ucn-1028-c and fura-2-am

To determine in human corneal epithelial cells (HCECs) the role of protein kinase C (PKC) in mediating epidermal growth factor (EGF)-induced stimulation of store-operated channel (SOC) activity and capacitative calcium entry (CCE).. Single-cell Ca2+ fluorescence imaging of fura2-loaded HCECs was used to evaluate CCE. PKC translocation induced by EGF or PDBu was monitored by Western blot analyses of four different subcellular fractions. Plasma membrane Ca2+ influx was measured by Mn2+ quench rates of fura2-fluorescence. The whole-cell patch clamp configuration was used to determine the SOC activation induced by EGF.. EGF-induced increases in SOC currents through PKC stimulation, since calphostin C inhibited this response. To determine which PKC isoforms mediated EGF-induced increases in CCE, the PKC isoform enrichment of a plasma membrane-containing fraction was determined. From 5 to 30 minutes, its rank order of enrichment was: delta > betaI > alpha approximately epsilon. Preferential PKCdelta and PKCbeta translocation was in accordance with other results showing that rottlerin and hispidin have the highest efficacy in suppressing EGF-induced CCE augmentation. Furthermore, after PKCbeta and PKCdelta siRNA knockdown of gene and protein expression, declines in EGF-induced increases in CCE matched those obtained after exposure to a corresponding selective PKC isoform inhibitor.. EGF-induced PKC stimulation in HCECs mediates SOC activation. This response contributes to CCE, which preferentially depends on PKCdelta and PKCbeta isoform stimulation. This rank order is based on the findings that either selective knockdown of their expression or exposure to PKCdelta and PKCbeta isoform inhibitors elicited the largest declines in EGF-augmented CCE.

Topics: Blotting, Western; Calcium; Calcium Channels; Cell Line; Epidermal Growth Factor; Epithelium, Corneal; Fura-2; Gene Deletion; Humans; Isoenzymes; Microscopy, Fluorescence; Naphthalenes; Patch-Clamp Techniques; Phorbol 12,13-Dibutyrate; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-delta; RNA, Small Interfering; Transfection

Insulin may decrease the contractile response of vascular smooth muscle to vasoactive agents. This could be due to interactions of insulin with the effects of vasoactive agonists on intracellular free calcium transients in vascular smooth muscle cells. This study assesses the effects of physiological doses of insulin (70 microU/mL) on calcium responses in cultured vascular smooth muscle cells (primary unpassaged and passaged) to angiotensin II (1 nmol/L), arginine vasopressin (10 nmol/L), and norepinephrine (10 mumol/L). Intracellular free Ca2+ concentrations in single cells were measured microphotometrically using fura 2-AM. Insulin, angiotensin II, arginine vasopressin, and norepinephrine significantly increased calcium (to 115 +/- 7, 183 +/- 20, 184 +/- 15, and 168 +/- 12 nmol/L, respectively, from basal calcium of 90 +/- 10 nmol/L). Insulin significantly attenuated the agonist-induced calcium responses. The effects of insulin were almost completely inhibited by diltiazem, staurosporine, calphostin C, and thapsigargin. In conclusion, insulin stimulates calcium transients but blunts agonist-mediated calcium rises in vascular smooth muscle cells. These responses are related to regulatory effects of insulin on cellular calcium homeostasis and may explain how insulin modulates vascular smooth muscle contraction.

Topics: Alkaloids; Analysis of Variance; Angiotensin II; Animals; Arginine Vasopressin; Calcium; Calcium-Transporting ATPases; Cells, Cultured; Diltiazem; Fluorescent Dyes; Fura-2; Humans; Insulin; Mesenteric Arteries; Microscopy, Fluorescence; Muscle, Smooth, Vascular; Naphthalenes; Norepinephrine; Polycyclic Compounds; Protein Kinase C; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Staurosporine; Terpenes; Thapsigargin