2 H
stricted to multiple dendritic mechanosensory neurons in named hgN-1, a glutathione S-transferase GST fusion
the Drosophila embryo also suggested a central role in protein containing the last 34 amino acids of the
mechanosensation [1,9]. We recently showed that the g C-terminus
of rgENaC
residues 616–649,
subunit of ENaC was located in baroreceptor nerve termi- GSTVPGTPPPRYNTLRLDRAFSSQLTDTQLTNEL na-
nals that innervate the aortic arch and carotid sinus [13]. med rgC-2, and a GST fusion protein containing the
Moreover, amiloride and its analog benzamil attenuated N-terminus of hbENaC HVKKYLLKGLHRLQKGPGY-
baroreceptor function. These data suggest that the g TYKELLVWYCDNTNTGHPKRIICEGPKK
named subunit of ENaC, and possibly other subunits, may form
hbN-1. The antibodies were generated in sheep by Elmira part of the mechanosensory complex. This led us to
Biologicals Iowa City, IA. Immunoreactive sera was consider the possibility that ENaC subunits may also
purified using cyanogen bromide-activated sepharose 4B contribute to peripheral mechanosensation in the skin. To
Pharmacia, Piscataway, NJ linked to the appropriate begin to evaluate this hypothesis, we asked if ENaC
antigen gN-terminus peptide, GST-bN-terminus or GST- subunits are located in touch receptors in the hairless skin
gC-terminus. Detection of ENaC subunits and antibody of the rat paw.
specificity were evaluated in COS-7 cells transfected with both human and rat a, b, and g ENaC [30]. Note that the
N-terminus of rat and human gENaC are 90 identical, the C-terminus of rat and human gENaC are 76 identical,
2. Materials and methods
and the N-terminus of rat and human bENaC are 94 identical, in the regions used as immunogens.
2.1. Reverse transcriptase–polymerase chain reaction To localize expression of bENaC and gENaC in touch
receptors, we used immunofluorescence in rat DRG and To determine if ENaC family members are expressed in
paw pad. Rats were anesthetized with halothane, perfused dorsal root ganglia DRG, we used reverse transcriptase–
transcardially with 60 ml ice cold PBS, followed by 60 ml polymerase chain reaction RT–PCR to detect a, b and
ice cold 4 paraformaldehyde. The lumbar and cervical gENaC transcripts. Spraque–Dawley rats, Harlan, In-
DRG and hind paws were removed and fixed for 20 min to dianapolis, IN were anesthetized with halothane, placed
2 h in 4 paraformaldehyde. The tissue samples were on ice, and cervical and lumbar DRG were removed. The
rinsed in PBS then stored overnight in 30 sucrose in ganglia were combined and flash frozen in EtOH and dry
PBS. Tissue samples were embedded in O.C.T. compound ice, then stored at 2708C. Total RNA was extracted using
Sakura Finetek, Torrance, CA media then 10–50 mm RNA STAT-60 TEL TEST ‘B’, Friendswood, TX and
frozen sections were obtained. The sections were incubated reverse transcribed using a random hexamer primer and
with the primary antibody solution for 72 h at 48C with 5 RNase H-murine leukemia virus reverse transcriptase
normal donkey serum and 0.1 Triton X-100. DRG tissue Superscript
Preamplification System,
Gibco BRL,
sections were double-labeled with a 1:100 dilution of the Gaithersburg, MD. Partial cDNA’s from DRG tissue
sheep antibodies hbN-1 or hgN-1 and a 1:100 dilution of were
amplified using
the following
primer pairs:
mouse anti-NF200 Neurofilament 200 kDa, Sigma Chemi- aENaC,
59-AGTACCTCAGCTACCCAGTGAGC-39, cal Co., St Louis, MO. The hgN-1 antibody produced a
59-TTGACCGGGACATCGCTACCAT-39; bENaC,
59- stronger signal-to-noise ratio only in DRG sections. Hin-
CTACACCTACAAGGAGCTGCTAG-39, 59-AAGTGC-
dpaw tissue sections were double- or triple-labeled with TTGACCTTGGAGTACTG-39; gENaC, 59-CCTCTGCT-
various combinations of the following antibodies: 1 hbN- GTGGATCGCGTTCAC-39, 59-CACAGCACTGTACTT-
1 1:100; 2 rgC-2 1:100; 3 rabbit anti-S-100 which GTAAGGGTTGAT-39. Samples were preheated to 948C
stains Schwann cells in the peripheral nervous system [23] for 3 min then cycled 40 times at 948C for 20 s, 628C for
1:100, Sigma Chemical Co; 4 mouse anti-cytokeratin 20 s and 728C for 20 s for b and gENaC; 948C for 45 s,
20 CK20, 1:100, Research Diagnostics Inc., Flanders, 638C for 45 s and 728C for 2 min for aENaC. After
NJ; 5 rabbit anti-protein gene product 9.5 PGP 9.5, cycling, all samples were held at 728C for 7 min. PCR
1:2,500, Chemicon, Temecula, CA; or 6 a cocktail of reactions in which reverse transcriptase was not added to
mouse anti-neuron antibodies that included anti-neurofila- RNA served as a negative control and lung RNA was a
ment 68 1:3, Roche, Indianapolis, IN, anti-neurofilament positive control. The products of PCR reactions were run
160 1:3, Roche, anti-NF200 1:100, Sigma, and anti- on a 1.5 agarose gel and visualized with ethidium
peripherin 1:100, Chemicon. The samples were rinsed in bromide.
PBS and exposed to the secondary antibody solution: donkey anti-sheep-Cy3 1:500, Jackson Immuno Research,
2.2. Antibodies and immunofluorescence West Grove, PA, donkey anti-rabbit-Cy5 1:200, Jackson
Immuno Research or donkey anti-mouse-Cy2 1:200, Immunogens for antibody generation included: a syn-
Jackson Immuno Research for 24–48 h at 48C. Samples thetic peptide containing the first 20 amino acids of the
were rinsed in PBS, mounted with Gel Mount Biomeda N-terminus of hgENaC MAPGEKIKAKIKKNLPVTGL
Corp., Foster City, CA and examined using a BioRad
H .A. Drummond et al. Brain Research 884 2000 1 –12
3
MRC1024 Hercules, CA laser scanning confocal micro- in small neurons was occasionally observed. Expression of
scope. bENaC and gENaC in some small neurons would not be
unexpected since subpopulations of type III and IV skeletal muscle afferents, whose cell bodies are also located in the
3. Results and discussion DRG, also respond to mechanical stimuli and may there-
