A rare missense mutation in CHRNA4 associates with smoking behavior and its consequences.

Using Icelandic whole-genome sequence data and an imputation approach we searched for rare sequence variants in CHRNA4 and tested them for association with nicotine dependence. We show that carriers of a rare missense variant (allele frequency=0.24%) within CHRNA4, encoding an R336C substitution, have greater risk of nicotine addiction than non-carriers as assessed by the Fagerstrom Test for Nicotine Dependence (P=1.2 × 10(-4)). The variant also confers risk of several serious smoking-related diseases previously shown to be associated with the D398N substitution in CHRNA5. We observed odds ratios (ORs) of 1.7-2.3 for lung cancer (LC; P=4.0 × 10(-4)), chronic obstructive pulmonary disease (COPD; P=9.3 × 10(-4)), peripheral artery disease (PAD; P=0.090) and abdominal aortic aneurysms (AAAs; P=0.12), and the variant associates strongly with the early-onset forms of LC (OR=4.49, P=2.2 × 10(-4)), COPD (OR=3.22, P=2.9 × 10(-4)), PAD (OR=3.47, P=9.2 × 10(-3)) and AAA (OR=6.44, P=6.3 × 10(-3)). Joint analysis of the four smoking-related diseases reveals significant association (P=6.8 × 10(-5)), particularly for early-onset cases (P=2.1 × 10(-7)). Our results are in agreement with functional studies showing that the human α4ß2 isoform of the channel containing R336C has less sensitivity for its agonists than the wild-type form following nicotine incubation.

Genes associated with polymorphic variants predicting lung function are differentially expressed during human lung development.

BACKGROUND: Recent meta-analyses of genome-wide association studies have identified single nucleotide polymorphisms (SNPs) within/near 54 genes associated with lung function measures. Current understanding of the contribution of these genes to human lung development is limited. We set out to further define i) the expression profile of these genes during human lung development using a unique set of resources to examine both mRNA and protein expression and ii) the link between key polymorphisms and genes using expression quantitative trait loci (eQTL) approaches. METHODS: The mRNA expression profile of lung function associated genes across pseudoglandular and canalicular stages of lung development were determined using expression array data of 38 human fetal lungs. eQTLs were investigated for selected genes using blood cell and lung tissue data. Immunohistochemistry of the top 5 candidates was performed in a panel of 24 fetal lung samples. RESULTS: Twenty-nine lung function associated genes were differentially expressed during lung development at the mRNA level. The greatest magnitude of effect was observed for 5 genes; TMEM163, FAM13A and HHIP which had increasing expression and CDC123 and PTCH1 with decreased expression across developmental stages. Focussed eQTL analyses investigating SNPs in these five loci identified several cis-eQTL's. Protein expression of TMEM163 increased and CDC123 decreased with fetal lung age in agreement with mRNA data. Protein expression in FAM13A, HHIP and PTCH1 remained relatively constant throughout lung development. CONCLUSIONS: We have identified that > 50 % of lung function associated genes show evidence of differential expression during lung development and we show that in particular TMEM163 and CDC123 are differentially expressed at both the mRNA and protein levels. Our data provides a systematic evaluation of lung function associated genes in this context and offers some insight into the potential role of several of these genes in contributing to human lung development.

The UK prevalence of hereditary haemorrhagic telangiectasia and its association with sex, socioeconomic status and region of residence: a population-based study.

BACKGROUND: Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant genetic disorder of aberrant blood vessel development characterised by arteriovenous malformations. HHT is associated with significant morbidity due to complications including epistaxis, gastrointestinal bleeding and stroke. We explored the hypothesis that a diagnosis of HHT is associated with sex, socioeconomic status and geographical location. METHODS: We used The Health Improvement Network, a longitudinal, computerised general practice database covering 5% of the UK population to calculate prevalence estimates for HHT stratified by age, sex, socioeconomic status and geographical location. RESULTS: The 2010 UK point prevalence for HHT was 1.06/10 000 person years (95% CI 0.95 to 1.17) or 1 in 9400 individuals. The diagnosed prevalence of HHT was significantly higher in women compared with men (adjusted prevalence rate ratio (PRR) 1.53, 95% CI 1.24 to 1.88) and in those from the most affluent socioeconomic group compared with the least (adjusted PRR 1.74, 95% CI 1.14 to 2.64). The PRR varied between different regions of the UK, being highest in the South West and lowest in the West Midlands (adjusted PRR for former compared with latter 1.86, 95% CI 1.61 to 2.15). CONCLUSIONS: HHT prevalence is more common in the UK population than previously demonstrated, though this updated figure is still likely to be an underestimate. HHT appears to be significantly under-diagnosed in men, which is likely to reflect their lower rates of consultation with primary care services. There is under-diagnosis in patients from lower socioeconomic groups and a marked variation in the prevalence of diagnosis between different geographical regions across the UK that requires further investigation.

Genome-wide association study to identify genetic determinants of severe asthma.

BACKGROUND: The genetic basis for developing asthma has been extensively studied. However, association studies to date have mostly focused on mild to moderate disease and genetic risk factors for severe asthma remain unclear. OBJECTIVE: To identify common genetic variants affecting susceptibility to severe asthma. METHODS: A genome-wide association study was undertaken in 933 European ancestry individuals with severe asthma based on Global Initiative for Asthma (GINA) criteria 3 or above and 3346 clean controls. After standard quality control measures, the association of 480 889 genotyped single nucleotide polymorphisms (SNPs) was tested. To improve the resolution of the association signals identified, non-genotyped SNPs were imputed in these regions using a dense reference panel of SNP genotypes from the 1000 Genomes Project. Then replication of SNPs of interest was undertaken in a further 231 cases and 1345 controls and a meta-analysis was performed to combine the results across studies. RESULTS: An association was confirmed in subjects with severe asthma of loci previously identified for association with mild to moderate asthma. The strongest evidence was seen for the ORMDL3/GSDMB locus on chromosome 17q12-21 (rs4794820, p=1.03×10((-8)) following meta-analysis) meeting genome-wide significance. Strong evidence was also found for the IL1RL1/IL18R1 locus on 2q12 (rs9807989, p=5.59×10((-8)) following meta-analysis) just below this threshold. No novel loci for susceptibility to severe asthma met strict criteria for genome-wide significance. CONCLUSIONS: The largest genome-wide association study of severe asthma to date was carried out and strong evidence found for the association of two previously identified asthma susceptibility loci in patients with severe disease. A number of novel regions with suggestive evidence were also identified warranting further study.

Discoidin domain receptor 1 regulates bronchial epithelial repair and matrix metalloproteinase production.

Discoidin domain receptor (DDR)1 is an extracellular matrix (ECM)-sensing receptor tyrosine kinase, which is activated by collagen and expressed in bronchial epithelium. DDR1 is responsible for maintaining the normal structure of skin and kidney epithelia and we hypothesised that DDR1 plays a regulatory role in bronchial epithelial integrity by transducing signals from the airway ECM. Effects of DDR1 depletion were studied using RNA interference in primary human bronchial epithelial cells (HBECs) and BEAS-2B cells. The effects of overexpression of DDR1a and DDR1b in BEAS-2B cells were studied using a plasmid vector. We measured the effects on epithelial repair using a scratch wounding model, and levels of matrix metalloproteinases (MMPs) by gelatin zymography (MMP-2 and -9) and ELISA (MMP-7). We showed that knockdown of DDR1 slowed epithelial repair by 50%, which was associated with a reduction in levels of MMP-7, whilst DDR1 overexpression enhanced epithelial repair. DDR1 knockdown reduced proliferation of HBECs, but had no significant effect on adhesion to collagen I or other matrix substrates. These data suggest that ECM signalling via DDR1 regulates aspects of bronchial epithelial repair, integrity and MMP expression in the airways.

Polymorphisms in IL13 pathway genes in asthma and chronic obstructive pulmonary disease.

BACKGROUND: Asthma and chronic obstructive pulmonary disease (COPD) are chronic respiratory diseases involving an interaction between genetic and environmental factors. Interleukin-13 (IL13) has been suggested to have a role in both asthma and COPD. We investigated whether single nucleotide polymorphisms (SNPs) in the IL13 pathway may contribute to the susceptibility and severity of asthma and COPD in adults. METHODS: Twelve SNPs in IL13 pathway genes -IL4, IL13, IL4RA, IL13RA1, IL13RA2 and STAT6- were genotyped in subjects with asthma (n = 299) and in subjects with COPD or healthy smokers (n = 992). Genetic association was evaluated using genotype and allele models for asthma severity, atopy phenotypes and COPD susceptibility. Linear regression was used to determine the effects of polymorphism on baseline lung function (FEV(1), FEV(1)/FVC). RESULTS: In asthmatics, three IL13 SNPs - rs1881457(-1512), rs1800925(-1111) and rs20541(R130Q) - were associated with atopy risk. One SNP in IL4RA1 [rs1805010(I75V)] was associated with asthma severity, and several IL13 SNPs showed borderline significance. IL13 SNPs rs1881457(-1512) and rs1800925(-1111) were associated with better FEV(1) and FEV(1)/FVC in asthmatics. IL13 SNPs rs2066960(intron 1), rs20541(R130Q) and rs1295685(exon 4) were associated with COPD risk and lower baseline lung function in the recessive model. In females, but not in males, rs2250747 of the IL13RA1 gene was associated with COPD and lower FEV(1). CONCLUSION: These data suggest that IL13 SNPs (promoter and coding region) and, to a lesser extent, IL4RA SNPs may contribute to atopy and asthma. We also provide tentative evidence that IL13 SNPs in the coding region may be of significance in COPD susceptibility.

Positionally cloned asthma susceptibility gene polymorphisms and disease risk in the British 1958 Birth Cohort.

OBJECTIVE: The aim of this study was to estimate the contribution of polymorphisms in the positionally cloned asthma candidate genes ADAM33, PHF11, DPP10, GPRA and PTGDR to the risk of asthma, total and specific immunoglobulin E level, lung function and wheezing in a large, nationally representative, population. METHODS: An association analysis was undertaken using genotype data for tagging and previously associated single nucleotide polymorphisms (SNPs) in regions of these genes and longitudinal phenotype data from singletons of white ethnicity in the British 1958 Birth Cohort DNA archive (n = 7703). Population-attributable risk fractions for SNPs showing association were calculated. RESULTS: Polymorphisms producing small but statistically significant increases in asthma risk (OR 1.1 per allele) were identified in DPP10 and ADAM33, with the strongest evidence being for SNPs tagging the DPP10 gene. No individual SNP in any gene under study markedly increased risk for any of the phenotypes in the population studied. CONCLUSIONS: These data suggest that DPP10 and ADAM33 influence asthma risk in the UK population. However, the effects driven by any given locus are small, and genotyping of multiple polymorphisms in many genes will be needed to define a full genetic profile for disease risk.

Regulation of proliferation of human colonic subepithelial myofibroblasts by mediators important in intestinal inflammation.

An increase in myofibroblast number may be necessary for wound healing but may also lead to postinflammatory scarring. We have, therefore, studied the role of mediators important in inflammatory bowel disease in regulating proliferation of human colonic myofibroblasts. Using primary cultures of these cells, we have shown increases in [3H]thymidine incorporation in response to platelet-derived growth factor (EC50 = 14 ng/ml), basic fibroblast growth factor (EC50 = 2.2 ng/ml), and epidermal growth factor (EC50 = 1.1 ng/ml). Coulter counting of cell suspensions demonstrated increases in cell number with these growth factors along with insulin-like growth factor-I and -II. In addition the proinflammatory cytokines IL-1beta and TNF-alpha produced increases in [3H]thymidine incorporation. IL-1beta and platelet-derived growth factor together produced an increase in [3H]thymidine greater than either agonist alone; this effect was not, however, seen when we examined changes in cell numbers. Finally, we demonstrate a mechanism whereby these responses may be downregulated: vasoactive intestinal peptide (1 microM) elevates cyclic AwMP in these cells 4. 2-fold over control and produces a dose-related inhibition of platelet-derived growth factor-driven proliferation with a maximum inhibition of 33% at 1 microM.

Beta-adrenergic agonists regulate KCa channels in airway smooth muscle by cAMP-dependent and -independent mechanisms.

Stimulation of calcium-activated potassium (KCa) channels in airway smooth muscle cells by phosphorylation-dependent and membrane-delimited, G protein actions has been reported (Kume, H. A. Takai, H. Tokuno, and T. Tomita. 1989. Nature [Lond.]. 341:152-154; Kume, H., M. P. Graziano, and M. I. Kotlikoff. 1992. Proc. Natl. Acad. Sci. USA. 89:11051-11055). We show that beta-adrenergic receptor/channel coupling is not affected by inhibition of endogenous ATP, and that activation of KCa channels is stimulated by both alpha S and cAMP-dependent protein kinase (PKA). PKA stimulated channel activity in a dose-dependent fashion with an EC50 of 0.12 U/ml and maximum stimulation of 7.38 +/- 2.04-fold. Application of alpha S to patches near maximally stimulated by PKA significantly increased channel activity to 15.1 +/- 3.65-fold above baseline, providing further evidence for dual regulatory mechanisms and suggesting that the stimulatory actions are independent. Analysis of channel open-time kinetics indicated that isoproterenol and alpha S stimulation of channel activity primarily increased the proportion of longer duration events, whereas PKA stimulation had little effect on the proportion of short and long duration events, but resulted in a significant increase in the duration of the long open-state. cAMP formation during equivalent relaxation of precontracted muscle strips by isoproterenol and forskolin resulted in significantly less cAMP formation by isoproterenol than by forskolin, suggesting that the degree of activation of PKA is not the only determinant of tissue relaxation. We conclude that beta-adrenergic stimulation of KCa channel activity and relaxation of tone in airway smooth muscle occurs, in part, by means independent of cyclic AMP formation.

Cell adhesion and mechanical properties of a flexible scaffold for cardiac tissue engineering.

Cardiac tissue engineering is focused on obtaining functional cardiomyocyte constructs to provide an alternative to cellular cardiomyoplasty. Mechanical stimuli have been shown to stimulate protein expression and the differentiation of mammalian cells from contractile tissues. Our aim was to obtain a flexible scaffold which could be used to apply mechanical forces during tissue regeneration. Poly(1,8-octanediol-co-citric acid) (POC) is an elastomer that can be processed into scaffolds for tissue engineering. We investigated the effect of modifying the porosity on the mechanical properties of the POC scaffolds. In addition, the effects of the storage method and strain rate on material integrity were assessed. The maximum elongation of POC porous films varied from 60% to 160% of their original length. A decrease in the porosity caused a rise in this elastic modulus. The attachment of HL-1 cardiomyocytes to POC was assessed on films coated with fibronectin, collagen and laminin. These extracellular matrix proteins promoted cell adhesion in a protein-type- and concentration-dependent manner. Therefore, POC scaffolds can be optimised to meet the mechanical and biological parameters needed for cardiac culture. This porous material has the potential to be used for cardiac tissue engineering as well as for other soft tissue applications.

Severe hyponatraemia in medical in-patients: aetiology, assessment and outcome.

BACKGROUND: Hyponatraemia is the most commonly identified electrolyte abnormality. Published data on severe hyponatraemia in general medical in-patients is lacking. AIM: To determine the aetiology, adequacy of assessment, and outcome of severe hyponatraemia in general medical in-patients. DESIGN: Retrospective case-note review. METHODS: All general medical in-patients (n = 108) with serum sodium < or =125 mmol/l were identified from the clinical chemistry database, over a six-month period. A full review of notes and computer records was undertaken at the index date and a pre-determined follow-up date. RESULTS: Follow-up data were available in 105 patients. There was a wide range of aetiologies: diuretic therapy (loop and thiazide), congestive cardiac failure and liver disease were the most common, and 75.3% of patients had multiple causes. None of the 48% of patients whose history suggested a possible diagnosis of the syndrome of inappropriate anti-diuretic hormone (SIADH) met the generally accepted diagnostic criteria. Overall mortality was 20% during the index admission and 44.6% at follow-up, vs. 7.1% and 22%, respectively, for other patients admitted to the same directorate over the same time period (p < 0.001). Mortality was linked to aetiology, but not to reduced absolute serum sodium concentration at admission. DISCUSSION: Severe hyponatraemia in general medical patients is associated with a complex, multifactoral aetiology and a very poor prognosis. Outlook is governed principally by aetiology, and not by serum sodium level. Assessment of patients with hyponatraemia requires a practical clinical algorithm for diagnosing SIADH.

Translating Lung Function Genome-Wide Association Study (GWAS) Findings: New Insights for Lung Biology.

Chronic respiratory diseases are a major cause of worldwide mortality and morbidity. Although hereditary severe deficiency of α1 antitrypsin (A1AD) has been established to cause emphysema, A1AD accounts for only ∼ 1% of Chronic Obstructive Pulmonary Disease (COPD) cases. Genome-wide association studies (GWAS) have been successful at detecting multiple loci harboring variants predicting the variation in lung function measures and risk of COPD. However, GWAS are incapable of distinguishing causal from noncausal variants. Several approaches can be used for functional translation of genetic findings. These approaches have the scope to identify underlying alleles and pathways that are important in lung function and COPD. Computational methods aim at effective functional variant prediction by combining experimentally generated regulatory information with associated region of the human genome. Classically, GWAS association follow-up concentrated on manipulation of a single gene. However association data has identified genetic variants in >50 loci predicting disease risk or lung function. Therefore there is a clear precedent for experiments that interrogate multiple candidate genes in parallel, which is now possible with genome editing technology. Gene expression profiling can be used for effective discovery of biological pathways underpinning gene function. This information may be used for informed decisions about cellular assays post genetic manipulation. Investigating respiratory phenotypes in human lung tissue and specific gene knockout mice is a valuable in vivo approach that can complement in vitro work. Herein, we review state-of-the-art in silico, in vivo, and in vitro approaches that may be used to accelerate functional translation of genetic findings.

Variations in the subunit content and catalytic activity of the cytochrome c oxidase complex from different tissues and different cardiac compartments.

The composition and activity of cytochrome c oxidase (COX) was studied in mitochondria from rat liver, brain, kidney and heart and also in different compartments of the bovine heart to see whether any correlation exists between known oxidative capacity and COX activity. Immunoblot analysis showed that the levels of ubiquitously expressed subunits IV and Vb are about 8-12-fold lower in liver mitochondria as compared to the heart, kidney and brain. The heart enzyme with higher abundance of COX IV and Vb showed lower turnover number (495) while the liver enzyme with lower abundance of these subunits exhibited higher turnover number of 750. In support of the immunoblot results, immunohistochemical analysis of heart and kidney tissue sections showed an intense staining with the COX Vb antibody as compared to the liver sections. COX Vb antibody stained certain tubular regions of the kidney more intensely than the other regions suggesting region specific variation in the subunit level. Bovine heart compartments showed variation in subunit levels and also differed in the kinetic parameters of COX. The right atrium contained relatively more Vb protein, while the left ventricle contained higher level of subunit VIa. COX from both the ventricles showed high Km for cytochrome c (23-37 microM) as compared to the atrial COX (Km 8-15 microM). These results suggest a correlation between tissue specific oxidative capacity/work load and changes in subunit composition and associated changes in the activity of COX complex. More important, our results suggest variations based on the oxidative load of cell types within a tissue.

Association analysis of beta2 adrenoceptor polymorphisms with hypertension in a Black African population.

OBJECTIVE: To determine whether or not beta2 adrenoceptor polymorphism is a risk factor for the development of hypertension in a Black South African population. BACKGROUND: Attenuated vasodilator responses to endogenous catecholamines may contribute to the aetiology of hypertension. Downregulation of beta2 adrenoreceptors (beta2AR) following stimulation with agonists is determined in part by variation at the beta2AR gene locus. The Glu27 beta2AR genotype results in attenuated downregulation compared with the wild-type Gln27 receptor, whereas Gly16 exhibits enhanced down-regulation compared to Arg16. Possible racial differences in the prevalence of the beta2AR polymorphisms may be an explanation for the blunted responses to isoprenaline and the increased prevalence of hypertension in Black African populations. METHODS: One hundred and ninety-two unrelated hypertensives and 123 normotensives of Black South African origin were studied. Hypertensives were recruited from hospital hypertension clinics in the province of Gauteng and if on treatment, had a 2-4 week washout period before 24-h ambulatory blood pressure assessment Normotensive controls were recruited from the same community. RESULTS: There was no significant association between either the Arg-Gly16 polymorphism or the Gln-Glu27 polymorphism and hypertension status. Furthermore, in the hypertensives, no significant association was seen between beta2AR genotype at either site and clinical blood pressure, 24-h blood pressure or left ventricular mass. A significant association was seen between Arg16 homozygotes and lower body mass index in hypertensives (P = 0.007) although this was not a primary end point. Interestingly, the Glu27 polymorphism was much rarer in this population (allelic frequency 17%) compared to a Caucasian population. CONCLUSION: These data suggest that beta2AR polymorphism is not a risk factor for hypertension per se in this defined population. The possibility that the decreased prevalence of Glu27 in black South African populations explains blunted vasodilator responses to isoprenaline requires further study.

Beta-adrenoceptor stimulation inhibits histamine-stimulated inositol phospholipid hydrolysis in bovine tracheal smooth muscle.

1. Histamine and carbachol produced concentration-related increases in the accumulation of 3H-inositol phosphates in slices of bovine tracheal smooth muscle. 2. Noradrenaline alone produced a small stimulation of 3H-inositol phosphate accumulation which was inhibited by the alpha-adrenoceptor antagonist phentolamine. In contrast, when noradrenaline (0.1 mM) was added simultaneously with histamine it significantly reduced the inositol phosphate response to high (greater than or equal to 0.1 mM) concentrations of histamine. However, noradrenaline had no inhibitory effect on the carbachol-induced inositol phosphate response. 3. The non-selective beta-agonist isoprenaline (IC50 = 0.08 microM) and the beta 2-selective agonist salbutamol (IC50 = 0.29 microM) both produced a dose-related inhibition of the inositol phosphate response to 0.1 mM histamine. The inhibitory effect of salbutamol was antagonized by propranolol (KA = 2.4 x 10(9) M-1) and the beta 2-selective adrenoceptor antagonist ICI 118551 (KA = 1.7 x 10(9) M-1). 4. The accumulation of 3H-inositol phosphates induced by histamine increased steadily over a 40 min period after an initial lag period of 3-4 min. Following the simultaneous addition of histamine and salbutamol there was a further delay of 3-4 min before the appearance of the inhibitory effect of salbutamol. 5. The effect of histamine on inositol phosphate accumulation was accompanied by a stimulation of [3H]-inositol incorporation into membrane phospholipids which was reduced by the presence of salbutamol. However, when histamine was used to stimulate maximally [3H]-inositol incorporation during the prelabelling period, salbutamol produced a marked inhibition of histamine-stimulated 3H-inositol phosphate accumulation under conditions in which there was no change in the level of incorporation.

Regulation of histamine H1 receptor coupling by dexamethasone in human cultured airway smooth muscle.

1. The regulation of histamine-induced [3H]-inositol phosphate and intracellular calcium responses in human cultured airway smooth muscle cells was studied. 2. Histamine induced concentration-dependent [3H]-inositol phosphate formation (EC50 4 microM). This response was inhibited by a range of selective H1 receptor antagonists but not by the H2-selective antagonist, tiotidone or the H3 receptor-selective antagonist, thioperamide, indicating that an H1 receptor is involved in this response in human cultured airway smooth muscle cells. 3. Preincubation of human cultured airway smooth muscle cells with concentrations of dexamethasone > 10 nM for 22 h produced concentration-dependent inhibition of histamine-induced inositol phosphate formation. The maximum inhibition observed was 45% of the response in control cells. The inhibitory effect of dexamethasone was itself reversed by prior exposure to the glucocorticoid receptor antagonist, RU38486 (10 microM). Preincubation for 22 h with 1 microM dexamethasone produced inhibition of the inositol phosphate response to histamine to all concentrations of histamine inducing significant inositol phosphate formation in these cells. In contrast, the response to the G protein activator, NaF (0.1-20 mM) was unaltered by preincubation with dexamethasone. 4. Preincubation of human airway smooth muscle cells with 1 microM dexamethasone for time periods of < 6 h failed to inhibit histamine-induced inositol phosphate formation in human airway smooth muscle cells. 5. Histamine also induced concentration-dependent elevation of intracellular calcium levels in Fura 2-loaded human airway smooth muscle cells. This response was inhibited by preincubation with 1 microM dexamethasone. 6. We conclude that signal transduction through the H1 receptor in human airway smooth muscle is subject to regulation by dexamethasone and that this may in part account for the protective effect of dexamethasone against spasmogen-induced contractile responses in the airways.

Agonist-induced desensitization of histamine H1 receptor-mediated inositol phospholipid hydrolysis in human umbilical vein endothelial cells.

1. The regulation of histamine-induced [3H]-inositol phosphate formation was studied in human cultured umbilical vein endothelial cells (HUVEC). 2. Histamine (EC50 4.8 microM) produced a 12.7 fold increase in [3H]-inositol phosphate formation over basal levels. Prior exposure to 0.1 mM histamine (2 h) produced a 78% reduction in the response to subsequent histamine (0.1 mM) challenge. The IC50 for this histamine-induced desensitization was 0.9 microM. 3. The inositol phosphate response to histamine (0.1 mM) was inhibited by phorbol dibutyrate (IC50 40 nM; maximal reduction 64%). This effect was antagonized by both staurosporine (100 nM) and Ro 31-8220 (10 microM). However, the histamine-induced desensitization of the H1-receptor-mediated inositol phosphate response was insensitive to the protein kinase inhibitors, staurosporine, Ro 31-8220, K252a and KN62. 4. Prior exposure to sodium nitroprusside (100 microM), forskolin (10 microM) or dibutyryl cyclic AMP (1 mM) had no effect upon histamine-induced [3H]-inositol phosphate formation. 5. NaF (20 mM) and thrombin (EC50 0.4 u ml-1) also induced inositol phosphate formation in HUVEC. Histamine pretreatment (0.1 mM, 10-120 min) failed to modify the inositol phosphate response to a subsequent NaF or thrombin challenge. 6. We conclude that the desensitization of histamine H1-receptor-mediated [3H]-inositol phosphate formation occurs at the level of the receptor and involves a mechanism independent of activation of protein kinase A, G, or C, or calcium calmodulin-dependent protein kinase II.

Modulation of fluoroaluminate-induced inositol phosphate formation by increases in tissue cyclic AMP content in bovine tracheal smooth muscle.

1. The effect of fluoroaluminate complexes (AlCl3 plus NaF) upon smooth muscle tone, [3H]-inositol phosphate accumulation and [3H]-cyclic AMP accumulation has been investigated in slices of bovine tracheal smooth muscle. 2. Fluoroaluminate (10 microM AlCl3 + various concentrations of NaF) elicited concentration-dependent contractions of bovine tracheal smooth muscle strips at concentrations of NaF in the range 1-10 mM. The resultant contractile response was reversed by isoprenaline (50 nM) and was preserved in calcium-free medium. 3. Fluoroaluminate stimulated [3H]-inositol phosphate formation at concentrations of NaF over 1 mM. The response to 20 mM NaF + 10 microM AlCl3 was 164 +/- 29% of the response to 1 mM histamine. Fluoroaluminate also increased the incorporation of [3H]-myo-inositol into membrane phospholipids. 4. Fluoroaluminate produced a small rise in [3H]-cyclic AMP levels (2.1 fold increase over basal with 20 mM NaF). The response to forskolin (1 microM, 8.6 fold over basal) was reduced by fluoroaluminate in a concentration-dependent manner, but still remained significantly (P less than 0.05) elevated over the response to fluoroaluminate alone. 5. The [3H]-inositol phosphate response to fluoroaluminate was inhibited by salbutamol (maximum inhibition 60%, IC50 = 0.08 microM), forskolin (1 microM, 46% inhibition) and isobutylmethylxanthine (1 mM, 73% inhibition). 6. These data suggest that inhibition of agonist-induced inositol phospholipid turnover by cyclic AMP in this tissue can occur at the post-receptor level.

Inhibition of histamine-stimulated inositol phospholipid hydrolysis by agents which increase cyclic AMP levels in bovine tracheal smooth muscle.

1. The effect on histamine-stimulated [3H]-inositol phosphate accumulation of a range of agents which increase the accumulation, or mimic the actions, of cyclic AMP has been investigated in bovine tracheal smooth muscle. 2. Salbutamol (1 microM), forskolin (1 microM) and vasoactive intestinal peptide (VIP, 1 microM) inhibited the inositol phosphate response to 0.1 mM histamine and increased the accumulation of [3H]-cyclic AMP in [3H]-adenine-labelled slices of bovine tracheal smooth muscle. The effect on inositol phospholipid hydrolysis was mimicked by the membrane permeant analogues of cyclic AMP, dibutrylcyclic AMP (1 mM) and 8-bromo-cyclic AMP (1 mM). 3. In contrast to salbutamol, which was equally effective at producing the two effects, forskolin produced large increases in [3H]-cyclic AMP accumulation (EC50 = 1.2 microM) at much higher concentrations than those required for inhibition of histamine-stimulated [3H]-inositol phosphate accumulation (EC50 = 0.09 microM). However, significant increases in [3H]-cyclic AMP accumulation, of similar magnitude to those obtained with salbutamol and VIP, were observed over the concentration range appropriate for inhibition of the inositol phosphate response to histamine. 4. In the presence of histamine (0.1 mM), isobutylmethylxanthine (IBMX, 1 mM) and rolipram (0.1 mM) both significantly (P less than 0.05) elevated tissue [3H]-cyclic AMP levels. IBMX, rolipram and (to a lesser extent) SKF 94120 significantly (P less than 0.05) reduced histamine-stimulated [3H]-inositol phosphate accumulation by 81%, 68% and 20%, respectively. M&B 22948 was without a significant effect on either [3H]-cyclic AMP or histamine-induced [3H]-inositol phosphate accumulation. 5. Both rolipram and forskolin reduced the increase in incorporation of [3H]-inositol into membrane phospholipids which followed stimulation with histamine. However, a significant inhibition of [3H]-inositol phosphate accumulation could be demonstrated under conditions in which there was no change in the level of [3H]-inositol incorporation.