Local inhibition of carbonic anhydrase does not decrease sweat rate.

BACKGROUND: The purpose of this study was to measure sweat rate during exercise in the heat after directly inhibiting carbonic anhydrase (CA) in eccrine sweat glands via transdermal iontophoresis of acetazolamide. It was hypothesized that if CA was important for sweat production, local administration of acetazolamide, without the confounding systemic effects of dehydration typically associated with past studies, would have a significant effect on sweat rate during exercise. METHODS: Ten healthy subjects volunteered to exercise in the heat following acetazolamide or distilled water iontophoresis on the forearm. RESULTS: The distilled water iontophoresis site had a mean sweat rate during exercise in the heat of 0.59±0.31 µL/cm2/min, while the acetazolamide iontophoresis site had a mean sweat rate of 0.63±0.36 µL/cm2/min (p>0.05). CONCLUSIONS: The most important finding of the current study was that iontophoresis of acetazolamide did not significantly decrease sweat rate during exercise in the heat. Such results suggest that in past studies it was systemic dehydration, and not CA inhibition at the level of the sweat gland, that caused the reported decreased sweat rate.

Peripheral choline acetyltransferase in rat skin demonstrated by immunohistochemistry.

Conventional choline acetyltransferase immunohistochemistry has been used widely for visualizing central cholinergic neurons and fibers but not often for labeling peripheral structures, probably because of their poor staining. The recent identification of the peripheral type of choline acetyltransferase (pChAT) has enabled the clear immunohistochemical detection of many known peripheral cholinergic elements. Here, we report the presence of pChAT-immunoreactive nerve fibers in rat skin. Intensely stained nerve fibers were distributed in association with eccrine sweat glands, blood vessels, hair follicles and portions just beneath the epidermis. These results suggest that pChAT-positive nerves participate in the sympathetic cholinergic innervation of eccrine sweat glands. Moreover, pChAT also appears to play a role in cutaneous sensory nerve endings. These findings are supported by the presence of many pChAT-positive neuronal cells in the sympathetic ganglion and dorsal root ganglion. Thus, pChAT immunohistochemistry should provide a novel and unique tool for studying cholinergic nerves in the skin.

Cytochemistry of sialoglycoconjugates, lysozyme, and ß-defensin in eccrine glands of porcine snout skin as studied by electron microscopy.

In most mammals except for humanoid primates, eccrine glands are confined to the skin of a series of specific body regions. Sialic acids and antimicrobial substances exhibit various functional properties and serve as a component of nonspecific defense against micro-organisms, respectively. In this study, the distribution of these moieties was studied by electron microscopic histochemical methods. The eccrine glandular acini consisted of two types of dark cells as well as clear cells. The secretory granules and Golgi apparatus of both types of dark cells contained sialic acid residues linked to α2-6Gal/GalNAc. On the other hand, sialoglycoconjugates with Siα2-3Galß1-4GlcNAc sequence were confined to those of the Type II dark cells. In addition, lysozyme and ß-defensin were mainly detected in the secretory granules of the Type II dark cells. These secretory products may create a defensive barrier against microbial invasion and play an essential role in preservation of the integrity of porcine snout skin as a sensory organ.

Differential expression of the antioxidant repair enzyme methionine sulfoxide reductase (MSRA and MSRB) in human skin.

Recently, the antioxidant repair enzymes methionine-S-sulfoxide reductase A (MSRA) and methionine-R-sulfoxide reductase B (MSRB) were described in human epidermal keratinocytes and melanocytes. Methionine sulfoxide reductases (MSRs) are thought to protect against reactive oxygen species-induced oxidative damage in many organs, including the most environmentally exposed organ, human skin. We sought to examine the expression and distribution of this enzyme family (MSRA, MSRB1, MSRB2, and MSRB3) within the various compartments of healthy and diseased human skin. Expression was assessed using polyclonal MSR antibodies and immunohistochemical staining of human skin biopsies from various anatomical sites. Remarkably, MSRA expression was not only found in the epidermis as previously described but also in hair follicles and eccrine glands and was most pronounced in sebaceous glands. Furthermore, MSRB2 expression was found in melanocytes while MSRB1 and MSRB3 were both expressed within vascular endothelial cells. In conclusion, MSR enzymes are differentially expressed in human skin. Thus, modulation of MSR repair antioxidants may have implications for cutaneous aging and carcinogenesis.

17beta-estradiol rapidly mobilizes intracellular calcium from ryanodine-receptor-gated stores via a PKC-PKA-Erk-dependent pathway in the human eccrine sweat gland cell line NCL-SG3.

We describe a novel rapid non-genomic effect of 17beta-estradiol (E2) on intracellular Ca2+ ([Ca2+]i) signalling in the eccrine sweat gland epithelial cell line NCL-SG3. E2 had no observable effect on basal [Ca2+]i, however exposure of cells to E2 in the presence of the microsomal Ca2+ ATPase pump inhibitor, thapsigargin, produced a secondary, sustained increase in [Ca2+]i compared to thapsigargin treatment alone, where cells responded with a transient single spike-like increase in [Ca2+]i. The E2-induced increase in [Ca2+]i was not dependent on the presence of extracellular calcium and was completely abolished by ryanodine (100 microM). The estrogen receptor antagonist ICI 182,780 (1 microM) prevented the E2-induced effects suggesting a role for the estrogen receptor in the release of [Ca2+]i from ryanodine-receptor-gated stores. The E2-induced effect on [Ca2+]i could also be prevented by the protein kinase C delta (PKCdelta)-specific inhibitor rottlerin (10 microM), the protein kinase A (PKA) inhibitor Rp-adenosine 3',5'-cyclic monophosphorothioate (200 microM) and the MEK inhibitor PD98059 (10 microM). We established E2 rapidly activates the novel PKC isoform PKCepsilon, PKA and Erk 1/2 MAPK in a PKCdelta and estrogen-receptor-dependent manner. The E2-induced effect was specific to 17beta-estradiol, as other steroids had no effect on [Ca2+]i. We have demonstrated a novel mechanism by which E2 rapidly modulates [Ca2+]i release from ryanodine-receptor-gated intracellular Ca2+ stores. The signal transduction pathway involves the estrogen receptor coupled to a PKC-PKA-Erk 1/2 signalling pathway.

Expression of the sarco/endoplasmic reticulum calcium ATPase type 2 and 3 isoforms in normal skin and Darier's disease.

BACKGROUND: Darier's disease (DD) is caused by mutations in ATP2A2, which encodes the sarco/endoplasmic reticulum calcium ATPase type 2 (SERCA2), a member of a family of calcium pumps important in intracellular calcium signalling. SERCA2 has two isoforms. SERCA2a occurs mainly in cardiac and skeletal muscle, whereas SERCA2b occurs ubiquitously and is coexpressed with the related SERCA type 3 (SERCA3) in many tissues. It is not known why mutations in the widely expressed SERCA2 manifest as a focal skin disease. OBJECTIVES: To provide insight into the pathogenesis of DD by examining SERCA isoform expression in normal skin and DD skin. METHODS: Using immunohistochemistry we studied SERCA2a, SERCA2b and SERCA3 expression in nonlesional and lesional skin from seven patients with DD and normal skin from seven control subjects. We quantified SERCA2a and SERCA2b staining intensity by grey scale analysis of fluorescence intensity. RESULTS: In normal and DD epidermis both SERCA2a and SERCA2b staining was seen. SERCA2a staining in epidermis was less intense relative to pilar muscle whereas SERCA2b staining in epidermis was of marginally greater intensity than in pilar muscle. SERCA3 was not expressed in normal or DD epidermis, but was found in eccrine glands and blood vessels. No reduction was detected in SERCA2a or SERCA2b staining intensity in DD nonlesional epidermis compared with control epidermis. In within-patient comparisons, SERCA2a and SERCA2b staining in lesional epidermis was less intense than in nonlesional epidermis. CONCLUSIONS: Both SERCA2a and SERCA2b are present in epidermis, although the latter may predominate. The absence of coexpressed SERCA3 in epidermis may explain the localization of DD. Comparable SERCA2 staining intensity in nonlesional DD and control epidermis, even in patients predicted to be haploinsufficient, suggests partial compensation by upregulation of the normal allele. Unknown additional factors may trigger focal lesions by overcoming this compensation. Reduced staining intensity in lesional tissue may be secondary, or may reflect local downregulation of SERCA2 expression predisposing to development of focal lesions.

PACAP activated adenylate cyclase in human sweat glands. An ultracytochemical study.

The ultracytochemical localization of adenylate cyclase (AC) was studied after stimulation with pituitary adenylate cyclase activating peptide (PACAP) in human sweat glands. PACAP stimulated AC in both eccrine and apocrine glands. In the secretory cells, enzymatic activity was associated with membranes involved in the secretory mechanism. In both glands, the cells of the excretory duct and myoepithelial cells presented AC activity. These localizations of enzymatic activity suggest a role for PACAP in regulating glandular secretion.

V-type H+-ATPase in the human eccrine sweat duct: immunolocalization and functional demonstration.

We investigated for the presence of a vacuolar-type H+-ATPase (V-ATPase) in the human eccrine sweat duct (SD). With the use of immunocytochemistry, an anti-V- ATPase antibody showed a strong staining at the apical membrane and a weaker one in the cytoplasm. Cold preservation followed by rewarming did not alter this staining pattern. With the use of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein on isolated and perfused straight SD under HCO-free conditions and in the absence of Na+, proton extrusion was determined from the recovery rate of intracellular pH (dpH(i)/dt) following an acid load. Oligomycin (25 microM), an inhibitor of F-type ATPases, decreased dpH(i)/dt by 88 +/- 6%, suggesting a role for an ATP-dependent process involved in pH(i) recovery. Moreover, dpH(i)/dt was inhibited at 95 +/- 3% by 100 nM luminal concanamycin A, a specific inhibitor of V-ATPases, whereas 10 microM bafilomycin A1, another specific inhibitor of V-ATPases, was required to decrease dpH(i)/dt by 73%. These results strongly suggest that a V-ATPase is involved in proton secretion in the human eccrine SD.

Vacuolar-type H+ -ATPase distribution in unstimulated and acetylcholine-activated isolated human eccrine sweat glands.

The presence and cellular distribution of subunits of the V1 sector of the vacuolar-type H+ -ATPase (V-ATPase) was investigated in isolated human eccrine sweat glands. In every instance, V-ATPase was located in the cytoplasm and apical membranes of the luminal cells of the reabsorptive duct segment. In the secretory coil, both diffuse and perinuclear staining was demonstrated in the secretory cells, with additional expression at the apical and basolateral membranes and on the intercellular canaliculi. There was no detectable difference in V-ATPase expression as a result of prior application of 100 microM acetylcholine.

Atrial natriuretic peptide and guanylin-activated guanylate cyclase isoforms in human sweat glands.

The ultracytochemical localization of membrane-bound guanylate cyclases A and C, stimulated by atrial natriuretic peptide and guanylin respectively, has been studied in human sweat glands. The results showed that the peptides stimulated guanylate cyclases A and C in both eccrine and apocrine glands. In the secretory cells, enzymatic activity was present on the plasma membranes and on intracellular membranes involved in the secretory mechanism. In eccrine glands, the cells of the excretory duct also presented enzymatic activity on the plasma membranes. In both glands, myoepithelial cells, surrounding the secretory cells, exhibited only guanylate cyclase A activity. These localizations of enzymatic activity suggest a role for both atrial natriuretic peptide and guanylin in regulating glandular secretion.

Expression of endothelial nitric oxide synthase in human eccrine clear cells.

Nitric oxide is generated from L-arginine by nitric oxide synthase (NOS), which has at least three isoforms; endothelial-type NOS (eNOS) and brain-type NOS (bNOS) are constitutive enzymes, and inducible-type NOS (iNOS) is expressed after stimulation. Studies by the avidin-biotin immunocomplex method, revealed eNOS immunoreactivity exclusively in the human eccrine clear cells. No eNOS immunoreactivity was observed in the eccrine dark cells or myoepithelial cells. No staining of iNOS or bNOS was observed in the eccrine gland. These findings indicate that NO plays a physiological part in the production and/or excretion of sweat in the human skin eccrine gland.

Immunohistochemical localization of nitric oxide synthase in normal human skin: expression of endothelial-type and inducible-type nitric oxide synthase in keratinocytes.

Nitric oxide (NO) is a critical mediator of various biological functions. NO is generated from L-arginine by nitric oxide synthase (NOS), which has three isoforms; endothelial-type NOS (eNOS) and brain-type NOS (bNOS) are constitutive enzymes, and inducible-type NOS (iNOS) is expressed after stimulation. We investigated the expression of NOS in normal human skin by an immunohistochemical technique and western blotting analysis. In human skin, epidermal keratinocytes and the outer root sheath were labeled with not only eNOS antibody but also with iNOS antibody. Both eNOS and iNOS protein in epidermal keratinocytes were confirmed by western blotting. eNOS immunoreactivity was observed in endothelial cells, fibroblasts, the arrector pili muscle, apocrine secretory gland, eccrine coiled duct, and eccrine secretory gland. bNOS immunoreactivity was observed in mast cells. No staining with anti-bNOS antibody was observed in any other cell type. Our present findings suggest that epidermal keratinocytes in normal human skin contain both eNOS and iNOS.

Localization of 11 beta-hydroxysteroid dehydrogenase type II in human epithelial tissues.

The enzyme 11 beta-hydroxysteroid dehydrogenase type II (11 beta HSD2) confers specificity on the renal mineralocorticoid receptor by inactivating glucocorticoids. Mutations in this gene give rise to the syndrome of apparent mineralocorticoid excess, a congenital condition characterized by sodium retention, severe hypertension, and often by growth retardation. It is not known whether 11 beta HSD2 or another enzyme confers specificity in nonrenal sodium transporting epithelia, such as those in the sweat gland, salivary gland, and gastrointestinal tract. We previously have used the HUH23 antibody to localize 11 beta HSD2 in the human kidney, vascular smooth muscle cells, and placenta. In the present study, we have examined a range of human epithelia for the presence of 11 beta HSD2. In the skin, staining was seen in eccrine sweat glands and arterioles, whereas weak HUH23 immunostaining was observed in the epidermis. Staining was absent from sebaceous glands and hair follicles. In the parotid gland, the 11 beta HSD2 enzyme was present in striated and excretory ducts, whereas in the submandibular gland, it was found in striated and interlobular ducts. Acini, adipocytes, and associated tumor tissue did not stain with the HUH23 antibody. In the gastrointestinal tract, HUH23 stained ileal enterocytes, colonic absorptive cells, and epithelial goblet cells, whereas the rectum contained areas of staining and nonstaining absorptive cells. Gastrointestinal structures, such as the lamina propria, Peyer's patch, and goblet cells within the crypts of Lieberkuhn did not stain with the antibody. This study demonstrates the presence of 11 beta HSD2 in nonrenal sodium-transporting epithelia and describes a range of tissues affected in the syndrome of apparent mineralocorticoid excess.

Ultrastructural localization of alkaline phosphatase activity in human eccrine and apocrine sweat glands.

Alkaline phosphatase (ALP) is a membrane-bound enzyme that catalyzes the hydrolysis of inorganic and organic monophosphate esters at alkaline pH. Although the functions of ALP are poorly understood, it is believed to be involved in membrane transport. Because little is known about the functions and distribution of ALP in the sweat glands, we studied the localization of ALP in human sweat glands with light and electron microscopic enzyme cytochemistry. In eccrine sweat glands, ALP was restricted to the cell membranes of intercellular canaliculi. Luminal cell membranes of secretory cells that are in continuity with intercellular canaliculi did not show ALP activity. These results suggest that ALP participates in the production of primary sweat at intercellular canaliculi. In apocrine sweat glands, basal cell membranes of secretory cells and myoepithelial cell membranes that were in apposition with each other showed ALP activity, where as no activity was seen in eccrine sweat glands. These differences in the distribution of ALP in myoepithelial cells between eccrine and apocrine sweat glands might be related to the functional differences of these sweat glands. ALP histochemistry could help to diagnose and to determine the direction of differentiation in sweat gland tumors.

Demonstration of NADPH-diaphorase (NO-synthase) in the apocrine and eccrine skin glands of domesticated mammals.

The study demonstrates a strong enzyme histochemical and immunohistochemical reaction staining for NADPH-diaphorase/NO-synthase in the secretory cells of the apocrine glands in the hairy skin, and the eccrine glands in the foot pads of domesticated mammals. The results obtained are discussed in view of a regulatory action of the NO generated by these enzyme activities, implying a direct influence of NO on the contractile properties of glandular myoepithelial cells. In this way, a basic and simple mechanism to couple secretion production and secretion extrusion can be proposed.

Properties of gamma-glutamyl transpeptidase in squamous cell carcinoma.

gamma-Glutamyl transpeptidase (GGT) was extracted from squamous cell carcinoma tissues of human skin (SCC) by Triton X-100 and bromelain treatment, and some of its biochemical properties were compared with those of GGT extracted from eccrine gland-rich tissue and normal kidney. GGT activity significantly increased in SCC, but there was no definitive differences in enzymological properties between GGT of SCC and normal tissue enzyme. However, GGT of SCC was distinguishable from those of normal tissues by isoelectric point, electrophoretic mobility, and sensitivity to neuraminidase treatment. These results indicate that GGT of SCC has some variant properties which may be related to skin carcinogenesis.

Immunohistochemical demonstration of peptidylarginine deiminase in human sweat glands.

Human skin is known to contain protein-bound citrulline. This is the product of enzymatic deimination of arginine residues catalyzed by peptidylarginine deiminase. We probed frozen sections of human skin with a rabbit antiserum raised to rat skeletal muscle peptidylarginine deiminase using the avidin-biotin-peroxidase complex technique. This led us to interesting findings. No staining was observed in epidermis, inner root sheaths of hair follicles, sebaceous glands, and hair erector muscle. However, we noticed specific staining of the cytoplasm of secretory and myoepithelial cells of both eccrine and apocrine sweat glands. The procedure also stained neoplastic cells present in specimens dissected from extramammary Paget's disease. The data mean that peptidylarginine deiminase may be used as a new marker in the classification of skin neoplasms showing sweat gland differentiation. Possible localization of multiple types of peptidylarginine deiminases in human skin is discussed.

Immunohistochemical study of lysozyme in human apocrine glands.

The localization of lysozyme in human apocrine glands was studied by adopting the avidin-biotin-peroxidase complex method. The results showed that the glands were enriched with lysozyme. The apical portion of secretory cells was most heavily stained. Eccrine glands did not stain for lysozyme. Although apocrine glands have been regarded as having no apparent function in man, it is suggested in the present report that they may have an excretory bactericidal role.

Ultrastructural localization of ouabain-sensitive, K-dependent p-nitrophenyl phosphatase activity in monkey eccrine sweat gland.

We studied the electron microscopic localization of ouabain-sensitive, potassium-dependent p-nitrophenyl phosphatase (K-pNPPase) activity of the Na K-ATPase complex in Rhesus monkey eccrine sweat gland by use of the one-step lead citrate method of Mayahara et al. (Histochemistry 1980; 67:125). Reaction product was observed predominantly in the cytoplasmic side of the basolateral membranes of clear (secretory) cells, especially in the interdigitating membrane folds in the basal labryinth, and were completely abolished by 10 mM ouabain or by removal of K+. Little or no enzyme activity was noted in membrane processes in the intercellular canaliculi and in the secretory coil lumen. Basolateral membranes of the dark cells also showed moderate enzyme activity. The myoepithelial cell membrane was devoid of reaction product, except in a few membrane processes arising from the inner aspect of myoepithelial cells. In the coiled duct, K-pNPPase activity was present predominantly in the entire cell membrane of the peripheral ductal cells. The predominantly basolateral distribution of Na-K-ATPase in the eccrine sweat secretory cells is consistent with the concept that a Na-K-Cl co-transport model may be involved in the mechanism of eccrine sweat secretion.