© 1996 American Heart Association, Inc.
Articles |
The 
3 Isoform Protein of the Na+,K+-ATPase Is Associated With the Sites of Cardiac and Neuromuscular Impulse Transmission
From the Departments of Internal Medicine, Physiology, Neurology, and Pathology, Yale University School of Medicine, New Haven, Conn.
Correspondence to Raphael Zahler, MD, PhD, Section of Cardiology, Fitkin 3, Yale University School of Medicine, 333 Cedar St, New Haven CT 06510.
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Abstract |
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Abstract The
(catalytic) subunit of the Na+
pump (Na+,K+-ATPase) has three isoforms:
1 is ubiquitous, skeletal muscle expresses predominantly 2, and
3 has been localized to specific types of neurons and, possibly, to
axonal processes. The 3 isoform mRNA is also expressed in the rat
cardiac conduction system. Thus, we studied rat heart and quadriceps
muscles by immunohistochemistry using isoform-specific antibodies
to the Na+ pump subunit and labeled
-bungarotoxin as a probe for the neuromuscular junction (NMJ).
We found that 3 pump protein is localized to three sites important
for impulse transmission: the junctional complex between cardiac
myocytes, the heart conduction system, and the NMJ. Specifically, all
levels of the conduction system expressed 3 immunoreactive protein,
as assessed by two isoform-specific antibodies and
histological conduction system markers. Specific
expression at the junctional complex was confirmed by immuno-EM.
Double-labeling and denervation analysis indicated that
3-positive areas in skeletal muscle were presynaptic and adjacent to
postsynaptic bungarotoxin-positive regions, which had the classic
morphology of NMJs. Thus, specific
Na+,K+-ATPase pump isoforms may be
adapted to maintenance of membrane potential and/or
intracellular ion concentrations required for impulse transmission in
both heart and presynaptic motor terminals contacting skeletal muscle.
Key Words: Na+,K+-ATPase isoform • neuromuscular junction • cardiac conduction system
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Na+,K+-ATPase activity is important for the maintenance of membrane potential in excitable tissues such as nerve, heart, and muscle. Of the three isoforms of the
(catalytic) subunit of the Na+ pump,
1 is found in virtually all mammalian cells, but 2 and 3 are
localized to excitable tissue. In the rat, moreover, 1 is highly
ouabain resistant, whereas 2 and 3 are relatively
sensitive. The 2- and 3-isoform mRNAs are concentrated in the
cardiac conduction system of the adult rat but not in working
myocardial fibers.1 In the brain, there is a complex
pattern of isoform distribution at the cellular level.2 3 4 5
These data, together with the fact that the -subunit genes are
strongly conserved in evolution,6 suggest that each
isoform may have a specific biological role.
The NMJ is the interface between motor neuron and skeletal muscle. The
Na+,K+-ATPase is important for the
generation of the membrane potential in both of these excitable
tissues, although the biochemical and electrical characteristics of
presynaptic and postsynaptic membranes are markedly
different.7 Skeletal muscle expresses predominantly the
2 isoform,8 whereas 3 is found in both cell bodies
and axonal processes of neurons.4 9 10 This suggests that
at the NMJ, a structure expressing predominantly 2 might be adjacent
to one expressing predominantly 3.
In the present study, we demonstrate that 3 is associated with
presynaptic motor efferents, whereas 2 localizes to the postsynaptic
NMJ as well as the rest of the skeletal muscle sarcolemma. We also show
that 3 protein is associated with the cardiac conduction system and
with the cardiac junctional complex, the site of impulse conduction
from one contractile myofiber to the next. Although the 1 isoform is
also detectable in these locations, it is found in essentially all
tissues, whereas the distribution of 3 is narrowly restricted. Thus,
these data suggest that 3 may be adapted to the maintenance
of membrane potential and ion distributions associated with conducting
tissue. Alternatively, however, differences in stability between the
isoforms or distinct roles in cellular adhesion could explain these
findings.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hearts were obtained from normal adult male Sprague-Dawley rats. Gluteus and soleus muscles were from normal Sprague-Dawley rats or from Wistar rats that had undergone right-sided sciatic nerve transection 7 days before they were killed. Specifically, 180-g Wistar rats were anesthetized with sodium pentobarbital (50 mg/kg), and the sciatic nerve was exposed at the pyriformis tendon, where it was ligated and cut distal to the ligation. After closure of the incision, the rats were placed individually in cages and allowed to recover from the anesthesia; they were able to eat and ambulate within hours, and no signs of distress were observed.
Tissue to be used for immunofluorescence was perfusion-fixed in 0.5% periodate-lysine-paraformaldehyde, washed in either PBS/10% dimethyl sulfoxide or PBS/15% sucrose for cryoprotection, rapidly frozen in liquid nitrogen or supercooled Freon-22, and stored at -70°C until use. Immediately before sectioning, tissue was thawed and refrozen on a cryostat chuck in OCT (Miles, Inc). For heart sections, care was taken to place the specimens on the chuck with proper orientation. Sections (8 to 10 µmol/L) were mounted on glass slides coated with poly-L-lysine (Sigma Chemical Co) and processed as described previously.11 Briefly, sections were blocked against nonspecific labeling using PBS, 1% BSA, and 0.1% gelatin at room temperature for at least 30 minutes. They were then exposed to primary antibody in PBS/BSA for at least 1 hour with gentle agitation every 15 minutes. Sections were then washed with PBS/BSA three times for 15 minutes each. Secondary antibody [FITC-conjugated, rhodamine-conjugated, or Texas red–conjugated F(ab')2; Cappel, Molecular Probes] diluted 1:200 in PBS/BSA was then added, and the sections were incubated for an additional hour.
Primary antibodies McK1 and McB2 were used at a 1:5 dilution, 6H at
1:50, F9G10 at 1:40, and CM at 1:10 (see below for more information
about the antibodies used). For the NMJ studies, double labeling with
FITC/-bungarotoxin (Molecular Probes), a specific marker for the
acetylcholine receptor,12 13 and Texas red–conjugated
F(ab')2 was performed. FITC/-bungarotoxin (700
µg/mL) was applied at 1:50 000 dilution. Single-label
experiments verified that at these fluorophore and antibody
concentrations there was no cross talk between FITC and Texas red
channels. To confirm conduction-system localization, we also used
the following antibodies: mouse monoclonal anti-connexin43 (Zymed
Laboratories) at 1:1000; anti-connexin40, residues 335 to 356 (kind
gift of M. Théveniau-Ruissy), at 1:814 ; and mouse
monoclonal anti-AChE antibody (Chemicon International) at 1:100. Nerve
fibers in muscle tissue were identified with rhodamine-labeled
recombinant tetanus toxin C fragment (Neurotag-red,
Boehringer Mannheim)15 according to the
manufacturer's directions.
Specimens were evaluated with a Zeiss Axiophot microscope with conventional epifluorescence illumination. Imaging was also performed with a BioRad MRC 600 laser confocal microscope, which eliminated out-of-focus flare inherent in the relatively thick cryostat sections. The illuminator and detection system were standardized to provide high-quality images exhibiting an acceptable signal-to-noise ratio. All positive findings presented below were found repeatedly in multiple specimens (obtained from >20 animals) that were processed on different dates. Also, each staining run included multiple slides of adjacent sections stained with secondary antibodies (anti-rabbit and anti-mouse) only, and positive results were not considered valid unless the corresponding same-run, adjacent-section control slides were negative.
Immunoelectron microscopy was performed as previously described.11 Briefly, after fixation, tissue was cryoprotected in 10% dimethyl sulfoxide and PBS for 15 minutes at 4°C and then frozen on a cryostat chuck. Cryosections (28 µm) were cut and collected in PBS with 1% BSA at 4°C. Sections were allowed to block for 1 hour in the same solution. They were then incubated in primary antibody overnight at 4°C on a rotational mixer. Sections were washed for 15 minutes three times and then incubated with peroxidase-conjugated sheep anti-mouse F(ab')2 secondary antibody at room temperature for 2 hours. Sections were washed three times and then fixed in 1.5% glutaraldehyde in 1 mol/L sodium cacodylate buffer with 5% sucrose (pH 7.4). Next, sections were washed in 0.1 mol/L sodium cacodylate buffer with 7.5% sucrose, followed by immersion in 0.1% diaminobenzidine/H2O2 with 50 mmol/L Tris-HCl/7.5% sucrose for 15 minutes. The sections were then washed, postfixed with OsO4, dehydrated in ethanol, and embedded in Epox 812 (E. Fullam, Inc). Semithin and thin sections were cut on an LKB 8800 ultramicrotome and viewed on a Zeiss EM10B operating at 60 kV.
The isoform specificity of the antibodies McK1 and 6H (anti-1), McB2
(anti-2), and CM (anti-3), generously supplied by Drs K.
Sweadner, Massachusetts General Hospital, and M. Caplan, Yale
University, has been previously verified in multiple
publications.8 10 16 CM, which was raised against a
synthetic peptide comprising amino acids 2 to 14 of the N-terminus of
rat 3, was affinity-purified before use on columns of peptide
immobilized on thiopropyl/Sepharose 6B (Pharmacia). We
checked to ensure that the monoclonal antibody F9G10 (generously
supplied by Dr K.P. Campbell, University of Iowa), which was raised
against dog cardiac microsomes boosted with dog cardiac wheat germ
agglutinin eluate, was specific for 3. This was done by performing
immunoblots on crude membrane preparations from rat kidney,
skeletal muscle, and brain (hippocampus) (Fig 1
); these
tissues were chosen because the major form in kidney is 1, the major
form in skeletal muscle is 2, and brain expresses all three
isoforms. Microsomes were first assayed for ouabain-inhibitable
Na+,K+-ATPase activity by the method of
Forbush.17 Equal amounts of ouabain-inhibitable
Na+,K+-ATPase activity from each tissue
preparation were then loaded on a 7% SDS-PAGE gel and transferred
electrically to Immobilon-P membrane (Millipore). The membrane was
blocked with 20 mmol/L Tris (pH 7.5)/150 mmol/L NaCl/5% nonfat dry
milk/0.1% Tween 20 at room temperature for 1 hour, incubated with
primary antibody, and washed. Secondary antibody
(peroxidase-conjugated sheep anti-mouse IgG or donkey
anti-rabbit IgG, Amersham) was diluted 1:5000 and signal detected
with the Amersham ECL kit.
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As a further check for isoform specificity of F9G10, we cocultured two
types of cells: one of which (SY5Y) expresses both 1 and 3
protein, and one of which (HeLa) expresses only 1.18
When this mixed culture was double-labeled with CM and F9G10, both
anti-3 antibodies labeled the same cells (which were morphologically
identifiable as SY5Y cells), and both failed to label HeLa cells (Fig 2). As a third check for specificity, we applied each
antibody to histological sections of rat kidney,
skeletal muscle, and hippocampus fixed as above. For these controls and
for controls using secondary antibody only, slides were viewed and
photographed at identical confocal settings. The expected patterns of
tissue-specific isoform expression were reproduced for McK1, 6H,
McB2, and CM (data not shown). F9G10 was positive on brain
(hippocampus) and negative on kidney, as expected; results on heart and
skeletal muscle are discussed below.
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Results |
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Comparison of Two Anti-
3 AntibodiesBoth
3 antibodies were found to be positive on hippocampus and
negative on kidney, by analysis of immunoblots (Fig 1
) and histological sections. When applied to a mixed
culture of two morphologically distinguishable types of cells that
express known different Na+,K+-ATPase
isoforms, the two anti-3 antibodies concordantly labeled the cells
(Fig 2). In brain and heart double-label
immunofluorescence experiments, cells that were
reactive with one antibody were also reactive with the other, and the
staining in each case was at the cell surface. In addition, F9G10 and
CM both gave no signal above background on Western blots of rat heart
ventricle and skeletal muscle, as expected. In
histological sections of skeletal muscle, however,
F9G10 stained discrete linear regions of cell surface membrane in
occasional (
1 in 20) muscle fibers, as did CM. Below, we show that
these regions correspond to NMJs.
Heart Conduction System
The cardiac conduction system transmits impulses from the SA node
through the AV node, His bundle, and Purkinje fibers to the working
myocardium. Immunofluorescence with
3 isoform–specific antibodies indicated that 3 pump protein
is expressed at all levels of this system in adult rat heart (Figs 3 through 7). Conduction tissue was identified by its anatomic location
and also by staining with specific antibodies (Figs 3 through 5). For
example, Purkinje fibers are found in the
interventricular septum, located subendocardially, and
(in rat) show immunoreactivity for connexin40 and
connexin43.14 19 Double-label experiments showed that
Purkinje fibers in normal rat heart, identified by connexin40
fluorescence, are also immunoreactive for 3 (Fig 3). Similarly, serial sectioning identified 3
immunofluorescence in subendocardial fibers, in
regions whose cells were strongly reactive for connexin43 along their
lateral surface (Fig 4). These findings are
representative of multiple specimens obtained from
different animals and processed on different dates (see
"Discussion").
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AV nodal tissue was sought in the area of the basal
interventricular septum, proximal to the tricuspid
valve insertion. Cells of the AV node were identified with antibody to
AChE; serial sections showed that cells in this region also expressed
immunoreactive 3 (Fig 5). Although cells in the AV
nodal region were reactive with both anti-3 antibodies (Fig 6), CM tended to stain discrete linear regions of the
cell surface (Fig 6A), whereas F9G10 labeled most of the sarcolemma of
AV nodal cells (Fig 6C). This pattern is similar to that observed at
the NMJ (see Figs 11 through 13 below and "Discussion").
Ventricular myocytes in the septum but away from the AV
nodal region were not stained (Fig 6B). Whereas the AV node is richly
innervated, NMJs do not exist in heart; instead, afferent
neurons form varicosities that release neurotransmitter diffusely. The
staining pattern in heart is thus suggestive of 3 expression in both
AV nodal conduction tissue (consistent with in situ
hybridization data indicating that cardiac-derived elements of the
AV node express 3 mRNA1 ) and in adjacent nerve
terminals.
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In addition, the SA node appeared as an area of bright 3 staining
adjacent to the superior vena cava and right atrium (Fig 7). The latter structures appeared dim and were
comparable to adjacent sections stained with secondary antibody only.
In contrast to conduction tissue, immunoreactivity for 3 was not
present in the sarcolemma of working ventricular
myocardial cells of adult rat heart (data not shown). In addition to
the conduction-system expression of the 3 isoform described in
the present study, we have demonstrated elsewhere that there is
increased expression of 2 mRNA and protein in the adult rat
conduction system.1 20
Junctional Complex
The cardiac impulse spreads from one working myocardial cell to
the next via the junctional complex, "the structure where the
stepped faces of the fiber ends interlock and interdigitate with
corresponding faces of its neighbors."21 The
characteristic electron microscopic morphology of this structure is
illustrated in the article by Sommer and Johnson.21
Although there was no detectable signal for 3 in the sarcolemmal
surface membrane of working ventricular myocytes, the
junctional complex of these cells reacted strongly with both anti-3
antibodies by immuno-EM (Fig 8A, 8B, and 8C). No such
labeling was seen with antibodies against 1 or 2, although these
antibodies did label other structures at the EM level (Fig 8D).
Immunoreactivity of the junctional complex with anti-3 antibody was
also seen at the light-microscopic level (Fig 9; see
also Fig 3). Taken together, these results suggest that 3 is the
predominant Na+,K+-ATPase isoform at the
junctional complex.
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NMJ
Although the monoclonal anti-3 antibody F9G10 was negative on
Western blots of skeletal muscle, histologically, F9G10
stained discrete linear regions of skeletal muscle cell surface
membrane, as did the polyclonal anti-3 antibody CM. This staining
pattern, together with previous data indicating that 3 is expressed
at high levels in the nervous system,23 suggested that the
regions of 3 reactivity might represent NMJs. Thus,
double-labeling studies were performed using
FITC/-bungarotoxin, which specifically identifies the
acetylcholine receptors at the postsynaptic NMJ.24
Simultaneous staining with Texas red–labeled secondary
antibody was used to identify
Na+,K+-ATPase protein isoforms. Care was
taken to ensure that the FITC signal did not bleed through to the Texas
red channel or vice versa (see "Materials and Methods"). In
addition, nerve axons traversing muscle were identified by double
labeling with recombinant tetanus toxin C fragment.
Whereas nerve axons expressed 3 protein (Fig 10),
there was also immunofluorescence for 3 at
discrete structures whose morphology was not axonal (Fig 11). Double labeling with -bungarotoxin
demonstrated that this staining represented 3 protein
expressed at the NMJ, in close proximity to the postsynaptic motor end
plate (Figs 11 through 13). Colocalization with bungarotoxin was seen
with both the F9G10 and the CM antibodies to 3 (Fig 12). Furthermore, 3 signal was present only at
the NMJs and in nerve fibers traversing the muscle but not elsewhere in
the skeletal muscle sections. In contrast, signal for the 2 isoform
was diffusely present in muscle cell surface membranes; in some
cases this fluorescence was more intense at NMJ sites, but most
NMJs did not have increased 2 signal (Fig 13). The
1 isoform was found diffusely in the sarcolemma, unlike 3;
however, 1 signal was more intense at NMJ sites (Fig 13).
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In longitudinal sections, the fluorescence for 3 appeared to
be offset slightly from that for bungarotoxin, suggesting that 3 is
on the presynaptic side of the NMJ. To confirm that the 3 isoform is
presynaptic, we studied lower-extremity muscle obtained from rats 7
days after unilateral denervation. At this time point, there is
predictable degeneration of the nerve terminal but not of the
postsynaptic structure. Fluorescence for 3 at each NMJ site
was evaluated, and right and left sides and muscles proximal and distal
to the denervation site were compared quantitatively. Although end
plates marked by FITC/-bungarotoxin fluorescence
persisted in the denervated limb,25 those in the muscle
groups distal to the cut nerve had markedly decreased-to-absent
Texas red fluorescence for 3. The difference from the
contralateral limb was highly statistically significant (Fig 14). However, fluorescence for the McB2
antibody to 2 was unaffected in the denervated limb. As an
additional control, right- and left-sided muscles proximal to the
cut nerve had identical staining scores for 3 (Fig 14).
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Discussion |
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The cardiac conduction system is composed of electrophysiologically specialized cells, with resting potentials and ion channels different from those found in working myocardial cells; it also has a unique pattern of innervation. Embryologically, the conduction system appears to be derived from the neural crest and thus may retain some neural-specific characteristics.26 Furthermore, the increase in K+ exchange during stimulation is seven times greater in Purkinje fibers than in ventricular muscle, and Purkinje fibers are more sensitive than ventricle to ouabain.27 28 The myocardium is also a functional syncytium in which the junctional complex provides a low-resistance pathway for longitudinal impulse transmission, so that longitudinal impulse conduction proceeds several times faster than transverse conduction.29 30 Therefore, it is reasonable that the conduction system and junctional complex would express a specialized Na+ pump isoform.
Although the finding of 3 protein expression in the cardiac
conduction system and NMJ is new, this localization is
consistent with physiological data in the
literature. Thus, ouabain administration leads to prolonged
end-plate potential augmentation in skeletal muscle31
but does not affect overall muscle function. This end-plate
potential augmentation is
electrophysiologically similar to delayed
afterdepolarizations in cardiac Purkinje fibers,32 another
tissue that is immunoreactive for the 3 isoform. Furthermore,
Na+ pump activity in skeletal muscle has been reported to
be nonuniform, with greater activity per pump unit, greater pump
density, or altered electrogenicity at the end-plate
region.33 These observations are consistent with
the presence of a different pump isoform adjacent to the motor end
plate, an isoform similar to that found in cardiac Purkinje fibers.
The presence of different pump isoforms on motor terminal and muscle
might also reflect differences in their requirement for maximal
Na+-K+ flux. Since several neurotransmitters
are cotransported with Na+, synaptic transmission
may be affected by changes in Na+ pump
physiology.34 Also,
Na+,K+-ATPase activity in nerve is
electrogenic; relatively short bursts of action potentials in
peripheral35 and optic36 nerves
result in a ouabain-sensitive
afterhyperpolarization. Axonal elements have a
much larger surface-to-volume ratio than do muscle fibers;
therefore, activity-dependent Na+ loading could more
easily depolarize the axon. Low levels of such depolarization would
tend to increase axonal excitability, whereas higher levels would lead
to conduction block.37 This hypothesis fits well with
recent work indicating that the rat 3 isoform has a lower
Na+ affinity than does the 1 or 2
isoform.18 38 Thus, 3 pumps would be expected to be
active only under conditions of high
[Na+]i, such as may occur at the nerve
terminal.
An alternative explanation for our findings relates to the cytoskeletal
and adhesion-related molecules that have been specifically
localized to the NMJ.13 23 39 In the heterodimeric
Na+,K+-ATPase, the 3 subunit in
pineal gland pairs preferentially with the ß2 subunit,40
which is identical to AMOG, an adhesion protein involved in
neural-glial recognition.41 Thus, 3 could be
related to the formation of the NMJ.13 Also, 3 may be
better adapted for axonal transport than the other
Na+,K+-ATPase
isoforms42 43 44 ; thus, presynaptic 3 may have been
transported to the NMJ from the neuronal cell body in preference to the
other isoforms.
Since our results are based on histological techniques,
which are subject to intersample variability, findings were
presented only if they were present repeatedly in multiple
specimens processed on different dates. Multiple controls were used,
including double labeling, comparison of multiple isoform-specific
antibodies on adjacent sections, and inclusion of multiple adjacent
sections stained with secondary antibody only with each specimen group.
For the double-label studies, care was taken to completely rule out
bleed-through between the FITC and Texas red channels. Furthermore,
the results show internal consistency as follows: (1) The
conduction-system isoform pattern is similar at the mRNA and
protein levels. (2) There is agreement between light- and
electron-microscopic data (in which a different staining technique
is used) regarding the junctional complex. (3) Results obtained using
two different anti-3 antibodies are the same.
Nevertheless, it is important to consider whether our findings could
have resulted from technical artifacts. Thus, the NMJ is known to stain
nonspecifically with sarcolemmal markers.13 24 In our
experiments, however, anti-3 and anti-1 antibodies reacted
preferentially with the NMJ, but anti-2 did not. In addition, our
denervation data indicate that the 3 signal is presynaptic (Figure 14), whereas most nonspecific NMJ labeling tends to be postsynaptic. In
the only previous published data relating
Na+,K+-ATPase isoforms to the
NMJ,24 there was uniform labeling of rabbit skeletal
muscle cell membranes with McB2 (anti-2) but more intense labeling
of the NMJ region. Although we also found uniform labeling of muscle
surface membrane with McB2, in our experience stronger 2 labeling of
NMJs was more the exception than the rule.
The reactivity of anti-3 antibodies with skeletal muscle could
conceivably be a spurious result caused by cross-reactivity with
the 2 isoform. This is unlikely, however, because (1) the anti-3
antibodies do not stain adipose tissue, which is known to express 2
protein (data not shown); (2) their staining patterns in heart closely
correlate with the distribution of 3 mRNA found by in situ
hybridization1 ; and (3) when the two anti-3 antibodies
are compared, the cell types positive for one antibody are always
positive for the other, with the staining at the cell surface (Fig 2).
Evidence suggests rather that these antibodies recognize 3
associated with neural tissue (Fig 10) and NMJ. The observed
fluorescence pattern in the AV node is also consistent
with the idea that CM is staining neurally derived elements in heart,
since previous workers have reported that 3 is present in nerve
axons.10 43
Although the existence of more than one isoform of the
Na+,K+-ATPase has been known since
1980,8 23 it is still controversial whether there are any
major physiological differences between the
isoforms. Although 3 has a lower affinity for
[Na+]i and a higher affinity for
[K]o than 1 or 2,18 38 45 the
clear-cut ouabain-sensitivity differences between the rat
isoforms are attenuated in larger animals, and transfection studies
indicate only modest differences in affinity for
ATP.18 46 47 Nevertheless, the complex pattern of isoform
distribution at the organ and cellular level4 suggests
that these isoforms are not functionally interchangeable. Since 3 in
adult rat is essentially restricted in its distribution to the nervous
system and tissues involved in cardiac conduction, the 3 isoform may
play a specific role in cardiac and neuromuscular impulse
conduction.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Dr Zahler was supported in part by the National Science Foundation, Patrick and Catherine Weldon Donaghue Foundation, BRSG fluid research fund, and the American Heart Association, Connecticut Affiliate, Inc; Drs Kocsis and Kashgarian were supported by the National Institutes of Health. We thank Dr E.J. Benz for his advice and support, Drs M. Brines, J. Sanes, F. Chen, and M. Caplan for helpful comments, and Drs Caplan, K. Sweadner, and K.P. Campbell for providing antibodies.
Received November 1, 1995; accepted February 12, 1996.
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