Gap junction-mediated contraction of myoepithelial cells induces the peristaltic transport of sweat in human eccrine glands

Nakashima, Kie; Kato, Hiroko; Kurata, Ryuichiro; Qianwen, Luo; Hayakawa, Tomohisa; Okada, Fumihiro; Fujita, Fumitaka; Nakagawa, Yukinobu; Tanemura, Atsushi; Murota, Hiroyuki; Katayama, Ichiro; Sekiguchi, Kiyotoshi

doi:10.1038/s42003-023-05557-9

Download PDF

Article
Open access
Published: 18 November 2023

Gap junction-mediated contraction of myoepithelial cells induces the peristaltic transport of sweat in human eccrine glands

Kie Nakashima ORCID: orcid.org/0000-0002-7255-6855¹,
Hiroko Kato¹,
Ryuichiro Kurata¹,
Luo Qianwen¹,
Tomohisa Hayakawa¹,
Fumihiro Okada²,
Fumitaka Fujita^1,2,
Yukinobu Nakagawa³,
Atsushi Tanemura ORCID: orcid.org/0000-0002-5239-8474³,
Hiroyuki Murota⁴,
Ichiro Katayama⁵ &
…
Kiyotoshi Sekiguchi ORCID: orcid.org/0000-0002-6433-1410⁶

Communications Biology volume 6, Article number: 1175 (2023) Cite this article

1049 Accesses
11 Altmetric
Metrics details

Subjects

Abstract

Eccrine sweat glands play an essential role in regulating body temperature. Sweat is produced in the coiled secretory portion of the gland, which is surrounded by obliquely aligned myoepithelial cells; the sweat is then peristaltically transported to the skin surface. Myoepithelial cells are contractile and have been implicated in sweat transport, but how myoepithelial cells contract and transport sweat remains unexplored. Here, we perform ex vivo live imaging of an isolated human eccrine gland and demonstrate that cholinergic stimulation induces dynamic contractile motion of the coiled secretory duct that is driven by gap junction-mediated contraction of myoepithelial cells. The contraction of the secretory duct occurs segmentally, and it is most prominent in the region surrounded by nerve fibers, followed by distension-contraction sequences of the excretory duct. Overall, our ex vivo live imaging approach provides evidence of the contractile function of myoepithelial cells in peristaltic sweat secretion from human eccrine glands.

Dynamic polyhedral actomyosin lattices remodel micron-scale curved membranes during exocytosis in live mice

Article 29 July 2019

An epifluidic electronic patch with spiking sweat clearance for event-driven perspiration monitoring

Article Open access 07 November 2022

A wearable patch for continuous analysis of thermoregulatory sweat at rest

Article Open access 23 March 2021

Introduction

Humans have two types of sweat glands: eccrine and apocrine. Eccrine glands are essential thermoregulatory organs that secrete a watery fluid, i.e., sweat, while apocrine glands are evolutionary remnants of odor-producing organs that secrete thick, milky mucus¹. Sweat from eccrine glands removes excess body heat from the skin surface through evaporative cooling. Eccrine gland dysfunction can cause either excessive or inadequate sweat secretion, commonly known as hyperhidrosis and hypohidrosis, respectively. Hyperhidrosis leads to physical discomfort, social stigmatization and even mental stress, whereas hypohidrosis can result in heatstroke, a form of life-threatening hyperthermia². These abnormalities in sweat production are caused by failures in either the hypothalamic thermoregulatory center or local sweat glands^3,4. Because the hypothalamus is a multimodal regulatory center, understanding the local mechanisms of sweat secretion in human skin is fundamental to developing site-specific treatments for sweating dysfunction.

The eccrine gland forms an unbranched, highly coiled tubule that extends into the deep dermis (Fig. 1a). The coiled portion of the eccrine gland consists of the secretory duct and a transitional region of the excretory duct. The excretory duct is formed by two layers of stratified cuboidal cells, i.e., the luminal and basal layers, while the secretory duct is composed of a monolayer of secretory luminal cells obliquely surrounded by spindle-shaped myoepithelial cells⁵ (Fig. 1b). Myoepithelial cells are filled with dense masses of myofilaments comprising smooth muscle actin^5,6, indicative of their ability to contract. Indeed, myoepithelial cells dissociated from the eccrine gland of primate palms are induced to contract upon cholinergic stimulation⁷, although the contraction of myoepithelial cells in situ and its relevance to sweat secretion have remained unexplored. The myoepithelial cells in the eccrine gland are arranged longitudinally around the entangled secretory duct and partially surrounded by autonomic nerve fibers in a bandage-like manner⁵, suggesting that the contraction of myoepithelial cells may be dominated by synchronous and directional neuronal activities to compress the secretory duct for sweat transport. Therefore, it is necessary to capture the dynamics of the contraction of myoepithelial cells as well as the changes in the lumen volume in secretory and excretory ducts to elucidate the in situ function of myoepithelial cells in eccrine glands.

**Fig. 1: Schematic illustration of a human eccrine gland.**

To demonstrate the contractile motion of the secretory ducts of eccrine glands, we established an ex vivo method of 3D live-tissue imaging of isolated secretory ducts that includes the simultaneous labeling of actin and the cell membrane of both luminal and myoepithelial cells. Time-lapse imaging allowed quantitative visualization of a series of contractile motions of the secretory ducts triggered by pharmacological stimulation. Moreover, 3D analysis of myoepithelial and luminal cells at a single-cell resolution corroborated the essential role of myoepithelial cells in the contractile motions of secretory ducts. We also demonstrated that cholinergic stimulation triggers the contraction of myoepithelial cells to compress secretory ducts and that the propagation of contraction in the myoepithelium requires cell-to-cell communication via gap junctions that are associated with sweat-expelling motions of the secretory duct; thus, we provide technological and conceptual advances for elucidating the molecular mechanisms underlying sweating in humans.

Results

3D live-tissue imaging of myoepithelial cells in isolated eccrine glands

The secretory duct of the sweat gland is difficult to observe in vivo and ex vivo because the secretory duct, which forms the coiled portion of the sweat gland, is localized in the deep dermis. To visualize the shape and location of myoepithelial cells of the secretory duct, we harvested eccrine glands from human skin and live-stained the whole eccrine coiled structure. Because myoepithelial cells contain abundant filamentous actin, we used Acti-stain 488 (Acti-stain), a fluorochrome-conjugated phalloidin that directly binds to filamentous actin. The staining patterns of Acti-stain for excretory and secretory ducts were distinguishable (Fig. 2a, b). Acti-stain signals were detected in submembranous regions of cuboidal cells in the excretory duct (Fig. 2c) and ubiquitously within spindle-shaped cells in the secretory duct (Fig. 2d). The Acti-stain signals in the spindle-shaped cells overlapped with the signals of α-smooth muscle actin (αSMA), a marker of myoepithelial cells (Fig. 2e, f), indicating that the spindle-shaped cells were myoepithelial cells longitudinally elongated along an entangled secretory tubule. Consistent with a previous report⁵, the cuboidal cells in the excretory duct lacked an αSMA signal (Fig. 2g). Furthermore, counterstaining with Hoechst 33342 revealed that nuclei in the excretory duct were densely packed because of compactly aligned luminal cuboidal cells (Fig. 2h, i; arrows), whereas nuclei in the secretory duct were rather sparse because of the elongated myoepithelial cells (Fig. 2h, i; arrowheads). In addition, the nuclei of the myoepithelial cells were typically elliptical (Fig. 2i, arrowheads in the right panel), while those of the luminal cuboidal cells were rounded (Fig. 2i, arrows in the right panel). These results indicate that myoepithelial cells are distinguishable from other cell types based on their cellular and nuclear morphologies on 3D live-tissue imaging.

**Fig. 2: Myoepithelial cells in eccrine glands visualized by actin staining.**

Cholinergic stimulation triggers eccrine gland contraction

Considering that nerve fibers wrap around the secretory duct in a bundle⁵, we hypothesized that the myoepithelium in the secretory duct would synchronously contract by autonomic nerve stimulation to extrude sweat from the secretory duct. Thus, we attempted to record the motions of the isolated eccrine glands after labeling their nuclei and actin cytoskeletons with Hoechst 33342 and Acti-stain, respectively. Eccrine glands are innervated by atypical sympathetic terminals, which release acetylcholine⁸. We tracked the motions of both the nuclei and the tissue structure of the coiled portion of the eccrine gland upon stimulation with pilocarpine, a cholinergic agonist, to mimic autonomic nervous stimuli for sweating⁹. To quantitatively describe the motions from time-lapse images, we adopted the color aberration measurement of two merged images pseudocolored green or magenta at different time points (Fig. 3a). In the control condition with phosphate-buffered saline (PBS), spontaneous motion of the Acti-stained myoepithelial cells was rarely observed, leaving the colors almost completely merged (white) for both myoepithelial actin structures and nuclei (Fig. 3b; Supplementary movie 1). Upon pilocarpine stimulation, however, spindle-shaped myoepithelial cells responded and moved slightly, as evidenced by color aberration from white to green-magenta (Fig. 3c; Supplementary movie 2). The round nuclei of luminal secretory cells were also dislodged (Fig. 3c). The pilocarpine-induced dislodgement of myoepithelial cells and luminal secretory cells was observed irrespective of the presence or absence of divalent metal ions in the extracellular milieu (Supplementary movie 3). Furthermore, the nuclei of myoepithelial cells without Acti-stain costaining showed greater displacement after pilocarpine treatment (Fig. 3d, e; Supplementary movie 4), suggesting that Acti-stain that directly binds to filamentous actin might interfere with the dynamic motion of actin filaments in myoepithelial cells. Thus, to visualize the skeletal frame of the eccrine gland components, we employed the alternative fluorescent dye CellMask^TM Deep Red (CMDR), a cell membrane marker¹⁰, which should not interfere with physiological actin dynamics. CMDR staining visualized the αSMA-positive myoepithelial cells of the secretory duct as thick striped streaks (Fig. 4a, b) and the αSMA-negative cuboidal cells of the excretory duct as cubic lattices by delineating the outlines of the cells (Fig. 4c). Therefore, the shape and location of the two duct types were distinctively specified by the CMDR staining patterns. The CMDR-stained eccrine glands contracted more dynamically after pilocarpine treatment than those stained with Acti-stain (Fig. 4d, e; Supplementary movie 5). Notably, contractile motions were most pronounced in the region where the eccrine gland displayed concertina-like CMDR signals (Fig. 4d, red arrowheads). Previously, we reported that PGP9.5-positive nerve fibers were predominantly wrapped around the secretory portions of sweat glands⁵. Consistent with this observation, double immunostaining for PGP9.5 and αSMA confirmed that the PGP9.5-positive nerve fibers exclusively surrounded the secretory ducts transversely, while αSMA-positive myoepithelial cells were longitudinally aligned like thick stripes on the secretory duct (Fig. 4f). In addition, the color aberration of Hoechst 33342 signals revealed nuclear displacement in the secretory duct that was triggered by pilocarpine (Fig. 5a, upper panels; Supplementary movie 6). Such nuclear displacement was almost completely blocked in the presence of atropine, an antagonist against muscarinic acetylcholine receptors (Fig. 5a, lower panels; Fig. 5b; Supplementary movie 7).

**Fig. 3: Cholinergic stimulation triggers contractile motion of Acti-stained eccrine glands.**

**Fig. 4: Dynamic contraction of the secretory duct occurs at the concertina-like region surrounded by coiled nerve fibers.**

**Fig. 5: Muscarinic stimulation triggers contractile motion of the secretory duct.**

Perspiration can also be induced via the activation of nicotinic acetylcholine receptors in vivo¹¹. To assess the contribution of nicotinic stimulation to the contraction of the human eccrine gland, we examined the effect of nicotine by 3D live-tissue imaging. The nuclei did not move after nicotine stimulation (Fig. 5c, d; Supplementary movie 8), indicating that muscarinic stimulation, but not nicotinic stimulation, induces the contractile motion of the human eccrine gland as the basis for sweat expulsion^12,13. Hereafter, we used muscarinic stimulation by pilocarpine to induce eccrine gland contraction in vitro.

Myoepithelial cells segmentally contract to compress the secretory duct

At low magnification, the secretory duct was visualized by CMDR staining in two distinctive patterns: a concertina-like pattern with nerve fibers wrapped around the duct in a bandage-like manner⁵ (Fig. 6a, magenta box) and a thick striped pattern with a faint contour of spindle-shaped myoepithelial cells (Fig. 6a, yellow box). In sharp contrast, the excretory duct was visualized in a latticework pattern (Fig. 6a, black arrowhead). Upon pilocarpine stimulation, only the concertina-like part of the secretory duct exhibited dynamic contractile motion (Fig. 6b, Supplementary movies 9-11). For quantification of contractile motion, we measured the length of two virtual reference points arbitrarily selected from the bright signals on the secretory duct. The spans of the virtual reference points on the concertina-like part began to shorten after pilocarpine application, reached maximum contraction after approximately 5 minutes, and then gradually returned to the initial length over the next 20 minutes (Fig. 6c). On the other hand, the spans of the virtual reference points on the secretory duct showing thick stripes/less concertina-like patterns remained unchanged (Fig. 6d). Such transient glandular contraction of the concertina-like part was also observed in another pilocarpine-induced contractile motion of the secretory duct (Supplementary movie 5 and Fig. S1). Thus, only the limited parts of the secretory duct characterized by concertina-like patterns engage in pilocarpine-induced contraction.

**Fig. 6: Myoepithelial contraction compresses the secretory duct.**

To further dissect the fine cellular motions within the secretory duct, the displacement of the nuclei of the myoepithelial and luminal cells was traced in magnified cross-sections of the duct. Focusing on the luminal cells, the radius of curvature was estimated from the position of three nuclei (dots in Fig. 6e). The radius of curvature of the duct decreased after pilocarpine treatment (Fig. 6f; P < 0.05). To further scrutinize the mode of nuclear displacement at the single-cell level, we also compared the direction of nuclear displacement in both myoepithelial and luminal cells. The elliptical myoepithelial nuclei diagonally slid along the perimeter of the duct (Fig. 6g, h). On the other hand, the round luminal nuclei slipped aside after the addition of pilocarpine, followed by a change in direction straight toward the center of the lumen (Fig. 6g, h), suggesting that myoepithelial displacement pushed luminal cells toward the lumen to compress the duct. There was no significant delay in the displacement of the nuclei between myoepithelial cells and their adjacent luminal cells (Fig. 6g, h), which was further supported by the measurement of cumulative distances of the displaced nuclei of myoepithelial cells and their adjacent luminal cells (Fig. 6i).

To substantiate the role of actin dynamics in myoepithelial cells, we pharmacologically inhibited the molecular machinery of smooth muscle contraction. By pretreating the isolated eccrine glands with cytochalasin D, an inhibitor of actin polymerization, the contraction of the secretory duct and the nuclear displacement of the myoepithelial and luminal cells were abolished, as the color aberration remained white even after pilocarpine stimulation (Fig. 6j, k; Supplementary movie 12). These results indicate that actin dynamics in myoepithelial cells play a pivotal role in the contractile motion of the limited parts of the secretory duct upon cholinergic stimulation.

Myoepithelial contraction causes reciprocal changes in the lumen volume of secretory and excretory ducts

To further address the role of the contractile motions of the eccrine gland in sweat secretion, we analyzed the deformation of the CMDR-stained cellular latticework, focusing on the changes in the volume of the luminal space within the duct of the coiled structure. Cell membrane staining by CMDR revealed temporal changes in the latticework of the cellular structure and the lumen volume as signal voids inside the secretory coil. We extracted the areas of the signal voids from the 3D time-lapse data to trace the successive changes in the lumen volume in both the secretory and excretory ducts, thereby capturing the motion of sweat transport driven by the contraction of the secretory duct (Fig. 7a). First, we disassembled the 3D live-tissue images along the axes of time and Z slices; then, the lumen area on each Z slice was extracted by masking the cellular signals and measured on the X-Y plane (Fig. 7b). The lumen volume was estimated from the summation of these X-Y plane areas. The reconstructed 3D image data showed that the lumen of the secretory duct was segmented by partition (Supplementary movie 13; Fig. 7c; yellow arrowhead at t = −8’00”). On pilocarpine administration, the narrow segment of the lumen started to shrink immediately (Fig. 7c, arrow at t = 0’00”; asterisk (*) at t = 2’00”, Fig. 7c, f) and then dilated even larger than the initial volume until the adjacent segments were recanalized (open triangle (Δ) at t = 9’20”, Fig. 7c, f). The lumen started to shrink again at 9’20”-10’40” (open diamond (◊), Fig. 7c, f) and continued to regress throughout the stimulation. In parallel with this second shrinking phase, the different parts of the excretory duct (designated ED1 and ED2) expanded sequentially with a time lag of 4–5 minutes (closed triangles (▲), Fig. 7d–f), followed by the gradual shrinking phase that was prolonged over 30 minutes. Thus, pilocarpine-stimulated dilation/constriction of the secretory duct was followed by sequential dilation/constriction of the different parts of the excretory duct, indicative of successive transport of sweat within the coiled secretory portion of the eccrine gland. The dynamics of luminal dilation and constriction support the view that the pilocarpine-induced contraction of myoepithelial cells generates a sweat-transporting force through the excretory duct.

**Fig. 7: Reciprocal changes in lumen volume of secretory and excretory ducts.**

Gap junction inhibitors block myoepithelial contraction

Although cholinergic stimulation was a trigger for myoepithelial contraction, not all myoepithelial cells contracted simultaneously. Rather, propagation of cholinergic stimulation on myoepithelial cells may be locally restricted along the longitudinal axis of the secretory duct and induce segmental contractions to generate force for sweat transport. In addition to involuntary control by the autonomic nervous system¹⁴, some smooth muscle cells are electrically interconnected via gap junctions to operate in a coordinated fashion as a functional cluster¹⁵. Accordingly, excitation can rapidly spread among unitary smooth muscle cells, resulting in segmental contraction. To determine whether the myoepithelial cells of the secretory duct are also interconnected by gap junctions, we examined the effects of carbenoxolone (CBX), a broad-spectrum inhibitor of gap junctions¹⁶, on the contractile motion of the secretory duct upon pilocarpine stimulation. In the presence of CBX, both luminal and myoepithelial nuclei scarcely moved from their initial positions after pilocarpine treatment (Fig. 8a, b; Supplementary movies 14, 15). The expansion of the excretory ducts was also inhibited in the presence of CBX. The blockade of nuclear displacement by CBX was of a magnitude similar to that imposed by atropine pretreatment (Fig. 8c).

**Fig. 8: Gap junctions mediate pilocarpine-induced contraction of isolated eccrine glands.**

To further explore the role of gap junctions in the segmental contraction of the secretory duct, we examined the expression levels of gap junction proteins, i.e., connexins (Cxs), in the myoepithelial cells of the secretory duct by quantitative reverse transcription polymerase chain reaction (qRT‒PCR). Because the myoepithelial cells in the eccrine sweat glands have been characterized as a CD29^high/CD49f^high subpopulation⁵, we dissociated the isolated eccrine glands with collagenase and obtained the myoepithelial cell–enriched CD29^high/CD49f^high subpopulation. qRT‒PCR analyses of a total of 17 connexins, namely, Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx31.9, Cx32, Cx37, Cx40, Cx40.1, Cx43, Cx45, Cx46, Cx47, Cx50, Cx58, and Cx62, showed that the connexins expressed in the CD29^high/CD49f^high subpopulation were Cx26, Cx30, Cx31, Cx37, Cx43, and Cx45, of which Cx43 was most abundant (Fig. 8d). Immunofluorescence staining of the isolated eccrine glands for the connexins expressed in the myoepithelial cells along with αSMA, a marker for myoepithelial cells, showed that Cx43 and Cx45 were detected on αSMA-positive myoepithelial cells as puncta (white arrowheads), although no such punctate signals were detected on myoepithelial cells for other connexins, including Cx26, Cx30, Cx31, and Cx37 (Fig. 8e–j). Punctate signals for other connexins were also detected in the luminal cells of the secretory duct at variable densities. Notably, the Cx43 signal was frequently localized between myoepithelial cells in small puncta (Supplementary Fig. S2), raising the possibility that Cx43 is the major gap junction constituent involved in the segmental contraction of myoepithelial cells in the secretory duct.

The role of Cx43 in the segmental contraction of myoepithelial cells was further addressed by using 2-aminoethoxydiphenylborane (2-APB) and Gap27, both of which inhibit Cx43^17,18, although 2-APB has been known to exhibit complex pharmacology, inhibiting not only Cx43 but also inositol trisphosphate receptors (IP3Rs) and transient receptor potential (TRP) channels and having a dose-dependent bimodal effect on store-operated calcium entry¹⁹. Both inhibitors attenuated the pilocarpine-induced contractile motion of the secretory duct and the nuclear displacement of the luminal cells (Fig. 8k; Supplementary movies 16, 17), supporting the involvement of Cx43 in the pilocarpine-induced segmental contraction of myoepithelial cells in the secretory duct. The inhibitory effect was more pronounced with 2-APB than with Gap27, an inhibitor more specific for Cx43, suggesting that the complex pharmacological effects of 2-APB on IP3Rs and store-operated calcium entry were also involved in the pronounced inhibition of contractile motion and nuclear displacement in the secretory duct.

Finally, we examined the contribution of gap junctions to sweating in vivo. We applied CBX to the human palm and measured the perspiration rate by the minor test, which visualizes the expulsion of sweat drops by the metachromasia of the iodine-starch reaction (Fig. 9a). Sweating was induced by autonomic stimulation by passive heating via a foot bath. Prior to the sweating test, we ran a pretest to visualize active sweat pores on the palms of the subjects. Palm sweating was locally inhibited by approximately 30% by pretreatment with 16 mM CBX for 30 minutes compared to the control in a nearby area (Fig. 9b). This result demonstrates that gap junction-mediated communication in eccrine glands is essential for the transportation of sweat to the skin surface. Taken together, our findings suggest that myoepithelial cells are partially connected through gap junctions to segmentally contract for sweat secretion upon autonomic stimulation.

**Fig. 9: CBX suppresses sweat secretion.**

Discussion

Although the motions of eccrine glands have been investigated by several methodologies^20,21,22, no study reported to date has captured the live motion of eccrine glands. In the present study, we established a method for ex vivo 3D live imaging to observe the overall contractile motions of isolated human eccrine glands. The dynamic motion of the coiled structure upon cholinergic stimulation was successfully captured, recapitulating the physiological contractile response to autonomic stimuli²³. The dynamic motion was verifiably the direct consequence of cholinergic stimulation because atropine, a cholinergic antagonist, blocked the motion of the coiled structure. We found that some parts of the secretory duct were visualized on CMDR staining in a concertina-like pattern, which likely reflects sympathetic nerve fibers surrounding the secretory duct. The secretory coils of eccrine glands have been shown to be innervated by sympathetic nerves with varicose nerve terminals^5,24. The concertina region in the secretory duct displayed more pronounced contractile motion upon cholinergic stimulation than the region with little or no concertina-like signal, consistent with previous ex vivo studies demonstrating glandular contraction²² and sweat secretion²⁵ induced by cholinergic stimuli. Our results also revealed that contractile motion of the secretory duct was blocked by gap junction inhibitors, including CBX, 2-APB, and Gap27. The inhibitory effect of CBX was also confirmed by the in vivo perspiration test on human palms. These results raise the possibility that gap junction-mediated communication between myoepithelial cells controls the effective propagation of contraction in a single long tubular structure. Our ex vivo 3D live imaging is therefore a useful model for deciphering the mechanisms that operate during sweat secretion by eccrine glands and the role of myoepithelial cell contraction in transporting sweat to the skin surface.

Several findings indicate that myoepithelial cells surrounding the exocrine secretory ducts and/or alveoli contract upon neuronal or hormonal stimulation^26,27,28,29. Myoepithelial cells are enriched in actin filaments and arranged longitudinally in parallel around the secretory duct of the eccrine gland. We demonstrated that cytochalasin D, an inhibitor of actin polymerization, blocked the pilocarpine-induced contractile motion of the secretory duct of the isolated coiled structure. Acti-stain, a fluorochrome-conjugated phalloidin, also reduced the contractile response to cholinergic stimulation, supporting the role of myoepithelial cells in the contractile motion of the isolated coiled structure. Previously, Sato et al. examined the contractile properties of an isolated secretory duct of the primate palm sweat gland using a sensitive mechanical strain gauge transducer and demonstrated that acetylcholine, but not adrenergic agonists or the calcium ionophore A23187, induced glandular contraction of the secretory duct²¹. Consistent with this observation, our 3D live imaging detected shortening of the secretory duct in the region showing a concertina-like pattern upon CMDR staining. Such shortening of the secretory duct was not significantly detected in the region that did not show a clear concertina-like pattern. Furthermore, the high-resolution analysis focusing on the displacement of the nuclei of luminal and myoepithelial cells in cross-sections indicated that the curvature radii of the secretory duct decreased after pilocarpine treatment in the following manner: myoepithelial nuclei slid diagonally along the perimeter of the secretory duct, followed by sideways sliding and subsequent downward displacement of luminal nuclei. These observations indicate that contraction of myoepithelial cells causes not only shortening but also narrowing of the secretory duct.

The contraction of myoepithelial cells is considered instrumental in the secretory functions of other exocrine organs, including mammary, salivary, and lacrimal glands^27,28,30,31. Myoepithelial cells in mammary glands contract in response to the pulsatile release of oxytocin from the pituitary gland to eject milk. Mice deficient in αSMA (ACTA2), the most abundant actin isoform in the mammary duct, exhibited no gross anatomical defects but presented lactation failure²⁶. The mammary gland myoepithelial cells from ACTA2-null dams contracted considerably less than those from wild-type dams²⁶. Similar lactation defects with reduced contractility of myoepithelial cells have been reported in mice deficient in Orai1, a store-operated Ca²⁺ channel subunit required for milk ejection³². It should be noted that isolated sweat glands from Orai1-deficient mice exhibited a comparable calcium release from ER stores upon acetylcholine stimulation with that from wild-type mice³³. In our study, we observed pilocarpine-induced contractile motion of isolated sweat glands in Ca²⁺-free PBS. Because intracellular calcium was not depleted by EGTA in our experimental conditions, such initial calcium release from ER stores by pilocarpine stimulation may be sufficient for the induction of the contractile motion of the isolated sweat glands. Notably, mammary myoepithelial cells have been shown to communicate with each other through gap junctions³⁴. Given that gap junction inhibitors attenuate the contractile motions of the isolated eccrine secretory coil, it is conceivable that the coordinated contraction of myoepithelial cells via gap junctions operates in both mammary and sweat glands to expel secretory products.

The mechanistic basis of how sweat is transferred from the secretory duct to the excretory duct remains to be elucidated. Our 3D image reconstruction analyses focusing on the cholinergic stimulation-induced changes in luminal volume demonstrated that the lumen of the secretory duct was segmented by partition and underwent biphasic contraction with a rebound relaxation period upon cholinergic stimulation, followed by the progressive dilation-regression cycle of the excretory duct. These results raise the possibility that the secretory duct of eccrine glands is divided into segments and contracts segmentally upon cholinergic stimulation to transport sweat to the excretory duct.

Consistent with the scheme, the nerve fibers wrap around the secretory coil in a discrete manner to yield CMDR staining patterns with and without (or less) concertina-like signals, thereby eliciting the segmental contraction of the highly entangled secretory coiled structure. Such segmental contraction may effectively summate the compression force of the segmented portion to transport the secreted sweat toward the excretory duct.

Previous in vivo studies demonstrated high-frequency pulsatile sweat expulsion in humans^35,36, which has been shown to occur in response to the firing rhythm of skin sympathetic nerve activity³⁷. Such pulsatile sweat expulsion can be elicited either by rhythmic contraction of myoepithelial cells or by sweat production by secretory cells in a pulsatile rhythm. Segmental contraction of the secretory duct, together with intermittent cholinergic stimulation, is instrumental to ensure high-frequency pulsatile sweat expulsion from the eccrine gland.

Perspiration can be induced not only by agonists of muscarinic acetylcholine receptors but also by an agonist of nicotinic acetylcholine receptors, i.e., nicotine¹¹. Both muscarinic and nicotinic acetylcholine receptors were detected in eccrine sweat glands by in situ hybridization and immunohistochemistry¹². Our results show that nicotinic stimulation does not trigger the contractile motion of the secretory duct, making it likely that nicotinic stimulation governs sweat production from secretory cells but not the contraction of myoepithelial cells. Consistent with this conclusion, Sato and Sato (1981) reported that isolated eccrine sweat glands contracted in response to muscarinic agonists (e.g., muscarine and pilocarpine) but not in response to nicotine²⁵. They concluded that sweating in response to nicotine was elicited through the axon reflex, as proposed by Coon and Rothman¹¹.

Our results show that the segmental contraction of myoepithelial cells requires gap junctional intercellular communication (GJIC), as CBX, 2-APB, and Gap27 inhibited the contractile motions of the isolated secretory coil upon cholinergic stimulation. qRT‒PCR analyses of the connexins expressed in the myoepithelial cell–enriched CD29^high/CD49f^high subpopulation indicate that the myoepithelial cells express Cx26, Cx30, Cx31, Cx37, Cx43, and Cx45, of which Cx43 is most abundant. Immunofluorescence staining of Cx43 in the secretory coil of the eccrine gland shows that Cx43 is expressed on the myoepithelial cells in small puncta that are frequently detected between the myoepithelial cells, making it likely that the gap junctions connecting the myoepithelial cells are predominantly composed of Cx43. In support of this possibility, Gap27, a peptide gap junction inhibitor derived from Cx43, blocked the pilocarpine-induced contractile motion of the secretory coil of the isolated eccrine gland. Consistent with this view, there is accumulating evidence that Cx43 is the major connexin type expressed in myoepithelial cells of various exocrine glands^34,38,39,40. In mammary glands, while Cx26, Cx30 and Cx32 are expressed in luminal cells, Cx43 is primarily expressed in myoepithelial cells⁴¹ and is involved in myoepithelial contraction for milk ejection^34,40,42. Thus, ectopic expression of Cx26 suppresses the expression of Cx43 in the myoepithelial cells of mammary glands, leading to impaired GJIC and defects in milk ejection³⁴. A similar defect in milk ejection has been reported in a mouse model harboring a missense mutation in the Cx43 gene, which resulted in reduced Cx43 expression in myoepithelial cells⁴⁰. Cx43 has also been observed at gap junctions connecting myoepithelial cells in sublingual glands and implicated in the contraction of myoepithelial cells³⁹. Given that Cx43 is also predominantly expressed in the smooth muscle cells of the aorta⁴³ and is involved in vasoconstriction by vasopressin⁴⁴, it is conceivable that GJIC through Cx43 underlies the effective propagation of contractile signals within a segmented cluster of myoepithelial cells in the secretory duct of eccrine glands.

The requirement of GJIC in sweat secretion in vivo was also demonstrated by the perspiration test on a human palm, as topical administration of CBX suppressed sweat secretion by 30%. From a clinical viewpoint, selective inhibitors of Cx43 may be promising as antiperspirants to halt sweating without the drawbacks of conventional treatments. In everyday life, abnormally excessive sweating causes various social problems, including mental stress caused by self-hatred and unnoticed harassment by others in addition to daily inconvenience⁴⁵. However, the sole antiperspirants available for the last century have been conventional metal colloid–type antiperspirants that mechanically plug sweat pores⁴⁶. These mechano-plug antiperspirants are convenient, but they are clinically and esthetically disadvantageous because they irritate the skin and stain clothes^47,48. Additionally, the sweat held in the ducts can pathologically leak into the dermis, causing acute exudative inflammation^49,50,51. In contrast, selective gap junction inhibitors in the form of antedrugs could be safe antiperspirants with minimal adverse effects on the skin. CBX is such a candidate ingredient for next-generation antiperspirants. Notably, chemotherapeutic drugs acting on the hypothalamic‒pituitary‒adrenal (HPA) axis are known to have an impact on perspiration. For example, many antidepressants that act on the HPA axis have a side effect of causing excessive sweating⁵². Antiepileptic drugs sometimes cause sweating disorders by acting on the hypothalamic–pituitary–somatotropic axis, resulting in diminished sweating and a risk of heatstroke^53,54. Conversely, it is known that anticholinergic drugs cause diminished sweating and have been used to treat hyperhidrosis, although these drugs cause side effects such as dry mouth, dry eyes and constipation⁵⁵. CBX can be applied as a topical therapeutic agent that acts on the sweat glands directly by blocking GJIC, thereby eliminating the side effects of anticholinergic drugs.

In conclusion, 3D live-tissue imaging of the whole eccrine coiled structure revealed the gap junction-mediated contraction of myoepithelial cells in the secretory duct and progressive dilation-regression cycle of the excretory duct for sweat expulsion. The contraction sequence is thus more complicated than simply compressing the secretory duct, requiring further investigation of the segmental contraction that occurs in the secretory duct.

Methods

Human skin specimens

Fresh human postoperative skin specimens were obtained with written informed consent from all subjects at Osaka University Medical School (Osaka, Japan), Kinugasa Clinic (Osaka, Japan), Seishin Plastic and Aesthetic Surgery Clinic Co., Ltd (Tokyo, Japan) and Bizcom Japan (Tokyo, Japan). Experiments using human skin samples were approved by the Ethics Committee of Osaka University (yakukumi-28-3, yakukumi-28-5, yakukumi-29-2, yakujin-2019-10, yakujin-2019-28). Dissected skin specimens were stored at 4 °C in saline for up to 72 hours before use.

Eccrine gland isolation

Human eccrine glands were isolated from fresh skin specimens by microsurgery as follows. Eccrine glands in the dermis were labeled using 10 mM neutral red (Sigma‒Aldrich, Gillingham, UK) in divalent cation-free PBS (PBS(-)) for 5–10 minutes prior to microsurgery⁵⁶. The labeled eccrine glands were gently picked up manually from the skin by pinching the surrounding tissues with forceps to minimize damage to the ducts under a stereomicroscope (M-125; Leica, Wetzlar, Germany) and placed in PBS(-) until use in subsequent experiments.

Staining and mounting live eccrine glands

Freshly isolated eccrine glands were incubated for 30 minutes at 25 °C in vital stain solution supplemented with either Acti-stain 488 (Alexa 488-conjugated phalloidin; Cytoskeleton, Inc., Denver, USA), CMDR (a cell membrane stain; Thermo Fisher Scientific, Massachusetts, USA) or Hoechst 33342 and then washed with PBS (-) for 10 minutes. The stained eccrine glands were mounted on glass-bottom dishes (µ-Slide 8-well Glass Bottom, #80826, ibidi, Martinsried, Germany) coated with collagen gel (type 1-A: Nitta Gelatin, Inc., Osaka, Japan), which stably held the isolated eccrine glands. To prevent the samples from drying, the mounted eccrine glands were immersed in Krebs–Ringer bicarbonate solution (KRS) supplemented with 11 mM glucose at 25 °C¹⁸, except that the mounted eccrine glands were immersed in Ca²⁺/Mg²⁺-free PBS in the experiments shown in Fig. 3 and Supplementary movies 1, 2, and 4.

Live imaging

For the time-lapse 3D visualization of eccrine gland dynamics, multicolor fluorescence images of labeled nuclei (excitation: 405 nm), cell membranes (excitation: 650 nm) and actin filaments (excitation: 488 nm) were recorded under a confocal microscope (IX83 with FV1200; Olympus Corporation, Tokyo, Japan) with a 20× objective lens (NA of 0.75; Olympus Corporation) at intervals of 30 or 40 seconds for 30 minutes, depending on the thickness of the samples. At each recording time, twelve to thirty Z slices at 2-µm steps were obtained and immediately stacked to reconstruct the 3D images of the ducts. The Z-stacked images were freely rotated, and any plane of interest was obtained as a single image using ImageJ (NIH, USA). To induce eccrine gland contraction, 10 mM pilocarpine (P6503, Sigma‒Aldrich, Missouri, USA) was added 3 or 10 minutes after the initiation of image recording, thereby confirming the absence of any significant spontaneous contraction with the gland tested. To examine the nicotinic effect on sweat gland contraction, 6 mM nicotine (140-01211, Wako, Japan) was added at 10 minutes to start the observation. To inhibit pilocarpine-induced contraction, the eccrine glands were treated with 10 µM atropine (A0257, Sigma‒Aldrich), 1 mM CBX (C4790, Sigma‒Aldrich), 50 µM 2-APB (013-24911, Wako), or 50 µM Gap27 (HY-P0139, MedChemExpress, USA) 30 minutes before stimulation with pilocarpine. Eccrine gland dynamics were calculated by the object-tracking function of ImageJ, focusing on nuclei. Short-interval time-lapse images of eccrine gland dynamics were captured by confocal microscopy with a spinning disk system (IX83 with CSU W-1; Olympus Corporation and Yokogawa, Tokyo, Japan) and a 20× or 40× objective lens (NA of 0.75 and 0.95, respectively).

Pharmacological inhibition of myoepithelial contraction

To confirm whether myoepithelial contraction induces gland dynamics, actin dynamics were inhibited by cytochalasin D (Cayman Chemical, USA), which was dissolved in dimethyl sulfoxide (DMSO, Thermo Fisher Science, USA) and used at 10 µM, where the DMSO concentration in the chamber was 0.1% after dilution with KRS. The mounted eccrine gland was pretreated with cytochalasin D 30 minutes before stimulation with pilocarpine. As the control, 0.1% DMSO was applied for pretreatment at the same time point.

Calculation of the luminal space from 3D cellular latticework signals

Original confocal images before Z-stacking were processed to detect the cellular latticework signals as follows. First, the CMDR cell membrane signals were selected and rendered into a continuous surface by Imaris software’s Surface tools (Oxford Instruments, Belfast, UK). Within the rendered surface, the areas with continuous signal intensities corresponding to the cell-rich walls of the duct were digitally masked by the Imaris Masking tool. Next, the masked areas were cut out from the images. Then, the remaining cell-free luminal areas were selected and integrated along the depth to calculate the lumen volume of the inner duct in each 3D view along the entire length of the time-lapse movie. The nuclear signals provided information regarding the position of individual cells in motion. The 3D luminal views were imposed on the original 3D data, and any planes of interest were obtained by the Oblique-slicer tool in Imaris. The displacement of the nuclei was analyzed by the distance-calculation function in Imaris.

Immunostaining and fluorescent labeling

Isolated eccrine glands with or without CMDR staining were fixed in 4% paraformaldehyde (PFA) in PBS (Wako Pure Chemical, Osaka, Japan) at 25 °C for 4 hours, washed three times with PBS supplemented with 0.5% Triton X-100 (PBS-T), and blocked with 5% bovine serum albumin (BSA: A7030-100G, Sigma‒Aldrich) in PBS-T at 4 °C for 12 hours. Subsequently, the eccrine glands were incubated with anti-αSMA antibody (1:200, ab5694, Abcam, MA) and anti-PGP9.5 antibody (1:200, ab8189, Abcam, MA) in the same blocking solution at 4 °C for 12 hours. After washing with PBS-T three times, the eccrine glands were incubated with Alexa594-conjugated anti-rabbit antibody (1:400, A11037, Thermo Fisher Scientific), Alexa 488-conjugated anti-mouse antibody (1:400, A28175, Thermo Fisher Scientific) and Hoechst 33342 (H3570, Life Technologies, USA) at 25 °C for 12 hours.

Immunostaining of frozen tissue sections

Skin tissues were fixed in 4% PFA in PBS at 4 °C overnight, embedded in optimal cutting temperature compound (4583, Sakura Finetek Japan Co., Japan) and frozen in liquid nitrogen–chilled 2-methylbutane. The samples were sectioned at 10 μm using a cryostat (CM3050S, Leica Microsystems, Wetzlar, Germany), fixed in 4% PFA in PBS and blocked with 5% BSA in PBS-T at 25 °C for 20 minutes. For Cx31 staining, frozen skin tissue blocks were used; after sectioning, they were fixed with ice-cold acetone for 15 minutes. Subsequently, the sections were incubated with primary antibodies in the same blocking solution at 4 °C for 12 hours. The antibodies used were anti-Cx26 (1:500, ab59020, Abcam, Cambridge, UK), anti-Cx30 (1:100, HPA014846, Sigma‒Aldrich), anti-Cx31 (1:50, LS-C375784, LSBio, WA, USA), anti-Cx37 (1:125, #40-4300, Invitrogen), anti-Cx43 (1:1000, ab11370, Abcam), anti-Cx45 (1:100, CX45B12-A, Alpha Diagnostics) and anti-αSMA (1:100, A2547, Merck). After being washed with PBS-T three times, the sections were incubated at room temperature for 2 hours with the following secondary antibodies and Hoechst 33342 (1:1000): Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 594-conjugated donkey anti-rabbit or anti-goat IgG (1:200, A21202, A21207, A11058, Thermo Fisher). After a final wash, the slides were mounted with Prolong Gold (P10144, Thermo Fisher).

Measurement of sweating

The activity of human eccrine glands was measured on the palm of six healthy volunteers (four males and two females) under passive heating by a foot bath with approval from the Ethics Committees of Mandom Corporation (Osaka, Japan). Prior to the heat stress test, active sweat pores were stained by the starch-iodine method (Minor test). Absorbent cotton soaked in 16 mM CBX in 20% ethanol or in 20% ethanol was placed on the palm and held in position for 30 minutes at 25 °C. Then, the absorbent cotton was removed, and the surface of the palm was dried. The palms were stained with 3% w/v iodine (092-05422, Wako Pure Chemical) in ethanol and 10% starch solution in castor oil (034-01586, Wako Pure Chemical). To induce sweating, the volunteers immersed their feet in hot water (42 °C) for 30 minutes⁵⁷. All ethical regulations relevant to human research participants were followed. Signed informed consent was obtained from all participants.

Statistical analysis

The statistical analyses were performed using either Microsoft Excel (Microsoft, WA, USA) or KaleidaGraph 4.5 (Synergy Software, PA, USA). All data are presented as the mean ± standard deviation (SD).

Measuring the radius of curvature

The coordinates of three continuous nuclei of luminal cells were chosen and plotted on an arbitrary Cartesian plane. These coordinates were applied to obtain the radius of curvature using the online calculation platform Wolfram Alfa (https://www.wolframalpha.com/widgets/view.jsp?id=b0d28dd78e48b231c995d69b91666803).

Flow cytometry for isolation of CD29^high/CD49f^high myoepithelial cells

Fresh skin tissues were minced and enzymatically disaggregated for 16 hours at 37 °C in Complete MammoCult Human medium (Stem Cell Technologies, Vancouver, BC, Canada) with 600 U/ml collagenase type II (Worthington Biochemicals, Freehold, NJ, USA) on a tube rotator. After centrifugation at 300× g for 5 minutes, a single-cell suspension was obtained by repeated pipetting for 3 minutes in prewarmed 0.5% trypsin/1 mM EDTA in PBS, followed by repeated pipetting for 1 minute in prewarmed 5 mg/ml dispase (Gibco, Paisley, UK)/0.1 mg/ml DNase I (Sigma). The resulting suspension was filtered through a 40-µm mesh (BD Biosciences, San Jose, CA, USA). Immunolabeling of cells was performed at 4 °C for 30 minutes in PBS containing 5% BSA with allophycocyanin-conjugated anti-CD29 (1:6, 559883, BD Pharmingen, San Diego, CA) and Brilliant Violet 421-conjugated anti-CD49f (1:20, 313624, BioLegend, San Diego, CA). After being washed twice with 5% BSA in PBS, the cells were resuspended in PBS with 7-AAD (1:100, BioLegend) for dead cell staining.

Flow cytometric analysis and cell sorting were performed with a FACSAria2 (BD Biosciences), and the CD29^high/CD49f^high population was sorted for further qRT‒PCR analysis.

Quantitative RT‒PCR (qRT‒PCR)

Total RNA was isolated using TRI Reagent LS (Molecular Research Center, Inc.). cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). Real-time quantitative PCR was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) and a ViiA7 real-time PCR system (Applied Biosystems, CA, USA). The expression levels of target genes were normalized to that of 18 S ribosomal RNA using a standard curve method. The primer sets for qRT‒PCR are listed in Table 1.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Numerical source data for all plots and graphs can be found in the supplementary data file.

References

Peterson, R. A. et al. Sweating the small stuff: Glycoproteins in human sweat and their unexplored potential for microbial adhesion. Glycobiology 26, 218–229 (2015).
PubMed Google Scholar
Cheshire, W. P. & Freeman, R. Disorders of sweating. Semin. Neurol. 23, 399–406 (2003).
Article PubMed Google Scholar
Shibasaki, M., Wilson, T. E. & Crandall, C. G. Neural control and mechanisms of eccrine sweating during heat stress and exercise. J. Appl. Physiol. 100, 1692–1701 (2006).
Article PubMed Google Scholar
Hu, Y., Converse, C., Lyons, M. C. & Hsu, W. H. D. Neural control of sweat secretion: a review. Br. J. Dermatol. 178, 1246–1256 (2018).
Article CAS PubMed Google Scholar
Kurata, R. et al. Three-dimensional cell shapes and arrangements in human sweat glands as revealed by whole-mount immunostaining. PLoS One 12, 1–17 (2017).
Article Google Scholar
Ellis, R. A. Fine structure of the myoepithelium of the eccrine sweat glands of man. Cell 27, 551–563 (1965).
Article CAS Google Scholar
Sato, K., Kang, W. H., Saga, K. & Sato, K. T. Biology of sweat glands and their disorders. II. Disorders of sweat gland function. J. Am. Acad. Dermatol. 20, 713–726 (1989).
Article CAS PubMed Google Scholar
Tian, H. et al. Catecholamines are required for the acquisition of secretory responsiveness by sweat glands. J. Neurosci. 20, 7362–7369 (2000).
Article CAS PubMed PubMed Central Google Scholar
Kahn, D. & Rothman, S. Sweat response to acetylcholine. J. Investig. Dermatol. 5, 431–444 (1942).
Article CAS Google Scholar
Van Der Linden, M. et al. Differential signalling and kinetics of neutrophil extracellular trap release revealed by quantitative live imaging. Sci. Rep. 7, 6579 (2017).
Google Scholar
Coon, J. M. & Rothman, S. The sweat response to drugs with nicotine-like action. J. Pharmacol. Exp. Ther. 73, 1–11 (1941).
CAS Google Scholar
Kurzen, H. et al. Phenotypical and molecular profiling of the extraneuronal cholinergic system of the skin. J. Investig. Dermatol. 123, 937–949 (2004).
Article CAS PubMed Google Scholar
Wada, M., Arai, T., Takagaki, T. & Nakagawa, T. Axon reflex mechanism in sweat responses to nicotine, acetylcholine and sodium chloride. J. Appl. Physiol. 4, 745–752 (1952).
Article CAS PubMed Google Scholar
Webb, R. C. Smooth muscle contraction and relaxation. Adv. Physiol. Educ. 27, 201–206 (2003).
Article PubMed Google Scholar
Taugner, R. & Schiller, A. Gap junctions on myoepithelial cells. Cell. Tissue Res. 206, 65–72 (1980).
Article CAS PubMed Google Scholar
Manjarrez-marmolejo, J. & Franco-pérez, J. Gap junction blockers: An overview of their effects on induced seizures in animal models. Curr. Neuropharmacol. 14, 759–771 (2016).
Article CAS PubMed PubMed Central Google Scholar
Neijssen, J. et al. Cross-presentation by intercellular peptide transfer through gap junctions. Nature 434, 83–88 (2005).
Article CAS PubMed Google Scholar
Wang, N. et al. Connexin targeting peptides as inhibitors of voltage- and intracellular Ca²⁺-triggered Cx43 hemichannel opening. Neuropharmacology 75, 506–516 (2013).
Article CAS PubMed Google Scholar
Prakriya, M. & Lewis, R. S. Store-Operated Calcium Channels. Physiol. Rev. 95, 1383–1436 (2015).
Article CAS PubMed PubMed Central Google Scholar
Sato, K. Pharmacology and function of the myoepithelial cell in the eccrine sweat gland. Experientia 1, 631–633 (1977).
Article Google Scholar
Sato, K., Nishiyama, A. & Kobayashi, M. Mechanical properties and functions of the myoepithelium in the eccrine sweat gland. Am. J. Physiol., Cell Physiol. 237, C177–C184 (1979).
Article CAS Google Scholar
Ohmi, M., Tanigawa, M., Wada, Y. & Haruna, M. Dynamic analysis for mental sweating of a group of eccrine sweat glands on a human fingertip by optical coherence tomography. Skin. Res. Technol. 18, 378–383 (2012).
Article PubMed Google Scholar
Sato, K. & Sato, F. Role of calcium in cholinergic and adrenergic mechanisms of eccrine sweat secretion. Am. J. Physiol. 241, C113–C120 (1981).
Article CAS PubMed Google Scholar
Uno, H. Sympathetic innervation of the sweat glands and piloarrector muscles of macaques and human beings. J. Investig. Dermatol. 69, 112–120 (1977).
Article CAS PubMed Google Scholar
Sato, K. & Sato, F. Pharmacologic responsiveness of isolated single eccrine sweat glands. Am. J. Physiol. 240, R44–R51 (1981).
CAS PubMed Google Scholar
Haaksma, C. J., Schwartz, R. J. & Tomasek, J. J. Myoepithelial cell contraction and milk ejection are impaired in mammary glands of mice lacking smooth muscle alpha-actin. Biol. Reprod. 85, 13–21 (2011).
Article CAS PubMed PubMed Central Google Scholar
Hawley, D. et al. Myoepithelial cell-driven acini contraction in response to oxytocin receptor stimulation is impaired in lacrimal glands of Sjögren’s syndrome animal models. Sci. Rep. 8, 9919 (2018).
Article PubMed PubMed Central Google Scholar
Redman, R. S. Myoepithelium of salivary glands. Microsc. Res. Tech. 27, 25–45 (1994).
Article CAS PubMed Google Scholar
Stevenson, A. J., Vanwalleghem, G., Stewart, T. A. & Davis, F. M. Multiscale imaging of basal cell dynamics in the functionally mature mammary gland. Proc. Natl Acad. Sci. USA. 117, 26822–26832 (2020).
Article CAS PubMed PubMed Central Google Scholar
Moumen, M. et al. The mammary myoepithelial cell. Int. J. Dev. Biol. 55, 763–771 (2011).
Article PubMed Google Scholar
Makarenkova, H. P. & Dartt, D. A. Myoepithelial cells: Their origin and function in lacrimal gland morphogenesis, homeostasis, and repair. Curr. Mol. Biol. Rep. 1, 115–123 (2015).
Article PubMed PubMed Central Google Scholar
Davis, F. M. et al. Essential role of Orai1 store-operated calcium channels in lactation. Proc. Natl Acad. Sci. USA 112, 5827–5832 (2015).
Article CAS PubMed PubMed Central Google Scholar
Concepcion, A. R. et al. Store-operated Ca2+ entry regulates Ca2+-activated chloride channels and eccrine sweat gland function. J. Clin. Investig. 126, 4303–4318 (2016).
Article PubMed PubMed Central Google Scholar
Mroue, R. et al. Asymmetric expression of connexins between luminal epithelial- and myoepithelial- cells is essential for contractile function of the mammary gland. Dev. Biol. 399, 15–26 (2015).
Article CAS PubMed Google Scholar
Nicolaidis, S. & Sivadjian, J. High-frequency pulsatile discharge of human sweat glands:myoepithelial mechanism. J. Appl. Physiol. 32, 86–90 (1972).
Article CAS PubMed Google Scholar
Sugenoya, J. & Ogawa, T. Characteristics of central sudomotor mechanism estimated by frequency of sweat expulsions. Jpn. J. Physiol. 35, 783–794 (1985).
Article CAS PubMed Google Scholar
Ogawa, T. & Sugenoya, J. Pulsatile sweating and sympathetic sudomotor activity. Jpn. J. Physiol. 43, 275–289 (1993).
Article CAS PubMed Google Scholar
Lee, C. Y., Muramatsu, T. & Shimono, M. Localization of Connexin 26, 32 and 43 in rat parotid glands. Acta Histochem. Cytochem. 31, 211–216 (1998).
Article CAS Google Scholar
Muramatsu, T., Hashimoto, S. & Shimono, M. Differential expression of gap junction proteins Connexin32 and 43 in rat submandibular and sublingual glands. J. Histochem. Cytochem. 44, 49–56 (1996).
Article CAS PubMed Google Scholar
Plante, I. & Laird, D. W. Decreased levels of connexin43 result in impaired development of the mammary gland in a mouse model of oculodentodigital dysplasia. Dev. Biol. 318, 312–322 (2008).
Article CAS PubMed Google Scholar
Teleki, I. et al. Correlations of differentially expressed gap junction Connexins Cx26, Cx30, Cx32, Cx43 and Cx46 with breast cancer progression and prognosis. PLoS One 9, 1–12 (2014).
Article Google Scholar
Stewart, M. K. G., Simek, J. & Laird, D. W. Insights into the role of connexins in mammary gland morphogenesis and function. Reproduction 149, R279–R290 (2015).
Article CAS PubMed Google Scholar
Haefliger, J., Nicod, P. & Meda, P. Contribution of connexins to the function of the vascular wall. Cardiovasc. Res. 62, 345–356 (2004).
Article CAS PubMed Google Scholar
Yang, G. et al. Involvement of connexin 43 phosphorylation and gap junctional communication between smooth muscle cells in vasopressin-induced ROCK-dependent vasoconstriction after hemorrhagic shock. Am. J. Physiol., Cell. Physiol. 313, 362–370 (2017).
Article Google Scholar
Doolittle, J., Walker, P., Mills, T. & Thurston, J. Hyperhidrosis: an update on prevalence and severity in the United States. Arch. Dermatol. Res. 308, 743–749 (2016).
Article PubMed PubMed Central Google Scholar
Srillians, A. W. M. D. The control of localized hyperhidrosis. JAMA LXVII, 2015–2016 (1916).
Article Google Scholar
Grote, B. I. W., Holbert, J. M. & Cross, P. W. Dichloro aluminum aminoacetate, a new anti- perspirant and deodorant. J. Am. Pharm. Assoc. Am. Pharm. Assoc. 38, 593–594 (1949).
Article CAS PubMed Google Scholar
Matthew, J. Z. & Jessica, M. Antiperspirant and deodorant allergy. J. Clin. Aesthet. Dermatol. 1, 38–43 (2008).
Google Scholar
Yamaga, K. et al. Claudin-3 loss causes leakage of sweat from the sweat gland to contribute to the pathogenesis of atopic dermatitis. J. Investig. Dermatol. 138, 1279–1287 (2018).
Article CAS PubMed Google Scholar
Wenzel, F. G. & Horn, T. D. Nonneoplastic disorders of the eccrine glands. J. Am. Acad. Dermatol. 38, 1–20 (1998).
Article CAS PubMed Google Scholar
Dobson, R. L. & Lobitz, W. C. Some histochemical observations on the human eccrine sweat glands: II. The pathogenesis of miliaria. Ama. Arch. Derm. 75, 653–666 (1957).
Article CAS PubMed Google Scholar
Beyer, C. et al. Meta-analysis: Risk of hyperhidrosis with second-generation antidepressants. Depress Anxiety 34, 1134–1146 (2017).
Article CAS PubMed Google Scholar
Cerminara, C. et al. Hypohidrosis during topiramate treatment: a rare and reversible side effect. Pediatr. Neurol. 34, 392–39 (2006).
Article PubMed Google Scholar
Canel, L. et al. Topiramate-induced severe heatstroke in an adult patient: a case report. J. Med. Case. Rep. 10, 95 (2016).
Article PubMed PubMed Central Google Scholar
Glaser, D. A. & Glaser, K. Use of systemic therapies to manage focal hyperhidrosis. Mol. Med. 112, 287–291 (2015).
Google Scholar
Brayden, D. J. & Fitzpatrick, J. Cultured human sweat gland epithelia: isolation of glands using neutral red. Pharm. Res. 12, 171–175 (1995).
Article CAS PubMed Google Scholar
Kondo, N. et al. Function of human eccrine sweat glands during dynamic exercise and passive heat stress. J. Appl. Physiol. 90, 1877–1881 (2001).
Article CAS PubMed Google Scholar

Download references

Acknowledgements

This work was supported by JSPS KAKENHI Grants-in Aid for Young Scientists (B) (grant number JP17K16337), Early-Career Scientists (grant number JP19K17771) and Grant-in-Aid for Scientific Research (C) (grant number JP22K08429) to K.N. and by the Collaborative Research Fund from Mandom Corporation to K.S. This work was also partly supported by MEXT KAKENHI for Transformative Research Area (A) (23721401) to K.S. We are grateful to Moe Nishizawa for assistance in tissue immunostaining and data acquisition. We thank American Journal Experts (https://www.aje.com/go/springernature) for providing professional manuscript editing.

Author information

Authors and Affiliations

Laboratory of Advanced Cosmetic Science, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
Kie Nakashima, Hiroko Kato, Ryuichiro Kurata, Luo Qianwen, Tomohisa Hayakawa & Fumitaka Fujita
Fundamental Research Institute, Mandom Corporation, Osaka, Japan
Fumihiro Okada & Fumitaka Fujita
Department of Dermatology, Graduate School of Medicine, Osaka University, Osaka, Japan
Yukinobu Nakagawa & Atsushi Tanemura
Department of Dermatology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
Hiroyuki Murota
Department of Dermatology, Graduate School of Medicine, Osaka City University, Osaka, Japan
Ichiro Katayama
Division of Matrixome Research and Application, Institute for Protein Research, Osaka University, Osaka, Japan
Kiyotoshi Sekiguchi

Authors

Kie Nakashima
View author publications
You can also search for this author in PubMed Google Scholar
Hiroko Kato
View author publications
You can also search for this author in PubMed Google Scholar
Ryuichiro Kurata
View author publications
You can also search for this author in PubMed Google Scholar
Luo Qianwen
View author publications
You can also search for this author in PubMed Google Scholar
Tomohisa Hayakawa
View author publications
You can also search for this author in PubMed Google Scholar
Fumihiro Okada
View author publications
You can also search for this author in PubMed Google Scholar
Fumitaka Fujita
View author publications
You can also search for this author in PubMed Google Scholar
Yukinobu Nakagawa
View author publications
You can also search for this author in PubMed Google Scholar
Atsushi Tanemura
View author publications
You can also search for this author in PubMed Google Scholar
Hiroyuki Murota
View author publications
You can also search for this author in PubMed Google Scholar
Ichiro Katayama
View author publications
You can also search for this author in PubMed Google Scholar
Kiyotoshi Sekiguchi
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

K.N. designed and performed ex vivo and in vivo experiments, analyzed the data and wrote the manuscript. H.K., L.Q. and T.H. performed FACS sorting of myoepithelial cells, qRT‒PCR analyses, and tissue immunostaining and analyzed the data. R.K. conceived the project, designed the in vivo perspiration test and analyzed the data. F.F. analyzed the data and wrote the manuscript. F.O. analyzed the data and financially supported the project. Y.N., A.T., H.M., and I.K. contributed to the sample preparation. K.S. conceived the project, analyzed the data, and wrote the manuscript. All authors contributed to the preparation of the manuscript.

Corresponding authors

Correspondence to Fumitaka Fujita or Kiyotoshi Sekiguchi.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Joao Valente. A peer review file is available. [Last Edited by Joao Manuel de Sousa].

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Peer review file

Supplementary Information

Description of Additional Supplementary Files

Supplementary Data 1

Supplementary movie 1

Supplementary movie 2

Supplementary movie 3

Supplementary movie 4

Supplementary movie 5

Supplementary movie 6

Supplementary movie 7

Supplementary movie 8

Supplementary movie 9

Supplementary movie 10

Supplementary movie 11

Supplementary movie 12

Supplementary movie 13

Supplementary movie 14

Supplementary movie 15

Supplementary movie 16

Supplementary movie 17

Reporting Summary

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Nakashima, K., Kato, H., Kurata, R. et al. Gap junction-mediated contraction of myoepithelial cells induces the peristaltic transport of sweat in human eccrine glands. Commun Biol 6, 1175 (2023). https://doi.org/10.1038/s42003-023-05557-9

Download citation

Received: 15 October 2021
Accepted: 08 November 2023
Published: 18 November 2023
DOI: https://doi.org/10.1038/s42003-023-05557-9

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.