(1/1461) Variations in 35SO4 incorporation into glycosaminoglycans along canine coronary arteries. A possible index of artery wall stress.
Focal areas of accentuated wall stress along the course of canine coronary arteries may be revealed by the level of 35SO4 incorporation into glycosaminoglycans (GAG). In the anterior descending artery, 35SO4 incorporation in higher in the proximal than in the distal region and may be extraordinarily high as the vessel enters a proximally located muscle bridge and at the takeoff region of multidirectional branches. In the circumflex artery, the incorporation also is higher in the proximal than in the distal region and is high at the genu where the posterior descending artery forms. There are differences in uptake of 35SO4 in vessels even when the arteries arise from the same vascular bed.this was shown by the higher incorporation in the left coronary artery than in the right coronary artery. A general anatomical agreement exists between these sites of high 35SO4 incorporation and previously described locations of interval elastic disruption ans proliferation of intimal connective tissue in the dog. (+info)
(2/1461) Sulphation and secretion of the predominant secretory human colonic mucin MUC2 in ulcerative colitis.
BACKGROUND: Decreased synthesis of the predominant secretory human colonic mucin (MUC2) occurs during active ulcerative colitis. AIMS: To study possible alterations in mucin sulphation and mucin secretion, which could be the cause of decreased mucosal protection in ulcerative colitis. METHODS: Colonic biopsy specimens from patients with active ulcerative colitis, ulcerative colitis in remission, and controls were metabolically labelled with [35S]-amino acids or [35S]-sulphate, chase incubated and analysed by SDS-PAGE, followed by quantitation of mature [35S]-labelled MUC2. For quantitation of total MUC2, which includes non-radiolabelled and radiolabelled MUC2, dot blotting was performed, using a MUC2 monoclonal antibody. RESULTS: Between patient groups, no significant differences were found in [35S]-sulphate content of secreted MUC2 or in the secreted percentage of either [35S]-amino acid labelled MUC2 or total MUC2. During active ulcerative colitis, secretion of [35S]-sulphate labelled MUC2 was significantly increased twofold, whereas [35S]-sulphate incorporation into MUC2 was significantly reduced to half. CONCLUSIONS: During active ulcerative colitis, less MUC2 is secreted, because MUC2 synthesis is decreased while the secreted percentage of MUC2 is unaltered. Furthermore, sulphate content of secreted MUC2 is unaltered by a specific compensatory mechanism, because sulphated MUC2 is preferentially secreted while sulphate incorporation into MUC2 is reduced. (+info)
(3/1461) Insulin and TSH promote growth in size of PC Cl3 rat thyroid cells, possibly via a pathway different from DNA synthesis: comparison with FRTL-5 cells.
In the rat thyroid cell lines PC Cl3, FRTL- 5 and WRT, proliferation is mainly regulated by insulin or IGF, and TSH. However, the mechanism regulating cell mass doubling prior to division is still unknown. Our laboratory has shown that in dog thyroid cells insulin promotes growth in size while TSH in the presence of insulin triggers DNA replication. In the absence of insulin, TSH has no effect on cell growth. In this report we investigated insulin action on both cell mass and DNA synthesis and its modulation by TSH and insulin in PC Cl3 and FRTL-5 cells. In PC Cl3 cells, insulin activated not only DNA synthesis but also protein synthesis and accumulation. Although TSH potentiated the stimulation of DNA synthesis induced by insulin, enhancement of protein synthesis by both agents was additive. All TSH effects were reproduced by forskolin. Similar effects were also obtained in FRTL-5 cells. This suggests that insulin and TSH, via cAMP, modulate both growth in size and DNA replication in these cell lines. Lovastatin, which blocks 3-hydroxy-3-methylglutaryl coenzyme A reductase, decreased the induction of DNA synthesis, but not of protein synthesis induced by insulin or TSH in PC Cl3 cells. In FRTL-5 cells, lovastatin reduced protein and DNA synthesis stimulated by insulin but not TSH-induced protein synthesis. Taking these data together, we propose that insulin and/or TSH both modulate cell mass doubling and DNA synthesis in these cell lines, presumably via different pathways, and that there are at least two pathways which regulate growth in size in FRTL-5 thyroid cells: one triggered by insulin, which is lovastatin sensitive, and the other activated by TSH, which is not sensitive to lovastatin. (+info)
(4/1461) G protein activation by human dopamine D3 receptors in high-expressing Chinese hamster ovary cells: A guanosine-5'-O-(3-[35S]thio)- triphosphate binding and antibody study.
Despite extensive study, the G protein coupling of dopamine D3 receptors is poorly understood. In this study, we used guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]-GTPgammaS) binding to investigate the activation of G proteins coupled to human (h) D3 receptors stably expressed in Chinese hamster ovary (CHO) cells. Although the receptor expression level was high (15 pmol/mg), dopamine only stimulated G protein activation by 1.6-fold. This was despite the presence of marked receptor reserve for dopamine, as revealed by Furchgott analysis after irreversible hD3 receptor inactivation with the alkylating agent, EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline). Thus, half-maximal stimulation of [35S]-GTPgammaS binding required only 11.8% receptor occupation of hD3 sites. In contrast, although the hD2(short) receptor expression level in another CHO cell line was 11-fold lower, stimulation by dopamine was higher (2.5-fold). G protein activation was increased at hD3 and, less potently, at hD2 receptors by the preferential D3 agonists, PD 128,907 [(+)-(4aR,10bR)-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H- benzopyrano[4,3-b]-1, 4-oxazin-9-ol] and (+)-7-OH-DPAT (7-hydroxy-2-(di-n-propylamino)tetralin). Furthermore, the selective D3 antagonists, S 14297 ((+)-[7-(N, N-dipropylamino)-5,6,7, 8-tetrahydro-naphtho(2,3b)dihydro-2,3-furane]) and GR 218,231 (2(R, S)-(dipropylamino)-6-(4-methoxyphenylsulfonylmethyl)-1,2,3,4- tetrahydronaphtalene), blocked dopamine-stimulated [35S]GTPgammaS binding more potently at hD3 than at hD2 sites. Antibodies against Galphai/alphao reduced dopamine-induced G protein activation at both CHO-hD3 and -hD2 membranes, whereas GalphaS antibodies had no effect at either site. In contrast, incubation with anti-Galphaq/alpha11 antibodies, which did not affect dopamine-induced G protein activation at hD2 receptors, attenuated hD3-induced G protein activation. These data suggest that hD3 receptors may couple to Galphaq/alpha11 and would be consistent with the observation that pertussis toxin pretreatment, which inactivates only Gi/o proteins, only submaximally (80%) blocked dopamine-stimulated [35S]GTPgammaS binding in CHO-hD3 cells. Taken together, the present data indicate that 1) hD3 receptors functionally couple to G protein activation in CHO cells, 2) hD3 receptors activate G proteins less effectively than hD2 receptors, and 3) hD3 receptors may couple to different G protein subtypes than hD2 receptors, including nonpertussis sensitive Gq/11 proteins. (+info)
(5/1461) Mixed agonist-antagonist properties of clozapine at different human cloned muscarinic receptor subtypes expressed in Chinese hamster ovary cells.
We recently reported that clozapine behaves as a partial agonist at the cloned human m4 muscarinic receptor subtype. In the present study, we investigated whether the drug could elicit similar effects at the cloned human m1, m2, and m3 muscarinic receptor subtypes expressed in the Chinese hamster ovary (CHO) cells. Clozapine elicited a concentration-dependent stimulation of [3H]inositol phosphates accumulation in CHO cells expressing either the m1 or the m3 receptor subtype. Moreover, clozapine inhibited forskolin-stimulated cyclic AMP accumulation and enhanced [35S] GTP gamma S binding to membrane G proteins in CHO cells expressing the m2 receptor. These agonist effects of clozapine were antagonized by atropine. The intrinsic activity of clozapine was lower than that of the full cholinergic agonist carbachol, and, when the compounds were combined, clozapine potently reduced the receptor responses to carbachol. These data indicate that clozapine behaves as a partial agonist at different muscarinic receptor subtypes and may provide new hints for understanding the receptor mechanisms underlying the antipsychotic efficacy of the drug. (+info)
(6/1461) Effects of gamma-tocotrienol on ApoB synthesis, degradation, and secretion in HepG2 cells.
gamma-Tocotrienol (gamma-T3), a naturally occurring analog of tocopherol (vitamin E), has been shown to have a hypocholesterolemic effect in animals and humans. Unlike tocopherol, it has also been shown to reduce plasma apoB levels in hypercholesterolemic subjects. The aim of this study was to define the mechanism of action of gamma-T3 on hepatic modulation of apoB production using cultured HepG2 cells as the model system. HepG2 cells preincubated with gamma-T3 were initially shown to inhibit the rate of incorporation of [14C]acetate into cholesterol in a concentration- and time-dependent manner, with a maximum 86+/-3% inhibition at 50 micromol/L observed within 6 hours. gamma-T3, on the other hand, had no significant effect on the uptake of [14C]glycerol into pools of cellular triacylglycerol and phospholipid relative to untreated control. The rate of apoB synthesis and secretion was then studied by an [35S]methionine pulse-labeling experiment and quantified by immunoprecipitating apoB on chasing up to 3 hours. An average reduction of 24+/-3% in labeled apoB in the media was apparent with gamma-T3 despite a 60+/-2% increase in apoB synthesis. Fractionation of secreted apoB revealed a relatively denser lipoprotein particle, suggesting a less stable particle. Using a digitonin-permeabilized HepG2 cell system, the effects of gamma-T3 on apoB translocation and degradation in the endoplasmic reticulum were further investigated. The generation of a specific N-terminal 70-kDa proteolytic fragment proved to be a sensitive measure of the rate of apoB translocation and degradation. The abundance of this fragment increased significantly in gamma-T3-treated cells relative to untreated control cells (50+/-21%) after 2 hours of chase. In addition, the presence of gamma-T3 resulted in an average decrease of 64+/-8% in intact apoB. Taken together, the data suggest that gamma-T3 stimulates apoB degradation possibly as the result of decreased apoB translocation into the endoplasmic reticulum lumen. It is speculated that the lack of cholesterol availability reduces the number of secreted apoB-containing lipoprotein particles by limiting translocation of apoB into the endoplasmic reticulum lumen. (+info)
(7/1461) Absence of G-protein activation by mu-opioid receptor agonists in the spinal cord of mu-opioid receptor knockout mice.
1. The ability of mu-opioid receptor agonists to activate G-proteins in the spinal cord of mu-opioid receptor knockout mice was examined by monitoring the binding to membranes of the non-hydrolyzable analogue of GTP, guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS). 2. In the receptor binding study, Scatchard analysis of [3H][D-Ala2,NHPhe4,Gly-ol]enkephalin ([3H]DAMGO; mu-opioid receptor ligand) binding revealed that the heterozygous mu-knockout mice displayed approximately 40% reduction in the number of mu-receptors as compared to the wild-type mice. The homozygous mu-knockout mice showed no detectable mu-binding sites. 3. The newly isolated mu-opioid peptides endomorphin-1 and -2, the synthetic selective mu-opioid receptor agonist DAMGO and the prototype of mu-opioid receptor agonist morphine each produced concentration-dependent increases in [35S]GTPgammaS binding in wild-type mice. This stimulation was reduced by 55-70% of the wild-type level in heterozygous, and virtually eliminated in homozygous knockout mice. 4. No differences in the [35S]GTPgammaS binding stimulated by specific delta1- ([D-Pen2,5]enkephalin), delta2-([D-Ala2]deltorphin II) or kappa1-(U50,488H) opioid receptor agonists were noted in mice of any of the three genotypes. 5. The data clearly indicate that mu-opioid receptor gene products play a key role in G-protein activation by endomorphins, DAMGO and morphine in the mouse spinal cord. They support the idea that mu-opioid receptor densities could be rate-limiting steps in the G-protein activation by mu-opioid receptor agonists in the spinal cord. These thus indicate a limited physiological mu-receptor reserve. Furthermore, little change in delta1-, delta2- or kappa1-opioid receptor-G-protein complex appears to accompany mu-opioid receptor gene deletions in this region. (+info)
(8/1461) Effects of azole antifungal drugs on the transition from yeast cells to hyphae in susceptible and resistant isolates of the pathogenic yeast Candida albicans.
Oral infections caused by the yeast Candida albicans are some of the most frequent and earliest opportunistic infections in human immunodeficiency virus-infected patients. The widespread use of azole antifungal drugs has led to the development of drug resistance, creating a major problem in the treatment of yeast infections in AIDS patients and other immunocompromised individuals. Several molecular mechanisms that contribute to drug resistance have been identified. In C. albicans, the ability to morphologically switch from yeast cells (blastospores) to filamentous forms (hyphae) is an important virulence factor which contributes to the dissemination of Candida in host tissues and which promotes infection and invasion. A positive correlation between the level of antifungal drug resistance and the ability to form hyphae in the presence of azole drugs has been identified. Under hypha-inducing conditions in the presence of an azole drug, resistant clinical isolates form hyphae, while susceptible yeast isolates do not. This correlation is observed in a random sample from a population of susceptible and resistant isolates and is independent of the mechanisms of resistance. 35S-methionine incorporation suggests that growth inhibition is not sufficient to explain the inhibition of hyphal formation, but it may contribute to this inhibition. (+info)