-MSH potentiates the responsiveness of mammotropes by
increasing Ca2+ entry
Lucia
Nuñez and
L. Stephen
Frawley
Laboratory of Molecular Dynamics, Department of Cell Biology and
Anatomy, Medical University of South Carolina, Charleston, South
Carolina 29425
 |
ABSTRACT |
It is well known
that the suckling stimulus renders mammotropes considerably more
responsive to prolactin (PRL)-releasing stimuli, and the
neurointermediate lobe peptide
-melanocyte-stimulating hormone
(
-MSH) has been proposed to play a pivotal role in this priming. The
objectives of the present study were to determine whether
-MSH could
act directly on pituitary cells to potentiate PRL release in response
to two physiologically relevant PRL secretagogues, thyrotropin-releasing hormone (TRH) and ATP, and, if so, to identify the mechanism by which this priming phenomenon is manifested. To this
end, we cultured anterior pituitary cells from lactating rats overnight
and then subjected them to a reverse hemolytic plaque assay for PRL to
evaluate their responses to various test agents. We found that
-MSH,
which had no effect on PRL export when tested alone, augmented by more
than threefold the secretory responses to TRH and ATP. Next, we
utilized digital-imaging fluorescence microscopy of fura 2 to evaluate
the role of intracellular Ca2+ in
this process. We found that PRL export induced by pharmacological activation of L-type voltage-operated calcium channels was also potentiated by
-MSH, as was
Ca2+ entry induced by TRH. Our
results indicate that
-MSH acts as a mammotrope-priming agent on a
subset of mammotropes by increasing Ca2+ entry induced by PRL
secretagogues.
prolactin; calcium; suckling; thyrotropin-releasing hormone; adenosine 5' -triphosphate
 |
INTRODUCTION |
IT HAS LONG BEEN ESTABLISHED that suckling is the most
potent physiological stimulus for prolactin (PRL) release in mammals (4, 23, 28), but the mechanisms by which its effects are transduced are
extremely complex and incompletely understood. A critical piece of the
mechanistic puzzle resides in the fact that the suckling stimulus
induces a potentiation of the secretory responsiveness of mammotropes
to physiologically relevant secretagogues such as thyrotropin-releasing
hormone (TRH) (14, 21, 24). This was first demonstrated almost two
decades ago by Grosvenor et al. (15), who showed that even a brief
nursing episode rendered mammotropes enormously sensitive and
responsive to the PRL-releasing effects of TRH administered in vivo.
More recently, conceptually similar results have been obtained with ex
vivo models (21, 22).
Attempts to identify the chemical agent that imparts responsiveness to
mammotropes have led to the proposal that the hypophysial neurointermediate lobe (NIL) plays a pivotal role in this process (10,
20). This view is based on observations that the NIL can communicate
directly with the anterior pituitary through the short portal
vasculature (1, 27) and that surgical removal of the NIL abolishes the
acute discharge of PRL evoked by nursing (20). At present, the most
likely candidate molecule to subserve the role of an NIL-derived
potentiator of mammotrope responsiveness is
-melanocyte-stimulating
hormone (
-MSH). This peptide is released abruptly after the suckling
stimulus (2, 9, 30), and its in vivo immunoneutralization leads to a
severe attenuation of suckling-induced PRL release (17). Moreover, the
peptide can substitute in vitro for the nursing stimulus by making
mammotropes more responsive to at least one PRL-releasing agent,
low-dose dopamine (18). These observations, coupled with our recent
report that a discrete subpopulation of rat mammotropes possesses
functional
-MSH receptors (35), comprise a convincing (albeit
indirect) case that
-MSH is the mammotrope responsiveness agent.
However, the final pieces of direct evidence in this regard are
currently lacking, as is information about the intracellular mechanisms that govern the potentiation response. Accordingly, the purpose of the
present study was to evaluate whether
-MSH could enhance the export
of PRL induced by two physiologically relevant secretagogues, TRH and
ATP. The former is a bona fide PRL-releasing factor of hypothalamic
origin, whereas the latter is released during PRL exocytosis and acts
in an autocrine/paracrine manner on mammotropes to induce additional
PRL release, thereby amplifying the secretory response (25). In
addition, we attempted in this study to determine whether the priming
of PRL release by
-MSH was mediated by the potentiation of
Ca2+ entry induced by PRL
secretagogues.
 |
MATERIALS AND METHODS |
Animals and primary pituitary cell culture.
Primiparous lactating female (days
6-10 postpartum) rats (Harlan Sprague
Dawley, Madison, WI) were provided with food and water ad libitum and
maintained under a standard 12:12-h light-dark cycle. Animal housing
and handling were performed in accordance with procedures approved by
our university animal care committee. Litter size was standardized to
eight pups from the first day postpartum. Before decapitation, the pups
were separated from their mothers for 4 h. Pituitaries were removed
aseptically and dispersed into single cells as described previously
(5). Unless otherwise stated, all cell culture reagents were obtained
from GIBCO/BRL (Grand Island, NY), and all treatments were purchased from Sigma Chemical (St. Louis, MO).
Reverse hemolytic plaque assay.
PRL release measurements from individual anterior pituitary cells were
performed by reverse hemolytic plaque assay (RHPA) in accordance with
the protocol described by Boockfor et al. (5). Briefly, monodispersed
pituitary cells were plated in DMEM supplemented with 0.1% BSA, 10%
fetal bovine serum (FBS), and antibiotics. The next day, cells were
resuspended by mild trypsinization, mixed with protein A-coated ovine
erythrocytes, and infused into Cunningham incubation chambers. After a
1-h attachment period, cells were rinsed with DMEM-0.1% BSA, and the
resulting monolayers were then flooded with 5 vol (150 µl) of medium
containing PRL antiserum (1:80), with or without treatments, and
incubated for specified times. Subsequently, guinea pig complement
(1:80, 50 min) was flooded into the chambers to develop the plaques.
Cells were then fixed and stained for storage and quantification. Three
replicates were performed per treatment in each experiment. Series of
chambers were incubated with antibody and/or treatments for 60, 90, or 120 min, and the percentage of PRL secretors was determined in each case. Inasmuch as the 90-min incubation time was sufficient to
detect a maximal percentage of plaque formers, we used this time frame
for quantification of plaque areas. At least 200 plaque areas were
quantified in each treatment group by use of a calibrated ocular
reticule to derive mean plaque area and frequency distributions of
plaque areas.
Digital-imaging fluorescence microscopy.
Monodispersed anterior pituitary cells were cultured overnight on
plastic Petri dishes in DMEM-10% FBS and subsequently subjected to
measurements of intracellular free calcium concentration
([Ca2+]i)
by digital-imaging fluorescence microscopy. This was accomplished essentially as reported elsewhere (35). Briefly, cells were loaded with
2 µM fura 2-acetoxymethyl ester (Molecular Probes, Eugene, OR) for 1 h in external medium containing (in mM) 145 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES (pH, 7.4) and 10 glucose. Then Petri dishes were mounted under the heated stage
(37°C) of an Axiovert 35 inverted microscope (Zeiss, Jena,
Germany). Cells were epi-illuminated at 340 and 380 nm. Light emitted
above 520 nm was recorded and analyzed using an Attofluor Ratio Vision
System (Atto Instruments, Rockville, MD). Four video frames of each
wavelength were averaged with an overall resolution time of 4 s. Ratios
for consecutive frames obtained at 340- and 380-nm excitation were calculated, and
[Ca2+]i
was estimated by comparison with fura 2 standards (16). During experiments, cells were bathed in 2 ml of external medium. Addition of
test substances was achieved by removing 1 ml of medium and adding 1 ml
of warm medium containing no secretagogue or a twofold concentration of
the required secretagogue.
To quantify
[Ca2+]i
oscillations, we used the Oscillation Index, a parameter developed
elsewhere (32, 33). The Oscillation Index represents the rate of change
for
[Ca2+]i
during the period of measurement and reflects the frequency and/or amplitude of
[Ca2+]i
oscillations. To calculate this parameter, absolute differences in
[Ca2+]i
levels between successive measurements (taken at 4-s intervals) were
averaged for the length of the entire sampling period.
Statistical analysis.
All data are reported as the result of at least three completely
independent experiments. A two-way ANOVA was employed to analyze the
data. Differences in mean plaque areas and Oscillation Index values
were compared by use of Bonferroni's multiple comparisons test.
Differences were considered significant at
P < 0.05.
 |
RESULTS |
-MSH potentiates the secretory response of a
discrete mammotrope subpopulation to TRH and ATP.
Our initial studies were aimed at determining whether
-MSH could
modulate the amount of PRL released by a consensus PRL-releasing factor, such as TRH, or by a more recently established
autocrine/paracrine stimulator of PRL export, ATP. The strategy was to
treat pituitary cells obtained from lactating rats with these
secretagogues and then to quantify PRL release from individual living
cells by RHPA. Plaque area, a reliable index of the relative amount of
hormone released (11, 19), was used as the biological end point for these studies. We found, as we had found previously (25), that TRH and
ATP (both at maximal doses) could each evoke a modest increase of PRL
release, whereas
-MSH alone was ineffective in this regard (Fig.
1). It is noteworthy that TRH and ATP
induced comparable increments (25 and 26% above control value,
respectively) of PRL release. Interestingly, coexposure to
-MSH
augmented the amount of PRL released by either TRH or ATP (Fig. 1,
A and
B). More specifically, the secretory
response induced by the combination of
-MSH and TRH was fourfold
higher than that elicited by TRH alone. Likewise, the combination of
-MSH and ATP led to a response 3.2 times that evoked by ATP alone.
Thus it is clear that the secretory responses of mammotropes to PRL
secretagogues are potentiated by concurrent exposure to
-MSH.

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Fig. 1.
-MSH potentiates effects of thyrotropin-releasing hormone (TRH) or
ATP on prolactin (PRL) secretion. Anterior pituitary cells derived from
lactating rats deprived of their pups for 4 h were cultured overnight
and then subjected to reverse hemolytic plaque assays (RHPA) for
determination of PRL release. Cells were stimulated for 90 min with TRH
(100 nM, A) or ATP (10 µM,
B) in the presence or the absence of
-melanocyte-stimulating hormone ( -MSH, 100 nM). Mean plaque areas
reflective of cumulative PRL release are expressed here as a percentage
of control. Values are means ± SE of 3 completely independent
experiments in which 3 replicates were measured per sample. Values
having different letters are significantly different
(P < 0.05) from each other or from
control group.
|
|
We established recently that only a subpopulation of mammotropes,
accounting for about one-fifth of the total, exhibits functional
-MSH receptors (35). Accordingly, we tested whether the potentiating effects of
-MSH could be localized to such a subpopulation. We found, as we had previously, that addition of either TRH or ATP induced
a unimodal shift to the right in the frequency distribution of plaque
areas, indicating that most if not all mammotropes released PRL in
response to these agents (data not shown). However, when each of these
secretagogues was applied in combination with
-MSH, the frequency
distribution of plaque areas shifted from unimodal to bimodal (Fig.
2, A and
B). The first of these modes
corresponded precisely to that found with TRH- or ATP-treated cells,
whereas the second mode (representing cells that released considerably more hormone) was found only when
-MSH was present. These data demonstrate that only a subpopulation of mammotropes was rendered much
more responsive to PRL secretagogues by
-MSH.

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Fig. 2.
Effects of -MSH on TRH- or ATP-induced PRL secretion are only
observed in a subpopulation of mammotropes. Frequency distributions of
PRL plaque areas after stimulation with TRH ( ) or ATP ( ) are
shown. Cotreatment with -MSH (solid symbols) evoked a shift from a
unimodal frequency distribution to a bimodal one (conditions as in Fig.
1). Distributions were generated by plotting areas of 3 replicates in 7 different groups. Values are the average of 3 independent
experiments.
|
|
-MSH potentiates PRL release induced by
Ca2+ entry
through L-type voltage-operated
Ca2+ channels.
Because it is well established that TRH and ATP induce PRL release, at
least in part, through calcium-dependent mechanisms (6, 13), we asked
whether
-MSH was able to potentiate
Ca2+-mediated PRL export. To this
end, we stimulated pituitary cells with each of two
Ca2+ agonists, either alone or in
combination with
-MSH. One of these was BAY K 8644 (BK), a
dihydropyridine derivative that induces Ca2+ entry into the cells through
L-type voltage-operated Ca2+
channels (VOCC) (12). The other was ionomycin, an ionophore that
induces a
[Ca2+]i
increase primarily by release from intracellular
Ca2+ stores and does not influence
L-type VOCC (3, 32). In preliminary experiments, we established the
doses of BK (1 nM) and ionomycin (200 nM) that could stimulate PRL
release to a degree indistinguishable from that of ATP, and this is
illustrated in Fig. 3. Interestingly, when
-MSH was coadministered with BK, it augmented PRL release just as it
had potentiated that of ATP. In striking contrast, cotreatment with
-MSH had no effect on the amount of PRL released by the
Ca2+ agonist ionomycin (Fig. 3).
These data indicate clearly that
-MSH augments the release of PRL
induced by Ca2+ entry through
L-type Ca2+ channels but not that
evoked by mobilization of intracellular Ca2+ stores.

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Fig. 3.
-MSH potentiates secretory response to an L-type calcium channel
agonist but not to a calcium ionophore. PRL secretion was measured by
RHPA in response to 90-min incubations with an L-type calcium channel
agonist, BAY K 8644 (BK, 1 nM), or to a calcium ionophore, ionomycin
(Iono, 200 nM), in the presence or the absence of -MSH (100 nM).
Values are means ± SE of 3 completely independent experiments in
which 3 replicates were measured per sample. Values having different
letters are significantly different
(P < 0.05) from each other
or from control group.
|
|
-MSH is able to potentiate
Ca2+ entry
induced by a PRL secretagogue.
Having established that
-MSH potentiates PRL release induced by
Ca2+ entry through L-type VOCC, we
asked whether the peptide interacts with a bona fide PRL secretagogue
via a similar mechanism. Accordingly, we made
[Ca2+]i
measurements during the exposure of pituitary cells to TRH (or BK),
administered alone or after priming with
-MSH. Figure 4 shows the averaged responses of
representative pituitary cells stimulated with TRH or BK at the same
concentrations employed in our measurements of PRL release by RHPA.
Consistent with our previous results (35), we found that a
subpopulation of pituitary cells (17%) exhibited a moderate increase
of
[Ca2+]i
secondary to activation of
-MSH binding [we had established elsewhere (35) that such binding was restricted to mammotropes]. However, this increase of
[Ca2+]i
was transient and returned to values indistinguishable from basal
levels within 5-10 min (Ref. 35 and unpublished observations). To
determine whether
-MSH could modify
Ca2+ responses to TRH and BK, we
averaged the
[Ca2+]i
profiles for those cells exhibiting a
[Ca2+]i
increase after
-MSH stimulation and compared them with the profiles
obtained in nontreated cells or in those cells that were not responsive
to the
-MSH challenge. As shown in Fig. 4, the transient increase of
[Ca2+]i
that occurred a few seconds after TRH administration (which is
traditionally attributed to Ca2+
release from intracellular stores) was similar for
-MSH-responsive cells, nontreated cells, and cells not responsive to
-MSH. However, the second phase of TRH action (widely attributed to enhanced [Ca2+]i
oscillations and Ca2+ entry
through VOCC) was quite apparent in
-MSH-responsive cells, as
opposed to nontreated or nonresponsive cells. A similar output was
obtained when the cells were stimulated with BK instead of TRH.
Specifically, the increase of
[Ca2+]i
induced by BK in
-MSH-responsive cells was larger than the rather
modest rise obtained in nontreated cells or those not responsive to
-MSH. These data clearly indicate that
Ca2+ entry induced by TRH or BK is
enhanced in a subpopulation of mammotropes bearing functional
-MSH
receptors. To quantify the potentiating effects of
-MSH on
Ca2+ entry in this subset of
mammotropes, we computed the Oscillation Index, a parameter that
reflects the frequency and/or amplitude of
[Ca2+]i
oscillations (32, 33). Because
[Ca2+]i
oscillations in mammotropes are driven by electrical activity and
Ca2+ influx through VOCC, the
magnitude of the Oscillation Index provides an indirect measure of the
rate of Ca2+ influx through these
channels. As shown in Fig. 5, the
Oscillation Index after treatment with TRH or BK was enhanced in
-MSH-responsive cells compared with nontreated (Control) cells or
cells that were not sensitive to
-MSH stimulation. These data
clearly indicate that
-MSH enhanced
Ca2+ entry induced by TRH or BK in
a subpopulation of mammotropes bearing functional
-MSH receptors.

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Fig. 4.
-MSH potentiates Ca2+ influx
induced by TRH and BK. Anterior pituitary cells were cultured
overnight, loaded with
Ca2+-sensitive probe fura 2, and
subjected to measurements of intracellular calcium concentration
([Ca2+]i)
by digital-imaging fluorescence microscopy. Cells were stimulated with
BK (1 nM) or TRH (100 nM). Traces shown are average responses to BK
(A, n = 50) or to TRH (C,
n = 42) in the absence of -MSH. In
C, only TRH-sensitive cells were taken
into account. Stimulation of cells with -MSH (100 nM) enhanced
[Ca2+]i
oscillations elicited by BK (B) or
TRH (D) only in those cells
exhibiting an increase of
[Ca2+]i
induced by -MSH (solid traces, n = 12 for B,
n = 11 for
D) but not in cells not responsive
to -MSH (dotted traces, n = 50 for
B, n = 31 for D). This experiment is
representative of 6 (BK) or 3 (TRH) others.
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Fig. 5.
-MSH potentiates oscillatory activity induced by TRH or BK. Effects
of TRH or BK on
[Ca2+]i
oscillations (Fig. 4) were analyzed further here by use of a parameter
previously developed for this purpose, the Oscillation Index.
A: Oscillation Index for 2nd phase of
TRH-induced
[Ca2+]i
response (100 s after addition of secretagogue). Only this phase was
selected for analysis because it corresponded to
Ca2+ influx through L-type
voltage-operated Ca2+ channels,
which are -MSH responsive. B:
results obtained with companion cultures treated with BK. For
A and
B, no. of observations is within
parentheses above each bar. Values are means ± SE of 3 (TRH) or 6 (BK) independent experiments. *P < 0.05 vs. other treatments. Note that TRH and BK increased Oscillation
Index only in cells responsive to -MSH.
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|
 |
DISCUSSION |
The results of the present study demonstrate unequivocally that
-MSH
acts on a discrete subset of mammotropes to augment their
responsiveness to two physiologically important PRL-releasing agents,
TRH and ATP. How do these findings contribute to and extend our current
understanding about the mechanisms governing suckling-induced PRL
release? It is well established that the suckling stimulus decreases
and increases, respectively, the amount of dopamine and TRH secreted by
the hypothalamus into the hypophysial portal blood that bathes
mammotropes within the adenohypophysis (7, 8, 26). However, the
magnitudes of these changes (when viewed either alone or collectively)
cannot begin to account for the massive discharge of PRL that occurs in
response to nursing. A suckling-induced change of mammotrope
responsiveness to these agents has been invoked as a possible
explanation for this dilemma, and, as indicated earlier, there is
considerable evidence derived from studies conducted in vivo and in
vitro to support the idea that
-MSH is the mammotrope responsiveness
agent (10). With respect to direct effects on the pituitary gland,
-MSH has been shown to act on mammotropes to override inhibition of
PRL release imposed by high concentrations of dopamine (18, 21, 22) and
to impart mammotrope responsiveness to the PRL-releasing actions of the
lower concentrations of dopamine that reach the pituitary gland
immediately after nursing (18). Our present efforts expand the scope of
this responsiveness phenomenon by demonstrating for the first time that
-MSH administered in vitro can substitute for the suckling stimulus
in vivo by rendering a subgroup of mammotropes considerably more
responsive to the PRL-releasing effects of TRH. Thus it appears that
suckling evokes the concurrent release of hypophysiotropic agents (TRH
and low-dose dopamine) that stimulate PRL export and a mammotrope
responsiveness factor, which optimizes and embellishes this response.
The net effect, then, is an increase in the rate of PRL release. How
does ATP fit into this picture? We have previously shown that ATP is
coreleased with PRL during exocytosis, that its rate of export is
modulated by TRH and the dopamine agonist bromocryptine in a
predictable manner (stimulation and inhibition, respectively), that
addition of ATP stimulates PRL release, and that removal of the purine
from medium bathing pituitary cells causes a diminution of basal and
regulated PRL secretion (25). On the basis of these observations, we
concluded that ATP acts as an autocrine/paracrine regulator of PRL
release that serves to initiate an autoamplification cascade that
prolongs the secretory response elicited by a given amount of
hypophysiotropic signal. Our present findings add a new dimension to
this process in that the PRL-releasing effects of the amplifying agent
(ATP) were also found to be augmented greatly in the presence of
-MSH. Taken together, it seems reasonable to conclude that
-MSH,
which by itself has no influence on PRL export, potentiates the acute release of PRL by modulating mammotrope responsiveness to at least a
trio of regulatory agents.
Having identified many of the major extracellular players involved in
elaboration of the responsiveness phenomenon, we next turned our
attention to the intracellular processes that might transduce the
response. Accordingly, we explored the possibility that changes in
[Ca2+]i
contribute to this phenomenon for reasons outlined earlier. Our
strategy here was to evaluate the potentiating effects of
-MSH with
agents that elevated
[Ca2+]i
by different mechanisms. Interestingly, we found that
-MSH greatly
augmented the amount of PRL released by BK, which elevates [Ca2+]i
by facilitating Ca2+ passage
across the cell membrane through L-type VOCC (12). In contrast,
-MSH
had no effect on the amount of PRL secreted in response to ionomycin, a
Ca2+ agonist that liberates the
ion from sequestered intracellular stores (3, 32). In light of these
findings, we proposed that
-MSH exerted its responsiveness effects
by potentiating, at least in part,
Ca2+ entry induced by
physiologically relevant secretagogues. Such a possibility seemed
viable, given reports that both TRH and ATP activate
Ca2+ entry through L-type VOCC
(29, 31, 34). In an attempt to test this idea experimentally, we
monitored
[Ca2+]i
during exposure of pituitary cells to TRH administered alone or after
priming with
-MSH. Our results demonstrate that
-MSH did indeed
augment TRH-induced
[Ca2+]i
dynamics within mammotropes, and that it did so with a profile (enhanced plateau phase of the
[Ca2+]i
trace) consistent with a primary effect on
Ca2+ entry through VOCC. Thus the
mammotrope responsiveness changes attributable to
-MSH appear to be
mediated by potentiation of extracellular
Ca2+ entry induced by other
secretagogues such as TRH.
In summary, we have provided compelling evidence that
-MSH acts as a
mammotrope priming agent by rendering these cells much more responsive
to physiologically relevant PRL secretagogues. This effect is
attributable to a direct action of
-MSH on a discrete subpopulation
of mammotropes bearing the corresponding receptor. In addition, we have
shown that the peptide's augmentation of secretory responsiveness is
mediated by potentiation of Ca2+
entry through L-type VOCC induced by at least one physiological secretagogue, TRH. It is noteworthy that a number of laboratories, including our own, have pursued for almost two decades the elusive prolactin-releasing factor (PRF) that mediates suckling-induced PRL
release. Although there is no shortage of candidate molecules to
subserve such a role, none of these is sufficiently potent to account
for the massive surge of PRL release that occurs after suckling.
Perhaps our search has been complicated by the fact that the
"real" PRF is actually the combination of a priming factor (e.g.,
-MSH) and other secretagogues that, in the absence of priming,
elicit a rather modest increase of PRL secretion. The present results
are entirely consistent with this possibility.
 |
ACKNOWLEDGEMENTS |
We thank W. J. Faught for expert technical assistance. We are also
grateful to J. Nicholson (Pathology Department, Medical University of
South Carolina) for access to the Attofluor Ratio Vision System.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-45216 (to L. S. Frawley) and a
Postdoctoral Fellowship from the Ministry of Education and Science of
Spain (to L. Nuñez).
Address for reprint requests: L. S. Frawley, Laboratory of Molecular
Dynamics, Dept. of Cell Biology and Anatomy, Medical Univ. of South
Carolina, 171 Ashley Ave., Charleston, SC 29425.
Received 30 October 1997; accepted in final form 16 February 1998.
 |
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