Abstract
Abstract: :
Purpose:Fluid secretion in lacrimal glands is an osmotic process driven by ion movement through the apical membrane. Our hypothesis is that receptor activation increases intracellular calcium which opens large calcium sensitive potassium channels (maxi–K) as well as chloride channels. The evidence for maxi–K channel involvement in lacrimal fluid secretion arises from electrophysiological data and from the high potassium concentration seen in the lacrimal gland fluid (∼40–50 mM). To more directly test this hypothesis, we measured lacrimal gland fluid secretion in knock–out mice lacking the Ca++ responsive beta subunit of the maxi–K channel. We also examined the effect of a maxi–K blocker, charybdotoxin, on carbachol induced shrinkage of isolated cells from normal mice. Methods:Fluid samples were collected into calibrated microcapillaries from the distal duct of exorbital lacrimal glands in situ in anesthetized mice. Fluid secretion was elicited by the addition of 0.1 mM carbachol to the fluid bathing the gland in situ. Knockout mice lacking the beta subunit (maxi–K ß–/–) were made in the C57 strain (maxi–K ß+/+). Cells were isolated from C57 exorbital lacrimal glands and allowed to rest for 24 hours on matrigel. Cells were then exposed to carbachol or carbachol and charybdotoxin and their volume was measured. Results:Maxi–K ß–/– mice, both male and female, showed significantly reduced flow responses compared to maxi–K ß+/+. Male maxi–K ß–/– stimulated flow was 169±29 (mean, SE) while maxi–K ß+/+ flow was significantly larger ( p<0.05) at 280±38 nl/min, n= 8,7 respectively. Female maxi–K ß–/– mice had a lower flow than the males of 91± 28 nl/min while the maxi–K ß+/+ were larger ( p<0.05) at 199±25 nl/min, n=9,7 respectively. Lacrimal gland weights, normalized for body weight, were similar between groups of maxi–K ß–/–and maxi–K ß+/+ mice, although gland weights in female mice were less than in the corresponding male groups. Charybodtoxin exposure reduced the carbachol induced shrinkage of cells isolated from normal mice by about 60%. Conclusions: These results provide new evidence that the calcium–activated maxi–K channels play an important role in fluid secretion by the mouse lacrimal gland.
Keywords: lacrimal gland • ion channels • transgenics/knock–outs