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Departments of
*
Pharmacology and
Obstetrics and Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
3To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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KEY WORDS: • metallothionein • zinc • nitric oxide • endothelium • myogenic reflex
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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). There are four known functional MT
genes in mice; MT-I, MT-II, MT-III and MT-IV. MT-I and
MT-II expression is restricted to the central nervous system and
stratified squamous epithelium, respectively, and accordingly accounts
for most of the systemic effects of functional MT (Liu et al. 1996
). Although precise physiological roles for members of this
multigene family remain unknown, it has been suggested that major
functions of MT-I and MT-II are to detoxify heavy metals,
regulate zinc and copper homeostasis and limit oxidative damage
(Hamer 1986
, Palmiter 1998
). A critical
role for MT in protection against toxic nonessential metals such as
cadmium is apparent (Klaassen et al. 1999
), and MT
appears to act as a component of cellular defense against partially
reduced oxygen (Kang 2000, Klaassen et al. 1999
, Lazo and Pitt 1995
, Sato and Bremner 1993
) and nitrogen (Schwarz et al. 1995
)
species. In vitro data support the hypothesis that MT is a critical
link between cellular redox state and metal ion homeostasis
(Jacob et al. 1998
, Jiang et al. 1998
,
Maret 1994
, 1995
, Maret and Vallee 1998
, Maret et al. 1999
). In this regard,
cysteines of metal thiolate clusters confer unique redox sensitivity to
an otherwise redox inert metal ligand (e.g., zinc) and facilitate the
potential for MT to participate in intracellular signal transduction
pathways.
Nitric oxide (NO) is an ubiquitous signaling molecule that is well
known to play a prominent role in vasomotor regulation. Indeed,
identification of the L-arginine–NO biosynthetic pathway
in endothelium was critical in the development of concepts regarding NO
and mammalian physiology. Although most bioregulatory targets of NO
contain either cysteines or iron at their allosteric or regulatory
sites, or both (Stamler 1994
), it is entirely plausible
that other molecular interactions contribute to the biology of NO. In
this regard, it is noteworthy that MT can react with NO by forming
electroparamagnetic resonance (EPR)-detectable
iron-dinitrosyl-sulfur complex (Kennedy et al. 1994
,
Schwarz et al. 1995
) and that NO causes the release of
cadmium (Misra et al. 1996
), Cu (Borisenko, G. G.,
Fabisiak, J. P., Lazo, J. S., Kagan V. E., Liu, S.-X.,
Pitt, B. R. & Tyurin, V., unpublished results) and zinc
(Kroncke et al. 1994
) from MT in vitro via reactions
that are critically dependent on oxygen or superoxide anion
(Aravindakumar et al. 1999
). A few studies indicate that
NO is capable of increasing the amount of labile zinc in cells of the
hippocampus (Cuajungco & Lees 1998
) and systemic
vascular endothelium (Berendji et al. 1997
,
Kroncke & Kolb-Bachofen 1999
), but little is known
regarding the function of MT and its impact on NO-mediated changes
in zinc homeostasis in vascular endothelium. In this review, we
consider the physiological significance of the interaction of NO (and
secondary reaction products) and MT on intracellular zinc homeostasis.
In particular, we focus on vascular endothelium, a critical locus of
L-arginine–NO biosynthetic pathway, and summarize our
recent observations supporting a role for MT (and zinc) in
NO-mediated vascular signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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). Furthermore, recent in
vitro studies by Maret and Vallee (1998
) strongly point
to a role of MT in coupling metal ion homeostasis to cellular redox
changes. In the current study, we describe a novel fluorescent
resonance energy transfer (FRET)-based genetic approach that allows
live cell imaging of the conformation of MT and thus facilitates formal
testing of these principles in intact cells and tissue. Cultured sheep pulmonary artery endothelial cells (SPAEC).
SPAEC were cultured from sheep pulmonary arteries obtained from a
nearby slaughterhouse as previously described (Hoyt et al. 1995
). The SPAEC were grown in OptiMEM supplemented with 10%
fetal bovine serum with endothelial cell growth supplement (15
µg/mL), 10 U/mL heparin sulfate, 100 U/mL penicillin and 100 µg/mL
streptomycin at 37°C in an atmosphere with 5% CO2.
MT-I and MT–II null mutant mice.
We imported breeding pairs of MT-I– and MT-II–deficient
(MT-/-) mice from A. E. Michalska & K. H. A. Choo
(Michalska and Choo 1993
). The mice are of a mixed
genetic background of OLA129 and C57BL6 strains. We bred MT-/- with
C57BL6 mice obtained from Jackson Immunoresearch Laboratories (West
Grove, PA) to generate a parental heterozygous chimera that in turn was
backbred to C57BL6 wild type. This backbreeding resulted in 
50%
offspring that were heterozygous mutants. These mutants were identified
through a genotyping protocol using polymerase chain reaction–based
restriction digestion strategy on novel sites within a murine
MT-II gene that was mutated. An additional round of interbreeding
and genotyping resulted in F2 generation of MT-/- and
MT+/+ mice, which allowed the establishment of breeding
colonies in which the genetic contributions of the two strains were
assumed to be similar. Mice were kept in specific pathogen–free animal
housing. All experiments were performed with male mice between 8 and 18
wk old.
FRET-MT.
The cDNA for yellow cameleon-2 [containing enhanced cyan fluorescent
protein (ECFP) and enhanced yellow fluorescent protein (EYFP);
Miyawaki et al. 1997
] was kindly provided by Dr. Roger
Tsien Howard Hughes Medical Institute, University of
California, San Diego), and the cDNA for human MT-IIa (hMTIIa) was
received from Dr. Jan Vilcek (New York University Medical Center). An
hMTIIa polymerase chain reaction product was ligated in-frame into
a pSP72 yellow cameleon-2 subclone, and the ECFP-hMTIIa-EYFP product
was then subcloned into an expression vector. At 2–4 d of transfection
with LipoFECTAMINE Plus (GIBCO BRL, Rockville, MD), SPAEC were imaged
on a Nikon inverted microscope with a Photometrics cooled CCD camera
(Quantix) controlled by ISEE software (Inovision, Raleigh, NC). The
dual emission imaging was accomplished by using a 440DF20 excitation
filter, a 455 DRLP dichroic mirror and alternating emission filters
(480DF30 for ECFP, 535DF25 for EYFP) as described previously
(Pearce et al. 2000
).
Pressurized arteriograph and myogenic reactivity.
Mesenteric resistance arteries (diameters of 200–250 µm at 60 mm Hg)
were removed from wild-type and MT-/- animals,
mounted on glass cannulas in pairs in a dual-chamber pressurized
arteriograph and placed on the stage of a compound microscope. A video
camera interfaced with a dimension analyzer was used to constantly
monitor arterial diameter during incremental changes in measured
intraluminal pressure (Gandley et al. 1997
).
Microspectrofluorometry.
SPAEC were plated onto polylysine-coated glass coverslips. Cells were washed with normal saline and incubated with buffer containing 10 µmol/L Zinquin for 30 min at room temperature. Cells were washed with saline. The plate was placed onto a Nikon Diaphot inverted microscope and Zinquin imaged with a DAPI dichroic mirror, and fluorescence images were collected with a Photometrics CCD camera using Ratio Tool software (Inovision Corp.)
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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, Kroncke & Kolb-Bachofen 1999
). In this regard, we used the
Zn-specific fluorophore Zinquin to image free zinc in cultured
pulmonary endothelial cells. In Figure 1
, we show that like cells of the central nervous system, free zinc
appears to partition into vesicles in SPAEC. We then used imaging to
examine relative changes in intracellular free zinc in response to an
NO donor. The exposure of SPAEC to S-nitrosocysteine
resulted in an abrupt increase in labile zinc that returned to levels
below control with TPEN, as shown in Figure 2
.
|
|
). Figure 3
shows that the FRET-MT signal from a representative transfected
SPAEC decreases in response to bath application of 100 µmol/L NO
donor S-nitrosylglutathione (Fig. 3A
), the
addition of buffer saturated with NO gas (Fig. 3B
) or in
response to the calcium ionophore bromo-A23187 (Fig. 3C
). A
summary of results from several subcultures of SPAEC is shown in Figure 3D
. In particular, the effect of bromo-A23187 was sensitive
to the calmodulin inhibitor
N-(6-aminohexyl)5-chloro-1-napthalenesulfonamide (W-7) and
the NO synthase (NOS) inhibitor
NG-nitro-L-arginine
methyl ester (L-NAME) and was restored by excess
L-arginine, strongly suggesting that endothelial
NOS–derived NO released metals from MT.
|
). In contrast, myogenic tone in MT-/- vessels
was significantly less than that in MT+/+ vessels
when endogenous NO was generated (Fig. 4B
).
|
, Kroncke & Kolb-Bachofen 1999
), stores Zinquin-sensitive labile
zinc in a vesicle-like compartment (Fig. 1)
. Labile zinc can be
rapidly mobilized by NO (Fig. 2)
and may arise from zinc bound to MT
(Fig. 3)
. This latter source of zinc is affected by NO donors (Fig. 3A, B
) as well as endothelial
NOS–derived NO (Fig. 3C, D
) suggesting
that MT may participate in NO signaling in the vascular wall. This was
further borne out by the lack of myogenic reflex in a resistance vessel
isolated from MT null mutant mouse (Fig. 4B
) unless an NOS
inhibitor is present (Fig. 4A
).
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FOOTNOTES |
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2 Supported in part by National Institutes of Health Grants HL32154 (to B.R.P.), GM53789 (to B.R.P.), NS32385 (to E.S.L.), HL55312 (to E.S.L.) and HL07563 (to L.L.P.).
4 Abbreviations used: ECFP, enhanced cyan fluorescent protein; EYFP, enhanced yellow fluorescent protein; FRET, fluorescence resonance energy transfer; hMTIIa, human metallothionein IIa; L-NAME, NG-nitro-L-arginine methyl ester; MT, metallothionein; NO, nitric oxide; NOS, nitric oxide synthase; SPAEC, sheep pulmonary artery endothelial cells.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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) and wild-type
MT+/+ (•) mice (n = 5) was similar in
the presence of the NOS inhibitor L-NAME. Panel B:
The removal of L-NAME revealed an NO-dependent significant
decrease in myogenic reactivity in MT-/- mice (modified
from Pearce et al. 2000