Biomedical subjects

J F Kronauge

Publications and source records attributed to J F Kronauge.

At least 19 recordsLinked to original sources

PubMed1995

Characterization of multidrug resistance P-glycoprotein transport function with an organotechnetium cation.

Multidrug resistance (MDR) in mammalian cells and tumors is associated with overexpression of an approximately 170 kDa integral membrane efflux transporter, the MDR1 P-glycoprotein. Hexakis (2-methoxyisobutyl isonitrile)technetium(I) (Tc-SESTAMIBI), a gamma-emitting lipophilic cationic metallopharmaceutical, has recently been shown to be a P-glycoprotein transport substrate. Exploiting the negligible lipid membrane adsorption properties of this organometallic substrate, we studied the transport kinetics, pharmacology, drug binding, and modulation of P-glycoprotein in cell preparations derived from a variety of species and selection strategies, including SW-1573, V79, Alex, and CHO drug-sensitive cells and in 77A, LZ-8, and Alex/A.5 MDR cells. Rapid cell accumulation (t1/2 approximately 6 min) of the agent to a steady state was observed which was inversely proportional to immunodetectable levels of P-glycoprotein. Many MDR cytotoxic agents inhibited P-glycoprotein-mediated Tc-SESTAMIBI efflux, thereby enhancing organometallic cation accumulation. Median effective concentrations (EC50; microM) were as follows: vinblastine, 13; daunomycin, 55; idarubicin, 65; actinomycin D, 235; colchicine, minimal inhibition; adriamycin, no effect. P-glycoprotein modulators generally demonstrated significantly greater potency (EC50; microM): SDZ PSC 833, 0.08; cyclosporin A, 1.3; verapamil, 4.1; quinidine, 6.4; prazosin, > 300. Modulator-induced enhancement up to 100-fold was observed with Hill coefficients approximately 1, consistent with simple Michaelis-Menten kinetics. Vanadate was an efficacious transport inhibitor, while agents usually not included in the MDR phenotype were without effect. Scatchard analysis showed quinidine to be a noncompetitive inhibitor of P-glycoprotein-mediated Tc-SESTAMIBI transport, indicating allosteric effector sites on P-glycoprotein. The lipid bilayer adsorbing agents tetraphenyl borate and phloretin induced large increases in final Tc-SESTAMIBI accumulation, showing maximal accumulations 2-fold greater than classic MDR modulators and Hill coefficients >> 2. In V79 and 77A cells, modulators of PKC activity altered Tc-SESTAMIBI accumulation, while there was no indication of modulation of P-glycoprotein-mediated Tc-SESTAMIBI transport by hypotonic buffer, extracellular ATP, Cl-, or K+ (membrane potential). While recognized and avidly transported by the P-glycoprotein at buffer concentrations as low as 7 pM, Tc-SESTAMIBI at up to 100 microM only minimally modulated the cytotoxic action of colchicine, doxorubicin, or vinblastine in MDR cells. In conclusion, transport analysis with Tc-SESTAMIBI is a sensitive assay for detecting functional expression of low levels of P-glycoprotein and for the quantitative characterization of transporter modulation and regulation. The biochemical data favor a high Km, high capacity allosterically modulated translocation mechanism for P-glycoprotein-mediated transport of this organometallic cation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Novel hexakis(areneisonitrile)technetium(I) complexes as radioligands targeted to the multidrug resistance P-glycoprotein.

Transport substrates and modulators of the human multidrug resistance (MDR1) P-glycoprotein (Pgp) are generally lipophilic cationic compounds, many with substituted aryl moieties. We sought to synthesize aromatic technetium-isonitrile complexes to enable functional detection in vivo of Pgp expression in tissues. A series of substituted aromatic isonitrile analogs were synthesized from their corresponding amines by reaction with dichlorocarbene under phase transfer-catalyzed conditions, and the non-carrier-added hexakis(areneisonitrile)Tc-99m(I) complexes were produced by reaction with pertechnetate in the presence of sodium dithionite. Cellular accumulation in vitro, whole body biodistribution, and the imaging properties of these lipophilic, monocationic organometallic complexes were determined in Chinese hamster lung fibroblasts expressing MDR Pgp, in normal rats, and in rabbits, respectively. For this initial series, verapamil (50 microM), the classical Pgp modulator, significantly enhanced cellular accumulation or displaced binding of Tc complexes of 1b, 1d, 1h, 2a, 2d, 3a, and 3b, indicative of targeted interactions with Pgp. Most complexes, despite their modestly high lipophilicity, were excluded by the blood/brain barrier, and several complexes displayed simultaneously high hepatobiliary and renal excretion in vivo, consistent with the physiological expression pattern of Pgp in these tissues. Selected Tc- and Re-areneisonitrile complexes of this class have potential applicability to the functional imaging and modulation, respectively, of MDR Pgp in human tissues.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Effect of histamine on haemorrhagic mucosal lesions is related to vascular permeability in rats: studies with histamine, H1-, H2- and H3-agonists and bradykinin.

OBJECTIVE: To test the hypothesis that an early increase in vascular permeability is correlated with later gastric mucosal protection in the rat. METHODS: Histamine, its agonists (H1, H2, H3) and bradykinin, were either given subcutaneously or intragastrically before the intragastric administration of ethanol. The extravasation of intravenously injected 99mTc-glucoheptonate into the gastric wall and into the gastric contents was used as an indicator of increased permeability. Gastric haemorrhagic lesions where measured by computerized planimetry and ethanol absorption was determined by an ACA Clinical Analyzer. RESULTS: Histamine and bradykinin increased vascular permeability in the glandular stomach and provided significant gastroprotection, similar to H1-, H2- and H3-agonists, against ethanol-induced gastric haemorrhagic lesions. This gastroprotection was accompanied by low blood levels of ethanol, probably indicating decreased ethanol absorption and the creation of a histodilutional barrier in the stomach by histamine. CONCLUSIONS: These data indicate that an increase in vascular permeability dissipates the concentration, and may delay the absorption, of ethanol in gastric mucosa by creating a perivascular histodilutional barrier. Vascular injury, which is an early pathogenetic factor in the development of ethanol-induced gastric haemorrhagic erosions, may thus be prevented.

Absorption↗

PubMed1994

Mitochondrial localization and characterization of 99Tc-SESTAMIBI in heart cells by electron probe X-ray microanalysis and 99Tc-NMR spectroscopy.

As the development of targeted intracellular magnetic resonance contrast agents proceeds, techniques for the quantitative analysis of the subcellular compartmentation and characterization of metallopharmaceuticals must also advance. To this end, the subcellular distribution and chemical state of hexakis (2-methoxyisobutyl isonitrile) technetium-99 (99Tc-SESTAMIBI), the ground state of the organotechnetium radiopharmaceutical used for the noninvasive evaluation of myocardial perfusion and viability by scintigraphy, has been determined by a novel application of electron probe X-ray microanalysis (EPXMA) and 99Tc-NMR spectroscopy. In cryopreserved cultured chick heart cells equilibrated in 36 microM 99Tc-SESTAMIBI, EPXMA imaging of mitochondria yielded a respiratory uncoupler-sensitive characteristic 99Tc X-ray peak representing 32.0 +/- 2.9 nmoles Tc/mg dry weight, while EPXMA of cytoplasm or nucleus showed no peak significantly greater than the threshold detectability limit of approximately 1 nmole/mg dry weight. Technetium-99 NMR spectroscopy of heart cells equilibrated with 99Tc-SESTAMIBI showed a single peak at -45.5 ppm with no evidence of significant line broadening or chemical shift compared to aqueous chemical standards, indicating that the majority of the complex exists unbound within the mitochondrial matrix. These data quantitatively demonstrate the localization of this lipophilic cationic organometallic complex within mitochondria in situ, consistent with a sequestration mechanism dependent on membrane potentials. Furthermore, this study establishes the general feasibility of combined EPXMA and NMR spectroscopy for the direct subcellular localization and characterization of metallopharmaceuticals, techniques that are readily applicable to MR contrast agents.

Animals↗

PubMed1994

Radiopharmacological evaluation in dogs of a new renal function agent.

The hydrophilic penta-anionic complex [Technetium (Carboxymethylisocyanide)6]-5, [Tc(CNCH2COO-)6]-5 (Tc-CAMI) was synthesized to evaluate its potential as a renal function imaging agent. The compound contains six distally arranged carboxyl groups that can act as substrates for the organic acid receptor of the renal cell to effect tubular secretion of this agent. Dynamic gamma-camera imaging of 99mTc-CAMI was performed in normal dogs to compare its bio-distribution and pharmacokinetics with those of proven tubular secretion (99mTc-MAG3) and globular filtration (99mTc-DTPA) agents. The relative difference between the observed mean renal transit times (MRTT) of 99mTc-CAMI and 99mTc-MAG3 was 0.15 compared with 1.24 for 99mTc-CAMI and 99mTc-DTPA. Pathological models of obstructive uropathy, renal arterial stenosis and renal denervation were produced in the same animals to demonstrate the diagnostic potential of the agent. These experiments and data showing that probenecid competes with 99mTc-CAMI for renal transport indicate that this compound functions as a tubular secretion agent and may be useful for monitoring renal function in various disease states.

Animals↗

PubMed1994

Expression of recombinant human multidrug resistance P-glycoprotein in insect cells confers decreased accumulation of technetium-99m-sestamibi.

UNLABELLED: The multidrug-resistant P-glycoprotein is a M(r) 170,000 plasma membrane protein encoded by the mammalian multidrug resistance gene (MDR) which appears to function as an efflux transporter of a variety of potent chemotherapeutic agents. METHODS: To directly demonstrate that 99mTc-sestamibi is recognized by the human P-glycoprotein, we overexpressed recombinant human MDR1 P-glycoprotein in host Sf9 insect cells using a baculoviral vector and correlated expression of the gene product with 99mTc-sestamibi accumulation. RESULTS: In parental Sf9 cells and in wild-type baculoviral infected (control) cells, 99mTc-sestamibi accumulation asymptotically approached a plateau of 650 fmoles (mg protein)-1 (nMo)-1 and 337 fmoles (mg protein)-1 (nMo)-1, respectively. In MDR1 baculoviral infected cells, P-glycoprotein expression was maximal at 72 hr postinfection, while 99mTc-sestamibi accumulation was reduced to 12 fmole (mg protein)-1 (nMo)-1. Verapamil (500 microM), the classical MDR modulator, produced an approximately 300% enhancement of 99mTc-sestamibi accumulation in Sf9 cells expressing MDR1 P-glycoprotein, but only a 50% enhancement in parental Sf9 cells, consistent with verapamil-induced inhibition of P-glycoprotein-mediated 99mTc-sestamibi efflux. CONCLUSIONS: These data demonstrate that the recombinant protein is transiently expressed in a functional state capable of drug transport in Sf9 cell membranes and that 99mTc-sestamibi is a transport substrate recognized by the human MDR1 P-glycoprotein. Technetium-99m-sestamibi may prove useful for functionally characterizing P-glycoprotein expression in human tumors in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Functional imaging of multidrug-resistant P-glycoprotein with an organotechnetium complex.

The multidrug-resistant P-glycoprotein (Pgp), a M(r) 170,000 plasma membrane protein encoded by the mammalian multidrug resistance gene (MDR1), appears to function as an energy-dependent efflux pump. Many of the drugs that interact with Pgp are lipophilic and cationic at physiological pH. We tested the hypothesis that the synthetic gamma-emitting organotechnetium complex, hexakis(2-methoxyisobutylisonitrile)technetium(I) ([99mTc]SESTAMIBI), a lipophilic cationic radiopharmaceutical, could be a suitable Pgp transport substrate capable of functional imaging of the MDR phenotype. The cellular pharmacological profile of [99mTc]SESTAMIBI transport was examined in Chinese hamster V79 lung fibroblasts and the 77A and LZ derivative cell lines which express modestly low, intermediate, and very high levels of Pgp, respectively. Steady-state contents of [99mTc]SESTAMIBI in V79, 77A, and LZ cells were 10.0 +/- 0.5 (SEM) (n = 9), 3.6 +/- 0.5 (n = 8), and 0.4 +/- 0.02 (n = 9) fmol.(mg protein)-1 (nMo)-1, respectively, consistent with enhanced extrusion of the imaging agent by Pgp-enriched cells. Maximal doses (> 100 microM) of the multidrug-resistant reversal agents verapamil and cyclosporin A enhanced [99mTc]SESTAMIBI accumulation in V79, 77A, and LZ cells by approximately 10-, 25-, and 200-fold, respectively. The median effective concentration values for tracer accumulation in the presence of verapamil in V79, 77A, and LZ cells were 4, 100, and 200 microM, and those for cyclosporin A were 0.9, 3, and > 25 microM, respectively. Pgp-mediated [99mTc]SESTAMIBI transport occurred against its electrochemical gradient and was found to be ATP dependent displaying an apparent Km of 50 microM. Carrier-added [99Tc]SESTAMIBI was 11- to 13-fold less toxic in multidrug-resistant cells, and inhibited photolabeling of Pgp by [125I]iodoaryl azidoprazosin in a concentration-dependent manner; half-maximal displacement was observed at approximately 100- to 1000-fold molar excess [99Tc]SESTAMIBI. Exploiting the favorable gamma emission properties of 99mTc, functional expression of Pgp was successfully imaged in human tumor xenographs in nude mice with pharmacologically inert tracer quantities of [99mTc]SESTAMIBI. Functional imaging with these organotechnetium complexes may provide a novel mechanism to rapidly characterize Pgp expression in human tumors in vivo, target reversal agents in vivo, and ultimately provide a means to direct patients to specific cancer therapies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Detection of adriamycin-induced cardiotoxicity in cultured heart cells with technetium 99m-SESTAMIBI.

Adriamycin, a broad-spectrum cytotoxic agent useful in cancer chemotherapy, is limited by a dose-dependent cardiomyopathy mediated in part by disruption of mitochondrial energetics. Hexakis(2-methoxyisobutyl isonitrile)technetium(I) (99mTc-SESTAMIBI) is a gamma-emitting radiopharmaceutical with myocellular accumulation properties dependent on mitochondrial membrane potential. To test the hypothesis that 99mTc-SESTAMIBI could monitor Adriamycin-induced alterations in cardiac energetics, cultured chick heart cells were treated with Adriamycin and 99mTc-SESTAMIBI tracer kinetics were determined. Concentration- and time-dependent depression of 99mTc-SESTAMIBI accumulation was evident within 60 min of treatment. The apparent Ki for acute Adriamycin inhibition of tracer accumulation was 82 microM. After 24 h of treatment, Adriamycin concentrations as low as 0.1 microM demonstrated detectable inhibitory effects. The apparent Ki for this subchronic Adriamycin inhibition of 99mTc-SESTAMIBI accumulation was 18 microM. Subchronic concentration-dependent increases in adriamycin-induced myocellular injury as reflected by lactate dehydrogenase (LDH) release correlated inversely with decreases in 99mTc-SESTAMIBI accumulation. These data further support a contribution from altered mitochondrial energetics to Adriamycin-induced injury and establish a pharmacological foundation for pursuing the possibility of noninvasive imaging of chronic Adriamycin cardiotoxicity in cancer patients using 99mTc-SESTAMIBI.

Analysis of Variance↗

PubMed1993

Radiopharmacological studies of 99mTc-CPI: experience with isolated rat atrial tissue.

Extraction, washout and chemical integrity of technetium(I) hexakis (2-carbomethoxy-2-isocyano propane), (99mTc-CPI) were evaluated in isolated contractile rat atrial tissue. 99mTc-CPI accumulated linearly over time at 32 degrees C with uptakes that were both concentration dependent and temperature sensitive. Uptake rates also increased with contractile strength, indicating a dependence of retention on metabolic status of the tissue. Retention indices showed that percent washout of 99mTc-CPI was much slower than control studies with 99mTcO4. HPLC analysis of retained radioactive products in atrial tissue showed most of the activity present as the 99mTc-CPI complex (75%). These observations in isolated rat atrial tissue help explain the interspecies differences in biodistribution of 99mTc-CPI and support the membrane potential dependent model for uptake and retention of technetium isonitrile complexes.

Animals↗

PubMed1993

Tumor uptake of 99mTc-MIBI and 201Tl by a 9L gliosarcoma brain tumor model in rats.

There have been several recent case reports of the accumulation of 99mTc-MIBI [hexakismethoxyisobutylisonitriletechnetium(I), Cardiolite, Sestamibi] in tumors, but no reports of the uptake of this radiopharmaceutical in an animal model. To address this question, the biodistributions of 99mTc-MIBI and 201Tl were compared in Fisher rats bearing 9L gliosarcomas. The results showed that, although the absolute uptake of the tracers by the tumor is relatively low (< 1% ID/g), the tumor-to-normal brain ratios are greater than 6:1 because of low uptake by normal brain. The tumor-to-normal brain ratio of 99mTc-MIBI exceeds that of other currently available 99mTc radiopharmaceuticals suggesting that 99mTc-MIBI may be of particular value in the clinical evaluation of brain tumors and that further investigation of this class of compounds as tumor-avid radiopharmaceuticals is necessary.

Animals↗

PubMed1992

Comparison of neutral and cationic myocardial perfusion agents: characteristics of accumulation in cultured cells.

Uptake and washout kinetics of two new neutral lipophilic technetium-99m-labeled boronic acid adducts of technetium tris(dioxime) (BATO complexes) were studied in monolayers of contractile chick heart cells and compared to the cationic myocardial perfusion agents, 99mTc(CNCH2C(CH3)2OCH3)6+ (Tc-MIBI) and 201Tl+. 99mTcCl(CDOH)2(CDO)(BCH3), where CDO = cyclohexanedione dioxime (CDO-MeB), had a 7-fold greater net accumulation than Tc-MIBI and the most rapid unidirectional washout with a fast initial phase and a slower secondary component. Incubation with cationic membrane transport inhibitors or metabolic inhibitors had little or modest influence, respectively, on uptake of these BATO complexes. Studies with NIH 3T3 fibroblasts indicated that the neutral complexes did not show myocyte specific accumulation.

3T3 Cells↗

PubMed1992

Comparative effects of neutral dipolar compounds and lipophilic anions on technetium 99m-hexakis (2-methoxyisobutyl isonitrile) accumulation in cultured chick ventricular myocytes.

RATIONALE AND OBJECTIVES: Non-flow-dependent myocellular accumulation and uptake kinetics of the myocardial perfusion and viability imaging agent, hexakis (2-methoxyisobutyl isonitrile) technetium 99m(I) (Tc-SESTAMIBI), are thermodynamically driven by large negative sarcolemmal and mitochondrial membrane potentials, and can be enhanced by addition of the lipophilic anion, tetraphenylborate (TPB). To further understand the general properties required of a co-administered compound for increasing the kinetic response of Tc-SESTAMIBI to membrane potential, a systematic appraisal of additional candidate lipid-soluble anions and neutral dipolar compounds was undertaken. METHODS: Each compound was biologically tested for its ability to enhance Tc-SESTAMIBI accumulation in a cultured heart cell model, and electronic dipole moments were evaluated using semi-empirical molecular orbital calculations. RESULTS: Of this series, phloretin (100 microM), TPB (10 microM), and to a lesser degree, 8-anilino-1-naphthalene sulfonate (100 microM) enhanced myocellular accumulation of Tc-SESTAMIBI. Phloretin enhancement was pH-dependent, showing maximal effect at pH 7.4, and was not additive to the augmentation induced by TPB. A series of additional lipid soluble anions and structural analogues of phloretin were without effect. CONCLUSION: Although selected compounds enhanced Tc-SESTAMIBI accumulation, overall, no direct relationship of dipole moment to biologic enhancement was demonstrated.

Anilino Naphthalenesulfonates↗

PubMed1992

Divergent kinetics of 201Tl and 99mTc-SESTAMIBI in cultured chick ventricular myocytes during ATP depletion.

BACKGROUND: Thallous chloride (201Tl) and hexakis(2-methoxyisobutyl isonitrile) technetium (I) (99mTc-SESTAMIBI) are myocardial perfusion imaging agents with biological properties that also reflect tissue viability. Initial myocellular uptake rates of 201Tl reflect activity of Na,K-ATPase, whereas those of 99mTc-SESTAMIBI reflect mean plasma membrane potential. METHODS AND RESULTS: To better understand the mechanistic responses of these tracers to myocellular injury, cultured chick embryo cardiac myocytes were metabolically inhibited in iodoacetate (1 mM) and rotenone (10 microM) for up to 2 hours, and initial uptake rates of each agent were determined at successive intervals along with correlative cellular contents of ATP, sodium, and potassium and lactate dehydrogenase release. ATP content fell from 30.5 +/- 1.4 to 2.7 +/- 0.9 nmol.(mg protein)-1 within 2 minutes, whereas sodium and potassium contents ran down their thermodynamic gradients more slowly (t 1/2 approximately 60 minutes). Modestly severe cell injury was produced at 2 hours as estimated by lactate dehydrogenase release (18% of total). Initial uptake rates of 201Tl declined from 6.9 +/- 0.8 to 4.0 +/- 0.4 fmol.(mg protein)-1.(nMo)-1.(min)-1 by 20 minutes and remained depressed and ouabain (100 microM)-insensitive at 30 +/- 13% of control. Conversely, initial uptake rates of 99mTc-SESTAMIBI increased from 10.6 +/- 0.8 to 15.0 +/- 0.6 fmol.(mg protein)-1.(nMo)-1.(min)-1 within 10 minutes, remained elevated for 40-60 minutes, and later declined to low values. Injury-induced enhancement of initial uptake rates of 99mTc-SESTAMIBI were insensitive to ouabain (100 microM), carbonyl cyanide-m-chlorophenyl hydrazone (5 microM), and valinomycin (1 microgram/ml) but were significantly inhibited by 130 mM Ko buffer, Ba2+ (1 mM), glybenclamide (100 microM), and quinacrine (10 microM). CONCLUSIONS: Uptake rates of 201Tl monotonically decline, correlating with Na-K pump inhibition from ATP depletion. Conversely, uptake rates of 99mTc-SESTAMIBI at first increase above control for 40-60 minutes, indicating a mean plasma membrane hyperpolarization possibly resulting from opening of ATP-sensitive and arachidonic acid-activated potassium channels, before declining to low values with more severe cell injury. Correlative non-flow-dependent relations between 201Tl and 99mTc-SESTAMIBI contain information regarding the degree of myocellular injury.

Adenosine Triphosphate↗

PubMed1992

Human pharmacokinetics and radiopharmacological studies of the myocardial perfusion agent: technetium (2-carbomethoxy-2-isocyano-propane)6+.

The myocardial perfusion agent technetium (2-carbomethoxy-2-isocyano-propane)6+ (99mTc-CPI) is unique from other cationic technetium isonitrile complexes in that it exhibits moderate washout from the heart and rapid hepatobiliary clearance in animal models and human volunteers. Dynamic imaging and HPLC analysis were performed in humans and guinea pigs to outline the pharmacological basis of its pharmacokinetics. Enzymatic hydrolysis of the terminal ester groups in blood was found to occur at a moderate rate producing new species that have been shown not to accumulate in heart tissue. However, after extraction by the heart, liver or kidneys, the 99mTc-CPI complex undergoes metabolism at a much slower rate than observed in the blood. Differences in hydrolysis rate and products obtained indicate separate mechanisms of hydrolysis occurring in blood and other organs. It is proposed that the heart washout occurring after hydrolysis produces a neutral compound which is no longer retained by the negative cytosolic and mitochondrial membrane potentials in myocardial tissue.

Adult↗

PubMed1992

Interspecies variation in biodistribution of technetium (2-carbomethoxy-2-isocyanopropane)6+.

The cationic complex technetium (2-carbomethoxy-2-isocyanopropane)6+ (99mTc-CPI) contains terminal ester groups that were included to provide a pathway for in-vivo metabolism of this compound, thereby enhancing its performance as a myocardial perfusion agent. Biodistribution studies of the compound demonstrated myocardial accumulation in rabbit, guinea pig, and chick, but not in rat and mouse. Radiochemical analysis by HPLC after in-vitro incubation of 99mTc-CPI in blood plasma from the various species confirmed enzymatic hydrolysis to numerous new compounds. Rat and mouse serum produced complete hydrolysis of this agent after incubation for less than 5 sec at 25 degrees C or rates greater than 500 times those observed in human, rabbit, guinea pig and chick serum. Chemical synthesis and isolation of the monohydrolyzed species with subsequent biodistribution studies in guinea pig and rabbit confirmed that this neutral lipophilic complex did not accumulate in heart tissue. It is concluded that varying rates of enzymatic in-vivo hydrolysis produce the interspecies biodistribution differences and may account for the moderate myocardial clearance relative to other isonitrile complexes.

Animals↗

PubMed1992

Subcellular distribution and analysis of technetium-99m-MIBI in isolated perfused rat hearts.

To address the apparent discrepancy between cultured cells and whole heart preparations, Langendorff-perfused rat hearts loaded with hexakis (2-methoxyisobutyl isonitrile) technetium (I) (99mTc-MIBI) were fractionated by a standard differential centrifugation method and fractional contents of 99mTc-MIBI were correlated with the mitochondrial marker, malate dehydrogenase (MDH), and mitochondrial substrates. The "cytosolic" fraction nominally contained 89% +/- 3% of total 99mTc-MIBI, but also contained 91% +/- 1% of total MDH activity by this method. Chromatographic analysis of activity in the "cytosolic" fraction demonstrated greater than 95% of the agent was present as the original free cationic complex; binding to a small molecular weight cytosolic protein was not involved in localization. Addition of the mitochondrial uncoupler CCCP (5 microM) to both "mitochondrial" and "cell fragment" pellets released up to 84% +/- 8% of 99mTc-MIBI content and addition of the mitochondrial substrate succinate (10 microM) in the presence of rotenone (1 microM) enhanced 99mTc-MIBI content by up to 139% +/- 52% over the control. These correlative data from rat hearts indicate that approximately 90% of 99mTc-MIBI activity in vivo is associated with mitochondria in an energy-dependent manner as a free cationic complex, but migrates during fractionation/centrifugation.

Animals↗

PubMed1991

Enhancement by tetraphenylborate of technetium-99m-MIBI uptake kinetics and accumulation in cultured chick myocardial cells.

Myocellular uptake and retention of technetium-99m-hexakis (2-methoxyisobutylisonitrile) (Tc-MIBI), a lipophilic cationic myocardial perfusion and viability imaging agent, is dependent on both mitochondrial and plasma-membrane potentials. To test for enhancement of uptake kinetics by lipophilic anions, cultured chick heart cells were exposed to tetraphenylborate (TPB), which produced a concentration-dependent maximal 15-fold increase in Tc-MIBI uptake kinetics (at 3 x 10(-5) M) and enhanced peak accumulation of Tc-MIBI from 165.4 +/- 26.3 to 705.6 +/- 61.3 fmoles/mg protein.nMo (P less than 0.001). Carbonyl cyanide-m-chloro phenylhydrazone (CCCP; 10(-5) M), a mitochondrial uncoupler, rapidly depleted cellular content of Tc-MIBI in the presence of TPB (10(-5) M) from 300.0 +/- 30.0 to 42.5 +/- 1.9 fmole/mg protein.nMo (p less than 0.001). TPB enhanced both uptake rates and net accumulation of Tc-MIBI at all buffer Ko concentrations between 130 mM and 0.54 mM. Tc-MIBI influx rates allowed estimation of plasma-membrane potential as a function of Ko in the presence of valinomycin with a slope of -67 mV/decade (r = -0.99). The results further support a potential-dependent mechanism for cell uptake of Tc-MIBI and suggest a rational approach for increasing tissue extraction fraction in vivo.

Animals↗

PubMed1990

Uptake and retention of hexakis (2-methoxyisobutyl isonitrile) technetium(I) in cultured chick myocardial cells. Mitochondrial and plasma membrane potential dependence.

The fundamental myocellular uptake and retention mechanisms of hexakis (2-methoxyisobutyl isonitrile) technetium(I) (Tc-MIBI), a technetium-99m-based myocardial perfusion imaging agent, are unresolved. Because of the lipophilic cationic nature of Tc-MIBI, it may be distributed across biological membranes in response to transmembrane potential. To test this hypothesis, net uptake and retention of Tc-MIBI in cultured chick embryo ventricular myocytes were determined under conditions known to alter mitochondrial and plasma membrane potentials. Isovolumic depolarization of plasma membrane potentials in 130 mM extracellular K (Ko) 20 mM extracellular Cl buffer reduced net accumulation of Tc-MIBI from 171 +/- 16 (control) to 29 +/- 3.3 fmol intracellular Tc-MIBI/mg protein.nM extracellular Tc-MIBI. Unidirectional influx of Tc-MIBI in cells depolarized in 30 mM Ko buffer was also reduced; a resting plasma membrane potential of -87 +/- 6 mV was calculated from the Goldman flux equation using normal Ko/high Ko Tc-MIBI influx ratios. Addition of the potassium ionophore valinomycin to cells incubated in 130 mM Ko buffer to additionally depolarize mitochondrial membrane potentials further reduced net uptake of Tc-MIBI to levels comparable to that found in nonviable freeze-thawed preparations ([Tc-MIBI]i/[Tc-MIBI]o = 1). By depolarizing mitochondrial (and in part plasma membrane) potentials with the protonophores 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone (CCCP) Tc-MIBI was rapidly depleted from 181 +/- 16 (control) to 16 +/- 2.6 and 31 +/- 4.2 fmol/mg protein.nMo, respectively, with kinetics that did not correlate with loss of cellular ATP content. CCCP alone inhibited 90 +/- 3% of net accumulation or 66 +/- 3% of unidirectional influx of Tc-MIBI in a concentration-dependent manner. By hyperpolarizing mitochondrial membrane potentials with the K+/H+ ionophore nigericin or the ATP synthase inhibitor oligomycin, net uptake and retention of Tc-MIBI were increased by 60 +/- 9% and 375 +/- 20%, respectively. Caffeine, as well as the respiratory chain electron transport inhibitor rotenone, did not significantly alter net cell uptake (p greater than 0.2). These data indicate that the fundamental myocellular uptake mechanism of Tc-MIBI involves passive distribution across plasma and mitochondrial membranes and that at equilibrium Tc-MIBI is sequestered within mitochondria by the large negative transmembrane potentials.

Animals↗