Mechanisms of neuronal chloride accumulation in intact mouse olfactory epithelium

Abstract

When olfactory receptor neurons respond to odours, a depolarizing Cl(-) efflux is a substantial part of the response. This requires that the resting neuron accumulate Cl(-) against an electrochemical gradient. In isolated olfactory receptor neurons, the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 is essential for Cl(-) accumulation. However, in intact epithelium, a robust electrical olfactory response persists in mice lacking NKCC1. This response is largely due to a neuronal Cl(-) efflux. It thus appears that NKCC1 is an important part of a more complex system of Cl(-) accumulation. To identify the remaining transport proteins, we first screened by RT-PCR for 21 Cl(-) transporters in mouse nasal tissue containing olfactory mucosa. For most of the Cl(-) transporters, the presence of mRNA was demonstrated. We also investigated the effects of pharmacological block or genetic ablation of Cl(-) transporters on the olfactory field potential, the electroolfactogram (EOG). Mice lacking the common Cl(-)/HCO(3)(-) exchanger AE2 had normal EOGs. Block of NKCC cotransport with bumetanide reduced the EOG in epithelia from wild-type mice but had no effect in mice lacking NKCC1. Hydrochlorothiazide, a blocker of the Na(+)-Cl(-) cotransporter, had only a small effect. DIDS, a blocker of some KCC cotransporters and Cl(-)/HCO(3)(-) exchangers, reduced the EOG in epithelia from both wild-type and NKCC1 knockout mice. A combination of bumetanide and DIDS decreased the response more than either drug alone. However, no combination of drugs completely abolished the Cl(-) component of the response. These results support the involvement of both NKCC1 and one or more DIDS-sensitive transporters in Cl(-) accumulation in olfactory receptor neurons.

Figure 1

Cartoon diagram of major cell types of the olfactory mucosa in vivo The cilia of the Cl⁻-accumulating ORNs are exposed to mucus. The mucus is generated by Bowman's glands (BG) and sustentacular cells (Sus) (Farbman, 1992). The ORN cell bodies sit in an extracellular space containing basal cells (not shown) and sustentacular cells. The Bowman's glands, ORN axons and basal feet of the sustentacular cells sit near or in a well-vascularized lamina propria. The ORN-containing olfactory epithelium and its underlying lamina propria comprise the olfactory mucosa.

Figure 2

mRNA for Cl⁻ transporters in nasal tissue containing olfactory mucosa assayed by RT-PCR A, RT-PCR for the Slc4a Cl⁻ transporters. B, RT-PCR for the Slc12a1–Slc12a5 Cl⁻ transporters. C, RT-PCR for the Slc12a6–Slc12a9 Cl⁻ transporters. D, RT-PCR for the Slc26a Cl⁻ transporters. The expression of genes for 21 Cl⁻ transporters was tested. The images of gels show the results of RT-PCR reactions with a conventional RNA concentration (3.7 ng RNA μl⁻¹). For all of the transporters except AE1 and CCC9(see Methods), the primers were designed to amplify a PCR product that would have the same sequence for all known variants of a given transporter. In the row ‘Results for Nasal Tissue’, ‘+’ (without a superscript) indicates that one or more of the variants for the Cl⁻ transporter was detected with a conventional RNA concentration and confirmed with sequencing; ‘−’ (without a superscript) indicates that kAE1 was not detected. ‘+¹’ indicates that while NKCC2 was negative at 3.7 ng RNA μl⁻¹, it was detected (data not shown) and confirmed with sequencing with 15 ng RNA μl⁻¹. ‘−²’ indicates that DRA was negative at 3.7 ng RNA μl⁻¹ but had a weak band (data not shown), which was not sequenced, at 15 ng RNA μl⁻¹. For NCBE and KCC4, the number of variants shown refers to the one variant that was known prior to the present study. For each transporter, nasal tissue (N), a ‘no-transcript’ (NT) control, and the appropriate positive control tissue (K, kidney; Sp, spleen; Ce, cerebellum; B, brain; Co, colon; SI, small intestine; St, stomach; and T, testis) were tested. In the first gel image of each section of the figure (A, B, C or D), the leftmost lane is a calibration lane; the values are in basepairs. Thereafter, the calibration marks are indicated by white dashes in the rightmost lane of each gel. The values for the calibration marks are the same for all gels in a section. A box around multiple lanes indicates that the lanes were part of the same gel. bp, basepair.

Figure 2

mRNA for Cl⁻ transporters in nasal tissue containing olfactory mucosa assayed by RT-PCR A, RT-PCR for the Slc4a Cl⁻ transporters. B, RT-PCR for the Slc12a1–Slc12a5 Cl⁻ transporters. C, RT-PCR for the Slc12a6–Slc12a9 Cl⁻ transporters. D, RT-PCR for the Slc26a Cl⁻ transporters. The expression of genes for 21 Cl⁻ transporters was tested. The images of gels show the results of RT-PCR reactions with a conventional RNA concentration (3.7 ng RNA μl⁻¹). For all of the transporters except AE1 and CCC9(see Methods), the primers were designed to amplify a PCR product that would have the same sequence for all known variants of a given transporter. In the row ‘Results for Nasal Tissue’, ‘+’ (without a superscript) indicates that one or more of the variants for the Cl⁻ transporter was detected with a conventional RNA concentration and confirmed with sequencing; ‘−’ (without a superscript) indicates that kAE1 was not detected. ‘+¹’ indicates that while NKCC2 was negative at 3.7 ng RNA μl⁻¹, it was detected (data not shown) and confirmed with sequencing with 15 ng RNA μl⁻¹. ‘−²’ indicates that DRA was negative at 3.7 ng RNA μl⁻¹ but had a weak band (data not shown), which was not sequenced, at 15 ng RNA μl⁻¹. For NCBE and KCC4, the number of variants shown refers to the one variant that was known prior to the present study. For each transporter, nasal tissue (N), a ‘no-transcript’ (NT) control, and the appropriate positive control tissue (K, kidney; Sp, spleen; Ce, cerebellum; B, brain; Co, colon; SI, small intestine; St, stomach; and T, testis) were tested. In the first gel image of each section of the figure (A, B, C or D), the leftmost lane is a calibration lane; the values are in basepairs. Thereafter, the calibration marks are indicated by white dashes in the rightmost lane of each gel. The values for the calibration marks are the same for all gels in a section. A box around multiple lanes indicates that the lanes were part of the same gel. bp, basepair.

Figure 3

Block of the NKCC1 cotransporter by bumetanide reduces the EOG amplitude in WT but not in NKCC1 KO mice The concentration of bumetanide was 50 μm. Mice were 45–86 days old. The WT series used 6 epithelia from 4 mice; the KO series used 4 epithelia from 2 mice.

Figure 5

Further pharmacological studies of the EOG in WT and KO mice A, hydrochlorothiazide, a blocker of the NCC transporter, has only small effects on the EOG amplitude in WT and NKCC1 KO mice. Hydrochlorothiazide concentration was 200 μm. Mice were 99–103 days old (WT) and 104–176 days old (KO). The WT series used 4 epithelia from 2 mice; the KO series used 6 epithelia from 4 mice. B, DIDS reduces the EOG amplitude in both WT and NKCC1 KO mice. DIDS concentration was 1 mm. Mice were 109–115 days old (WT) and 111–115 days old (KO). For each of the WT and KO series, 5 epithelia from 3 mice were used. C, bumetanide reduces the EOG amplitude in both WT and AE2 KO mice. The bumetanide concentration was 50 μm. Mice were 13–16 days old (WT) and 15–16 days old (KO). AE2 KO mice do not survive beyond weaning. For the WT series, 6 epithelia from 5 mice were used; for the AE2 KO series, 7 epithelia from 5 mice were used. D, application of bumetanide and DIDS to both surfaces of the mucosa does not eliminate the EOG. Responses are from septal mucosa that was removed from underlying cartilage and mounted on nylon mesh. Bumetanide concentration was 50 μm; DIDS concentration was 500 μm. All mice were adult WT aged 79–80 days (bumetanide only), 106–144 days (DIDS only) or 109–128 days (bumetanide plus DIDS). The first series (bumetanide only) used 5 epithelia from 3 mice; the second series (DIDS only) used 7 epithelia from 4 mice; the third series (bumetanide and DIDS) used 5 epithelia from 3 mice. Residual responses were strongly decreased or abolished by 300 μm niflumic acid (data not shown).

Figure 4

Simultaneous block of multiple Cl⁻ uptake systems reduces the EOG amplitude but does not abolish the response in WT mice Mice were all WT, 77–87 days old. Five epithelia from 3 mice were used. A, normalized EOG amplitudes before and after application of the All Block mixture of three transport blocking drugs (50 μm bumetanide, 500 μm DIDS and 200 μm hydrochlorothiazide). B, example EOG responses recorded in the following order: control; after application of the All Block mixture; and after application of niflumic acid (NFA, 300 μm).