The simultaneous measurement of epithelial ion transport and intracellular free Ca2+ in cultured equine sweat gland secretory epithelium.

We explored the relationship between nucleotide-evoked changes in intracellular free calcium ([Ca2+]i) and anion secretion by measuring [Ca2+]i and I(SC) simultaneously in Fura-2-loaded, cultured equine sweat gland epithelia. Apical ATP, UTP or UDP elicited sustained increases in [Ca2+]i that were initiated by the mobilization of cytoplasmic Ca2+ but maintained by Ca2+ influx. However, although these nucleotides also increased I(SC), this response was transient whereas the [Ca2+]i signals were sustained. Experiments in which external Ca2+ was removed/replaced showed that Ca2+ entering nucleotide-stimulated cells elicited very little change in ISC. Cross desensitization experiments showed that UTP-stimulated epithelia became insensitive to ATP but that UTP could increase both [Ca2+]i and ISC in ATP-stimulated cells by activating 'pyrimidinoceptors' essentially insensitive to ATP. Thapsigargin evoked a sustained rise in [Ca2+]i that was accompanied by a maintained increase in ISC. However, this increase in ISC was dependent upon external Ca2+ and so the responses to nucleotides and thapsigargin have different properties. ATP increased ISC in thapsigargin-treated cells without causing any rise in [Ca2+]i while ionomycin increased both parameters. The data therefore show that apical P2Y receptors allow nucleotides to increase ISC via two mechanisms, one of which appears to be [Ca2+]i-independent control of anion channels.