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Results for “Drug Collateral Sensitivity”

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PubMed1987

Caffeine inhibits post-tetanic potentiation but does not alter long-term potentiation in the rat hippocampal slice.

The effects of caffeine were investigated on the extracellular excitatory postsynaptic potentials (EPSPs) recorded in the stratum radiatum of CA1 of the rat hippocampal slice in response to stimulation of the Schaffer collaterals. Caffeine in concentrations from 500 microM to 10 mM caused an increase in the amplitude of the EPSP, which reached a peak after 5-10 min perfusion. This increase was antagonized by pretreatment with 20 microM trifluoperazine. Paired-pulse facilitation, augmentation and potentiation were strongly inhibited by caffeine. Augmentation was most sensitive to caffeine, being abolished by 500 microM caffeine. Long-term potentiation (LTP) induced by high-frequency stimulation was not significantly inhibited by caffeine either by 5-10 min or by 60 min perfusion with 10 mM caffeine. Moreover, the caffeine-induced increase in the low-frequency EPSP could be reversed by 30 min washout, demonstrating that it was not an LTP type increase.

Afferent Pathways↗

PubMed2002

The nicotinic allosteric potentiating ligand galantamine facilitates synaptic transmission in the mammalian central nervous system.

In this study, the patch-clamp technique was used to determine the effects of galantamine, a cholinesterase inhibitor and a nicotinic allosteric potentiating ligand (APL) used for treatment of Alzheimer's disease, on synaptic transmission in brain slices. In rat hippocampal and human cerebral cortical slices, 1 microM galantamine, acting as a nicotinic APL, increased gamma-aminobutyric acid (GABA) release triggered by 10 microM acetylcholine (ACh). Likewise, 1 microM galantamine, acting as an APL on presynaptically located nicotinic receptors (nAChRs) that are tonically active, potentiated glutamatergic or GABA-ergic transmission between Schaffer collaterals and CA1 neurons in rat hippocampal slices. The cholinesterase inhibitors rivastigmine, donepezil, and metrifonate, which are devoid of nicotinic APL action, did not affect synaptic transmission. Exogenous application of ACh indicated that high and low levels of nAChR activation in the Schaffer collaterals inhibit and facilitate, respectively, glutamate release onto CA1 neurons. The finding then that the nAChR antagonists methyllycaconitine and dihydro-beta-erythroidine facilitated glutamatergic transmission between Schaffer collaterals and CA1 neurons indicated that in a single hippocampal slice, the inhibitory action of strongly, tonically activated nAChRs in some glutamatergic fibers prevails over the facilitatory action of weakly, tonically activated nAChRs in other glutamatergic fibers synapsing onto a given neuron. Galantamine is known to sensitize nAChRs to activation by low, but not high agonist concentrations. Therefore, at 1 microM, galantamine is likely to increase facilitation of synaptic transmission by weakly, tonically activated nAChRs just enough to override inhibition by strongly, tonically activated nAChRs. In conclusion, the nicotinic APL action can be an important determinant of the therapeutic effectiveness of galantamine.

Action Potentials↗

PubMed2006

Paclitaxel-resistant cells have a mutation in the paclitaxel-binding region of beta-tubulin (Asp26Glu) and less stable microtubules.

Resistance to paclitaxel-based therapy is frequently encountered in the clinic. The mechanisms of intrinsic or acquired paclitaxel resistance are not well understood. We sought to characterize the resistance mechanisms that develop upon chronic exposure of a cancer cell line to paclitaxel in the presence of the P-glycoprotein reversal agent, CL-347099. The epidermoid tumor line KB-3-1 was exposed to increasing concentrations of paclitaxel and 5 micromol/L CL-347099 for up to 1 year. Cells grown in 15 nmol/L paclitaxel plus CL-347099 (KB-15-PTX/099) developed 18-fold resistance to paclitaxel and were dependent upon paclitaxel for maximal growth. They grew well and retained resistance to paclitaxel when grown in athymic mice. Cross-resistance (3- to 5-fold) was observed in tissue culture to docetaxel, the novel taxane MAC-321, and epothilone B. Collateral sensitivity (approximately 3-fold) was observed to the depolymerizing agents vinblastine, dolastatin-10, and HTI-286. KB-15-PTX/099-resistant cells did not overexpress P-glycoprotein nor did they have an alteration of [14C]paclitaxel accumulation compared with parental cells. However, a novel point mutation (T to A) resulting in Asp26 to glutamate substitution in class I (M40) beta-tubulin was found. Based on an electron crystallography structure of Zn-stabilized tubulin sheets, the phenyl ring of C-3' NHCO-C6H5 of paclitaxel makes contact with Asp26 of beta-tubulin, suggesting a ligand-induced mutation. Optimized model complexes of paclitaxel, docetaxel, and MAC-321 in beta-tubulin show a novel hydrogen bonding pattern for the glutamate mutant and rationalize the observed resistance profiles. However, a mutation in the paclitaxel binding pocket does not explain the phenotype completely. KB-15-PTX/099 cells have impaired microtubule stability as determined by a reduced percentage of tubulin in microtubules and reflected by less acetylated tubulin. These results suggest that a mutation in tubulin might affect microtubule stability as well as drug binding and contribute to the observed resistance profile.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

Modulation of cytosine arabinoside toxicity by 3-deazauridine in a murine leukemia model.

3-Deazauridine (DAUrd), a competitive inhibitor of CTP synthetase, inhibits both RNA and DNA synthesis. Murine leukemia cells resistant to cytosine arabinoside (ara-C) due to a deletion of deoxycytidine kinase are collaterally sensitive to DAUrd, which inhibits the de novo production of CTP and hence results in dCTP depletion. We evaluated DAUrd in combination with the palmitate derivative of ara-C (palmO-ara-C) in mice bearing L1210 leukemia cells with a subpopulation resistant to ara-C. Both simultaneous administration and a sequential schedule of palmO-ara-C at its maximally tolerated dose (MTD), followed by DAUrd treatment, failed to produce a therapeutic gain. We also studied whether non-toxic doses of DAUrd (15-250 mg/kg i.p. at h 0 and 6 on days 4 and 8) could modulate the antileukemic activity of palmO-ara-C (7.5-120 mg/kg i.p. at h 3 on days 4 and 8). The addition of DAUrd produced a modest (but statistically significant) prolongation of life span and a further 2-log10 reduction in tumor burden compared to the same dose of palmO-ara-C alone, and resulted in long-term survivors in five of 30 treated animals. Two-dimensional dose-response analysis of the survival data indicated a positive drug interaction (p less than or equal to 0.01) when the dosage of DAUrd was modeled to reflect an apparent threshold effect. Cyclopentenyl cytosine (CPE-C; 0.625-2.5 mg/kg i.p. at h 0 and 6 on days 4 and 8), a more potent inhibitor of CTP synthetase, was also given with palmO-ara-C. This combination resulted in an additional 2-6 log10 units of cell kill and occasional long-term survivors at palmO-ara-C dosages that alone resulted in no more than 2 log10 units of cell kill and no long-term survivors. However, DAUrd and CPE-C given with palmO-ara-C increased host toxicity, compromising the tolerable dose of palmO-ara-C. Single-agent palmO-ara-C given at its MTD produced a similar reduction in tumor burden and increase in life span compared to the highest palmO-ara-C dose that could be given in combination with either modulator.

3-Deazauridine↗

PubMed2004

Small conductance Ca2+-activated K+ channel knock-out mice reveal the identity of calcium-dependent afterhyperpolarization currents.

Action potentials in many central neurons are followed by a prolonged afterhyperpolarization (AHP) that influences firing frequency and affects neuronal integration. In hippocampal CA1 pyramidal neurons, the current ascribed to the AHP (IAHP) has three kinetic components. The IfastAHP is predominantly attributable to voltage-dependent K+ channels, whereas Ca2+-dependent and voltage-independent K+channels contribute to the ImediumAHP (ImAHP) and IslowAHP (IsAHP). Apamin, which selectively suppresses a component of the mAHP, increases neuronal excitability and facilitates the induction of synaptic plasticity at Schaffer collateral synapses and hippocampal-dependent learning. The Ca2+-dependent components of the AHP have been attributed to the activity of small conductance Ca2+-activated K+ (SK) channels. Examination of transgenic mice, each lacking one of the three SK channel genes expressed in the CNS, reveals that mice without the SK2 subunit completely lack the apamin-sensitive component of the ImAHP in CA1 neurons, whereas the IsAHP is not different in any of the SK transgenic mice. In each of the transgenic lines, the expression levels of the remaining SK genes are not changed. The results demonstrate that only SK2 channels are necessary for the ImAHP, and none of the SK channels underlie the IsAHP.

Animals↗

PubMed2003

Allergic and pseudoallergic reactions induced by glucocorticoids: a review.

Glucocorticoids (GCs) represent the most effective treatment for autoimmune and allergic diseases, even if collateral effects are not rare, especially endocrine and immunosuppressive manifestations. Moreover, these drugs can develop adverse immunological reactions of I, III or IV type. Though immediate adverse reactions caused by systemic therapy with GCs are not very frequent, the possible beginning of anaphylactic and pseudo-anaphylactic manifestations in patients undergoing therapy with these drugs has to be considered. It has been observed that immediate adverse reactions usually are happened in asthmatic patients and in patients obliged to assume GCs again and again because of their pathology (e.g, kidney transplant). Other risk factors resulted to be female sex and hypersensibility to acetylsalicylic acid (ASA). Both in the cases of pseudo-allergic and allergic reactions, the pharmacological principle is hardly the responsible agent for the reaction; instead the excipients in drugs are often implicated (succinate salt, sulphites and carboxy-methyl-cellulose). It is possible that the IgE-response is highly specific for a fixed GC molecule as well depending on the way of administration and its salification. Moreover, it has been hypothesized that in patients with a first type allergic reaction to GCs there is a fourth type, sensitization to GCs, which is not usually diagnosed and even comes before IgE sensitization. Third type hypersensibility reactions may occur, too. Since GCs are large-scale drugs, also in emergency medicine and reanimation, allergic sensitization towards them, although infrequent, gives many interventionist problems. In the light of this feature, it seems of crucial importance to verify the tolerance toward other GC molecules. And in particular, it has been noted that patients presenting immediate reactions to hydrocortisone (HC) and methylprednisolone (MP) could tolerate prednisone and prednisolone per os and second-generation GCs, such as desamathazone and betamethazone. Nevertheless, second-generation GCs must not be considered safe; in fact, the beginning of allergic manifestations has been pointed out even towards them.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2002

Alterations of intestinal motor responsiveness in a model of mild mesenteric ischemia/reperfusion in rats.

In this study we investigate the changes in intestinal motor responsiveness after mild mesenteric ischemia/reperfusion in anaesthetized rats. Motor responsiveness to pharmacological/electrical stimulation was studied in isolated ileum excised from sham-operated rats or animals which underwent occlusion of superior mesenteric artery (1 h) plus interruption of collateral blood flow and reperfusion for 0, 24, 72 h. Only 24 h reperfusion resulted in a significant suppression in acetylcholine induced contractile response and in indomethacin induced relaxation. In the presence of adrenergic and cholinergic blockade a greater relaxant response to field stimulation (trains 10 s every min, 120 mA, 1 ms and 10 Hz) was unmasked in all groups except 24 h reperfused rats. Such effect was sensitive to N(G)-Nitro-L-arginine methyl ester (NOS unselective inhibitor) and the proteolytic enzyme alpha-chymotrypsin but resistant to aminoguanidine (iNOS selective inhibitor). In conclusion, in this rat model, intestinal mild ischemia/24 h reperfusion induces reversible changes in enteric motility attributable to a decrease in eicosanoids, nitric oxide and neuropeptides availability.

Acetylcholine↗

PubMed1991

Cross-resistance patterns related to glutathione metabolism in primary human renal cell carcinoma.

In 59 cases of primary human renal cell carcinoma (RCC), cross-resistance and collateral susceptibility patterns were determined in an MTT microculture assay. Concomitantly, the glutathione (GSH) content and the enzymatic activity of gamma-glutamyl transpeptidase (GGT) were measured as distinct resistance characteristics. Resistance or chemoresponse towards Vinca alkaloids and anthracyclines were found to be highly coincident, suggesting that the classical multidrug resistance mechanism is active in human RCC. Strong resistance to platinum complexes combined with relative sensitivity to bleomycin was significantly associated with elevated glutathione levels, providing evidence for another pathway instigating chemoresistance. In contrast, despite substantial enzymatic activity, GGT effects revealed no correlation to the chemoresistance pattern. This result implies that it is the GSH-linked binding and reduction potential rather than the GGT-associated transportation capacity that has an impact on the expression of chemoresistance in human RCC.

Antineoplastic Agents↗

PubMed1991

Chronic theophylline treatment in vivo increases high affinity adenosine A1 receptor binding and sensitivity to exogenous adenosine in the in vitro hippocampal slice.

The present investigation examined the effects of chronic treatment with the adenosine receptor antagonist theophylline in vivo, on in vitro hippocampal electrophysiology and adenosine A1 receptor binding in the same animals. Adult rats were injected once daily (i.p.) with theophylline for 1 week at 75 mg/kg, followed by an additional week at 100 mg/kg, or with saline for the same 2-week period. Two days following the last injection, hippocampal slices were prepared and population spikes recorded from the pyramidal cell layer of area CA1 were elicited by Schaffer collateral-commissural fiber stimulation. The degree of inhibition caused by superfused adenosine was compared between hippocampal slices from theophylline- and saline-treated rats. Tissue from the contralateral hippocampus was used in [3H]cyclohexyladenosine ([3H]CHA) receptor binding. Hippocampi from theophylline-treated animals showed a significantly greater number of [3H]CHA binding sites (apparent Bmax; 125% of control, P less than 0.05), without a significant change in binding affinity, and were more sensitive than controls to the inhibitory effects of adenosine on the population spike response. These results suggest that chronic adenosine receptor antagonism results in the up-regulation of adenosine A1 receptors which are functional and physiologically relevant in the in vitro hippocampus, and further supports the hypothesis that methylxanthine tolerance is mediated, at least in part, by an increase in adenosine receptor density.

Adenosine↗

PubMed2006

Different anticonvulsive effects of hesperidin and its aglycone hesperetin on electrical activity in the rat hippocampus in-vitro.

This study investigated the possibility that hesperidin or hesperetin might interact directly with brain matter in a physiological manner. The effects of both compounds were followed in the in-vitro hippocampus preparation by continuous superfusion in a concentration-dependent manner in the presence of single stimuli and theta-burst stimulation of the Schaffer Collaterals. Hesperidin increased the population spike response at a concentration up to 10 microM, especially after induction of long-term potentiation, but attenuated it significantly at higher concentrations of up to 60 microM. Hesperetin only attenuated the response within the same concentration range. Modulation of the pyramidal cell response in the presence of tetraethylammonium (TEA) and pentylentetrazol on one hand and 4-aminopyridine (4-AP) and bicuculline on the other was influenced in a different way. Whereas hesperidin attenuated the response to 4-AP and bicuculline but not to TEA or pentylentetrazol, hesperetin was able to attenuate the response to TEA and pentylentetrazol, but not to 4AP or bicuculline. This feature was reproduced and confirmed ex-vivo after repetitive administration of hesperidin and hesperetin in-vivo for one week (500 mg kg(-1) orally) before in-vitro testing against the challenging effects of 4-AP and TEA. Since the action of hesperidin was sensitive to the presence of iberiotoxin, the involvement of a large conductance calcium-dependent potassium channel might be assumed. In summary, the results provide the possibility for use of both compounds to control pathophysiological disturbances of brain excitability in drug abuse, migraine and epilepsy.

4-Aminopyridine↗

PubMed1988

New findings in the pharmacokinetic, metabolic, and drug-resistance aspects of mitomycin C.

The pharmacokinetics of mitomycin C are reviewed from reports using specific and sensitive high-performance liquid chromatography (HPLC) assays. These studies demonstrate a rapid, biphasic elimination pattern for the drug: alpha half-life of 8 minutes and beta, or terminal, half-life of 48 minutes. Urinary elimination of intact drug is minimal (8% to 10% of a dose), whereas biliary drug levels may exceed those in the plasma. There was no evidence of dose dependent pharmacokinetics over a wide range of doses and patient populations, including pediatric solid tumor patients. Metabolic studies with mitomycin C have demonstrated an absolute requirement for reductive enzymatic activation of the drug to mono- and bifunctional alkylating species. The preferred DNA target for covalent attachment by mitomycin C was found to be the N2 position of guanine. Chemical metabolites of activated mitomycin C were also demonstrated to include 2, 7 diaminomitosene and its cis- and trans-1-hydroxy or 1-phosphate analogues. Also, there was no evidence for hypoxic cell selectivity for mitomycin C in several human tumor cell lines, although some animal tumors unequivocally display this phenomenon. Finally, there are new observations of the development of the multidrug-resistance phenotype in mitomycin C-treated L-1210 cells in vitro. These cells became collaterally resistant to anthracyclines and vinca alkaloids and expressed the P-glycoprotein in cell membranes. The implications of these findings for mitomycin C use in solid tumors in humans is discussed.

Biotransformation↗

PubMed2001

Steroids affect collateral sensitivity to gemcitabine of multidrug-resistant human lung cancer cells.

Gemcitabine is phosphorylated by deoxycytidine kinase and thymidine kinase 2 and during S-phase incorporated into DNA. The steroids cortisol and dexamethasone, which regulate cell proliferation and gene expression, are pumped out of the cell by the membrane efflux pumps P-glycoprotein and multidrug resistance-associated protein (MRP), which are blocked by verapamil. In parental non-small cell lung cancer (NSCLC) cells (SW1573), 5 microM cortisol and 100 nM dexamethasone decreased sensitivity to gemcitabine. However, both cortisol and dexamethasone only decreased sensitivity with verapamil in MRP (2R120) and P-glycoprotein (2R160) overexpressing variants. Cortisol decreased deoxycytidine kinase activity in SW1573 cells and cortisol with verapamil in 2R120 and 2R160 cells. Dexamethasone with verapamil decreased deoxycytidine kinase activity in 2R160. Cortisol decreased thymidine kinase 2 activity in 2R120 and 2R160 cells. Dexamethasone decreased thymidine kinase 2 activity in SW1573, 2R120 and 2R160 cells. In conclusion, since dexamethasone is frequently used to treat side effects of oncolytic therapy, a decrease of sensitivity to gemcitabine by steroids might be clinically relevant.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Cross-resistance studies of folylpolyglutamate synthetase-deficient, methotrexate-resistant CCRF-CEM human leukemia sublines.

CCRF-CEM human leukemia sublines resistant to short-term methotrexate (MTX) exposure as a result of decreased folylpolyglutamate synthetase (FPGS) activity were examined for their response to other cytotoxic agents. The R3/7 and R30dm sublines display 25 and 1%, respectively, of the FPGS activity of CCRF-CEM cells as measured with MTX in vitro. Response to agents in outgrowth experiments was examined under both continuous exposure (120 h, where MTX resistance is not observed) and short-term (6-14.5 h) exposure. During continuous exposure to various classes of agents, cross-resistance of R3/7 and R30dm that correlated with FPGS level was not observed, although some minor (< or = 3-fold) stochastic variations in sensitivity were noted. These agents included actinomycin D, Adriamycin, etoposide, vincristine, cisplatin, cytosine arabinoside, 5-fluorouracil, and some other antifolates. Cross-resistance during continuous exposure that did correlate with FPGS level was noted, however, to glutamate-containing thymidylate synthase inhibitors (including ICI D1694) and, to a minor extent, to 6-mercaptopurine and 5-fluorodeoxyuridine. Slight collateral sensitivity during continuous exposure that apparently correlated with FPGS level was noted to the lipid-soluble antifolate trimetrexate and to 5,8-dideazapteroyl-L-ornithine, an FPGS-specific inhibitor. In short-term exposures (where MTX resistance of the sublines is observed), the resistant sublines displayed sensitivity or cross-resistance to each agent that was qualitatively similar to that observed for the same agent in continuous exposure. Because of the requirement for reduced folates in the anti-DNA mechanism of action of fluoropyrimidines and the current clinical use of leucovorin (LV) to enhance their effects, the interaction of LV and fluoropyrimidines was examined. The results suggest that even highly FPGS-deficient cells are as sensitive to the effects of LV modulation as are wild-type cells even at fluoropyrimidine exposure times as short as 4 h.

Antimetabolites, Antineoplastic↗

PubMed1999

Effect of Tween-80 on cell killing by etoposide in human lung adenocarcinoma cells.

PURPOSE: The non-ionic detergent Tween-80, a surface-active agent, has been shown to modulate the cytocidal effect of certain antitumor agents. In the present study, we sought to determine whether or not Tween-80 could enhance the antitumor effect of etoposide (VP16) in human lung cancer cells in vitro. METHODS: Survival fractions were measured by growth inhibiton assays of PC14, H69, KB, and PC14/CDDP (the corresponding cisplatin-resistant subline of PC14) cells. An in vitro clonogenic assay of PC14 and PC14/CDDP cells was undertaken after incubation for 10-12 days in RPMI-1640 medium with 20% fetal calf serum and 1.72% methyl cellulose, plus continuous exposure to VP16 with Tween-80. We also investigated the direct toxicity of Tween-80 to PC14 and PC14/CDDP cells using a clonal assay. The intracellular accumulation of VP16 was further analyzed using [3H]VP16 in PC14, PC14/CDDP, A549, KB and H69 cells, and compared with that of daunorubicin (DNR), a hydrophilic anti-cancer agent, using [3H]DNR in PC14, A549 and KB cells. RESULTS: It was found that PC14/CDDP had collateral sensitivity to VP16 and Tween-80 markedly enhanced the killing effect of VP16 not only of PC14 cells but also of PC14/CDDP cells while exerting little cytotoxic effect. Moreover, Tween-80 increased the intracellular accumulation of VP16 in PC14, PC14/CDDP and A549 cells, and not in KB and H69 cells. Tween-80 did not increase the intracellular DNR levels in PC14, A549 and KB cells. CONCLUSIONS: Tween-80 was shown to potentiate the cytotoxicity of VP16 against several human lung adenocarcinoma cells by increasing the accumulation of VP16 in vitro. Tween-80-mediated sensitization of lung adenocarcinoma cells to VP16 is considered to be related to both the characteristics of the cell membrane in adenocarcinoma cells and the lipotropic properties of VP16. These results suggest that this combination might have the potential to improve the therapeutic index of VP16 in human lung adenocarcinoma.

Adenocarcinoma↗

PubMed1996

BMS-180448, a novel glyburide-reversible cardioprotective agent, enhances postischemic recovery of contractile function in dogs.

BMS-180448 has been found to retain the cardioprotective potency of its chemically related analog, cromakalim, although having significantly less peripheral vasodilating activity. The effect of the ATP-sensitive potassium channel opener, BMS-180448, on postischemic recovery of function (segmental shortening) was determined in open chested, anesthetized dogs instrumented with ultrasonic crystals. The plasma concentration of the effective and ineffective doses of BMS-180448 was compared to concentrations used in isolated rat hearts. BMS-180448 was given as a total dose of 4.2, 1.4 or 0.5 mg/kg over 30 min, starting 15 min before ischemia. Ischemia was initiated by a complete occlusion of the left anterior descending coronary artery for 15 min. Reperfusion was maintained for 3 hr and segmental shortening was measured. During ischemia, systolic bulging was observed in the ischemic region in drug- and vehicle-treated groups. Upon reperfusion, some contractile functional recovery was observed in vehicle-treated controls within minutes, but quickly decreased so that slight bulging was observed up to 3 hr into reperfusion. High dose BMS-180448 significantly improved the recovery of contractile function such that, by 3 hr after reperfusion, segmental shortening had recovered to 60% of base line. The 1.4-mg/kg dose also significantly improved reperfusion function, but 0.5 mg/kg of BMS-180448 was without effect. None of the doses of BMS-180448 significantly affected peripheral hemodynamic status or collateral blood flow. The plasma concentration of the 1.4-mg/kg dose was approximately 3 microM during ischemia. In isolated rat hearts, BMS-180448 significantly increased postischemic function at 3 microM and higher concentrations, which agrees with the dog data. BMS-180448 was protective in a dose-dependent manner in a canine model of stunned myocardium, and the concentrations necessary for protection are similar to that for rats.

Animals↗

PubMed1995

Markedly decreased expression of glutathione S-transferase pi gene in human cancer cell lines resistant to buthionine sulfoximine, an inhibitor of cellular glutathione synthesis.

Buthionine sulfoximine (BSO) is a synthetic amino acid that irreversibly inhibits an enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), which is a critical step in glutathione biosynthesis. We isolated three BSO-resistant sublines, KB/BSO1, KB/BSO2, and KB/BSO3, from human epidermoid cancer KB cells. These cell lines showed 10-to 13-fold higher resistance to BSO, respectively, and had collateral sensitivity to cisplatin, ethacrynic acid, and alkylating agents such as melphalan and nitrosourea. Cellular levels of glutathione S-transferase pi (GST-pi) and its mRNA in BSO-resistant cell lines were less than 10% of the parental cells. Nuclear run-on assay showed that the transcriptional activity of GST-pi was decreased in BSO-resistant cells, and transient transfection of GST-pi promoter-chloramphenicol acetyltransferase constructs revealed that the sequences between -130 and -80 base pairs of the 5'-flanking region wer at least partially responsible for the decreased expression of the GST-pi gene. By contrast, gamma-GCS mRNA levels were 3-to 5-fold higher in resistant cell lines than in KB cells, and the gamma-GCS gene was found to be amplified in the BSO-resistant cells lines. GST-pi mRNA levels appeared to be inversely correlated with gamma-GCS mRNA levels in BSO-resistant cells. We further established the transfectants, KB/BSO3-pi1 and KB/ BSO2-pi2, that overexpressed GST-pi, from KB/BSO3, after introducing a GST-pi expression plasmid. These two transfectants had similar levels in gamma-GCS mRNA, drug sensitivity to alkylating agents, and glutathione content at those of KB cells. These findings suggest that the cellular levels of GST-pi and gamma-GCS might be co-regulated in these novel BSO-resistant cells.

Blotting, Southern↗

PubMed1993

Role of nitric oxide, muscarinic receptors, and the ATP-sensitive K+ channel in mediating the effects of acetylcholine to mimic preconditioning in dogs.

The aims of the current study were to examine the efficacy of acetylcholine (ACh) to mimic ischemic preconditioning in dogs and to determine the role of nitric oxide (NO), muscarinic receptors, and ATP-sensitive K+ (KATP) channels in mediating its effects. Barbital-anesthetized open-chest dogs were subjected to 60 minutes of left anterior descending coronary artery (LAD) occlusion followed by 4 hours of reperfusion. Preconditioning was elicited by 10 minutes of LAD occlusion followed by 10 minutes of reperfusion before the 60-minute occlusion period. ACh (3 or 10 micrograms/min) or an equivalent volume of saline was infused into the LAD for 10 minutes, followed by a 10-minute drug-free period before the 60-minute ischemic insult. In other groups, the specific NO synthesis inhibitor NG-monomethyl-L-arginine (L-NMMA, 4 mg/min), the muscarinic receptor antagonist-NO synthesis inhibitor nitro-L-arginine methyl ester (L-NAME, 3 mg/min), or the specific KATP channel blocker 5-hydroxydecanoate (5-HD, 3 mg/min) was infused with ACh into the LAD for 10 minutes. The infusion of L-NAME, L-NMMA, or 5-HD was started 2 minutes before ACh infusion. Transmural myocardial blood flow was measured at 5 minutes of occlusion, and infarct size was determined by triphenyltetrazolium staining and expressed as a percentage of the area at risk. There were no significant differences in collateral blood flow or the area at risk between groups. Preconditioning produced a marked reduction (P < .05) in infarct size (6.2 +/- 3.0% versus 26.1 +/- 5.7% in the control group).(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1997

An ATP-sensitive potassium (KATP) channel opener, nicorandil, lowers the threshold for ischemic preconditioning in barbital-anesthetized dogs.

To determine whether the adenosine triphosphate (ATP)-sensitive potassium channel (KATP channel) opener, nicorandil, lowers the threshold for the infarct-reducing effect of preconditioning (PC), barbital-anesthetized dogs were subjected to 60-min occlusion (60'OC) of left anterior descending coronary artery (LAD) followed by 3-h reperfusion (3h-RP). At the end of the 3h-RP, heart was resected to measure infarct size. Infarct size was determined by triphenyltetrazolium chloride (TTC) staining, calculated gravimetrically and was expressed as a percent age of area at risk (AAR). Myocardial blood flow was measured by radioactive microspheres. Single 5-min LAD occlusion 10 min before 60'OC (5'PC group) markedly reduced infarct size, compared to the controls (11.8 +/- 3.7% versus 26.8 +/- 4.7%; P < 0.05). Neither 2.5-min PC (2.5'PC group), nor 2.5-min intracoronary infusion with 0.5 microgram/kg per min of nicorandil followed by a 10-min drug-free period (2.5'NC group), showed a cardioprotective effect (26.5 +/- 1.7% and 20.7 +/- 2.8%, respectively). However, when nicorandil was administered during the 2.5-min LAD occlusion period (2.5'PC + 2.5'NC group), infarct size was significantly reduced to an extent similar to that in 5'PC group (13.6 +/- 1.2%). There were no significant differences in hemodynamics, collateral circulation, or AAR between groups. These results suggest that nicorandil lowers the threshold for the infarct-reducing effect of PC in dogs by, at least in part, activation of myocardial KATP channels.

Animals↗