1 Center for Oral Biology, Aab Institute of Biomedical Sciences, 2 Eastman Department of Dentistry, University of Rochester Medical Center, Rochester, New York 14642; and 3 Division of Developmental Biology, Children's Hospital Research Foundation, Cincinnati, Ohio 45229
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ABSTRACT |
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Chronic 1-adrenergic receptor activation results
in hypertrophy and hyperplasia of rodent salivary gland acinar cells.
Na+/H+ exchanger isoform 1 (NHE1) regulates
cell volume and the induction of cell proliferation in many tissues. To
investigate the relationship between NHE1 and the response of parotid
glands to
1-adrenergic agonists, we examined by Northern
blot analysis NHE1 expression in saline-treated mice and mice 30 min
and 2, 6, and 24 h after isoproterenol injection. NHE1 transcripts
increased ~50% by 2 h, and a more than twofold increase was
noted at 24 h. Isoproterenol did not acutely increase
Na+/H+ exchanger activity; however, exchanger
activity was significantly elevated by 24 h. To test whether NHE1
activity is essential for inducing salivary gland hypertrophy in vivo,
mice with targeted disruption of Nhe1 were treated with
isoproterenol. Na+/H+ exchanger activity was
absent in acinar cells from Nhe1
/
mice,
nevertheless, the lack of NHE1 failed to inhibit isoproterenol-induced hypertrophy. These data directly demonstrate that acinar cell hypertrophy induced by chronic
1-adrenergic receptor
stimulation occurs independently of NHE1 activity.
Na+/H+ exchanger activity; salivary gland; acinar cells
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INTRODUCTION |
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IN RESPONSE TO
CHRONIC 1-adrenergic receptor stimulation, rodent
parotid glands undergo a 3- to 5-fold increase in mass (6, 34). Gland enlargement is due to both hyperplasia and
hypertrophy of the secretory acinar cells (2, 34). The
early responses to
1-adrenergic receptor stimulation
include increased expression of transcription factors and signal
transduction molecules involved in RNA transcription (21,
43), DNA synthesis (3, 8, 42), and RNA synthesis of
salivary gland-specific proteins (1, 9). However, the
mechanism by which
1-adrenergic receptor activation
initiates gland hypertrophy remains unclear.
An increase in the intracellular pH of mammalian cells is often
mediated by stimulation of Na+/H+ exchanger
(NHE) activity. This enhanced Na+/H+ exchange
may be necessary for initiating proliferation in many, but not all,
tissues and cell lines (16, 17, 25, 30, 37, 38). Cells
lacking NHE activity fail to grow in media of low pH (19)
or when NHE activity is inhibited (12). In addition to its
role in cell proliferation, activation of
Na+/H+ exchanger activity has also been linked
to cell hypertrophy (15). Although considerable evidence
supporting the involvement of Na+/H+ exchange
in the initiation of cell proliferation/hypertrophy has been generated,
this relationship is primarily based on indirect evidence
derived from experiments that (necessarily) employed solutions lacking
a HCO
The mammalian NHE gene family consists of six isoforms
(10, 23). Of these, the ubiquitously expressed NHE1
isoform is the major regulator of the intracellular pH in rodent
salivary gland acinar cells (14, 20, 22, 24, 28). NHE1 is
involved in cell volume regulation (18, 19) and is a
target for growth factor-induced cell proliferation (31,
41). Indeed, cell proliferation correlates with increased levels
of NHE1 mRNA (13, 26), which is likely due to direct
activation of the NHE1 promoter (5). Thus NHE1 may play a
key role in initiating both the hypertrophy and hyperplasia of salivary
acinar cells associated with chronic 1-adrenergic
receptor activation, although this relationship has never been directly tested.
To examine the potential connection among
1-adrenergic receptor stimulation, intracellular pH
homeostasis, and gland hypertrophy, we studied by Northern blot
analysis NHE1 expression and the effects of Nhe1 gene
disruption on mouse parotid gland hypertrophy. An early response to
1-adrenergic receptor activation was enhanced expression
of NHE1 transcripts, and this increase correlated with increased
Na+/H+ exchanger activity. Nevertheless, the
extent of salivary gland enlargement in
Nhe1
/
mice in response to
1-adrenergic receptor stimulation was comparable to that
observed in wild-type mice, clearly demonstrating that functional NHE1
protein is not required for in vivo induction of acinar cell
proliferation and/or hypertrophy.
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METHODS |
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Materials. Collagenase P was purchased from Boehringer Mannheim (Indianapolis, IN), and 2',7'-bis(carboxyethyl)-5-carboxyfluorescein-pentaacetoxymethyl ester (BCECF-AM) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were from Molecular Probes (Eugene, OR). All other chemicals were obtained from Sigma Chemical (St. Louis, MO). Six- to seven-week-old male C57BL/6 mice were obtained from Harlan (Indianapolis, IN). Targeted disruption of the murine Nhe1 gene was performed as previously described (4). Heterozygous offspring were used to establish a breeding colony in the University of Rochester vivarium. Experiments were performed on animals aged between 1.5 and 4 mo. All animals were fed ad libitum on a standard diet and water.
Isoproterenol treatment. Mice were given a single intraperitoneal injection of (±)-isoproterenol hydrochloride (25 mg/kg prepared in 140 mM NaCl). Control mice received vehicle only. After 30 min, 2, 6, and 24 h, and 7 days of isoproterenol exposure, mice were euthanized by exsanguination after CO2 anesthesia, and the parotid glands were removed and snap frozen in liquid nitrogen for subsequent RNA isolation. For functional studies, parotid glands were removed from mice treated for 24 h or 7 days with either isoproterenol or vehicle, and acinar cells were isolated as previously described (14). For morphological analysis, wild-type, heterozygous, and mice with targeted disruption of the Nhe1 gene were treated daily with saline or isoproterenol for 7 days. Twenty-four hours after the final injection, mice were euthanized and parotid, submandibular, and sublingual gland weights were measured.
Morphology. Parotid glands were fixed in 10% formalin, paraffin imbedded, sectioned at 10 µm, and stained with hematoxylin and eosin. Images were generated using a SPOT digital camera (Diagnostics Instruments) with a Nikon Plan Apo ×10/0.3 objective or a Nikon Plan Apo 60×/1.4 oil objective and a Nikon Eclipse E800 microscope. The ratio of acinar cells to duct cells was quantitated essentially as previously described (33). In brief, intersecting gridlines were superimposed on randomly selected computer images generated at ×100 magnification, and the intersections over acinar, ductal, and nonparenchymal tissues were recorded. The ratio of acini to ducts in the gland equals the total number of points over acini divided by the total number of points over ducts. To determine the size of the acinar elements, the long and short axes were measured on randomly selected computer images generated at ×600 magnification and converted to cross-sectional area.
Intracellular pH measurements. The acinar cell preparation was loaded with intracellular pH-sensitive fluoroprobe by incubation for 30 min at room temperature with BCECF-AM (2 µM). BCECF-loaded acinar cells were allowed to adhere to the base of a superfusion chamber mounted on a Nikon Diaphot 200 microscope interfaced with an imaging workbench (Axon Instruments, Foster City, CA). Cells were excited at 490 and 440 nm, and emitted fluorescence was measured at 530 nm. Solutions contained (in mM): 135 NaCl, 5.4 KCl, 0.4 KH2PO4, 0.33 NaH2PO4, 0.8 MgSO4, 1.2 CaCl2, 10 glucose, and 20 HEPES, pH 7.4, with Tris base. To induce an intracellular acid load, 10 mM NaCl was replaced with NH4Cl (29). Solutions were gassed with 100% O2.
Intracellular pH was estimated by in situ calibration of the F490/F440 fluorescence ratio with the use of the nigericin-high K+ method of Thomas et al. (39). The high K+ solution contained (in mM): 120 KCl, 20 NaCl, 0.8 MgCl2, 20 HEPES, and 0.005 nigericin, and the pH was adjusted from 5.6 to 8. Data presented in the figures are from single representative experiments. Values quoted are the means ± SE for the number of acinar aggregates examined. All experiments were performed with three or more separate preparations.Northern blot analysis.
Total RNA was isolated from parotid glands with TRIzol reagent (Life
Technologies, Rockville, MD) according to the manufacturer's instructions, fractionated by electrophoresis in a 1%
formaldehyde-agarose gel (20 µg per lane), and transferred to
Hybond-XL nylon membranes (Amersham Pharmacia, Piscataway, NJ). Parotid
glands from the five animals comprising each group were combined to
generate the total RNA. The blot was hybridized first with a
32P-labeled cDNA probe containing nucleotides 803-1393 of
the mouse NHE1 open reading frame (ORF) in ExpressHyb solution
(Clontech Laboratory, Palo Alto, CA) by use of the hybridization and
wash conditions recommended by the manufacturer. After autoradiography, the blot was stripped in 0.1% SDS at 90°C for 20 min and then hybridized as above to a 32P-labeled cDNA probe containing
nucleotides 2451-2720 of the rat -actin ORF. Finally, to normalize
RNA expression between preparations, the blot was restripped and then
hybridized to an end-labeled oligonucleotide that recognizes mouse 18S
ribosomal RNA (5'-TATTGGAGCTGGAATTACCGCGGCTGCTGG-3'). Quantitation
of the autoradiographs was performed by densitometry using the Alpha
Imager system (Alpha Innotech, San Leandro, CA).
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RESULTS |
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Enhanced expression of NHE1 transcripts after
1-adrenergic receptor stimulation.
One potential mechanism for inducing parotid gland hypertrophy in
response to isoproterenol stimulation is to increase the expression of
NHE1 in acinar cells, the major exchanger isoform expressed in this
cell type (14, 20, 22, 24, 28). Northern blot analysis of
total RNA with the use of a cDNA probe verified that expression of the
major 2.9-kb NHE1 mRNA was enhanced in parotid glands stimulated with
isoproterenol (Fig. 1A, top).
No detectable change was observed in NHE1 expression after 30-min exposure to isoproterenol, but after 2 h an increase was noted, and expression appeared to increase further 24 h after injection of isoproterenol. This blot was then rehybridized with
-actin and
18S probes. Figure 1A (middle) demonstrates that
the level of 1.9-kb transcripts for the structural protein
-actin
increased in a comparable fashion to NHE1 in the parotid glands of
stimulated mice, whereas 18S expression was stable (±5% of saline
treated; Fig. 1A, bottom). After normalization of expression
of NHE1 to 18S (Fig. 1B), no change was observed for NHE1
mRNA expression after 30-min stimulation, but an ~50% increase was
noted after 2 h, and NHE1 transcripts increased more than twofold
after 24 h of stimulation. Although the mechanism for enhanced
expression is unclear, these results demonstrate that NHE1 expression
in mouse parotid glands is upregulated within 2 h after a single injection of isoproterenol.
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1-adrenergic receptor stimulation increases
Na+/H+
exchanger activity in parotid acinar cells.
The increased expression of NHE1 mRNA suggests that this
Na+/H+ exchanger may be required for the
isoproterenol-induced gland hypertrophy (Fig. 1).
Consequently, if NHE1 plays such a role, it was predicted that enhanced
expression would result in increased activity. In agreement with this
hypothesis, the Na+/H+ exchanger activity in
acinar cells isolated from isoproterenol-treated mice was greater than
the exchanger activity in cells from saline-treated animals (Fig.
2A). The rectangular area in
Fig. 2A was enlarged (inset) to show clearly that
the initial rate of the recovery on extracellular Na+
addition was about twofold faster for isoproterenol-treated mice than
for saline-treated controls (Fig. 2B). Figure 2C
also shows that the intracellular pH "set point" was raised ~0.15
pH unit in acinar cells 24 h after isoproterenol stimulation. The
aforementioned results are consistent with previous studies in which
NHE1 activity was enhanced in a similar fashion after exposure to
mitogenic agents in a heterologous expression system (41).
Thus acinar cells were exposed to isoproterenol for 5 min to determine
whether activation of acinar Na+/H+ exchange
occurs acutely or requires chronic
1-adrenergic receptor stimulation. Figure 3A shows
that acute exposure to
1-adrenergic agonist did not
increase Na+/H+ exchanger activity in vitro,
indicating that chronic exposure is required for the
isoproterenol-induced response. Indeed, under these experimental
conditions, isoproterenol acutely inhibited activity ~15%. In
contrast, the Ca2+-mobilizing agonist carbachol or cell
shrinkage acutely upregulated NHE1 activity in mouse parotid acinar
cells (14), and this response is comparable in magnitude
to that detected during chronic isoproterenol treatment (Fig. 2).
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Loss of Nhe1 gene expression fails to disrupt
1-adrenergic receptor stimulation-induced gland
hypertrophy.
To examine the effects of Nhe1 gene disruption on the
1-adrenergic receptor-stimulated gland hypertrophy,
salivary gland wet weights from isoproterenol- and saline-treated null
mutant, heterozygous, and wild-type mice were determined. On the basis of the enhanced Na+/H+ exchanger activity in
parotid glands of isoproterenol-treated mice, our prediction was that
hypertrophy would be inhibited in knockout mice. However, Fig.
4 illustrates that the wet weights of
parotid and submandibular glands (Fig. 4, A and
B, respectively) from NHE1-deficient mice were comparable to
those in wild-type and heterozygous littermate mice after treatment
with isoproterenol for 7 days. In agreement with previous results
(32), no significant change in sublingual gland weight was
induced by chronic isoproterenol treatment (Fig. 4C).
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Morphology of the parotid gland in NHE1-deficient mice after
isoproterenol treatment.
It has previously been reported that chronic isoproterenol treatment
increases gland mass by increasing both the number and the size of
acinar cells (2, 3, 6, 32, 34). One possibility is that
the increase in the wet weight observed in NHE1-deficient mice
represents expansion of a nonacinar cell type. To test this hypothesis,
parotid glands of Nhe1+/+ and
Nhe1/
mice were examined by light microscopy.
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DISCUSSION |
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Several lines of evidence suggest that
Na+/H+ exchange plays an active role in the
induction of the hyperplastic response to mitogenic agents in many
different cell types (5, 13, 16, 25, 30, 36, 38, 41). The
mechanism through which Na+/H+ exchangers are
thought to regulate this response is to maintain an alkaline
cytoplasmic pH (17); however, direct verification of this
proposed mechanism has never been reported. Indeed, current evidence
supporting the involvement of Na+/H+ exchange
in the initiation of cell proliferation/hypertrophy relies on indirect
evidence in which HCO1-adrenergic receptor stimulation-induced salivary gland
hypertrophy. The functional effects of disrupting the expression of the
murine Nhe1 gene have been described in several tissues
(4, 11), including salivary glands (14, 22).
It is interesting to note that NHE1 expression appears not to be
critical during early development, but after birth, knockout mice begin
to grow slower than their wild-type and heterozygous littermates
and seizures and ataxia develop.
1-Adrenergic receptor stimulation increased NHE1 mRNA
levels in the parotid gland within 2 h (Fig. 1). Many mitogenic
agents have been shown to increase Na+/H+
exchanger activity and NHE1 expression in other systems as well (5, 7, 13, 25, 27, 35-37, 40, 41). Enhanced NHE1 expression in the parotid gland is possibly due to increased
transcription. A similar phenomenon has been noted in NIH/3T3 cells
expressing the mouse NHE1 promoter, in which a variety of mitogenic
factors activated the NHE1 promoter, linking
Na+/H+ exchanger activity to cell growth and
proliferation (5). Regardless of the mechanism that
mediates the increase in NHE1 transcript expression,
1-adrenergic receptor stimulation also produced an alkaline shift in Na+/H+ exchanger activity,
generating an increase in the intracellular pH (Fig. 2). This increased
Na+/H+ exchanger activity was due to
upregulation of NHE1, because knockout of the Nhe1 gene
virtually eliminated exchanger activity (Fig. 3). These results are
consistent with the observation that the Nhe1 gene product
is the major regulator of intracellular pH in this cell type (14,
22) and also demonstrates that chronic isoproterenol treatment
does not induce the expression of another Na+/H+ exchanger isoform to compensate for the
loss of NHE1. Moreover, muscarinic receptor activation and cell
shrinkage induce upregulation of Na+/H+
exchanger activity, and this enhanced activity is inhibited in parotid
acinar cells isolated from NHE1-deficient mice (14).
In Na+/H+ exchanger-deficient Chinese hamster
ovary cells, other NHE isoforms can support cell proliferation
(19); however, NHE1 appears to be the major, if not the
only, regulator of intracellular pH in this cell type (Fig. 4; see
Refs. 14, 22). Despite the lack of
upregulation of Na+/H+ exchanger activity after
1-adrenergic receptor stimulation in NHE1-deficient
mice, chronic isoproterenol treatment produced salivary gland
enlargement (Fig. 5), largely due to acinar cell hypertrophy (Table 1).
These results clearly demonstrate that upregulation of NHE activity is
not necessary for the isoproterenol-induced hyperplasia/hypertrophy.
Although Na+/H+ exchanger activity may be
permissive in this regard in some cell types (12, 15, 19),
this is clearly not the case in parotid acinar cells. Activation of
other factors that regulate salivary gland-specific gene expression may
lead to
1-adrenergic receptor-stimulated gland
hypertropy (21, 42, 43).
In conclusion, chronic 1-adrenergic receptor stimulation
increased the Na+/H+ exchanger activity in
mouse parotid acinar cells by enhancing the expression of NHE1
transcripts. Nevertheless, Nhe1 knockout mice clearly
demonstrated that, in vivo, isoproterenol-induced salivary gland
hypertrophy does not require the intracellular alkalinization
associated with expression of this gene. Thus the factor(s) responsible
for the hyperplasia/hypertrophy induced by isoproterenol in parotid
acinar cells remains unknown. Future studies in this area may benefit
from the recent development of microarray technologies that provide a
simultaneous quantitative readout of thousands of gene transcripts.
This latter approach will likely uncover the transcription factors,
signaling pathways, structural proteins, and other elements involved in
the development of salivary gland hypertrophy.
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ACKNOWLEDGEMENTS |
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We are indebted to Drs. Lawrence Tabak and Art Hand for their input and support during the course of these studies. We thank Linda Richardson and Marlene Balys for technical assistance with genotyping, injection of the animals, and isolation of the salivary gland tissues.
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FOOTNOTES |
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This work was supported in part by National Institutes of Health Grants DE-13539, DE-08921, and DE-09692 (J. E. Melvin).
Address for reprint requests and other correspondence: J. E. Melvin, Center for Oral Biology, Univ. of Rochester, Medical Center Box 611, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: james_melvin{at}urmc.rochester.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 31 July 2000; accepted in final form 25 October 2000.
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