Ultrastructure of the hyperhidrotic eccrine sweat gland.

BACKGROUND: Hyperhidrosis is the secretion of inappropriately large amounts of sweat by eccrine glands; it can be very debilitating. Little is known of the causes of primary hyperhidrosis. OBJECTIVES: To determine whether the glands exhibit any structural abnormality in primary hyperhidrosis. METHODS: Skin biopsies were obtained from the axilla (n = 6) or neck (n = 2) of individuals aged 26-62 years with primary hyperhidrosis and from five age- and sex-matched normal individuals, with informed consent and ethical committee approval. Samples were prepared by standard methods for light and electron microscopic examination. RESULTS: All characteristics observed in the hyperhidrotic specimens were consistent with the changes seen in normal glands following strong activation: degranulation of the granular (dark) cells, dilatation of the basolateral infoldings and the canaliculi of the non-granular (clear) cells, contraction of the myoepithelial cells and thickening of the basal lamina, and presence of cellular debris including lipid droplets in the gland lumen. Pathological changes were not observed. CONCLUSIONS: The present finding of the absence of structural defects in the glands indicates that future studies should concentrate on the investigation of neurohumoral or secretory cell metabolic abnormalities.

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.

Nucleotide-evoked ion transport and [Ca(2+)](i) changes in normal and hyperhidrotic human sweat gland cells.

Apical and basolateral application of ATP and UTP evoked [Ca(2+)](i) and short circuit current (Isc) increases in normal and hyperhidrotic human eccrine sweat gland cells grown into functionally polarised epithelia on permeable supports. Basolateral application to hyperhidrotic cells exhibited a markedly greater increase in Isc than in normal cells. Hyperhidrotic cells also demonstrated differences from the normal in [Ca(2+)](i) and Isc responses to ATP when pre-treated with thapsigargin. The data demonstrate the presence of apical and basolateral receptors that allow nucleotides to increase [Ca(2+)](i) and Isc. The results suggest that changes from the normal in transepithelial ion transport contribute to the characteristic excessive fluid production of hyperhidrotic sweat glands.

Activation of apical P2U purine receptors permits inhibition of adrenaline-evoked cyclic AMP accumulation in cultured equine sweat gland epithelial cells.

Experiments were undertaken using cultured equine sweat gland epithelial cells that express purine receptors belonging to the P2U subclass which allow the selective agonist uridine triphosphate (UTP) to increase the concentration of intracellular free Ca2+ ([Ca2+]i). Experiments using pertussis toxin (Ptx), which inactivates certain guanine-nucleotide-binding proteins (G-proteins), showed that this response consisted of Ptx-sensitive and Ptx-resistant components, and immunochemical analyses of the G-protein alpha subunits present in the cells showed that both Ptx-sensitive (alpha i1-3) and Ptx-resistant (alpha q/11) G-proteins were expressed. P2U receptors may, therefore, normally activate both of these G-protein families. Ptx-sensitive, alpha i2/3 subunits permit inhibitory control of adenylate cyclase, and UTP was shown to cause Ptx-sensitive inhibition of adrenaline-evoked cyclic AMP accumulation, suggesting that the receptors activate Gi2/3. Experiments using cells grown on permeable supports suggested that P2U receptors became essentially confined to the apical membrane in post-confluent cultures. Polarised epithelia may, therefore, express apical P2U receptors which influence two centrally important signal transduction pathways. It is highly improbable that these receptors could be activated by nucleotides released from purinergic nerves, but they may be involved in the autocrine regulation of epithelial function.

The regulation of membrane 125I- and 86Rb+ permeability in a virally transformed cell line (NCL-SG3) derived from the human sweat gland epithelium.

We have explored the factors that may regulate membrane permeability in a cell line (NCL-SG3) derived from the human sweat gland epithelium. Ionomycin increased the rate of 125I-efflux from preloaded cells and this action appeared to be due to an increase in intracellular free calcium ([Ca2+]i). The ionomycin-evoked increase in 125I- efflux was reduced in cells that were exposed either to barium or to valinomycin in the presence of a high concentration of external potassium. It thus appears that a fraction of the ionomycin-evoked increase in 125I- efflux is due to the activation of potassium channels and experiments using 86Rb+ also suggested that ionomycin increased the rate of potassium efflux, an effect which was totally abolished by barium. Blockade of Na(+)-K(+)-2Cl- cotransport and of Cl- -HCO3- exchange reduced the basal rate of 125I- efflux and the ionomycin-evoked increase in 125I-efflux from control cells and from cells depolarized by valinomycin. These transport systems thus contribute to anion efflux, although [Ca2+]i-dependent chloride channels also appear to be present. Acetylcholine increases [Ca2+]i in the secretory cells of human sweat glands, but this neurotransmitter did not increase [Ca2+]i in NCL-SG3 cells and so membrane permeability was not under cholinergic control. Adrenaline did not increase [Ca2+]i, but this hormone did evoke cyclic-3',5'-adenosine monophosphate (cyclic AMP) production. However, membrane permeability was not under adrenergic control, as the cells did not appear to express functional, cyclic AMP-dependent anion channels. This may be because they were not fully differentiated under the culture conditions. ATP consistently evoked a dose-dependent increase in anion efflux that appeared to be mediated by [Ca2+]i. The increase in [Ca2+]i was initiated by the release of calcium from a limited internal store and was subsequently sustained by calcium influx. UTP and ADP also increased [Ca2+]i, whereas adenosine, AMP and alpha,beta-methylene ATP were without effect. These data thus suggest that a subclass of type 2 purine receptor, which is functionally coupled to phosphoinositidase C, is present in these cells.

Extracellular ATP can activate autonomic signal transduction pathways in cultured equine sweat gland epithelial cells.

Changes in intracellular free calcium concentration ([Ca2+]i) were monitored in a cell line that was derived from the equine sweat gland epithelium. ATP and closely related compounds could increase [Ca2+]i with a rank order of potency of UTP > or = ATP > ADP >> AMP = adenosine = alpha,beta-methylene-ATP. The responses to ATP and to UTP were initiated by the release of calcium from an internal store and subsequently sustained by calcium influx. The rise in [Ca2+]i thus seems to be mediated by P2U receptors that are coupled to phosphoinositidase C. Some desensitisation of this response developed during repeated stimulation with ATP and this was blocked by staurosporine, an inhibitor of protein kinase C, and augmented by a phorbol ester which acts as an exogenous activator of this enzyme. A protein-kinase-C-dependent inhibitory pathway thus seems to become active during repeated stimulation with ATP. ATP and related compounds could also raise cellular cyclic AMP content. The order of potency was ATP > ADP = AMP = adenosine >> UTP, suggesting that this response is mediated via a separate subclass of P2 receptor. The present results demonstrate that ATP can activate autonomic signal-transduction pathways in cultured equine sweat gland cells and suggest that there may be a purinergic component to the control of secretory activity in the equine sweat gland.

The effect of removing external sodium upon the cholinergic regulation of potassium permeability in the rat submandibular gland in vitro.

Fragments of rat submandibular gland were loaded with 86Rb+ and superfused so that the rate of 86Rb(+)-efflux could be quantified as an indicator of potassium permeability. Acetylcholine evoked an increase in permeability consisting of a transient, calcium-independent response and a sustained, calcium-dependent. Total removal of external sodium significantly inhibited both phases of this response. The results thus confirm that the cholinergic regulation of potassium permeability is compromised by removal of external sodium but do not support the view that this is due, exclusively, to an effect on calcium influx.

Investigation of stimulus-secretion coupling in equine sweat gland epithelia using cell culture techniques.

When sweat glands isolated from samples of horse skin were explanted and cultured under favourable conditions, they could exhibit cellular outgrowth. This growth could be maintained for 2-4 weeks and these primary cultures were then disaggregated and the resultant cell suspensions used to initiate epithelial cell lines. Secretion from intact equine sweat glands is regulated by beta 2-adrenoceptors and appears to be mediated by cyclic AMP, but there is evidence that calcium may also play a role. Adrenaline could increase the cyclic AMP content of the cultured cells and this response was mediated by beta 2-adrenoceptors. Adrenaline was also able to evoke a small increase in intracellular free calcium ([Ca2+]i) but the pharmacology of this response remains obscure. Adrenaline thus activates at least two potentially important second-messenger signalling pathways which have the capacity to interact, because adrenaline-evoked cyclic AMP formation was inhibited if [Ca2+]i was raised with ionomycin. The chloride permeability of mammalian epithelial cells characteristically rises during secretion, and adrenaline could increase chloride permeability in the cultured epithelia but the cells did not contain cyclic-AMP-dependent chloride channels and so this response was mediated by [Ca2+]i.

Temporal changes in the granulocytic responses to experimental infection of the skin of mice and sheep with Dermatophilus congolensis.

The patterns of dermal inflammatory cell response to infection with Dermatophilosis congolensis were determined in mice and sheep from histological samples taken before and at intervals after topical application of infective zoospores to ether-swabbed skin. Neutrophils, eosinophils, basophils and mast cells were identified by histochemical staining. Temporal changes in the B cell, T cell, and MHC Class II+ dendritic cell populations form part of a separate report. The filamentous stages of the bacterium were observed in the stratum corneum of both species; in the sheep they were also found in the outer layers of the living epidermis. In both species, large numbers of neutrophils and some lymphocytes penetrated the epidermis and entered the infected surface region. Within the underlying dermis there was an accumulation of dendritic cells immediately below the infected epidermis and evidence of mast cell degranulation; the basophils and eosinophils did not appear to be actively involved. The striking difference between the two species was the duration of the infection and the associated response which, in the mouse, lasted about five days in comparison with over 21 days in the sheep. Neutrophil numbers in the mouse for example were elevated by 12 h and had peaked at 60 h after infection, while in the sheep they did not peak until about 120 h.

Amiloride impairs the cholinergic regulation of potassium permeability in the human sweat gland but not in the rat submandibular gland.

Potassium permeability was monitored in human sweat glands and rat submandibular glands. Acetylcholine increased permeability in both tissues and the responses consisted of transient, calcium-independent and sustained, calcium-dependent components. Amiloride, a drug which inhibits Na(+)-H+ countertransport, impaired the regulation of potassium permeability in sweat glands but not in the submandibular gland. It is suggested that the stimulus-permeability coupling process in the sweat gland may be sensitive to the lowering of internal pH.

Histological and electron microprobe studies of mineralisation in aluminium-related osteomalacia.

AIMS: To determine a possible mechanism to explain the presence of aluminium lines within fully calcified bone in aluminium-related osteomalacia. METHODS: Fifty five bone cases shown by bone biopsy to be aluminium-related osteomalacia were studied. In 38 specimens aluminium lines were identified within calcified bone by means of the Aluminon stain and a characteristic form of patchy mineralisation was seen within thickened osteoid seams. Five representative examples were analysed quantitatively by histomorphometry and electronprobe X-ray microanalysis and compared with five cases of vitamin D deficiency-related osteomalacia which also had patchy mineralisation. RESULTS: The patchy calcification occupied 40 +/- 8% (mean +/- SEM) of the osteoid and consisted of small focal deposits (less than 40 microns diameter), often (52%) around osteoid osteocytes (probably an underestimate of the association), and larger areas that extended to the aluminium lines at the underlying mineralisation front. Small and large mineralisation nuclei were seen ultrastructurally in the patchy calcification. Quantitative electronprobe X-ray microanalysis showed that calcium concentrations and calcium:phosphorus ratios in the mineralisation nuclei and in the superficial layer of the fully calcified bone of the aluminium-related osteomalacia cases were significantly less than values measured at similar sites in the vitamin D deficiency-related osteomalacia cases. Furthermore, aluminium could not be detected by means of this technique at the mineralisation front or along cement lines in these specimens. CONCLUSIONS: Calcification can occur in thickened osteoid seams in osteomalacia. It can begin around osteoid osteocytes as small deposits that enlarge within the osteoid and extend to the underlying mineralisation front or cement line where aluminium lines may become trapped. Complete calcification of osteoid could account for the presence of aluminium lines within fully calcified bone. The Aluminon stain appears to be a more sensitive method for the detection of aluminium in bone than electronprobe X-ray microanalysis.

The role of potassium and other ions in the control of aldosterone synthesis.

Fast and slow K+ efflux components, independently regulated by angiotensin II (AII), have been identified in bovine adrenocortical cells. We have further investigated the role of potassium in the control of aldosterone synthesis in two ways. Firstly, isotopic tracers, in conjunction with channel modulators, have been used to study the interrelationship of K+ and Ca2+ in the control of AII-stimulated aldosterone synthesis. Secondly, electron probe X-ray microanalysis (EPXMA) was used to quantify potassium, sodium, chlorine and phosphorous in control and AII-stimulated cells. The effects of verapamil on 43K efflux were measured at two stages during AII stimulation. During the first ten minutes of treatment, when efflux via the fast component predominates, AII and verapamil both slowed efflux and their effects were additive. If verapamil was added later, at the time when efflux by the fast component appeared exhausted and the stimulatory effect of AII on the slow efflux component was apparent, it again slowed efflux. These data suggest that verapamil prevents calcium-gated K+ channels from opening by blocking Ca2+ channels. However, verapamil had no effect on AII-stimulated calcium efflux. In addition to blocking Ca2+ channels, verapamil may directly inhibit potassium efflux. EPXMA showed a bimodal distribution of potassium concentrations in control cells. However, in cells stimulated with AII for five minutes, the mean potassium content was less than in controls and was not bimodally distributed. Sodium content was increased by AII-treatment, chlorine was lowered and phosphorus remained unchanged. The data confirm previous observations that AII inhibits Na+/K+ ATPase activity.

The effects of removing external sodium upon the control of potassium (86Rb+) permeability in the isolated human sweat gland.

The changes in cytoplasmic free calcium ([Ca2+]i) which occur in isolated human sweat glands during cholinergic stimulation have been studied indirectly by monitoring potassium permeability. The acetylcholine-evoked permeability increase normally consists of transient and sustained phases which are attributed to the mobilization of intracellular calcium stores and to calcium influx respectively. Such consistent responses to acetylcholine could not be obtained during superfusion with bicarbonate-free, HEPES-buffered solutions. The human sweat gland in vitro therefore appears to have a strict requirement for bicarbonate. The sustained component of the response was not affected by total removal of external sodium, suggesting that calcium influx does not occur via a sodium-dependent system. The transient component, however, was abolished when external sodium was replaced by N-methyl-D-glucammonium (NMDG+). It therefore appears that secretagogue-evoked mobilization of cytoplasmic calcium is dependent, in some way, upon external sodium. This dependence is not, however, absolute as the response was essentially normal when sodium was replaced by lithium.

Automated image segmentation and serial section reconstruction in microscopy.

A technique for automatic 3-D reconstruction of specifically stained features in televised serial histological sections has been developed using an image analyser. Images which included these features were individually converted to binary images, compiled, displayed to show the 3-D morphology and used to quantify the structure. The criteria necessary for producing valid reconstructions and the problems associated with the manipulation of images of fine detail, particularly those containing several thousand features, are illustrated by examples from skin.

Potassium (86Rb+) efflux from the rat submandibular gland under sodium-free conditions in vitro.

1. Fragments of rat submandibular gland were pre-loaded with 86Rb+, an isotopic marker of potassium transport, and rate constants for 86Rb+ efflux were determined during superfusion with a physiological salt solution. 2. In sodium-containing solutions acetylcholine evoked a rapid and immediate increase in efflux rate. After reaching a peak value, the efflux rate initially declined rapidly, but a second, slowly declining phase to the response was also evident. The response could be resolved into Ca2(+)-independent and Ca2(+)-dependent phases. 3. The basal efflux rate was elevated during superfusion with solutions in which sodium had been replaced with either lithium or N-methyl-D-glucammonium (NMDG+). Although lithium had a greater effect, which was absent under calcium-free conditions, addition of calcium to initially calcium-free, lithium-containing solutions did not affect the rate of efflux. 4. In the presence of calcium the response to acetylcholine was augmented during exposure to lithium-containing, sodium-free solutions but, in contrast, slightly inhibited when NMDG+ was used as a sodium substituent. 5. The transient, calcium-independent component of the response to acetylcholine was unaffected by exposure to lithium, whereas the calcium-dependent phase of the response was inhibited. 6. Responsiveness to acetylcholine was reduced during superfusion with a calcium-free, NMDG+-containing solution. The response normally observed when extracellular Ca2+ was subsequently elevated, in the continued presence of acetylcholine, was also inhibited. Sensitivity to acetylcholine was retained, however, when the tissue was initially exposed to a solution containing approximately 20 mumol l-1 Ca2+. The response was smaller than that evoked in sodium-containing solutions. 7. The use of lithium as a sodium substituent presents special problems, possibly related to the effects of this ion on the metabolic cycling of phosphatidylinositol-4,5-bisphosphate metabolites.

The control of potassium (86Rb+) efflux in the isolated human sweat gland.

Sweat glands, isolated from strips of human skin and pre-loaded with 86Rb+, a marker of potassium transport, were superfused with physiological saline and rate constants for 86Rb+ efflux calculated. The rate of efflux during superfusion with Ca2+-free saline was lower than that measured in the presence of calcium (2.56 mM). Acetylcholine increased the rate of 86Rb+ efflux and this response could be resolved into two components: an initial transient phase which was Ca2+-independent and a slowly declining Ca2+-dependent phase. Adrenaline only caused a Ca2+-dependent increase in efflux. It is suggested that the potassium permeability of the secretory cells increases during activity.

Effects of season and lower ambient temperature on the structure of the sweat glands in anhidrotic horses.

Histological studies of the sweat glands of anhidrotic horses in the Hong Kong summer and under conditions of reduced thermal stress, both natural and controlled, were undertaken to determine if glandular regeneration occurs. Clinical data were collected for comparison with the histological results in each instance. Horses were assigned to one of three categories on the basis of the resulting change in the number of thin glandular profiles in a cooler environment. Group 1, which was classed as normal, had a low initial value, which was maintained. Group 2, typical of mild and moderately affected animals, had a high initial value, which fell markedly after as little as six weeks in the cool environment. Animals in Group 3, classed as severely affected, had a high initial value which remained high even after prolonged exposure to the cool environment. Light microscopical examination of the sweat glands in the heat, and after six weeks in a cool environment, provided a means of predicting the degree of anhidrotic severity and the potential for recovery in a cool climate. This was superior to clinical observation, although a diagnostic test based on glandular function is still required.

Effects of thermal stimulation on intracellular sodium, potassium and chlorine in the sweat glands of the cow, sheep and goat.

The intracellular concentrations of electrolyte elements in the sweat glands of the cow, sheep and goat provide evidence of altered secretory activity upon thermal stimulation in the cow but give no indication of such change in the sheep and goat. The results support the view that increased secretory function contributes to the pattern of sweat output in the cow.

Effects of stimulation on the ultrastructure and Na, K, Cl composition of the fundus of the rat plantar sweat gland.

Initial stimulation of the rat plantar sweat gland with pilocarpine caused a variable degree of distension of the apical membrane of the secretory cell. This appeared to be a process of filtration of secretory cell cytoplasm through the apical terminal web. Further stimulation resulted in luminal dilatation, cytoplasmic depletion, and morbidity of some cells. These morphological changes in the footpad gland, which thus can no longer be considered as eccrine, were accompanied by a fall in potassium and a rise in sodium concentration within the secretory cells. The mode of secretion induced by pharmacological stimulation was fundamentally the same as that in the glands of species responsive to thermal stimulation.