![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 291, Issue 2, 456-463, November 1999
Department of Pharmacology, Cornell University, Weill Medical College, New York, New York
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
In myocardial ischemia, adrenergic terminals undergo ATP depletion,
hypoxia, and intracellular pH reduction, causing the accumulation of
axoplasmic norepinephrine (NE) and intracellular Na+ [via
the Na+-H+ exchanger (NHE)]. This forces the
reversal of the Na+- and Cl
-dependent NE
transporter (NET), triggering massive carrier-mediated NE release and,
thus, arrhythmias. We have now developed a cellular model of
carrier-mediated NE release using an LLC-PK1 cell line stably transfected with human NET cDNA (LLC-NET). LLC-NET cells transported [3H]NE and
[3H]N-methyl-4-phenylpyridinium
([3H]MPP+) in an inward direction. This
uptake was abolished by the NET inhibitors desipramine (100 nM) and
mazindol (300 nM) and by extracellular Na+ removal.
Na+-gradient reversal induced an efflux of
3H-substrate from preloaded LLC-NET cells. Desipramine and
mazindol blocked this efflux. Because of its greater intracellular
stability and higher sensitivity to Na+-gradient reversal,
[3H]MPP+ proved preferable to
[3H]NE as an NET substrate; therefore, only
[3H]MPP+ was used for subsequent studies. The
K+/H+ ionophore nigericin (10 µM) evoked a
large efflux of [3H]MPP+. This efflux was
potentiated by the Na+,K+-ATPase inhibitor
ouabain (100 µM), was sensitive to desipramine, and was blocked by
the NHE inhibitor
5-(N-ethyl-N-isopropyl)-amiloride (EIPA;
10 µM). In contrast, EIPA failed to inhibit the
[3H]MPP+ efflux elicited by the
Na+ ionophore gramicidin (10 µM). Furthermore,
[3H]MPP+ efflux induced by the NHE-stimulant
proprionate (25 mM) was negatively modulated by imidazoline receptor
activation. Our findings suggest that LLC-NET cells are a sensitive
model for studying transductional processes of carrier-mediated NE
release associated with myocardial ischemia.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Any
condition, physiological or pathophysiological, that causes a
Na+ increase in adrenergic neurons can trigger
the reversal of the norepinephrine (NE) transporter (NET; Paton, 1973
).
Accumulation of intracellular Na+ increases the
availability of the NET to the inside of the axonal membrane and
enhances the affinity of axoplasmic NE for the carrier (Sammet and
Graefe, 1979). In protracted myocardial ischemia, ATP depletion,
hypoxia, and proton accumulation impede NE storage and stimulates
Na+-H+ exchanger (NHE),
which exchanges intracellular H+ for
extracellular Na+ (Nao).
Thus, in myocardial ischemia, sympathetic nerve endings contain an
increased level of both Na+ and free axoplasmic
NE, triggering a reversal of the neuronal uptake system and, hence, a
massive release of NE (Schömig et al., 1991; Kubler and Strasser,
1994; Imamura et al., 1996; Kurz et al., 1996; Hatta et al., 1997).
Previous work from our laboratory demonstrated a direct correlation
between the magnitude of NE overflow from guinea pig hearts subjected
to global ischemia and the severity of reperfusion arrhythmias. A
variety of agents that inhibit or potentiate NE overflow were shown to
significantly reduce and prolong, respectively, the duration of
ventricular fibrillation (Imamura et al., 1996; Hatta et al., 1999).
Although isolated organ and tissue preparations allow the investigation
of many regulatory processes of carrier-mediated NE release, the
complexity of tissue responses to ischemia and reperfusion may mask
important transductional processes involved in the outward transport of
NE.
Accordingly, the purpose of the present study was to develop a simpler
cellular model of carrier-mediated transport. Cloning of the NET
(Pacholczyk et al., 1991) has permitted the stable expression of NET
cDNA in cell systems that are unequipped to store NE, allowing the
investigation of the plasma membrane transporter without interference
from vesicular storage. LLC-PK1 cells are a
rapidly growing, pig kidney-derived epithelial cell line (Haggerty et
al., 1985). In addition to being devoid of the machinery required for
the uptake and vesicular storage of NE, LLC-PK1
cells express two plasma membrane proteins that have pivotal roles in
myocardial ischemia-induced carrier-mediated NE release: the
Na+,K+-ATPase and an
amiloride-sensitive NHE (Chakraborty et al., 1994). Our findings with
an LLC-PK1 cell line stably transfected with human NET (hNET) cDNA demonstrate the potential of recombinant cell lines to study mechanisms and regulatory processes of
carrier-mediated NE release, as seen in pathophysiological conditions,
such as advanced myocardial ischemia.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Cell Culture.
LLC-PK1 cells stably transfected
with cDNA for the hNET (LLC-NET cells, established by Dr. G. Rudnick)
were maintained in 
-modified Eagle's medium supplemented with 10%
FBS, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml
streptomycin, and 450 µg/ml geneticin at 37°C with 5%
CO2. Parent LLC-PK1 cells were maintained in
the same medium without geneticin. When confluent, cells were passaged 1 to 10 by trypsinization.
Influx Studies. Cells were grown in 24-well plates at 37°C until confluent (this was usually 48 h after plating). After rinsing once with HEPES buffer (containing 25 mM HEPES, 125 mM NaCl, 2.6 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 4.8 mM KCl, and 5.6 mM glucose, pH 7.4), cells were incubated at 37°C for 30 min in 0.405 ml of HEPES buffer containing various drugs. Influx of [3H]NE or [3H]N-methyl-4-phenylpyridinium (MPP+) was initiated by the addition of 45 µl of a 10× solution (to give a final concentration of 40 nM [3H]NE and 20 nM [3H]MPP+) of the 3H-substrate. After 5 min of influx, buffer was removed, and cells were rinsed rapidly with ice-cold HEPES buffer. Cells were then lysed with 0.45 ml of 0.3% Triton X-100 for 30 min. A 0.3-ml aliquot of cell lysate was then counted in 4 ml of Bio-Safe II scintillation cocktail in a scintillation counter (Beckman LS 6000) for 3 min.
Efflux Studies.
LLC-NET cells were grown as described for
the influx studies. After rinsing once with HEPES buffer, cells were
incubated at 37°C for 60 min in 0.23 ml of HEPES buffer containing 40 nM [3H]NE or 20 nM [3H]MPP+. In
some experiments, the catechol-O-methyl transferase
(COMT) inhibitor tropolone (1 mM) was used during the loading period and throughout the rest of the experiment. At the end of the incubation period, cells were washed twice with prewarmed oxygenated HEPES buffer
and then incubated for a further 30 min in HEPES buffer at 37°C.
Efflux was initiated by replacing the incubation buffer with 0.45 ml of
release buffer for a given time. Release buffer consisted of HEPES
buffer with an altered NaCl concentration, ionophore (nigericin or
gramicidin, 10µM), or the Na+ salt of proprionate (25 mM;
Schlatter et al., 1997) in the absence or presence of drugs. LiCl was
used as a substitute for NaCl to maintain osmolarity. A 0.3-ml aliquot
of buffer containing released 3H-substrate was removed from
each well and transferred to a scintillation vial containing 4 ml of
scintillation cocktail. The remainder of the release buffer was
immediately removed from each well, and the amount of substrate
remaining was determined by lysing the cells with 0.45 ml of 0.3%
Triton X-100 for 30 min. A 0.3-ml aliquot of cell lysate was
transferred to a scintillation vial containing 4 ml of scintillation
cocktail. The release buffer and cell lysate were counted in a
scintillation counter (Beckman LS 6000) for 3 min. The amount of
3H released from LLC-NET cells was calculated as a
percentage of the total content of 3H.
Statistical Analysis. Values are expressed as mean ± S.E. Student's t test was performed for experiments with two groups. Comparison of more than two groups was performed by one-way ANOVA, with the Bonferroni t test used for post hoc analysis. A value of P < .05 was considered statistically significant.
Drugs. [3H]MPP+ (82.0 Ci/mmol) was purchased from NEN Life Science Products (Boston, MA). [3H]NE (32.0 Ci/mmol) was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). Desipramine hydrochloride (DMI), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and idazoxan were purchased from Research Biochemicals Inc. (Natick, MA). Gramicidin, mazindol, nigericin, ouabain, pargyline, reserpine, and tropolone were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Rilmenidine hemifuramate was purchased from Tocris-Cookson (Ballwin, MO). EIPA, mazindol, and rilmenidine were dissolved in 99.8% dimethyl sulfoxide. Gramicidin and nigericin were dissolved in 95% ethanol. Further dilutions of these drugs were made with HEPES buffer. At the concentration used (i.e., 0.1%), neither dimethyl sulfoxide nor ethanol had any effect on influx or efflux studies. All other drugs were dissolved in distilled water.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Uptake of [3H]NE and
[3H]MPP+ by LLC-NET Cells.
LLC-NET cells
transported both [3H]NE and
[3H]MPP+ in an inward direction under normal
physiological conditions. As shown in Fig.
1, uptake of both substrates was
abolished by preincubation of LLC-NET cells with the neuronal uptake
inhibitors DMI (100 nM) and mazindol (300 nM) or when experiments were
performed in a modified HEPES buffer in which Na+ was
replaced with Li+. Reserpine (100 nM), an inhibitor of the
vesicular monoamine transporter, had no inhibitory action on either
[3H]NE or [3H]MPP+ influx. The
amiloride derivative EIPA had no effect on uptake at 10 µM (data not
shown). Inhibitors of the NET also abolished the prolonged (60 min)
influx of [3H]NE and [3H]MPP+.
Parent LLC-PK1 cells were unable to accumulate
[3H]NE or [3H]MPP+.
|
Outward Transport of [3H]NE and
[3H]MPP+ from LLC-NET Cells.
Incubation
of preloaded LLC-NET cells in a modified HEPES buffer containing 5 mM
Na+ resulted in a significant time-dependent efflux of
[3H]NE (Fig. 2A) and
[3H]MPP+ (Fig. 2B). Spontaneous efflux of
[3H]NE from preloaded LLC-NET was very high (~22% in
20 min). In contrast, only a very small amount of
[3H]MPP+ efflux occurred in control
conditions and reached a maximum value of ~6% within 10 min (Fig.
2B). The total amount of 3H-substrate released from LLC-NET
cells, induced by Na+ gradient reversal, differed greatly
between [3H]NE and [3H]MPP+.
After a 20-min incubation with 5 mM Na+, ~27 and 68% of
the total cellular content of [3H]NE and
[3H]MPP+ were released into the extracellular
buffer, respectively (Fig. 2). The neuronal uptake inhibitors DMI (100 nM) and mazindol (300 nM) significantly blocked the efflux of
[3H]MPP+ (Figs. 2B and
3B) but not that of [3H]NE
(Fig. 3A) overflow. An IC50 value of ~55 nM was
calculated for the inhibitory action of DMI on the low
Na+-induced [3H]MPP+ efflux.
|
|
|
|
|
Nigericin-Induced [3H]MPP+ Efflux from
LLC-NET Cells.
Nigericin (10 µM), a
K+/H+ ionophore, induced a time-dependent
efflux of [3H]MPP+ from preloaded LLC-NET
cells (Fig. 7). The
Na+,K+-ATPase inhibitor ouabain (100 µM),
while having no effect of its own, significantly potentiated the
nigericin-induced efflux of [3H]MPP+ from
LLC-NET cells incubated in a modified HEPES buffer (40 mM Na+; Fig. 7). In both control and ouabain-treated
conditions, efflux of [3H]MPP+ from preloaded
LLC-NET cells during a 20-min period was ~20% of the total cellular
content of [3H]MPP+. This value increased to
~40 and 50% in the presence of nigericin and nigericin plus ouabain,
respectively (Fig. 7). In experiments performed in a physiological
HEPES buffer (125 mM Na+), prolonged inhibition of the
Na+,K+-ATPase with ouabain induced a
significant efflux of [3H]MPP+ (from
~8.0 ± 0.21 to ~11.7 ± 0.18% in a 30-min period).
|
|
|
|
Proprionate-Induced [3H]MPP+ Efflux from LLC-NET Cells: Modulation by Imidazoline Receptors. As an alternative to nigericin, we used proprionate to stimulate carrier-mediated release of [3H]MPP+ from LLC-NET cells. As illustrated in Fig. 10C, proprionate (25 mM) induced an efflux of [3H]MPP+ from LLC-NET cells that was markedly inhibited by the imidazoline receptor agonist rilmenidine (10 µM). The inhibitory effect of rilmenidine was antagonized by the imidazoline receptor blocker idazoxan (10 µM). Rilmenidine had no effect on the nigericin- or gramicidin-induced [3H]MPP+ efflux.
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Our findings demonstrate that LLC-PK1 cells
stably transfected with hNET cDNA (LLC-NET cells) inherit the ability
to transport [3H]NE and
[3H]MPP+ in an inward and
outward direction. Inward transport of both substrates was exclusively
NET mediated, as demonstrated by its dependence on extracellular
Na+ (Iversen and Kravitz, 1966), its complete
blockade by the NET inhibitors DMI and mazindol (Gu et al., 1994), and
its insensitivity to reserpine and EIPA, inhibitors of the vesicular
monoamine transporter (Scherman and Henry, 1984) and NHE (Kleyman and
Cragoe, 1988), respectively. Furthermore, analogous to carrier-mediated
NE release in advanced myocardial ischemia, which is known to be NHE
dependent (Imamura et al., 1996; Kurz et al., 1996; Schömig et
al., 1991), the efflux of
[3H]MPP+ was stimulated
by the K+/H+ ionophore
nigericin, an action that was blocked by the NHE inhibitor EIPA. These
LLC-PK1 cells, therefore, represent a useful
model for future studies of transductional processes involved in
carrier-mediated NE release in protracted myocardial ischemia.
Efflux of both [3H]NE and [3H]MPP+ from LLC-NET cells was stimulated by a reversal of the Na+ gradient. However, there were differences in the stability (normal HEPES buffer) and specific carrier-mediated transport (low Na+ buffer) of the two substrates from LLC-NET cells. The spontaneous efflux of 3H-substrate from preloaded LLC-NET cells was significantly larger for [3H]NE than for [3H]MPP+. In contrast, the maximal amount of low Na+-induced efflux of 3H-substrate was much greater for [3H]MPP+ than for [3H]NE, suggesting that [3H]MPP+ is the more suitable substrate for studying the carrier-mediated release process.
To elucidate the large spontaneous loss of 3H
radiolabel in the [3H]NE release experiments,
we assessed [3H]NE efflux in the presence of
the COMT inhibitor tropolone. We found that metabolism of
[3H]NE by COMT is a major pathway for the loss
of 3H radiolabel from LLC-NET cells. In a recent
study (Eshleman et al., 1997), it was found that COMT inhibitors
greatly elevate the apparent uptake of
[3H]dopamine by cell lines expressing
recombinant catecholamine transporters. The effect of COMT inhibition
on [3H]NE uptake, although significant, was
considerably less pronounced. In our study, LLC-NET cells were exposed
to [3H]NE for a much longer time period,
dramatically increasing the potential for metabolism by COMT. The
EC50 value for the action of tropolone in our
experiments (~39 µM) is similar to the IC50 value of COMT inhibition by tropolone (~22 µM; Guldberg and
Marsden, 1975). The use of tropolone enabled us to uncover a
significant inhibitory response to DMI. The instability of
3H-catecholamines, and thus the spontaneous loss
of radiolabel from preloaded cells expressing recombinant catecholamine
transporters, has been demonstrated elsewhere (Wall et al., 1995). Loss
of radiolabel from
[3H]MPP+-preloaded
LLC-NET cells was almost negligible, consistent with transport of
MPP+ being critically dependent on a carrier,
such as the NET (Buck and Amara, 1994; Pifl et al., 1996). By using a
stable substrate of the NET (i.e., one that is resistant to metabolism
by COMT), we significantly enhanced the sensitivity of our model and
therefore the potential to study regulatory mechanisms of
carrier-mediated release.
Notably, even with COMT inhibition, the amount of
[3H]NE released by reversal of the
Na+ gradient was less than that of
[3H]MPP+. This may
reflect differences in affinity of NE and MPP+
for the transporter. Indeed, in a study of the cloned NET, it was found
that the transporter had a higher apparent affinity (Km) for dopamine and
MPP+ than for NE (0.42, 0.64, and 1.92 µM,
respectively; Pifl et al., 1996). Under conditions in which the
Na+ concentration within the axoplasm is
relatively low, the 3-fold difference in Km
may be more critical for transport than under conditions of high
Na+ concentration (inward transport). It is well
documented that prior loading of the NET with Na+
increases the affinity of the transporter for substrate (Trendelenburg, 1991).
LLC-NET cells preloaded with [3H]MPP+ were very sensitive to the Nao concentration. Above 30 mM Nao, only a small amount of [3H]MPP+ efflux was seen. Lower concentrations of Na+ induced a marked release of [3H]MPP+ from LLC-NET cells. When the Nao is reduced below a critical value (<30 mM in our model), fewer transporters are immobilized on the extracellular side of the plasma membrane, making more transporters freely mobile and available to the intracellular side. Reuptake of released [3H]MPP+ will be less favorable in a reduced Na+ buffer, augmenting the release process.
Nigericin is a commonly used ionophore that mediates the exchange of
external H+ for internal K+
(Erecinska et al., 1993). Incubation of cells with nigericin lowers
both the intracellular pH (pHi) and
K+ concentration (Erecinska et al., 1993).
Therefore, exposure of cells to nigericin results in a depolarization
of the plasma membrane, triggering the opening of voltage-operated ion
channels and the subsequent influx of Na+ and
Ca2+ (Erecinska et al., 1993; Rodriguez and
Sitges, 1996). The influx of Ca2+ induced by
nigericin has been shown to stimulate the exocytotic release of
neurotransmitters (Erecinska et al., 1993; Rodriguez and Sitges, 1996),
whereas the nigericin-induced reduction in pHi
has been demonstrated to activate the NHE (Erecinska et al., 1993). The
influx of Na+ via NHEs and voltage-sensitive
Na+ channels could lead to the reversal of the
NET and the release of axoplasmic substrate. Indeed, nigericin elicits
a Ca2+-dependent and -independent release of
neurotransmitter from synaptosomes, neurons, and C6 glioma cells
(Erecinska et al., 1993; Rodriguez and Sitges, 1996) and has been shown
to induce the release of MPP+ from preloaded
platelets (Cesura et al., 1987). We found that nigericin induced a
large increase in
[3H]MPP+ release from
preloaded LLC-NET cells, indicating that this compound does indeed
stimulate the influx of Na+ and, consequently, a
reversal of the NET.
Under physiological conditions,
Na+,K+-ATPase is
responsible for maintaining the intracellular concentration of
Na+ and K+ by exchanging
extracellular K+ for intracellular
Na+ (Stute and Trendelenburg, 1984). ATP
depletion in various pathophysiological conditions (e.g., protracted
myocardial ischemia) leads to an augmentation of intracellular
Na+ accumulation, as
Na+,K+-ATPase activity is
inhibited. We found that inhibition of
Na+,K+-ATPase with ouabain
potentiates the nigericin-induced release of
[3H]MPP+ from LLC-NET
cells. These experiments were performed in a
reduced-Na+ buffer to minimize the effect of
ouabain alone. Prolonged inhibition of the
Na+,K+-ATPase can itself
induce neurotransmitter release (Stute and Trendelenburg, 1984), as
indeed we found in our experiments with LLC-NET cells in a
physiological HEPES buffer. In a 40 mM Na+ HEPES
buffer, ouabain did not stimulate
[3H]MPP+ efflux, but did
potentiate the release induced by nigericin.
DMI inhibited the prolonged (>10 min) nigericin plus ouabain-induced
efflux of [3H]MPP+,
clearly demonstrating that this release process is mediated by a
reversal of the NET. Interestingly, DMI markedly potentiated the efflux
of [3H]MPP+ during the
early phase (<10 min) of the nigericin plus ouabain-induced efflux.
These experiments were performed in a physiological buffer, which has a
high Na+ concentration. Accordingly, in the
experiments with nigericin plus ouabain alone,
[3H]MPP+ released from
LLC-NET cells was subject to reuptake, as the high Nao concentration favored inward transport.
Because DMI inhibits the reuptake process, the concentration of
extracellular [3H]MPP+
was higher than that in the absence of DMI. In contrast, with prolonged
exposure to nigericin plus ouabain, more Na+
accumulates within the cell, and thus the Na+
gradient favoring inward transport is reduced. In addition, more transporters will have been immobilized by DMI, reducing the number of
transporters available for outward transport. Hence, DMI would be
expected to reduce, as it did, the extracellular concentration of
[3H]MPP+ during prolonged
nigericin plus ouabain treatment (>10 min). The
IC50 value for DMI in our experiments with
nigericin plus ouabain was notably lower than that calculated for our
efflux studies with Na+-gradient reversal (~15
nM compared with ~55 nM). This probably reflects the
Na+ dependence of DMI on binding to the NET (Pifl
et al., 1997), as seen for NE itself (Trendelenburg, 1991).
A recent study in brain synaptosomes demonstrated that at a low
concentration (0.5 µM), nigericin behaves as a
Na+/H+ ionophore under
physiological conditions (Rodriguez and Sitges, 1996). In that study,
nigericin caused an increase in pHi (suggesting a
loss of H+), intracellular
Ca2+, and intracellular
Na+. It was concluded that the influx of
Na+ was mediated through the ionophore itself. In
our studies, no release of
[3H]MPP+ was seen at
concentrations of nigericin less than 3 µM (data not shown).
Furthermore, Rodriguez and Sitges used synaptosomes, rather than cells.
It is probable that these different systems respond differently to the
ionophore. In fact, in another study, it was found that 5 µM
nigericin caused a continuing increase of H+ and
thus lowered pHi in C6 glioma and cultured neuron
cells (Erecinska et al., 1993). Our experiments with the amiloride
derivative EIPA (a potent inhibitor of the NHE; Kleyman and Cragoe,
1988) clearly suggest that the nigericin plus ouabain-induced efflux of
[3H]MPP+ from preloaded
LLC-NET cells is a consequence of Na+ influx via
the NHE. Unlike DMI, EIPA inhibited both the early and the late phase
of the nigericin plus ouabain-induced
[3H]MPP+ efflux. This not
only indicates an inhibitory action of EIPA at the NHE but also
eliminates the possibility that EIPA is acting at the NET
(Schömig et al., 1989). Indeed, if EIPA were to act at the NET,
one would expect a potentiation of
[3H]MPP+ release in the
early phase of nigericin plus ouabain-induced efflux. The inability of
EIPA to inhibit gramicidin (a Na+ ionophore)
induced [3H]MPP+ efflux
further supports an action of EIPA at the NHE.
To determine the influence of voltage-dependent Na+ channels on the nigericin plus ouabain-induced efflux, we performed experiments in the presence of the Na+ channel blocker TTX. We found that TTX had very little effect on [3H]MPP+ efflux, suggesting that the opening of voltage-sensitive Na+ channels has only a minor role in stimulating [3H]MPP+ efflux in our model.
To further assess the usefulness of our model in studies of
receptor-mediated regulation of carrier-mediated NE release, we opted
to use a less powerful stimulant of reverse transport, namely, proprionate, which has previously been shown to stimulate NHE in
LLC-PK1 cells (Schlatter et al., 1997). We found
that proprionate elicits an efflux of
[3H]MPP+ from preloaded
LLC-NET cells. Imidazoline receptors are known to be coupled to the NHE
in LLC-PK1 cells (Schlatter et al., 1997). When
we activated these receptors with rilmenidine (Evinger et al., 1995),
we found that the proprionate-induced release of
[3H]MPP+ was markedly
inhibited. The imidazoline receptor antagonist idazoxan (Regunathan et
al., 1995) blocked the inhibitory effect of rilmenidine, confirming the
specificity of the rilmenidine response. The nigericin-induced efflux
of [3H]MPP+ was
unaffected by rilmenidine. This may be due to the greater amount of
[3H]MPP+ efflux induced
by nigericin compared with that of proprionate.
In conclusion, our data illustrate the potential of recombinant cell lines to study mechanisms and regulatory processes of carrier-mediated NE release. Similar to that seen in advanced myocardial ischemia, we were able to stimulate carrier-mediated efflux of an NET substrate from LLC-NET cells by activating the NHE. Furthermore, we could modulate this release process with inhibitors of the NET, NHE, and via a receptor-operated pathway. Because excessive NE release contributes to severe, sometimes fatal myocardial arrhythmias, an improved understanding of the carrier-mediated NE release process will ultimately enhance our ability to intervene and prevent the deleterious effects of excessive NE release.
| |
Acknowledgments |
|---|
We gratefully acknowledge our colleague Dr. Miklos Toth for the donation of LLC-NET cells that were established by Dr. Gary Rudnick with hNET cDNA cloned by Dr. Susan G. Amara.
| |
Footnotes |
|---|
Accepted for publication July 6, 1999.
Received for publication March 18, 1999.
1 This work was supported by National Institutes of Health Grants HL34215 and HL46403. A preliminary version of these findings was presented at the 71st Scientific Sessions of the American Heart Association, November 1998, and was published in abstract form in Circulation (1998) 98:I-681.
Send reprint requests to: Roberto Levi, M.D., Department of Pharmacology, Cornell University, Weill Medical College, 1300 York Ave., New York, NY 10021. E-mail: rlevi{at}med.cornell.edu
| |
Abbreviations |
|---|
NE, norepinephrine; NHE, Na+-H+ exchanger; COMT, catechol-O-methyl transferase; DMI, desipramine; hNET, human NET; EIPA, 5-(N-ethyl-N-isopropyl)-amiloride; LLC-NET, LLC-PK1 cells transfected with human norepinephrine transporter cDNA; MPP+, N-methyl-4-phenylpyridinium; Nao, extracellular Na+; TTX, tetrodotoxin; NET, norepinephrine transporter; pHi, intracellular pH.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
2-adrenoceptors.
Circ Res
78:
475-481 of cocaine sensitive carrier-mediated efflux of amines from cells transfected with the cloned human catecholamine transporters.
Br J Pharmacol
121:
205-212[Medline].This article has been cited by other articles:
|
|
 |
|
  A. C. Reid, C. J. Mackins, N. Seyedi, R. Levi, and R. B. Silver Coupling of angiotensin II AT1 receptors to neuronal NHE activity and carrier-mediated norepinephrine release in myocardial ischemia Am J Physiol Heart Circ Physiol, April 1, 2004; 286(4): H1448 - H1454. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. Schwartz, R. D. Blakely, and L. J. DeFelice Binding and Transport in Norepinephrine Transporters. REAL-TIME, SPATIALLY RESOLVED ANALYSIS IN SINGLE CELLS USING A FLUORESCENT SUBSTRATE J. Biol. Chem., March 7, 2003; 278(11): 9768 - 9777. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. B. Silver, C. J. Mackins, N. C. E. Smith, I. L. Koritchneva, K. Lefkowitz, T. W. Lovenberg, and R. Levi Coupling of histamine H3 receptors to neuronal Na+/H+ exchange: A novel protective mechanism in myocardial ischemia PNAS, February 15, 2001; (2001) 51599198. [Abstract] [Full Text]
|
|
|
|
|
|
R. B. Silver, C. J. Mackins, N. C. E. Smith, I. L. Koritchneva, K. Lefkowitz, T. W. Lovenberg, and R. Levi Coupling of histamine H3 receptors to neuronal Na+/H+ exchange: A novel protective mechanism in myocardial ischemia PNAS, February 27, 2001; 98(5): 2855 - 2859. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
