Limbal epithelial stem cell (LSC) transplantation is a prevalent therapeutic method for patients with LSC deficiency. The maintenance of stem cell characteristics in the process of culture expansion is critical for the success of ocular surface reconstruction. Pigment epithelialderived factor (PEDF) increased the numbers of holoclone in LSC monolayer culture and preserved the stemness of LSC in suspension culture by evidence of DNp63a, Bmi-1, and ABCG2 expression. BrdU pulse-labeling assay also demonstrated that PEDF stimulated LSCs proliferation. In air-lift culture of limbal equivalent, PEDF was capable of increasing the numbers of DNp63a-positive cells. The mitogenic effect of PEDF was found to be mediated by the phosphorylations of p38 MAPK and STAT3 in LSCs. Synthetic 44-mer PEDF (residues 78-121) was as effective as the full length PEDF in LSC expansion in suspension culture and limbal equivalent formation, as well as the activation of p38 MAPK and STAT3. In mice subjecting to mechanical removal of cornea epithelium, 44-mer PEDF facilitated corneal wound healing. Microscopically, 44-mer PEDF advanced the early proliferative response in limbus, increased the proliferation of DNp63a-positive cells both in limbus and in epithelial healing front, and assisted the repopulation of limbus in the late phase of wound healing. In conclusion, the capability of expanding LSC in cell culture and in animal indicates the potential of PEDF and its fragment (e.g., 44-mer PEDF) in ameliorating limbal stem cell deficiency; and their uses as therapeutics for treating corneal wound.

To study the role of intracellular pH (pHi) in catecholamine secretion and the regulation of pHi in bovine chromaffin cells, the pH-sensitive fluorescent indicator [2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein] was used to monitor the on-line changes in pHi. The pHi of chromaffin cells at resting state is approximately 7.2. The pHi was manipulated first by incubation of the cells with NH4+, and then the solution was replaced with a NH4(+)-free solution to induce acidification of the cytoplasm. The pHi returned toward the basal pH value after acidification within 5-10 min in the presence of Na+ or Li+, but the pHi stayed acidic when Na(+)-free buffers were used or in the presence of amiloride and its analogues. These results suggest that the pH recovery process after an acid load is due to the Na+/H+ exchange activity in the plasma membrane of the chromaffin cells. The catecholamine secretion evoked by carbachol and Na+ removal was enhanced after the cytoplasm had been made more acidic. It appears that acidic pH favors the occurrence of exocytosis.

Dopamine (DA) acts directly on pituitary lactotropes to inhibit the release of PRL. Removal of DA elicits a pronounced transient rise in PRL release to values exceeding pre-DA rates (PRL rebound). Electrophysiological studies have shown that lactotropes exhibit a period of increased Ca2+ action potential activity after DA withdrawal, leading to the proposal that enhanced Ca2+ influx during this period may support the rebound secretion of PRL. In the present studies, we investigated the effect of DA application and removal on the cytosolic free calcium concentration ([Ca2+]i) monitored by fura-2 in single rat lactotropes. Unchallenged lactotropes fell into two functionally distinct groups: those with stable [Ca2+]i that was not acutely sensitive to extracellular Ca2+, and those with spontaneous fluctuations in [Ca2+]i that were dependent upon influx of external Ca2+. There was striking variability in the [Ca2+]i patterns of the latter group, ranging from irregular, low amplitude fluctuations to rhythmic, repetitive oscillations with definable rise and decay kinetics. Application of DA resulted in a rapid decrease in [Ca2+]i concomitant with the cessation of these spontaneous [Ca2+]i fluctuations. After DA removal, these cells resumed oscillatory [Ca2+]i activities similar to those observed before DA application. In quiescent lactotropes, acute application of DA exerted no effect on resting [Ca2+]i, but quiescent cells could be activated to produce [Ca2+]i fluctuations by the application and withdrawal of DA. Again, the character of the induced [Ca2+] activity showed significant cell to cell variation. In contrast, the pattern of [Ca2+]i fluctuations was remarkably characteristic in a given cell in response to repeated challenges. A composite [Ca2+]i profile of 13 cells paralleled the PRL secretory rebound after application and removal of DA. The oscillatory rise in [Ca2+]i is functionally linked to the rebound release of PRL after DA removal, as both were immediately abolished by blockade of Ca2+ influx. These data demonstrate that the rebound secretion of PRL is dependent upon enhanced influx of extracellular Ca2+ after cells recover from DA-induced hyperpolarization and support the hypothesis that a population of inactivated Ca2+ channels has been recruited in response to application and withdrawal of DA.

Dopamine (DA) acts directly on pituitary lactotropes to inhibit the release of PRL. Removal of DA elicits a pronounced transient rise in PRL release to values exceeding pre-DA rates (PRL rebound). Electrophysiological studies have shown that lactotropes exhibit a period of increased Ca2+ action potential activity after DA withdrawal, leading to the proposal that enhanced Ca2+ influx during this period may support the rebound secretion of PRL. In the present studies, we investigated the effect of DA application and removal on the cytosolic free calcium concentration ([Ca2+]i) monitored by fura-2 in single rat lactotropes. Unchallenged lactotropes fell into two functionally distinct groups: those with stable [Ca2+]i that was not acutely sensitive to extracellular Ca2+, and those with spontaneous fluctuations in [Ca2+]i that were dependent upon influx of external Ca2+. There was striking variability in the [Ca2+]i patterns of the latter group, ranging from irregular, low amplitude fluctuations to rhythmic, repetitive oscillations with definable rise and decay kinetics. Application of DA resulted in a rapid decrease in [Ca2+]i concomitant with the cessation of these spontaneous [Ca2+]i fluctuations. After DA removal, these cells resumed oscillatory [Ca2+]i activities similar to those observed before DA application. In quiescent lactotropes, acute application of DA exerted no effect on resting [Ca2+]i, but quiescent cells could be activated to produce [Ca2+]i fluctuations by the application and withdrawal of DA. Again, the character of the induced [Ca2+] activity showed significant cell to cell variation. In contrast, the pattern of [Ca2+]i fluctuations was remarkably characteristic in a given cell in response to repeated challenges. A composite [Ca2+]i profile of 13 cells paralleled the PRL secretory rebound after application and removal of DA. The oscillatory rise in [Ca2+]i is functionally linked to the rebound release of PRL after DA removal, as both were immediately abolished by blockade of Ca2+ influx. These data demonstrate that the rebound secretion of PRL is dependent upon enhanced influx of extracellular Ca2+ after cells recover from DA-induced hyperpolarization and support the hypothesis that a population of inactivated Ca2+ channels has been recruited in response to application and withdrawal of DA.