(Circulation. 1998;98:2591-2597.)
© 1998 American Heart Association, Inc.
Basic Science Reports |
Cellular Uptake Mechanisms of 99mTcN-NOET in Cardiomyocytes From Newborn Rats
Calcium Channel Interaction
From the Laboratoire d'Etudes des Radiopharmaceutiques, ESA CNRS 5077, Faculté de médecine, Université Joseph Fourier, Grenoble (L.R., C.G., O.M., J.-P.M., M.C., D.F.), and Cis Bio International, Gif-sur-Yvette (R.P.), France.
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Abstract |
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Background—Bis[N-ethoxy,N-ethyl(dithiocarbamato)]nitrido Tc (V) (TcN-NOET) is a new technetium complex proposed as a tracer of myocardial perfusion. However, its cellular uptake mechanisms are unknown, although membrane localization on rat heart preparations and preferential binding to polymorphonuclear neutrophils (PMNs) have been reported. Because of the central role of calcium in PMN actions, a relationship was hypothesized between this ion flux and TcN-NOET cellular uptake.
Methods and Results—The mechanisms of cellular uptake of TcN-NOET were investigated in newborn rat cardiomyocytes by study of the effect of calcium channel modulators on tracer binding. Nifedipine had no effect on tracer uptake at 1 minute. However, verapamil 0.1 µmol/L and diltiazem 0.5 µmol/L induced a 40% decrease in uptake. Conversely, Bay K 8644 0.25 µmol/L increased TcN-NOET uptake by 73%. Alterations in other membrane ion transports failed to modify tracer uptake, indicating the specificity of the relationship between TcN-NOET uptake and calcium channels. Kinetic studies indicated that cellular net accumulation of the tracer was slow (t1/2=28.5 minutes) and retention was prolonged (84% of initial activity retained after 120 minutes of washout). The energy dependence of TcN-NOET uptake was investigated after 60 minutes of metabolic inhibition by iodoacetic acid plus rotenone. The ATP decrease was not associated with reduction in tracer uptake at 1 minute (114.9±21.9% of control, P=NS).
Conclusions—The decrease in uptake observed with verapamil and diltiazem, the increase with Bay K 8644, and the lack of effect with nifedipine suggest that TcN-NOET binds to L-type calcium channels in the open configuration, without entering cardiomyocytes. The kinetics of TcN-NOET accumulation and retention are slow, and the mechanism for cellular uptake is not energy-dependent. From a clinical point of view, the effect of concurrent treatment by calcium inhibitors on myocardial binding of TcN-NOET should be taken into account.
Key Words: technetium • myocytes • calcium channels
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Introduction |
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The substance TcN-NOET {bis[N-ethoxy,N-ethyl(dithiocarbamato)]nitrido Tc (V)} is a neutral, lipophilic technetium agent of myocardial perfusion presenting high myocardial uptake in numerous animal species.1 In the dog, TcN-NOET is an excellent tracer of coronary flow2 and presents a redistribution phenomenon equivalent to that of 201Tl.3 4 A preliminary clinical study showed no significant differences between TcN-NOET and 201Tl in sensitivity and specificity values in the detection of coronary stenosis >70%.5 Although TcN-NOET is currently in phase II and III clinical trials, its cellular uptake mechanisms need to be examined. Results of previous studies suggest that TcN-NOET binds to cellular membranes,6 7 8 but they do not indicate the fundamental mechanisms of cellular uptake of the tracer.
TcN-NOET, like teboroxime, is lipophilic and neutral, but the 2 tracers present different biological behavior: TcN-NOET does not bind to pig heart,1 whereas teboroxime presents no differences in uptake between species.9 Moreover, teboroxime uptake does not vary from one cell type to another,10 which is not the case for TcN-NOET.11 In fact, the study of TcN-NOET interaction with different types of blood cells in vitro11 has shown that 75% of the radioactivity is bound to polymorphonuclear neutrophils (PMNs), suggesting that there must be mechanisms that cause TcN-NOET to be sequestered by a specialized structure present in PMNs but not in other blood cells.
Calcium plays a central role in PMN functions,12 and calcium antagonists have powerful and varied inhibiting effects on these functions.13 14 These considerations led to a working hypothesis concerning a possible relationship between cellular binding of TcN-NOET and calcium fluxes.
Cell culture models have been used previously to determine the myocardial uptake mechanisms of other radiolabeled perfusion tracers.15 16 17 Thus, cellular uptake of TcN-NOET was studied on newborn rat cardiomyocytes in the presence of modulators of L-type calcium channels and other drugs that inhibit specific membrane ion transports. Cellular accumulation and retention kinetics were also conducted, as well as studies on the influence of the energy state of the cell on cellular uptake of the tracer.
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Methods |
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Cell Culture
Monolayer cultures of cardiomyocytes from newborn rats were prepared according to the method described by Harary and Farley,18 as modified by Blondel et al.19 The cells (106 cells/mL in 35-mm Petri dishes) were maintained in 95% air/5% CO2 for 3 days.
Preparation of TcN-NOET
99mTcN-NOET was synthesized as previously
described4 with a kit donated by Cis Bio
International (France). The solubilizer dimethyl-ß-cyclodextrin was
not used because the concentration of tracer
(10-12 mol/L) did not pose any problem of
solubilization. The radiochemical purity, checked by silica thin-layer
chromatography (TLC) with dichloromethane as the mobile
phase, exceeded 95%. The complex remained stable for 
6 hours.
Solutions
The composition of the cell culture medium was as follows:
Ham's F10 medium, 80% (vol/vol); FCS, 20% (vol/vol);
penicillin-streptomycin 0.1% (vol/vol);
CaCl2 · 2H2O 1
mmol/L; pH 7.4. Thirty minutes before the addition of the tracer, the
culture medium was replaced by the same medium free of FCS. Potassium
cyanide (KCN), tetraethylammonium
chloride (TEA), ouabain, diltiazem, and TcN-NOET were prepared
in a physiological medium containing (mmol/L): NaCl
127, KCl 5.4, KH2PO4 1.1,
Na2HPO4 ·
2H2O 1.1, glucose 6.1, at pH 7.4. Trypsin (1
g · L-1, pH 7.6) was made up in the same
solution. Rotenone, iodoacetic acid (IAA), amiloride, bumetanide,
disopyramide, A 23187, verapamil, and
nifedipine were dissolved in DMSO, the final concentration
of which was always <0.5% (vol/vol). Bay K 8644 had to be dissolved
with this solvent at 1.4% (vol/vol).
To ensure that TcN-NOET was maintained in a free form after incubation in the different media or drugs used in the experiments, controls were performed with TLC to measure the migration rate (Rf) of the tracer.
Uptake and Retention of TcN-NOET
The TcN-NOET solution was prepared to obtain a final
concentration of 10-12 mol/L by addition of 1
µCi (37 kBq) of TcN-NOET (2 mCi/mL and 0.5 Ci/nmol) in 2 mL of
culture medium.
Uptake and retention experiments were initiated by adding the tracer to each dish. Aliquots of the loading buffer were obtained to normalize the cellular uptake values to the extracellular concentration of TcN-NOET.
Tracer uptake was stopped by quickly rinsing the cells 3 times with
ice-cold buffer solution and adding 2 mL per dish of a 1% SDS solution
containing 10 mmol/L sodium borate. The cellular protein content
was determined in these samples according to the method of
Lowry.20 Radioactivity was assayed with a gamma
scintillation detector (Novelec). Knowledge of the elution history of
the generator allowed the determination of the quantity and absolute
concentration of TcN-NOET in the solutions used. Tracer uptake values
were then expressed in femtomoles of cellular TcN-NOET per milligram of
proteins normalized to the extracellular concentration of TcN-NOET
(fmol · mg protein-1 ·
nmol/L-1) (Figure 1
).
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To demonstrate a potential relationship between calcium transport and TcN-NOET uptake, cardiomyocytes were incubated for 15 minutes with L-type calcium channel inhibitors: nifedipine 0.5, 1, and 10 µmol/L, verapamil 0.01, 0.1, 0.5, and 1 µmol/L, and diltiazem 0.1, 0.2, 0.5, 5, and 10 µmol/L, or with an activator of these channels, Bay K 8644, at 0.1, 0.25, 1, and 10 µmol/L. The calcium ionophore A 23187 10 µmol/L, amiloride 100 µmol/L (inhibitor of Na/Ca and Na/H exchange), ouabain 100 µmol/L (inhibitor of Na,K-ATPase), bumetanide 10 µmol/L (inhibitor of Na/K/2Cl cotransport), TEA 100 µmol/L (inhibitor of potassium channels), and disopyramide 100 µmol/L (inhibitor of sodium channels) were also tested. These concentrations were those generally used to demonstrate tracer uptake mechanisms.17 21 22 Uptake of TcN-NOET was measured for 1 minute.
Net accumulation of the tracer was measured after incubation periods of 1, 5, 15, 30, 45, 60, 70, 80, 90, 120, or 180 minutes.
Washout kinetics were studied after preincubation with the tracer for 30 minutes, before the cells were rinsed and an isotope-free solution was again added for periods of 0, 5, 15, 30, 60, or 120 minutes. In some experiments, this solution contained verapamil, diltiazem, or nifedipine at a concentration of 0.5, 10, and 1 µmol/L, respectively. Elution for 120 minutes in the presence of BSA at various concentrations was also performed.
To determine the effect of metabolic inhibition on TcN-NOET uptake, other cells were preincubated for 60, 90, 150, or 180 minutes with 10 µmol/L KCN, 10 µmol/L rotenone, 100 µmol/L IAA, or IAA plus rotenone. Intracellular ATP content and LDH release were measured at each preincubation time. Tracer uptake at 1 minute was measured after 60 minutes of preincubation with the different metabolic inhibitors.
Analytical Measurements
Cellular ATP was extracted as previously
described.21 The sample was assessed for
radioactivity and frozen to allow subsequent ATP measurements by
high-performance liquid chromatography at 254
nm with a column of the inverse-phase type (Waters µBondapak C18,
3.9x300 mm). The eluent was a solution of sodium pyrophosphate
0.01 mol/L 95%, methanol 5%, at pH 6. Standards showed good
reproducibility of measurements. Cell ATP content was expressed in
nmol · (mg protein)-1.
LDH release was measured according to the method of Wroblesky and La Due23 with a standard Boehringer LDH-P kit and an Eppendorff PCP 6121 robot. Total cellular LDH was measured in preparations disrupted in distilled water. LDH release (U/mL) was normalized to milligrams of cell protein and expressed as a percentage of total LDH.
Materials
DMSO, ATP, BSA, and all drugs except KCN were obtained from
Sigma Aldrich Chimie SARL; Ham's F10 medium and penicillin
(10 000 U/mL)–streptomycin (10 mg/mL) from Techgen International; KCN
and TLC silica gel plates from Merck-Clevenot SA; trypsin from DIFCO
Laboratories; and FCS from Gibco BRL.
Statistical Analysis
Results were expressed as mean±SD. Statistical significance was
determined by Student's t test for nonpaired series. The
significance threshold was fixed at P<0.05.
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Results |
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Stability and Interaction of TcN-NOET With the Drugs and Media Used
None of the drugs used, apart from KCN, altered the TcN-NOET chemical entity in vitro. With KCN, the technetium atom is separated from its ligand. KCN was thus removed before the addition of the tracer.
The Rf of TcN-NOET was always 0.9, irrespective of conditions (data not shown), indicating that TcN-NOET remained in a free form. The only binding sites for TcN-NOET were therefore cellular.
Control experiments showed that DMSO 1.4% (vol/vol) had no effect on cellular uptake of TcN-NOET.
Relationship Between Uptake and Retention of TcN-NOET and
Calcium Channels
Uptake kinetics were determined for a tracer concentration of
1.36±0.09x10-12 mol/L (ie,
2.72±0.18x10-15 mol/dish). Net accumulation by
cardiomyocytes reached a steady state after a 90-minute
incubation period, with maximum uptake representing 59% of
the injected dose. The time required to reach half of maximum uptake
(t1/2) was 28.5 minutes.
Nifedipine had no effect on TcN-NOET uptake at any of the
doses tested (Figure 2A). However,
verapamil 0.1 µmol/L and diltiazem 0.5 µmol/L
caused a marked reduction of 30% to 40% in TcN-NOET binding (Figures 2B and 2C). Bay K 8644 induced an increase in tracer uptake of 
40%,
75%, 50%, and 40% at concentrations of 0.1, 0.25, 1, and 10
µmol/L, respectively (Figure 2D). The calcium ionophore A 23187 and
the other ion transport inhibitors had no effect on
TcN-NOET uptake (Figure 3).
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Washout kinetics indicate that, after a 120-minute rinsing period, the TcN-NOET activity bound to cells represented 84% of the initial activity. This retention was not modified by verapamil, diltiazem, or nifedipine (data not shown).
On the other hand, cellular retention of TcN-NOET after 120 minutes of
washout decreased in a dose-dependent manner with BSA concentrations of
0.17, 0.33, 0.50, 0.75, 1, and 1.5 mg/mL: this retention was 96%,
89%, 82%, 64%, 45% and 35%, respectively, of the retention
observed without BSA (Figure 4).
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Uptake of TcN-NOET and Metabolic Inhibitors
To validate the metabolic inhibition model, the ATP
content of cells and LDH release by cardiomyocytes were
determined according to the type and duration of inhibition (Figure 5).
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With a 60-minute incubation period, the various metabolic
inhibitors permitted different intracellular ATP levels to
be obtained without damaging the cells, as evidenced by LDH release.
When intracellular ATP content was intact, uptake at 1 minute was
increased by 136% with KCN (P<0.001) and 38% with
rotenone (P<0.05). When intracellular ATP content was
decreased by 47% (with IAA), 1-minute uptake was increased by 78%
(P<0.001). However, when ATP was totally depleted (with IAA
plus rotenone), uptake remained unchanged
(Table).
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Discussion |
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TcN-NOET is a new tracer of myocardial perfusion. Its macroscopic behavior in dogs and humans has been well characterized,2 5 and its clinical use can now be envisaged. However, its detailed cellular binding mechanisms are unknown. Current data indicate that TcN-NOET binds to cell membranes.6 7 8 The observations made by Moisan et al,11 whereby TcN-NOET binds preferentially to neutrophils in the presence of whole blood, suggest that there is a structure in the PMN membranes that causes the tracer to bind to this cell type. Given the preponderant role of calcium in neutrophil function, newborn rat cardiomyocytes were used to determine whether a modification of calcium fluxes could influence cellular uptake of TcN-NOET.
Uptake and Retention of TcN-NOET Relationship With Calcium
Channels
TcN-NOET net accumulation reached a plateau after a 90-minute
incubation period, with a t1/2 of 28.5 minutes.
This value is similar to that of Tc-MIBI (35
minutes)24 and significantly longer than that of
201Tl (4.95±0.51
minutes)15 and teboroxime (<2
minutes).24 However, this similarity between
TcN-NOET and Tc-MIBI uptake does not mean that their cellular uptake
mechanisms are identical. Intracellular accumulation of Tc-MIBI is made
possible essentially through its positive charge, allowing its
sequestration within mitochondria after diffusion through the membranes
because of its lipophilic nature.16 This
mechanism cannot be envisaged for TcN-NOET, which is a neutral
molecule. The rapid extraction of 201Tl is
because its entry into the cells is specifically via
Na,K-ATPase.15 The fact that the
t1/2 of uptake of teboroxime, which is
distributed nonspecifically in the cell
membranes,17 is lower than that of TcN-NOET may
be due to the greater lipophilicity of teboroxime (partition
coefficient octanol/water, 40 000 [Reference 2525 ] compared with 3100
for TcN-NOET, unpublished data), which could result in faster
distribution between the aqueous culture medium and the cell
membranes.
Verapamil and diltiazem decreased TcN-NOET uptake by 40%. The dihydropyridine nifedipine had no effect, whereas an activator of the same class, Bay K 8644, increased TcN-NOET uptake by 70%. There is thus an important relationship between L-type calcium channels and TcN-NOET uptake. The lack of effect of nifedipine on TcN-NOET uptake and the fact that TcN-NOET is a neutral complex do not argue in favor of cellular entry of TcN-NOET via calcium channels. Conversely, all of the experimental data can be explained by binding of TcN-NOET to the channel protein in its open configuration.
The path of access of verapamil and diltiazem (phenylalkylamine and benzothiazepine family, respectively) to their binding site is the lumen of the channel.26 By contrast, nifedipine, which is neutral and lipophilic, can access its binding site through either the channel opening or the lipid bilayer.27 Furthermore, nifedipine acts allosterically at a distance from its binding site, whereas verapamil interferes presumably directly with calcium ion fluxes.28 The lack of effect of nifedipine on TcN-NOET uptake could therefore reflect only the lack of interaction between their respective binding sites. Finally, Bay K 8644, which increases the probability of opening of the calcium channel,29 would facilitate the presentation of the binding site of TcN-NOET, thus increasing uptake. The lack of effect of A 23187 on TcN-NOET uptake means that TcN-NOET cannot be complexed like calcium by this ionophore.
Other ion transport inhibitors had no effect on TcN-NOET uptake, highlighting the specific nature of the interaction between TcN-NOET and L-type calcium channels.
Cellular clearance of TcN-NOET is very slow, whereas teboroxime is rapidly released from the cells by elution with an aqueous solution (t1/2 of washout, 13 minutes).24 This difference cannot currently be explained but suggests quite different binding and/or retention mechanisms between the 2 tracers.
The addition of verapamil, diltiazem, or nifedipine to the rinsing solution did not accelerate washout of the molecule. This result is not contrary to the hypothesis that TcN-NOET binds to the calcium channel in its open configuration but indicates that the binding sites for TcN-NOET and the 3 calcium inhibitors are different.
Cellular retention of TcN-NOET decreased after 120 minutes of elution with a medium supplemented with BSA, this effect being dose-dependent. These results suggest that addition of BSA led to the introduction of nonspecific binding sites into the elution solution, thereby allowing nonspecific distribution of TcN-NOET between cell membranes and culture medium. Without BSA, the aqueous nature of the culture medium promoted retention of the tracer in the membrane. These results can be compared with those obtained on isolated perfused rat hearts, which show that myocardial retention of TcN-NOET was decreased only when blood elements were added to the perfusion medium.8
The results obtained to date indicate the existence of a relationship between cellular binding of TcN-NOET and calcium channels. On the basis of the maximum effect obtained with verapamil and diltiazem, this relationship would involve 40% of total cellular uptake of TcN-NOET. Binding would take place on the calcium channel in an open configuration at a different binding site from that of nifedipine, verapamil, or diltiazem. For the remaining 60% of cellular uptake, TcN-NOET would be distributed nonspecifically between the cell membranes and the proteins present in the extracellular medium.
Uptake of TcN-NOET: Effect of Metabolic Inhibitors
Although KCN is a known metabolic
inhibitor, it had no effect on ATP at the concentration
used (10 µmol/L). Similar results were observed by
others30 for cyanide concentrations of 1
mmol/L. This drug abolishes oxygen consumption, but the cells
compensate for the ATP deficit of mitochondrial origin through an
increase in glycolysis.30
Rotenone had no effect on the ATP content of cardiomyocytes. First, although this drug blocks complex I of the mitochondrial electron chain, the reduced cofactor FADH2 could be oxidized downstream of this complex, allowing the necessary proton gradient for ATP synthesis to be maintained. Second, cardiomyocytes would be able to compensate for the reduced energy production by increasing anaerobic glycolysis.
IAA progressively depletes cells of ATP by inhibiting anaerobic glycolysis.
Finally, incubation of cardiomyocytes with IAA plus rotenone led to total depletion of ATP as early as the 60th minute of incubation. In this case, IAA prevented any increase in anaerobic glycolysis secondary to the inhibition of the respiratory chain by rotenone, and energy production was no longer possible.
When metabolic inhibition lasted <90 minutes, energy depletion was not associated with significant LDH release. A 60-minute incubation period with the different metabolic inhibitors was therefore chosen for the study.
For a negligible amount of intracellular ATP (IAA plus rotenone), uptake of TcN-NOET remain unchanged, whereas for an intermediate ATP amount (IAA), uptake was increased. These results indicate that a reduction in intracellular ATP was not associated with a reduction in tracer binding. Furthermore, the increase in uptake at 1 minute, which reached 136% and 38% with KCN and rotenone, respectively, took place at an intracellular ATP content that did not differ significantly from that of controls. This increase can therefore only be the result of a particular effect of these drugs. The effect of cyanide could be explained by the alterations it induces on transmembrane calcium movements. Indeed, cyanide induces an increase in intracellular calcium in various models, which could be explained by activation of L-type calcium channels.31 32 The increase observed with IAA and rotenone remains to be elucidated, especially because uptake of TcN-NOET was not changed when these 2 inhibitors were used together.
Clinical Implications
In clinical practice, the blood concentrations obtained during
treatment with calcium inhibitors are within the range of
10-7 mol/L.26 A reduction
in myocardial uptake of TcN-NOET in humans taking calcium channel
blockers can thus be envisaged, which would be detrimental to the
quality of the images obtained and could thus lead to clinical
misinterpretation.
A further finding is that a reduction in intracellular ATP does not lead to a reduction in TcN-NOET uptake. Unlike 201Tl or Tc-MIBI, TcN-NOET is not a tracer of "cellular viability," but this does not mean that this complex cannot be used as a tracer of "myocardial viability" in clinical practice. The cell system is a pure flow model with a relatively high and steady input function that does not correspond to in vivo kinetics. TcN-NOET redistributes in dogs3 4 and humans,5 so that there is likely to be a metabolic component involved with differential uptake and washout. Further experiments are necessary to help understand this phenomenon.
In summary, on newborn rat cardiomyocytes, the kinetics of TcN-NOET uptake were slow and not energy-dependent. The working hypothesis, namely, that there is a relationship between cellular binding of TcN-NOET and calcium movements, would seem to have been confirmed for this cell type. Indeed, the activation or inhibition of L-type calcium channels affects tracer binding. It seems that TcN-NOET does not enter into cardiomyocytes but binds to plasma membranes with a greater affinity for L-type calcium channels in an open configuration than other membrane proteins. This specific binding to L-type calcium channels in our model does not exclude specific binding to other types of calcium channel. Indeed, TcN-NOET binds preferentially to PMNs when incubated with whole blood,11 whereas calcium movements for this cell type do not take place through L-type calcium channels.12
These results must be taken into account if TcN-NOET is used in clinical practice. The action of calcium inhibitor treatments on myocardial uptake of TcN-NOET may have an effect on the quality of the scintigraphic images and thus on the clinical interpretation of these images.
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Footnotes |
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Reprint requests to Daniel Fagret, LER-ESA CNRS 5077, Faculté de Médecine, Université de Grenoble, 38700 La Tronche, France.
Received March 14, 1998; revision received July 26, 1998; accepted July 30, 1998.
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References |
|---|
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Pasqualini R, Duatti A, Bellande E, Comazzi V, Brucato V, Hoffschir D, Fagret D, Comet M. Bis (dithiocarbamato) nitrido technetium-99m radiopharmaceuticals: a class of neutral myocardial imaging agents. J Nucl Med. 1994;35:334–341.
2.
Ghezzi C, Fagret D, Arvieux CC, Mathieu JP, Bontron R,
Pasqualini R, de Leiris J, Comet M. Myocardial kinetics of TcN-NOET: a
neutral lipophilic complex tracer of regional myocardial blood flow.
J Nucl Med. 1995;36:1069–1077.
3. Ghezzi C, Fagret D, Brichon PY, Mathieu JP, Bontron R, Pasqualini R, de Leiris J, Comet M. Redistribution of bis (N-ethoxy, N-ethyl dithiocarbamato) nitrido technetium-99-m-(V), a new myocardial perfusion imaging agent: comparison with 201 thallium redistribution. Circulation. 1996;94(suppl I):I-302. Abstract.
4.
Vanzetto G, Calnon DA, Ruiz M, Xatson DD, Pasqualini
R, Beller GA, Glover DK. Myocardial uptake and redistribution of
99mTc-N-NOET in dogs with either sustained
coronary low flow or transient coronary occlusion:
comparison with 201Tl and myocardial blood flow.
Circulation. 1997;96:2325–2331.
5.
Fagret D, Marie PY, Brunotte F, Giganti M, Le Guludec
D, Bertrand A, Wolf JE, Piffanelli A, Chossat F, Bekhechi D, Pasqualini
R, Machecourt J, Comet M. Myocardial perfusion imaging with
technetium-99m-Tc-NOET: comparison with thallium 201 and
coronary angiography. J Nucl Med. 1995;36:936–943.
6.
Uccelli L, Giganti M, Duatti A, Bolzati C, Pasqualini
R, Cittanti C, Colamussi P, Piffanelli A. Subcellular distribution of
technetium-99m-TcN-NOET in rat myocardium.
J Nucl Med. 1995;36:2075–2079.
7. Johnson GJ, Allton IL, Nguyen KN, Lauinger JM, Beju D, Pasqualini R, Duatti A, Okada RD. Clearance of 99mTcN-NOET in normal, ischemic-reperfused and membrane-disrupted rat myocardium. J Nucl Cardiol. 1996;3:42–54.[Medline] [Order article via Infotrieve]
8.
Johnson G III, Nguyen KN, Pasqualini R, Okada RD.
Interaction of technetium-99m-N-NOET with blood elements:
potential mechanism of myocardial redistribution. J Nucl
Med. 1997;38:138–143.
9. Narra RK, Feld T, Wedeking P, Matyas J, Nunn AD. SQ 30217, a technetium 99m labeled myocardial imaging agent which shows no interspecies differences in uptake. J Nucl Med. 1986;27:1051. Abstract.
10.
Maublant JC, Zhang Z, Rapp M, Ollier M, Michelot J,
Veyre A. In vitro uptake of
technetium-99-m-teboroxime in carcinoma cell lines and
normal cells: comparison with technetium-99m-sestamibi and
thallium 201. J Nucl Med. 1993;34:1949–1952.
11. Moisan A, Pasqualini R, Devillers A, Trichet A, Maros S, Quillien V, Dazord L, Bourguet P. Granulocyte cell labeling with TcN-NOET. In: Radiolabeled Blood Elements: Recent Advances in Techniques and Applications. New York: Plenum Press; 1994;262:33–37.
12. Von Tscharner V, Prod'hom B, Baggiolini M, Reuter H. Ion channels in human neutrophils activated by a rise in free cytosolic calcium concentration. Nature. 1986;324:369–372.[Medline] [Order article via Infotrieve]
13.
Rosales C, Brown EJ. Calcium channel blockers
nifedipine and diltiazem inhibit Ca2+
release from intracellular stores in neutrophils. J Biol
Chem. 1992;267:1443–1448.
14. Khalfi F, Gressier B, Brunet C, Dine T, Luyckx M, Cazin M, Cazin JC. Effects of calcium antagonist diltiazem on leukocyte elastase and on reactive oxygen species production in human neutrophils. Pharmacol Res. 1996;33:117–122.[Medline] [Order article via Infotrieve]
15.
McCall D, Zimmer LJ, Katz AM. Kinetics of thallium
exchange in cultured myocardial cells. Circ Res. 1985;56:370–376.
16.
Piwnica-Worms D, Kronauge JF, Chiu ML. Uptake and
retention of hexakis (2-methoxyisobutyl isonitrile) technetium
(I) in cultured chick myocardial cells: mitochondrial and plasma
membrane potential dependence. Circulation. 1990;82:1826–1838.
17.
Maublant JC, Moins N, Gachon P, Renoux M, Zhang Z,
Veyre A. Uptake of technetium-99m-teboroxime in cultured
myocardial cells: comparison with thallium-201 and
technetium-99m-sestamibi. J Nucl Med. 1993;34:255–259.
18. Harary I, Farley B. In vitro studies on single beating rat heart cells, I: growth and organization. Exp Cell Res. 1963;29:451–465.[Medline] [Order article via Infotrieve]
19. Blondel B, Roijen I, Cheneval JP. Heart cells in culture: a simple method for increasing the proportion of myoblasts. Experientia. 1970;27:356–358.
20.
Lowry OH, Rosebrough NJ, Lewis Farr A, Randall
RJ. Protein measurement with the Folin phenol reagent. J
Biol Chem. 1951;193:265–275.
21.
Piwnica-Worms D, Kronauge JF, Delmon L, Holman BL,
Marsh JD, Jones AG. Effect of metabolic inhibition on
technetium-99m-MIBI kinetics in cultured chick myocardial
cells. J Nucl Med. 1990;31:464–472.
22. Kronauge JF, Chiu ML, Cone JS, Davison A, Holman BL, Jones AG, Piwnica-Worms D. Comparison of neutral and cationic myocardial perfusion agents: characteristics of accumulation in cultured cells. Nucl Med Biol. 1992;19:141–148.
23. Wrobleski F, La Due JS. Lactic dehydrogenase activity in blood. Proc Soc Exp Biol Med. 1955;90:210–213.
24. Maublant JC, Moins N, Gachon P. Uptake and release of two new 99mTc labeled myocardial blood flow imaging agents in cultured cardiac cells. Eur J Nucl Med. 1989;15:180–182.[Medline] [Order article via Infotrieve]
25. Ramalingam K, Jurisson SS, Kuczynski BL, Di Rocco R, Narra RK, Nowotnik DP, Nunn AD. BATO complexes derived from dimethoxy dioximes: synthesis, characterization and biodistribution. Nucl Med Biol. 1995;22:625–634.[Medline] [Order article via Infotrieve]
26. Nayler WG. Calcium Antagonists. San Diego, Calif: Academic Press/Harcourt, Brace, Jovanovich; 1988.
27. Herbette LG, Vant Erve YMH, Rhodes DG. Interaction of 1,4 dihydropyridine calcium channel antagonists with biological membranes: lipid bilayer partitioning could occur before drug binding to receptors. J Mol Cell Cardiol. 1989;21:187–201.[Medline] [Order article via Infotrieve]
28. Catterall A, Striessnig J. Receptor sites for Ca2+ channel antagonists. Trends Pharmacol Sci. 1992;13:256–262.[Medline] [Order article via Infotrieve]
29. Gomez JP, Fares N, Potreau D. Effects of Bay K 8644 on L-type current from newborn rat cardiomyocytes in primary culture. J Mol Cell Cardiol. 1996;28:2217–2219.[Medline] [Order article via Infotrieve]
30.
Webster KA, Discher DJ, Bishopric NH. Regulation of fos
and jun immediate-early genes by redox or metabolic stress
in cardiac myocytes. Circ Res. 1994;74:679–686.
31. Johnson JD, Conroy WG, Isom GE. Alteration of cytosolic calcium levels in PC 12 cells by potassium cyanide. Toxicol Appl Pharmacol. 1987;88:217–224.[Medline] [Order article via Infotrieve]
32. Sato M, Ikeda K, Yoshizaki K, Koyano H. Response of cytosolic calcium to anoxia and cyanide in cultured glomus cells of newborn rabbit carotid body. Brain Res. 1991;551:327–330.TcN-NOET is a new technetium complex proposed as a tracer of myocardial perfusion. The mechanisms of its cellular uptake were studied on newborn rat cardiomyocytes. Verapamil and diltiazem induced a significant decrease (40%) in 1-minute TcN-NOET uptake (P<0.01). Conversely, Bay K 8644 increased uptake by 73% (P<0.001). Other membrane transport inhibitors and metabolic inhibitors did not decrease this uptake. Cellular uptake of TcN-NOET in this model occurred at L-type calcium channels in their open configuration and was not energy-dependent.[Medline] [Order article via Infotrieve]
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