in response to chronic β₁-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 β₁-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 β₁-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${}_3^{-}$-CO₂ buffering system and/or, in some cases, inhibitors to infer the role of Na⁺/H⁺ exchanger function (17). Therefore, the correlation of enhanced Na⁺/H⁺exchanger gene expression and activity to cell proliferation and hypertrophy remains to be proven.

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 β₁-adrenergic receptor activation, although this relationship has never been directly tested.

To examine the potential connection among β₁-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 β₁-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 inNhe1^−/− mice in response to β₁-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.

METHODS

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 Nhe1gene 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 a³²P-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 β-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).

RESULTS

Enhanced expression of NHE1 transcripts after β₁-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 β-actin and 18S probes. Figure 1A (middle) demonstrates that the level of 1.9-kb transcripts for the structural protein β-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.

β₁-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 2Calso 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 β₁-adrenergic receptor stimulation. Figure 3A shows that acute exposure to β₁-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).

To verify that the increased Na⁺/H⁺ exchanger activity shown in Fig. 2 was associated with NHE1 expression, NHE1-deficient mice were treated with isoproterenol. Figure3B shows that 24 h after a single injection and after treatment for 7 days with isoproterenol, parotid acinar cells isolated from Nhe1^−/− mice lacked Na⁺/H⁺ exchanger activity (>95% loss of activity). Thus these results clearly demonstrate that increased Na⁺/H⁺ exchanger activity correlated with NHE1 expression and that chronic β₁-adrenergic receptor stimulation did not induce expression of another Na⁺/H⁺ exchanger in theNhe1^−/− mice to compensate for the loss of this pH regulatory pathway.

Loss of Nhe1 gene expression fails to disrupt β₁-adrenergic receptor stimulation-induced gland hypertrophy.

To examine the effects of Nhe1 gene disruption on the β₁-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 andB, 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).

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^+/+ andNhe1^−/− mice were examined by light microscopy.

In agreement with previous studies (4, 14), homozygousNhe1^−/− mice exhibited decreased rates of postnatal growth, resulting in significantly lower body weights than their wild-type littermates. In the present experiments, the mean body weight was 27.0 ± 1.3 g for wild-type animals (n = 6), 26.2 ± 1.3 g for heterozygous mice (n = 6), and 17.2 ± 0.9 g forNhe1 mutant mice (n = 7; P< 0.01 vs. Nhe1^+/+ orNhe1^+/−, Student's t-test). However, the reduced body weight of NHE1-deficient mice was not associated with a decrease in parotid gland weight (Fig. 4): parotid weights were 48.2 ± 5.6 (Nhe1^+/+,n = 12), 49.7 ± 2.8 (Nhe1^+/−, n = 12) and 50.5 ± 3.4 mg (Nhe1^−/−, n = 14). No obvious morphological differences were observed betweenNhe1^+/+ or Nhe1^−/−parotid glands (Fig. 5, Aand C, respectively) from saline-treated mice. Morphometric analyses were performed to verify these observations. Table 1 shows that the ratios of acinar to ductal elements in the glands were equivalent. Moreover, a comparable degree of acinar cell hypertrophy was clearly evident inNhe1^+/+ andNhe1^−/− parotid glands after chronic treatment with isoproterenol for 7 days (Fig. 5, B andD, respectively, and Table 1). Because the acinar-to-duct ratio increased about threefold in both wild-type and knockout mice, this suggests that the number and/or the size of the acinar cells increased dramatically with isoproterenol treatment. In fact, the cross-sectional area of acinar cells increased about fivefold inNhe1^+/+ and Nhe1^−/−parotid glands, demonstrating that the increase in the acinar-to-duct ratio was due primarily to an increase in the size of the acinar cells. Consistent with the glandular hypertrophy correlating with acinar cell enlargement, the percentage of nonparenchymal tissue decreased after chronic β₁-adrenergic receptor stimulation.

DISCUSSION

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 HCO${}_3^{-}$-CO₂-free buffering systems or amiloride analogs were used to test the role of Na⁺/H⁺ exchanger function (17). Thus it not clear whether Na⁺/H⁺ exchange plays an active role in the induction of hyperplasia under physiological conditions or is independent of nonspecific effects of inhibitors on cell proliferation. Thus the objective of the current study was to determine, with the use of knockout mice, the role of NHE1 in the β₁-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.

β₁-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, β₁-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 β₁-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 β₁-adrenergic receptor-stimulated gland hypertropy (21, 42, 43).

In conclusion, chronic β₁-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.

FOOTNOTES

• 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:[email protected]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.