A role for protein kinase C (PKC) isotypes is implicated in the activation of phagocytic cell functions. An antisense approach was used to selectively deplete ␤-PKC, both ␤I-and ␤II-PKC, but not ␣-PKC, ␦-PKC, or -PKC in HL60 cells differentiated to a neutrophil-like phenotype (dHL60 cells . generation, degranulation, adherence, and actin filament assembly (1-8). These functions are essential for the microbicidal activity of phagocytic cells and are also proinflammatory. PKC, a phospholipid-dependent family of serine/threonine kinases, acts in multiple signal transduction pathways. The cofactor requirements differ between different classes of PKC isotypes. Classical ␣-, ␤-, and ␥-PKC are acidic phospholipid, diglyceride (DG), and Ca 2ϩ -dependent; novel forms ␦-, ⑀-, -, and -PKC also require acidic phospholipid and DG, but are Ca 2ϩ independent. The atypical PKC isotypes, -and -PKC, require PS but are DG and Ca 2ϩ independent (9 -14). PKC isotypes differ in their tissue distribution and localization within the cell, suggesting that each isotype plays a specific role in signal transduction.Neutrophils, monocytes/macrophages, and dHL60 cells contain multiple isotypes of PKC, including Ca 2ϩ -dependent isotypes ␣-PKC, ␤I-PKC, and ␤II-PKC, Ca 2ϩ

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Tumor necrosis factor-alpha (TNF-alpha) triggers degranulation and oxygen radical release in adherent neutrophils. The p60TNF receptor (p60TNFR) is responsible for proinflammatory signaling, and protein kinase C (PKC) is a candidate for the regulation of p60TNFR. Both TNF-alpha and the PKC-activator phorbol 12-myristate 13-acetate triggered phosphorylation of p60TNFR. Receptor phosphorylation was on both serine and threonine but not on tyrosine residues. The PKC-delta isotype is a candidate enzyme for serine phosphorylation of p60TNFR. Staurosporine and the PKC-delta inhibitor rottlerin inhibited TNF-alpha-triggered serine but not threonine phosphorylation. Serine phosphorylation was associated with receptor desensitization, as inhibition of PKC resulted in enhanced degranulation (elastase release). After neutrophil activation, PKC-delta was the only PKC isotype that associated with p60TNFR within the correct time frame for receptor phosphorylation. In vitro, only PKC-delta, but not the alpha-, betaI-, betaII-, or zeta-isotypes, was competent to phosphorylate the receptor, indicating that p60TNFR is a direct substrate for PKC-delta. These findings suggest a selective role for PKC-delta in negative regulation of the p60TNFR and of TNF-alpha-induced signaling.

Amiloride-sensitive, epithelial Na(+) channel (ENaC)-mediated, active absorption of Na(+) is elevated in the airway epithelium of cystic fibrosis (CF) patients, resulting in excess fluid removal from the airway lumen. This excess fluid/volume absorption corresponds to CF transmembrane regulator-linked defects in ENaC regulation, resulting in the reduced mucociliary clearance found in CF airways. Herein we show that INO-4995, a synthetic analog of the intracellular signaling molecule, D-myo-inositol 3,4,5,6-tetrakisphosphate, inhibits Na(+) and fluid absorption across CF airway epithelia, thus alleviating this critical pathology. This conclusion was based on electrophysiological studies, fluid absorption, and (22)Na(+) flux measurements in CF airway epithelia, contrasted with normal epithelia, and on electrophysiological studies in Madin-Darby canine kidney cells and 3T3 cells overexpressing ENaC. The effects of INO-4995 were long-lasting, dose-dependent, and more pronounced in epithelia from CF patients vs. controls. These findings support preclinical development of INO-4995 for CF treatment and demonstrate for the first time the therapeutic potential of inositol polyphosphate derivatives.