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Results for “Drug Collateral Sensitivity”

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PubMed1989

Multidrug resistance in mitoxantrone-selected HL-60 leukemia cells in the absence of P-glycoprotein overexpression.

A multidrug-resistant variant of the human HL-60 promyelocytic leukemia cell line (HL-60/MX2) has been isolated in vitro by subculturing these cells in progressively increasing concentrations of mitoxantrone. The MX2 cells are cross-resistant to etoposide, teniposide, bisantrene, dactinomycin, 4'-(9-acridinylamino)methanesulfon-m-anisidide, and the anthracyclines daunorubicin and doxorubicin but retain sensitivity to the Vinca alkaloids melphalan and mitomycin C. In addition, the MX2 cells display slight collateral sensitivity to bleomycin. Despite being 30-35-fold less sensitive to mitoxantrone, net [14C]mitoxantrone accumulation at 60 min was reduced by only 10% in the mitoxantrone-resistant cells compared to the parental line. Furthermore, at later time points, e.g., 120 and 180 min, mitoxantrone accumulation in the MX2 cells exceeded that in HL-60 cells by 8.5 and 6.4%, respectively. No significant differences were observed between the sensitive and resistant cell lines in the initial (first 60 s) accumulation of mitoxantrone, and only minor (3-6%) enhancement of mitoxantrone efflux was detected in the resistant cell type. Monoclonal antibodies to P-glycoprotein had no detectable reactivity with membrane vesicles from either the sensitive or resistant cell types as determined by standard immunoblotting techniques. The mitoxantrone-resistant cells displayed a reciprocal translocation [rcpt(1;3)-(q21;p23)] not found in the sensitive parent, but there were no demonstrable double minute chromosomes or homogeneous staining regions in cells from either line. Thus, these mitoxantrone-resistant human leukemia cells display many features which are atypical for the "classic" multidrug resistance phenotype and should provide a useful model for the study of multidrug resistance which is not mediated by P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Adenosine receptor blockade reveals N-methyl-D-aspartate receptor- and voltage-sensitive dendritic spikes in rat hippocampal CA1 pyramidal cells in vitro.

The present study was done to determine the possible effects of endogenous adenosine, present in the extracellular fluid of the hippocampal slice, on pyramidal cells in the CA1 region using intracellular recording techniques. Administration of 5 microM of the adenosine receptor antagonist, 8-sulfophenyltheophylline (n=11), induced a depolarization (2.6+/-0.4 mV, mean+/-S.E.M.) with an increase in input resistance (6.7+/-2.1%) in pyramidal cells, and increased the amplitude of the excitatory postsynaptic potentials elicited by stimulation of Schaffer collateral afferents; 50 microM 8-sulfophenyltheophylline (n=68) produced a similar depolarization (3.4+/-1.7 mV) and an increase in input resistance (26+/-3.0%), but also produced spontaneous, synchronized giant excitatory postsynaptic potentials which could generate bursts of spikes. These effects lasted more than 10 min after washout. In the presence of 20 microM 6-cyano-7-nitro-quinoxaline-2,3-dione, a non-N-methyl-D-aspartate receptor antagonist, and 50 microM D-2-amino-5-phosphonovalerate, an N-methyl-D-aspartate receptor antagonist, 50 microM 8-sulfophenyltheophylline (n=4) induced only depolarization (3.1+/-1.3 mV) and an increase in input resistance (23+/-3.8%). In the presence of 20 microM 6-cyano-7-nitro-quinoxaline-2,3-dione only, 50 microM 8-sulfophenyltheophylline (n=7) induced not only the depolarization with an increase in input resistance, but also the occurrence of small-amplitude (11+/-5.6 mV), fast rising, all-or-none, voltage-sensitive spikes of 2-3 ms duration, which were attributed to a dendritic origin. The latency of these dendritic spikes in response to stimulation of Schaffer collateral afferents lasted up to 21 ms. These dendritic spikes could generate one or more action potentials, depending on the resting membrane potential and the frequency of the dendritic spikes. In the presence of 50 microM 8-sulfophenyltheophylline plus 20 microM 6-cyano-7-nitro-quinoxaline-2,3-dione, 50 microM D-2-amino-5-phosphonovalerate blocked the spontaneous dendritic spikes (n=4). In the presence of 5 microM 8-sulfophenyltheophylline, 200 microM N-methyl-D-aspartate (n=5) increased the occurrence of dendritic spikes. These data indicate that adenosine present in the extracellular fluid of the hippocampal slice tonically inhibits not only (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-mediated synaptic transmission, but also voltage- and N-methyl-D-aspartate receptor-sensitive dendritic spikes. Endogenous adenosine acting on adenosine A(1) receptors is thus visualized as a control to prevent the genesis of synchronized giant excitatory postsynaptic potentials. In our experiments, blockade of this tonic activation of adenosine receptors appears to have altered the origins of action potentials and led to epileptiform firing in CA1 pyramidal cells.

Action Potentials↗

PubMed2003

Optical detection of dendritic spike initiation in hippocampal CA1 pyramidal neurons.

Previous studies have shown that spikes can be generated in the dendrites of CA1 pyramidal neurons. Some have suggested that, in response to synaptic inputs, spikes are initiated near the soma and propagate back into the dendrites, but some recent studies have shown that intense synaptic inputs initiate spikes in the dendrite. Here, we report the optical detection of spike propagation along the apical dendrites of hippocampal pyramidal neurons. Rat hippocampal slices were stained with the fluorescent voltage-sensitive dye, JPW1114, and optical signals monitored using a 16 x 16 photodiode array system at a frame rate of 4 kHz. A stimulating electrode was placed at the boundary between the stratum (str.) lacnosum-moleculare and the str. radiatum to stimulate the Schaffer collateral, and fast and slow signal components were detected in the dendritic and somatic regions. By comparing the optical signals with whole-cell recordings, we confirmed that the fast component was due to a population of dendritic spikes in pyramidal neurons. The fast component appeared in dendritic locations near the input sites in response to synaptic activation, and signal onset at the soma was delayed by a few milliseconds compared with that at the input sites. Local perfusion of a Na(+) channel blocker near the soma eliminated the fast component at the soma, but had no effect on the fast component at the input sites. Our results indicate that dendritic spikes can be initiated in dendrites near the input site and propagate orthodromically toward the proximal dendrites and the soma.

Anesthetics, Local↗

PubMed1991

Hypersensitivity of rat glioma sublines with acquired ACNU resistance to L-asparaginase.

Cell lines resistant to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3- (2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) show a high degree of collateral sensitivity to L-asparaginase. The mechanism for this phenomenon was investigated by comparing the nutritional requirements and asparagine synthetase activity of the resistant sublines to those of parent cells. Nine ACNU-resistant sublines were isolated from rat glioma 9L cells after incubation with various concentrations of ACNU in Ham's F-12 medium. The 9L cells grew independently of asparagine, developing well in asparagine-deficient Dulbecco's modified Eagle's medium. In contrast, the growth rates of all nine ACNU-resistant sublines decreased under the same conditions and required the addition of 10(-4) M asparagine for maximum growth. Asparagine synthetase activity in the ACNU-resistant cells was much lower than in the 9L cells, suggesting that the requirement for asparagine in the resistant sublines was due to reduced activity of this enzyme. A growth-inhibition assay showed that the ACNU-resistant sublines were more sensitive to L-asparaginase than 9L cells by up to 2 x 10(5)-fold. These results suggest that L-asparaginase therapy has the potential to become a new approach for treating acquired ACNU resistance.

Animals↗

PubMed1996

Reduced probability of orthodromically evoked action potential firing in CA1 pyramidal cells of guinea pig hippocampal slices after acute thallium exposure.

We investigated the effect of thallium ions on extracellular field potentials in the CA1 region of guinea pig hippocampal slices in a matched-pair experimental setup. Somatic and dendritic responses evoked by paired-pulse stimulation of the Schaffer collateral-commissural pathway were recorded before, during and after acute thallium exposure and compared to field potentials from nontreated control slices recorded simultaneously. Thallium reduced the orthodromically evoked population spike reversibly in a clear concentration-effect relationship. In contrast, the field excitatory postsynaptic potential fEPSP, as well as the presynaptic fiber volley of the afferent pathway, were not affected by thallium. Furthermore, the paired-pulse facilitation was reversibly reduced during thallium exposure. Input-output relations clearly demonstrated that thallium did not interfere with the presynaptic transmitter release mechanisms or the postsynaptic transmitter receptor sensitivity, but had a predominant postsynaptic target site. Additionally, any influence of thallium ions on the somatic and/or axonal membrane excitability could be excluded, as the antidromically evoked responses after alvear stimulation were not diminished by thallium. Therefore, the main effect of thallium was a decoupling of the somatic from the dendritic activity at the CA1 pyramidal cells. We conclude that the toxic influence of thallium ions in the guinea pig hippocampus must be confined to intracellular somatic mechanisms. Interactions with intracellular organelles and an impairment of their calcium storage capacity are supposed.

Action Potentials↗

PubMed2003

The echinocandin antifungals: an overview of the pharmacology, spectrum and clinical efficacy.

For over four decades, the principal target of antifungal therapy has been the fungal cell membrane sterol ergosterol. Although this has proven to be a successful and relatively selective antifungal target, collateral toxicity to mammalian cells (amphotericin B) and drug interactions (azoles) have been by-products of agents that target the fungal cell membrane. In the 1970s, the echinocandins were identified during the screening of fungal fermentation products for new antibiotic agents. These agents were subsequently shown to inhibit production of beta(1,3)-glucan, a key structural component of the fungal cell wall. Subsequent chemical modification of these natural products has led to the development of safer, semi-synthetic beta(1,3)-glucan synthase inhibitors with enhanced microbiological and clinical efficacy against infections caused by Candida and Aspergillus species. In this review, the pharmacology, spectrum and clinical efficacy of the three leading beta(1,3)glucan synthase inhibitors (caspofungin, micafungin and anidulafungin), which have completed phase III clinical trials, will be discussed and a perspective for the role of these agents in the management of life-threatening mycoses will be offered.

Animals↗

PubMed2000

Late gestational changes in sympathomimetic sensitivity in primagravid rabbit ligaments.

The adrenoceptor profile of blood vessels supplying the medial collateral ligament (MCL) of virgin and primagravid (day 29 of pregnancy) rabbit knees was examined. Topical bolus administration of the alpha1-adrenoceptor agonists methoxamine and phenylephrine, and the alpha2-adrenoceptor agonist clonidine, to exposed knee joints resulted in a dose-dependent vasoconstriction of ligamentous vessels. The rank order of potency for the alpha-agonists was found to be phenylephrine > methoxamine > clonidine. Dobutamine, which acts on beta1-adrenoceptors, and ritodrine, which is a beta2-agonist, imparted a mild vasodilatatory effect on MCL blood vessels with the efficacy of ritodrine being greater than that of dobutamine. In primagravid rabbits, the constrictor effects of methoxamine and phenylephrine were significantly attenuated compared with virgin control animals (P < 0.0001), whereas clonidine-mediated vasoconstriction was unaltered in the gravid animal (P = 0.3957). With respect to beta-adrenoceptor activity, the dilatational effect of dobutamine was the same as in controls (P = 0.5294), while ritodrine vasoactivity was completely abolished in primagravid knees (P < 0.005). These findings suggest that the adrenergic control of rabbit MCL blood flow is predominantly mediated by postjunctional alpha1 and beta2-adrenoceptors. Pregnancy leads to either downregulation and (or) desensitisation of alpha1 and beta2-adrenoceptors in the ligament which could disrupt normal joint homeostasis.

Animals↗

PubMed2006

Novel mechanisms of platinum drug resistance identified in cells selected for resistance to JM118 the active metabolite of satraplatin.

PURPOSE: The goal of this study was to identify molecular determinants of sensitivity and resistance to JM118, the active metabolite of satraplatin, an orally bioavailable cisplatin analog that has activity in prostate cancer. EXPERIMENTAL DESIGN: Human ovarian carcinoma 2008/JM118 cells were derived from parental 2008 cells by repeated exposure to JM118; the revertant 2008/JM118/REV subline was isolated from the 2008/JM118 cells by growth in the absence of drug. Drug sensitivity was determined by clonogenic assay and Pt levels were measured by ICP-MS. RESULTS: Eight sequential rounds of selection yielded the 2008/JM118 subline that was 4.9-fold resistant to JM118 and cross-resistant at varying levels to satraplatin, cisplatin, carboplatin, and oxaliplatin. Cross-resistance to the other Pt drugs was lost as resistance to JM118 waned. The same parental 2008 cells selected for resistance to cisplatin were partially cross-resistant to JM118. The 2008/JM118 cells accumulated significantly more Pt than the 2008 cells when exposed to low concentrations of either JM118 or cisplatin indicating a detoxification process that involves intracellular sequestration. In contrast, 2008 cells selected for cisplatin resistance accumulated less cisplatin and less JM118 reflecting a mechanism involving reduced accumulation. The 2008 and 2008/JM118 cells did not differ in their uptake or efflux of 64Cu, expression of Cu efflux transporters ATP7A or ATP7B or their glutathione content. The 2008/JM118 cells exhibited 3.0-7.7-fold hypersensitivity to docetaxel, paclitaxel and doxorubicin. Expression profiling identified 4 genes that were significantly up-regulated and 19 that were down-regulated in the 2008/JM118 cells at a false discovery rate of 1 gene. CONCLUSIONS: While the cellular defense mechanisms that protect cells against JM118 also mediate resistance to the other Pt drugs, these mechanisms are quite different from those commonly found in cells selected for resistance to cisplatin. JM118-resistant cells accumulate more rather than less Pt and rely on an intracellular detoxification mechanism different from that involved in cisplatin resistance. This is consistent with clinical evidence suggesting that satraplatin has activity in diseases in which cisplatin does not. In this model, JM118 resistance is associated with substantial collateral hypersensitivity to docetaxel, paclitaxel, and doxorubicin.

Antineoplastic Agents↗

PubMed2006

Collateral sprouting of human epidermal nerve fibers following intracutaneous axotomy.

Despite the clinical need, there are no therapeutic compounds available to promote peripheral nerve regeneration. In part, this may be due to a lack of sensitive measures of nerve growth. Here, we describe a novel approach of measuring collateral sprouting of epidermal nerve fibers (ENF) in human subjects and describe the effect of the neuroimmunophilin ligand timcodar dimesylate on collateral nerve sprouting. The objective of this study was to describe a model of intracutaneous axotomy and evaluate the ability of timcodar dimesylate to accelerate human cutaneous nerve regeneration through collateral sprouting. Subjects were randomized to receive placebo, 12.5 or 50 mg/day timcodar dimesylate in a prospective, two-center, double-blind, placebo-controlled trial. A 3-mm distal thigh punch skin biopsy was performed at baseline, and a 4-mm overlapping concentric biopsy was taken after 56 days of treatment. Biopsies were processed to visualize ENF, and the collateral sprouting distance (CSD) was measured. Sixty-two subjects completed the trial, and the CSD was measurable in 52. The CSD (mean +/- SEM) was 474.5 microm +/- 38.3, 473.4 microm +/- 28.4, and 450.8 microm +/- 26.5 for the placebo, low and high dose groups, respectively (p = 0.84). The baseline ENF density was associated with the CSD (p = 0.02). Collateral sprouting was efficiently measured using an intracutaneous axotomy model and suggests a collateral sprouting rate of 8.5 microm/day in healthy subjects. The model was consistent across treatment groups and had a low coefficient of variation. Timcodar dimesylate treatment was safe over an 8-week period but did not improve collateral sprouting among healthy subjects.

Adult↗

PubMed2003

Overexpression of mutated MRP4 in cisplatin resistant small cell lung cancer cell line: collateral sensitivity to azidothymidine.

Cisplatin (CDDP) resistance is one of the major impediments in cancer chemotherapy. In an attempt to define this complex mechanism(s) of resistance, we have identified 7 cDNA fragments which are overexpressed in CDDP resistant small cell lung cancer cell line (SR-2) using PCR selected cDNA subtraction. One of these fragments was identical with nucleotide 3657-4042 of MRP4. The other fragments share sequence homology with elongation factor alpha, human placenta villi cDNA, heat shock protein (Hsp70), ribosomal RNA, BNP1 brain specific Na-dependent inorganic phosphate cotransporter and telomeric catalytic subunit. Examination of other MRP members (MRP1, 2, 3, 5, 6) did not show discernable differences in their expression between the parental (SCLC1) and the CDDP-resistant variant (SR-2). Full length MRP4 cDNA was obtained from SCLC1 and SR-2. Both cell lines carry a point mutation at nucleotide 3532 while SR-2 carries two additional mutations at 3228 and 3246. Since MRP4 is known to transport azidiothymidine (AZT) and overexpression of MRP4 confers AZT resistance, we have studied growth inhibitory effects of AZT and [3H]-AZT accumulation. Interestingly, SR-2 is more sensitive to AZT while accumulating lesser amounts of [3H]-AZT. The thymidine kinase activity is similar in both cell lines. Thus, the increased sensitivity to AZT in SR-2 could not be solely due to mutation of MRP4. These findings are most likely due to the inhibitory effects of telomere catalytic subunit by AZT. Thus, certain biochemical changes induced by CDDP can be explored for future treatment to overcome this form of resistance.

Blotting, Northern↗

PubMed1994

cis-Diamminedichloroplatinum(II) resistant human tumor cell lines are collaterally sensitive to PtCl4(Rh-123)2: evidence for mitochondrial involvement.

Three human tumor cell lines made resistant to cis-diamminedichloroplatinum(II) (CDDP), SCC-25/CP, MCF-7/CP, and C13, are more sensitive to rhodamine-123 [tetrachloroplatinum(II)] [(PtCl4(Rh-123)2] than are the corresponding parental cell lines. The CDDP-resistant cells have higher intracellular concentrations of PtCl4(Rh-123)2 for the same exposure than do the parent cells. Each of the CDDP-resistant cell lines has an increased level of cytochrome c oxidase activity compared with the parent cell lines, indicating that the resistant cells have greater mitochondrial mass or activity than the parent cells. In fact, there was a linear correlation between the increase in cytochrome c oxidase activity and the increased sensitivity to PtCl4(Rh-123)2 in the CDDP-resistant lines. Exposure of the cells to each of the mitochondrial effectors, chloramphenicol, FCCP, oligomycin, or antimycin prior to and during exposure to CDDP or PtCl4(Rh-123)2 had variable effects on the cytotoxicity of the platinum complexes in the parental lines. However, there was a consistent decrease in the cytotoxicity of PtCl4(Rh-123)2 in the CDDP-resistant cells in the presence of the mitochondrial effectors such that, in some cases, the CDDP-resistant lines were now less responsive to PtCl4(Rh-123)2 than were the parent cell lines. These studies indicate that mitochondrial alterations may be an important component of CDDP resistance in these cell lines and that PtCl4(Rh-123)2 may represent a prototype platinum complex useful in the treatment of CDDP resistant tumors.

Adenocarcinoma↗

PubMed1986

Cross-resistance of nocodazole-resistant mutants of CHO cells toward other microtubule inhibitors: similar mode of action of benzimidazole carbamate derivatives and NSC 181928 and TN-16.

Stable mutants which are between 1.6- and 2.2-fold resistant to the microtubule inhibitor, nocodazole (NocR mutants) have been isolated in Chinese hamster ovary cells after a single-step selection. The different NocR mutants exhibit specific changes in their cross-resistance or collateral sensitivity toward various microtubule inhibitors (viz. colchicine, podophyllotoxin, taxol, vinblastine, and maytansine), but they show no change in resistance toward unrelated compounds, indicating that the genetic lesions in these mutants are microtubule related. The set of NocR mutants examined and a second-step podophyllotoxin-resistant cell line, PodRII6 (which is highly resistant to nocodazole) were found to exhibit proportionately increased cross-resistance toward various benzimidazole carbamate derivatives (viz. mebendazole, fenbendazole, carbendazim, parbendazole, oxibendazole, albendazole, benomyl, and cambendazole) as well as two additional microtubule inhibitors, NSC 181928 (ethyl 5-amino-1,2-dihydro-3[N-methylanilino)methyl]pyrido [3,4-b]-pyrazin-7-ylcarbamate) and TN-16 [3-(1-anilinoethylidene)-5-benzyl-pyrrolidine-2,4-dione]. These results indicate that the mechanism of action of these latter two inhibitors is very similar to that of nocodazole. The lack of cross-resistance of the NocR and Podrii6 cell lines to thiabendazole provides evidence that the mechanism of action of this compound is different from that of other benzimidazole carbamate derivatives. Based on structure-activity relationship studies between various nocodazole-like compounds, a number of structural features of these compounds which appear important/essential for this type of biological activity have been identified.

Animals↗

PubMed1995

Bimakalim, an ATP-sensitive potassium channel opener, mimics the effects of ischemic preconditioning to reduce infarct size, adenosine release, and neutrophil function in dogs.

BACKGROUND: The primary goal of the present study was to determine whether the infarct size-reducing effect of preconditioning is associated with an increase in adenosine release from the ischemic myocardium during a prolonged occlusion period or the subsequent reperfusion period and by a decrease in neutrophil infiltration. A second objective was to determine whether bimakalim, a KATP channel opener, mimics the effects of ischemic preconditioning. METHODS AND RESULTS: Barbital-anesthetized open-chest dogs were subjected to 60 minutes of left anterior descending coronary artery (LAD) occlusion followed by 3 hours of reperfusion. In the preconditioning group, 5 minutes of LAD occlusion followed by 10 minutes of reperfusion was elicited before the 60-minute occlusion period. In two other groups, bimakalim 1 microgram/kg bolus followed by a 0.05 micrograms.kg-1.min-1 infusion or an equivalent volume of saline was administered intravenously 15 minutes before occlusion and continued until the time of reperfusion. In a final group, bimakalim was administered 10 minutes before reperfusion and continued until the end of the experiment. To measure the release of adenosine from the ischemic region, coronary venous blood samples were collected at various times during ischemia and after reperfusion, and the concentration of adenosine was measured. Myocardial infarct size was determined by triphenyl tetrazolium chloride; transmural myocardial blood flow, by radioactive microspheres. Transmural myeloperoxidase (MPO) activity, an index of neutrophil infiltration in the area at risk, was also measured. Preconditioning produced a marked reduction in infarct size (9.8 +/- 3.0% versus 28.6 +/- 5.2% in the control group, mean +/- SEM); adenosine release at 5, 10, 15, and 30 minutes of the 3-hour reperfusion period; and transmural MPO activity in the risk area. Similarly, pretreatment with bimakalim resulted in reductions in infarct size, adenosine release, and transmural MPO activity to an extent almost identical to that of preconditioning. When bimakalim was administered 10 minutes before reperfusion, the drug also produced a significant reduction in infarct size and transmural MPO activity; however, no significant reduction in coronary venous adenosine concentrations was observed. There were no significant differences in collateral blood flow between groups. CONCLUSIONS: These results indicate that myocardial preconditioning in the canine heart produced by a short period of ischemia or a KATP channel opener is not mediated by an increase in adenosine release, as measured by coronary venous adenosine concentrations, during 60 minutes of occlusion or the initial 30 minutes of reperfusion. A significant reduction in transmural MPO activity in the ischemic area also appears to result from KATP channel activation and may play a role, at least in part, in the reduction in infarct size observed, particularly when a KATP channel opener is administered just before reperfusion.

Adenosine↗

PubMed1993

Effects of halothane, propofol, and thiopental on peripheral airway reactivity.

BACKGROUND: General anesthetics modify airway responsiveness by several mechanisms, including direct effects on airway smooth muscle and reductions in neural reflex activity. Halothane has been shown to reduce responsiveness through both of these mechanisms. The airway effects of barbiturates are controversial, and the effects of propofol are unknown. METHODS: To compare the direct effects of halothane, thiopental, and propofol in vivo, canine peripheral airways were constricted with two stimuli, histamine and hypocapnia, which are thought to directly contract smooth muscle. The authors then investigated the role of ATP-sensitive potassium (KATP) channels as a mechanism for attenuating these responses. Basenji-Greyhound (BG) dogs were anesthetized with either halothane (1.5 MAC), thiopental (7.5 mg.kg-1 x min-1 intravenously) plus fentanyl (25 micrograms intravenously every 20-30 min), or propofol (0.6 mg.kg-1 x min-1 intravenously). A wedged bronchoscope technique was used to measure peripheral airway resistance (RP). After a stable baseline was obtained, dose-response curves to histamine (50, 100, or 200 micrograms intravenous bolus) or hypocapnia (0% CO2 for 2 min with 100, 200, or 400 ml/min collateral flow) were constructed. On separate occasions, the same sublobar segments were pretreated with glibenclamide (2 mg/ml aerosol), a KATP channel blocker, and dose-response curves to hypocapnia were repeated. RESULTS: Dose-response curves to histamine were similar during all three anesthetics. Halothane decreased airway responsiveness to hypocapnia, compared with either thiopental or propofol (P < 0.05). Pretreatment with glibenclamide abolished the effect of halothane on hypocapnia-induced airway constriction. CONCLUSIONS: These results indicate that propofol afforded no benefit over thiopental or halothane in reducing peripheral airway responsiveness. Furthermore, the beneficial effects of halothane in reducing responsiveness to hypocapnia appear to be mediated by the opening of KATP channels.

Airway Resistance↗

PubMed1994

P2Y purinoceptor and nucleotide receptor-induced relaxation of precontracted bovine aortic collateral artery rings: differential sensitivity to suramin and indomethacin.

We have examined a series of adenine nucleotides and UTP for their ability to cause relaxation of precontracted bovine aortic collateral artery rings. The overall rank order of agonist potency for relaxation was 2-methylthioadenosine 5'-triphosphate (2MeSATP) > adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) > UTP > ADP > ATP. These responses were endothelium-dependent. Interaction studies showed that responses to the selective P2Y purinoceptor agonist 2MeSATP, and to ADP, were mediated by different receptors from those mediating responses to UTP. Suramin, a P2 purinoceptor antagonist that binds to diverse sites for ATP, produced a concentration-dependent shift in the agonist concentration-effect curve to 2MeSATP, with a pKB of 5.45 +/- 0.15 and a slope of 0.94 +/- 0.09. Suramin produced a small, nonsignificant shift in the UTP response curve and had little effect on responses to ATP. Indomethacin (2.8 x 10(-6) M) had no effect on concentration-effect curves to UTP but almost abolished the relaxations produced by 2MeSATP and ADP. The concentration-effect curves to ATP and ATP gamma S showed a significant (P < .05) rightward shift in the presence of indomethacin. These results suggest the presence of separate P2Y purinoceptor and nucleotide receptors mediating endothelium-dependent relaxations of bovine aortic collateral artery smooth muscle. ATP acts at both receptors, whereas ADP acts at only one (P2Y). The effects of indomethacin show that these receptors differentially modulate the release of cyclooxygenase-derived mediators of relaxation.

Adenosine Triphosphate↗

PubMed1974

Nucleo-cytoplasmic interaction between oligomycin-resistant mutations in Saccharomyces cerevisiae.

1.A single-gene nuclear mutant of Saccharomyces cerevisiae, isolated as oligomycin-resistant, exhibits in vivo cross-resistance to venturicidin and collateral sensitivity to Synthalin. All three compounds are inhibitors of mitochondrial oxidative phosphorylation. Oligomycin resistance and Synthalin sensitivity are recessive, while venturicidin resistance is dominant. 2. Acytoplasmic mutant, also isolated as oligomycin-resistant, shows collateral sensitivity to both Synthalin and venturicidin. All three traits undergo mitotic segregation in diploids formed by crossing mutant and normal halpoids. 3. A novel nucleocytoplasmic interaction is observed in diploids formed by crossing haploid strains containing the nuclear and the cytoplasmic mutations, respectively. The dominant venturicidin resistance determined by the nuclear gene undergoes mitotic segregation, which results from a suppression of the nuclear phenotype by the cytoplasmic mutation. When a diploid mitotic segregant contains primarily mutant-type mitochondria, venturicidin resistance is completely suppressed. In haploids containing both the nuclear and cytoplasmic mutations, suppression is only partial. 4. Oxidative phosphorylation and ATPase in mitochondrial fractions isolated fromcytoplasmic mutant cells are less sensitive to inhibition by oligomycin than normal, but in vitro sensitivity to venturicidin is not significantly changed. In similar mitochondrial fractions isolated from normal and nuclear mutant cells, no significant differences in sensitivity to either inhibitor are detected. 5. The molecular basis for the nucleocytoplasmic suppression of venturicidin resistance may involve participation of mitochondrial membrane, plasma membrane or both. Either mitochondria can undergo changes in venturicidin sensitivity upon isolation, or the molecular entity which controls access of venturicidin to the mitochondria resides outside of the organelles. 6. Our data establish that aspects of the response in vivo of both venturicidin and Snythalin are controlled by the mitochondrial genome. 7. The nucleocytoplasmic interaction described here is the first example in which a specific restricted mitochondrial mutation modifies the phenotypic expression of a nuclear gene.

Antifungal Agents↗

PubMed1996

2-deoxy-D-glucose toxicity and transport in human multidrug-resistant KB carcinoma cell lines.

It is shown that a series of colchicine-selected multidrug-resistant (MDR) human KB carcinoma cell lines displayed increasing 2-deoxy-D-glucose collateral sensitivity, which correlated with increasing multidrug resistance. The relative resistance of MDR cell lines to 2-deoxy-D-glucose was reduced to 0.73 (KB-8-5), 0.3 (KB-8-5-11) and 0.2 (KB-C1) when compared with parental KB-3-1 (1.0). 2-Deoxy-D-glucose accumulation was found to be reduced in the MDR cell lines in a manner that correlated with 2-deoxy-D-glucose collateral sensitivity. At 30 min 2-deoxy-D-glucose accumulation was reduced to 0.61 (KB-8-5), 0.41 (KB-8-5-11) and 0.22 (KB-C1) relative to KB-3-1 uptake (1.0). The efflux of 2-deoxy-D-glucose was not significantly different between resistant and sensitive cell lines. Analysis of 2-deoxy-D-glucose uptake kinetics, by initial rate measurements, showed alterations in K(t) and J(max) for MDR when compared with KB-3-l cells. The levels of GLUT-1 facilitative transporter were found to be reduced significantly in the MDR cell lines in total cell homogenate and plasma membrane fractions by using Western blot analysis. Changes in the plasma membrane level of GLUT-1 correlated with 2-deoxy-D-glucose toxicity and uptake for MDR cell lines, where relative GLUT-i levels were reduced to 0.71 (KB-8-5), 0.43 (KB-8-5-1 1) and 0.27 (KB-Cl) relative to KB-31(1.0). It is concluded that the response of human KB MDR cells to 2-deoxy-D-glucose involved alterations in the level and activity of the facilitative glucose transporter, GLUT-1, in a manner that is associated with the degree of multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Seizure-induced cell death produced by repeated tetanic stimulation in vitro: possible role of endoplasmic reticulum calcium stores.

Seizures may cause brain damage due to mechanisms initiated by excessive excitatory synaptic transmission. One such mechanism is the activation of death-promoting intracellular cascades by the influx and the perturbed homeostasis of Ca2+. The neuroprotective effects of preventing the entry of Ca2+ from voltage-dependent Ca2+ channels, NMDA receptors, and non-NMDA receptors, is well known. Less clear is the contribution to excitotoxicity of Ca2+ released from endoplasmic reticulum (ER) stores. We produced epileptiform discharges in combined entorhinal cortex/hippocampus slices using repeated tetanic stimulation of the Schaffer collaterals and assessed cell death after 1, 3, or 12-14 h with gel electrophoresis of genomic DNA and immunohistologically using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine 5'-triphosphate (dUTP) nick end labeling (TUNEL) staining. We manipulated ER Ca2+ stores using two conventional drugs, dantrolene, which blocks the Ca2+ release channel, and thapsigargin, which blocks sarco-endoplasmic reticulum Ca2+-ATPases resulting in depletion of ER Ca2+ stores. To monitor epileptogenesis, and to assess effects attributable to dantrolene and thapsigargin on normal synaptic transmission, extracellular potentials were recorded in stratum pyramidale of the CA1 region. Repeated tetanic stimulation reliably produced primary afterdischarge and spontaneous epileptiform discharges, which persisted for 14 h, the longest time recorded. We did not observe indications of cell death attributable to seizures with either method when assessed after 1 or 3 h; however, qualitatively more degraded DNA always was observed in tetanized slices from the 12- to 14-h group compared with time-matched controls. Consistent with these data was a significant, fourfold, increase in the percentage of TUNEL-positive cells in CA3, CA1, and entorhinal cortex in tetanized slices from the 12- to 14-h group (16. 5 +/- 4.4, 33.7 +/- 7.1, 11.6 +/- 2.1, respectively; means +/- SE; n = 7) compared with the appropriate time-matched control (4.1 +/- 2.2, 7.3 +/- 2.0, 2.8 +/- 0.9, respectively; n = 6). Dantrolene (30 microM; n = 5) and thapsigargin (1 microM; n = 4) did not affect significantly normal synaptic transmission, assessed by the amplitude of the population spike after 30 min of exposure. Dantrolene and thapsigargin also were without effect on the induction or the persistence of epileptiform discharges, but both drugs prevented seizure-induced cell death when assessed with gel electrophoresis. We suggest that Ca2+ entering a cell from the outside, in addition to the Ca2+ contributed from ryanodine-sensitive stores (i.e., Ca2+-induced Ca2+ release), may be necessary for seizure-induced cell death.

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