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Introduction: The diagnosis of bile acid diarrhea is often missed because the availability of the seleno-taurohomocholic acid (SeHCAT) test is limited. We aimed to compare the biomarkers 7α-hydroxy-4-cholesten-3-one (C4) and fibroblast growth factor 19 (FGF19) with the SeHCAT test., Methods: Patients with chronic diarrhea without intestinal resection referred for SeHCAT were prospectively recruited for this diagnostic accuracy study. Blood was sampled at fasting and after a stimulation meal with chenodeoxycholic acid. SeHCAT retention ≤10% defined bile acid diarrhea and >10% defined miscellaneous diarrhea. Receiver operating characteristics (ROC) were analyzed with SeHCAT as the gold standard. www.clinicaltrials.gov (NCT03059537)., Results: Patients with bile acid diarrhea (n = 26) had mean C4 of 30 ng/mL (95% confidence interval: 19-46) vs 8 (7-11; P < 0.001) in the miscellaneous diarrhea group (n = 45). Area under the ROC curve (ROCAUC) for C4 was 0.83 (0.72-0.93). C4 < 15 ng/mL had 85% (74%-96%) negative predictive value; C4 > 48 ng/mL had 82% (59%-100%) positive predictive value. Twenty patients had C4 values 15-48 ng/mL, of whom 11/20 had SeHCAT ≤10%. Median fasting FGF19 was 72 pg/mL (interquartile range: 53-146) vs 119 (84-240) (P = 0.004); ROCAUC was 0.71 (0.58-0.83). Stimulated FGF19 responses did not differ (P = 0.54)., Discussion: We identified C4 thresholds with clinically useful predictive values for the diagnosis of and screening for bile acid diarrhea in patients with chronic watery diarrhea. Further validation of the cutoff values with the placebo-controlled effect of sequestrant therapy is warranted (see Visual Abstract, Supplementary Digital Content 2, http://links.lww.com/AJG/B603).

Background: Bile acid diarrhoea is underdiagnosed and better diagnostic tests are needed. Fasting serum fibroblast growth factor-19 (FGF19) has insufficient diagnostic value, but this may be improved by stimulation., Aim: To explore if an impaired FGF19 response identifies primary bile acid diarrhoea., Methods: Eight patients with primary bile acid diarrhoea and eight healthy volunteers ingested (i) a meal plus 1250 mg chenodeoxycholic acid (CDCA), (ii) 1250 mg CDCA or (iii) the meal. Blood was sampled at fasting and repeatedly after stimulation. We analysed FGF19 by enzyme-linked immunosorbent assay and bile acids including 7α-hydroxy-4-cholesten-3-one by liquid chromatography-tandem mass spectrometry., Results: Stimulation with the meal plus CDCA increased median FGF19 in healthy volunteers from fasting 62 pg/mL [interquartile range (IQR): 41-138] to 99 pg/mL (IQR: 67-147; P = 0.012) after 90 min and peaked after 150 min at 313 pg/mL (IQR: 54-512). This response was impaired in primary bile acid diarrhoea patients [fasting 56 pg/mL (IQR: 42-79); 90 min: 48 pg/mL [IQR: 37-63); 150 min: 57 pg/mL (48-198)]. Receiver operating characteristics (ROC AUC ) for fasting FGF19 was 0.55 (P = 0.75) and at 90 min 0.84 (P = 0.02). The difference in FGF19 from fasting to 90 min after the meal plus CDCA separated the groups (ROC AUC 1.0; P = 0.001). 7α-hydroxy-4-cholesten-3-one was elevated in primary bile acid diarrhoea (P = 0.038) and not significantly affected by stimulation., Conclusions: The FGF19 response following chenodeoxycholic acid plus meal is impaired in primary bile acid diarrhoea. This may provide a biochemical diagnostic test., (© 2017 John Wiley & Sons Ltd.)

Background: Patients with cystic fibrosis (CF) have a higher prevalence of asthma than the background population, however, it is unclear whether heterozygous CF carriers are susceptible to asthma. Given this, a meta-analysis is necessary to determine the veracity of the association of CF heterozygosity with asthma., Methods: We screened the medical literature from 1966 to 2015 and performed a meta-analysis to determine the risk of asthma in CF heterozygotes vs. non-carriers., Results: Aggregating data from 15 studies, the odds ratio for asthma in CF heterozygotes compared with non-carriers was significantly elevated at 1.61 (95% CI: 1.18-2.21). When analyzing the studies considered of high quality in which asthma was diagnosed by a physician, the patients were >18years, or study size was ≥500, the trend remained the same, that heterozygous carriers of CF had elevated risk for asthma., Conclusions: The results show that heterozygous carriers for CF have a higher risk of asthma than non-carriers., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Purpose: A 15-gene hypoxia profile has previously demonstrated to have both prognostic and predictive impact for hypoxic modification in squamous cell carcinoma of the head and neck. This gene expression profile may also have a prognostic value in other histological cancer types, and could potentially have a function as a universal hypoxia profile. The hypoxia induced upregulation of the included genes, and the validity of the previously used reference genes was established in this study, in a range of different cell lines representing carcinomas of the prostate, colon, and esophagus., Materials and Methods: Eleven adenocarcinoma and one squamous cell lines: Six colon carcinomas (HTC8, HT29, LS174T, SW116, SW948 and T48), 3 prostate carcinomas (LNCaP, DU-145 and PC-3) and 3 esophagus carcinoma cell lines (OE19, OE21 and OE33) were cultured under normoxic or hypoxic conditions (0% O2) for 24hours. Total RNA was extracted and gene expression levels measured by qPCR. For each tissue type, individual reference genes were selected and applied in the normalization of the relative expression levels., Results: In all three tissue types, individual, optimal, reference genes were selected. In the analysis of the hypoxia induced genes, both the original reference genes and the new selected reference genes were used. There was no significant difference in the obtained data. The gene expression analysis demonstrated cell line specific differences in the hypoxia response of the 15 genes, with BNIP3 not being upregulated at hypoxic conditions in 3 out of 6 colon cancer cell lines, and ALDOA in OE21 and FAM162A and SLC2A1 in SW116 only showing limited hypoxia induction. Furthermore, in the esophagus cell lines, the normoxic and hypoxic expression levels of LOX and BNIP3 were below the detection limit in OE19 and OE33, respectively. However, a combined analysis of the 15 genes in both adenocarcinoma cell lines and squamous carcinoma cell lines demonstrated a very consistent expression pattern in hypoxic induced gene expression across all cell lines., Conclusion: This study addressed the tissue type dependency of hypoxia induced genes included in a 15-gene hypoxic profile in carcinoma cell lines from prostate, colon, and esophagus cancer, and demonstrated that in vitro, with minor fluctuations, the genes in the hypoxic profile are hypoxia inducible, and the hypoxia profile may be applicable in other sites than HNSCC., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Objective: To examine 24-h blood pressure (24BP), systemic haemodynamics and the effect of sodium intake on 24BP in obese patients before and after gastric bypass surgery [laparoscopic Roux-en-Y gastric bypass (LRYGB)], and to determine whether weight loss from LRYGB might be related to an increase in plasma concentrations of atrial natriuretic peptide., Methods: Twelve hypertensive and 12 normotensive morbidly obese patients underwent LRYGB: 24BP, systemic haemodynamics and mid-regional pro-atrial natriuretic peptide (MRproANP) were assessed before, 6 weeks and 12 months after surgery. The effect of high versus low sodium intake on 24BP was evaluated before and 12 months after LRYGB., Results: Six weeks after LRYGB, the average weight loss was 20 kg, with a further 21 kg weight loss 1 year after surgery. In hypertensive patients, 24BP was significantly reduced at 6 weeks, but not 1 year after LRYGB. However, antihypertensive medications were successively reduced from baseline to 1 year after surgery. In normotensive patients, there was no change in 24BP 6 weeks after LRYGB, but a tendency towards a reduction 1 year after the operation. Plasma concentrations of MRproANP were subnormal prior to surgery in hypertensive patients and increased by 77% 1 year after the operation. In normotensive patients, preoperative concentrations were normal and increased only by 6%. High sodium intake induced plasma volume expansion, increased stroke volume and cardiac output, but no significant change in 24BP - neither before nor after LRYGB., Conclusions: LRYGB resulted in a significant 24BP reduction and a substantial increase in MRproANP plasma concentrations in hypertensive, obese patients 6 weeks after surgery, suggesting a causal link between obesity-hypertension and altered release/degradation of cardiac natriuretic peptides.

Objective. To investigate if antibodies towards biological TNF-α inhibitors (anti-TNFi Abs) are present in patients with rheumatoid arthritis (RA) in clinical remission and to relate any anti-TNFi Abs to circulating level of TNF-α inhibitor (TNFi). Methods. Patients with RA, treated with infliximab or adalimumab, and in clinical remission (DAS28(CRP) < 2.6) were included from 6 out-patient clinics. In blood samples, presence of anti-TNFi Abs was determined by radioimmunoassay, and concentration of bioactive TNFi was measured by a cell-based reporter gene assay. Results. Anti-TNFi Abs were present in 8/44 patients (18%) treated with infliximab and 1/49 patients (2%) treated with adalimumab (p = 0.012). In the former group, anti-TNFi Abs corresponded with low levels of TNFi (p = 0.048). Anti-TNFi Ab-positive patients had shorter disease duration at initiation of TNFi therapy (p = 0.023) but were similar for the rest of the compared parameters. Conclusions. In RA patients in clinical remission, anti-TNFi Abs occur frequently in patients treated with infliximab, while they occur rarely in patients treated with adalimumab. Presence of anti-infliximab Abs is accompanied by low or undetectable levels of infliximab. These data suggest that continued infliximab treatment may be redundant in a proportion of RA patients treated with infliximab and in clinical remission.

Introduction: The revised Scandinavian Neurotrauma Committee (SNC) guidelines on management of patients with head trauma include an option for measurement of S100B in peripheral blood with 100% sensitivity for neurosurgical intervention. A medical technology assessment was conducted to evaluate any impact of using S100B on the use of computed tomographies (CT) of the brain and admission for observation., Material and Methods: Patients referred for assessment of head injury over a period of 1.5 months had their blood sampled for measurement of S100B in serum. Results were not available to the treating physician and treatment was conducted according to existing practice. Patient records were reviewed retrospectively and post hoc divided into two groups depending on whether the SNC criteria for taking the blood sample were met. The use of CT and admission was analysed., Results: A total of 39 patients had their blood sampled for analysis. In all, 12 patients were excluded in pursuance of SNC guidelines, which left 27 patients for analysis. A total of 15 patients had abnormally high S100B levels. Using the SNC criteria, only eight of these qualified a priori for blood sampling. Furthermore, seven of the 11 patients who were admitted had normal S100B levels., Conclusion: The number of patients with an above-threshold concentration of S100B was almost equally distributed between those fulfilling the SNC criteria for S100B assessment and those who could have been discharged without further evaluation. Using S100B as a screening tool may lead to an increase in the use of CTs of the brain. In relation to admission, measurement of S100B may contribute to the adoption of an appropriate observation strategy., Funding: not relevant., Trial Registration: not relevant.

Objectives: To summarize the empirical evidence regarding the effect of treatment intensification on clinical outcomes in patients with rheumatoid arthritis treated with one of the TNF-α-inhibitors, adalimumab, etanercept or infliximab., Methods: A systematic search of the bibliographic databases Embase, Medline, Web of Science and Cochrane Central identifying articles concerning treatment with adalimumab, etanercept or infliximab in adult patients with rheumatoid arthritis exposed to dose increase or shortening of dosing intervals was performed. Longitudinal cohorts, both clinical trials and observational studies, were included. ACR and EULAR response criteria and DAS28 were the preferred outcome measures., Results: Out of 1135 records, eleven studies were included in the final evidence synthesis. One article concerned all the three TNF-α-inhibitors, eight used infliximab, one adalimumab and one etanercept. According to GRADE, evidence was weakened in particular by the lack of control groups, and for treatment intensification with adalimumab and etanercept, no conclusions could be drawn. With infliximab, two trials of high quality revealed contradictory results, but six studies described an improved clinical outcome following intensified treatment strategies. Some studies (2/2) also indicated that for infliximab, frequency increase was superior to dose increase., Conclusions: Available studies indicate that intensifying treatment with infliximab in rheumatoid arthritis patients, preferably by increasing the frequency of drug administration, may lead to improved clinical outcome in some patients, but the evidence is weak. There is an urgent need for prospectively designed cohort studies to be able to draw a final conclusion., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aim: To investigate the effect of 4,5-dichloro-1,3-diethyl-1,3-dihydro-benzoimidazol-2-one (NS4591), a novel SK/IK channels positive modulator, on human myometrial activity., Methods: Organ bath studies were performed on myometrial preparations obtained from women undergoing elective caesarean section at term (N = 11) or hysterectomy (N = 11). NS4591 was added cumulatively in the concentration range of 0.3-30 μm. In separate experiments, the effects of pre-incubation of muscle preparation with the SK or IK channel blockers apamin (1 μm) and TRAM34 (10 μm) on the outcomes of NS4591 were evaluated. Simultaneous vehicle controls were performed for all experiments. The effects of drugs were studied on spontaneous contractions., Results: NS4591 exerted an inhibitory effect on myometrial contractions in muscle strips from non-pregnant and pregnant women. The contractility in non-pregnant and pregnant myometrium was reduced to the following values respectively: amplitude 20.65 ± 7.38% (P < 0.001) and 42.85 ± 11.04% (P < 0.05) and area under the curve 11.72 ± 7.39% (P < 0.001) and 34.84 ± 10.50% (P < 0.001) and are reflective of 30 μm NS4591 compared to vehicle control. In non-pregnant tissue, apamin partially reduced the inhibitory effects of NS4591, but we observed relaxation mediated by NS4591 despite pre-incubation with TRAM34. In contrast, in pregnant tissue, neither apamin nor TRAM34 could reverse the relaxatory effects of NS4591., Conclusion: Our findings imply that SK/IK channels are present and functional in myometrium from pregnant and non-pregnant women. The SK/IK channel-positive modulator NS4591 exerts relaxation of human myometrium in vitro, and this may have implications for the clinical management of preterm labour., (© 2011 The Authors Acta Physiologica © 2011 Scandinavian Physiological Society.)

In the present study, we investigated the effect of interferon-gamma (IFN-gamma) on cellular inositol phosphate formation and cellular calcium ion concentration [Ca2+]i in human renal proximal tubular (HRPT) cells. We also examined the possible role of the inositol phosphate-Ca2+ signalling pathway during IFN-gamma-induced intercellular adhesion molecule-1 (ICAM-1) antigen expression. IFN-gamma caused an increase in the formation of inositol 1-monophosphate (Ins 1-P), inositol 1,4-bisphosphate (Ins 1,4-P2), inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) and inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5-P4). A rapid time-dependent rise in [Ca2+]i was observed upon IFN-gamma stimulation, with maximal levels reached after 1 min. A lower rise in [Ca2+]i was observed when cells were stimulated in Ca2+-free medium. This correlated with the generation of Ins 1,4,5-P3 by IFN-gamma, a well-known secondary messenger capable of releasing Ca2+ from intracellular stores. The induction of ICAM-1 antigen expression was enhanced by IFN-gamma, 4-bromocalcium ionophore A23187 (Bromo-A23187), and their combinations. However, the calcium antagonist diltiazem and calcium chelator EGTA had no effect on IFN-gamma antigen induction. In conclusion, our data suggest that IFN-gamma stimulation of HRPT cells results in the cleavage of phosphatidylinositol bisphosphate by phospholipase C, generating inositol phosphates, of which Ins 1,4,5-P3 probably releases Ca2+ from intracellular stores. A further increase in [Ca2+]i upon IFN-gamma stimulation results from influx of extracellular Ca2+. IFN-gamma signal transduction in HRPT cells may not be limited to the inositol phosphate-Ca2+ pathway since IFN-gamma-induced ICAM-1 antigen expression was unaffected by calcium antagonist/chelator.

Objective: To investigate the leukotriene B4 (LTB4) signal transducing mechanism in polymorphonuclear neutrophils (PMNs) from patients with Crohn's disease., Methods: Cytosolic free calcium ([Ca2+]i), inositol (1,4,5)-trisphosphate [(1,4,5)-IP3] chemotaxis, LTB4 receptor number and affinity were investigated in peripheral PMNs from 11 patients with Crohn's disease and 11 healthy controls., Results: There was a slight reduction (P = 0.31) in the number of LTB4 receptor sites per cell expressed on PMNs (mean Bmax 931) from nine of the 11 patients studied compared with the healthy controls (mean Bmax 1095). LTB4-mediated (1,4,5)-IP3 formation and the increase in [Ca2+]i were markedly decreased in PMNs from the 11 patients with Crohn's disease [(1,4,5)-IP3, mean +/- SEM 12 +/- 0.84 and 27.4 +/- 1.4 pmol/l/tube for patients and controls, respectively; [Ca2+]i, mean +/- SEM 295 +/- 2.75 and 598 +/- 4.7 nmol/l for patients and controls, respectively]. The decrease in calcium might be related to the decrease in Bmax (P < 0.05). Ionomycin, a calcium ionophore which bypasses the initial steps of LTB4 receptor activation, showed only a minor difference in peak [Ca2+]i between PMNs from patients and controls. LTB4-directed chemotaxis showed that the sensitivity to suboptimal concentrations of LTB4 (1.0 nmol/l) was significantly depressed in PMNs from patients (P < 0.05)., Conclusion: Peripheral PMNs from patients with Crohn's disease had a small deficit in the expression of LTB4 receptors. This deficiency was paralleled by marked alterations in cellular signalling. Whether these results are specific to Crohn's disease or simply result from the exposure of circulating PMNs to elevated levels of LTB4 remains to be established.

Activated polymorphonuclear leukocytes (PMNs), which are found in the inflammatory lesions of chronic inflammatory bowel disease, produce tissue-destructive oxygen-derived free radicals. The influence of 5-aminosalicylic acid (5-ASA), its acetylated metabolite (Ac-5-ASA), sulfasalazine (SAZ), and olsalazine (OLZ) (5-ASA dimer linked by an azo group) in pharmacologically relevant concentrations (0.1-10 mM) were tested on PMN superoxide production with either the receptor-specific agent formyl-methionyl-leucyl-phenylalanine (fMLP) or the protein kinase C activator phorbol myristate acetate (PMA). Inhibition of receptor-specific superoxide production occurred at 0.07, 0.32, and 0.63 mM (IC50 values) for 5-ASA, SAZ, and OLZ, respectively. No inhibitory effects of SAZ and OLZ were observed when PMA was applied as stimulus for PMN superoxide production. The results indicate that the signal to which PMNs respond by generating superoxide is primarily due to calcium release from intracellular stores. They further suggest that SAZ and OLZ may affect the oxygen-derived free radical production in human PMNs by unspecific cytotoxicity or by interference with the nicotinamide adenine dinucleotide phosphate, reduced (NADPH) oxidase system, whereas 5-ASA itself is a free radical scavenger.

Incubation of the human renal carcinoma cell line CaKi-1 with interferon-gamma (IFN-gamma) or the phorbol ester, phorbol-12-myristate 13-acetate (PMA) strongly stimulated the immunocytochemical expression of the intercellular adhesion molecule-1 (ICAM-1) in a dose-dependent manner. Since PMA is capable of activating the Ca2+/phospholipid-dependent protein kinase C (PKC), we investigated the role of this kinase during IFN-gamma signal transduction. Calcium ionophore A23187 significantly enhanced IFN-gamma- and PMA-induced ICAM-1 staining. While staurosporine, H7 and sphingosine, three known PKC inhibitors, blocked the PMA effect, only staurosporine abrogated the action of IFN-gamma. Finally, 24 h of PMA pretreatment with subsequent IFN-gamma stimulation enhanced ICAM-1 staining above values from cultures where IFN-gamma was omitted. This occurred despite the fact that 24 h of PMA pretreatment abolished the effect of IFN-gamma on PKC activation, as determined by acetylated myelin basic protein 4-14 phosphorylation. In conclusion, these results suggest that additional events other than PKC activation are required for complete regulation of ICAM-1 antigen by IFN-gamma in the whole cell population. Hence, other Ca(2+)-dependent signalling pathway(s) mediated by IFN-gamma receptors must act. Further studies are needed to elucidate these specific pathway(s) activated during IFN-gamma stimulation in our model.

We have recently shown that interferon-gamma (IFN-gamma) stimulated immunocytochemical staining of the intercellular adhesion molecule ICAM-1 may be dependent on inositol phosphate formation in the human renal carcinoma cell line CaKi-1. In the present study we investigated the possible role of GTP-binding proteins (G-proteins) during IFN-gamma signalling. Preincubation of CaKi-1 cells for 24 h with increasing amounts of pertussis toxin (PT) or cholera toxin (CT), two regulators of G-protein activity, inhibited IFN-gamma induced ICAM-1 staining. Preincubation with PT or CT for 24 h also inhibited IFN-gamma induced inositol 1-monophosphate (Ins 1-P), inositol 1,4 bisphosphate (Ins 1,4-P2) and inositol 1,4,5 trisphosphate (Ins 1,4,5-P3) formation. Our findings suggest that IFN-gamma induced ICAM-1 staining and inositol phosphate formation in CaKi-1 cells is dependent on a PT and CT sensitive signalling pathway. This may reflect a role for G-proteins in the coupling of IFN-gamma receptor activation and phospholipase C catalyzed phosphoinositide hydrolysis.

Quantitative Fluorescence Microscopy is a powerful analytical tool for the in vivo determination of molecules and ions concentrations in cell populations. Until recently the results obtained represent the average value from a large number of cells under the microscope. The recent development of ratiometric ion-sensitive fluorochromes has provided us with a molecular stopwatch, with which we can monitor microscopic and submicroscopic cellular events in individual cells. These probes can be introduced into the cytoplasm of living cells and as the ionic concentration of, i.e., calcium and hydrogen in the cell interior changes, the dyes undergo changes in fluorescence consisting of wavelength shifts and quantum efficiency. Combining this powerful new technique with ultra-sensitive low-light-level video cameras and digital image processing to the fluorescence microscope permits the study of both spatial and temporal distribution of ions localized on or within intact single cells. Details of the procedures and the equipment required for such a fluorescence ratio imaging system are described. A review of some of the fluorescent probes used for measuring intracellular free Ca2+ and pH will be illustrated with examples of data obtained with a Digital Image Processing System in our laboratory. The use of quantitative video fluorescence microscopy is proposed as a means of studying intracellular chemical pathology both for the dynamic investigations of intracellular processes and for diagnosis of diseases.

Melittin, the predominant fraction of bee venom proteins, was studied in an experimental model of human neutrophil granulocytes to reveal its influence on eicosanoid release, metabolism and receptor function in relation to intracellular calcium metabolism. Melittin (2 mumol/l) was as potent as the calcium ionophore A23187 (10 mumol/l) for activation of 5-lipoxygenase, releasing arachidonate only from phosphatidyl-choline and phosphatidyl-ethanolamine of cellular membranes, as judged from the decreases in radioactivity by 15.4% and 30.5%, respectively. The mechanism responsible for the release of arachidonate from cellular membranes is closely coupled to cellular calcium metabolism, and melittin was found to promote calcium entry through receptor gated calcium channels, probably due to an activation of phospholipase A(2). Furthermore, a down-regulation of leukotriene B(4) receptors was seen. The maximal number of binding sites per cell was reduced from a median of 1520 to 950 with melittin (1 mumol/l). The study has revealed some factors important for the inflammatory mechanisms mediated by melittin.

Previous work from this laboratory has demonstrated that stimulation of human polymorphonuclear leukocytes (PMN) with the peptidyl leukotriene D4 (LTD4) results in the formation of second messenger signals, i.e. mobilization of intracellular free Ca2+ ([Ca2+]i) and hydrolysis of phosphoinositides (PIP2). Based on these earlier results we have employed radioligand binding techniques to study the presence of LTD4 receptors in intact human PMN leukocytes. The binding of [3H]-LTD4 to LTD4 receptors is rapid, reversible, specific and saturable. Scatchard analysis of the binding data indicates the presence of 116-275 identical receptors per neutrophil with an apparent dissociation constant (KD) of 1,1-2,3 nM. Only one class of binding sites was identified. The LTD4 receptors are located in the plasma membrane and are specific for LTD4 since binding is unaffected by other leukotrienes. Furthermore, LTD4 induces a rapid and persistent translocation of Protein Kinase C (PKC) from the cytosol to the membrane. The LTD4 binding and PKC translocation could be blocked in a concentration dependent manner by the new and specific LTD4 receptor antagonist ICI 198,615. These observations strongly suggest that human PMN might possess specific LTD4 receptors which are coupled to the inositol trisphosphate pathway resulting in a rise in the cytoplasmic free Ca2+ and redistribution of protein kinase C. A mechanism of signal transduction for leukotriene D4 is proposed.

Interleukin 1 (IL-1) exerts both stimulatory and inhibitory (cytotoxic) effects on insulin-producing beta cells in isolated pancreatic islets. Since alteration in ion fluxes is crucial for endocrine cell activation and is a denominator of cell death, and since IL-1 was recently shown to increase the total sodium content in a murine pre-B-lymphocyte cell line, we investigated the effect of recombinant human IL-1 beta (rhIL-1 beta) on the cytosolic free sodium concentration (fNa+i) in rat islets. Furthermore, long-term rhIL-1 beta effects on islet cell function were studied during exposure of islets to amiloride, a blocker of the plasma membrane Na+/H+ exchange. One hour of islet exposure to 60 U/ml of rhIL-1 beta caused a threefold increase in fNa+i in islet cells, and this effect was abolished by depletion of extracellular sodium. Blockade of Na+/H+ exchange with amiloride abolished the inhibitory effect of rhIL-1 beta on insulin release. In conclusion, rhIL-1 beta was found to increase sodium influx in pancreatic islet cells. This might underlie the widespread effects of rhIL-1 beta on beta-cell function and morphology, possibly related to IL-1-mediated toxic free radical formation.

LTD4 increased the level of free intracellular calcium ([Ca++]i) and stimulated the production of inositol phosphates (IP) in human polymorphonuclear neutrophils (PMN). Calcium was predominantly mobilized from intracellular pools. After a single stimulus, the cells were refractory to a second challenge with the same concentration of LTD4, but the calcium response to LTB4 was normal. The rise in [Ca++]i as well as the stimulated production of IP was inhibited by the novel LTD4 antagonist, SR2640. SR2640 also abolished the attenuation by LTD4 of LTB4-directed PMN chemotaxis. The results suggest that human PMN contain specific LTD4 receptor that trigger phosphatidyl inositol hydrolysis by activation of phospholipase C, leading to intracellular calcium mobilization, which may be involved in modulation of chemotaxis.

Stimulation of murine L-M and 3T3 fibroblasts with human recombinant tumour necrosis factor (rTNF) resulted in an increase in the cytosolic free Ca2+ concentration ([Ca2+]i). In 3T3 cells rTNF also induced release and metabolization of arachidonic acid, whereas in L-M cells rTNF provoked rapid increases in the levels of inositol mono-, bis- and trisphosphates (IP1, IP2 and IP3). In these cells the Ca2+ response was also observed in Ca2+ free medium, suggesting that rTNF promotes mobilization of Ca2+ from intracellular stores. In 3T3 cells, however, Ca2+ originated from the extracellular space, since the response was abolished in medium containing 1 mM EGTA. Both rTNF-induced calcium responses were inhibited by a specific rabbit IgG antibody to rTNF but not by 1-verapamil, a blocker potential-operated calcium channels. These results suggest that increased formation of inositol phosphates, arachidonic acid release and increased cytosolic free Ca2+ are involved in the biological effects of rTNF. However, rTNF generate these signals by different mechanisms depending upon the target cell.

Changes in the concentration of cytosolic free calcium ((Ca2+)i) in response to purified blood monocyte IL-1 and human rIL-1 alpha and rIL-1 beta (17.5 kDa) were measured in murine L-M fibroblasts and in human foreskin fibroblasts using the fluorescent Ca2+ indicator, fura-2. In L-M fibroblasts, each of these IL-1 species, but not a recombinant 24-kDa precursor of the predominant IL-1 beta, produced a prompt, dose-related, and transient increase in (Ca2+)i. The effect was smaller but not eliminated when the cells were stimulated in EGTA-containing calcium, suggesting that the rise in (Ca2+)i was due to influx from both intracellular and extracellular Ca2+ pools. In human fibroblasts, however, the (Ca2+)i increased gradually, reaching a maximum after 1 hr of incubation with IL-1 and returning slowly to near basal levels in the following 2 hr. In contrast to the L-M cells, this accumulation of Ca2+ was abolished by EGTA, suggesting that in human fibroblasts, Ca2+ is mobilized solely from the extracellular space. Addition of the Ca2+ channel blockers verapamil and nifedipine was ineffective. IL-1 alpha and IL-1 beta both induced a dose-related increase in prostaglandin E2, but only in the human fibroblasts. These findings indicate that an increase in (Ca2+)i may be an important second mediator by which IL-1 initiates cell activation, but the signal may differ between cells.

Rabbit gall-bladder epithelial cells were isolated by a combination of Ca2+ omission, enzymatic treatment, and mechanical detachment and had a viability of 96-98% and well preserved morphology. Measurements of cytosolic free Ca2+ concentration ([Ca2+]i) in these cells with the Ca2+-fluorescent indicator fura-2 demonstrated a resting [Ca2+]i level of 115 +/- 12 nM. When used in concentrations which inhibit rabbit gall-bladder isosmotic NaCl absorption (1-100 microM), the Ca2+-channel activator BAY K 8644 caused a dose-dependent increase in the epithelial [Ca2+]i to a maximal value of 850 nM. The effect was dependent on extracellular Ca2+, and was not altered by 1 microM L-verapamil. Depolarization of the epithelial cells with KCl had no effect on [Ca2+]i. The results suggest that BAY K 8644 activates a Ca2+ influx which is not dependent on voltage-gated channels. Cytosolic Ca2+ may be involved in the regulation of isosmotic NaCl absorption in the mammalian gall-bladder.