Potential costs of B-type natriuretic peptide for the identification of people with heart failure in primary care in Scotland - a pilot study.

BACKGROUND AND AIMS: The utility of B-type natriuretic peptide as a screening test for heart failure has been proven in a number of clinical trials. The aims of this study were to assess the utility of the measurement of B-type natriuretic peptide in a 'real life' setting and to estimate the potential costs of implementing its use in primary care in Scotland. METHODS AND RESULTS: Eight general practitioner practices with a combined population of approximately 62,000 were invited to participate. During the 9-month study period, 82 samples for B-type natriuretic peptide measurement were requested. The negative predictive value for B-type natriuretic peptide was 96.9%. Compared with electrocardiography, B-type natriuretic peptide reduced the need for echocardiography by 308 tests per million population per year. The estimated cost of implementation in Scotland is approximately £220,000 per annum, equating to £64.93 per patient correctly diagnosed with heart failure, with a potential saving in echocardiography of £110,800. CONCLUSION: In this pilot study, measurement of plasma B-type natriuretic peptide in a 'real life' setting in primary care had a similar sensitivity, specificity and negative predictive value to that observed in trial populations. B-type natriuretic peptide aids early diagnosis of heart failure in primary care and may help to facilitate prompt introduction of evidence based therapies to modify patient outcomes. The costs of measuring plasma B-type natriuretic peptide in suspected cases of heart failure are modest, and its use would increase the diagnostic capacity of primary care if supported by local cardiology services.

Persistently elevated troponin I in paracetamol hepatotoxicity: association with liver injury, organ failure, and outcome.

CONTEXT: An elevated troponin I (TnI) is associated with a poorer prognosis during critical illness. OBJECTIVE: Our aims were to determine whether significant paracetamol-induced hepatotoxicity was associated with an elevated TnI; if this elevation was persistent and was associated with worse clinical outcomes. MATERIALS AND METHODS: In this retrospective cohort study, the requirement for orthotopic liver transplantation (OLT) or death and/or the development of multiorgan failure (MOF) was evaluated for 48 consecutive patients admitted to the Royal Infirmary of Edinburgh (a university tertiary referral centre) with acute liver injury or acute liver failure secondary to paracetamol overdose. RESULTS: TnI was elevated (≥ 0.05 ng/L) in 13/48 patients (27%). This appeared to be sustained for at least 6 days which has not been shown previously in the context of Acute Liver Injury (ALI). Elevated TnI was strongly associated with MOF, with the requirement for inotropic support being the strongest predictor (p = 0.003, OR 9.00, 95% CI 2.13-37.98). TnI elevations also correlated strongly with Acute Physiology and Chronic Health Evaluation (APACHE) II scores (p = 0.0006, r = 0.482, 95% CI 0.22-0.68) and with interleukin 6 (IL-6) levels (p = 0.0001, r = 0.55, 95% CI 0.29-0.73). Although a raised TnI was associated with a markedly increased risk of death or orthotopic liver transplant (p = 0.005, OR 7.73, 95% CI 1.87-32.05) on univariate analysis, this was primarily seen in the context of MOF (SOFA score p = 0.003, OR 1.23, 95% CI 1.07-1.41) and was not an independent predictor of death. There was no correlation between TnI or outcome with other cardiac biomarkers and markers of cardiovascular risk. DISCUSSION AND CONCLUSION: An elevated TnI in the context of acute liver injury or liver failure following paracetamol overdose is associated with a significantly worse patient outcome but it is not an independent prognostic factor. Further studies should be undertaken to investigate the mechanism behind this elevated troponin association.

Genetic mutations in patients with possible familial hypercholesterolaemia in South East Scotland.

Familial hypercholesterolaemia (FH) is one of the most common genetic disorders in the general population. Genetic testing of this condition is increasingly available in the UK to confirm its diagnosis, but the strategies of genetic testing vary. In this pilot study, we sought to investigate whether a strategy that focuses on the low-density lipoprotein receptor (LDLR) and apolipoprotein B (APOB) genes can identify the majority of genetic variants in patients with possible FH in South East Scotland. Forty patients with a clinical diagnosis of possible FH according to the Simon Broome criteria were recruited in a lipid clinic serving South East Scotland. All 18 exons of the LDLR gene were sequenced and multiplex ligation probe amplification was performed to identify major deletions and duplications. Variants of the APOB gene at codon 3527 were investigated by direct sequencing. Genetic mutations were detected in 45% of the patients. Sixteen patients (40%) were found to have mutations in their LDLR gene, whereas two other patients (5%) were identified as heterozygous for the APOB variant commonly associated with FH (c.10580G>A; p.R3527Q). None of these genetic variants were detected in more than two patients. Multiple genetic mutations are associated with a clinical phenotype of FH in South East Scotland. A genetic testing strategy which focuses on a limited number of mutations is unlikely to confirm the diagnosis of FH in the majority of patients in this part of Scotland.

International retrieval of adults on extracorporeal membrane oxygenation support.

A retrieval service was established in New South Wales to provide mobile extracorporeal membrane oxygenation support to patients with severe, acute cardiac or respiratory failure. This service has also retrieved four adult patients from Nouméa, New Caledonia to Sydney on extracorporeal membrane oxygenation support, which are the first international retrievals of this type from Australia. We discuss our experience with these patients, three of whom survived to hospital discharge. However, one patient referred from New Caledonia died before extracorporeal membrane oxygenation could be established.

Vasoactive intestinal peptide (VIP) stimulates cortisol secretion from the H295 human adrenocortical tumour cell line via VPAC1 receptors.

Vasoactive intestinal peptide (VIP) shows a wide tissue distribution and exerts numerous physiological actions. VIP was shown in a dose-dependent manner to increase cortisol secretion in the NCI-H295R human adrenocortical carcinoma (H295) cell line (threshold dose 3.3x10(-10) M, maximal dose 10(-7) M), coupled with a parallel increase in cAMP accumulation. Receptor-specific agonists were employed to determine which of the two known VIP receptor subtypes was involved in cortisol secretion. Treatment with the VPAC1 receptor agonist, [K(15), R(16), L(27)]VIP(1-7)/GRF(8-27), produced a dose-dependent increase in H295 cell cortisol secretion (threshold dose 10(-11) M, maximal dose 10(-7) M) similar to that seen with VIP. Meanwhile, the high-affinity VPAC2 receptor agonist, RO-25-1553, failed to stimulate significantly cortisol or cAMP production from H295 cells. Inhibition of VIP-mediated H295 cell cortisol secretion by PG97-269, a competitive VPAC1-specific antagonist, produced parallel shifts of the dose-response curve and a Schild regression slope of 0.99, indicating competitive inhibition at a single receptor subtype. VIP is known also to interact with the PAC1 receptor, albeit with lower affinity (EC(50) of approximately 200 nM) than the homologous ligand, PACAP (EC(50) of approximately 0.5 nM). PACAP stimulated cortisol secretion from H295 cells (EC(50) of 0.3 nM), suggesting the presence of functional PAC1 receptors. However, stimulation of cortisol secretion by nanomolar concentrations of VIP (EC(50) of 5 nM), coupled with real-time PCR estimation that VPAC1 receptor transcripts appear 1000-fold more abundant than PAC1 transcripts in H295 cells, makes it unlikely that VIP signals via PAC1 receptors. Together, these data suggest that VIP directly stimulates cortisol secretion from H295 cells via activation of the VPAC1 receptor subtype.

Selenoprotein expression in endothelial cells from different human vasculature and species.

Selenium (Se) can protect endothelial cells (EC) from oxidative damage by altering the expression of selenoproteins with antioxidant function such as cytoplasmic glutathione peroxidase (cyGPX), phospholipid hydroperoxide glutathione peroxidase (PHGPX) and thioredoxin reductase (TR). If the role of Se on EC function is to be studied, it is essential that a model system be chosen which reflects selenoprotein expression in human EC derived from vessels prone to developing atheroma. We have used [75Se]-selenite labelling and selenoenzyme measurements to compare the selenoproteins expressed by cultures of EC isolated from different human vasculature with EC bovine and porcine aorta. Only small differences were observed in selenoprotein expression and activity in EC originating from human coronary artery, human umbilical vein (HUVEC), human umbilical artery and the human EC line EAhy926. The selenoprotein profile in HUVEC was consistent over eight passages and HUVEC isolated from four cords also showed little variability. In contrast, EC isolated from pig and bovine aorta showed marked differences in selenoprotein expression when compared to human cells. This study firmly establishes the suitability and consistency of using HUVEC (and possibly the human cell line EAhy926) as a model to study the effects of Se on EC function in relation to atheroma development in the coronary artery. Bovine or porcine EC appear to be an inappropriate model.

Myocardial infarction redefined: the new ACC/ESC definition, based on cardiac troponin, increases the apparent incidence of infarction.

OBJECTIVES: To investigate the impact of the redefinition of the diagnostic criteria for myocardial infarction on its apparent incidence in a non-selected and representative series of patients admitted with acute chest pain. DESIGN: Single centre prospective study. SETTING: Medical assessment unit and cardiology wards of an inner city university hospital. PATIENTS: 80 consecutive patients aged over 25 years admitted with suspected ischaemic acute chest pain (excluding those where the ECG indicated definite myocardial infarction). INTERVENTIONS: Measurement of concentrations of conventional cardiac biomarkers (creatine kinase and its MB isoenzyme, CK-MB) and concentrations of the highly specific diagnostic indicator of myocardial damage, cardiac troponin I (cTnI) 12-24 hours after the onset of acute chest pain. MAIN OUTCOME MEASURES: Frequency of myocardial infarction as assessed by conventional diagnostic criteria (creatine kinase and CK-MB) plus clinical symptoms of infarction, versus frequency of infarction based on high sensitivity troponin assays. RESULTS: Among patients with acute coronary syndromes but non-diagnostic ECG changes, 40% (32/80) fulfilled the new criteria for myocardial infarction using high sensitivity cTnI measurement, compared with 29% (23/80) using the conventional diagnostic criteria for myocardial infarction. CONCLUSIONS: The implications of the redefinition of myocardial infarction on patients, their care, and the use of health care resources are substantial.

Thioredoxin reductase and cytoplasmic glutathione peroxidase activity in human foetal and neonatal liver.

Cytosolic thioredoxin reductase (TR) is an FAD-containing homodimeric selenoenzyme which, together with thioredoxin (Trx) and NADPH, forms a powerful oxidoreductase system. Cytoplasmic glutathione peroxidase (GPX-1) is a selenoprotein with antioxidant activity. The TR/Trx system has been associated with cellular processes including regulation of cell growth, and modification of activity of transcription factors. TR may also act as an antioxidant. We have measured TR activity, TR concentration, and GPX-1 activity in human hepatic cytosols from foetuses and neonates. The concentration of TR was significantly greater (P<0.05) in foetal (43.6, 37.9-50.8 microg/g protein, median, interquartile range) than in neonatal liver (11.6, 8.70-15.0 microg/g). This was also true of TR activity which was 2.1, 1.8-2.5 U/g protein in foetal, and 0.65, 0.44-0.74 U/g protein in neonatal liver (P<0.0005). Similarly, GPX-1 activity was significantly higher (P<0.005) in the foetal (199.7, 144.0-227.9 U/g protein) than in neonatal (77.0, 58.4-110.3 U/g protein) hepatic cytosol. Overall, foetal liver expressed approx. 3-fold higher activities of TR and GPX-1 than neonatal liver.

Selenite protects human endothelial cells from oxidative damage and induces thioredoxin reductase.

The ability of selenium to protect cultured human coronary artery endothelial cells (HCAEC), human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC) from oxidative damage induced by 100 microM t-butyl hydroperoxide (t-BuOOH) was compared. Preincubation of human endothelial cells for 24 h with sodium selenite at concentrations as low as 5 nM provided significant protection against the harmful effects of 100 microM t-BuOOH, with complete protection being achieved with 40 nM selenite. The preincubation period was required for selenite to exert this protective effect on endothelial cells. When compared with selenium-deficient cells, the activities of cytoplasmic glutathione peroxidase (GPX-1), phospholipid hydroperoxide glutathione peroxidase (GPX-4) and thioredoxin reductase (TR) were each induced approx. 3--4-fold by 40 nM selenite. HCAEC and HUVEC showed great similarity in their relative abilities to resist oxidative damage in the presence and absence of selenite, and the activities of TR and the GPXs were also similar in these cell types. BAEC were more susceptible to damage by 100 microM t-BuOOH than were human endothelial cells, and could not be protected completely by incubation with selenite at concentrations up to 160 nM. The activity of TR in human endothelial cells was approx. 25-fold greater than that in BAEC of a similar selenium status, but GPX-1 and GPX-4 activities were not significantly different between the human and bovine cells. These studies, although performed with a small number of cultures, show for the first time that selenium at low doses can provide significant protection of the human coronary artery endothelium against damage by oxidative stress. TR may be an important antioxidant selenoprotein in this regard, in addition to the GPXs. The data also suggest that HUVEC, but not BAEC, represent a suitable model system in which to study the effects of selenium on the endothelium of human coronary arteries.

An improved ex vivo method of primary porcine hepatocyte isolation for use in bioartificial liver systems.

INTRODUCTION: Primary porcine hepatocytes are commonly, used in bioartificial liver devices and for in vitro studies of hepatocyte function. Although in vivo isolation of porcine hepatocytes can give high yield and viability, such methods are time-consuming and expensive, requiring specialist surgical facilities. AIM: To develop a simple, low-cost, high viability, high yield, reproducible ex vivo method for obtaining functional porcine hepatocytes for use in bioartificial liver systems. METHODS: Weanling piglets (12 kg) were killed with pentobarbitone sodium, the infra-hepatic inferior vena cava was clamped and the supra-hepatic inferior vena cava cannulated. The whole liver was retrogradely perfused in situ with cold saline and excised, followed by an ex vivo open-loop and re-circulating perfusion method (at 37 degrees C) in five steps. The liver was disrupted, sequentially filtered in washing buffer, purified by centrifugation and resuspended in Williams E medium. Viability and cell number were assessed using trypan blue exclusion. The cells were subsequently cultured in serum-free chemically-defined medium and function was assessed. RESULTS: The time interval from when the animals were killed to the final cell wash was 105+/-5 min (n = 20). Cell viability was 85+/-6% with a yield of (2.4+/-0.5) x 10(10) from 12+/-1 kg piglets using 0.03% (w/v) collagenase (n = 20). Hepatocytes from all isolations were successfully plated and grown in monolayer culture. In freshly isolated hepatocytes (day 0) total protein content (TP) was 1.2+/-0.1 mg/10(6) cells (n = 5) and 1.2+/-0.3 mg/10(6) cells (n = 5) for day 2 monolayer cultures, corresponding to approximately 9x10(6) hepatocytes per dish. The percentage of total LDH released into the medium was 13+/-4% for day 0 and 8+/-4% at day 2; conversely, intracellular LDH activities were 87+/-4% and 92+/-4% of the total, respectively. The urea synthesis rate was 196+/-36 nmol/h/mg total protein at day 0 (n = 5) and 292+/-62 nmol/h/mg protein (n = 9) at day 2. The total P450 content was 99+/-11 pmol/mg total protein for fresh cells (n = 5) and maintained at 89+/-35 pmol/mg total protein in day 2 cultures. CONCLUSIONS: This ex vivo method provides a high viability, high yield, cost-effective and rapid technique for isolating functional porcine hepatocytes with high plating efficiency, which compares favourably with results obtained using complex in vivo techniques.

Role of arachidonic acid metabolism in ACTH-stimulated cortisol secretion by bovine adrenocortical cells.

We have studied the effects of inhibitors of arachidonic acid (AA) metabolism, nordihydroguaiaretic acid (NDGA), a lipoxygenase (LPX) inhibitor, and indomethacin (INDO), a cyclooxygenase (COX) inhibitor, on cortisol secretion and StAR protein in primary cultures of bovine adrenal zona fasciculata (ZF) cells. NDGA inhibited cortisol secretion in response to both 10(-12) M and 10(-8) M ACTH. AA (10(-4) M) partially reversed the inhibition of cortisol secretion by NDGA at 10(-12) M ACTH but not at 10(-8) M ACTH. On the other hand, INDO potentiated the cortisol response to 10(-12) M ACTH. Neither NDGA nor INDO significantly affected StAR protein levels. These results suggest a StAR protein-independent role for the LPX and COX pathways in acute cortisol secretion, and support the hypothesis that LPX products of AA metabolism are key cellular signals when bovine ZF cells are acutely stimulated by physiological concentrations of ACTH (10(-12) M).

Thioredoxin reductase is the major selenoprotein expressed in human umbilical-vein endothelial cells and is regulated by protein kinase C.

Damage to the endothelium by reactive oxygen species favours atherogenesis. Such damage can be prevented by selenium, which is thought to exert its actions through the expression of selenoproteins. The family of glutathione peroxidases (GPXs) may have antioxidant roles in the endothelium but other intracellular and extracellular selenoproteins with antioxidant actions may also be important. The selenoproteins expressed by cultured human umbilical-vein endothelial cells (HUVECs) were labelled with [(75)Se]selenite and separated using SDS/PAGE. HUVECs secreted no extracellular selenoproteins. There were distinct differences between the intracellular selenoprotein profile of (75)Se-labelled HUVECs and those of other tissues. A single selenoprotein with a molecular mass of 58 kDa accounted for approx. 43% of the intracellular (75)Se-labelled proteins in HUVECs. This protein was identified by Western blotting as the redox-active lipid-hydroperoxide-detoxifying selenoprotein, thioredoxin reductase (TR). TR expression in HUVECs was down-regulated by transiently exposing cells to the phorbol ester PMA for periods as short as 1 min. However, there was a delay of 48 h after PMA exposure before maximal down-regulation of TR was observed. The protein kinase C (PKC) inhibitor bisindolylmaleimide I hydrochloride had no effect on TR expression when added alone, but the agent prevented the down-regulation of TR expression seen with PMA. The calcium ionophore A23187 increased TR expression in HUVECs after a 12-h exposure, but the maximal effect was only observed after a 35-h exposure. These findings suggest that TR may be an important factor in the known ability of Se to protect HUVECs from peroxidative damage. Furthermore, the results also suggest that TR expression can be negatively regulated through PKC. It is possible that TR expression may be positively regulated by the calcium-signalling cascade, although TR induction by A23187 may be due to toxicity.

Contractile response of isolated human hepatic arteries to alpha-adrenoceptor agonists is not impaired in patients with cirrhosis.

1. Impaired vasoconstriction in animals with cirrhosis is maintained in isolated vessels in vitro, indicating an intrinsic alteration in function or structure of the cells in the vascular wall. This may be due to receptor down-regulation, a defect in post-receptor signal transduction or overproduction of vasodilator compounds. This investigation examined the role of these mechanisms in modulating alpha-adrenoceptor-mediated contraction in hepatic arteries from patients with advanced cirrhosis. 2. Hepatic arteries were obtained from subjects with and without cirrhosis for functional investigation in vitro. Endothelial cell function was assessed using endothelium-dependent (acetylcholine) and independent (3'-morpholinosydnonimine) vasodilators. alpha-Adrenoceptor-mediated contraction was assessed by constructing cumulative concentration-response curves to the alpha1-selective agonist phenylephrine, the non-selective adrenoceptor agonist noradrenaline and the receptor-independent vasoconstrictor potassium chloride. 3. None of the vessels used in this study had an intact endothelium but endothelium-independent relaxation was not different in arteries from subject with (79.5+/-10.16%; n=23) and without (84.45+/-18%; n=20) cirrhosis. Phenylephrine, noradrenaline and potassium chloride produced contractions that were of similar size (P>0.05) in arteries from subjects with (10.10+/-0.97 g, 8.85+/-1.03 g and 8.56+/-0.65 g respectively) and without (10.42+/-1.23 g, 9.58+/-1.39 g and 8. 62+/-0.98 g respectively) cirrhosis. The sensitivities (pD2) of the responses to these agonists were also similar (P<0.05) in arteries from patients with cirrhosis (5.45+/-0.10, 5.60+/-0.12 and 1.57+/-0. 03 respectively) and those from non-cirrhotic donors (5.58+/-0.11, 5. 67+/-0.11 and 1.54+/-0.05 respectively).4. Contraction of the denuded hepatic artery was unaffected by cirrhosis indicating that vascular abnormalities in this condition in man are not due to an intrinsic alteration of smooth muscle cell function in hepatic conduit arteries.

Identification of a 57-kilodalton selenoprotein in human thyrocytes as thioredoxin reductase and evidence that its expression is regulated through the calcium-phosphoinositol signaling pathway.

Human thyrocytes incubated with the phorbol ester, phorbol 12-myristate 13-acetate (PMA; 10(-5)-10(-8) mol/L) and the calcium ionophore A23187 (10(-5)-10(-8) mol/L) showed a marked increase in the expression of a 57-kDa selenoprotein identified as thioredoxin reductase (TR). After the addition of A23187 with PMA, a significant induction in TR expression was observed after 6 h, with maximal induction occurring by 24 h. The addition of 8-bromo-cAMP (10(-4) mol/L) or TSH (10 U/L) alone had no effect on TR expression, nor did these agents influence the induction of TR brought about by the addition of A23187 and PMA. These data show that the calcium-phosphoinositol second messenger cascade that controls hydrogen peroxide generation in the human thyrocyte is also an important stimulator of TR expression. The role of TR in the thyrocyte is unclear, but the selenoenzyme has a high capacity to detoxify compounds, such as hydrogen peroxide and lipid hydroperoxides, that are produced in high concentration during thyroid hormone synthesis.

Simplified detection of a mutation causing familial hypercholesterolaemia throughout Britain: evidence for an origin in a common distant ancestor.

Familial hypercholesterolaemia (FH) is an inherited autosomal codominant disorder caused by many different mutations in the low-density lipoprotein receptor (LDLR) gene. The one described most frequently in patients with FH from England, arises from a G-->A transition at the first nucleotide of codon 80, resulting in the substitution of lysine for glutamic acid at residue 80 of the mature protein, FH E80K. We describe a simple method to detect this mutation in genomic DNA using the polymerase chain reaction (PCR). A 69 base pair (bp) fragment of exon 3 of the LDLR gene is amplified using a mutagenic upstream PCR primer. This substitutes a T for an A residue in the amplified product, 2 bp upstream from the mutant site, generating a restriction site for the endonuclease Taq I, in normal, but not in mutant DNA. Following digestion of amplified DNA with Taq I, normal but not mutant DNA is cut into two fragments of 29 and 40 bp, which are readily identified by polyacrylamide gel electrophoresis. Using this method, 410 patients with clinically diagnosed FH, attending lipid clinics in Edinburgh (72), Newport (158), Walsall (30) and Southampton (150), were screened for the mutation. Five individuals tested positive as heterozygotes, one from Edinburgh, three from Newport and one from Southampton. This finding was confirmed by DNA sequence analysis. We conclude that FH due to this mutation occurs in individuals throughout Great Britain and that it can be detected accurately using this simple technique. DNA from these and other individuals previously identified to be heterozygous for FH E80K, was then studied using PCR of highly informative microsatellite markers flanking the LDLR gene. Sixteen of 17 apparently unrelated individuals heterozygous for FH E80K also were heterozygous for an identical size (239 nucleotide) allele, of polymorphic microsatellite D19S394, located approximately 250 kb away from the LDLR gene. This supports the hypothesis that FH E80K in these 16 individuals arose from a single ancestor less than 1000 years ago.

The expression of steroidogenic acute regulatory protein (StAR) in bovine adrenocortical cells.

StAR protein may facilitate rapid transfer of cholesterol from the outer to the inner mitochondrial membrane, the site at which cholesterol is converted to pregnenolone by the cholesterol side chain cleavage complex. We have studied the effect of ACTH treatment on StAR mRNA and protein levels in bovine adrenocortical cells in primary culture. Cells were initially cultured for 3 days after isolation, and then treated with ACTH (10(-8) M) for various times up to 24 hours. Northern analysis of total BAC mRNA, using a [alpha32P]-labelled cDNA probe encoding a 5' region of bovine StAR mRNA, revealed two principal hybridising species of 1.6 and 3.0 kb. Western immunoblot analysis revealed a principal band at 30 kDa. Levels of both StAR mRNA and protein showed an increase at 1 hour, reached a maximum at around 6 hours and declined to basal levels at 24 hours. Cortisol secretion (measured by RIA) showed a similar change over the same period. From these results it appears that StAR mRNA and protein levels in BAC are acutely regulated in concert with ACTH-stimulated cortisol secretion.

Dopaminergic stimulation of cortisol secretion from bovine zfr cells occurs through nonspecific stimulation of adrenergic beta-receptors.

Previous reports have suggested a possible dopaminergic inhibition of the actions of AII on aldosterone secretion via adenylate-cyclase inhibitory 'D2' receptors. Others suggest a possible stimulation of aldosterone secretion via a stimulatory 'D1' receptor/cAMP pathway. We have examined the actions of dopamine on basal and AII-stimulated cortisol secretion by cultured bovine zfr cells. Dopamine alone caused a dose-dependent increase in cortisol secretion at doses >10(-5) M, and also enhanced steroidogenic output in response to submaximal (10(-10) M) but not maximal (10(-8) M) stimulatory doses of AII. The stimulatory action of dopamine alone on cortisol secretion was not, however, reproduced by the 'D1' agonist fenoldopam, and was fully blocked by propranolol. Dopamine had neither a stimulatory effect on basal phosphoinositol production nor an inhibitory effect on AII-stimulated phosphoinositol production. Our findings are therefore inconsistent with the activation of a 'D1' or 'D2' class receptor, and suggest the stimulation of cortisol secretion occurred nonspecifically through a beta-adrenergic receptor.

Familial ligand-defective apolipoprotein B-100: detection, biochemical features and haplotype analysis of the R3531C mutation in the UK.

Familial ligand-defective apolipoprotein (apo) B-100 (FDB) is an autosomal codominant disorder which may give rise to hypercholesterolaemia. It is caused by the substitution of glutamine for arginine at codon 3500 of the apo B gene (apo B R3500Q), resulting in decreased binding of low density lipoprotein (LDL) to the LDL receptor. In order to search for other mutations in this region of the apo B gene, we have screened genomic DNA, obtained from 412 hypercholesterolaemic individuals, using heteroduplex analysis. Additional heteroduplex bands were observed following analysis of DNA from 11 individuals, nine of whom were heterozygous for apo B R3500Q. The two remaining individuals, both of Celtic origin, were shown by DNA sequencing to be heterozygous for a C-->T transition at nucleotide 10800 of the apo B gene, resulting in the substitution of cysteine for arginine at codon 3531 (apo B R3531C). Both had a strong family history of atherosclerosis and family studies revealed a further four individuals heterozygous for the mutation, three of whom were hypercholesterolaemic. Individuals heterozygous for apo B R3531C and R3500Q had mean +/- S.E.M. cholesterol concentrations of 7.82 +/- 0.68 and 8.53 +/- 0.31 mmol/l, respectively. These values were significantly higher than the value of 5.51 +/- 0.23 mmol/l observed in their unaffected relatives. These findings suggest that apo B R3531C is both less common in the UK and gives rise to a less severe form of hypercholesterolaemia than the classical 3500 mutation. In one of the families, the R3531C mutation occurred on a haplotype, compatible with that previously assigned to the mutation in a North American family also of Celtic origin. This is consistent with the mutation having been inherited from a common distant ancestor in individuals of Celtic origin.

Direct stimulation of cortisol secretion from the human NCI H295 adrenocortical cell line by vasoactive intestinal polypeptide.

OBJECTIVE: To investigate a possible direct action of vasoactive intestinal polypeptide (VIP) on adrenal cortisol secretion and to define its mechanism of action. DESIGN: The human adrenocortical carcinoma cell line NCI H295, which is not contaminated by medullary chromaffin cells, was used to aid distinction between a direct action of VIP on adrenocortical cells and an indirect mechanism involving VIP-stimulated release of catecholamines. METHODS: NCI H295 cells were challenged with 10(-11)-10(-7) mol/l VIP for 4 h, with or without prior exposure for 72 h to 10 micromol/l forskolin. Cortisol and cyclic AMP contents of the overlying media were measured using in-house radioimmunoassays. Cells were treated with 10(-8)-10(-6) mol/l adrenaline or 3.3 x 10(-8) mol/l VIP with and without 10(-8)-10(-6) mol/l propranolol to exclude the possibility that an indirect mechanism of action involving beta-adrenoceptors was operating. RESULTS: VIP treatment produced an increase in cortisol secretion without pre-incubation, but this was markedly enhanced by prior exposure of cells to forskolin. VIP was potent, with a threshold of 10(-11) mol/l (n = 4), reaching a maximum 3.9+/-0.9-fold increase in effect on cells pre-exposed to forskolin (n = 4) by 3.3 x 10(-8) mol/l. This increase matched the 4 h response to 10 micromol/l forskolin. Cortisol secretion was accompanied by a parallel, dose-dependent increase in accumulation of cAMP. CONCLUSIONS: VIP potently and directly stimulates secretion of cortisol from these adrenocortical cells of human origin via an adenylate cyclase-coupled VIP receptor. These findings raise the possibility of a significant and direct effect of VIP in the control of steroid secretion from the adrenal cortex in humans.