Increased microvascular permeability in mice lacking Epac1 (Rapgef3).

AIM: Maintenance of the blood and extracellular volume requires tight control of endothelial macromolecule permeability, which is regulated by cAMP signalling. This study probes the role of the cAMP mediators rap guanine nucleotide exchange factor 3 and 4 (Epac1 and Epac2) for in vivo control of microvascular macromolecule permeability under basal conditions. METHODS: Epac1-/- and Epac2-/- C57BL/6J mice were produced and compared with wild-type mice for transvascular flux of radio-labelled albumin in skin, adipose tissue, intestine, heart and skeletal muscle. The transvascular leakage was also studied by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) using the MRI contrast agent Gadomer-17 as probe. RESULTS: Epac1-/- mice had constitutively increased transvascular macromolecule transport, indicating Epac1-dependent restriction of baseline permeability. In addition, Epac1-/- mice showed little or no enhancement of vascular permeability in response to atrial natriuretic peptide (ANP), whether probed with labelled albumin or Gadomer-17. Epac2-/- and wild-type mice had similar basal and ANP-stimulated clearances. Ultrastructure analysis revealed that Epac1-/- microvascular interendothelial junctions had constitutively less junctional complex. CONCLUSION: Epac1 exerts a tonic inhibition of in vivo basal microvascular permeability. The loss of this tonic action increases baseline permeability, presumably by reducing the interendothelial permeability resistance. Part of the action of ANP to increase permeability in wild-type microvessels may involve inhibition of the basal Epac1-dependent activity.

Origin of Swedish hemophilia B mutations.

BACKGROUND: More than 1100 mutations that cause hemophilia B (HB) have been identified. At the same time, specific F9 mutations are present at high frequencies in certain populations, which raise questions about the origin of HB mutations. OBJECTIVES: To describe the mutation spectrum of all HB families in Sweden and investigate if mutations appearing in several families are due to independent recurrent mutations (RMs) or to a common mutation event (i.e. are identical by descent (IBD)). PATIENTS/METHODS: The registered Swedish HB population consists of patients from 86 families. Mutations were identified by resequencing and identical haplotypes were defined using 74 markers and a control population of 285 individuals. The ages of IBD mutations were estimated using ESTIAGE. RESULTS: Out of 77 presumably unrelated patients with substitution mutations, 47 patients (61%) had mutations in common with other patients. Haplotyping of the 47 patients showed that 24 patients had IBD mutations (51%) with estimated ages of between two and 23 generations. A majority of these patients had mild disease. Eight of the 15 mutations observed in more than one family were C>T transitions in CpG sites and all eight were RMs. CONCLUSIONS: The association of IBD mutations with a mild phenotype is similar to what has been previously observed in hemophilia A. Noteworthy features of the mutations that are common to more than one family are the equal proportions of patients with RM and IBD mutations and the correlation between the occurrence of RMs and C>T transitions at CpG sites.

Origin of Swedish hemophilia A mutations.

BACKGROUND: Hemophilia A (HA) has a high level of variation within the disease class, with more than 1000 mutations being listed in the HAMSTeRS database. At the same time a number of F8 mutations are present in specific populations at high frequencies. OBJECTIVES: The simultaneous presence of large numbers of rare mutations and a small number of high-frequency mutations raises questions about the origins of HA mutations. The present study was aimed at describing the origins of HA mutations in the complete Swedish population. The primary issue was to determine what proportion of identical mutations are identical by descent (IBD) and what proportion are attributable to recurrent mutation events. The age of IBD mutations was also determined. PATIENTS/METHODS: In Sweden, the care of HA is centralized, and the Swedish HA population consists of ~ 750 patients from > 300 families (35% severe, 15% moderate, and 50% mild). Identical haplotypes were defined by single-nucleotide polymorphism and microsatellite haplotyping, and the ages of the mutations were estimated with estiage. RESULTS: Among 212 presumably unrelated patients with substitution mutations, 97 (46%) had mutations in common with other patients. Haplotyping of the 97 patients showed that 47 had IBD mutations (22%) with estimated ages of between two and 35 generations. The frequency of mild disease increased with an increasing number of patients sharing the mutations. CONCLUSIONS: A majority of the IBD mutations are mild and have age estimates of a few hundred years, but some could date back to the Middle Ages.

The alpha11beta1 integrin has a mechanistic role in control of interstitial fluid pressure and edema formation in inflammation.

OBJECTIVE: Collagen-binding integrins may be involved in controlling interstitial fluid pressure (Pif), transcapillary fluid flux, and tissue fluid volume. Our aim was to explore whether the newly discovered collagen binding alpha11beta1 integrin has a mechanistic role in inflammatory edema formation. METHODS AND RESULTS: In collagen matrices seeded with a mixture of mast cells and fibroblasts, fibroblasts lacking the alpha11 integrin subunit (alpha11(-/-)) contracted collagen gels less efficiently than control fibroblasts, suggesting that the alpha11beta1 integrin is able to mediate tensile force in connective tissues. In alpha11(-/-) mice, control Pif in skin did not differ from the pressure found in wild-type mice. Whereas a reduction in Pif was found in control mice after inducing inflammation, thereby contributing to fluid extravasation and edema formation, such a reduction was not seen in alpha11(-/-) mice. That this effect is mediated through the extracellular compartment is suggested by a similar plasma protein extravasation ratio in alpha11(-/-) and wild-type mice. CONCLUSIONS: Our data suggest that alpha11beta1 integrins on dermal fibroblasts mediate collagen lattice remodeling and have a mechanistic role in controlling Pif in inflammation and thereby fluid extravasation and edema formation in vivo.

Lowered albumin extravasation rate in heart but not in other organs in beta3-integrin-deficient mice.

AIM: The vascular protein permeability is dependent on the integrity of the vascular wall. The heart capillaries in male mice lacking beta3 integrins have an immature phenotype. Previously, we have demonstrated a role for alphavbeta3 integrins in control of interstitial fluid pressure (Pif) and thereby in the fluid flux during inflammation. We wanted to explore a possible role for alphavbeta3 integrins in controlling capillary protein permeability during control situation and inflammation. METHODS: We performed double-tracer and microdialysis experiments on beta3-integrin-deficient mice and wild type control mice. We also measured blood pressure and heart rate in the two mice strains. RESULTS: We found reduced albumin extravasation (during 25 min) in the heart capillaries (0.053 +/- 0.003 vs. 0.087 +/- 0.009 mL g(-1) dw, P < 0.05), and an increased cardiac mass/body weight (5.3 x 10(-3) +/- 0.3 x 10(-3) vs. 3.8 x 10(-3) +/- 0.1 x 10(-3), P < 0.01) in the beta3-integrin-deficient mice (n = 6) compared with the controls (n = 6). Heart rate and blood pressure were the same in mice with and without beta3-integrins. No difference in permeability was found in other tissues studied, or under local inflammation. CONCLUSION: These results show a function for the alphavbeta3 integrin in the regulation of protein permeability, selective for the heart capillaries.

Colorectal cancer screening with flexible sigmoidoscopy-participants' experiences and technical feasibility.

AIM: To evaluate tolerability and technical feasibility of colorectal cancer screening with flexible sigmoidoscopy. METHODS: One thousand men and women aged 59-61 years, randomly selected from the population register of Uppsala, Sweden, were invited by mail. After random allocation, half of them were called up by a nurse (group 1), while the other half were asked to call themselves (group 2) to book a sigmoidoscopy. After the examination, the participants anonymously answered a questionnaire about their subjective experiences. Endoscopists and their assisting nurse filled out structured forms documenting various technical aspects including an estimation of the subjects' discomfort. RESULTS: Four hundred and sixty-nine subjects participated. Mean intubation depth was 59 cm (range 28-60) and mean duration 5.8 min (range 2-23). On average, participants reported low degrees of discomfort and feeling of exposure, but 19 and 27% rated pain and distension, respectively, on the upper half of a visual analogue scale (VAS). Most subjects found the duration acceptable. Patient discomfort, as appraised by the endoscopists, was lower in men than in women, positively linked to duration of the procedure, but inversely associated with intubation distance. However, the overall differences between strata of participants were small. Among self-reported variables, group 1 and 2 differed significantly only with regard to 'other discomfort'. All but six subjects would accept a repeat examination. Failures, resulting in incomplete examinations, occurred in 14 subjects. CONCLUSIONS: Flexible sigmoidoscopy is generally well tolerated and technically feasible in screening for colorectal cancer. A more personalised invitation did not have any important effects on the subjective experience.

Quality of life in patients with multiple endocrine neoplasia type 1 (MEN 1).

To study quality of life among patients living with a hereditary tumor syndrome, the small group with multiple endocrine neoplasia type 1 (MEN1) was selected. It is characterized by multifocal adenomas of the pancreas, parathyroid, anterior pituitary and other endocrine glands. Patients were assessed at an in-hospital stay and six months later at home. Patients at a specialist ward for MEN1 were recruited consecutively (n = 36) during one year. Eighty-one percent participated (n = 29). Four questionnaires were used: the Hospital Anxiety and Depression Scale (HADS), the Impact of Event Scale (IES), the Life Orientation Test (LOT) and the Short Form-36 (SF-36). Psychosocial outcome measures (anxiety, depression, intrusion, avoidance) changed only marginally between the in hospital stay and six months later at home. However, depression increased for patients categorized as having a high burden of disease and treatment. Compared to population-based norm values, the SF-36 scores of the patient group MEN1were lower for General Health and Social Functioning. Optimism assessed at the hospital was a predictor of Mental Health six months later. Most MEN 1 patients (70%) were pessimists. Patients having a higher burden of disease and treatment are in need of support after discharge. Patients could easily be monitored with questionnaires and, when indicated, offered help for their psychosocial distress.

Compliance and findings in a Swedish population screened for colorectal cancer with sigmoidoscopy.

AIM: The aim of this study was to evaluate the patterns of compliance and the frequency of adenomas and neoplasms in a Swedish population. METHODS: In 1996, 2000 men and women born in 1935 or 1936 were selected at random from the population registers of Uppsala and Malmö/Lund. All subjects were invited by mail to participate. In a randomised study design, subjects were either called up by a nurse to schedule the appointment for sigmoidoscopy or instructed to call themselves. At sigmoidoscopy subjects with a cancer, an adenoma (neoplastic polyp) or more than three hyperplastic polyps were scheduled for a complete colonoscopy. RESULTS: Thirty-nine percent (770/1988) of all the invited subjects had a sigmoidoscopy. The participation differed between the two centres, 47% at the Uppsala centre and 30% at the Malmö/Lund centre (P<0.01). There was no statistically significant difference between the two different invitation groups. In all, 98 subjects (13%) were planned for colonoscopy. Thirty-one (35%) of the subjects having a colonoscopy were women and 57 (65%) were men. Fifty-five true adenomas were found in 46 subjects. All together, six subjects had proximal adenomas. Five adenocarcinomas were diagnosed, all within the reach of the sigmoidoscope. CONCLUSIONS: The compliance was lower and the adenomas were fewer than expected. To increase compliance it is necessary with rigorously controlled invitation routines.

Food intake and the presystemic metabolism of single doses of amitriptyline and nortriptyline.

The influence of food on presystemic metabolism of single doses of amitriptyline (AMI) and nortriptyline (NT) was examined. In randomised order 25 mg tablets of each drug was given to 9 healthy, female volunteers both in the fasting state and together with a standardised breakfast. Concentrations of the drugs and of their dealkylated, hydroxylated and conjugated metabolites were measured by gas chromatography--mass spectrometry (AMI experiment) or high-pressure liquid chromatography (NT experiment). Standard pharmacokinetic parameters were calculated. Food intake did not consistently or significantly influence the bioavailability of either AMI or NT, nor the demethylation of AMI, nor the hydroxylation or the primary or secondary conjugation of NT. There were large interindividual changes in AUC of AMI after food (+94% to -44%). A significant negative correlation between AUC of AMI but not of NT during fasting conditions and per cent change in AUC after food was found (r = -0.72, P = 0.029). The implication of this (negative) correlation for an individual patient might be to keep the intake of the drug in standardised relation to food to avoid undue heavy changes in drug concentration, which might just occur with a change in time relation between intake of drug and food. From a mechanistic view the results argue against a direct and selective influence of food on the presystemic oxidation and conjugation of weakly basic drugs but does not exclude that food may reduce the presystemic metabolism of some such drugs indirectly, by enhancing their rate of hepatic delivery. Presentation of data from food interaction studies should not be restricted to general descriptions. It seems equally important to present the variability of individual data to allow inspection of the extent and direction of effects. This should be of interest for patient, prescriber as well as the regulatory agency.

Effect of beta-glucuronidase on urinary benzodiazepine concentrations determined by fluorescence polarization immunoassay.

In samples from patients treated with oxazepam, beta-glucuronidase increased the immunoreactivity of urinary benzodiazepines analyzed by fluorescence polarization immunoassay (FPIA). Increasing concentrations of beta-glucuronidase added to samples from drug-free controls did not influence the results. In the absence of beta-glucuronidase, 22 of 35 samples from patients undergoing detoxification gave positive results at a cutoff concentration of 200 micrograms/L. Pretreatment with beta-glucuronidase increased the number of drug-positive samples to 33. The drug-negative samples were obtained from two patients who had been oxazepam-free for at least 1 week. Thus, beta-glucuronidase can be used to increase the sensitivity of the urinary benzodiazepine FPIA without reducing the specificity of the method.

Pharmacokinetic interactions of alcohol and acetylsalicylic acid.

This study assessed the influence of acetylsalicylic acid (ASA, 1.0 g), ibuprofen (0.8 g) and paracetamol (1.0 g) on the single-dose kinetics of ethanol in 12 healthy volunteers ingesting the drug and a standardised 1840-kJ breakfast 1 h before intake of ethanol. It also assessed the influence of ethanol on the single-dose kinetics of 1.0 g ASA in ten fasting healthy volunteers. Plasma concentrations of ethanol were measured by gas chromatography, and those of the drugs by liquid chromatography. There was no effect of ASA, ibuprofen or paracetamol on the single-dose kinetics of ethanol, but concurrent intake of ethanol reduced the peak concentration of ASA by 25%.

Chronic treatment with a classical neuroleptic alters excitatory amino acid and GABAergic neurotransmission in specific regions of the rat brain.

The purpose of the following experiments was to describe some of the neurochemical changes that occur in the basal ganglia of rats exposed chronically to a classical neuroleptic, fluphenazine, and to relate these changes to extrapyramidal motor dysfunction. For these studies a combination of behavioural, receptor autoradiographic and in situ hybridization methods were employed. Preliminary pharmacological studies on GABA receptors showed that incubation in Tris-acetate rather than Tris-citrate buffer increased the number of binding sites labelled by [3H]muscimol by over 120% without affecting binding affinity or selectivity. The results of experiments with fluphenazine showed that treatment for six months increased the frequency of vacuous chewing movements compared to controls. In the striatum, changes in GABA transmission were observed in fluphenazine-treated rats with increases in glutamate decarboxylase mRNA levels in the caudate nucleus, dorsal shell and core of the accumbens and decreases in [3H]muscimol binding in the caudate and dorsal shell regions. These data suggest that fluphenazine treatment increased GABA transmission in specific subregions of the caudate and accumbens nuclei. In addition, glutamate decarboxylase mRNA levels were elevated in the entopeduncular nucleus of fluphenazine-treated animals. Autoradiographic analysis of excitatory amino acid binding showed that fluphenazine exposure decreased [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding in entopeduncular nucleus and in the ventrolateral thalamic nucleus and decreased [3H]dizocilpine maleate binding in the medial geniculate nucleus. These experiments show that in addition to altering GABA transmission, chronic neuroleptic exposure alters excitatory amino acid transmission in specific regions of the basal ganglia-thalamocortical motor system. The neuroleptic dependent increases in glutamate decarboxylase mRNA levels in the entopeduncular nucleus may reflect changes in neurotransmission in the indirect pathway connecting the major input and output nuclei of the basal ganglia. Changes in some of these brain regions may be related to the occurrence of extrapyramidal motor disturbances.

Brain cortical tissue levels of noradrenaline and its glycol metabolites: effects of ischemia and postischemic administration of idazoxan.

The brain noradrenaline (NA) system is known to modulate ischemic neuronal damage, and the turnover of NA has been suggested to increase in the early recovery period following cerebral ischemia. Using HPLC and gas chromatography-mass spectrometry we analyzed the tissue levels of NA and its metabolites, 3,4-dihydroxyphenylethyleneglycol (DHPG) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), in rat brain cortex after 10 min of forebrain ischemia followed by 1 h of recirculation. The effect of idazoxan, given in cerebro-protective doses, as a bolus of 0.1 mg.kg-1 immediately after ischemia followed by 10 micrograms.kg-1.min-1 for 1 h, was also investigated. Ischemia decreased basal NA cortical levels from 384 ng/g tissue in control animals to 214 ng/g, while DHPG increased from 74 to 103 ng/g (+39%) and MHPG from 82 to 154 ng/g (+88%). Conjugated but not free DHPG increased, while both free and conjugated MHPG increased equally. The findings indicate an enhanced postischemic NA turnover with a major proportion of uptake and metabolism occurring extraneuronally, possibly secondary to a saturation of neuronal NA uptake in the postischemic phase. Idazoxan further increased NA turnover, as evidenced by higher postischemic levels of free MHPG and a higher MHPG/NA ratio. A correlation may exist between the protective action of idazoxan and its effect on NA turnover.

Carbamazepine kinetics and adverse effects during and after ethanol exposure in alcoholics and in healthy volunteers.

The influence of ethanol on the single-dose kinetics of carbamazepine (400 mg syrup) was assessed in 7 alcoholics after a debauche (mean daily consumption 240 g ethanol) and after 9 days of controlled abstinence, and in 8 healthy volunteers after intake of the drug with and without a single dose of ethanol (25 g). Twelve h after the first test dose of carbamazepine the alcoholics were treated with the drug for 4 days (200 mg tablet b.d.). Carbamazepine was then withheld until a single test dose was given on day 9. Serum levels of carbamazepine and its 10,11-epoxide metabolite were measured by liquid chromatography. Carbamazepine absorption appeared to be delayed in alcoholics, both after debauche and withdrawal, but its bioavailability did not seem to be reduced. Carbamazepine levels were higher and those of its metabolite lower in alcoholics after a debauche than after 9 days of controlled abstinence, but neither was changed in healthy volunteers after the ingestion of carbamazepine together with a single dose of ethanol. The difference may have been due to inhibition of carbamazepine metabolism by ethanol at the high levels attained in alcoholics but not in volunteers. However, it could also be an expression of the unmasking of enzyme induction after ethanol withdrawal. None of the alcoholics had any withdrawal seizures. Despite similar carbamazepine levels, side effects occurred in all volunteers but in none of the alcoholics, indicating that long-term ethanol exposure may promote central nervous adaptation to the acute untoward effects of carbamazepine.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma levels of thioridazine and metabolites are influenced by the debrisoquin hydroxylation phenotype.

The pharmacokinetics of thioridazine and its metabolites were studied in 19 healthy male subjects: 6 slow and 13 rapid hydroxylators of debrisoquin. The subjects received a single 25 mg oral dose of thioridazine, and blood samples were collected during 48 hours. Concentrations of thioridazine and metabolites in serum were measured by HPLC. Slow hydroxylators of debrisoquin obtained higher serum levels of thioridazine with a 2.4-fold higher Cmax and a 4.5-fold larger AUC(0-infinity) associated with a twofold longer half-life compared with that of rapid hydroxylators. The side-chain sulphoxide (mesoridazine) and sulphone (sulphoridazine), which are active metabolites, appeared more slowly in serum and had lower Cmax values, but comparable AUC. The thioridazine ring-sulphoxide attained higher Cmax and 3.3-fold higher AUC in slow hydroxylators than in rapid hydroxylators of debrisoquin. Thus the formation of mesoridazine from thioridazine and the 4-hydroxylation of debrisoquin seem to be catalyzed by the same enzyme, whereas the formation of thioridazine ring-sulphoxide is probably formed mainly by another enzyme.

Pharmacokinetics of alprazolam in geriatric patients with neurotic depression.

The pharmacokinetics of alprazolam was studied in 10 geriatric patients (5 males, 5 females) with neurotic depression during a 6-week period. After 0.5 mg of alprazolam on Day 1 the mean elimination half-life was 11.1 hr and Cmax 12.3 ng/ml. The pharmacokinetic evaluation on Day 1 (t 1/2, tmax) did not differ significantly from the evaluation on Day 42. The mean daily dosage on Day 42 was 1.6 mg alprazolam. The concentrations of the metabolites alpha-OH alprazolam and 4-OH alprazolam were less than 10% of that of alprazolam. All the patients improved clinically. The most common side effect was drowsiness, more often during the first week than the last week.

Disposition of perphenazine is related to polymorphic debrisoquin hydroxylation in human beings.

The pharmacokinetics of a single oral dose of 6 mg perphenazine was studied in a group of six slow and six rapid hydroxylators of debrisoquin. Peak serum concentrations of perphenazine were significantly higher in slow hydroxylators than they were in rapid hydroxylators (2.4 +/- 0.6 versus 0.7 +/- 0.3 nmol/L, p less than 0.001). The AUC(0-12) was also higher in slow hydroxylators than it was in rapid hydroxylators (18.5 +/- 6.2 versus 4.5 +/- 2.5 nmol.L-1.hr, p less than 0.001). The data suggest that the disposition of the antipsychotic drug perphenazine covaries with polymorphic debrisoquin hydroxylation.