In situ determination of [Ca2+]i threshold for translocation of the alpha-protein kinase C isoform

Abstract

Because of inherent difficulties in maintaining physiological conditions in biochemical assays, the intracellular free Ca2+ concentration ([Ca2+]i) required for activation of protein kinase C (PKC) in intact cells remains unclear. In the present study, [Ca2+]i was measured in freshly isolated vascular smooth muscle cells loaded with fura 2 while, in parallel, the distribution of the Ca(2+)-dependent alpha-PKC isoform was monitored using digital imaging microscopy. The [Ca2+]i alpha-PKC translocation threshold was determined by changing extracellular free Ca2+ concentration in steps while monitoring [Ca2+]i. In the absence of agonists, increasing [Ca2+]i caused < 25% of maximal translocation. In the presence of phenylephrine, maximum translocation occurred at [Ca2+]i > or = 198 nM. Phenylephrine augmented translocation of alpha-PKC primarily by increasing the slope of the [Ca2+]i-PKC translocation relationship. These results indicate that the [Ca2+]i threshold of alpha-PKC translocation in situ is less than that reported in most in vitro assays and are consistent with an effect of agonist-induced generation of other second messengers that cause cooperative interactions leading to translocation.

Dynamics of Glucose-induced Membrane Recruitment of Protein Kinase C βII in Living Pancreatic Islet β-Cells

“…6) that cytosolic [Ca 2ϩ ] and PKC membrane localization could be dissociated in the same single cell are perhaps most simply explained by the fact that in the absence of generation of DAG a "threshold" concentration of Ca 2ϩ , probably Ն 1 M, is required to ensure the binding of the C2 domain of PKC␤II to membrane phospholipids (44) as previously proposed for PKC␣ (56). Interestingly, the concentrations of Ca 2ϩ measured here immediately beneath the membrane of stimulated MIN6 ␤-cells (2-3 M) are similar to, if somewhat lower than, those previously reported at greater distances from the plasma membrane (0.5-1.0 m) of ␤-cells using diffusible dyes (6 -10 M) (57).…”

Cytoskeletal targeting of calponin in differentiated, contractile smooth muscle cells of the ferret

“…This procedure has previously been shown not to interfere with the signal transduction in smooth muscle cells, as measured by depolarization and agonist-induced increases in intracellular [Ca¥] and PKC translocation (Khalil, Lajoie, Resnick & Morgan, 1992;Khalil & Morgan, 1993;Khalil, Lajoie & Morgan, 1994;Khalil & Morgan, 1996) and is thought to prevent contraction at the crossbridge level (Cecchi & Bagni, 1994). Also, this procedure has previously been shown not to affect CaP localization in resting cells or mean cell length in the resting state (Parker et al 1994).…”

Regulation of arterial diameter and wall [Ca2+] in cerebral arteries of rat by membrane potential and intravascular pressure

“…R is the ratio between emission signals, when exciting with 340 and 380 nm light; Rmin is R under Ca¥-free conditions (5 mÒ EGTA); Rmax is R under Ca¥-saturated conditions (2·4 mÒ calcium); â is the ratio between the emission signals when exciting with 380 nm light under Ca¥-free and Ca¥-saturated conditions. 1988; Williams & Fay, 1990;Jensen, Mulvany, Aalkjaer, Nilsson & Yamaguchi, 1993;Khalil, Lajoie & Morgan, 1994;Morgan & Jacob, 1994;Nilsson, Jensen & Mulvany, 1994;Chen & Rembold, 1995). Extracellular free [Ca¥] in this bathing solution was changed by altering the level of total Ca¥, and calculated using an EGTA stability constant of 6·79 (Martell & Smith, 1974;Perrin, 1979).…”