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Transcriptome profiling of anhidrotic eccrine sweat glands reveals that olfactory receptors on eccrine sweat glands regulate perspiration in a ligand-dependent manner
Correspondence to: Hiroyuki Murota, 1-7-1 Sakamoto, Nagasaki city, Nagasaki 8528501, Japan. Telephone: +81958197331; Fax: +81958497335; [email protected]
Affiliations
Department of Dermatology, Nagasaki University Graduate School of Biomedical SciencesLeading Medical Research Core Unit, Life Science Innovation, Nagasaki University Graduate School of Biomedical Sciences
Sweat maintains systemic homeostasis in humans. Although sweating disorders may cause multifaceted health problems, therapeutic options for sweat disorders have not yet been established. To gain new insight into the mechanism underlying the regulation of perspiration, we compared eccrine sweat gland transcriptomes from hidrotic and anhidrotic lesions from anhidrosis patients and found out that olfactory receptors (ORs) were expressed differentially in anhidrotic and hidrotic eccrine sweat glands. We then confirmed OR51A7 and OR51E2 expression in human eccrine sweat glands by in situ hybridization and immunohistochemistry. An alkaline phosphatase (AP)-transforming growth factor (TGF) α shedding assay revealed that β-ionone activates G proteins through OR51A7 or OR51E2. The effect of topically applied β-ionone on sweating was examined with the quantitative sudomotor axon reflex test, which showed that responses to β-ionone differed between genders. Topical β-ionone attenuated female sweating and augmented male sweating. Taken together, this study suggests that ORs expressed in eccrine sweat glands may regulate sweating in response to odorous ligands based on gender. These unexpected results indicate that ORs may modulate sweating and that OR modulators may contribute to the management of sweat disorders.
In humans, perspiration is essential to maintain skin homeostasis. Sweat contains many substances, including metabolites, antibiotic peptides, and electrolytes. Sweat regulates body temperature, immune defense, and moisture retention (
). Therefore, a reduction in sweating may affect wellness in various ways.
Acquired idiopathic generalized anhidrosis (AIGA) is extensive anhidrosis of unknown etiology characterized by fatal heatstroke and skin dryness. Although AIGA symptoms impair patient quality of life, there are few promising treatment strategies for the disease (
). AIGA is diagnosed in patients presenting with diffuse hypohidrosis or anhidrosis detected by the sweat test (e.g., starch-iodine technique, Minor’s method). AIGA patients have no apparent abnormalities in the central, peripheral, and autonomic nervous systems (
). It has been hypothesized that increased blood carcinoembryonic antigen levels in some AIGA patients may contribute to the destruction of eccrine sweat glands (
). Histopathological observation showed no specific patterns associated with eccrine sweat gland atrophy or massive lymphocyte infiltration around sweat glands (
To investigate the etiology of AIGA, we analyzed gene expression profiles of eccrine sweat glands in hidrotic and anhidrotic skin specimens from AIGA patients. Coincidently, our RNA seq-based transcriptome analysis found that several ORs were expressed in eccrine sweat glands in hidrotic areas but downregulated in anhidrotic areas. ORs are generally expressed in nasal mucosa and are involved in olfactory functions. ORs expressed in other organs are expected to have different functions (
). It was also reported that an odorant factor induces wound healing in human keratinocytes via OR2AT4. Several ORs have been found to be expressed in HaCaT cells (
UV-Irradiation- and Inflammation-Induced Skin Barrier Dysfunction Is Associated with the Expression of Olfactory Receptor Genes in Human Keratinocytes.
). OR 2AT4/7 and OR51B5 are expressed on suprabasal keratinocytes, with OR 2AT4/7 involved in IL-1 production and OR51B5 in keratinocyte migration and IL-6 production (
). To the best of our knowledge, there have been no reports of OR expression in sweat glands. In this study, we showed that OR51A7 and OR51E2 are expressed in sweat glands and that they regulate sweating via β-ionone.
RESULTS
RNA sequencing-based transcriptome analysis of eccrine sweat glands from anhidrotic patients
Existing stored tissue samples derived from patients with AIGA, a rare disease, were used in this study to complement the small number of patients. Eccrine sweat glands in hidrotic and anhidrotic skin areas were excised from paraffin-embedded skin specimens from four AIGA patients, and gene expression was profiled by RNA sequencing. Because RNA could not be extracted from one patient due to lack of sample material in the hidrotic lesion, we analyzed RNA derived from three hidrotic and four anhidrotic lesions. We identified 102 genes by filtering transcripts with significant differences (< 0.01) and with > 2-fold differences in gene expression. OR51A7, OR6C74, and OR4A15 were expressed highly in sweat glands in hidrotic lesions, whereas their expression levels were downregulated in anhidrotic sweat glands (Figure 1). Because ORs are members of the G protein-coupled receptor superfamily, which transduces cellular signals, ORs may function in sweating in addition to odor sensory systems.
Figure 1Transcriptome analyses of skin lesions from AIGA patients. (a) Schematic representation of the strategy for identifying genes responsible for sweating. Hidrotic and anhidrotic lesions were identified by the conventional Minor’s test. We obtained written informed consent from him for the publication of clinical images (left). Skin tissues were biopsied from the hidrotic and anhidrotic lesions, and sweat glands were excised from the tissues by standard laser microdissection (middle). RNAs were extracted from the samples and subjected to transcriptome analyses (right). (b) Classification of genes identified by transcriptome analyses. One hundred two genes were upregulated in the hidrotic lesions and downregulated in the anhidrotic lesions (p <0.01). (c) Identification of ORs as factors that contribute to sweating. Three ORs (OR51A7, OR6C74, and OR4A15) were downregulated in sweat glands from anhidrotic lesions.
To determine expression and localization of OR mRNAs in anhidrotic and hidrotic specimens from an AIGA patient, in situ hybridization (ISH) was performed with digoxigenin-labeled synthetic oligo-DNAs. As shown in Figure 2, OR51A7 mRNA was detected in the cytoplasm of acinar cells in sweat glands. OR51A7 mRNA was also detected in the myoepithelial cells surrounding the sweat glands (Figure 2b). OR4A15 and OR6C74 mRNAs were not detected in eccrine sweat glands from AIGA patients and healthy donors under the conditions used in this study (data not shown).
Figure 2OR51A7 mRNA levels in sweat glands of AIGA patients and a healthy donor. (a) ISH analyses of ORs in sweat glands. Of the three ORs evaluated, only OR51A7 mRNA was detected in sweat glands from sweating areas of AIGA patients and the healthy donor. Scale bar: 20 μm. (b) Expression of OR51A7 in myoepithelial cells surrounding sweat glands. OR51A7 mRNA was expressed in the acinar cells of sweat glands and in the myoepithelial cells surrounding sweat glands (red arrow). Scale bar: 20 μm.
To further evaluate OR expression, conventional immunohistochemistry with antibodies against OR51A7 was performed on anhidrotic and hidrotic skin specimens from AIGA patients. OR51A7 protein was detected in eccrine sweat glands of AIGA patients, and there was no apparent difference in the staining intensities of OR51A7 in the hidrotic and anhidrotic areas (Figure 3). OR51A7 expression was also detected in a specimen derived from a healthy donor. Because the ligands for OR51A7 have not yet been identified, we compared the expression pattern and ligand recognition of OR51A7 with OR51E2, a member of the OR51 receptor family whose ligand has been identified as β-ionone. We examined OR51E2 protein expression in eccrine sweat glands from AIGA patients and a healthy donor by immunohistochemistry. OR51E2 protein was expressed in eccrine sweat glands from both hidrotic and anhidrotic skin lesions from AIGA patients. OR51E2 protein was also expressed in eccrine sweat glands of the healthy donor. These data suggest that OR51A7 and OR51E2 may play roles in sweating from eccrine sweat glands.
Figure 3Immunohistochemical analyses of OR51A7 and OR51E2. Immunohistochemical staining was performed to confirm expression and localization of OR51A7 protein. OR51E2 protein expression was also examined because it has been shown to recognize the chemically defined odorous compound β-ionone. Both OR51E2 and OR51A7 proteins were expressed in the sweat glands of AIGA patients and a healthy donor. OR51E2 and OR51A7 were expressed in both sweating and non-sweating areas of AIGA patients. Scale bar: 100 μm.
Effect of β-ionone on OR51A7-mediated signal transduction
We examined the effect of β-ionone on OR51A7- or OR51E2-mediated signal transduction based on the redundant recognition of odor ligands by ORs. We used an alkaline phosphatase-transforming growth factor (AP-TGF) α shedding assay system with HEK293T reporter cells (Figure 4a). In this assay system, when odorous ligands bind to ORs, a signal is transduced via G-proteins, such as Golf and Gαq/i1. Gαq/i1 is a chimera Gαq-protein with the six C-terminal Gαq amino acid residues replaced with the six C-terminal amino acid residues of Gαi resulting in ectodomain shedding of the AP-TGFα reporter by tumor necrosis factor α-converting enzyme (TACE, also known as ADAM17). AP activity is measured with the substrate p-nitrophenyl phosphate (p-NPP).
Figure 4Recognition of β-ionone by OR51A7 in an AP-TGFα shedding assay. (a) Schematic of the AP-TGFα shedding assay. When an odorous ligand binds to an OR, such as OR51A7 or OR51E2, a signal is transduced through a G-protein, such as Golf or Gαq/i1, and TACE is activated resulting in the liberation of AP-TGFα. AP activity was assessed using the AP substrate p-NPP. (b) Specific recognition of β-ionone by OR51A7 in a G-protein- and dose-dependent manner. After HEK293T cells were transiently transfected with AP-TGFα, ORs, Golf, and/or Gαq/i1, the transfectants were challenged with 0, 50, 100, 150, or 200 μM β-ionone, and the liberated AP-TGFα was measured at 405 nm using a spectrophotometer. Phorbol 12-myristate 13-acetate (PMA) was used as a positive control. Experiments were performed in triplicate, and data are shown as mean ± s.d. (n = 3).
After HEK293T cells were transiently transformed with plasmids containing AP-TGFα, OR51A7 or OR51E2, Gαq/il, and/or Golf, the cells were challenged with β-ionone, and shedding of the extracellular domain of the AP-TGFα fusion protein was measured. As shown in Figure 4b, β-ionone activated G proteins in a concentration-dependent manner in the presence of OR51A7 or OR51E2, Golf and Gαq/i1. We increased the β-ionone dose up to 300 μM and found that the minimum dose for OR51A7 was 100 μM and the minimum dose for OR51E2 was 250 to 300 μM in the presence of both Golf and Gαq/il. These results indicated that OR51E2 could serve as the positive control as reported previously even though its sensitivity to β-ionone was weaker than that of OR51A7. Expression of both Golf and Gαq in human eccrine sweat glands was confirmed by immunohistochemical staining for GNAL (Golf subunit α) and GNAQ (Gαq) (Figure 5).
Figure 5Expression of Gαq and Golf in sweat glands. Human skin samples were examined for protein expression of Gαq, which is encoded by GNAL, and Golf, which is encoded by GNAQ, by immunohistochemistry. Scale bar: 100 μm.
Effect of β-ionone on the sudomotor axon reflex and odor perception
Based on the above findings, topical β-ionone likely affects sweating in humans through OR51A7 or OR51E2. Thus, we examined the effect of topically applied β-ionone on human perspiration using the quantitative sudomotor axon reflex test (QSART) with glycerol as the control (Table 1). When β-ionone was applied to the skin of female donors, sweating decreased. By contrast, lower axon reflex-mediated sweating increased in male donors in response to the same odorant demonstrating a gender-based difference in OR signal transduction in response to β-ionone. This prompted us to examine the effect of β-ionone on odor perception in males and females. A β-ionone whiff test performed among the same subjects revealed that all the female subjects could smell the β-ionone, whereas most of the male subjects (5/7）could not, suggesting a gender-based difference in the sensing of β-ionone through ORs.
Table 1QSART analyses of forearm skin areas treated with topical β-ionone.
Age
Gender
QSARTl(mg/5min)
daily exercise habit
smell
impact of topical β-ionone on sweating
β-ionone
control
50
F
0.039
0.305
+
+
decrease
49
F
0.29
0.242
-
+
not apparent
47
F
0.403
0.995
-
+
decrease
46
F
0.077
0.27
-
+
decrease
62
F
0.308
0.456
-
+
decrease
41
F
0.516
0.807
+
+
decrease
51
M
1.862
1.52
-
-
increase
35
M
1.424
0.456
+
+
increase
30
M
0.59
0.257
+
-
increase
41
M
1.484
0.219
+
+
increase
39
M
1.463
0.695
-
-
increase
39
M
0.528
0.573
+
-
not apparent
28
M
0.327
0.546
-
-
decrease
The effect of β-ionone on sweating was examined using conventional QSART. The integrated volumes of sweating for 5 min after application of β-ionone and the control are shown. A difference of < 0.05 mg/5 min compared to the control was not considered significant. The subjects' exercise habits and their sensitivities to β-ionone as an odor are also shown.
In this study, we conducted transcriptome analysis to identify genes responsible for AIGA, and we unexpectedly found OR expression in eccrine sweat glands. Histochemical and functional experiments confirmed OR expression in eccrine sweat glands and OR ligand-dependent activation of perspiration. This finding identifies an unprecedented mechanism underlying perspiration.
There are diverse causes of anhidrosis, including neurological trauma, angiopathy, sweat gland disorder, and decreased responses to acetylcholine (
). Historically, tropical anhidrotic asthenia, which is characterized by extended lesions of anhidrosis accompanied with miliaria, has been thought to be caused by obstruction of sweat ducts due to miliaria (
Histamine, mast cells, blood vessels, microneuropathies, sweat leakage from sweat glands, and a high state of anxiety have also been shown to be involved in AIGA pathogenesis (
). Thus, the etiological picture of AIGA varies by case. Decreased sweating function leads to difficulties in acclimation to heat and causes heat retention and heat stroke. Usually, patients with AIGA complain that their sweating stopped suddenly despite sweaty conditions. According to clinical guidelines, AIGA is diagnosed by the presence of an area of reduced sweating accompanied by the absence of neurological symptoms (
) Thus, disorders in sweat glands or their microenvironments may contribute to the development of AIGA. Originally, this study aimed to delineate the molecular mechanism of anhidrosis by comparing gene expression profiles between hidrotic and anhidrotic eccrine sweat glands. During the gene expression analysis, we found OR expression in eccrine sweat glands in AIGA patients. This serendipitous finding expands the role of odorants on sweat regulation.
In the RNA sequencing-based transcriptome analysis, an individual patient was used to compare anhidrotic and hidrotic lesions to reduce non-specific gene expression differences resulting from individual differences. The weak point of this procedure was that we could not ensure the persistency of the sweating ability in each collected sample. Nonetheless, skin samples were collected from regions in which sweating ability was confirmed just before the biopsy. Statistical analyses of RNA sequencing-based transcriptomes led us to focus on ORs that were expressed highly in hidrotic eccrine sweat glands when compared to anhidrotic sweat glands. ORs were cloned originally by Buck and Axel and were found to discriminate odors by interacting with odorous ligands (
) . To the best of our knowledge, expression of ORs in human eccrine sweat glands has not yet been reported. Although we cannot prove that both transcriptome analysis and ISH achieved with highest accuracy, we focused on expression level of OR51A7 which showed consistency between those assays, while other ORs showed inconsistency. Because OR51A7 ligands had not been identified before this study, we set out to identify the odorous compounds recognized by this receptor. Based on receptor family analysis, we hypothesized that β-ionone is an odorous ligand of OR51A7. It was previously shown that OR51E2 is expressed on cancer cells and that β-ionone promotes cancer cell migration via OR51E2, indicating that β-ionone is an OR51E2 ligand (
). In this study, the HEK293T cell-based shedding assay system using AP-TGFα- and G-protein-containing plasmids confirmed the recognition of β-ionone by OR51E2 in the presence of Gαq/il and/or Golf. It is possible that the OR51A7 and OR51E2 signaling pathways may be the same. Notably, sweat glands express both Gαq and Golf at the transcriptional level suggesting that β-ionone may transduce perspiration signals through OR51A7 and/or OR51E2.
Consistent with the hypothesis that ORs deliver sweat gland signals in response to β-ionone, QSART analyses showed that topically applied β-ionone induces skin perspiration in humans. Thus, β-ionone may affect axon reflex-mediated perspiration. Unexpectedly, QSART measurements revealed a gender-based difference in the induction of perspiration in response to β-ionone. Sweat volumes decreased in female subjects upon topical application of β-ionone to the skin, whereas sweat volumes increased in male subjects under the same conditions. In addition, all the female subjects could smell the aroma of β-ionone, but most of the male subjects could not. It has been reported that genetic and demographic phenotypes contribute to gender variance in human odorant perception (
). In a study of mice that were maintained separately based on their sex, extensive differences in olfactory sensory receptor repertoires between the genders were found (
). These findings suggest that gender variance in odorous ligand-mediated sweating associates with odor perception. In this study, the effect of odor perception of β-ionone via the nasal cavity on the QSART results cannot be excluded. Although a side-by-side comparison of β-ionone and glycerol was performed simultaneously, the effect of the topical glycerol control may have also been influenced by odor perception. Thus, topical β-ionone may affect sweating through percutaneous penetration.
This study has its limitations. The primary limitation was the small number of human samples because AIGA is a rare intractable disease. In addition, the in vitro reporter cell assay was established using HEK293T cells rather than sweat gland cells because we have not yet established a physiologically relevant sweat gland acinar cell line. Further, β-ionone dose dependence was not investigated in the sweat test due to ethical concerns regarding the burden on the study subjects. Moreover, our results cannot explain the gender-based difference in perspiration observed upon β-ionone application. Despite these limitations, our findings showed that perspiration can be regulated via a unknown pathway involving activation of ORs by odorous substances. The etiology and nature of AIGA remains unknown, and information on regional differences and similarities of anhidrosis are lacking and should be elucidated in the future. The involvement of ORs in AIGA pathogenesis was not proven in this study and should be addressed in future studies.
MATERIALS AND METHODS
Preparation of skin samples for transcriptome analyses
AIGA patients and healthy donors were recruited after the study was approved by the Institutional Review Board of Osaka University Hospital (653-4) and Nagasaki University Hospital (ID 20042025). Participants provided written informed consent, and one of them also agreed to publish the image in Figure 1a. Biopsy samples were manipulated according to the Declaration of Helsinki protocols. Subjects with anhidrosis were diagnosed based on the conventional Minor’s test, also called the starch-iodine sweat test, in which 2% iodine tincture was applied evenly to the skin followed by coating with a mixed suspension of starch-castor oil (50–100 g and 100 g, respectively). Sweating was induced by sauna bathing at 60°C for 10 min. After the Minor’s test confirmed that subjects suffered from AIGA, skin punch biopsies (4–5 mm in diameter) were taken from anhidrotic (uncolored) and hidrotic (colored) areas. RNA for transcriptomic analysis was prepared from stored biopsy samples collected from four patients after the Minor’s test as described above. Details of the cases are described in table 2. During this process, sweat glands, isolated from those specimens by using laser microdissection (LMD 7000, Leica), were used as the source of RNA-preparation.
Table 2Demographic and clinical characteristics of the four AIGA patients.
case
Age/gender
dulation of disease
treatment
occupation
underlying comorbidities
No sweat area
Sweating area
1
43/M
1 year
antihistamine
construction worker
none
back
forearm
2
32/M
1 year
steroid pulse
construction worker
none
back
chest
3
49/F
5 months
-
exercise instructor
cholinergic urticaria
upper arm
thigh
4
43/F
17 years
herbal medicine
-
none
right back
left back
Four AIGA patients were recruited into this study after approval from the Institutional Review Board of Nagasaki University Hospital. Two of the patients were male and two were female, and all provided written informed consent.
Total RNA was extracted from cells using an miRNeasy FFPE kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. Next-generation sequencing library preparation was conducted using the SMARTer ® Stranded Total RNA Sample Prep Kit-Pico Input Mammalian (Clontech, TaKaRa) kit according to the manufacturer’s instructions. Sequencing was performed on an Illumina HiSeq 2500 platform in 75-base single-end mode. Illumina Casava1.8.2 software was used for base calling. Sequenced reads were mapped to the human reference genome sequence (hg19) using TopHat v2.0.13 software in combination with Bowtie2 ver. 2.2.3 and SAMtools ver. 0.1.19 software. Fragments per kilobase of exon per million mapped fragments were calculated with Cuffnorm version 2.2.1. Raw data were deposited in the NCBI Gene Expression Omnibus database (GSE 193125).
Localization of estrogen receptor messenger ribonucleic acid in rhesus monkey uterus by nonradioactive in situ hybridization with digoxigenin-labeled oligodeoxynucleotides.
). Briefly, sections were deparaffinized, rehydrated, treated with 0.2 N HCl for 20 min, and then treated with 50 μg/ml proteinase K (Wako, Osaka, Japan) at 37°C for 15 min. After fixation with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 5 min, sections were immersed in 2 mg/ml glycine in PBS for 15 min and then maintained in 40% deionized formamide in 4x SSC (1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate, pH 7.0) until hybridization. Hybridization was carried out at 37°C overnight with 2 μg/ml digoxigenin-labeled sense or antisense oligo-DNAs (Thermo-Fisher, Waltham, Massachusetts) dissolved in hybridization medium containing 10 mM Tris-HCl (pH 7.4), 1 mM ethylenediaminetetraacetic acid (EDTA), 0.6 M NaCl, 1x Denhardt’s solution, 250 mg/ml yeast transfer RNA, 125 mg/ml salmon sperm DNA, and 40% deionized formamide. After hybridization, sections were washed four times with 40% deionized formamide in 2x SSC for 1 h at 37°C, immersed in blocking solution for 1 h, treated with horseradish peroxidase (HRP)-conjugated goat anti-digoxigenin antibody overnight, and washed three times with 0.075% Brij 35 in PBS for 15 min. After rinsing with PBS, visualization was performed with 3,3’-diaminobenzidinetetrahydrochloride (DAB; Dojindo, Kumamoto, Japan), H2O2, CoCl2, and NiSO4. Antisense oligo-DNA sequences complementary to portions of human OR51A7, OR6C74, and OR4A15 sense sequences were selected (Table 3). We also prepared oligo-DNA complementary to part of human 28S ribosomal RNA (rRNA) as a positive control probe (
Total RNA sequences were searched for using the NCBI database. ISH probes were designed based on the number of bases and the GC ratio. 28S rRNA was used as the positive control.
The skin samples used in the immunohistological study were obtained from a different subject than the samples used in the transcriptome assay. Skin samples were fixed in a 10% formalin neutral buffer solution overnight. For staining, paraffin-embedded skin samples were sectioned (4 μm), deparaffinized, and dehydrated. Some sections were stained with HE, and other sections underwent antigen retrieval in 10 mM sodium citrate buffer pH 6.0 at 121°C for 3 min. Samples were blocked with 5% NGS/TBST for 60 min, incubated with primary antibodies at 4°C overnight, and then washed three times with PBS. The following antibodies were used: OR51A7 (Invitrogen, Waltham, Massachusetts), OR51E2 (Invitrogen, Waltham, Massachusetts), GNAL (abcam, Cambridge, England), and GNAQ (abcam, Cambridge, England). Then, samples were incubated with secondary antibodies (Dako, LSAB2 system-HRP, Agilent, Santa Clara, California) at room temperature for 30 min and washed three times with PBS. Signals were visualized using the DAB substrate kit (BD, Franklin Lakes, New Jersey). As a negative control, 5% bovine serum albumin in PBS was used instead of the primary antibody (data not shown).
Construction of OR51A7 and OR51E2 expression vectors
Full-length canonical human OR51A7 and OR51E2 cDNAs were purchased from GenScript Biotech and cloned into the pCAGGS vector (Riken, Tokyo, Japan) containing the cytomegalovirus immediate-early enhancer, chicken β-actin, and the rabbit β-globulin (CAG) heterozygous promoter for expression in mammalian cells. Sequences are shown in Table 4.
) with minor modifications. HEK293T cells were cultured in RPMI1640 medium supplemented with 10% FCS, 100 IU/ml penicillin, and 100 μg/ml of streptomycin at 37°C in a humidified atmosphere with 5% CO2. Cells (8 × 105 cells/4 ml) were transfected with the AP-TGFα-encoding plasmid (4 μg), the OR-encoding plasmid (3.2 μg), and/or G-protein-encoding plasmids (1.6 μg) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 12 h, the transfected cells were harvested using trypsin/EDTA, pelleted by centrifugation at 600 g for 5 min at 4°C, washed once with Dulbecco’s modified Eagle’s medium, and resuspended in Hanks’ balanced salt solution (HBSS). Cell suspensions (180 μl each) were seeded into a 96-well culture plates, 20 μl of β-ionone solution (10×, diluted in HBSS containing 0.01% (w/v) fatty acid-free and protease-free grade bovine serum albumin; Sigma-Aldrich, St. Louis, Missouri).was added to each well, and the plates were incubated for 60 min at 37°C. Then, the plates were centrifuged at 600 g for 2 min at 4°C, cell supernatants (80 μl) were transferred to fresh 96-well plates, and 80 μl of AP reaction solution (10 mM p-NPP, 120 mM Tris-HCl buffer pH 9.5, 40 mM NaCl, and 10 mM MgCl2) was added to each well. Before and after a 2 h incubation at 37°C, absorbances at 405 nm were measured using a microplate reader.
Impact of topical β-ionone on sweating measured by the Quantitative Sudomotor Axon Reflex Test (QSART)
In this study, the volume of acetylcholine-induced sweating was measured quantitatively by QSART based on the method established by Lee et al (
Tropical Malaysians and temperate Koreans exhibit significant differences in sweating sensitivity in response to iontophoretically administered acetylcholine.
). Briefly, subjects were asked to remain quiet for 20 min before undergoing QSART in a hospital outpatient clinic at constant temperature (20°C) and humidity (60%). The multicompartmental sweat capsule used in QSART consists of two independent compartments. Acetylcholine (100 mg/ml) applied iontophoretically to the skin from the outer compartment stimulates the underlying sweat glands directly; simultaneously, the central compartment of the capsule collects the sweat on the skin surface and measures sweat volume during the 5 min of iontophoresis. The integrated value of the sweat volume during the 5 min was regarded as the sweating ability (
The effect of β-ionone topical application was evaluated in a side-by-side comparison with glycerol on both forearms of healthy subjects. This study was approved by the Institutional Ethical Committee of Nagasaki University (ID 20062602), and written informed consent was obtained from all study subjects. Briefly, 100 μl of β-ionone or glycerol (control) was applied on each forearm topically and separately. Immediately after topical application, ventilated sweat capsules that functioned in iontophoresis of acetylcholine (100 mg/ml) and that were attached to an additional outer compartment of sponge were placed onto the application areas. Integrated values of sweat volumes during the 5 min acetylcholine iontophoresis after β-ionone and glycerol treatment were compared.
QSART activates postganglionic nerve fibers by both acetylcholine and electric stimulation, and once an axon is activated, the effect will be sustained for a certain period. Therefore, this test was performed once on the same subject to avoid erroneous results.
UV-Irradiation- and Inflammation-Induced Skin Barrier Dysfunction Is Associated with the Expression of Olfactory Receptor Genes in Human Keratinocytes.
Localization of estrogen receptor messenger ribonucleic acid in rhesus monkey uterus by nonradioactive in situ hybridization with digoxigenin-labeled oligodeoxynucleotides.
Tropical Malaysians and temperate Koreans exhibit significant differences in sweating sensitivity in response to iontophoretically administered acetylcholine.
Acknowledgements We thank Ms. Mariko Yozaki for her technical assistance with the immunohistochemistry.
Conflict of Interest
HM, NM, and YT have a patent pending. This study was supported by the Platform Project for Supporting Drug Discovery and Life Science Research, Japan Agency for Medical Research and Development, under grant number JP17am0101001 (support number 2259) and a grant from the Ministry of Education, Culture, Science and Technology under grant number 20K17320.
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