Relationships between body mass index and short-circuit current in human duodenal and colonic mucosal biopsies.

AIM: Retrospectively, to investigate the relationship between body mass index (BMI) and basal electrogenic transport as measured by short-circuit current (SCC) in human duodenal and colonic mucosal biopsies. METHODS: The study included biopsies from mucosa of normal appearance in the sigmoid colon and/or distal duodenum. Patients were referred for routine endoscopy (predominantly for monosymptomatic abdominal pain) and had normal endoscopic findings. Biopsies were mounted in miniaturized Ussing chambers and basal SCC was recorded. Patients were divided into two groups on the basis of BMI (≤25 and >25 kg m⁻²). Statistical significance was assessed by the unpaired t-test or Wilcoxon rank-sum test. Correlation coefficients were calculated by Pearson product moment correlation. RESULTS: In colonic biopsies, basal SCC (mean ± standard deviation) was significantly higher in 59 biopsies from 30 patients with low BMI than in 32 biopsies from 23 patients with high BMI (45 ± 29 µA cm⁻² vs. 27 ± 21 µA cm⁻², P = 0.016). In duodenal biopsies, mean basal SCC was numerically lower in 38 biopsies from 15 patients with low BMI than in 46 biopsies from 19 patients with high BMI (54 ± 26 µA cm⁻² vs. 74 ± 39 µA cm⁻², P = 0.069). The correlation coefficient between BMI and SCC was -0.26 (P = 0.06) in colonic biopsies and +0.44 (P = 0.001) in duodenal biopsies. CONCLUSION: Basal intestinal active electrogenic transport is related to BMI and this relationship may differ in different segments of the intestinal tract.

Duodenal secretion in humans mediated by the EP4 receptor subtype.

AIM: Assessment of functional EP receptor subtypes involved in PGE2-induced secretion in human duodenum. The spectrum of activities by PGE2 in mammals, including cytoprotective bicarbonate secretion in duodenum, is mediated through four G protein-coupled receptor subtypes (EP1-EP4). METHODS: Biopsies from the second part of duodenum from patients undergoing endoscopy were mounted in modified Ussing chambers. Basal and stimulated short circuit current (SCC) and slope conductance (SG) were measured. Dose-response relations for PGE2 and subtype receptors EP1/EP3 (sulprostone), EP2 (butaprost), and EP4 (1-OH PGE1) were assessed by cumulated doses of single agonists. RESULTS: PGE2 caused a dose-dependent increase in SCC, maximum 101 +/- 20 microA cm(-2) with an EC50 of 35.6 +/- 5.8 nm (n = 3). Likewise 1-OH PGE1 caused a dose-dependent SCC increase, maximum 63.3 +/- 28.6 microA cm(-2) with an EC50 of 56.7 +/- 7.2 nm (n = 3). 1-OH PGE1 at 500 nm increased SCC by 18.0 +/- 3.0 microA cm(-2) (n = 10) and SG by 2.9 +/- 0.4 mS cm(-2) (n = 6). Sulprostone (n = 6) and butaprost (n = 6) had no effects on SCC or SG. SCC was inhibited 31.4 +/- 13.2% (n = 10) by bumetanide (25 microM serosa) and 18.6 +/- 5.8% (n = 10) by acetazolamide (250 microM lumen). Diphenylamine-2-carboxylate (DPC) (250 microM mucosa) and SITS (10 microM mucosa) had almost no effect. CONCLUSIONS: Effects of PGE2 on secretion in the second part of human duodenum is mediated through the EP4 receptor and not through EP1, EP2, or EP3.

Functional characterization of muscarinic receptor subtypes in human duodenal secretion.

AIM: Acetylcholine (ACh) stimulates ion secretion in the small intestine and colon. The purpose of the present study was to characterize the ACh-induced electrogenic ion transport in human duodenum and determine the muscarinic receptor subtypes functionally involved. METHODS: Biopsies from the second part of duodenum were obtained from 28 patients during endoscopy. Biopsies were mounted in modified Ussing chambers with air-suction for measurements of short-circuit current by a previously validated technique. Short-circuit current was measured after application of chloride/bicarbonate transport inhibitors bumetanide, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), diphenylamine-2-carboxylate (DPC), and acetazolamide. 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and two mamba toxins MT3 and MT7 were used to characterize the mAChR receptor subtypes involved. The effects of transport inhibitors and receptor antagonists were measured by comparing two consecutive responses of ACh on short-circuit current in the same biopsy specimen. RESULTS: Bumetanide and 4-DAMP significantly inhibited ACh-induced short-circuit current, whereas SITS, DPC, acetazolamide, mamba toxin MT3, and mamba toxin MT7 all failed to show any significant effect. CONCLUSION: In conclusion, our results indicate that muscarinic receptor subtype M3 acts as the main mediator of bumetanide-sensitive ACh-induced secretion in human duodenum.

Novel modified Ussing chamber for the study of absorption and secretion in human endoscopic biopsies.

The aim of this study was to design and evaluate a modified Ussing chamber, that makes use of constant air suction (modified Ussing air suction chamber, MUAS) for fixation of biopsy specimens. Standard size forceps biopsies were taken from the descending part of duodenum from patients undergoing endoscopy. Short circuit current (SCC) and conductance (G) were measured during basal conditions and after addition of different sugars and secretagogues. Histologic examination was performed to determine the degree of tissue damage after study in the chamber. Basal SCC was 54.7 +/- 4.3 microA x cm(-2) and G was 58.7 +/- 4.7 mS x cm(-2) (mean +/- SEM, n=48) and steady values of these parameters were observed for at least 2 h. Reproducible and steady responses in SCC were obtained with D-glucose (SCCmax=172 +/- 22.1 microA x cm(-2); EC50=6.9 +/- 0.7 mM, n=5) and D-galactose (SCCmax=233 +/- 55.7 microA x cm(-2); EC50=9.2 +/- 0.7 mM, n=3), and secretory responses with 5-hydroxytryptamine, 100 microM (DeltaSCC= 16.1 +/- 3.8 microA x cm(-2), n=10), histamine, 100 microM (DeltaSCC=24.0 +/- 4.1 microA x cm(-2), n=10) and prostaglandin E2, 1 microM (DeltaSCC=30.3 +/- 5.4 microA x cm(-2), n=6). Experimental biopsy specimens showed intact surface epithelium by histologic examination and did not differ from controls apart from minor indications of edge damage. No difference in basal electrical parameters and D-glucose fluxes were found between Helicobacter pylori positive and negative patients. Our data suggests that the MUAS chamber represents a promising alternative approach to measure transport processes in intestinal endoscopic biopsies.

Functional characterisation of tachykinin receptors mediating ion transport in porcine jejunum.

In the present study, tachykinin receptors (designated NK 1, NK2 and NK3) involved in regulation of ion transport in porcine jejunum were characterised. Stripped tissue preparations were mounted in Ussing chambers and short-circuited. Substance P produced a concentration dependent increase in short-circuit current, the relationship showing a double sigmoidal form. The non-peptide NK1 receptor antagonist, CP 99,994 ((2S,3S)-3-(2-methoxybenzyl)amino-2-phenylpiperidine), caused a dextral shift of the first sigmoidal response, indicating the involvement of an NK1 receptor. This was further supported by a concentration-dependent response of the NK1 receptor agonist [Sar9Met(O2)11]substance P with an EC50 value of 235.0+/-53.9 nM. Increasing concentrations of CP 99,994 (0.1, 0.3 and 1 microM) produced a parallel dextral shift of the [Sar9Met(O2)11]substance P curve with a slope of the Schild regression significantly different from unity (1.59). The neurokinin A concentration-response curve, with an EC50 value of 68.87+/-16.23 nM, was not significantly changed by the non-peptide NK2 receptor antagonist SR 48,968 ((S)-N-methyl-N-(4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophe nyl)butyl)bezamide). In additional studies, the peptide NK2 receptor antagonists, GR 94,800 (PhCO-Ala-Ala-DTrp-Phe-DPro-Pro-NleNH2) and PD 147,714 ((2,3-diOMeZ)-(S)Trp(S)alphaMePheGlyNH2), did not change the response to neurokinin A. However, CP 99,994 totally inhibited neurokinin A responses at 0.5 microM and above. The NK2 receptor agonist, [beta-Ala8]neurokinin A-(4-10), caused only an increase in short-circuit current in microM concentrations, whereas the NK3 receptor agonist, senktide, did not elicit a response. These results indicate, that substance P and neurokinin A mediate ion transport in porcine jejunum through NK1 receptors. However, tachykinins seem to activate another receptor. Two active conformers of the NK1 receptor might be present.

Tachykinins mediate changes in ion transport in porcine jejunum through release of prostaglandins and neurotransmitters.

In the present study, we investigated the mediators involved in substance P (SP) and neurokinin A (NKA) induced ion transport. Stripped preparations of porcine jejunal tissue were mounted in Ussing-chambers and short-circuited. The cyclo-oxygenase inhibitor, piroxicam (10 microM) and the neuronal conduction blocker, tetrodotoxin (TTX) (0.1 microM) both significantly decreased the SP (0.1 microM) (66% and 36%, respectively) and NKA (1 microM) (64% and 31%, respectively) induced increase in short-circuit current (SCC). Pretreatment with both piroxicam and TTX totally abolished the SP and NKA response. SP (0.1 microM) caused a significant release of prostaglandin E2 (PGE2), whereas the release of PGE2 induced by NKA was not significant. Experiments were performed to clarify if vasoactive intestinal polypeptide (VIP) was mediating SP or NKA responses. VIP caused a TTX-insensitive and a concentration-dependent increase in SCC. Two VIP antagonists did not change the response to VIP (10 nM and 0.1 microM). Thus, these antagonists could not be used to further elucidate the role of VIP. We were unable to measure a significant release of VIP after SP or NKA treatment. These results indicate, that SP and NKA regulate ion transport in porcine jejunum, entirely through the release of prostaglandins and enteric neurotransmitters.

[Pretreatment with prednisolone enhances the effect of human lymphoblastoid interferon in chronic hepatitis B]. / Prednisolon-forbehandling øger effekten af humant lymfoblastinterferon ved kronisk hepatitis B. INTERPRED Trial Group.

Patients (n = 213) with chronic hepatitis B were randomised to prednisolone (two weeks of 0.6 mg/kg/day, one week of 0.45 mg/kg/day and one week of 0.25 mg/kg/day) or placebo followed by two weeks rest, and were then given human lymphoblastoid interferon 10 MU daily for five days followed by 10 MU thrice weekly for 11 weeks. There were statistically significant effects of prednisolone pre-treatment on both HBeAg disappearance and HBeAg to anti-HBe seroconversion (log rank test statistics 5.43; p = 0.02 and 4.75; p = 0.03). HBeAg disappearance and HBeAg to anti-HBe seroconversion rates were 28 vs. 44% and 23 vs. 38% (placebo vs. prednisolone). Fifteen patients (7.5%) lost HBsAg. Three out of 22 cirrhotic patients (14%), one of whom received prednisolone pre-treatment, developed hepatic decompensation with a fatal outcome. Prednisolone pre-treatment, enhances the effect of lymphoblastoid interferon in chronic hepatitis B. Interferon treatment (with and without prednisolone) should be used with caution in patients with cirrhosis and avoided in patients with evidence of hepatic decompensation.

Novel protein kinase C, nPKC, inhibition of murine bumetanide-sensitive Na+,K+,2Cl- cotransporter BSC1 in Xenopus oocyte.

In vitro transcribed RNAs obtained for the absorptive type of Na,K, 2Cl cotransporter BSC1, isoform F, and either of three deletion mutants were injected into oocytes, while oocytes injected with water served as controls. Wild-type cRNA induced a bumetanide-sensitive Rb flux after 18 h which rose to a maximum of about 600 pmol . (h.oocyte)-1 during the next 24 h. This level of flux lasted for at least 31/2 days. All deletion mutants were either not expressed and/or were non-functional. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) eliminated the induced flux, with an apparent dissociation constant (aKi) of 6 nM. A 15 nM PMA-elicited inhibition of Rb flux was blocked by the PKC inhibitor calphostin C (200 nM) to approximately 80%, while PKC inhibitors staurosporine (40 nM) and Gö6976 (50 nM) were ineffective. The phosphatase inhibitor okadaic acid (4.6 nM) did not influence PMA-induced flux inhibition. The PKC activator sn-1, 2-dioctanoyl glycerol (DOG), even at 2 microM, did not inhibit bumetanide-sensitive Rb influx. The induced Rb fluxes were not affected by PKA or PKG, as stimulation by either 0.5 mM dibutyryl-cAMP, 0.5 mM dibutyryl-cGMP, 10 microM forskolin, and/or 0. 5 mM theophylline had no effect on bumetanide-sensitive Rb fluxes. Activating endogenous muscarinic receptors with 20 microM acetylcholine had no effect on expressed BSC1 fluxes. The sensitivity to bumetanide of induced Rb flux had an aKi of around 70 nM. Attempts to follow the expression of BSC1 in plasma membranes with either immunoblotting or radio-methionine pulse-chase were unsuccessful. We conclude that a PKC, likely of the novel type, nPKC, seems to be involved in PMA-induced reduction of expressed BSC1 and/or its ion transport function.

Na/D-glucose cotransport and SGLT1 expression in hen colon correlates with dietary Na+.

We have tested whether separately varying the content of either Na or Cl in diets causes earlier observed increase in Na-coupled sugar and amino acid transport induced by high NaCl diets in hen colon. A comparison was also made between the dependence of the Na-coupled transport on a pure wheat/barley/soya diet against a diet with supplements of essential amino acids, fatty acids, vitamins, and trace elements, as a test for possible elimination of the cotransporters due to a deficient diet. Na/nutrient-coupled transport was measured as changes in short circuit current. The level of expressed Na/glucose cotransporters, SGLT1, due to dietary alterations was followed by quantitative Western blot and immunodetection of SGLT1 in colon, and the dietary effects on plasma aldosterone were assessed as well. An observed switch in transport from amiloride-sensitive electrodiffusive Na transport to phlorizin-sensitive Na/D-glucose cotransport and Na/amino acid-coupled transport is caused solely by increasing Na+ in the diet. Thus, neither dietary Cl- nor the dietary supplements altered the expression of Na(+)-coupled nutrient transport processes. Corroborating these findings, only Na+ in the diet increased the expression of SGLT1 in colon epithelium and suppressed aldosterone level in plasma.

Relation between treatment efficacy and cumulative dose of alpha interferon in chronic hepatitis B. European Concerted Action on Viral Hepatitis (Eurohep).

BACKGROUND/AIMS: Alpha interferon (IFN) is an established treatment of chronic hepatitis B. The effect has been shown to be dose related, recommended dose regimens being associated with a doubling of the spontaneous, baseline HBeAg to anti-HBe seroconversion rate. However, the efficacy of IFN treatment in relation to the dose of IFN actually received remains to be established. The aim of this study was to estimate the relative efficacy of IFN as a function of the cumulative IFN dose. In addition we determined if and when a patient returns to his baseline chance of seroconversion after stopping IFN therapy. MATERIALS AND METHODS: Individual patient data from 10 clinical controlled trials were available for the present analysis, in all, 746 patients, of whom 491 received IFN and 255 were untreated controls. The data were analyzed performing a time-dependent Cox regression analysis of the relative efficacy of IFN using the cumulative IFN dose administered up to any given time during the observation period and the time after termination of therapy as explanatory variables. RESULTS: In the proposed model, the chance of HBeAg disappearance for a treated patient relative to no therapy was estimated to 2.1 at a cumulative dose of 100 MU and leveled out at about 2.8 at a cumulative dose of 500 MU. The effect of IFN was shown to decay rapidly after discontinuation and after 3 months a patient could be considered to be back to his baseline chance of HBeAg disappearance. These findings show that IFN administered at a dose of 15-30 MU/week should be considered effective (relative efficacy approximately 2) already after 1-2 months of treatment. CONCLUSIONS: The present findings do not lend any support to the concept that IFN treatment becomes less effective when a certain total dose of IFN has been administered or that the treatment effect reaches beyond 3 months after stopping IFN.

Prednisolone withdrawal therapy enhances the effect of human lymphoblastoid interferon in chronic hepatitis B. INTERPRED Trial Group.

BACKGROUND/AIMS: The aim of this multicentre, randomised, controlled, clinical trial was to evaluate the effect of prednisolone followed by lymphoblastoid interferon treatment in chronic hepatitis B. METHODS: Two hundred and thirteen patients with chronic hepatitis B were randomised to either prednisolone (2 weeks of 0.6 mg/kg/day, 1 week of 0.45 mg/kg/day and 1 week of 0.25 mg/kg/day) or matching placebo followed by a 2-week rest phase and then human lymphoblastoid interferon 10 MU daily for 5 days followed by 10 MU thrice weekly for 11 weeks. Of 200 evaluable patients, 33 (16.5%) were females, and 50 (25%) were male homosexuals. Thirty three patients (16.5%) had chronic persistent hepatitis, 145 (72.5%) had chronic active hepatitis and 22 (11%) had active cirrhosis. RESULTS: Survival analysis disclosed statistically significant effects of prednisolone pre-treatment on both HBeAg disappearance and HBeAg to anti-HBe seroconversion (log-rank test statistics 5.43; p = 0.02 and 4.75; p = 0.03). Observed HBeAg disappearance and HBeAg to anti-HBe seroconversion rates (placebo vs. prednisolone patients) were 28% vs. 44% and 23% vs. 38%. Six months after stopping interferon, HBV DNA was negative in 51% of prednisolone patients vs. 28% of placebo patients (Chi-square test statistic 6.13; p = 0.013). Prednisolone pre-treatment tended to be more effective in patients with higher transaminase levels and in patients with low levels of HBV DNA. Fifteen patients (7.5%) (13 within 1 year of follow-up) eventually lost HBsAg; 14 of these subsequently developed anti-HBs. Interferon treatment was modified in 102 patients (51%). Three out of 22 patients with cirrhosis (14%), one of whom received prednisolone pre-treatment, developed hepatic decompensation with a fatal outcome while on interferon treatment. CONCLUSIONS: Prednisolone pre-treatment significantly enhanced the treatment effect of lymphoblastoid interferon in terms of HBeAg clearance and seroconversion to anti-HBe. Treatment should be used with caution in patients with cirrhosis and avoided in patients showing signs, or with a history, of decompensated cirrhosis.

The treatment effect of alpha interferon in chronic hepatitis B is independent of pre-treatment variables. Results based on individual patient data from 10 clinical controlled trials. European Concerted Action on Viral Hepatitis (Eurohep).

Alpha interferon induces HBeAg seroconversion in about one third of treated patients and has become an established treatment of chronic hepatitis B. A number of smaller studies have suggested that response to treatment is more likely to occur in patients with higher levels of transaminases, with recent (adult) onset, a history of acute hepatitis, low levels of HBV DNA and in heterosexual males. The aim of this European co-operative study was to estimate the effect of alpha interferon more accurately and to evaluate the influence of host pre-treatment variables on the effect of interferon. Individual data were collected from 751 patients from 10 controlled clinical trials on alpha interferon (lymphoblastoid or recombinant) treatment for chronic hepatitis B. Alpha interferon was administered to 496 patients, while 255 were untreated controls. Individual patient data were analysed by survival analysis (log rank test and Cox regression analysis), stratified by trial, with the disappearance of HBeAg as the major endpoint. The results showed that the HBeAg disappearance rate with or without interferon treatment was higher in patients with high aminotransferase levels, with a history of acute hepatitis and in male heterosexual patients disregarding HIV status. If HIV-positive patients were excluded, the effect of sexual orientation was not significant. Therapy with alpha interferon increased the a priori HBeAg disappearance rate by a factor of 1.76; the relative treatment effect of alpha interferon was independent of the tested pretreatment host variables, but dependent on the total (intended) interferon dose (low dose < or = 200 MU/m2 increased HBeAg disappearance by a factor 1.37; medium/high dose > or = 200 MU/m2 increased HBeAg disappearance by a factor 2.05). In conclusion, this meta-analysis suggests that the effect of alpha interferon is less than previously assumed and independent of pretreatment host variables tested. It confirms the higher therapeutic benefit of a total dose exceeding 200 MU/m2 and of selection of patients based on disease activity and immune reactivity. Although all patient seem to have the same relative benefit, the absolute benefit of alpha interferon treatment seems to be greatest in patients with high transaminase levels and with a history of acute hepatitis.

Serotonin-induced short-circuit current in pig jejunum.

This paper presents the effects of serotonin (5-HT) on short-circuit current (SCC), sodium and chloride fluxes, and prostaglandin E2 release in pig jejunum, using the Ussing-chamber technique. 5-HT elicited a dose-dependent increase in SCC, yielding an EC50 of 6 +/- 4 microM and EMAX of 77 +/- 8 microA.cm-2 using about 100 microM. Inhibited sodium absorption and stimulated chloride secretion carried part of the 5-HT-induced SCC. 5-HT caused a dose-independent PGE2 release, and indomethacin reduced the SCC-inducing effect of 5-HT by 40%. Octreotide, a long-lasting somatostatin analogue, also reduced 5-HT-induced SCC by about 40%, while tetrodotoxin (TTX) did not alter the effect of 5-HT. In conclusion, 5-HT causes a dose-dependent indomethacin and octreotide-sensitive, and TTX-insensitive increase in SCC, and a chloride secretion and inhibited sodium absorption and an increased release of PGE2 in pig jejunum in vitro.

Desensitization and reactivation of ACh-regulated exocrine secretion in hen tracheal epithelium.

We have studied acetylcholine (ACh) desensitization of Cl- secretion and its reactivation by atropine in the hen tracheal epithelium employing voltage-clamp technique. Adding 4 microM ACh to the serosal side induces a maximal bumetanide-sensitive Cl- secretion. The ACh-induced Cl- secretion is desensitized nearly 100% at 512 microM ACh. The desensitization of Cl- secretion may be reduced 30-40% by 0.32 microM atropine (reactivation). From a kinetic model for desensitization and reactivation with two muscarinic receptors (mAChR), we have estimated the maximal secretory response, the apparent dissociation coefficients, and the apparent Hill coefficients for ACh and atropine at the two receptors. Based on the kinetic model we can predict an optimal fixed concentration ratio (Ofcor) between ACh and atropine that will maintain Cl- secretion above 40% of maximum over a wide concentration range, ACh 1-4,100 microM, and we have confirmed the predicted Ofcor experimentally. A cellular model is proposed with two mAChR, one stimulatory and another inhibitory, that accounts for an observed dissociation between desensitization in short-circuit current and conductance.

Second messengers and ion channels in acetylcholine-induced chloride secretion in hen trachea.

1. Hen tracheal epithelium can be stimulated by serosal application of acetylcholine (ACh) to secrete Cl- equal to approximately 60-90 microA/cm2. 2. Radio-ligand-displacement for IP3, cAMP and cGMP and ion channel selective drugs in voltage clamp set-ups were employed to characterize second messengers and Cl-, K+ and Ca2+ channels involved in the ACh response. 3. ACh induced a significant rise in IP3 in isolated tracheocytes, while ACh did not influence the production of cAMP in whole tissue, isolated tracheocytes or basolateral cell membrane vesicles. Further ACh desensitization did not effect cAMP level in tracheocytes. In addition neither ACh stimulation nor desensitization interfered with cAMP production in presence of 4.5 microM forskolin in tracheocytes, a level of forskolin rising base level cAMP by around five fold. 4. Around 35% of ACh Cl- secretion depends on Ca2+ mobilization from internal stores and about 65% on Ca2+ influx over basolateral membrane. The activated Ca2+ channel is insensitive to class I, II, III and IV Ca2+ antagonists. 5. A 23187 can mimic the ACh effect although 30% is indomethacin-sensitive demonstrating a prostaglandin activated adenylyl cyclase. 6. Two K+ channels are involved in ACh secretion, one sensitive to Ba2+ and quinine and both insensitive to 4-aminopyridine, apamin, charybdotoxin and TEA. 7. Flufenamate and triaminopyrimidine block a non-selective ion channel likely involved in the ACh response. An ACh activated apical Cl- channel is NPPB-sensitive.

An eight-month controlled study of a low-fat high-fibre diet: effects on blood lipids and blood pressure in healthy young subjects.

The effects on blood lipids and blood pressure of a diet corresponding to present Nordic Nutrition Recommendations, i.e. less than 30% of energy from fat and with a fibre content exceeding 3 g/MJ, were studied in 18 men and 12 women (mean age, 24 years) under strict dietary control over 8 months. Blood sampling, blood pressure and body weight measurement were performed at four occasions on their habitual diet and once a month during the intervention period. An age-matched control group (17 men, 8 women) was followed with monthly measurements parallel to the intervention group. The habitual diets, assessed by 7-day records, showed an average fat content corresponding to 36% of energy. Initial levels of total cholesterol and HDL cholesterol (X +/- SD) were 4.21 +/- 0.61 and 1.23 +/- 0.23 mmol/l for the men in the intervention group; 4.35 +/- 0.79 and 1.21 +/- 0.26 mmol/l for the male controls; 4.61 +/- 0.59 and 1.46 +/- 0.31 mmol/l for the women in the intervention group and 4.48 +/- 0.64 and 1.48 +/- 0.29 mmol/l for the female controls. Significantly decreased levels of total cholesterol and HDL cholesterol throughout the experimental period were seen for both sexes in the intervention group. Total cholesterol fell 0.49 mmol/l (95% CI: 0.41-0.56) in the male subjects and 0.49 mmol/l (95% CI: 0.39-0.59) in the female subjects. The fall in HDL cholesterol was 0.16 mmol/l (95% C: 0.13-0.18) and 0.18 mmol/l (95% CI: 0.12-0.23), respectively. Total cholesterol changes were independent of initial values. All subjects were normotensive at the start of the study with an average blood pressure of 122/68 mmHg for men and 112/68 mmHg for the women. Systolic blood pressure dropped gradually and significantly in the male subjects of the intervention group. A minimum of 6 mmHg below initial values was noted after six months of dietary intervention. No significant changes in dietary intake and blood lipids were observed in the control group. Thus, changes of present dietary habits of young healthy Danish subjects to an intake in accordance with the Nordic Nutrition Recommendations 1989 will favourably affect suggested risk factors for disease.

Transport of the multidrug resistance modulators verapamil and azidopine in wild type and daunorubicin resistant Ehrlich ascites tumour cells.

Verapamil has been proposed to modulate the multidrug resistance phenotype by competitive inhibition of an energy dependent efflux of cytotoxic drug. However, the accumulation of both 14C-verapamil and 3H-verapamil was similar in wild type EHR2 and multidrug resistant EHR2/DNR+ Ehrlich ascites cells, and was much less in both cell lines in energy deprived medium than in medium containing glucose. Azidopine accumulation was also similar in both EHR2 and EHR2/DNR+ cells but, in contrast to verapamil, did not differ significantly with changes in cellular energy levels. Azidopine photolabelled a 170 kDa protein in EHR2/DHR+ plasma membrane vesicles which was immunoprecipitated by monoclonal antibody towards P-glycoprotein. Azidopine increased daunorubicin accumulation and modulated vincristine resistance in EHR2/DNR+ cells in a similar fashion to verapamil. Azidopine photolabelling was inhibited by vincristine and verapamil, but not by daunorubicin. Vincristine, but not daunorubicin, was able to increase both azidopine and verapamil accumulation in EHR2/DNR+ cells only. Finally, though both verapamil and azidopine are a substrate for P-glycoprotein in EHR2/DNR+ cells, they do not themselves appear to be transported by the multidrug resistance efflux mechanism to any significant extent in these cells.

Characterization of a muscarinic receptor controlling Cl- secretion in hen trachea.

We have attempted to characterize a muscarinic receptor subtype involved in Cl- secretion in isolated epithelium of hen trachea, taking advantage of drugs developed in the last 15 yr. Hen trachea can be stimulated to secrete Cl- equal to approximately 80-90 microA/cm2 by application of acetylcholine (ACh) to the serosal side. The process has an apparent dissociation constant (Kd) for ACh of 740 nM. The Cl- secretion is completely inhibited by 20 microM bumetanide at the serosal side. Of five selective antagonists for muscarinic receptors, pirenzepine, hexahydrosiladifenidol, dicyclomine, 11-([2-[(diethylamino)-methyl]-1-piperidinyl]acetyl)-5,11- dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one, and 4 diphenyl acetoxy-N-methylpiperidine methobromide, only the latter had a high affinity for the functional receptor with an apparent Kd of 3 nM. The receptor may be classified as an M4-muscarinic receptor subtype and probably belongs to a group of muscarinic receptors on exocrine glands and mucosal cells involved in ion transport. All the functional responses caused by muscarinic agonists and antagonists tested exhibited exponents (apparent Hill coefficients) in the range from 1.3 to 2.4, indicating a gain in the stimulus secretion coupling mechanism, an aspect of muscarinic receptor function that is not revealed in radioligand binding studies.

Inhibition of vincristine binding to plasma membrane vesicles from daunorubicin-resistant Ehrlich ascites cells by multidrug resistance modulators.

The multidrug resistance (MDR) phenotype is presumed to be mostly dependent on changes in the resistant cell plasma membrane, notably the emergence of a 170 kDa glycoprotein called P-glycoprotein, which facilitate increased drug efflux. We have previously demonstrated that ATP-enhanced binding of vincristine (VCR) to plasma membrane vesicles is much greater in MDR than in wild type cells. The present study has shown that VCR binding to MDR Ehrlich ascites tumour cell plasma membrane vesicles is inhibited 50% most efficiently by quinidine (0.5 microM) followed by verapamil (4.1 microM) and trifluoperazine (23.2 microM). This is the reverse order of the effect on whole cells where a ranking of efficiency in terms of enhancement of VCR accumulation, inhibition of VCR efflux, DNA perturbation and modulation of resistance in a clonogenic assay, was trifluoperazine greater than or equal to verapamil much greater than quinidine. The detergent Tween 80 inhibited VCR binding to plasma membrane vesicles at 0.001% v/v which agreed with the level which modulated resistance and increased VCR accumulation in whole cells. No effect was observed on daunorubicin binding to MDR plasma membrane vesicles after incubation with either Tween 80 (up to 0.1% v/v) or verapamil (up to 25 microM). We conclude that the effect of a modulating drug in reversing resistance to VCR correlates with its ability to raise intracellular VCR levels but not with its capability to inhibit VCR binding to the plasma membrane. Thus, enhancement of VCR accumulation in MDR cells is hardly solely due to competition for a drug binding site on P-glycoprotein. Furthermore, the lack of a demonstrable effect on daunorubicin binding to the plasma membrane by modulators points to transport mechanisms which do not utilise specific drug binding to the plasma membrane.