8% in control cells; P<0.01) and markedly increased the formation of lamellipodia. In addition, adhesion of Isreco1 cells to collagen I-coated culture dishes was stimulated in the presence of 1 nM bombesin (69+/-6% versus 42+/-1% in control cells; P<0.01). Finally, bombesin significantly increased [3H]thymidine uptake by Isreco1 cells in a dose-dependent manner, with a first significant response at 0.1 nM and a maximal effect at 100 nM bombesin (192.2+/-9.7% of control). These results clearly indicate that bombesin exerts morphological, adhesive, and proliferative effects on Isreco1 cells, suggesting that expression of the bombesin/GRP-R may contribute to the malignant properties of colon carcinoma cells.

Raised serum alkaline phosphatase activity in one family.

Eleven members of the same family were studied after an incidental detection of raised serum alkaline phosphatase activity in one of them without any apparent underlying cause. Three other members were found to have the same abnormality; none of them had an associated disease. In the four cases with elevated serum alkaline phosphatase levels, its activity showed a preponderance of the bone isoenzyme. Studies of the erythrocyte and histocompatibility antigens in nine members of the family, as well as idiograms of karyotypes of four of them, did not show any relation between histocompatibility antigens and the raised levels of serum alkaline phosphatase. Also, no chromosomal abnormality is shown from karyotypes. The data suggest a probable autosomal dominant pattern of inheritance.

Regulation of cholecystokinin secretion by peptones and peptidomimetic antibiotics in STC-1 cells.

Peptones are potent stimulants of cholecystokinin (CCK) release in rats, both in vivo and ex vivo in a model of isolated vascularly perfused duodeno-jejunum preparation and in vitro in the intestinal CCK-producing cell line STC-1. The underlying mechanisms were here investigated with this cell line. Protein hydrolysates from various origins (meat, casein, soybean, and ovalbumin; 0.5-1%, wt/vol) dose dependently increased CCK release. Cephalosporin antibiotics, which mimic tripeptides, also stimulated the release of CCK over the concentration range 1-20 mM. The study of concentration dependence of cephalosporin uptake indicated a passive diffusion process at either pH 7.4 or pH 6.0, thus arguing against the involvement of a peptide transporter in CCK secretion. After pertussis toxin treatment (200 ng/ml; 5 h), the peptone- and cephalexin-induced CCK secretion was significantly reduced, suggesting the involvement of pertussis toxin-sensitive heterotrimeric G protein(s) in the secretory activity of STC-1 cells. Consistent with this was the identification by Western blot of G(i2)alpha, G(i3)alpha, and G(o)alpha immunoreactivities in STC-1 cell extracts. Additionally, peptones and cephalexin increased the cellular content in inositol phosphates, whereas a mild increase in cAMP content was restricted to peptone-treated cells. Protein kinase A or C inhibition did not modify peptone- or antibiotic drug-evoked CCK release. The extracellular Ca2+ chelator EGTA (500 microM) and the intracellular Ca2+ chelator BAPTA-AM [1,2-bis-(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; 20 microM] abolished the peptone- and antibiotic drug-induced CCK release. Nifedipine and verapamil (10 microM) reduced by about 50% the CCK secretion evoked by these two secretagogues. In conclusion, peptones and some cephalosporins are potent stimulants of CCK release in the STC-1 cell line. The cellular mechanisms involve pertussis toxin-sensitive G protein(s) and are dependent on Ca2+ availability. We suggest that the STC-1 cell line is a useful model to study the molecular basis of peptone-induced CCK secretion.

Stimulation of glucagon-like peptide-1 secretion by muscarinic agonist in a murine intestinal endocrine cell line.

Studies on the cholinergic regulation of intestinal L-cells have been focused on the release of enteroglucagon, but the signal transduction pathways were not defined. These were here investigated by using as index the release of immunoreactive glucagon-like peptide-1 (GLP-1) from the endocrine cell line STC-1, that has been shown to contain proglucagon mRNA transcripts. Abundant GLP-1 immunoreactivity was revealed in STC-1 cells at immunocytochemistry and by RIA. The cell content was 4927 +/- 689 pg/10(6) cells, as measured with antiserum 199D that recognizes specifically the C-terminal amidated forms of GLP-1. The secretion of GLP-1 over a 2-h incubation period amounted to 1.4 +/- 0.3% of the total GLP-1 cell content and was significantly increased by 10 microM forskolin and 100 nM 12-O-tetradecanoylphorbol 13-acetate to 206% and 574% of control values, respectively. The cholinergic agonist carbachol stimulated GLP-1 secretion in a concentration-dependent manner, maximal release was observed at 1 mM carbachol (228% of the control value). Binding of the muscarinic antagonist [N-methyl-]scopolamine ([3H]NMS) on cell homogenates was time dependent, specific, and saturable. Scatchard analysis revealed one class of receptors (Kd, 14 pM; binding capacity, 20 fmol/mg protein). Carbachol (0.1 microM to 1 mM) dose dependently displaced [3H] NMS binding and increased the intracellular calcium concentration without modification of adenylate cyclase activity. The order of potency of different antagonists, showing a preferential affinity for M1, M2, and M3 muscarinic receptor subtypes, to inhibit [3H]NMS binding, the carbachol-induced increase in intracellular calcium, and carbachol-stimulated GLP-1 secretion, was as follows: atropine (nonselective) > 4-diphenylacetoxy-N-methylpiperidine methiodide (M3) > pirenzepine (M1) > AF-DX 116 (M2). The results of the present study, therefore, demonstrate that secretion of GLP-1 induced by cholinergic agonist depends on muscarinic M3-subtype receptors in the endocrine intestinal cell line STC-1. This system may prove useful to study the cellular mechanisms of GLP-1 secretion.

Peptones stimulate cholecystokinin secretion and gene transcription in the intestinal cell line STC-1.

In rats, protein hydrolysates (peptones) stimulate cholecystokinin (CCK) release both in vivo and in a model of isolated vascularly perfused duodeno-jejunum. However, the mechanisms involved in peptone-induced stimulation of CCK cells are not well understood. In particular, the possibility that peptones may directly interact with CCK-producing cells to stimulate CCK release and gene transcription has not yet been examined. To test this hypothesis, we used the enteroendocrine cell line STC-1. Incubation of STC-1 cells for 2 h with albumin egg hydrolysate over the concentration range 0.01-1% (wt/ vol) caused a dose-dependent release of CCK, with a maximal increase at 1420% of the control value. In contrast, BSA (1%, wt/vol) or a mixture of amino acids (1%, wt/vol) induced a modest rise in CCK secretion. A dose-dependent, hydrolysate-specific, increase in the CCK steady state RNA level was also observed. It was detectable by 2-4 h of peptone treatment and sustained until 24-48 h. Peptones did not increase the CCK RNA level in the colonic CCK-producing cell line GLUTag or in nonintestinal CCK-expressing cell lines, namely the pancreatic cell line RINm5F and the medullar thyroid carcinoma cell line CA77. The peptone-induced increase in the CCK RNA level resulted from enhanced gene transcription, because labeled CCK transcripts from nuclear run-on incubations increased 3-fold when cells were incubated with peptones, whereas the level of beta-actin transcripts was not modified. Finally, peptones dose-dependently stimulated the transcriptional activity of an 800-bp fragment of CCK gene promoter transfected in STC-1 cells. These studies indicate that peptones specifically stimulate CCK secretion and gene transcription in the intestinal cell line STC-1, and that cis-acting elements conferring peptone inducibility are located in the first 800 bp of the 5'-flanking region of the CCK gene.

Characterization of muscarinic acetylcholine receptors on the rat pancreatic gastrin-producing cell line B6 RIN.

The mechanisms of cholinergic stimulation of gastrin cells were studied in the rat pancreatic cell line B6 RIN. Carbachol induced an increase in intracellular Ca2+ and stimulated gastrin release in a dose-dependent manner over the range 10(-5)-10(-3) M. These effects were completely abolished by atropine, suggesting the implication of muscarinic cholinergic receptors. The binding properties of these receptors were investigated. [N-Methyl-3H]scopolamine [( 3H]NMS) binding on cell homogenates was time-dependent, saturable and consistent with a single high-affinity binding class (Kd = 39.5 pM, and Bmax = 7.9 fmol/mg DNA). Carbachol competitively inhibited [3H]NMS binding. The potency of inhibition of [3H]NMS binding by subtype selective antagonists was hexahydrodifenidol greater than pirenzepine greater than AF-DX 116. These results suggest the M3 muscarinic receptors may be involved in the carbachol-induced gastrin release from B6 RIN cells.

Expression of SNARE proteins in enteroendocrine cell lines and functional role of tetanus toxin-sensitive proteins in cholecystokinin release.

In neurons, synaptic vesicle exocytosis involves the formation of a core complex particle including syntaxin-1, synaptosomal-associated protein of 25 kDa (SNAP-25) and vesicle-associated membrane protein (VAMP)-2/synaptobrevin. The expression of these proteins was investigated in a panel of cell lines, including lines of endocrine and intestinal origin, by Western blotting and/or immunocytochemistry. The three core complex proteins were detected in the enteroendocrine, cholecystokinin (CCK)-secreting, cell lines STC-1 and GLUTag, and in the endocrine non-intestinal cell lines CA-77 and HIT-T15. In contrast, SNAP-25 and syntaxin-1 were undetected in the intestinal non-endocrine cell lines IEC-6, HT-29 and Caco-2, whereas a slight expression of VAMP-2 was documented in IEC-6 and HT-29 cells. Co-immunoprecipitation experiments indicated that syntaxin-1, SNAP-25 and VAMP-2 were present in a complex similar to that identified in brain. In the STC-1 cell line, treatment of streptolysin-O-permeabilized cells with tetanus toxin (Tetx) selectively cleaved VAMP-2 and VAMP-3/cellubrevin, and simultaneously abolished Ca2+-induced CCK secretion (IC50 approximately 12 nM). These results show that endocrine cell lines of intestinal origin express syntaxin-1, SNAP-25 and VAMP-2, and suggest a key role for a Tetx-sensitive protein (for example VAMP-2 and/or VAMP-3) in the CCK secretion by STC-1 cells.

Rab3a controls exocytosis in cholecystokinin-secreting cells.

The expression of rab3A and rab3D isoforms in the enteroendocrine, cholecystokinin-secreting, cell lines STC-1 and GLUTag is here demonstrated. In contrast, rab3B is undetectable in these two cell lines, and rab3C is only slightly expressed in GLUTag cells. Using a transient co-transfection system with human growth hormone as reporter protein, we show that overexpression of the GTPase-deficient mutant rab3AQ81L, but not rab3DQ81L, significantly decreases human growth hormone secretory responses to various agonists in STC-1 cells. These results indicate that endocrine cell lines of intestinal origin express rab3A and rab3D proteins, but the GTP-bound form of rab3A only acts as a negative modulator in the control of cholecystokinin secretion from STC-1 cells.

High gastrin releasing peptide receptor mRNA level is related to tumour dedifferentiation and lymphatic vessel invasion in human colon cancer.

The neuropeptide bombesin stimulates tumour cell proliferation in vitro. Through pharmacological testing, 20-40% of human colorectal tumours have been shown to be equipped with bombesin/gastrin releasing peptide receptor (GRP-R). The aim of the present study was to test whether GRP-R expression is correlated with tumour characteristics and usual prognostic factors in colorectal adenocarcinomas. A sensitive reverse transcription (RT)-competitive polymerase chain reaction (PCR) method was validated by studying GRP-R mRNA in separated layers of normal colonic wall, and GRP-R mRNA levels (in parallel with binding studies) in colon cancer cell lines LoVo and Caco-2. GRP-R mRNA levels were then determined in 29 surgical tumour specimens and the results compared with tumour histology and, using histochemistry, with the accumulation of p53 protein and a Ki-67 cell proliferation index. The mRNA was not detected in normal colonic epithelium, whereas a distinct signal was observed after amplification in 27/29 (93%) tumour specimens. Estimates of mRNA levels in the 27 positive tumours ranged from 52 to 8000 amol/0.25 microgram total RNA, and were significantly higher in poorly/moderately differentiated tumours (P < 0.05) and in tumours with lymphatic vessel invasion (P < 0.01). There was no relationship with p53 accumulation or to the proliferation index. Our results show that GRP-R mRNA can be detected in most colorectal tumour specimens, and suggest a link between high mRNA levels and both tumour dedifferentiation and lymph vessel invasion, but not proliferation.

Galanin inhibits glucagon-like peptide-1 secretion through pertussis toxin-sensitive G protein and ATP-dependent potassium channels in rat ileal L-cells.

The neuropeptide galanin is widely distributed in the gastrointestinal tract and exerts several inhibitory effects, especially on intestinal motility and on insulin release from pancreatic beta-cells. The presence of galanin fibres not only in the myenteric and submucosal plexus but also in the mucosa, prompted us to investigate the regulatory role of galanin, and its mechanism of action, on the secretion of the insulinotropic hormone glucagon-like peptide-1 (GLP-1). Rat ileal cells were dispersed through mechanical vibration followed by moderate exposure to hyaluronidase, DNase I and EDTA, and enriched for L-cells by counterflow elutriation. A 6- to 7-fold enrichment in GLP-1 cell content was registered after elutriation, as compared with the crude cell preparation (929 +/- 81 vs 138 +/- 14 fmol/10(6) cells). L-cells then accounted for 4-5% of the total cell population. Bombesin induced a time-(15-240 min) and dose- (0.1 nM-1 microM) dependent release of GLP-1. Glucose-dependent insulinotropic peptide (GIP, 100 nM), forskolin (10 microM) and the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA, 1 microM) each stimulated GLP-1 secretion over a 1-h incubation period. Galanin (0.01-100 nM) induced a dose-dependent inhibition of bombesin- and of GIP-stimulated GLP-1 release (mean inhibition of 90% with 100 nM galanin). Galanin also dose-dependently inhibited forskolin-induced GLP-1 secretion (74% of inhibition with 100 nM galanin), but not TPA-stimulated hormone release. Pretreatment of cells with 200 ng/ml pertussis toxin for 3 h, or incubation with the ATP-sensitive K+ channel blocker disopyramide (200 microM), prevented the inhibition by galanin of bombesin- and GIP-stimulated GLP-1 secretion. These studies indicate that intestinal secretion of GLP-1 is negatively controlled by galanin, that acts through receptors coupled to pertussis toxin-sensitive G protein and involves ATP-dependent K+ channels.

CCK expression in enteroendocrine cell is regulated by soluble factor(s) from underlying fibroblasts.

Studies on the cross-talk between the intestinal epithelium and the underlying connective tissue have concentrated on enterocytes. In contrast, little is known about the interactions between the mesenchymal compartment and the enteroendocrine cells, scattered among the other cell types of the epithelium. To address this question, a panel of coculture systems between the enteroendocrine STC-1 cell line and three intestinal myofibroblastic cell lines (MIC) was used in order to assess different levels of regulation, namely cell-cell and cell-matrix interactions, and the role of diffusible factors. We demonstrate that the expression of cholecystokinin, a typical intestinal hormone produced by STC-1 cells, is up-regulated in the presence of a fibroblastic environment through a paracrine pathway involving FGF2. Concomitantly, STC-1 cell morphology and proliferation were also modulated, but through distinct mechanisms according to the origin of fibroblasts. The results reveal definite epithelio-mesenchymal interactions that may be critical for the maintenance of phenotype and function of enteroendocrine cells.

Variable stimulation of adenylate cyclase activity by vasoactive intestinal-like peptides and beta-adrenergic agonists in murine T cell lymphomas of immature, helper, and cytotoxic types.

We examined several cultured murine T cell lymphomas, induced by a radiation leukemia virus MuRadLV, including cell lines derived from immature T cells (5 clones of the BL/VL3 cell line), antigen-specific T helper cells (5 lines of the TL2 series), and one T cytotoxic cell line (NS8). With one exception (the TL2-9 cell line), these cells showed common characteristics: 1) an efficient adenylate cyclase system; 2) increased cyclic AMP production in response to at least one type of neurotransmitter, i.e., to the catecholamine isoproterenol and/or the neuropeptide VIP; 3) on the basis of adenylate cyclase stimulation, beta-adrenoceptors were of the beta 2 subtype and VIP receptors were of a "helodermin-preferring" subtype previously encountered in a human T lymphoblast cell line. Although we analyzed only a limited number of cell lines, it appeared that the immature T BL/VL3 clones responded to peptides of the VIP family with higher potency and efficacy than T helper and T cytotoxic cells. The membranes from the specific TL2-9 helper cell line were without adenylate cyclase activity in the presence of Gpp[NH]p, NaF, and GTP alone or GTP in the presence of isoproterenol or VIP. They produced cyclic AMP in the presence of Mn2+ and forskolin only, suggesting a defect in Gs as in S49 cyc- mouse lymphosarcoma cells. This was further demonstrated by the absence of cholera toxin-stimulated ADP-ribosylation in TL2-9 membranes.

Recovery of VIP/helodermin- and prostaglandin E1-stimulated adenylate cyclase activities in desensitized SUP-T1 human lymphoblasts.

VIP/helodermin receptors and PGE1 receptors coupled to adenylate cyclase underwent rapid homologous desensitization and/or down regulation in the human lymphoma SUP-T1 cell line: helodermin- and PGE1-stimulated adenylate cyclase activities in membranes decreased by 75% and 80%, respectively, after a 16-hr incubation of cells with 30 nM VIP or 0.1 microM PGE1. The adenylate cyclase response to helodermin doubled within 120 min of incubation with fresh medium, this part of the resensitization process being not significantly reduced by cycloheximide. The second slower phase of recovery attained 80% of control values after 8 hr and was significantly affected by cycloheximide added at time 0. These data were corroborated by our observations on [125I]helodermin binding to intact cells. In the case of functional PGE1 receptors, sixty percent of the adenylate cyclase response reappeared within 30-60 min, with the second phase of recovery leading, after 2-3 hr to 80-85% of control values of PGE1-stimulated enzyme activity. This resensitization process to PGE1 was, as a whole, cycloheximide sensitive.

Release of ileal neurotensin in the rat by neurotransmitters and neuropeptides.

To investigate the functional relationship between the enteric nervous system and the intestinal neurotensin (N) cells, the release of neurotensin (NT) was measured upon vascular 8-min infusion periods of various neurotransmitters and neuropeptides in an isolated vascularly perfused rat jejunoileum. NT-like immunoreactivity (NT-LI) was measured with an antiserum that specifically recognizes intact NT. The cholinergic agonists methacholine and carbachol produced a strong release of NT-LI (250% and 700% of basal, respectively at 10(-5) M). The infusion of a lower dose (10(-7) M) was less effective in both cases. The nicotinic receptor agonist DMPP (10(-4) M) had no significant effect on NT-LI release. Norepinephrine (10(-6) M) produced a moderate and well-sustained secretion of NT (200% of basal). Infusion of higher doses of these neurotransmitters dramatically increased the arterial pressure. G-amino-n-butyric acid (GABA), histamine, serotonin and dopamine administered at final concentrations up to 10(-5) M had no effect on NT-LI release. In contrast, gastrin-releasing peptide and bombesin induced a dose-dependent transient increase of portal NT-LI (maximal value at 10(-7) M: 1000% of basal) followed by a rapid return to near basal values. Substance P (10(-7) M) evoked a prompt release of NT-LI with a peak at 600% of basal followed by a decline to 200% of basal at the end of the session. Leu-enkephalin and calcitonin-gene-related-peptide (CGRP, 10(-7) M) produced a small rise in portal NT-LI, while Met-enkephalin, dynorphin, vasoactive intestinal peptide (VIP), galanin, neuropeptide Y (NPY), peptide histidine isoleucine (PHI), neuromedin U and thyrotropin releasing hormone (TRH) had no stimulatory effect. Our results indicate that additionally to the secretion of NT induced by cholinergic agents and bombesin, substance P and to a lesser extent Leu-enkephalin are capable of stimulating NT release in the rat.

Characterization of calcitonin gene-related peptide receptors and adenylate cyclase response in the murine macrophage cell line P388 D1.

Specific receptors for calcitonin gene-related peptide (CGRP) were identified and characterized on plasma membranes from the interleukin-1 secreting murine macrophage-like cells line P388 D1. The binding of [125I]-rat CGRP I was time-dependent, reversible and the rate of dissociation of [125I]-rat CGRP I increased in the presence of GTP. Scatchard analysis was consistent with a single class of binding sites, with an apparent dissociation constant of 1.76 nM and a maximal binding capacity of 85.48 fmol/mg protein. In competitive displacement studies, rat CGRP I, human CGRP I and human CGRP II were equipotent to inhibit the binding of [125I]-rat CGRP I (IC50 = 4 nM) while rat CGRP II and the synthetic analogue [tyr(o)]-human CGRP I were ten-fold less potent. Porcine calcitonin and VIP did not inhibit tracer binding. In the presence of GTP, CGRP stimulation of adenylate cyclase was dose-dependent and strongly correlated with receptor occupation. These results indicate that the P388 D1 macrophage-like cell line expresses CGRP specific receptors functionally coupled to adenylate cyclase, which may be involved in CGRP-mediated macrophage immune response.