Morphogenetic plasticity of adult human pancreatic islets of Langerhans.

The aim of this study was to investigate the phenotypic plasticity of pancreatic islets of Langerhans. Quiescent adult human islets were induced to undergo a phenotypic switch to highly proliferative duct-like structures in a process characterized by a loss of expression of islet-specific hormones and transcription factors as well as a temporally related rise in the expression of markers of both duct epithelial and progenitor cells. Short-term treatment of these primitive duct-like structures with the neogenic factor islet neogenesis-associated protein (INGAP104-118) induced their reconversion back to islet-like structures in a PI3-kinase-dependent manner. These neoislets resembled freshly isolated human islets with respect to the presence and topological arrangement of the four endocrine cell types, islet gene expression and hormone production, insulin content and glucose-responsive insulin secretion. Our results suggest that adult human islets possess a remarkable degree of morphogenetic plasticity. This novel observation may have important implications for understanding pancreatic carcinogenesis and islet neogenesis.

Pharmacokinetic and pharmacodynamic interactions between diltiazem and quinidine.

OBJECTIVES: To examine the pharmacokinetic and pharmacodynamic interactions between quinidine and diltiazem because both drugs can inhibit drug metabolism. METHODS: Twelve fasting, healthy male volunteers (age, 24 +/- 5 years; weight, 75 +/- 10 kg) received a single oral dose of diltiazem (60 mg) or quinidine (200 mg), alone and on a background of the other drug, in a crossover study. Background treatment consisted of 100 mg quinidine twice a day or 90 mg sustained-release diltiazem twice a day for 2 day before the study day. RESULTS: Pretreatment with diltiazem significantly (p < 0.05) increased the area under the curve of quinidine from 7414 +/- 1965 to 11,213 +/- 2610 ng.hr/ml and increased its terminal elimination half-life (t1/2) from 6.8 +/- 1.1 to 9.3 +/- 1.5 hours. Its oral clearance was decreased from 0.39 +/- 0.1 to 0.25 +/- 0.1 L/hr/kg, whereas the maximal concentration was not significantly affected. Diltiazem disposition was not significantly affected by pretreatment with quinidine. Diltiazem pretreatment increased QTc and PR intervals and decreased heart rate and diastolic blood pressure. No significant pharmacodynamic differences were shown for diltiazem alone versus quinidine pretreatment. CONCLUSION: Diltiazem significantly decreased the clearance and increased the t1/2 of quinidine, but quinidine did not alter the kinetics of diltiazem with the dose used. No significant pharmacodynamic interaction was shown for the combination that would not be predicted from individual drug administration.

Effect of chronic food restriction in aging rats. I. Liver subcellular membranes.

To assess which membrane properties are modulated by the action of food restriction, characteristics of liver membrane structures of ad libitum-fed and food restricted Fischer 344 rats were analyzed over a wide age range. The results show that the yields of mitochondrial and microsomal membranes decreased in ad libitum-fed rats, but this age-related loss did not occur in food restricted rats until 30 months. Changes in membrane fatty acid composition which occurred with age were substantially modified by food restriction. Linoleic acid content progressively decreased in the membranes of ad libitum-fed rats with a concomitant increase of docosapentaenoic acid while an opposite pattern of change occurred in food restricted rats. Furthermore, food restriction maintained a low docosahexaenoic acid level in microsomes at all ages studied. While serum tocopherol increased markedly with age, there was little change in membrane tocopherol content in ad libitum-fed rats.

Effect of chronic food restriction in aging rats. II. Liver cytosolic antioxidants and related enzymes.

The cytosolic status during aging of several antioxidants and enzymatic activities which protect the cell from oxidative damage was explored in the liver of ad libitum-fed and food restricted rats. Restricting calories effectively prevented the age-related decrease in cellular glutathione that occurs in ad libitum-fed rats. Although glutathione reductase exhibited little change with age in ad libitum-fed rats, dietary restriction resulted in greater activity of this enzyme than that of ad libitum-fed animals. Glutathione S-transferase activity of ad libitum-fed rats decreased significantly with age in ad libitum-fed rats but not in food restricted rats. The glutathione peroxidase activity which increased until 12 months in the ad libitum-fed rats declined by 24 months; there was little change with adult age in this enzymatic activity in food restricted rats. Catalase activity declined steadily from 3-24 months in the ad libitum-fed rats, and food restriction prevented this age-related decline. The significance of antioxidants and the related protective enzymes is discussed relative to membrane alterations and the anti-oxidative action of food restriction in relation to age-related degenerative damages.

Acute and chronic studies with the anticholinesterase Huperzine A: effect on central nervous system cholinergic parameters.

High affinity choline transport, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were assessed in rats after acute and chronic administration of the AChE inhibitor Huperzine A. Acute treatment: Forty-five min after a single injection of Huperzine A (0.5 mg/kg i.p.) the activity of AChE was significantly decreased by 15-30% in hippocampus, striatum and septum. The activity of ChAT was not altered. In the hippocampus high affinity choline transport was attenuated by 25%, whereas no effect in the striatum was observed. After 90 min, both inhibition of AChE and attenuation of high affinity choline transport had returned to control values. A dose of 0.1 mg/kg (i.p.) did not produce significant effects. Similar results were obtained with physostigmine (0.25 mg/kg), although the duration of inhibition of AChE was shorter than that with Huperzine A. Chronic treatment: After 5 days (twice a day), at 0.5 mg/kg, the activity of AChE was significantly reduced by 20-30% in every region of the brain studied. High affinity choline transport in the hippocampus was reduced by 28%, 45 min after the last injection, but in the striatum there was no effect. The activity of ChAT was not affected in any region of the brain studied. Thus, acute or chronic treatment with Huperzine A: did not alter ChAT; reduced high affinity choline transport in the hippocampus in a transient manner; and had a longer duration of action as an AChE inhibitor than physostigmine. Moreover, tolerance to low-toxicity doses of Huperzine A was minimal, contrary to what has been observed with other inhibitors of AChE.

Sector-dependent neurotoxicity of ethylcholine aziridinium (AF64A) in the rat hippocampus.

The present study was aimed at measuring the distribution of ethylcholine aziridinium (AF64A)-induced cholinotoxicity within the hippocampus 6 days after bilateral (icv) administration of 1, 2 or 3 nmol, or vehicle. The dissected hippocampus was sectioned with a vibratome into 5 parallel sectors distributed along its long axis from its thalamic surface (medial) to its cortical surface (lateral). In vehicle-treated rats, the high affinity cholinergic transport (HAChT), choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were distributed according to a gradient of increasing activity, extending from the lateral to the medial surface of the hippocampus. After treatment with AF64A, the normal gradient of enzyme activity was profoundly disrupted at all doses of AF64A and the core sectors of the hippocampus were significantly more affected than the superficial sectors. The HAChT gradient was progressively abolished with increasing doses of toxin, and the effect was maximal at 2 nmol.

Alteration in the disposition and metabolism of S(-)-propranolol in rats with active respiratory viral infection.

Viral illness elicited marked changes in the disposition and metabolism of S(-)-propranolol in rats during the course of a recent viral outbreak in our rodent colony. A slower rate of gastrointestinal absorption and a much higher clearance of orally-administered S(-)-propranolol were observed. The apparent increase in metabolic clearance is partly attributed to a decrease in the serum protein binding of S(-)-propranolol. A direct stimulation of microsomal oxidative metabolism was also suggested by an increase in urinary recovery of the monohydroxyl metabolites of S(-)-propranolol. In addition, a greater portion of the phenolic metabolites were present in urine as the glucuronic acid conjugates, which may be explained by a concomitant stimulation in conjugative reaction. These observations are unusual in that existing reports indicate that viral illness in man and animals generally leads to an inhibition of oxidative drug metabolism.

Study on the lipid composition of aging Fischer-344 rat lymphoid cells: effect of long-term calorie restriction.

Long-term calorie restriction (LCR) is widely known to increase the survival rate of laboratory rodents and appears to retard the aging and senescence process. The present study was undertaken in Fischer-344 male rats maintained on ad libitum (AL) or LCR (40% less food intake than AL starting at 6 weeks of age). Age-associated changes in the proliferative response of lymphoid cells to mitogenic stimuli were studied in relation to alterations in the fatty acid composition of adherent and non-adherent-enriched subpopulations of spleen cells. Increases in spleen cell long-chain highly unsaturated fatty acids (20:4, 22:4 and 22:5) were accompanied by decreases in linoleic acid (18:2) in aging AL-fed rats. However, LCR stabilized levels of 18:2 and prevented the rise in highly unsaturated fatty acids. In addition, LCR markedly modulated the fatty acid profiles of thymocytes and bone marrow cells. A 70% decline in concanavalin A (Con A) stimulated [3H]thymidine uptake of spleen cells from AL animals was normalized by LCR. Splenic reduced glutathione (GSH), a potential modulator of the mitogenic response, was unaffected by age and nutritional regimen. Thus, normalization of lymphoid cell fatty acid composition by LCR parallels the preservation of mitogenic responsiveness to Con A.

Amrinone and N-acetylamrinone assay in human plasma using solid-phase extraction and reversed-phase chromatography.

A simple and sensitive liquid chromatographic assay for simultaneous quantitation of amrinone and N-acetylamrinone in human plasma was developed. The method involves extraction of samples via activated solid-phase extraction Bond Elut C18 disposable columns, followed by chromatographic separation on a reversed-phase phenyl column using isocratic condition and UV detection. The assay can measure concentrations of both compounds over the range 0.075-10 micrograms ml-1. The injection interval is 11 min. The inter-day relative standard deviation (RSD) for replicate analysis of spiked samples is less than 10% and the accuracy more than 94% for both compounds over the standard curve range. The assay has been successfully applied to pharmacokinetic studies in humans.

Studies on membrane lipid peroxidation in omega-3 fatty acid-fed autoimmune mice: effect of vitamin E supplementation.

Enzyme-dependent and non-enzymatic in vitro lipid peroxidation was studied in autoimmune prone B/W mice fed diets containing high levels of dietary corn oil (CO) or menhaden fish oil (FO) as lipid source since weaning. Lipid analysis revealed that FO-fed mouse liver mitochondrial and microsomal membrane fractions incorporated 20:5 omega 3 and 22:6 omega 3 in replacement of 18:2 omega 6 and 20:4 omega 6 found in corn oil (CO) fed control animals reflecting the composition of the dietary oils. Lower concentrations of vitamin E were found in the FO-fed mouse membranes and serum than those of CO-fed mice when diets were supplemented with a standard 75 I.U. alpha-tocopheryl acetate/kg diet. The rate and extent of membrane lipid peroxidation was greatly increased in FO-fed, vitamin-E-depleted membranes. Full repletion of membrane vitamin E levels by supplementation with 500 I.U./kg of FO diet for 30 days significantly decreased lipid peroxidation and showed that in FO-fed mice, membrane peroxidation is inversely proportional to vitamin E content. However, due to a lower ratio of vitamin E and highly unsaturated fatty acids, FO-fed mouse membranes were more sensitive to pro-oxidant stimulus than were those from CO-fed mice. These findings illustrate the action of vitamin E against membrane lipid peroxidation and stress the importance of adequate supplementation of antioxidant with high omega-3 fatty acids intake.

Modulation of membrane phospholipid fatty acid composition by age and food restriction.

Phospholipids from liver mitochondrial and microsomal membrane preparations were analyzed to further assess the effects of age and lifelong calorie restriction on membrane lipid composition. Results showed that the major phospholipid classes, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol and cardiolipin did not vary significantly with age or diet. The fatty acid composition of the phospholipids was determined in PC and PE and ages of 6, 12 and 24 months. The data revealed characteristic patterns of age-related changes in ad libitum (AL) fed rats: membrane levels of long-chain polyunsaturated fatty acids, 22:4 and 22:5, increased progressively, while membrane linoleic acid (18:2) decreased steadily with age. Levels of 18:2 fell by approximately 40%, and 22:5 content almost doubled making the peroxidizability index increase with age. In addition, levels of 16:1 and 18:1 decreased significantly with age, indicating a possible change in delta 9-desaturase activity coefficient. Food restriction resulted in a significant increase in levels of essential fatty acids while attenuating levels of 22:4, 22:5, 22:6 and peroxidizability. We concluded that the membrane-stabilizing action of long-term calorie restriction relates to the selective modification of membrane long-chain polyunsaturated fatty acids during aging.

High-performance liquid chromatographic assay for sematilide in plasma using solid-phase extraction microcolumn technology.

A simple and sensitive high-performance liquid chromatographic assay for quantification of sematilide in rabbit plasma was developed. After extraction of samples via solid-phase extraction on C8 microcolumns, baseline resolution was achieved on a reversed-phase 5 microns Inertsil ODS-2 column using isocratic conditions with mobile phase consisting of water-glacial acetic acid-acetonitrile-methanol-triethylamine (93.5:4.0:1.5:0.5:0.5) and UV detection at 254 nm. The assay did not require evaporation or reconstitution steps. The injection interval was 8 minutes. The inter-day coefficient of variation for replicate analysis of spiked samples was less than 7.6% and the accuracy was more than 97% over the standard curve range (0.128 to 3.191 microM) using 0.5 ml of plasma. The assay has been successfully applied to pharmacokinetic studies in rabbits.

Adaptation of intestinal sugar and amino acid transport to gastric hyperalimentation.

These studies were undertaken to analyze the effects in dogs of the administration of liquid polymeric diets by gastric tube for 14 days on brush-border absorptive functions measured in vitro. Transport studies of D-glucose, L-leucine and L-alanine have been performed by a rapid filtration technique using brush border membrane vesicles isolated from different segments of the small intestine in order to get insights about events occurring directly at the membrane level. Our results clearly show that only the Na+ -gradient dependent pathways for sugars and neutral amino acids transport were modified in treated dogs as compared to chow-fed controls. The adaptation to the diets varied according to the intestinal segment considered and the effects were dependent on both the nature of the diet and the transport function analysed. These effects cannot be due to heterogeneity in the populations of vesicles isolated in the different situations and cannot be attributed to a modification of either the passive permeability of the membrane to solutes or ions or the membrane potential. Our results are best explained by changes in carrier densities along the small intestine in response to modification in the local concentrations of nutrients sequential to faster flow rates in the small intestine. The liquid nature of the polymeric diets, combined with their high fat and low residue content as compared to dog chow, could act in concert to promote faster rates of gastric emptying and intestinal transit, thus leading to modifications in nutrient availability along the small intestine.

Anti-lipoperoxidation action of food restriction.

Chronic food restriction inhibited the age-related increase of malondialdehyde production and lipid hydroperoxides in liver mitochondrial and microsomal membranes of ad libitum fed Fischer 344 rats. The anti-lipoperoxidation action of food restriction could not be attributable to the changes in membrane lipid content nor vitamin E status. Restricting calories modified membrane fatty acid composition by increasing linoleic acid and decreasing docosapentaenoic acid content in both membranes. The significance of the fatty acid modification was discussed in terms of anti-lipoperoxidation and membrane fluidity.

Altered S(--)-propranolol disposition in bilateral ureter-ligated rats.

Previous clinical studies indicate that the metabolic clearance of oral (+/-)-propranolol is reduced in end-stage renal failure patients. Animal models are needed to explore the mechanism(s) underlying the observed metabolic inhibition in man. The disposition kinetics of S(-)-propranolol were characterized after intravenous and peroral administration in rats with acute renal failure induced by bilateral ureteral ligation (BUL). No alteration in either the systemic clearance or the apparent volumes of distribution of S(-)-propranolol was observed in renal failure animals after a single intravenous dose of 1.5 mg/kg. In contrast, acute uremia did elicit a change in the bioavailability of orally administered S(-)-propranolol. At 36 h after ureteral ligation, the area under the serum concentration-time curve after a 6 mg/kg oral dose of S(-)-propranolol was significantly elevated in renal failure animals, which corresponded to an approximate two fold increase in its systemic availability (from 7.7 to 20.5%). Such an effect could not be demonstrated at times earlier than 36 h after ureteral ligation. Additional experiments were performed to evaluate whether concomitant changes in gastrointestinal absorption or serum protein binding of S(-)-propranolol could have contributed to the apparent increase in oral availability. The results lead to the hypothesis of an inhibited first-pass hepatic metabolism of S(-)-propranolol in acute renal failure and suggest a significant time delay in the onset of inhibition.

Digestive and absorptive functions along dog small intestine: comparative distributions in relation to biochemical and morphological parameters.

The digestive (hydrolytic enzymes) and absorptive (sugar and amino acid transport) functions of dog small intestine have been evaluated in different segments and analysed in relation to morphometric and biochemical parameters. The dog small intestine is a cylinder of decreasing diameter in which the underlying mucosa thins down from duodenum to ileum, though maintaining its cellular homogeneity as revealed by measuring the mucosal weight, the total DNA and protein content and the protein content of the brush border membrane. Sucrase, gamma-glutamyltranspeptidase, leucylnaphthylamidase and alkaline phosphatase specific activities, measured both in homogenates of the mucosa and purified brush border membrane fractions, were found distributed along proximo-distal gradients of activity. However, different patterns were obtained which are specific for the enzyme considered. Kinetic parameters, Vmax and Km, were estimated for sucrase and alkaline phosphatase in purified brush border membrane fractions. It appeared that Vmax correlated well with the observed distribution of catalytic sites along the small intestine. Sugar (glucose) and amino acid (alanine and leucine) transport capacities were also distributed according to specific proximo-distal gradients but passive and facilitated diffusions were not affected. Only the active, Na+ -dependent component of transport was sensitive to position along the small intestine and we postulated that this adaptation should involve variations in carrier densities. It is therefore concluded that absorbo-digestive functions are intrinsic characteristics of the brush border membrane which are regulated according to the position along the small intestine.

Simultaneous determination of diltiazem and quinidine in human plasma by liquid chromatography.

A novel method for simultaneous determination of diltiazem and quinidine in human plasma is described. Plasma is alkalinized and extracted with methyl tert.-butyl ether. The ether phase is separated and evaporated. The residue is reconstituted in 0.2 ml of mobile phase containing 56 mM octanesulfonic acid then washed twice with n-hexane. Aliquots are chromatographed on a silanol-deactivated reversed-phase column using a mobile phase containing aqueous H2SO4 (0.01 M, pH 2)-methanol-acetonitrile (45:45:10) and 10 mM octanesulfonic acid. Peaks are monitored with a UV detector set at 237 nm and a fluorescence detector using an excitation set at 247 nm and a 270 nm UV cut-off filter at the emission. Calibration and standard curves were linear from 1 to 130 ng on-column for diltiazem and from 2 to 600 ng on-column for quinidine. Limits of quantitation were 2 and 4 ng/ml for diltiazem and quinidine, respectively. Recoveries from spiked plasma were 94.0 to 102.5% (R.S.D. 6.0-11.4%) for diltiazem and 98.5% to 104.1 (R.S.D. 7.7-8.7%) for quinidine over the ranges studied. In vitro stability was studied in spiked plasma samples stored at -80 degrees C for sixteen months. Both diltiazem and quinidine remained within 10% from nominal values. For ex vivo stability at -80 degrees C, a plasma sample obtained from a volunteer 2 h after oral administration of diltiazem (60 mg) was analysed for two days after sampling and eighteen months later. The mean deviation from initial measured was 4.7%.

Intravenous nifedipine for prevention of myocardial ischaemia after coronary revascularization.

We sought to determine the pharmacokinetic and pharmacodynamic behaviour of a continuous infusion of nifedipine given for prevention of myocardial ischaemia following coronary artery bypass graft (CABG) surgery. Patients scheduled for elective CABG, who had good left ventricular function, were included. Only normotensive patients who did not require treatment with vasoactive drugs and were bleeding less than 100 ml.hr-1 following surgery were included. The patients were randomly distributed into two groups: a control group not receiving any treatment and a treated group receiving a bolus (3 micrograms.kg-1.min-1 for 5 min) and maintenance (0.2 micrograms.kg-1.min-1) infusion of nifedipine, starting upon arrival in the recovery room and continuing for four hours. Patients given nifedipine were compared with control patients in order to determine the effects of nifedipine on haemodynamic function and on the postoperative incidence of hypotension, hypertension, myocardial ischaemia and infarction. Continuous 2-lead Holter monitoring was used to detect myocardial ischaemia. Infarction was diagnosed by 12-lead ECGs and by assessment of the MB-isoenzyme creatine kinase. The infusion of nifedipine rapidly achieved and maintained plasma concentrations between 30 and 40 ng.ml-1. The pharmacokinetic studies revealed a systemic clearance of nifedipine of 0.371 +/- 0.101 L.hr-1.kg-1, an apparent volume of distribution of 0.764 +/- 0.288 L.kg-1 and an elimination half-life of 1.4 +/- 0.6 hr. No correlation was found between plasma concentration of nifedipine and mean arterial pressure (MAP). The incidence of postoperative hypotension (MAP < 70 mmHg) and hypertension (MAP > 100 mmHg) was comparable between the groups. All haemodynamic variables were similar in both groups during the study period. Of 23 patients who received nifedipine, none showed evidence of ischaemia within six hours of starting the infusion. During the same period, five of 24 patients in the control group had ST-segment deviation suggestive of myocardial ischaemia (P = 0.05, Fisher's exact test). Three patients in the control group and none in the nifedipine group suffered perioperative myocardial infarction (P = NS). In conclusion, the continuous infusion of nifedipine used in this study is safe and reduces the incidence of myocardial ischaemia in normotensive patients with good left ventricular function following CABG. Further studies of larger number of patients are required to determine the role of calcium entry blockers following coronary artery surgery.

Stereoselective high-performance liquid chromatographic assay for propranolol enantiomers in serum.

A simple and sensitive stereoselective high-performance liquid chromatographic assay for the quantitation of propranolol enantiomers in serum is described. The method involves conversion of the propranolol enantiomers to diastereomeric urea derivatives by reaction with the clinical reagent (+)-phenylethylisocyanate, followed by chromatographic separation of the diastereomeric products. Conditions of the derivatization reaction were optimized to achieve rapid and quantitative yield with either of the enantiomers. Baseline resolution of the diastereomers was achieved on a reversed phase C8 column with an isocratic mobile phase. Fluorescence detection afforded an absolute on-column detection limit of 100 pg. The assay has been applied to pharmacokinetic studies in humans and small laboratory animals.

Proarrhythmia of a class Ic drug: suppression by combination with a drug prolonging repolarization in the dog late after infarction.

Encainide treatment in patients after myocardial infarction is associated with increased risk of sudden cardiac death. This may relate to drug-induced changes in the electrophysiologic milieu, thus predisposing the patient to sustained ventricular tachyarrhythmias. The goals of this study were to first develop a model of class Ic-induced ventricular tachycardia (VT) and then to design treatments to oppose this prodysrhythmic activity. Dogs with time-dependent loss of inducible sustained VT in the antiarrhythmic drug-free state were studied late after infarction. These dogs received a series of three loading and maintenance infusions of O-demethyl encainide (ODME) to achieve concentrations of 60 +/- 31, 136 +/- 46 and 339 +/- 171 ng/ml. Drug maintenance continued until programmed stimulation induced monomorphic sustained VT. When ODME infusion allowed this induction, barium chloride infusions were added. ODME treatment allowed induction of monomorphic sustained VT in 9 of 10 dogs studied. Prodysrhythmic monomorphic VT was significantly related (P < .01) to prolongation of conduction velocity in the peri-infarct zone. ODME modestly increased ventricular refractoriness at some but not all peri-infarct sites. Infusion of barium chloride in the above nine dogs caused their hearts to return to the noninducible state. Prolongation of refractoriness in the peri-infarct zone was correlated to this suppression of prodysrhythmia. Prolongation of conduction velocity in the absence of substantial prolongation of refractoriness may underlie ODME-facilitated induction of monomorphic VT. Prolongation of refractoriness in the peri-infarct zone by combination treatment with barium chloride reversed prodysrhythmic VT in all of the dogs.