Purpose: Calcium signalling receptor systems are not uniformly distributed in the intact human lens (Collison & Duncan, IOVS. 2001;42:2356-2363). This study therefore investigated the regional distribution of membrane-associated, signal-amplifying calcium entry pathways. Methods: The lens was dissected from donor eyes (20-80 yrs), placed anterior face down in a plastic chamber and perifused with artificial aqueous humour at 30C. Following Fura-2 incorporation into anterior and equatorial epithelial cells, cytosolic calcium levels were monitored using epifluorescence techniques. Calcium entry was monitored in the resting lens, and following activation of G-protein and tyrosine-kinase signalling systems. The blocking effects of LaCl on store-mediated calcium entry, flufenamic acid on non-selective cation entry, and nifedipine on voltage-gated calcium channels were also investigated. Results: In the anterior region, sustained G-protein receptor activation (e.g. histamine, 10-100 µM) led to a maintained increase in cytosolic calcium that was inhibited by addition of LaCl (10 µM), but insensitive to nifedipine (10 µM). In the equatorial region, both G-protein (histamine, 10-100 µM) and tyrosine-kinase (EGF, 10 ng/ml) receptor activation gave rise to maintained calcium increases that were abolished by LaCl, but not by nifedipine. In this region the resting calcium level was significantly reduced in the presence of flufenamic acid (50 µM), indicating the presence of a calcium entry pathway in unstimulated cells. LaCl or nifedipine had no effect on the resting calcium levels in either anterior or equatorial epithelial cells. Furthermore, exposure to flufenamic acid had only a minor effect on resting levels in anterior cells. Conclusion: The stimulated store-mediated calcium entry pathway amplifies receptor-mediated calcium signals in both anterior and equatorial cells. However, a novel, resting, flufenamic acid-sensitive pathway plays a major role only in the equatorial region and will contribute to ion current asymmetries in the intact lens.
|Investigative Ophthalmology and Visual Science
|Published - Dec 2002