Inhibition of NHE-1 Na+/H+ exchanger by natriuretic peptides in ocular nonpigmented ciliary epithelium

Pawel Fidzinski,1 Mercedes Salvador-Silva,1 Lars Choritz,1 John Geibel,2,3 and Miguel Coca-Prados1

1Department of Ophthalmology and Visual Science, 2Department of Surgery, and 3Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510

Submitted 5 December 2003 ; accepted in final form 22 April 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The natriuretic peptides (NPs) atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) display hypotensive effects in the mammalian eye by lowering the intraocular pressure (IOP), a function that is mediated by the bilayer ocular ciliary epithelium (CE), in conjunction with the trabecular meshwork. ANP regulates Na+/H+ exchanger (NHE) activity, and inhibitors of NHE have been shown to lower IOP. We examined whether NPs influence the NHE activity of the CE, which is comprised of pigmented (PE) and nonpigmented (NPE) epithelial cells, by directly recording the rate of intracellular pH (pHi) recovery from its inner NPE cell layer. NPs inhibited, in a dose-dependent manner (1–100 nM), the rate of pHi recovery with the order of potency CNP > ANP > BNP, indicative that this inhibition is mediated by the presence of NPR type B receptors. 8-Bromo-cGMP (8-BrcGMP), a nonhydrolyzable analog of cGMP, mimicked NPs in inhibiting the rate of Na+-dependent pHi recovery. In contrast, ethylisopropyl amiloride (EIPA, 100 nM) or amiloride (10 µM) completely abolished the pHi recovery by NHE. 18{alpha}-Glycyrrhetinic acid (18{alpha}-GA), a gap junction blocker, attenuated the inhibitory effect of CNP on the rate of pHi recovery, suggesting that NHE activity in both cell layers of the CE is coregulated. This interpretation was supported, in part, by the coexpression of NHE-1 isoform mRNA in both NPE and PE cells. The mechanism by which the inhibitory effect of NPs on NHE-1 activity might influence the net solute movement or fluid transport by the bilayer CE remains to be determined.

Na+/H+ exchanger type 1; intracellular pH; aqueous humor


NATRIURETIC PEPTIDES (NPs) are a family of bioactive polypeptides that includes three different prohormones: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) (34, 42). NPs fulfill functions in fluid homeostasis, vasodilatation, and contraction and are involved in the pathophysiology of several diseases, including hypertension, congestive heart failure, renal and cardiovascular diseases (26, 47), and perhaps glaucoma (46). The function of NP is mediated by the activation of three distinct receptors (NPR-A, NPR-B, and NPR-C) of which NPR-A and NPR-B are known to elicit intrinsic guanylyl cyclase activity (45). NPR-C, in contrast, lacks the COOH-terminal guanylyl cyclase domain that characterizes NPR-A and NPR-B and functions primarily as a clearance receptor. Interestingly, NPR-C-selective analogs (i.e., C-ANP4–23) also inhibit adenylate cyclase activity in a cGMP-independent fashion. The NP binding of NPR-A and NPR-B causes the generation of intracellular cGMP that leads to the activation of a protein kinase G (PKG). Downstream kinase activities include regulation of ion channels, substrate protein phosphorylation, and cellular proliferation (26).

In glaucoma, elevation of the intraocular pressure (IOP) has been reported to be an important risk factor in the development of the disease, one of the leading causes of blindness in the world (41). A balance between aqueous humor secretion by the ciliary epithelium (CE) and its drainage throughout the trabecular meshwork regulates IOP. Previous findings indicate that NPs reduce IOP in several species including monkey, rabbit, and bovine (14, 37, 39). In clinical studies, a hypotensive effect has been reported in humans and in experimental animals with elevated IOP (14, 20, 48). It is generally believed that NPs exert their effects on IOP by reducing the rate of aqueous humor secretion by the CE, although involvement of the trabecular meshwork and the outflow pathway has also been proposed (7, 32). To date, many of the components that comprise the NP system have been identified in the CE, including NPs and their cognate receptors (15, 31). In the aqueous humor ANP and BNP have been found in both healthy and glaucomatous eyes, with elevated levels in the former (35, 36). In the rabbit glaucoma eye model (buphthalmus), downregulation of NPR receptors has been reported in ciliary processes (16).

Aqueous humor formation is a complex process of ion, water, and protein transport through the CE in a transcellular and paracellular manner. Net secretion of the aqueous fluid is considered to occur toward the posterior chamber in the pars plicata region of the CE. However, it was also recently suggested to contain the necessary mechanisms supporting fluid reabsorption (5). In part this view is supported by anatomic, biochemical, and physiological differences along the CE (18, 19, 28).

The current understanding of pivotal ionic transport mechanisms involved in aqueous humor secretion includes a variety of channels and transporters, including the paired Na+/H+ exchanger (NHE) and Cl/HCO3 exchangers (AE), the bumetanide-sensitive Na+-K+-2Cl cotransporter, and Na+-K+-ATPase (5).

Each of these ion transport proteins is believed to play a role in the modulation and maintenance of IOP. Recently, Avila et al. (3) found that inhibitors of the NHE lower IOP when applied to anesthetized mice. Little, however, is known about the nature of the endogenous effector molecules that regulate IOP, and in particular whether NPs may serve as signaling molecules in this mechanism. Earlier studies have been suggestive of the effect of NPs on Na+ and K+ channels and their inhibitory effect on aquaporin channels (4, 23). In kidney and heart, NPs influence ion transport activities of the NHE and/or Na+-K+-2Cl cotransporter (26). These transport systems are also believed to contribute to fluid secretion in the CE. In the present study we examined the physiological role of NPs on the NHE expressed in the ocular CE. The NHE are among the major transporters involved in cell volume regulation. Their activation leads to a cellular influx of Na+ and extrusion of H+, resulting in a net import of Na+. In many systems NHE functions in parallel to AE, resulting in the uptake of NaCl and osmotically obliged water. The export of protons may link NHE activation to changes in intracellular pH (pHi), and the NHE can independently respond to alterations of pHi and cell volume. NP has been shown to either stimulate or inhibit NHE. In the present work we set out to determine the pHi responses of bovine and rat nonpigmented epithelial (NPE) cells in intact ciliary processes. We identified and characterized a functional NHE isoform in this tissue, NHE-1, and we showed that NPs exhibit a profound effect on the rate of pHi recovery.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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All studies were approved by the Yale University Animal Care and Use Committee and followed the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society.

Source of tissue. Bovine eyes were obtained from 6- to 12-wk-old calves through a local abattoir, and rat eyes were enucleated from Sprague-Dawley rats weighing 150–250 g (Charles River Laboratories, Wilmington, MA). Single strips of ciliary processes from the pars plicata region of the CE were microdissected from the ciliary body as previously described (18).

Chemicals and solutions. The following solutions were used in perfusion experiments (in mM): 1) HEPES-buffered Ringer solution: 115 NaCl, 5 KCl, 1 CaCl2, 1.2 MgSO4, 2 NaH2PO4, 32.2 HEPES, and 10 glucose; 2) HEPES-buffered NH4Cl solution: 20 NH4Cl, 95 NaCl, 5 KCl, 1 CaCl2, 1.2 MgSO4, 2 NaH2PO4, 32.2 HEPES, 10 glucose, and 8 mannitol; 3) HEPES-buffered Na+-free solution: 122 N-methyl-D-glucamine (NMDG), 3 KCl, 1 CaCl2, 1.2 MgSO4, 2 KH2PO4, 32.2 HEPES, 10 glucose, and 15 mannitol. All solutions were preheated to 37°C and adjusted to a pH of 7.4 before the experiment. The following drugs (obtained from Sigma, St. Louis, MO) were added to the solutions at concentrations indicated in Figs. 25: ANP, CNP, amiloride, bumetanide, benzamil, 8-bromo-cGMP (8-BrcGMP), lysophosphatidic acid (LPA) 18{alpha}-glycyrrhetinic acid (18{alpha}-GA). BNP was purchased from Bachem (Bubendorf, Switzerland), ethylisopropyl amiloride (EIPA) and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein were obtained from Molecular Probes (Eugene, OR), HOE-694 was a gift from Carsten Wagner (University of Zurich, Zurich, Switzerland), and A-71915 was a gift from Abbott Laboratories. With the exception of bumetanide, which was added after the NH4+ pulse (because of inhibition of NH4+ uptake; see Fig. 1), all drugs were added over the entire course of the experiment. The concentration of solvent reagents used for dissolving the drugs (dimethyl sulfoxide or ethanol) never exceeded 0.1%.



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Fig. 2. Expression and functional properties of Na+/H+ exchanger isoform 1 (NHE-1) in bovine ciliary NPE cells. A: detection of NHE-1 transcripts by RT-PCR in the bovine ciliary processes (CB) and cultured CE cells (NPE and PE). The predicted 359-bp DNA product (arrow) amplified by PCR from CB, NPE, and PE cells was resolved in a 1.2% agarose gel and stained with ethidium bromide. B: the NHE-1-specific blocker HOE-694 (1 µM) completely inhibited the Na+-dependent pHi recovery in bovine NPE cells. Similar results were obtained in 4 other experiments. Filled bars at top are as described in Fig. 1. Inhibitor was added to the cells over the entire experiment. C: effects of NHE inhibitors HOE-694 (1 µM), ethylisopropyl amiloride (EIPA; 100 nM and 10 µM), and amiloride (10 µM) compared with control on the NHE activity calculated as change of pHi per minute ({Delta}pHi/min). Arrows indicate NHE inhibitors that at higher concentrations can also block NHE-2/NHE-3 activities. Data are presented as means ± SE; n = 4–7 independent experiments with a total of 29–64 cells/experiment. *Significant difference (P < 0.001) vs. control.

 


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Fig. 5. Summary of effects by CNP and NP receptor (NPR) antagonists on NHE-1 activity in NPE cells. The inhibitory effect elicited by CNP (100 nM) on the NHE-1 activity is given a value of 1 and that in the absence of CNP (control of inhibition) a value of 0. In the presence of A-71915 (1 µM), a NPR-A blocker, there is a significant reversal effect on the inhibition of NHE-1 by CNP. However, lysophosphatidic acid (LPA; 10 µM), a NPR-B blocker, completely reversed the effect of CNP when added together (CNP + LPA) or when added with CNP plus A-71915 (CNP + A-71915 + LPA). Interestingly, LPA, but not A-71915, when added alone exhibited a stimulatory effect on NHE-1 activity.

 


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Fig. 1. Representative intracellular pH (pHi) recorded from a single bovine ciliary nonpigmented epithelial (NPE) cell under steady-state conditions. NPE cells in the intact ciliary epithelium (CE) were loaded with protons by an NH4+ pulse (phase a). Subsequent removal of NH4+ and Na+ led to acidification (phase b), which dropped the baseline to an even lower pHi level. Addition of Na+ (phase c) resulted in alkalinization (phase d) reversing pHi to a level observed before the NH4+ pulse (phase a). Filled bars indicate length of exposure (in s) to buffer solutions indicated.

 
Reverse transcription-polymerase chain reaction. Total RNA was isolated from bovine processes and from cultured ciliary pigmented epithelial (PE) and NPE cells with TRIzol reagent (Invitrogen, Carlsbad, CA), and cDNA was synthesized in vitro with a reverse transcription-polymerase chain reaction (RT-PCR) kit (Stratagene, La Jolla, CA). PCR was carried out with sets of primers based on published cDNA nucleotide sequences of bovine NHE-1 (GenBank accession no. AJ131763) and NHE-3 (GenBank accession no. AJ131764). The oligonucleotide primers used in PCR were forward: (nt 47–69) 5'-TCAAAATGTGGAGCA GCGTCA GT-3' and reverse: (nt 389–405) 5'-CAGGGGCCGAATGGTCA-3' for NHE-1 and forward: (nt 65–88) 5'-GTGTACAGGGCCATAGGGGTCATC'-3 and reverse: (nt 605–624) 5'-TTGTCTGTGCTGGGGGAACG-3' for NHE-3. The expected DNA size products in PCR reactions were 359 bp for NHE-1 and 560 bp for NHE-3. The predicted DNA products amplified by PCR were gel purified and sequenced in an automated DNA sequencer ABI PRISM 310 Genetic Analyzer, with the rhodamine terminator cycle sequencing ready reaction from a DNA sequencing kit (PE Applied Biosystems, Foster City, CA). Nucleotide sequences were aligned and verified to share 100% homology with the corresponding published bovine cDNA sequences in GenBank.

pHi measurements and BCECF fluorescence imaging. Freshly microdissected ciliary processes from bovine and rat eyes were placed on top of coverslips precoated with CellTAK tissue adhesive (BD Bioscience, Bedford, MA) and incubated with 10 M BCECF-acetoxymethyl ester (Molecular Probes) for 10 min. Coverslips were then transferred to a preheated (37°C) perfusion chamber on an Olympus IX70 inverted microscope with a x20 objective and washed with HEPES-buffered Ringer solution to remove any residual deesterified dye. After excitation at 490 and 440 nm with a Lambda DG-4 light source (Sutter Instrument, Novato, CA), the fluorescent signal emission was recorded at 535 nm with a Quantix camera (Roper Scientific, Tucson, AZ). Recordings were made from the NPE cell layer only because the PE layer did not show an uptake of the BCECF dye and the pigment absorbed the fluorescent signal. Data was collected every 15 s and processed with MetaFluor software (Universal Imaging, Downingtown, PA). pHi was calculated from the 490 nm-to-440 nm intensity ratio with the high K+/nigericin calibration method (40).

In the course of the experiment the cells were acidified by perfusion with NH4Cl-HEPES-buffered solution for 4 min followed by Na+-free HEPES solution. After readdition of HEPES-buffered Ringer solution the realkalinization rate was monitored to calculate Na+-dependent pHi recovery. NHE activity was calculated as change of pHi per minute ({Delta}pH/min) from the gradient during Na+-dependent pHi recovery. Because the activity of NHE transporters is pHi dependent (2) and the cells varied in pHi after acidification, we chose a fixed pHi value for all cells that was used as a start point for calculation of activity and thus avoided changing intracellular buffering power. The fixed pHi values were 6.80 for bovine NPE cells and 7.04 for rat NPE cells.

Indirect immunofluorescence. Cryostat sections (1–2 µm thick) of the bovine and rat CE were prepared as described previously (18). Sections were incubated with polyclonal antibodies against ANP, BNP, or CNP (Phoenix Pharmaceuticals) diluted 1:100 in 10% normal horse serum containing 1% BSA in PBS for 2 h at 37°C in a humidified atmosphere. After being washed with 1% BSA in PBS, the sections were incubated for 1 h at 37°C with the secondary antibody (rhodamine-conjugated anti-rabbit immunoglobulin) followed by three 10-min washes in PBS. The sections were mounted in a solution of glycerol (pH 7.0) and analyzed in a Zeiss microscope equipped with epifluorescent microscopy. Antibodies against the NHE-1 isoform were the generous gift of Dr. Sergio Grinstein (Division of Cell Biology, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada) and were characterized previously (21). Antibodies to Na+-K+-ATPase at a dilution of 1:100 were used as previously described (19).

Statistical analysis. Unpaired Student's t-test was used to determine significance between two experiments; P values <0.05 were considered to be statistically significant. If not otherwise stated, data are given as means ± SE. The number of cells recorded (n) during one experiment varied between 8 and 12, and each experiment was conducted at least four times.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
pHi recording of NPE cells in ciliary epithelium. The pHi of bovine and rat NPE cells was recorded as described in MATERIALS AND METHODS. Under control conditions, strips of ciliary processes were incubated with pH-sensitive dye (BCECF) and the pHi was monitored and recorded from single cells in the intact CE under HCO3-free conditions with a digital imaging system. Figure 1 shows a representative profile of the pHi recorded from a bovine NPE cell in response to an acid load with 20 mM NH4Cl, followed by Na+-free solution and subsequent return to a Na+-containing solution. The rise in pHi induced by a NH4+ pulse (phase a) led to an acidification (phase b) in the absence of Na+ (Fig. 1). The addition of Na+ (phase c) resulted in a realkalinization (phase d) and recovery of pHi to control normal values (Fig. 1).

Under these conditions the mean pHi in the bovine NPE cells was 7.096 ± 0.018 (n = 64), whereas the mean pHi was 7.392 ± 0.031 in rat NPE cells (n = 48). After acidification, bovine NPE cells reached a pHi between 6.90 and 7.10, whereas rat NPE cells showed a pHi between 6.65 and 6.85. The alkalinization rate was calculated on the basis of the fixed set points after readdition of Na+ as indicated in MATERIALS AND METHODS. The alkalinization rate was estimated at 0.162 ± 0.005 pH/min (n = 48) in bovine NPE cells and 0.234 ± 0.007 pH/min (n = 48) in rat NPE cells.

Molecular identity and functional characteristics of NHE expressed in bovine NPE cells. To characterize the molecular and functional properties of the NHE subtypes expressed in bovine CE, we first carried out RT-PCR on cDNA synthesized in vitro. At least nine NHE isoforms have been cloned so far, of which only NHE-1 and NHE-3 bovine sequences are available. Sets of oligonucleotide pair primers were selected for bovine NHE-1 and NHE-3 (see MATERIALS AND METHODS) and annealed to bovine cDNA prepared from bovine ciliary processes and bovine NPE and PE cells. Of the two sets of pair primers used, only the NHE-1 primers amplified a DNA product of the expected size (359 bp) in intact CE and NPE and PE cells (Fig. 2A). No PCR DNA product was amplified with the NHE-3 primers. Nucleotide sequencing of the 359-bp DNA product confirmed that it shared 100% homology with the published cDNA sequence for the bovine NHE-1 isoform.

We next investigated functional properties of the NHE-1 isoform expressed in bovine NPE cells. We found that the NHE-1-specific inhibitor HOE-694 (1 µM) (9, 30) completely inhibited the Na+-dependent pHi recovery of NPE cells (Fig. 2B). Less specific NHE-1 inhibitors including EIPA (100 nM to 10 µM) and amiloride (10 µM) (24, 25) also inhibited the pHi recovery in NPE cells at higher concentrations (Fig. 2C).

NPs inhibit Na+-dependent pHi recovery in bovine and rat NPE cells. Earlier studies indicated that among the physiological effect of NPs figured their ability to inhibit cellular NHE activity (22). Before examining the effect of NPs on NHE-1 activity in bovine NPE cells, we verified whether NP treatment modified the intrinsic buffering capacity of the cells. In the absence of NP, the start pHi in bovine NPE cells was 7.096 ± 0.018, and in the presence of CNP, the pHi was 7.12 ± 0.020. Similarly, in the presence of ANP, the pHi in bovine NPE cells was 7.14 ± 0.018. When rat NPE cells were analyzed, the start pHi was 7.392 ± 0.031 and pHi in the presence of CNP or ANP was 7.45 ± 0.025 or 7.48 ± 0.010, respectively. These results suggested that NP did not change the buffering capacity of NPE cells significantly. ANP, BNP, and CNP added separately at a concentration of 10–7 M significantly (P < 0.001) reduced the alkalinization rate in the order of potency CNP > ANP > BNP (Fig. 3A).



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Fig. 3. Natriuretic peptides (NPs) inhibited NHE-1 activity in the bovine NPE. A: merged pHi recordings of single experiments with atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and control are shown. Curves correspond to phases c and d shown in Fig. 1. NPE cells in the intact CE were treated with NP (100 nM) over the entire course of the experiment. NPs inhibited the pHi recovery in the following order of potency: CNP > ANP > BNP. B: effects on NHE-1 activity for each hormone and the control. Data are presented as means ± SE; n = 4–7 independent experiments with a total of 28–64 cells/group. *Significant difference (P < 0.001) vs. control (symbols over bars) or between peptides (symbols over braces).

 
CNP (10–7 M) inhibited the rate of Na+-dependent pHi recovery (0.051 ± 0.003 pHi/min; n = 40) in a more potent fashion than ANP (10–7 M; 0.076 ± 0.004 pHi/min; n = 28) or BNP (10–7 M; 0.096 ± 0.006 pHi/min; n = 39) (Fig. 3B). Interestingly, this order of potency to inhibit Na+-dependent pHi recovery correlates with the NP ability to activate cGMP synthesis in NPE cells (10, 31). This indicates that the NP type B receptor (NPR-B) is the primary receptor mediating NP cellular effects in NPE cells (10, 13). The inhibitory effect of CNP on the rate of Na+-dependent pHi recovery in NPE cells was dose dependent within the range of 10 pM to 100 nM (Fig. 4A) with a log EC50 = –8.931 (Fig. 4B).



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Fig. 4. Inhibition of NHE-1 activity in NPE cells by CNP is dose dependent. A: merged pHi responses, corresponding to phases c and d in Fig. 1, of single NPE cells in the intact CE in the presence of increasing concentrations of CNP (1, 10, and 100 nM). B: inhibition of NHE-1 activity ({Delta}pHi) in NPE cells by CNP ranging from 10–11 to 10–6 M. Data points refer to summarized data for each concentration used and are given as means ± SE (n = 5–7 experiments/series with a total number of 27–64 cells/group). Plotted curve was fitted against the data points with an equation for nonlinear regression with GraphPad Prism 3.02 software and revealed a log EC50 = –8.984 for the inhibition of NHE-1 by CNP in bovine NPE cells.

 
8-BrcGMP (1 mM), a hydrolysis-resistant cGMP analog, mimicked the effect of NP in bovine NPE cells by inhibiting the rate of Na+-dependent pHi recovery at 0.113 ± 0.011 pHi/min (n = 12). This level of inhibition was comparable to the inhibitory effect elicited by CNP at 1 nM.

We next tested whether NPs exhibit species differences in their inhibitory effects on NHE activity. For this purpose we determined pHi responses of rat NPE cells to CNP and ANP and compared their effects with those of bovine NPE cells. CNP (10–7 M) reduced the pHi recovery of rat NPE cells to 0.147 ± 0.005 pHi/min (n = 40), whereas ANP (10–7 M) inhibited the response to 0.187 ± 0.006 pHi/min (n = 75). These values were significantly lower (P < 0.001) than the control 0.234 ± 0.007 pHi/min (n = 48). However, the magnitude of inhibition of NHE activity by CNP and ANP was significantly higher in bovine NPE cells than in rat NPE cells. Despite the species differences in the inhibitory effect on NHE activity of CNP and ANP, rat NPE cells showed the same phase shift of NHE activity as bovine cells.

To verify whether the rate of pHi recovery in rat NPE cells in the presence of CNP reflects a contribution of NHE activity in PE cells, we used the gap junction blockers 18{alpha}-GA and heptanol (17, 30). In the presence of 18{alpha}-GA (100 µM) alone, the rate of pHi recovery in rat NPE cells was 0.144 ± 0.013 pHi/min (n = 30), whereas in the presence of heptanol (2 mM), it was 0.153 ± 0.005 pHi/min (n = 19). However, when 18{alpha}-GA was added together with CNP (100 nM), the pHi recovery was 0.184 ± 0.006 pHi/min (n = 47). This indicated that 18{alpha}-GA attenuates the inhibitory effect of CNP on the rate of pHi recovery. Table 1 summarizes the effect of NP on the pHi recovery in bovine and rat NPE cells.


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Table 1. Effect of NP on pHi and on rate of Na+-dependent pHi recovery in bovine and rat NPE cells

 
Antagonists of NPR-B receptors can block inhibitory effect of NP on NHE-1. To verify that the inhibitory effect of CNP on NHE-1 activity in NPE cells is mediated by NPR-B receptors, we attempted to block the NHE inhibition by A-71915 and LPA. A-71915, an analog of ANP, is known to exhibit an inhibitory effect on the cGMP-mediated stimulation of ANP and BNP on NPE cells (10, 11, 31). In contrast, LPA has been shown to block the effect of CNP by binding NPR-B (1). In the presence of A-71915 (1 µM), the inhibitory effect of CNP (100 nM) on NHE-1 activity was attenuated by 66.8 ± 3.8% (n = 46) and completely abolished in the presence of LPA (10 µM; n = 42). The addition of both A-71915 and LPA completely reversed the inhibitory effect of CNP on NHE activity (n = 54; Fig. 5). Surprisingly, LPA (10 µM) alone led to a relative activation of NHE-1 up to 29.2 ± 2.7% (n = 36; represented as negative value in Fig. 5), whereas A-71915 (n = 42) alone had no influence on the pHi recovery (Fig. 5).

Inhibitors of Na+ transport do not influence NHE-1 activity in NPE cells. We studied the influence of other Na+ transporters on NHE-1 activity expressed in NPE cells. For this purpose we determined the NHE-1 activity in NPE cells on inhibition of the bumetanide-sensitive Na+-K+-2Cl cotransporter or the epithelial Na+ channel (ENaC), which have been reported to be present and functional in NPE cells (6, 12). Application of bumetanide (100 µM) or benzamil (10 µM) to bovine NPE cells did not reveal significant differences in pHi recovery compared with control (data not shown). Furthermore, the simultaneous addition of bumetanide or benzamil with ANP (100 nM) did not alter the inhibitory effect elicited by ANP alone.

Indirect immunofluorescence of NP and NHE-1 in bovine and rat ciliary processes. We examined the pattern of NP labeling with antibodies to ANP, BNP, and CNP on bovine semithin cryostat sections of ciliary processes. ANP and BNP antibodies preferentially labeled the cytoplasm of the NPE cell layer (Fig. 6, A–D). In contrast, the CNP antibody labeled the vascular endothelium in the stroma of the ciliary processes (Fig. 6, E and F).



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Fig. 6. Indirect immunofluorescence of NP antibodies on cryostat sections of bovine ciliary processes. Polyclonal antibodies against ANP (A), BNP (C) and CNP (E) were used to label bovine cryostat sections. B, D, and F are corresponding phase contrasts of the fluorescence photographs in A, C, and E, respectively. Arrows indicate NPE, pigmented CE cells (PE), and stroma (S). Bar = 100 µM.

 
We also verified and compared the pattern of immunolabeling of NHE-1 in the CE of bovine and rat cryostat sections. Tissues were incubated with a polyclonal antibody against NHE-1 (21) at a 1:100 dilution and processed as indicated for the NP antibodies. The immunostaining pattern observed indicates that the antibody preferentially labels the basal plasma membrane of NPE cells in the rat (Fig. 7, A and B) and bovine (Fig. 7, C and D) CE. The immunostaining signal at the basal plasma membrane of the PE cells ranged from absent in rat to low in bovine. This pattern of labeling contrasted with the profile of staining with a Na+-K+-ATPase antibody, which equally labeled both basal membranes in PE and NPE cells in the bovine CE (Fig. 7, E and F). No signal was detected when normal serum or secondary antibody alone was used (Fig. 7, G and H).



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Fig. 7. Cellular distribution of the NHE-1 isoform in the rat and bovine CE. Semithin cryostat sections of ciliary processes were labeled with an NHE-1 antibody (gift of Dr. Sergio Grinstein; Ref. 24). In rat ciliary processes the immunolabeling of NHE-1 is restricted to the basal membrane of NPE cells (A). In contrast, in bovine ciliary processes the antibody labeled more intensely the NPE than the PE basal membrane (C). An antibody to Na+-K+-ATPase (19) was used to demonstrate equal accessibility to the basolateral plasma membrane domain of PE and NPE cells in the bovine ciliary epithelium (E). A secondary antibody alone did not label the NPE or PE cells (G). B, D, F, and H are phase contrast photographs of the corresponding fluorescent views in A, C, E, and G, respectively. Bar = 100 µM.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Aqueous humor secretion is a complex process that takes place in the ocular CE, a bilayer of neuroepithelial polarized cells. The present model of aqueous humor secretion contemplates at least three steps: 1) uptake of solute and water at the stromal surface by PE cells, involving a Na+-K+-2Cl symport and the parallel Cl/HCO3 and Na+/H+ antiports, 2) transfer from PE to NPE cells through gap junctions, and 3) transfer of solute and water from NPE cells into aqueous humor in the posterior chamber of the eye (5). Of the multiple transporters described in the CE the Na+/H+ antiport (NHE) plays an important role.

NHE-1 is considered a molecular sensor to changes in pHi and in cell volume regulation. The NHE catalyzes the electroneutral exchange of extracellular Na+ and intracellular H+. This results in a net import of extracellular Na+ into the cell. In many systems, including the CE, the NHE operates in parallel with a Cl/HCO3 exchanger, resulting in the uptake of NaCl, and the influx of osmotically obliged water will follow. This effect will consequently lead to an increase in cell volume. Out of the multiple NHE isoforms so far identified, we focused in this study on the NHE-1 that is ubiquitously expressed and highly restricted to the basal membrane of the NPE CE. The diminished (to absent) NHE-1 immunostaining signal along the basal plasma membrane of PE cells on cryostat sections was unexpected, because by RT-PCR amplification NHE-1 mRNA was detected in both cell types of the CE. One possible interpretation of this distinct labeling of NHE-1 in NPE and PE cells is that the epitope that recognizes the NHE antibody is masked in the PE cells. Alternatively, this difference could simply reflect a higher abundance of NHE-1 expression in NPE than in PE cells. Additional antibodies to NHE-1 protein could help to corroborate this finding.

In the present study we attempted to determine whether NPs, which exhibit hypotensive effects in lowering IOP, mediate their effect by regulating NHE activity in the CE. The NPE and PE cells in the CE are coupled to each other by numerous gap junctions (8).

The main effect of NPs on the NHE-1 activity in NPE cells was their ability to inhibit the Na+-dependent pHi recovery on an acid load. The level of inhibition of NHE-1 by NPs followed an order of potency (CNP > ANP > BNP) that was similar to their ability to stimulate cGMP in cultured NPE cells (31). These results indicate that NPR-B receptors mediate both the inhibition of Na+-dependent pHi recovery and the stimulation of cGMP in NPE cells. On the basis of the reversing effect of A-71915 on the inhibition of NHE-1 activity by CNP, we could not rule out that NP type A receptor is not involved in the NHE-1 inhibition. However, the complete reversal by LPA of the inhibitory effect of CNP on NHE supports that NPR-B in NPE cells are primarily involved in this response. On the other hand, the ability of LPA to enhance, when added alone, the NHE activity in NPE was unexpected, although not unique, because a similar effect has been described in tumor cells and smooth muscle cells (33, 38).

The present study also suggests that NPs, which are naturally occurring hormones locally synthesized within the ciliary body (15, 31), could modulate IOP through NHE-1 activity in the CE. Although the present studies clearly demonstrate that NPs exhibited an inhibitory effect on the NHE-1 activity expressed in the NPE cells, we do not know how much the PE cell layer contributes to the overall activity or how the NHE activity is coregulated by these two cell layers. Additional work is needed before a comprehensive working model can be suggested to explain the potential interrelation between NP receptors in the tissues (CE and trabecular meshwork) of the anterior segment that govern IOP and NHE. However, recent findings may provide a possible physiological clue to the role of NHE in IOP (3). In these studies it has been shown that inhibitors of NHE-1 activity [i.e., dimethylamiloride (DMA), EIPA, and BIIB723] lower IOP when applied topically to the eye (3).

The colocalization of ANP and BNP along the NPE cell layer is consistent with the observation that they are secreted into aqueous humor. The level of NPs in aqueous humor is approximately sixfold higher than in plasma, BNP being the most abundant, followed by CNP and, at very low levels, ANP (14). The immunostaining of the vascular endothelium rather than the CE with CNP antibodies is consistent with the cell distribution of this NP in the endothelium of the blood vessels (43). Another site of action of NP released by the ciliary processes in the aqueous humor may be the trabecular meshwork and the Schlemm canal at the outflow system where NP receptors are present (7, 44).

It has been suggested that the ciliary bilayer functions as a syncytium (27). In this type of anatomic configuration, there is a possibility that the Na+-dependent pHi recovery recorded in NPE cells might reflect the sum of NHE activities in NPE and PE cell layers. One limitation of the present work is the inability to assess NHE activity directly at the PE cell side in the intact CE, and therefore the inability to determine whether PE cells contribute to the overall NHE-1 activity recorded on NPE cells. However, we attempted to address this question indirectly, by using uncoupling agents of intercellular junctions, including 18{alpha}-GA and heptanol. We observed that in the presence of CNP, 18{alpha}-GA was able to attenuate the inhibitory effect elicited by CNP when added alone on the rate of pHi recovery. This effect suggested that the overall NHE-1 activity recorded in NPE cells may reflect the contribution of NHE-1 activity from PE cells as well, supporting the view that the NHE-1 activity in the CE is coregulated. Future studies using stroma-free preparations of CE will allow comparison of the NHE-1 activity of both PE and NPE cell sides of the bilayer.

The activity of NHE-1 was studied by pharmacological means using specific blockers such as EIPA and amiloride. The inhibition of ENaC and of the Na+-K+-2Clcotransporter, respectively, did not alter the Na+-dependent pHi recovery of the NPE cells, suggesting that these transport systems are not likely to affect the NHE activity.

We think that an endocrine/paracrine cell mechanism could explain how NPs may influence NHE activity in the CE by creating a more hypotensive environment in the eye. CNP, the most potent of the three NPs in lowering IOP, presumably acts in a paracrine fashion on NPR-B receptors in the NPE-PE cells and presumably in cells of the outflow system. The activation of NPR-B receptors, leading to an increase in intracellular cGMP, will negatively affect the NHE in both NPE and PE cells, which could result in a decrease in ion and fluid transport into the aqueous humor. We believe that the source of NP in the aqueous humor is the CE for ANP and BNP and the vascular endothelium for CNP. The vascular endothelium of the ciliary processes may fulfill a storage function for CNP rather than being the site of synthesis. We think that ANP and BNP, on their secretion by the NPE cells, may target NPR-A or NPR-C by an autocrine mechanism in the same hormone-producing cells (NPE cells). Alternatively, ANP and BNP may target NPR-A in the paracrine vascular endothelial cells and enhance CNP release. Our observation that 8-Br-cGMP mimicked NP's effect on NHE activity, as well as previous studies, suggests that CNP could activate NPR-B and presumably NPR-A in NPE cells, inducing an increase in intracellular cGMP production and thereby modulating NHE activity (28a, 44). Although cGMP-induced activation of PKG appears to be the most likely intracellular mechanism of NHE inhibition, alternate pathways cannot be ruled out. CNP is known to exhibit a vasodilatory effect in the cardiovascular system and to exhibit a more lasting effect than BNP or ANP in lowering IOP in experimental animals. Levels of NP in the aqueous humor are likely to be modulated by the clearance activity of the NPR-C receptors in the NPE cells (31).

The present findings open up at least two potential ways in which a decrease in NHE activity could influence aqueous humor formation and possibly lower IOP: 1) NHE is involved in regulation of both cell volume and intracellular pHi, both of which have been identified as influencing transport activity across membranes, and 2) NPs likely influence other transport proteins through the second messenger cGMP. Which of these possible mechanisms, that is, a decrease in pHi, a change in cell volume, and/or an increase of intracellular cGMP levels, may have the greatest effect on aqueous humor formation and IOP will be the subject of further studies.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Institutes of Health Grants EY-04873 and EY-00785 (for core facilities), Research to Prevent Blindness, The Connecticut Lions Foundation, Research to Prevent Blindness Lew Waserman Merit Award (to M. Coca-Prados), and NIH Grants DK-50230, DK-17433, and DK-14669 (to J. Geibel).


    ACKNOWLEDGMENTS
 
We thank Dr. Sergio Grinstein for the generous gift of the NHE-1 antisera.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. Coca-Prados, Dept. of Ophthalmology and Visual Science, Yale Univ. School of Medicine, New Haven, CT 06510 (E-mail: miguel.coca-prados{at}yale.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.


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