Measurement of intercellular electrical coupling in guinea-pig detrusor smooth muscle

Ropivacaine, a new long-acting local anesthetic, is currently being investigated for the treatment of ulcerative colitis. In view of the increased incidence of dysplasia and neoplasia associated with ulcerative colitis, it is important that the medical treatment of these patients does not stimulate cell proliferation further. This study was performed to evaluate the effect of ropivacaine on the proliferation of human colon adenocarcinoma cells (HT-29 and Caco-2) in vitro. A serum-induced proliferation assay of human colon adenocarcinoma cells was used. Ropivacaine inhibited the growth of HT-29 and Caco-2 cells in a dose-dependent manner. Fifty percent inhibition of growth was found at a ropivacaine concentration of 250 microM when the HT-29 cells were cultured in 1% fetal calf serum and of 550 microM when the HT-29 cells were cultured in 10% serum. The effective concentrations are within the range of the therapeutic concentrations obtained in the colon of patients treated rectally with ropivacaine. Lidocaine, hydrocortisone, and 5-aminosalicylic acid were found to be less potent than ropivacaine in inhibiting proliferation. Ropivacaine caused a dose-dependent membrane depolarization that appeared to correlate with the inhibited cell proliferation, whereas the effect was not related to inhibition of leukotriene B4 or prostaglandin E2. In conclusion, the antiproliferative activity of ropivacaine, combined with previously reported anti-inflammatory activities, makes this drug an interesting new alternative for the local treatment of ulcerative colitis.     

Growth of rabbit aortic smooth muscle cells in serum from patients with juvenile diabetes

The effect of human diabetic serum on the growth of rabbit arterial smooth muscle cell cultures was studied in the stationary phase of growth. The serum was obtained from young, male, non-obese, juvenile diabetics and non-diabetics. The experiments were carried out using dialysed as well as non-dialysed serum. The concentration of cholesterol and triglycerides were equal in normal and diabetic serum. Media supplemented with diabetic serum from both short term and long term diabetics stimulated the outgrowth of the smooth muscle cells significantly (2p less than 0.01). A statistically significantly stimulation of growth was also observed using dialysed human diabetic serum (2p less than 0.05). Autoradiographic studies showed that the number of 3H-thymidine labelled cells and of cells in mitosis increased appreciably after incubation in diabetic human serum (2p less than 0.005). The present data show that human serum from juvenile diabetics contains a factor or factors which promote an excessive growth of arterial medial cells. The factor(s) is not lipids as hyperlipemia was not present nor is it glucose, aminoacids, fructose or ketones, as the growth effect remained after dialysis.     

Calcium transients and the effect of a photolytically released calcium chelator during electrically induced contractions in rabbit rectococcygeus smooth muscle

Intracellular Ca2+ was determined with the fura-2 technique during electrically induced contractions in the rabbit rectococcygeus smooth muscle at 22 degreesC. The muscles were electrically activated to give short, reproducible contractions. Intracellular [Ca2+] increased during activation; the increase in [Ca2+] preceded force development by approximately 2 s. After cessation of stimulation Ca2+ fell, preceding the fall in force by approximately 4 s. The fluorescence properties of fura-2 were determined with time-resolved spectroscopy using synchrotron light at the MAX-storage ring, Lund, Sweden. The fluorescence decay of free fura-2 was best described by two exponential decays (time constants approximately 0.5 and 1.5 ns) at low Ca2+ (pCa 9). At high Ca2+ (pCa 4.5), fluorescence decay became slower and could be fitted by one exponential decay (1.9 ns). Time-resolved anisotropy of free fura-2 was characteristic of free rotational motion (correlation time 0.3 ns). Motion of fura-2 could be markedly inhibited by high concentrations of creatine kinase. Time-resolved spectroscopy measurements of muscle fibers loaded with fura-2 showed that the fluorescence lifetime of the probe was longer, suggesting an influence of the chemical environment. Anisotropy measurements revealed, however, that the probe was mobile in the cells. The Ca2+-dependence of contraction and relaxation was studied using a photolabile calcium chelator, diazo-2, which could be loaded into the muscle cells in a similar manner as fura-2. Photolysis of diazo-2 leads to an increase in its Ca2+-affinity and a fall in free Ca2+. When muscles that had been loaded with diazo-2 were illuminated with UV light flashes during the rising phase of contraction, the rate of contraction became slower, suggesting a close relation between intracellular Ca2+ and the cross-bridge interaction. In contrast, photolysis during relaxation did not influence the rate of force decay, suggesting that relaxation of these contractions is not determined by the rate of Ca2+ removal or due to an increased Ca2+ sensitivity, but instead is limited by other processes such as deactivation by dephosphorylation or detachment of tension-bearing cross-bridges, possibly regulated by thin filament systems.     

Measurement of plasma total homocysteine by HPLC with coulometric detection

Vertebrate MitBASE is a specialized database where all the vertebrate mitochondrial DNA entries from primary databases are collected, revised and integrated with new information emerging from the literature. Variant sequences are also analyzed, aligned and linked to reference sequences. Data related to the same species and fragment can be viewed over the WWW. The database has a flexible interface and a retrieval system to help non-expert users and contains information not currently available in the primary databases. Vertebrate MitBASE is now available through the MitBASE home page at URL: http://www.ebi.ac.uk/htbin/Mitbase/mitb ase.pl. This work is part of a larger project, MitBASE which is a network of databases covering the full panorama of knowledge on mitochondrial DNA from protists to human sequences.     

A procedure for the determination of 5-fluorouracil in tissue using microbore HPLC and fluorescence detection

For the determination of the cytotoxic drug 5-fluorouracil in tissue, a sensitive and selective assay has been developed based on microbore high-performance liquid chromatography and fluorescence detection after pre-column derivatization with 4-bromomethyl-7-methoxycoumarin. 5-Chlorouracil was found to be an appropriate internal standard. A column switching protocol has been devised to separate the analyte from late eluting matrix components and the natural uracil. The drug can be determined at concentrations in the low ng/g wet tissue range with a detection limit of 3 ng/g; furthermore, the same protocol can be applied to analyze serum and plasma samples. The procedure was employed to determine the drug in samples taken from human liver tumors and metastases after an intra-arterial bolus administration and from epithelial carcinomas after systemic continuous application.     

Determination of SDZ ICM 567 in blood and muscle microdialysis samples by microbore liquid chromatography with ultraviolet and fluorescence detection

Fast, simple and accurate methods for the determination of SDZ ICM 567, the 7-methoxy derivative of tropisetron, in microdialysates have been developed. Sampling by microdialysis from freely moving rats in the portal and jugular vein offers a new technology for pharmacokinetic studies by direct and continuous measurement of unbound drug concentrations with time. SDZ ICM 567 can be identified in small sample volumes of dialysates on a microbore high-performance liquid chromatography column-switching system with ultraviolet detection. In addition, determination of SDZ ICM 567 by fluorimetric detection has been developed for muscle microdialysates from rats. [14C]SDZ ICM 567 was used as reference substance for the estimation of the amount of substance transferred through the dialysis membrane. The radioactive measurement (RA) gave the recovery information, whereas the liquid chromatographic method detected the sum of [14C]SDZ ICM 567 and dialyzed SDZ ICM 567.     

Specific detection and quantification of palmitoyl-stearoyl-phosphatidylserine in human blood using normal-phase liquid chromatography coupled with electrospray mass spectrometry

We have made an immunohistochemical study of the vomeronasal (VN) complex of 12-day-old rats to characterize the innervation of its blood vessels. The VN complex can be subdivided into rostral, middle and caudal segments, each one with a particular vascularization pattern. Several small vessels were associated with the rostral segment, whereas a large venous sinus ran along the middle and caudal segments. Immunostaining for alpha-smooth muscle actin demonstrated that the muscular sheath was asymmetric, with more cells layers in its lateral than in its medial walls. Nerves were demonstrated with antisera against protein gene product 9.5 (PGP), and against several molecules associated with specific classes of nerve fibers: the C-terminal peptide of neuropeptide Y (CPON), calcitonin gene-related peptide (CGRP), substance P (SP), galanin (GAL), vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (NOS). The latter, was also studied with NADPH-diaphorase. Vascular associated fibers exhibited NOS-, CPON-, GAL-, CGRP-, SP- and VIP-immunoreactivity. Only the vessels of the rostral segment showed VIP-immunoreactive fibers. Each wall of the venous sinus exhibited different types of nerve fibers. CPON-, GAL-, CGRP- and SP-immunoreactive fibers concentrated in the medial wall, whereas NOS-immunoreactive ones concentrated in the lateral wall. This distribution of vascular fibers, plus the presence of sensory fibers exhibiting CGRP-, SP- and GAL-immunoreactivity within the pseudostratified epithelium of the VN tube, would be relevant to understand the operation of the pumping mechanism regulating influx and efflux from the VN tube.     

Simultaneous activation of Ca(2+)-dependent K+ and Cl- currents by various forms of stimulation in the membrane of smooth muscle cells from the rabbit basilar artery

In 1997, the Harvard School of Public Health College Alcohol Study resurveyed colleges that participated in a 1993 study. The findings revealed little change in binge drinking: a slight decrease in percentage of binge drinkers and slight increases in percentages of abstainers and frequent binge drinkers. Two of 5 students were binge drinkers (42.7%); 1 in 5 (19.0%) was an abstainer, and 1 in 5 was a frequent binge drinker (20.7%). As was true in 1993, 4 of 5 residents of fraternities or sororities were binge drinkers (81.1%). Asian students showed a greater increase and White students a greater decrease in binge drinking from 1993 to 1997, compared with all other students. Among students who drank alcohol, increases in frequency of drinking; drunkenness; drinking to get drunk; and alcohol-related problems, including drinking and driving, were reported. Binge drinkers in both 1993 and 1997 were at increased risk of alcohol-related problems, and nonbingers at colleges with high binge drinking rates had increased risks of encountering secondhand effects of binge drinking.     

Increased gene expression after liposome-mediated arterial gene transfer associated with intimal smooth muscle cell proliferation. In vitro and in vivo findings in a rabbit model of vascular injury

Arterial gene transfer represents a novel strategy that is potentially applicable to a variety of cardiovascular disorders. Attempts to perform arterial gene transfer using nonviral vectors have been compromised by a low transfection efficiency. We investigated the hypothesis that cellular proliferation induced by arterial injury could augment gene expression after liposome-mediated gene transfer. Nondenuded and denuded rabbit arterial strips were maintained in culture for up to 21 d, after which transfection was performed with a mixture of the plasmid encoding firefly luciferase and cationic liposomes. In non-denuded arteries, the culture interval before transfection did not affect the gene expression. In contrast, denuded arteries cultured for 3-14 d before transfection yielded 7-13-fold higher expression (vs. day 0; P < 0.005). Transfection was then performed percutaneously to the iliac arteries of live rabbits with or without antecedent angioplasty. Gene expression increased when transfection was performed 3-7 d postangioplasty (P < 0.05). Proliferative activity of neointimal cells assessed in vitro by [3H]thymidine incorporation, and in vivo by immunostaining for proliferating cell nuclear antigen, increased and declined in parallel with gene expression. These findings thus indicate that the expression of liposome-mediated arterial gene transfer may be augmented in presence of ongoing cellular proliferation.     

Measurement of the extracellular matrix in glomeruli from patients with diabetic nephropathy using an automatic image analyzer

To elucidate the characteristics of glycoproteins in the mesangial matrix in glomeruli from patients with diabetic nephropathy (DN) in non-insulin-dependent diabetes mellitus (NIDDM), we examined periodic acid-Shiff (PAS) and immunofluorescence staining of types III (III-C), IV (IV-C), V (V-C) and VI (VI-C) collagen, fibronectin (FN), fibrinogen (FBG) and laminin (LN) in the glomeruli of 16 patients with DN and 12 patients with diffuse proliferative glomerulonephritis (DPGN). The percentage of positively staining areas in the glomeruli were calculated using an automatic image analyzer. The percentages of areas positive for PAS, III-C, IV-C, V-C, VI-C or FN in DN were significantly greater than those in DPGN. Moreover, the percentages of PAS-positive areas were significantly correlated with not only IV-C-positive areas, but also FN-positive areas in patients with DN. The percentages of PAS-positive areas were also significantly correlated with the levels of serum creatinine and the degree of proteinuria in these patients. It was concluded that mesangial expansion in this disease might be associated mainly with an increase in IV-C and FN. These changes appear to affect the deterioration of renal function and the appearance of proteinuria, and vice versa.     

Qualitative analysis of potential metabolites and degradation products of a new antiinfective drug in rat urine, using HPLC with radiochemical detection and HPLC-mass spectrometry

High-performance liquid chromatography (HPLC) was used in combination with radioactivity detection and mass spectrometry (MS) to elucidate the metabolic fate of GV104326, a novel tricyclic beta-lactam antibacterial agent. Metabolic profiles were obtained by analysis of rat urine samples collected after intravenous administration of the 14C-labelled drug at the dose of 50 mg kg-1 (12.95 MBq kg-1). Methods for solid-phase extraction from urine samples and for reversed-phase chromatographic separation of drug related material were developed. HPLC-MS was used to confirm that the parent compound corresponded to the principal peak in the chromatograms, and two minor peaks were identified as potential metabolites of GV104326. They were shown to be an open beta-lactam ring derivative (GV173923) and a dimeric compound (GV196359).     

A specific and rapid method for determination of adenosine 3'-monophosphate (3'-AMP) content and 3'-AMP forming enzyme activity in rat liver mitochondria, using reversed-phase HPLC with fluorescence detection

To study the physiological significance of adenosine 3'-monophosphate (3'-AMP), an intracellular P-site inhibitor of adenylate cyclase, in rat liver mitochondria, a specific, rapid and reliable assay method for determination of 3'-AMP and the activity of its forming enzyme is required. 3'-AMP in rat liver was determined to be ca. 23+/-7 nmol/g wet weight, but no 2-deoxy-3'-AMP, another P-site inhibitor of adenylate cyclase, was detected, even when using a reversed-phase HPLC column with a fluorescent-reaction, as established in this study. By using the optimized assay method developed here, 3'-AMP forming enzyme activity in rat crude mitochondrial extract was found to be enhanced by EDTA and inhibited by p-chloromercurybenzoate. The optimum pH was ca. 5.8 and no divalent cation was required for activity. From these results, 3'-AMP forming enzyme(s) in rat liver mitochondria could be classified as acid exoribonuclease, which mainly existed in an active form. The results obtained in this study will help to gain more insight into the physiological roles of 3'-AMP in living systems.     

A sensitive procedure for the quantitation of free and N-(2-hydroxypropyl) methacrylamide polymer-bound doxorubicin (PK1) and some of its metabolites, 13-dihydrodoxorubicin, 13-dihydrodoxorubicinone and doxorubicinone, in human plasma and urine by reversed-phase HPLC with fluorimetric detection

A high-performance liquid chromatographic assay has been developed and validated for the determination in plasma and urine of doxorubicin (DXR) and some of its metabolites released in vivo from an N-(2-hydroxypropyl)methacrylamide (HPMA) polymer containing DXR linked through its aminosugar moiety to the polymer via an oligopeptide spacer (PK1). The method also allows measurement of the DXR still bound to the polymer. Following addition of two internal standards, the free compounds were extracted twice with isopropanol-chloroform (25:75, v/v). The first extraction was performed at physiological pH and the second after buffering at pH 8.4, in order to extract the aglycones and the glycosides, respectively. Determination of total DXR (polymer-bound plus free DXR) was performed, after quantitative acid hydrolysis to release doxorubicinone from free or polymer-bound DXR, by extraction with the same solvent mixture at pH 7.4. In both cases the organic phase was evaporated to dryness; the compounds were then separated by reversed-phase high-performance liquid chromatography (HPLC) under isocratic conditions and quantitated by fluorimetric detection. In the chromatograms all the analytes appeared to be separated at the baseline and no interference from blank human plasma and urine was observed. The suitability of the method for in vivo samples was checked by the analysis of plasma and urine samples obtained from a cancer patient who had received a single intravenous dose of the test compound.