BMC Pharmacology[JOURNAL]
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200 papers
The aminoguanidine carboxylate BVT.12777 activates ATP-sensitive K+ channels in the rat insulinoma cell line, CRI-G1.
Kinsella JM, Laidlaw HA, Tang T … +3 more , Harvey J, Sutherland C, Ashford ML
BMC Pharmacol · 2004 Aug · PMID 15329154 · Full text
BACKGROUND: 3-guanidinopropionic acid derivatives reduce body weight in obese, diabetic mice. We have assessed whether one of these analogues, the aminoguanidine carboxylate BVT.12777, opens KATP channels in rat insulino...
BACKGROUND: 3-guanidinopropionic acid derivatives reduce body weight in obese, diabetic mice. We have assessed whether one of these analogues, the aminoguanidine carboxylate BVT.12777, opens KATP channels in rat insulinoma cells, by the same mechanism as leptin. RESULTS: BVT.12777 hyperpolarized CRI-G1 rat insulinoma cells by activation of KATP channels. In contrast, BVT.12777 did not activate heterologously expressed pancreatic beta-cell KATP subunits directly. Although BVT.12777 stimulated phosphorylation of MAPK and STAT3, there was no effect on enzymes downstream of PI3K. Activation of KATP in CRI-G1 cells by BVT.12777 was not dependent on MAPK or PI3K activity. Confocal imaging showed that BVT.12777 induced a re-organization of cellular actin. Furthermore, the activation of KATP by BVT.12777 in CRI-G1 cells was demonstrated to be dependent on actin cytoskeletal dynamics, similar to that observed for leptin. CONCLUSIONS: This study shows that BVT.12777, like leptin, activates KATP channels in insulinoma cells. Unlike leptin, BVT.12777 activates KATP channels in a PI3K-independent manner, but, like leptin, channel activation is dependent on actin cytoskeleton remodelling. Thus, BVT.12777 appears to act as a leptin mimetic, at least with respect to KATP channel activation, and may bypass up-stream signalling components of the leptin pathway.
Extrinsic factors regulate partial agonist efficacy of strychnine-sensitive glycine receptors.
Farroni JS, McCool BA
BMC Pharmacol · 2004 Aug · PMID 15301692 · Full text
BACKGROUND: Strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha2 + beta heteromeric channels. This subunit composition is distinct from the alpha1 + beta channels found throughout the...
BACKGROUND: Strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha2 + beta heteromeric channels. This subunit composition is distinct from the alpha1 + beta channels found throughout the adult spinal cord. Unfortunately, the pharmacology of forebrain alpha2beta receptors are poorly defined compared to 'neonatal' alpha2 homomeric channels or 'spinal' alpha1beta heteromers. In addition, the pharmacologic properties of native alpha2beta glycine receptors have been generally distinct from receptors produced by heterologous expression. To identify subtype-specific pharmacologic tools for the forebrain alpha2beta receptors, it is important to identify a heterologous expression system that closely resembles these native glycine-gated chloride channels. RESULTS: While exploring pharmacological properties of alpha2beta glycine receptors compared to alpha2-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. The beta-amino acid taurine possessed 30-50% efficacy for alpha2-containing receptor isoforms when expressed in HEK 293 cells. However, taurine efficacy was dramatically reduced in L-cell fibroblasts. Similar results were obtained for beta-alanine. The efficacy of these partial agonists was also strongly reduced by the beta subunit. There were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. Nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. Finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion. CONCLUSIONS: Our results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. In the systems examine here, these effects are independent of both absolute expression level and any system-related alterations in the agonist binding site. We conclude that complex interactions between receptor composition and extrinsic factors may play a significant role in determining strychnine-sensitive glycine receptor partial agonist pharmacology.
Proteasome inhibitors: their effects on arachidonic acid release from cells in culture and arachidonic acid metabolism in rat liver cells.
Levine L
BMC Pharmacol · 2004 Aug · PMID 15296516 · Full text
BACKGROUND: I have postulated that arachidonic acid release from rat liver cells is associated with cancer chemoprevention. Since it has been reported that inhibition of proteasome activities may prevent cancer, the effe...
BACKGROUND: I have postulated that arachidonic acid release from rat liver cells is associated with cancer chemoprevention. Since it has been reported that inhibition of proteasome activities may prevent cancer, the effects of proteasome inhibitors on arachidonic acid release from cells and on prostaglandin I2 production in rat liver cells were studied. RESULTS: The proteasome inhibitors, epoxomicin, lactacystin and carbobenzoxy-leucyl-leucyl-leucinal, stimulate the release of arachidonic acid from rat glial, human colon carcinoma, human breast carcinoma and the rat liver cells. They also stimulate basal and induced prostacycin production in the rat liver cells. The stimulated arachidonic acid release and basal prostaglandin I2 production in rat liver cells is inhibited by actinomycin D. CONCLUSIONS: Stimulation of arachidonic acid release and arachidonic acid metabolism may be associated with some of the biologic effects observed after proteasome inhibition, e.g. prevention of tumor growth, induction of apoptosis, stimulation of bone formation.
A study comparing the actions of gabapentin and pregabalin on the electrophysiological properties of cultured DRG neurones from neonatal rats.
McClelland D, Evans RM, Barkworth L … +2 more , Martin DJ, Scott RH
BMC Pharmacol · 2004 Aug · PMID 15294026 · Full text
BACKGROUND: Gabapentin and pregabalin have wide-ranging therapeutic actions, and are structurally related to the inhibitory neurotransmitter GABA. Gabapentin, pregablin and GABA can all modulate voltage-activated Ca2+ ch...
BACKGROUND: Gabapentin and pregabalin have wide-ranging therapeutic actions, and are structurally related to the inhibitory neurotransmitter GABA. Gabapentin, pregablin and GABA can all modulate voltage-activated Ca2+ channels. In this study we have used whole cell patch clamp recording and fura-2 Ca2+ imaging to characterise the actions of pregabalin on the electrophysiological properties of cultured dorsal root ganglion (DRG) neurones from neonatal rats. The aims of this study were to determine whether pregabalin and gabapentin had additive inhibitory effects on high voltage-activated Ca2+ channels, evaluate whether the actions of pregabalin were dependent on GABA receptors and characterise the actions of pregabalin on voltage-activated potassium currents. RESULTS: Pregabalin (25 nM - 2.5 microM) inhibited 20-30% of the high voltage-activated Ca2+ current in cultured DRG neurones. The residual Ca2+ current recorded in the presence of pregabalin was sensitive to the L-type Ca2+ channel modulator, Bay K8644. Saturating concentrations of gabapentin failed to have additive effects when applied with pregabalin, indicating that these two compounds act on the same type(s) of voltage-activated Ca2+ channels but the majority of Ca2+ current was resistant to both drugs. The continual application of GABA, the GABAB receptor antagonist CGP52432, or intracellular photorelease of GTP-gamma-S had no effect on pregabalin-induced inhibition of Ca2+ currents. Although clear inhibition of Ca2+ influx was produced by pregabalin in a population of small neurones, a significant population of larger neurones showed enhanced Ca2+ influx in response to pregabalin. The enhanced Ca2+ influx evoked by pregabalin was mimicked by partial block of K+ conductances with tetraethylammonium. Pregabalin produced biphasic effects on voltage-activated K+ currents, the inhibitory effect of pregabalin was prevented with apamin. The delayed enhancement of K+ currents was attenuated by pertussis toxin and by intracellular application of a (Rp)-analogue of cAMP. CONCLUSIONS: Pregabalin reduces excitatory properties of cultured DRG neurones by modulating voltage-activated Ca2+ and K+ channels. The pharmacological activity of pregabalin is similar but not identical to that of gabapentin. The actions of pregabalin may involve both extracellular and intracellular drug target sites and modulation of a variety of neuronal conductances, by direct interactions, and through intracellular signalling involving protein kinase A.
8-Cl-Adenosine enhances 1,25-dihydroxyvitamin D3-induced growth inhibition without affecting 1,25-dihydroxyvitamin D3-stimulated differentiation of primary mouse epidermal keratinocytes.
Bollag WB, Zhong X, Josephson S
BMC Pharmacol · 2004 Jul · PMID 15279680 · Full text
BACKGROUND: Epidermal keratinocytes continuously proliferate and differentiate to form the mechanical and water permeability barrier that makes terrestrial life possible. In certain skin diseases, these processes become...
BACKGROUND: Epidermal keratinocytes continuously proliferate and differentiate to form the mechanical and water permeability barrier that makes terrestrial life possible. In certain skin diseases, these processes become dysregulated, resulting in abnormal barrier formation. In particular, skin diseases such as psoriasis, actinic keratosis and basal and squamous cell carcinomas are characterized by hyperproliferation and aberrant or absent differentiation of epidermal keratinocytes. We previously demonstrated that 8-Cl-adenosine (8-Cl-Ado) can induce keratinocyte growth arrest without inducing differentiation. RESULTS: To determine if this agent might be useful in treating hyperproliferative skin disorders, we investigated whether 8-Cl-Ado could enhance the ability of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], a known keratinocyte differentiating agent and a clinical treatment for psoriasis, to inhibit keratinocyte growth. We found that low concentrations of 8-Cl-Ado and 1,25(OH)2D3 appeared to act additively to reduce proliferation of primary mouse epidermal keratinocytes. However, another agent (transforming growth factor-beta) that triggers growth arrest without inducing differentiation also coincidentally inhibits differentiation elicited by other agents; inhibition of differentiation is suboptimal for treating skin disorders, as differentiation is often already reduced. Thus, we determined whether 8-Cl-Ado also decreased keratinocyte differentiation induced by 1,25(OH)2D3, as measured using the early and late differentiation markers, keratin 1 protein levels and transglutaminase activity, respectively. 8-Cl-Ado did not affect 1,25(OH)2D3-stimulated keratin 1 protein expression or transglutaminase activity. CONCLUSIONS: Our results suggest that 8-Cl-Ado might be useful in combination with differentiating agents for the treatment of hyperproliferative disorders of the skin.
A protease activated receptor-2 (PAR-2) activating peptide, tc-LIGRLO-NH2, induces protease release from mast cells: role in TNF degradation.
Alshurafa HN, Stenton GR, Wallace JL … +3 more , Hollenberg MD, Befus AD, Vliagoftis H
BMC Pharmacol · 2004 Jul · PMID 15265236 · Full text
BACKGROUND: Mast cell (MC)-derived serine proteases have been implicated in a variety of inflammatory processes. We have previously shown that rat peritoneal MC (PMC) express mRNA for protease activated receptor 2 (PAR-2...
BACKGROUND: Mast cell (MC)-derived serine proteases have been implicated in a variety of inflammatory processes. We have previously shown that rat peritoneal MC (PMC) express mRNA for protease activated receptor 2 (PAR-2), a G-coupled receptor activated by trypsin-like proteases. Recent evidence also suggests that MC-induced inflammation can be mediated through PAR. Therefore, we hypothesized that specific PAR-2 agonist peptides (PAR-2ap) induce protease release from PMC. RESULTS: Western blot analysis of PMC supernatants revealed that a PAR-2ap, tc-LIGRLO (10 microM), stimulated the release of rat MC protease (RMCP)-1, RMCP-5 and carboxypeptidase-A. The release was evident by 20 min but further increased up to 8 h. To study the biological effects of protease release we tested supernatants from tc-LIGRLO, tc-OLRGIL (inactive control peptide) and antigen-activated PMC for proteolytic activity by seeding with TNF (150 pg/ml), incubating for 8 h at 37 degrees C, and measuring TNF remaining in the supernatants. Supernatants from tc-LIGRLO-stimulated PMC degraded 44 % of seeded TNF (n = 5). Moreover, this TNF proteolysis was dependent on the concentration of tc-LIGRLO used to stimulate PMC, and was significantly inhibited (94 %) by soybean trypsin inhibitor. Antigen and tc-OLRGIL induced no significant release of such proteolytic activity. CONCLUSIONS: These data indicate that a PAR-2ap induces the release of proteases from mast cells, which may degrade extracellular cytokines and other substrates thus modulating the inflammatory response.
A homotropic two-state model and auto-antagonism.
Bindslev N
BMC Pharmacol · 2004 Jul · PMID 15257759 · Full text
BACKGROUND: Bell-shaped and terraced dose-response relations have been observed in single ligand application for enzymes, carriers, transporters, G protein-coupled receptors as well as for other receptive units. It seems...
BACKGROUND: Bell-shaped and terraced dose-response relations have been observed in single ligand application for enzymes, carriers, transporters, G protein-coupled receptors as well as for other receptive units. It seems that there is still a need for new models as analytical tools for such dose-responses, especially in the light of expanding di- and multi-merization of the receptive units for functionality. RESULTS: Self-inhibition by drugs is analyzed in the frame-work of a theoretical homotropic two-state model, HOTSM. The model is a cubic reaction scheme based on a combination of conformational isomerization between two states within a receptive unit and ternary-complexing of two identical agonist molecules with the receptor. Concepts and terms related to self-inhibition are presented. HOTSM has seven independent parameters. Making a few simplifying assumptions narrows its analysis to initially look at four parameters. Some conclusions to be drawn are that a first level of spontaneous activity is solely determined by an isomerization constant, L. As ligand concentration rises, all seven parameters influence a second level of activity. At high ligand concentrations, a third level of activity is determined by only four of the seven constants, viz. the L constant and three intrinsic efficacy related constants, a, b, and d. The third level is given by 1/[1 + 1/(L.a.b.d)]. The third level may be above, at, or below the first and second levels. When the third level is above the first level, dose-responses may be bell-shaped, terraced, or reversed bell-shaped while when it is below the first level, dose-responses can attain forms of bell-shapes, reverse terraces, or reverse bell-shapes. To exemplify its use, the HOTSM is fitted to experimental dose-responses from sources in the literature. Development of the HOTSM is reviewed. CONCLUSIONS: The homotropic two-state model, HOTSM, is a novel model for analyses of dose-responses at equilibrium that are co-operative or show bell-shapes of auto-antagonism.
EMD273316 & EMD95833, type 4 phosphodiesterase inhibitors, stimulate fibroblastic-colony formation by bone marrow cells via direct inhibition of PDE4 and the induction of endogenous prostaglandin synthesis.
Scutt A, Beier N, Fittschen C
BMC Pharmacol · 2004 Jun · PMID 15219232 · Full text
BACKGROUND: Type 4 phosphodiesterase (PDE4) inhibitors have been shown to stimulate bone formation in vivo and to stimulate osteoblastic differentiation in vitro. As one possible mechanism for the stimulation of bone for...
BACKGROUND: Type 4 phosphodiesterase (PDE4) inhibitors have been shown to stimulate bone formation in vivo and to stimulate osteoblastic differentiation in vitro. As one possible mechanism for the stimulation of bone formation is the recruitment of osteoprogenitor cells from the bone marrow, we have investigated the effect of the PDE4 inhibitors EMD273316, EMD95833, EMD249615 and EMD 219906 on fibroblastic colony formation by whole bone marrow cells and on the ability of these colonies to adopt an osteoblastic phenotype. RESULTS: All four agents stimulated colony formation in a concentration dependent manner, however, in the case of EMD273316 & EMD95833, the effect was evident at lower concentrations and the addition of prostaglandin E2 (PGE2) was not necessary for maximal stimulation. It was subsequently found that co-incubation with indomethacin reduced the stimulatory effects of EMD273316 & EMD95833 but had no effect on the actions of EMD249615 and EMD 219906 and that EMD273316 & EMD95833 stimulated the synthesis of endogenous PGE2 by whole bone marrow cells whereas EMD249615 and EMD 219906 had no significant effect. CONCLUSIONS: These data suggest that EMD249615, EMD 219906, EMD273316 & EMD95833 can promote the recruitment of bone marrow osteoprogenitor cells leading to a stimulation of bone formation via their direct inhibitory effects on PDE4. The actions of EMD273316 & EMD95833 however, are augmented by their ability to stimulate endogenous prostanoids synthesis which acts synergistically with their direct effects on PDE4.
Hypoglycemic and anti-lipemic effects of the aqueous extract from Cissus sicyoides.
Viana GS, Medeiros AC, Lacerda AM … +3 more , Leal LK, Vale TG, Matos FJ
BMC Pharmacol · 2004 Jun · PMID 15182373 · Full text
BACKGROUND: Cissus sicyoides (Vitaceae) is a medicinal plant popularly known in Brazil as "cipó-pucá, anil-trepador, cortina, and insulina". The plant is used in several diseases, including rheumatism, epilepsy, stroke a...
BACKGROUND: Cissus sicyoides (Vitaceae) is a medicinal plant popularly known in Brazil as "cipó-pucá, anil-trepador, cortina, and insulina". The plant is used in several diseases, including rheumatism, epilepsy, stroke and also in the treatment of diabetes. In the present work, we studied the hypoglycemic and anti-lipemic effects of the aqueous extract prepared from fresh leaves of the plant (AECS), in the model of alloxan-induced diabetes in rats. In addition, hepatic enzyme levels were also determined. RESULTS: Results showed that the daily treatment of diabetic rats with AECS for 7 days (100 and 200 mg/kg, p.o.) significantly decreased blood glucose levels in 25 and 22% respectively, as compared to the same groups before AECS treatment. No significant changes were seen in control diabetic rats before (48 h after alloxan administration) and after distilled water treatment. While no changes were seen in total cholesterol levels, a significant decrease was observed in plasma triglyceride levels, in the alloxan-induced diabetic rats after AECS treatment with both doses, as compared to the same groups before treatment. Significant decreases in blood glucose (25%) and triglyceride levels (48%) were also observed in the alloxan-induced diabetic rats after 4 days treatment with AECS (200 mg/kg, p.o.). Aspartate (AST) and alanine (ALT) aminotransferases levels, in diabetic controls and AECS-treated rats, were in the range of reference values presented by normal rats. CONCLUSIONS: The results justify the popular use of C. sicyoides, pointing out to the potential benefit of the plant aqueous extract (AECS) in alternative medicine, in the treatment of type 2 diabetes mellitus.
Resistance to gemcitabine in a human follicular lymphoma cell line is due to partial deletion of the deoxycytidine kinase gene.
Galmarini CM, Clarke ML, Jordheim L … +4 more , Santos CL, Cros E, Mackey JR, Dumontet C
BMC Pharmacol · 2004 May · PMID 15157282 · Full text
BACKGROUND: Gemcitabine is an analogue of deoxycytidine with activity against several solid tumors. In order to elucidate the mechanisms by which tumor cells become resistant to gemcitabine, we developed the resistant su...
BACKGROUND: Gemcitabine is an analogue of deoxycytidine with activity against several solid tumors. In order to elucidate the mechanisms by which tumor cells become resistant to gemcitabine, we developed the resistant subline RL-G from the human follicular lymphoma cell line RL-7 by prolonged exposure of parental cells to increasing concentrations of gemcitabine. RESULTS: In vitro, the IC50 increased from 0.015 microM in parental RL-7 cells to 25 microM in the resistant variant, RL-G. Xenografts of both cell lines developed in nude mice were treated with repeated injections of gemcitabine. Under conditions of gemcitabine treatment which totally inhibited the development of RL-7 tumors, RL-G derived tumors grew similarly to those of untreated animals, demonstrating the in vivo resistance of RL-G cells to gemcitabine. HPLC experiments showed that RL-G cells accumulated and incorporated less gemcitabine metabolites into DNA and RNA than RL-7 cells. Gemcitabine induced an S-phase arrest in RL-7 cells but not in RL-G cells. Exposure to gemcitabine induced a higher degree of apoptosis in RL-7 than in RL-G cells, with poly-(ADP-ribose) polymerase cleavage in RL-7 cells. No modifications of Bcl-2 nor of Bax expression were observed in RL-7 or RL-G cells exposed to gemcitabine. These alterations were associated with the absence of the deoxycytidine kinase mRNA expression observed by quantitative RT-PCR in RL-G cells. PCR amplification of désoxycytidine kinase gene exons showed a partial deletion of the dCK gene in RL-G cells. CONCLUSIONS: These results suggest that partial deletion of the dCK gene observed after selection in the presence of gemcitabine is involved with resistance to this agent both in vitro and in vivo.
A novel method for evaluation and screening of caspase inhibitory peptides by the amino acid positional fitness score.
Yoshimori A, Takasawa R, Tanuma S
BMC Pharmacol · 2004 May · PMID 15154972 · Full text
BACKGROUND: Since caspases are key executioners of apoptosis in cases of severe diseases including neurodegenerative disorders such as Alzheimer's disease and Huntington's disease, and viral infection diseases such as AI...
BACKGROUND: Since caspases are key executioners of apoptosis in cases of severe diseases including neurodegenerative disorders such as Alzheimer's disease and Huntington's disease, and viral infection diseases such as AIDS and hepatitis, potent and specific inhibitors of caspases have clinical potential. A series of peptide inhibitors has been designed based on cleavage sites of substrate proteins. However, these peptides are not necessarily the most potent to each caspase. Moreover, so far, it has proved to be difficult to design potent and specific peptide inhibitors of each caspase from sequence data of known cleavage sites in substrate proteins. We have attempted to develop a computational screening system for rapid selection of potent and specific peptide inhibitors from a comprehensive peptide library. RESULTS: We developed a new method for rapid evaluation and screening of peptide inhibitors based on Amino acid Positional Fitness (APF) score. By using this score, all known peptide inhibitors of each caspases-3,-7,-8, and -9 were rapidly selected in their enriched libraries. In this libraries, there were good correlations between predicted binding affinities of the known peptide inhibitors and their experimental Ki values. Furthermore, a novel potent peptide inhibitor, Ac-DNLD-CHO, for caspase-3 was able to be designed by this method. To our knowledge, DNLD is a first reported caspase-3 inhibitory peptide identified by using the computational screening strategy. CONCLUSION: Our new method for rapid screening of peptide inhibitors using APF score is an efficient strategy to select potent and specific peptide inhibitors from a comprehensive peptide library. Thus, the APF method has the potential to become a valuable approach for the discovery of the most effective peptide inhibitors. Moreover, it is anticipated that these peptide inhibitors can serve as leads for further drug design and optimization of small molecular inhibitors.
15-deoxy-delta 12, 14-Prostaglandin J2 prevents reactive oxygen species generation and mitochondrial membrane depolarization induced by oxidative stress.
Garg TK, Chang JY
BMC Pharmacol · 2004 May · PMID 15149553 · Full text
BACKGROUND: With the use of cultured human retinal pigment epithelial cells, we have previously described a number of cellular responses to oxidative stress caused by H2O2. We also demonstrated that the cytotoxicity caus...
BACKGROUND: With the use of cultured human retinal pigment epithelial cells, we have previously described a number of cellular responses to oxidative stress caused by H2O2. We also demonstrated that the cytotoxicity caused by H2O2 could be prevented by the prostaglandin derivative, 15-deoxy-delta 12, 14-Prostaglandin J2 (15d-PGJ2). RESULTS: Further characterization of the experimental system indicated that the half-life of H2O2 in cultures was ~1 hour. At a fixed H2O2 concentration, the cytotoxicity was dependent on the volume of H2O2 solution used in the culture, such that higher volume caused more cytotoxicity. Most cells were committed to die if the culture was treated for 2 hours with a cytotoxic concentration of H2O2. The prostaglandin derivative, 15d-PGJ2, could prevent oxidative damage caused by t-butyl hydroperoxide, in addition to H2O2. Further studies indicated that both H2O2 and tBH caused an increase in reactive oxygen species and depolarization of mitochondrial membrane potential. Pretreatment of cells with 1 microM 15d-PGJ2 led to a modest decrease in reactive oxygen species generation, and a significant restoration of mitochondrial membrane potential. CONCLUSION: This agent may be used in the future as a pharmacological tool for preventing cellular damage caused by oxidative stress.
Inhibition of pancreatic cholesterol esterase reduces cholesterol absorption in the hamster.
Heidrich JE, Contos LM, Hunsaker LA … +2 more , Deck LM, Vander Jagt DL
BMC Pharmacol · 2004 Apr · PMID 15096274 · Full text
BACKGROUND: Pancreatic cholesterol esterase has three proposed functions in the intestine: 1) to control the bioavailability of cholesterol from dietary cholesterol esters; 2) to contribute to incorporation of cholestero...
BACKGROUND: Pancreatic cholesterol esterase has three proposed functions in the intestine: 1) to control the bioavailability of cholesterol from dietary cholesterol esters; 2) to contribute to incorporation of cholesterol into mixed micelles; and 3) to aid in transport of free cholesterol to the enterocyte. Inhibitors of cholesterol esterase are anticipated to limit the absorption of dietary cholesterol. RESULTS: The selective and potent cholesterol esterase inhibitor 6-chloro-3-(1-ethyl-2-cyclohexyl)-2-pyrone (figure 1, structure 1) was administered to hamsters fed a high cholesterol diet supplemented with radiolabeled cholesterol ester. Hamsters were gavage fed 3H-labeled cholesteryl oleate along with inhibitor 1, 0-200 micromoles. Twenty-four hours later, hepatic and serum radioactive cholesterol levels were determined. The ED50 of inhibitor 1 for prevention of the uptake of labeled cholesterol derived from hydrolysis of labeled cholesteryl oleate was 100 micromoles. The toxicity of inhibitor 1 was investigated in a 30 day feeding trial. Inhibitor 1, 100 micromoles or 200 micromoles per day, was added to chow supplemented with 1% cholesterol and 0.5% cholic acid. Clinical chemistry urinalysis and tissue histopathology were obtained. No toxicity differences were noted between control and inhibitor supplemented groups. CONCLUSIONS: Inhibitors of cholesterol esterase may be useful therapeutics for limiting cholesterol absorption.
Gender comparison in a murine model of allergen-driven airway inflammation and the response to budesonide treatment.
Corteling R, Trifilieff A
BMC Pharmacol · 2004 Apr · PMID 15086961 · Full text
BACKGROUND: Evidence suggests that gender differences exist in the severity of many immunological diseases and their response to glucocorticosteroid treatment. In this report, we have used a murine model of ovalbumin-ind...
BACKGROUND: Evidence suggests that gender differences exist in the severity of many immunological diseases and their response to glucocorticosteroid treatment. In this report, we have used a murine model of ovalbumin-induced lung inflammation to address whether gender could affect the systemic response, airway inflammation and hyperreactivity and their responses to budesonide. RESULTS: Following an acute ovalbumin challenge, actively sensitised BALB/c mice developed a time-dependent increase in interleukin-4 and interleukin-5 production and inflammatory cell influx into bronchoalveolar lavage fluid. Apart from an increased number of lymphocytes in female mice at day 3 post-challenge, none of the above parameters were affected by gender. Blood leukocyte numbers were also unaffected, whereas a two-fold increase in total serum immunoglobulin E was observed in female mice. Budesonide, given intranasally, did not affect the blood parameters, but dose-dependently inhibited the pulmonary inflammation and airway hyperreactivity in both male and female mice. Female mice were slightly less sensitive to budesonide's inhibitory action on interleukin-5 production and the development of airway hyperreactivity. CONCLUSIONS: Our results suggest that, apart from a 2-fold increase in serum immunoglobulin E levels observed in female mice, gender is not a major factor in the present murine model of ovalbumin-induced lung inflammation. In contrast, gender might slightly influence the potency of test compounds such as steroids.
Possible mechanism(s) for relaxant effect of aqueous and macerated extracts from Nigella sativa on tracheal chains of guinea pig.
Boskabady MH, Shirmohammadi B, Jandaghi P … +1 more , Kiani S
BMC Pharmacol · 2004 Feb · PMID 15070429 · Full text
BACKGROUND: In previous studies, the relaxant, anticholinergic (functional antagonism) and antihistaminic effects of Nigella sativa have been demonstrated on guinea pig tracheal chains. To elucidate the other mechanisms...
BACKGROUND: In previous studies, the relaxant, anticholinergic (functional antagonism) and antihistaminic effects of Nigella sativa have been demonstrated on guinea pig tracheal chains. To elucidate the other mechanisms responsible for the relaxant effect of this plant, its inhibitory effect on the calcium channel was examined in this study. RESULTS: The inhibitory effects of both concentrations of diltiazem in all three groups of experiments were significantly greater than those of saline (p < 0.01 to P < 0.001). The inhibitory of two larger concentrations of aqueous extracts in group 1 and 2 were significantly greater than those of saline (p < 0.01 to P < 0.001). The effect of two larger concentrations of macerated extract in group 1 and all concentrations of this extract in group 2 were also significantly greater than those of saline (p < 0.01 to P < 0.001). However, the extract of Nigella sativa did not show any inhibitory effect in group 3. There was a significant correlation between inhibitory effect and increasing concentrations for both extracts and diltiazem in groups 1 and 2 (p < 0.05 to p < 0.005). CONCLUSION: Although the extracts of Nigella sativa showed inhibitory effects on pre-contracted tracheal chains in the presence of both ordinary and calcium free Krebs solution, the absence of inhibitory effects of the extracts on KCl induced contraction of tracheal chains suggest that the calcium channel blocking effect of this plant dose not contribute to the relaxant effect of this plant on the tracheal chains of guinea pigs.
Triptolide, an active compound identified in a traditional Chinese herb, induces apoptosis of rheumatoid synovial fibroblasts.
Kusunoki N, Yamazaki R, Kitasato H … +3 more , Beppu M, Aoki H, Kawai S
BMC Pharmacol · 2004 Feb · PMID 15040811 · Full text
BACKGROUND: Extracts of Tripterygium wilfordii Hook F (TWHF), a traditional Chinese herb, have been reported to show efficacy in patients with rheumatoid arthritis (RA). Since RA is not only characterized by inflammation...
BACKGROUND: Extracts of Tripterygium wilfordii Hook F (TWHF), a traditional Chinese herb, have been reported to show efficacy in patients with rheumatoid arthritis (RA). Since RA is not only characterized by inflammation but also by synovial proliferation in the joints, we examined whether triptolide (a constituent of TWHF) could influence the proliferation of rheumatoid synovial fibroblasts (RSF) by induction of apoptosis. RESULTS: RSF were obtained from RA patients during surgery and were treated with triptolide under various conditions. The viability and proliferation of RSF were measured by the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay and by 5-bromo-2'-deoxyuridine incorporation, respectively. Apoptosis was identified by detection of DNA fragmentation using an enzyme-linked immunosorbent assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). The role of caspases in apoptosis of RSF was analyzed by measuring caspase-3 activity. Activation of the peroxisome proliferator-activated receptor (PPAR) gamma was assessed by a luciferase reporter gene assay using RSF transfected with a plasmid containing the peroxisome proliferator response element. Triptolide decreased viability, inhibited proliferation, and induced apoptosis of RSF in a concentration-dependent manner at very low (nM) concentrations. Caspase-3 activity was increased by treatment with triptolide and was suppressed by caspase inhibitors. Although PPARgamma activation was induced by 15-deoxy-Delta12,14-prostaglandin J2, triptolide did not induce it under the same experimental conditions. An extract of TWHF also induced DNA fragmentation in RSF. CONCLUSION: The mechanism of action remains to be studied; however, triptolide may possibly have a disease-modifying effect in patients with RA.
Relationship between PPARalpha activation and NO on proximal tubular Na+ transport in the rat.
Newaz MA, Ranganna K, Oyekan AO
BMC Pharmacol · 2004 Feb · PMID 15018640 · Full text
BACKGROUND: Nitric oxide (NO) regulates renal proximal tubular (PT) Na+ handling through modulation of Na+-K+ ATPase. Peroxisome Proliferator Activated Receptor alpha (PPARalpha), a nuclear transcription factor, is expre...
BACKGROUND: Nitric oxide (NO) regulates renal proximal tubular (PT) Na+ handling through modulation of Na+-K+ ATPase. Peroxisome Proliferator Activated Receptor alpha (PPARalpha), a nuclear transcription factor, is expressed in PTs and has been reported to influence NO generation/activity in renal tissues. This study tested the hypothesis that PPARalpha interacts with NO and thereby affects renal tubular Na+ transport. Urinary excretion of nitrite (UNOXV) and Na+ (UNaV) and PT Na+ transport (Na+-K+ ATPase activity) were determined in rats treated with clofibrate (250 mg/kg i.p) or WY14643 (45 mg/kg; i.p.), a PPARalpha ligand, 2% NaCl (orally), clofibrate/NaCl, L-NAME, an inhibitor of NO production (100 mg/kg; orally), L-NAME/Clofibrate. RESULTS: Clofibrate or WY14643 increased PPARalpha expression by 106 +/- 7% (p < 0.05) and 113 +/- 8% (p < 0.05), respectively. Similarly, clofibrate and WY14643 increased expression of MCAD, a downstream target protein of PPARalpha by 123 +/- 8% (p < 0.05) and 143 +/- 8% (p < 0.05), respectively. L-NAME attenuated clofibrate-induced increase in PPARalpha expression by 27 +/- 2% (p < 0.05) but did not affect MCAD expression. UNOXV excretion increased 3-4 fold in rats treated with clofibrate, WY14643 or NaCl from 44 +/- 7 to 170 +/- 15, 144 +/- 18 or 132 +/- 11 nmol/24 hr, respectively (p < 0.05). Similarly, clofibrate, WY14643 or NaCl elicited a 2-5 fold increase in UNaV. L-NAME significantly reduced basal UNOXV and UNaV and abolished the clofibrate-induced increase. Clofibrate, WY14643, NaCl or clofibrate + NaCl treatment reduced Na+-K+-ATPase activity in the PT by 89 +/- 23, 62 +/- 10, 43 +/- 9 and 82 +/- 15% (p < 0.05), respectively. On the contrary, L-NAME or ODQ, inhibitor of sGC, abolished the inhibition of Na+-K+-ATPase activity by clofibrate (p < 0.05). Clofibrate either alone or with NaCl elicited approximately 2-fold increase in the expression of the alpha1 subunit of Na+-K+ ATPase in the PT while L-NAME abolished clofibrate-induced increase in Na+-K+ ATPase expression. CONCLUSION: These data suggest that PPARalpha activation, through increased NO generation promotes renal excretion of Na+ through reduced Na+-K+ ATPase activity in the PT probably via post translational modification of Na+-K+-ATPase.
Protection against acute adriamycin-induced cardiotoxicity by garlic: role of endogenous antioxidants and inhibition of TNF-alpha expression.
Mukherjee S, Banerjee SK, Maulik M … +3 more , Dinda AK, Talwar KK, Maulik SK
BMC Pharmacol · 2003 Dec · PMID 14687418 · Full text
BACKGROUND: Oxidative stress is the major etiopathological factor in adriamycin-induced cardiotoxicity. Relatively low amounts of endogenous antioxidant makes the heart vulnerable to oxidative stress-induced damage. Chro...
BACKGROUND: Oxidative stress is the major etiopathological factor in adriamycin-induced cardiotoxicity. Relatively low amounts of endogenous antioxidant makes the heart vulnerable to oxidative stress-induced damage. Chronic oral administration of garlic has been reported to enhance the endogenous antioxidants of heart. We hypothesized that garlic-induced enhanced cardiac antioxidants may offer protection against acute adriamycin-induced cardiotoxicity. RESULTS: Rats were either administered freshly prepared garlic homogenate (250 and 500 mg/kg daily, orally, for 30 days) or probucol (cumulative dose, 120 mg/kg body weight divided in 12, i.p. over a period of 30 days) or double distilled water (vehicle), followed by a single dose of adriamycin (30 mg/kg i.p.). In the adriamycin group, increased oxidative stress was evidenced by a significant increase in myocardial TBARS (thiobarbituric acid reactive substances) and decrease in myocardial SOD (superoxide dismutase), catalase and GPx (glutathione peroxidase) activity. Histopathological studies showed focal as well as subendocardial myocytolysis with infiltration of macrophages, lymphocytes and edema. Immunocytochemistry showed marked expression of TNF-alpha (tumor necrosis factor-alpha) in the myocardium. Increase in myocardial TBARS and decrease in endogenous antioxidants by adriamycin was prevented significantly in the garlic treated rat hearts, which was comparable to the probucol-treated group. Histopathological evidence of protection was also evident in both garlic-treated and probucol-treated groups. Probucol, 250 mg/kg and 500 mg/kg of garlic reduced adriamycin induced TNF-alpha expression in the myocardium and was associated with reduced myocyte injury. CONCLUSIONS: It is concluded that chronic garlic administration prevents acute adriamycin-induced cardiotoxicity and decreases myocardial TNF-alpha expression.
Enhanced beta2-adrenergic receptor (beta2AR) signaling by adeno-associated viral (AAV)-mediated gene transfer.
Jones SM, Hiller FC, Jacobi SE … +3 more , Foreman SK, Pittman LM, Cornett LE
BMC Pharmacol · 2003 Dec · PMID 14656380 · Full text
BACKGROUND: Beta2-adrenergic receptors (beta2AR) play important regulatory roles in a variety of cells and organ systems and are important therapeutic targets in the treatment of airway and cardiovascular disease. Prolon...
BACKGROUND: Beta2-adrenergic receptors (beta2AR) play important regulatory roles in a variety of cells and organ systems and are important therapeutic targets in the treatment of airway and cardiovascular disease. Prolonged use of beta-agonists results in tolerance secondary to receptor down-regulation resulting in reduced therapeutic efficiency. The purpose of this work is to evaluate the signaling capabilities of the beta2AR expressed by a recombinant adeno-associated viral (AAV) vector that also included an enhanced green fluorescent protein (EGFP) gene (AAV-beta2AR/EGFP). RESULTS: By epifluorescence microscopy, approximately 40% of infected HEK 293 cells demonstrated EGFP expression. beta2AR density measured with [3H]dihydroalprenolol ([3H]DHA) increased either 13- or 77-fold in infected cells compared to mock infected controls depending on the culture conditions used. The [3H]DHA binding was to a single receptor population with a dissociation constant of 0.42 nM, as would be expected for wild-type beta2AR. Agonist competition assays with [3H]DHA showed the following rank order of potency: isoproterenol>epinephrine> norepinephrine, consistent with beta2AR interaction. Isoproterenol-stimulated cyclic AMP levels were 5-fold higher in infected cells compared to controls (314 +/- 43 vs. 63.4 +/- 9.6 nmol/dish; n = 3). Receptor trafficking demonstrated surface expression of beta2AR with vehicle treatment and internalization following isoproterenol treatment. CONCLUSIONS: We conclude that HEK 293 cells infected with AAV-beta2AR/EGFP effectively express beta2AR and that increased expression of these receptors results in enhanced beta2AR signaling. This method of gene transfer may provide an important means to enhance function in in vivo systems.
Enhanced spontaneous activity of the mu opioid receptor by cysteine mutations: characterization of a tool for inverse agonist screening.
Brillet K, Kieffer BL, Massotte D
BMC Pharmacol · 2003 Dec · PMID 14641935 · Full text
BACKGROUND: The concept of spontaneous- or constitutive-activity has become widely accepted and verified for numerous G protein-coupled receptors and this ligand-independent activity is also acknowledged to play a role i...
BACKGROUND: The concept of spontaneous- or constitutive-activity has become widely accepted and verified for numerous G protein-coupled receptors and this ligand-independent activity is also acknowledged to play a role in some pathologies. Constitutive activity has been reported for the mu opioid receptor. In some cases the increase in receptor basal activity was induced by chronic morphine administration suggesting that constitutive activity may contribute to the development of drug tolerance and dependence. Constitutively active mutants represent excellent tools for gathering information about the mechanisms of receptor activation and the possible physiological relevance of spontaneous receptor activity. The high basal level of activity of these mutants also allows for easier identification of inverse agonists, defined as ligands able to suppress spontaneous receptor activity, and leads to a better comprehension of their modulatory effects as well as possible in vivo use. RESULTS: Cysteines 348 and 353 of the human mu opioid receptor (hMOR) were mutated into alanines and Ala348,353 hMOR was stably expressed in HEK 293 cells. [35S] GTPgammaS binding experiments revealed that Ala348,353 hMOR basal activity was significantly higher when compared to hMOR, suggesting that the mutant receptor is constitutively active. [35S] GTPgammaS binding was decreased by cyprodime or CTOP indicating that both ligands have inverse agonist properties. All tested agonists exhibited binding affinities higher for Ala348,353 hMOR than for hMOR, with the exception of endogenous opioid peptides. Antagonist affinity remained virtually unchanged except for CTOP and cyprodime that bound the double mutant with higher affinities. The agonists DAMGO and morphine showed enhanced potency for the Ala348,353 hMOR receptor in [35S] GTPgammaS experiments. Finally, pretreatment with the antagonists naloxone, cyprodime or CTOP significantly increased Ala348,353 hMOR expression. CONCLUSION: Taken together our data indicate that the double C348/353A mutation results in a constitutively active conformation of hMOR that is still activated by agonists. This is the first report of a stable CAM of hMOR with the potential to screen for inverse agonists.
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