nystatin-a1 and rottlerin

We previously designed a novel transdermal formulation containing ketoprofen solid nanoparticles (KET-NPs formulation), and showed that the skin penetration from the KET-NPs formulation was higher than that of a transdermal formulation containing ketoprofen microparticles (KET-MPs formulation). However, the precise mechanism for the skin penetration from the KET-NPs formulation was not clear. In this study we investigated whether energy-dependent endocytosis relates to the transdermal delivery from a 1.5% KET-NPs formulation. Transdermal formulations were prepared by a bead mill method using additives including methylcellulose and carbopol 934. The mean particle size of the ketoprofen nanoparticles was 98.3 nm. Four inhibitors of endocytosis dissolved in 0.5% DMSO (54 μM nystatin, a caveolae-mediated endocytosis inhibitor; 40 μM dynasore, a clathrin-mediated endocytosis inhibitor; 2 μM rottlerin, a macropinocytosis inhibitor; 10 μM cytochalasin D, a phagocytosis inhibitor) were used in this study. In the transdermal penetration study using a Franz diffusion cell, skin penetration through rat skin treated with cytochalasin D was similar to the control (DMSO) group. In contrast to the results for cytochalasin D, skin penetration from the KET-NPs formulation was significantly decreased by treatment with nystatin, dynasore or rottlerin with penetrated ketoprofen concentration-time curves (

Topics: Acetophenones; Administration, Cutaneous; Animals; Anti-Inflammatory Agents, Non-Steroidal; Benzopyrans; Cells, Cultured; Cytochalasin D; Drug Liberation; Endocytosis; Hydrazones; Ketoprofen; Male; Nanoparticles; Nystatin; Rats; Rats, Wistar; Skin; Skin Absorption

A role for protein kinase C (PKC)-delta and -zeta isotypes in alpha 1-adrenergic regulation of human tracheal epithelial Na-K-2Cl cotransport was studied with the use of isotype-specific PKC inhibitors and antisense oligodeoxy-nucleotides to PKC-delta or -zeta mRNA. Rottlerin, a PKC-delta inhibitor, blocked 72% of basolateral-to-apical, bumetanide-sensitive 36Cl flux in nystatin-permeabilized cell monolayers stimulated with methoxamine, an alpha 1-adrenergic agonist, with a 50% inhibitory concentration of 2.3 microM. Methoxamine increased PKC activity in cytosol and a particulate fraction; the response was insensitive to PKC-alpha and -beta II isotype-specific inhibitors, but was blocked by general PKC inhibitors and rottlerin. Rottlerin also inhibited methoxamine-induced PKC activity in immune complexes of PKC-delta, but not PKC-zeta. At the subcellular level, methoxamine selectively elevated cytosolic PKC-delta activity and particulate PKC-zeta activity. Pretreatment of cell monolayers with antisense oligodeoxynucleotide to PKC-delta for 48 h reduced the amount of whole cell and cytosolic PKC-delta, diminished whole cell and cytosolic PKC-delta activity, and blocked methoxamine-stimulated Na-K-2Cl cotransport. Sense oligodeoxynucleotide to PKC-delta and antisense oligodeoxynucleotide to PKC-zeta did not alter methoxamine-induced cotransport activity. These results demonstrate the selective activation of Na-K-2Cl cotransport by cytosolic PKC-delta.

Topics: Acetophenones; Base Sequence; Benzopyrans; Biological Transport; Bumetanide; Calcium-Calmodulin-Dependent Protein Kinases; Carrier Proteins; Cells, Cultured; Chlorides; Enzyme Inhibitors; Humans; Isoenzymes; Kinetics; Methoxamine; Mucous Membrane; Naphthalenes; Nystatin; Oligonucleotides, Antisense; Protein Kinase C; Protein Kinase C-delta; Receptors, Adrenergic, alpha-1; RNA, Messenger; Sodium-Potassium-Chloride Symporters; Staurosporine; Trachea; Transcription, Genetic