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.

Rate-dependent effects of sematilide on ventricular monophasic action potential duration and delayed rectifier K+ current in rabbits.

Our objective was to define the actions of sematilide in rabbits and to assess the contribution of the delayed rectifier (IK) to rate dependence of action potential duration (APD) in rabbit ventricular myocardium. In studies in vivo, New Zealand White rabbits were used to obtain dose/response curves of the effects of sematilide on APD from contact monophasic action potentials (MAP), ventricular effective refractory period (VERP), and ECG. Sematilide or placebo was administered as an i.v. bolus followed by a 45-min infusion in the following cumulative manner: infusion 1 (1 mg/kg bolus + 8 micrograms/kg/min); infusion 2 (2 mg/kg + 20 micrograms/kg/min); and infusion 3 (7 mg/kg + 68 micrograms/kg/min). At each infusion level, VERP and APD at 75% repolarization (APD75) were measured during cardiac pacing between 200- and 400-ms cycle length (CL). Serum sematilide levels were analyzed by high-performance liquid chromatography (HPLC). In studies in vitro, sematilide's effects on the delayed rectifier were assessed in isolated rabbit ventricular myocytes by using patch-clamp techniques. Sematilide infusion in vivo resulted in stable serum levels of 1.3 +/- 0.5, 3.7 +/- 1.4, and 13.4 +/- 1.8 micrograms/ml during infusions 1, 2, and 3, respectively. Maximal effects occurred at infusion 2, such that at 400 ms CL, sematilide widened predrug APD75 (145 +/- 5 ms) by 27 +/- 4% (p < 0.001 vs. placebo), and at 200-ms CL, sematilide prolonged predrug APD75 (115 +/- 10 ms) by only 18 +/- 4% (p < 0.001 vs. placebo; p < 0.05 vs. 400-ms CL). Similar effects were observed in VERP. Sematilide enhanced rate dependence of APD and produced the same degree of APD prolongation at a given CL, during accommodation to and recovery from rapid pacing. Rabbit ventricular myocytes appeared to have at least two types of delayed rectifier. Sematilide selectively blocked IKr, and block was not relieved by repetitive stimulation. In conclusion, the APD-widening effect of sematilide was independent of previous pacing history. Sematilide had little influence on background processes likely responsible for shortening APD because of rapid repetitive stimulation.

Hepatic sinusoidal fibrosis induced by cholesterol and stilbestrol in the rabbit: 2. Hemodynamic and drug disposition studies.

We assessed hepatic functions and systemic and splanchnic hemodynamics in a new model of hepatic sinusoidal fibrosis. Fibrosis was induced by the simultaneous administration for 8 weeks of diethyl-stilbestrol (DES) (10 mg twice weekly, subcutaneously) and cholesterol-supplemented diet (1%) in rabbits. A marked and progressive impairment of hepatic function was observed during the 8 weeks of treatment with a significant decrease in indocyanine green (ICG) systemic clearance (-89%; P < .001) and aminopyrine elimination (-69%; P < .001). In fibrotic animals, hyperdynamic circulation was found with an increased cardiac output (+73%, P < 0.01) and a decreased peripheral vascular resistance (-50%; P < .005), as evaluated by the microsphere technique in animals that were awake. The total portal venous inflow was not significantly modified in fibrotic rabbits. However, since there was a marked increase in the liver weight, the portal venous inflow was significantly decreased when expressed per gram of liver weight (-30%; P < .05). In contrast, the hepatic artery blood flow was markedly increased, even when expressed per gram of liver weight (+95%; P < .01). Portal pressure was significantly increased in treated rabbits (from 7.4 +/- .4 to 14.4 +/- .6 mm Hg, P < .01). This new experimental model could prove useful to evaluate the influence of extensive perisinusoidal fibrosis on exchanges between plasma and hepatocytes, particularly of protein-bound substances.

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.

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%.

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.

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.

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.

A comparison of lumbar epidural and intravenous fentanyl infusions for post-thoracotomy analgesia.

This double-blind randomised study compared the analgesic efficacy, respiratory effects, side effects, and pharmacokinetic disposition of 24 hr lumbar epidural and intravenous infusions of the same dosage regimen of fentanyl (1.5 micrograms.kg-1 bolus then 1 microgram.kg-1.hr-1 infusion) in 50 patients after thoracotomy. Patients received either epidural fentanyl and intravenous normal saline, or epidural normal saline and intravenous fentanyl, for postoperative analgesia, after a standard low-dose alfentanil and isoflurane general anaesthetic. Visual analogue pain scores were lower in the epidural group (P < 0.05) only at two hours postoperatively, and there was no difference in the amount of supplementary morphine self-administered by patient-controlled analgesic pump. A mainly spinal analgesic effect probably occurred in the first few hours since fentanyl was not detectable in the plasma of patients in the epidural group until two hours after bolus injection; its concentration was less at that time than after intravenous injection (P < 0.05). Thereafter there was no difference in the plasma concentration profiles between the two groups. Seven patients in the epidural group and ten patients in the intravenous group received naloxone for PaCO2 > 50 mmHg, and one patient in the intravenous group had the infusions stopped because of PaCO2 elevation and somnolence. In patients who did not receive naloxone, the epidural route produced better analgesia throughout the study period (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Intravenous nifedipine to treat hypertension after coronary artery revascularization surgery. A comparison with sodium nitroprusside.

We administered sodium nitroprusside (SNP) or nifedipine intravenously to patients who became hypertensive after elective coronary revascularization and compared their effects on hemodynamics and the electrocardiogram in a parallel, randomized, open-label study. Four of 21 patients treated with nifedipine required the addition of SNP to maintain mean arterial pressure less than 90 mm Hg, compared with 4 of 28 patients in the SNP group who required the addition of nifedipine. The success rates of nifedipine (81%) and SNP (86%) were not significantly different. There was no difference in the incidence of adverse ST-segment changes during drug infusion (4% versus 5%) or perioperative myocardial infarction (9.5% versus 10.7%) in the nifedipine versus SNP groups, respectively. The plasma nifedipine concentration (mean value +/- SD) at steady state for 21 patients receiving nifedipine was 119 +/- 42.5 ng/mL. The pharmacokinetic variables for nifedipine were as follows (mean values +/- SD): systemic clearance, 0.525 +/- 0.228 L.h-1.kg-1; apparent volume of distribution, 0.738 +/- 0.446 L/kg; and elimination half-life, 1.02 +/- 0.51 h. These values are similar to those reported previously in healthy volunteers. We conclude that intravenous nifedipine can be used safely to control hypertension after coronary revascularization but were unable to demonstrate an advantage of nifedipine compared with SNP in preventing postoperative ischemia or infarction in this group of patients who had good left ventricular function.

Effect of an acute dose of alcohol on the pharmacokinetics of oral nifedipine in humans.

Pharmacokinetic and pharmacodynamic interactions of alcohol and nifedipine were assessed in 10 healthy human volunteers. Doses of 20 mg (2 x 10-mg capsules) of nifedipine were administered with either 150 ml of orange juice or 75 ml of alcohol (94%) in 75 ml of orange juice according to a crossover randomized design. Plasma nifedipine levels were monitored for 16 hr after each dosing, along with pulse rate and blood pressure. The relative bioavailability of nifedipine, measured as AUC, was increased by 54% (533 vs 346 ng.hr/ml) after the dose of alcohol (P less than 0.0002). However, there were no significant differences between treatments in Cmax, tmax, or t1/2. Although there was no difference in the systolic and diastolic blood pressure and pulse rate between the two treatment groups, the time to reach peak heart rate was significantly faster in the group treated with alcohol (1.4 vs 2.2 hr). This study shows that ethanol increases the bioavailability of nifedipine and decreases the time for onset of increased heart rate.

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.

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.

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.

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.