S. M. Wilson

Purine and pyrimidine nucleotide receptors in the apical membranes of equine cultured epithelia

The short circuit current (ISC) technique was used to quantify electrolyte transport by equine cultured sweat gland epithelia. Adenosine 5′‐triphosphate (ATP) and certain related compounds, caused transient increases in ISC when added to the apical solution. The order of potency was uridine triphosphate (UTP)>ATP>ADP>>AMP=adenosine. The responses to apical nucleotides were due to chloride and bicarbonate secretion and were reduced in pertussis toxin‐treated cells. P2‐receptors sensitive to uridine 5′‐triphosphate (UTP), that interact with inhibitory G proteins, therefore appear to be present in the apical membrane. Responses to ATP and UTP were reduced in cells loaded with BAPTA, a calcium chelator. BAPTA attenuated the response to ATP more than the response to UTP suggesting that these nucleotides may not act via a common pathway. Cross‐desensitization experiments indicated that two populations of UTP‐sensitive receptor were present. One was sensitive to UTP and ATP, whereas the second was sensitive only to UTP. Uridine diphosphate appeared to activate the ATP‐insensitive receptor population selectively. These data suggest that apical pyrimidinoceptors may be expressed by these cells. The physiological role of these receptors is unknown but they may allow the autocrine regulation of epithelial function.

Nucleotide‐evoked calcium signals and anion secretion in equine cultured epithelia that express apical P2Y2 receptors and pyrimidine nucleotide receptors

Experiments with a spontaneously transformed equine epithelial cell line showed that certain nucleotides increased intracellular free calcium ([Ca2+]i) in cells plated on glass coverslips. The rank order of potency was ATP=UTP>5‐Br‐UTP, whilst UDP and ADP were ineffective. The response thus appears to be mediated by P2Y2 receptors. Nucleotides also increased short circuit current (ISC) in cells grown into epithelial monolayers and the rank order of potency was UDP>UTP>5‐Br‐UTP>ATP>ADP. The increase in [Ca2+]i and the rise in ISC thus have different pharmacological properties. Cross‐desensitization experiments indicated that, as well as P2Y2 receptors, the monolayer cultures express at least one additional receptor population that allowed nucleotides to increase ISC. The UDP‐evoked increase in ISC was essentially abolished in BAPTA‐loaded epithelia suggesting that this response is dependent upon increased [Ca2+]i. Moreover, experiments in which ISC and [Ca2+]i were measured simultaneously showed that the UDP‐ and ADP‐evoked increases in ISC were accompanied by increases in [Ca2+]i. When grown under conditions which favour the development of a polarized phenotype, these epithelial cells thus appear to express [Ca2+]i‐mobilizing receptors sensitive to UDP and ADP that are not present in non‐polarized cells on coverslips.

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.

P2Y receptor-mediated Ca2+ signalling in cultured rat aortic smooth muscle cells.

ATP, UTP, ADP and ADP‐β‐S elicited Ca2+‐signals in cultured aortic smooth muscle cells although ADP, UDP and ADP‐β‐S gave ∼40% of the maximal response seen with ATP and UTP. Adenosine, AMP or α,β‐methylene‐ATP had no effect. These responses were attributed to P2Y2/4 and P2Y1 receptors, which we assumed could be selectively activated by UTP and ADP‐β‐S respectively. The response to UTP was reduced (∼50%) by pertussis toxin, whilst this toxin had no effect upon the response to ADP‐β‐S. This suggests P2Y2/4 receptors simultaneously couple to pertussis toxin‐sensitive and ‐resistant G proteins whilst P2Y1 receptors couple to only the toxin‐resistant proteins. Repeated stimulation with UTP or ADP‐β‐S caused desensitization which was potentiated by 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA) and attenuated by staurosporine. TPA completely abolished sensitivity to ADP‐β‐S but the response to UTP had a TPA‐resistant component. In pertussis toxin‐treated cells, however, TPA could completely abolish sensitivity to UTP and so the TPA‐resistant part of this response seems to be mediated by pertussis toxin‐sensitive G proteins. Loss of sensitivity to UTP did not occur when pertussis toxin‐treated cells were repeatedly stimulated with this nucleotide, suggesting that pertussis toxin‐sensitive G proteins mediate this effect. The toxin did not, however affect desensitization to ADP‐β‐S.

Calcium-dependent regulation of membrane ion permeability in a cell line derived from the equine sweat gland epithelium

We measured the rates of 125I- and 86Rb+ efflux from preloaded, cultured equine sweat gland cells. The calcium ionophore ionomycin increased the efflux of both isotopes. Anion efflux was unaffected by Ba2+, but this cation inhibited 86Rb(+)-efflux, suggesting that [Ca2+]i-activated potassium channels were present. Activation of these channels was not, however, important for the efflux of anions. We measured 125I- efflux from valinomycin-depolarised cells in which anion cotransport was inhibited. Changes in 125I- efflux reflect changes in anion permeability under these conditions, and ionomycin caused a clear permeability increase that was abolished by the anion channel blocker diphenylamine-2-carboxylate. ATP and UTP increased the efflux of both isotopes, suggesting that type P2U purine receptors allow these nucleotides to regulate membrane permeability.

Effects of topically‐applied antiperspirant on sweat gland function

In subjects exposed to a hot environment, short‐term topical pretreatment with aluminium zirconium tetrachlorhydrate delayed the onset of visible sweating although it failed to prevent the response. The delay was considered most probably to be due to the occlusivc action, in the duct within the upper epidermis, of aluminium‐containing conglomerates, which disappear after continuous sweating. However, microanalytical evidence indicated that ionic transport within the fundus secretory cells was also modified.

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.

Sr2+ can become incorporated into an agonist-sensitive, cytoplasmic Ca2+ store in a cell line derived from the equine sweat gland epithelium

We have explored the properties of a Ca2+-dependent cell-signalling pathway that becomes active when cultured equine sweat gland cells are stimulated with ATP. The ATP-regulated, Ca2+-influx pathway allowed Sr2+ to enter the cytoplasm but permitted only a minimal influx of Ba2+. Experiments in which cells were repeatedly stimulated with ATP suggested that Sr2+, but not Ba2+, could become incorporated into the agonist-sensitive, cytoplasmic Ca2+ store. Further evidence for this was provided by experiments using ionomycin, a Ca2+ ionophore which has no affinity for Sr2+.

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 effects of thermally-induced activity in vivo upon the ultrastructure and Na, K and Cl composition of the epithelial cells of sweat glands from patients with cystic fibrosis

The secretory cells of the fundus of sweat glands from cystic fibrosis (CF) patients had higher Na and Cl contents and showed more granule depletion, cellular disruption and dilated intercellular canaliculi than normal. The cells of the coiled duct also had higher cytoplasmic levels of Na and Cl but were structurally normal. Thermal stimulation produced ultrastructural changes in the CF fundus comparable to normal, including further dilatation of the basolateral clefts, but did not induce the marked changes in the coiled duct which normally occur. The elevated Na and fall in K in the fundus and raised Na and Cl in the coiled duct upon activation, were not observed in the CF glands in which no significant changes were detected.