DOI: 10.1111/exd.12595
Commentary
www.wileyonlinelibrary.com/journal/EXD
Sweating chloride bullets: understanding the role of calcium in eccrine sweat glands and possible implications for hyperhidrosis Thad E. Wilson and Kristen Metzler-Wilson Marian University College of Osteopathic Medicine, Indianapolis, IN, USA Correspondence: Thad E. Wilson, PhD, Marian University College of Osteopathic Medicine, 3200 Cold Spring Rd, Indianapolis, IN 46222-1997, USA, Tel./Fax: 317-955-6256, e-mail: [email protected] Key words: Ca2+-activated Cl channels – hyperhidrosis – NCL-SG3 – secretory clear cells
Accepted for publication 10 November 2014
Eccrine (atrichial) sweat glands are large, active cutaneous endorgans vital to human thermal and fluid homeostasis, but regional overactivity of these glands can lead to focal hyperhidrosis. Eccrine sweat glands can expulse over 10 nl/min in a single isolated gland and as much as 3.7 l/h systemically in response to thermal and non-thermal stimuli (1, S1, S2). How do these glands produce such copious secretions? Sweating involves a two-stage process of first producing an isotonic primary solution within the bulbous coiled region of the gland and then modifying (primarily via reabsorbing NaCl) this fluid within the ductal portion as it travels to the skin surface. Thus, production of this primary fluid requires careful scrutiny to understand hyperhidrosis. Epithelial transport in these glands appears to be primarily mediated via clear (agranular) cells, which are also the proposed cell type associated with hyperhidrosis (2). In the current understanding of clear cell function, Cl is transported through the clear cell, Na+ is transported through tight junctions via a transepithelial voltage gradient, and finally water is pulled through aquaporin channels via osmotic forces. Thus, Cl is a principal player in the formation of the isotonic primary solution. It is, however, another ion, Ca2+, that appears to be the primary regulator within clear cells. The majority of sudorific agonists (e.g. acetylcholine, norepinephrine via a1-adrenergic receptors and ATP) increase cytosolic Ca2+ via efflux from cellular stores and influx from extracellular fluid. Ca2+ release from intracellular stores is derived via IP3-activated Ca2+ and Ca2+-induced Ca2+ release channels. The influx into the clear cells is a bit more complex, likely involving TRPV1, store-operated Ca2+ entry (Orai1 and TRPC1) and L-type voltagegated Ca2+ channels (3–5, S3). Highlighting the importance of Ca2+ influx, secretions are abated when isolated glands are placed into Ca2+-free bath (S4, S5), and recent in vivo experiments identified right shifts in the cholinergic agonist to sweating relation with both EDTA (used as an interstitial Ca2+ chelator) and verapamil (L-type channel blocker) (4). The regulatory role of cytosolic Ca2+ is to activate many of the ion channels involved in epithelial transport.
intracellular Ca2+ as well as being voltage-activated at low Ca2+ concentrations (S6). Ertongur-Fauth and colleagues (6) identified 3 TMEM16A splice variants in the NCL-SG3 sweat gland epithelial cell line as well as in hyperhidrotic and control skin. Investigating the function of a novel splice variant, TMEM16A(acDe3), authors found it lacks the dimerization domain but still forms a functional channel. Previous studies indicated that TMEM16A subunits appeared to dimerize before reaching the plasma membrane and that dimerization was required for functionality (7). Overexpression of TMEM16A(acDe3) increased basal Cl transport even without the dimerization domain (6). This may indicate that the novel TMEM16A splice variant is more constitutively active and thus could possibly produce primary solution during non-stimulated conditions. This, in theory, could lead to increases in production of primary fluid. Alternately, it is possible that sudorific modulators such as galanin and CGRP (8–10), which appear to accentuate sweat formation once transport is initiated, may now be more functionally important due to this increased basal Cl secretion. Overexpression of the novel TMEM16A splice variant had another interesting result: unlike in basal conditions, expressing either the novel TMEM16A(acDe3) or the canonical TMEM16A (ac) splice variant enhanced Ca2+-induced Cl secretion during activated conditions, as compared to the parental cells containing only endogenously expressed TMEM16A splice variants (6). Overexpression of TMEM16A(acDe3), however, decreased Ca2+-dependent Cl transport compared to the canonical variant (6). This seems to suggest that a certain mix of TMEM16A splice variants may enhance Ca2+-regulated transport and that the novel variant may be less regulated and less responsive to activators than other splice variants. Thus, overexpression of either splice variant in disease states, such as hyperhidrosis, could yield greater sweating per agonist activation of Ca2+.
Role of TMEM16A splice variants in sweat gland epithelial cells This is where the study by Ertongur-Fauth and colleagues (6) enters the picture. Anoctamin 1 (ANO1, also termed TMEM16A), is a Ca2+-activated Cl channel, which is an anion-selective membrane protein that allows passive Cl flow and is activated by
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 177–178
Role of Ca2+ in regulating apical Cl secretion in eccrine clear cells
TMEM16A Cl channels are not the only channels involved in the formation of isotonic primary solution. Another Ca2+-activated Cl channel, bestrophin 2 (Best2), is expressed in basolateral and apical membranes of sweat gland secretory cells (S7) and among other functions regulates L-type voltage-gated Ca2+ channels (S8). Clear cells are arranged with a basolateral membrane that contains Na+-K+ ATPase, Na+-Cl -K+ cotransporters (NCCK1), Ca2+activated K+ channels, Best2, aquaporin-5 channels (AQP5) and
177
Commentary
The NCL-SG3 cell line (S9) has dramatically increased our understanding of sweating, but there are some inherent limitations in the use of the cell line. In addition to not knowing precisely which eccrine sweat gland cells are included in the line, cultured cells cannot interact with surrounding cells of other types in cell– cell interactions as would occur in intact human skin. Cultured cells also do not have the neural influences that drive responses in vivo. This specifically relates to agonist-induced sweating in which the traditional neural agonists such as acetylcholine do not cause secretion (S9, S10). Thus, the normal IP3 response to release Ca2+ from intracellular stores cannot occur. While in vitro work such as that described by Ertongur-Fauth and colleagues (6) tells an important story, there is also a need for in vivo studies to determine whether agonist-induced Cl secretion is similar in intact human skin.
Conclusions Figure 1. Conceptual model of the role of Ca2+ in regulating Cl release in eccrine clear cells. Muscarinic receptor activation engages cellular processes which cause the release of Ca2+ and potentially cell swelling, activating basolateral NCCK1 channels. At the apical membrane, release of Ca2+ from internal stores and Ca2+ influx from extracellular sources activates TMEM16A and Best2 Cl channels to allow efflux of Cl which then combines with Na+ and water into the lumen to form primary isotonic solution. NCCK1: Na+-Cl -K+ cotransporter; Cl/ HCO3: Cl /HCO3 exchanger; AQP5: Aquaporin 5; IK: intermediate conductance Ca2+-activated K+ channel; BK: large conductance Ca2+-activated K+ channel; VGCC: voltage-gated Ca2+ channel (L-type); Best2: bestrophin 2; Na/H: Na+/H+ exchanger; TRPV1: transient receptor potential (vanilloid) channel, type 1; Orai: Orai1 channel; TRPC: transient receptor potential (canonical) channel; IP3: IP3activated Ca2+ channel; CICR: Ca2+-induced Ca2+ release channel; SOC: storeoperated channel; SERCA: sarco/endoplasmic reticulum Ca2+ ATPase; TMEM16A: Anoctamin 1; H-ATPase: vacuolar H+-ATPase. Red spheres indicate Ca2+.
various channels that allow for the Ca2+ influx discussed above. The apical membrane contains TMEM16A and Best2 Ca2+-activated Cl channels, Ca2+-activated K+ channels, vacuolar H+-ATPase and AQP5. A conceptual model of the role of Ca2+ in activating basolateral Cl transport, primarily via NCCK1, and then apical Cl transport, via TMEM16A and Best2, is depicted in Fig. 1. Both known and proposed transporters in clear cells are included in the model to show not only ion transport but also the potential interactions, regulatory points and redundancies in the system.
It would have been nice for the purpose of telling this story if the novel TMEM16A splice variant would have only been expressed in hyperhidrotic skin and if overexpression would have yielded extremely high agonist-induced Cl secretion, but, alas, it appears more complex. It is interesting that a combination of isoforms may cause greater function (more Cl transport and therefore more sweating) than any one isoform. Speculations on the data from Ertongur-Fauth and colleagues (6) could include TMEM16A splice variant differences leading to different rates of sweating and perhaps even differences between hyperhidrotic and normal skin. One of the most effective hyperhidrosis treatments consists of blocking neural activation of sweat glands through cholinergic presynaptic release inhibitors like botulinum toxin, but perhaps the root cause of focal hyperhidrosis is not only at the neural activation level but also includes downstream components such as at the Ca2+-activated Cl or other Ca2+-activated ion channel level.
Acknowledgements Both authors contributed to all aspects of background research, interpretation and manuscript preparation.
Conflicts of interest The authors have declared no conflict of interests.
References 1 Shibasaki M, Wilson T E, Crandall C G. J Appl Physiol 2006: 100: 1692–1701. 2 Bovell D L, MacDonald A, Meyer B A et al. Exp Dermatol 2011: 20: 1017–1020. 3 Ambudkar I S. Cell Calcium 2014: 55: 297–305. 4 Metzler-Wilson K, Sammons D L, Ossim M A et al. Exp Physiol 2014: 99: 393–402. 5 Stander S, Moormann C, Schumacher M et al. Exp Dermatol 2004: 13: 129–139.
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6 Ertongur-Fauth T, Hochheimer A, Buescher J et al. Exp Dermatol 2014: 23: 825–831. 7 Sheridan J T, Worthington E N, Yu K et al. J Biol Chem 2011: 286: 1381–1388. 8 Schlereth T, Dittmar J O, Seewald B et al. J Physiol 2006: 576: 823–832. 9 Schlereth T, Breimhorst M, Werner N et al. Exp Dermatol 2013: 22: 299–301. 10 Bovell D L, Holub B S, Odusanwo O et al. Exp Dermatol 2013: 22: 141–143.
Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web site: Data S1-S10: Supplemental References.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 177–178
Commentary
www.wileyonlinelibrary.com/journal/EXD
Sweating chloride bullets: understanding the role of calcium in eccrine sweat glands and possible implications for hyperhidrosis Thad E. Wilson and Kristen Metzler-Wilson Marian University College of Osteopathic Medicine, Indianapolis, IN, USA Correspondence: Thad E. Wilson, PhD, Marian University College of Osteopathic Medicine, 3200 Cold Spring Rd, Indianapolis, IN 46222-1997, USA, Tel./Fax: 317-955-6256, e-mail: [email protected] Key words: Ca2+-activated Cl channels – hyperhidrosis – NCL-SG3 – secretory clear cells
Accepted for publication 10 November 2014
Eccrine (atrichial) sweat glands are large, active cutaneous endorgans vital to human thermal and fluid homeostasis, but regional overactivity of these glands can lead to focal hyperhidrosis. Eccrine sweat glands can expulse over 10 nl/min in a single isolated gland and as much as 3.7 l/h systemically in response to thermal and non-thermal stimuli (1, S1, S2). How do these glands produce such copious secretions? Sweating involves a two-stage process of first producing an isotonic primary solution within the bulbous coiled region of the gland and then modifying (primarily via reabsorbing NaCl) this fluid within the ductal portion as it travels to the skin surface. Thus, production of this primary fluid requires careful scrutiny to understand hyperhidrosis. Epithelial transport in these glands appears to be primarily mediated via clear (agranular) cells, which are also the proposed cell type associated with hyperhidrosis (2). In the current understanding of clear cell function, Cl is transported through the clear cell, Na+ is transported through tight junctions via a transepithelial voltage gradient, and finally water is pulled through aquaporin channels via osmotic forces. Thus, Cl is a principal player in the formation of the isotonic primary solution. It is, however, another ion, Ca2+, that appears to be the primary regulator within clear cells. The majority of sudorific agonists (e.g. acetylcholine, norepinephrine via a1-adrenergic receptors and ATP) increase cytosolic Ca2+ via efflux from cellular stores and influx from extracellular fluid. Ca2+ release from intracellular stores is derived via IP3-activated Ca2+ and Ca2+-induced Ca2+ release channels. The influx into the clear cells is a bit more complex, likely involving TRPV1, store-operated Ca2+ entry (Orai1 and TRPC1) and L-type voltagegated Ca2+ channels (3–5, S3). Highlighting the importance of Ca2+ influx, secretions are abated when isolated glands are placed into Ca2+-free bath (S4, S5), and recent in vivo experiments identified right shifts in the cholinergic agonist to sweating relation with both EDTA (used as an interstitial Ca2+ chelator) and verapamil (L-type channel blocker) (4). The regulatory role of cytosolic Ca2+ is to activate many of the ion channels involved in epithelial transport.
intracellular Ca2+ as well as being voltage-activated at low Ca2+ concentrations (S6). Ertongur-Fauth and colleagues (6) identified 3 TMEM16A splice variants in the NCL-SG3 sweat gland epithelial cell line as well as in hyperhidrotic and control skin. Investigating the function of a novel splice variant, TMEM16A(acDe3), authors found it lacks the dimerization domain but still forms a functional channel. Previous studies indicated that TMEM16A subunits appeared to dimerize before reaching the plasma membrane and that dimerization was required for functionality (7). Overexpression of TMEM16A(acDe3) increased basal Cl transport even without the dimerization domain (6). This may indicate that the novel TMEM16A splice variant is more constitutively active and thus could possibly produce primary solution during non-stimulated conditions. This, in theory, could lead to increases in production of primary fluid. Alternately, it is possible that sudorific modulators such as galanin and CGRP (8–10), which appear to accentuate sweat formation once transport is initiated, may now be more functionally important due to this increased basal Cl secretion. Overexpression of the novel TMEM16A splice variant had another interesting result: unlike in basal conditions, expressing either the novel TMEM16A(acDe3) or the canonical TMEM16A (ac) splice variant enhanced Ca2+-induced Cl secretion during activated conditions, as compared to the parental cells containing only endogenously expressed TMEM16A splice variants (6). Overexpression of TMEM16A(acDe3), however, decreased Ca2+-dependent Cl transport compared to the canonical variant (6). This seems to suggest that a certain mix of TMEM16A splice variants may enhance Ca2+-regulated transport and that the novel variant may be less regulated and less responsive to activators than other splice variants. Thus, overexpression of either splice variant in disease states, such as hyperhidrosis, could yield greater sweating per agonist activation of Ca2+.
Role of TMEM16A splice variants in sweat gland epithelial cells This is where the study by Ertongur-Fauth and colleagues (6) enters the picture. Anoctamin 1 (ANO1, also termed TMEM16A), is a Ca2+-activated Cl channel, which is an anion-selective membrane protein that allows passive Cl flow and is activated by
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 177–178
Role of Ca2+ in regulating apical Cl secretion in eccrine clear cells
TMEM16A Cl channels are not the only channels involved in the formation of isotonic primary solution. Another Ca2+-activated Cl channel, bestrophin 2 (Best2), is expressed in basolateral and apical membranes of sweat gland secretory cells (S7) and among other functions regulates L-type voltage-gated Ca2+ channels (S8). Clear cells are arranged with a basolateral membrane that contains Na+-K+ ATPase, Na+-Cl -K+ cotransporters (NCCK1), Ca2+activated K+ channels, Best2, aquaporin-5 channels (AQP5) and
177
Commentary
The NCL-SG3 cell line (S9) has dramatically increased our understanding of sweating, but there are some inherent limitations in the use of the cell line. In addition to not knowing precisely which eccrine sweat gland cells are included in the line, cultured cells cannot interact with surrounding cells of other types in cell– cell interactions as would occur in intact human skin. Cultured cells also do not have the neural influences that drive responses in vivo. This specifically relates to agonist-induced sweating in which the traditional neural agonists such as acetylcholine do not cause secretion (S9, S10). Thus, the normal IP3 response to release Ca2+ from intracellular stores cannot occur. While in vitro work such as that described by Ertongur-Fauth and colleagues (6) tells an important story, there is also a need for in vivo studies to determine whether agonist-induced Cl secretion is similar in intact human skin.
Conclusions Figure 1. Conceptual model of the role of Ca2+ in regulating Cl release in eccrine clear cells. Muscarinic receptor activation engages cellular processes which cause the release of Ca2+ and potentially cell swelling, activating basolateral NCCK1 channels. At the apical membrane, release of Ca2+ from internal stores and Ca2+ influx from extracellular sources activates TMEM16A and Best2 Cl channels to allow efflux of Cl which then combines with Na+ and water into the lumen to form primary isotonic solution. NCCK1: Na+-Cl -K+ cotransporter; Cl/ HCO3: Cl /HCO3 exchanger; AQP5: Aquaporin 5; IK: intermediate conductance Ca2+-activated K+ channel; BK: large conductance Ca2+-activated K+ channel; VGCC: voltage-gated Ca2+ channel (L-type); Best2: bestrophin 2; Na/H: Na+/H+ exchanger; TRPV1: transient receptor potential (vanilloid) channel, type 1; Orai: Orai1 channel; TRPC: transient receptor potential (canonical) channel; IP3: IP3activated Ca2+ channel; CICR: Ca2+-induced Ca2+ release channel; SOC: storeoperated channel; SERCA: sarco/endoplasmic reticulum Ca2+ ATPase; TMEM16A: Anoctamin 1; H-ATPase: vacuolar H+-ATPase. Red spheres indicate Ca2+.
various channels that allow for the Ca2+ influx discussed above. The apical membrane contains TMEM16A and Best2 Ca2+-activated Cl channels, Ca2+-activated K+ channels, vacuolar H+-ATPase and AQP5. A conceptual model of the role of Ca2+ in activating basolateral Cl transport, primarily via NCCK1, and then apical Cl transport, via TMEM16A and Best2, is depicted in Fig. 1. Both known and proposed transporters in clear cells are included in the model to show not only ion transport but also the potential interactions, regulatory points and redundancies in the system.
It would have been nice for the purpose of telling this story if the novel TMEM16A splice variant would have only been expressed in hyperhidrotic skin and if overexpression would have yielded extremely high agonist-induced Cl secretion, but, alas, it appears more complex. It is interesting that a combination of isoforms may cause greater function (more Cl transport and therefore more sweating) than any one isoform. Speculations on the data from Ertongur-Fauth and colleagues (6) could include TMEM16A splice variant differences leading to different rates of sweating and perhaps even differences between hyperhidrotic and normal skin. One of the most effective hyperhidrosis treatments consists of blocking neural activation of sweat glands through cholinergic presynaptic release inhibitors like botulinum toxin, but perhaps the root cause of focal hyperhidrosis is not only at the neural activation level but also includes downstream components such as at the Ca2+-activated Cl or other Ca2+-activated ion channel level.
Acknowledgements Both authors contributed to all aspects of background research, interpretation and manuscript preparation.
Conflicts of interest The authors have declared no conflict of interests.
References 1 Shibasaki M, Wilson T E, Crandall C G. J Appl Physiol 2006: 100: 1692–1701. 2 Bovell D L, MacDonald A, Meyer B A et al. Exp Dermatol 2011: 20: 1017–1020. 3 Ambudkar I S. Cell Calcium 2014: 55: 297–305. 4 Metzler-Wilson K, Sammons D L, Ossim M A et al. Exp Physiol 2014: 99: 393–402. 5 Stander S, Moormann C, Schumacher M et al. Exp Dermatol 2004: 13: 129–139.
178
6 Ertongur-Fauth T, Hochheimer A, Buescher J et al. Exp Dermatol 2014: 23: 825–831. 7 Sheridan J T, Worthington E N, Yu K et al. J Biol Chem 2011: 286: 1381–1388. 8 Schlereth T, Dittmar J O, Seewald B et al. J Physiol 2006: 576: 823–832. 9 Schlereth T, Breimhorst M, Werner N et al. Exp Dermatol 2013: 22: 299–301. 10 Bovell D L, Holub B S, Odusanwo O et al. Exp Dermatol 2013: 22: 141–143.
Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web site: Data S1-S10: Supplemental References.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 177–178
