The ultrastructure of monkey eccrine sweat glands

Summary The ultrastructure of monkey eccrine sweat glands is described. The secretory portion of the sweat gland is discussed in detail. The morphological differences in the secretory coil using three different fixatives and fixative combinations are emphasized. The secretory product of dark cells is seen to have three distinct appearances depending upon the fixative used. The biochemical significance of the latter finding is discussed. The appearance of clear cell cytoplasmic processes is described using the different fixatives. The similarity of adjacent clear cell processes to those of avian salt glands is pointed out and discussed. Evidence is presented to indicate that dark cells arise from clear cells via an intermediate cell type. The appearance of the clear cell plasma membrane is described and the necessity for the use of the general term multilaminar plasma membrane is discussed.Supported by U.S.P.H.S. grant 5 T 1-GM-29 F-04 AS. The author would like to express his gratitude to the Lederle Laboratories and in particular to Dr.James Vickers for providing the tissue. Sincere thanks is given to Mrs.Dagmar Graham and Mrs.Ditza Springer for technical assistance and also to MissMary Lorenc for preparation of the diagram. In addition, I would like to thank Dr.J. A. G. Rhodin for his criticism and advice.     

Ultrastructural localization of dipeptidyl peptidase IV in rat salivary glands by immunocytochemistry

Summary The ultrastructural localization of dipeptidyl peptidase IV (DPP IV) (EC 3.4.14.5) in rat submandibular and parotid glands was studied immunocytochemically by the peroxidase-antiperoxidase (PAP) method, using a monospecific antiserum against rat kidney DPP IV. There were no differences in the immunocytochemical localization of DPP IV between submandibular and parotid glands. In these glands, DPP IV was primarily found to be associated with the luminal and intercellular canalicular plasma membranes of acinar cells and with the luminal plasma membranes of intercalated and striated duct cells. Occasionally, immunoreaction of DPP IV was detected in cytoplasmic vesicles (vacuoles), lysosomes, and multivesicular bodies in some acinar cells as well as in ductal epithelial cells. Furthermore, the reaction product was also found within the lumina of peri-acinar and peri-ductal capillaries and in the cytoplasm of some fibroblasts in the interstitial connective tissue. These data suggest that DPP IV in the submandibular and parotid glands may play some role in the secretion or reabsorption processes of secretory proteins and peptides in these glands.     

Lectin binding pattern and proteoglycan distribution in human eccrine sweat glands

The distribution pattern of glycoconjugates in human eccrine sweat glands has been studied by the binding of newly discovered lectins and by antibodies against a chondroitin sulphate proteoglycan and chondroitin sulphate glycosaminoglycans. Mannose-specific lectins labelled large intracellular granules, part of which could be extended cisternae of the endoplasmic reticulum or Golgi apparatus. In contrast, lectins specific for terminal mannose/glucose residues predominantly labelled basement membranes and the glycocalyx. Lectins recognizing terminal N-acetylgalactosamine groups left most parts of the glands unstained, but stained some dark cells intensely. These last cells were also intensively labelled by N-acetylglucosamine-specific and by fucose-specific lectins. Sialic acid residues were preferentially located in luminal borders of secretory coils. No terminal galactose residues were detected. All antibodies against chondroitin glycoconjugates stained large granules similar to those revealed by the mannose-specific lectins in the secretory cells. The basement membrane is only stained by the proteoglycan antibody and the chondroitin-6-sulphate antibody.Thus, a complex composition of glycoconjugates exists not only in matrix elements but also in the cells of eccrine glands of the human skin. A possible secretion of glycoconjugates is discussed.     

Purification and properties of dipeptidyl peptidase IV from human urine

Dipeptidyl peptidase IV (DPP-IV) (EC 3.4.14.5) has been purified from normal human urine using immunoaffinity chromatography. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular mass of urinary DPP-IV was estimated to be 280 kDa by gel filtration. The Km value for the hydrolysis of (Gly-L-Pro)-4-methylcoumaryl-7-amide tosylate was calculated to be 1.25 +/- 0.25 X 10(-4) M; the pH optimum and pI were 8.7 and 6.5, respectively. These properties were the same as those of renal DPP-IV. Both renal and this urinary DPP-IV were inhibited by diisopropylfluorophosphate and activated by phospholipid. From these results we suppose that urinary DPP-IV may be derived from the kidney rather than from the blood     

Thermogenic and psychogenic recruitment of human eccrine sweat glands: Variations between glabrous and non-glabrous skin surfaces

Human eccrine sweat-gland recruitment and secretion rates were investigated from the glabrous (volar) and non-glabrous hand surfaces during psychogenic (mental arithmetic) and thermogenic stimuli (mild hyperthermia). It was hypothesised that these treatments would activate glands from both skin surfaces, with the non-thermal stimulus increasing secretion rates primarily by recruiting more sweat glands. Ten healthy men participated in two seated, resting trials in temperate conditions (25–26 °C). Trials commenced under normothermic conditions during which the first psychogenic stress was applied. That was followed by passive heating (0.5 °C mean body temperature elevation) and thermal clamping, with a second cognitive challenge then applied. Sudomotor activity was evaluated from both hands, with colourimetry used to identify activated sweat glands, skin conductance to determine the onset of precursor sweating and ventilated sweat capsules to measure rates of discharged sweating. From glandular activation and sweat rate data, sweat-gland outputs were derived. These psychogenic and thermogenic stimuli activated sweat glands from both the glabrous and non-glabrous skin surfaces, with the former dominating at the glabrous skin and the latter at the non-glabrous surface. Indeed, those stimuli individually accounted for ~90% of the site-specific maximal number of activated sweat glands observed when both stimuli were simultaneously applied. During the normothermic psychological stimulation, sweating from the glabrous surface was elevated via a 185% increase in the number of activated glands within the first 60 s. The hypothetical mechanism for this response may involve the serial activation of additional eccrine sweat glands during the progressive evolution of psychogenic sweating.     

Reduced cell cohesiveness of outgrowths from eccrine sweat glands delays wound closure in elderly skin

Human skin heals more slowly in aged vs. young adults, but the mechanism for this delay is unclear. In humans, eccrine sweat glands (ESGs) and hair follicles underlying wounds generate cohesive keratinocyte outgrowths that expand to form the new epidermis. Here, we compared the re‐epithelialization of partial‐thickness wounds created on the forearm of healthy young (< 40 yo) and aged (> 70 yo) adults. Our results confirm that the outgrowth of cells from ESGs is a major feature of repair in young skin. Strikingly, in aged skin, although ESG density is unaltered, less than 50% of the ESGs generate epithelial outgrowths during repair (vs. 100% in young). Surprisingly, aging does not alter the wound‐induced proliferation response in hair follicles or ESGs. Instead, there is an overall reduced cohesiveness of keratinocytes in aged skin. Reduced cell–cell cohesiveness was most obvious in ESG‐derived outgrowths that, when present, were surrounded by unconnected cells in the scab overlaying aged wounds. Reduced cell–cell contact persisted during the repair process, with increased intercellular spacing and reduced number of desmosomes. Together, reduced outgrowths of ESG (i) reduce the initial number of cells participating in epidermal repair, (ii) delay wound closure, and (iii) lead to a thinner repaired epidermis in aged vs. young skin. Failure to form cohesive ESG outgrowths may reflect impaired interactions of keratinocytes with the damaged ECM in aged skin. Our findings provide a framework to better understand the mediators of delayed re‐epithelialization in aging and further support the importance of ESGs for the repair of human wounds.     

Preparative purification of dipeptidyl peptidase IV.

Dipeptidyl-Peptidase IV was purified from pig kidney by ammonium sulfate fractionation, gel filtration, QAE-cellulose chromatography and affinity columns with Gly-Pro- and Concanavalin A-Sepharose. The specific activity of the purified enzyme is 41.8 units/mg. Polyacrylamide gel electrophoresis and silver staining show a single band. The enzyme preparation is free of aminopeptidase and dipeptidase activity, proved fluorimetrically and by gas chromatography/mass spectrometry. The most important procedure for removal of contaminating enzyme activities is a stepwise NaCl-gradient on a QAE-ZetaPrep ion exchange disk.     

Three-dimensional culture and identification of human eccrine sweat glands in matrigel basement membrane matrix

Interactions between the extracellular matrix (ECM) and epithelial cells are necessary for the proper organization and function of the epithelium. In the present study, we show that human eccrine sweat gland epithelial cells cultured in matrigel, a representation of ECM components, constitute a good model for studying three-dimensional reconstruction, wound repair and regeneration and differentiation of the human eccrine sweat gland. In matrigel, epithelial cells from the human eccrine sweat gland form tubular-like structures and then the tubular-like structures coil into sphere-like shapes that structurally resemble human eccrine sweat glands in vivo. One sphere-like shape can be linked to another sphere-like shape or to a cell monolayer via tubular-like structures. Hematoxylin and eosin staining has revealed that the tubular-like structures have a single layer or stratified epithelial cells located peripherally and a lumen at the center, similar to the secretory part or duct part, respectively, of the eccrine sweat gland in sections of skin tissue. Immunohistochemical analysis of the cultures has demonstrated that the cells express CK7, CK19, epithelial membrane antigen and actin. Thus, matrigel promotes the organization and differentiation of epithelial cells from the human eccrine sweat gland into eccrine sweat gland tissues.     

Immunohistochemical localization of activated EGF receptor in human eccrine and apocrine sweat glands.

Epidermal growth factor (EGF) is secreted into sweat from secretory cells of human sweat glands. The function of EGF in sweat is poorly understood. The biological function of EGF is exerted by the binding of EGF to the receptor (EGFR) and its activation. Therefore, we immunohistochemically localized the activated form of EGFR in human eccrine and apocrine sweat glands to assess the functional importance of the EGF-EGFR system in human sweat glands. Frozen sections of human skin were stained with a monoclonal antibody (MAb) specific for tyrosine-phosphorylated (activated) EGFR and with an MAb that stains both activated and non-activated EGFR. In the secretory portion of eccrine sweat glands, nuclei of the secretory cells were stained with the anti-activated EGFR MAb. In coiled and straight portions of eccrine sweat ducts, nuclei of luminal and peripheral cells were stained with the antibody specific for activated EGFR. Luminal cell membranes and luminal cytoplasm of inner ductal cells possessed non-activated EGFR. In the secretory portion of apocrine sweat glands, activated EGFRs were present in cytoplasm and nuclei of secretory cells. These data suggest that EGF, already known to be present in the cytoplasm of secretory cells in eccrine and apocrine sweat glands, activates EGFR in the nuclei of secretory cells themselves in an intracrine manner. Because ductal cells do not express EGF, EGF in the sweat secreted from the secretory cells should activate EGFR in the ductal cells in a paracrine manner. (J Histochem Cytochem 49:597-601, 2001)     

Demonstration of two molecular forms of dipeptidyl peptidase IV in normal human serum

The heterogeneity of dipeptidyl peptidase IV (EC 3.4.14.5) was investigated in normal human serum. Thin-layer analytical isoelectric focusing revealed the presence of multiple molecular forms of the enzyme, their isoelectric points being in the pH range of 3.30-4.25. The maximum of enzyme activity appeared around pH 3.50. After treatment with neuraminidase the pI shifted to 4.70-5.40 with two maxima at pH 5.00 and 5.15. The Triton X-100 solubilized as well as the papain-treated-Triton X-100 solubilized enzyme from the whole human adult jejunal biopsy were also found to be heterogeneous. They focused--both before and after neuraminidase treatment--at pH values different from those of the enzyme of normal human serum. There was almost no pI shift after neuraminidase treatment of the intestinal enzyme from adult enterobiopsy. Electrophoresis in continuous polyacrylamide gradient gels as well as gel chromatography on Bio-Gel A-1.5m revealed two molecular forms of dipeptidyl peptidase IV in normal human serum. The estimated relative molecular mass of the major enzyme form was 250 000 in both the separation techniques used. On the other hand, the apparent relative molecular mass of the minor enzyme form was 450 000 as assessed by gradient gel electrophoresis, and 550 000, when estimated by gel chromatography. The Km values for glycyl-L-proline-4-nitroanilide as substrate with the major and minor forms of the serum enzyme were 1.60 +/- 0.39 X 10(-4) mol/l and 1.60 +/- 0.13 X 10(-4) mol/l, respectively. Our results indicate that the dipeptidyl peptidase IV in normal human serum is a heterogeneous enzyme as far as its charge and molecular size are concerned.     

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 100µM acetylcholine.     

Purification and characterization of barley dipeptidyl peptidase IV

Barley (Hordeum vulgare L.) storage proteins, which have a high content of proline (Pro) and glutamine, are cleaved by cysteine endoproteases to yield peptides with a Pro next to the N-terminal and/or C-terminal amino acid residues. A peptidase cleaving after Xaa-Pro- at the N terminus of peptides was purified from green barley malt. It was identified as a serine-type dipeptidyl peptidase (DPP), based on inhibitor studies, and the nature of the cleavage product. It is a monomeric glycoprotein with an apparent molecular mass of 105 kD (85 kD after deglycosylation), with a pI of 3.55 and a pH optimum at 7.2. Substrate specificity was determined with a series of fluorogenic peptide substrates with the general formula Xaa-Pro-AMC, where Xaa is an unspecified amino acid and AMC is 7-amino-4-methylcoumarin. The best substrates were Xaa = lysine and arginine, while the poorest were Xaa = aspartic acid, phenylalanine, and glutamic acid. The K(m) values ranged from 0.071 to 8.9 microM, compared with values of 9 to 130 microM reported for mammalian DPP IVs. We discuss the possible role of DPP IV in the degradation of small Pro-containing peptides transported from the endosperm to the embryo of the germinating barley grain.     

The protease corin regulates electrolyte homeostasis in eccrine sweat glands

Sweating is a basic skin function in body temperature control. In sweat glands, salt excretion and reabsorption are regulated to avoid electrolyte imbalance. To date, the mechanism underlying such regulation is not fully understood. Corin is a transmembrane protease that activates atrial natriuretic peptide (ANP), a cardiac hormone essential for normal blood volume and pressure. Here, we report an unexpected role of corin in sweat glands to promote sweat and salt excretion in regulating electrolyte homeostasis. In human and mouse eccrine sweat glands, corin and ANP are expressed in the luminal epithelial cells. In corin-deficient mice on normal- and high-salt diets, sweat and salt excretion is reduced. This phenotype is associated with enhanced epithelial sodium channel (ENaC) activity that mediates Na⁺ and water reabsorption. Treatment of amiloride, an ENaC inhibitor, normalizes sweat and salt excretion in corin-deficient mice. Moreover, treatment of aldosterone decreases sweat and salt excretion in wild-type (WT), but not corin-deficient, mice. These results reveal an important regulatory function of corin in eccrine sweat glands to promote sweat and salt excretion.

Sweating is a basic skin function in body temperature control, and salt excretion and reabsorption in sweat glands are essential for salt-water balance. This study identifies corin, a transmembrane protease that activates atrial natriuretic peptide, as a key enzyme in regulating salt excretion in the skin.