Masahiko Ogihara

Poly-l-Arginine Enhances Paracellular Permeability via Serine/Threonine Phosphorylation of ZO-1 and Tyrosine Dephosphorylation of Occludin in Rabbit Nasal Epithelium

AbstractPurpose. The purpose of the present study is to explore whether a poly-l-arginine (poly-l-Arg)-induced increase in tight junctions (TJ) permeability of fluorescein isothiocyanate-labeled dextran (MW 4.4 kDa, FD-4) is associated with the Ca2+-dependent signaling and occurs following the phosphorylation/dephosphorylation of TJ proteins. Methods. Excised rabbit nasal epithelium was mounted in an Ussing-type chamber for measurement of FD-4 transport and membrane conductance (Gt) in the presence of various inhibitors that are involved in the Ca2+-dependent pathway and the phosphorylation/dephosphorylation of TJ proteins. The resultant distribution of TJ proteins was observed using confocal laser scanning microscopy (CLSM) in an immunostaining. Results. The increase in TJ permeability of FD-4 induced by 0.2 mg/ml poly-l-Arg was not altered by treatment with inhibitors of possible Ca2+ mobilization pathways followed by exposure of poly-L-Arg, suggesting that the promoting effect of poly-l-Arg is independent of Ca2+-related signaling. On the other hand, the protein kinase C (PKC) and tyrosine phosphatase inhibitors suppress the increase in TJ permeability by poly-l-Arg, indicating that serine/threonine phosphorylation by way of Ca2+-independent PKC and tyrosine dephosphorylation of junction proteins may have occurred. Furthermore, immunofluorescent monitoring of ZO-1, a TJ associated protein, and occludin, an integral membrane protein localizing at TJ, after preincubation with PKC and tyrosine phosphatase inhibitors followed by poly-l-Arg treatment has shown that the internalization of ZO-1 and occludin occurred by way of serine/threonine phosphorylation by PKC activation and by way of tyrosine dephosphorylation, respectively, providing TJ disassembly. Conclusions. We conclude that poly-l-Arg enhances the paracellular permeability of FD-4 (i.e., macromolecules), at least, by way of both serine/threonine phosphorylation of ZO-1 and tyrosine dephosphorylation of occludin in rabbit nasal epithelium.

Density-dependent proliferation of adult rat hepatocytes in primary culture induced by epidermal growth factor is potentiated by cAMP-elevating agents

We investigated whether or not epidermal growth factor (EGF) and cAMP-elevating agents induce the proliferation of adult rat hepatocytes during the early (4 h after adding EGF) and late phases (21 h after adding EGF) of primary cultures. Adult rat hepatocytes did not significantly proliferate after culture with 20 ng/ml EGF for 4 h at a density of 1 X 10(5) cells/cm2. In contrast, when the density was decreased by about one-third to 3.3 X 10(4) cells/cm2, the number of nuclei increased about 1.2-fold after culture with 10-20 ng/ml EGF for 4 h. Under these culture conditions, DNA synthesis began within 2-4 h of exposure to 20 ng/ml of EGF, although at the high cell density, DNA was not synthesized during this period. The beta-adrenoceptor agonists, metaproterenol and isoproterenol, and other cAMP-elevating agents, such as glucagon, forskolin, and dibutyryl cAMP, potentiated both hepatocyte DNA synthesis and proliferation about 1.4-fold when cultured in combination with 20 ng/ml EGF. The stimulatory effects of metaproterenol and other cAMP-elevating agents were specifically blocked by the cAMP-dependent protein kinase inhibitor, H-89 (10(-7) M). The effect of EGF was almost completely suppressed by genistein (5 X 10(-6) M) and rapamycin (10 ng/ml), but it was unaffected by wortmannin (10(-7) M). These results demonstrate that mature rat hepatocytes can proliferate very rapidly in low-density cultures with EGF, the effects of which were potentiated by beta-adrenoceptor agonists and cAMP-elevating agents. In addition, the activation of receptor tyrosine kinase and p70 ribosomal protein S6 kinase may be involved in EGF-induced hepatocyte DNA synthesis and proliferation.

Stimulation of DNA synthesis and proliferation by prostaglandins in primary cultures of adult rat hepatocytes

We studied the effects of several prostaglandins on DNA synthesis and proliferation in serum-free primary cultures of adult rat hepatocytes. Maintained in short-term cultures (i.e., 3.5 h), the hepatocyte parenchymal cells synthesized DNA and proliferated in the presence of various prostaglandins in a dose-dependent manner. The half-maximal effective concentrations (ED(50)) of prostaglandin F(2alpha), prostaglandin E(1), prostaglandin E(2) and prostaglandin I(2) for proliferation were estimated to be 1.7 x 10(-9), 2.3 x 10(-8), 2.7 x 10(-8) and 3.3 x 10(-9) M, respectively. Prostaglandin E(2) and prostaglandin I(2) produced greater maximal responses than did either prostaglandin E(1) or prostaglandin F(2alpha). The cells responded only weakly to prostaglandin D(2). The stimulatory effects of 10(-6) M prostaglandin E(1) and 10(-6) M prostaglandin E(2) on hepatocyte DNA synthesis and proliferation were inhibited by a specific antagonist of the EP(1) receptor, 8-chlorodibenz[b, f][1, 4]oxazepine-10(11H)carboxylic acid, 2-[3-[(2-furanylmethyl)-thio]-1-oxopropyl]hydrazide (SC-51322; 10(-6) M). Specific inhibitors of signal transducing elements (e.g., 1-[6-[17beta-3-methoxyestra-1, 3, 5(10)-trien-17-yl]amino] hexyl]-1H-pyrrol-2,5-dione (U-73122); 10(-6) M), 10(-6) M verapamil, 5 x 10(-6) M genistein) almost completely blocked the growth-promoting effects of the prostaglandins. These results suggest that prostaglandins stimulate hepatocyte DNA synthesis and proliferation by their own receptors and exert their effects through both phospholipase C/Ca(2+) and receptor tyrosine kinase pathways.

Prostaglandin E2 (EP1) Receptor Agonist-Induced DNA Synthesis and Proliferation in Primary Cultures of Adult Rat Hepatocytes: The Involvement of TGF-α

We investigated the effects of prostaglandin (EP) receptor subtype agonists on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes to elucidate their mechanisms of action. Maintained in short-term cultures (i.e. 3.5 h) in a serum-free, defined medium, hepatocyte parenchymal cells underwent DNA synthesis and proliferation in the presence of sulprostone (10−6 m), PGE2 (10−6 m), and 17-phenyl-trinor-PGE2 (10−9 m) in a time- and dose-dependent manner. PGE2 was less potent than 17-phenyl-trinor-PGE2 in stimulating hepatocyte mitogenesis. Sulprostone (10−6 m) and 11-deoxy-PGE1 (10−6 m) showed weak and insignificant stimulation, respectively, for hepatocyte mitogenesis. These effects of PGE2, 17-phenyl-trinor-PGE2, and sulprostone were abolished by treatment with a specific EP1 receptor antagonist, SC-51322, or the PLC inhibitor U-73122. The effects of these EP1 receptor agonists were potentiated by ionomycin and blocked by verapamil. Hepatocyte mitogenesis was almost completely blocked ...

Proliferation of adult rat hepatocytes in primary culture induced by insulin is potentiated by cAMP-elevating agents

We investigated whether or not insulin and cAMP-elevating agents induce the proliferation of adult rat hepatocytes during the early and late phases of primary culture. Adult rat hepatocytes synthesized a significant amount of DNA when cultured in the presence of 10(-7) M insulin for 3 h. Under these conditions, the number of nuclei increased within 4 h. Hepatocyte DNA synthesis and proliferation were not essentially affected by the initial plating densities. Other cAMP-elevating agents, such as glucagon, forskolin and dibutyryl cAMP, as well as beta-adrenoceptor agonists (i.e., metaproterenol and isoproterenol) alone had no effect on either hepatocyte DNA synthesis or proliferation in primary culture. In contrast, these agents potentiated both processes at concentrations as low as 10(-7) M when cultured in combination with 10(-7) M insulin. The stimulatory effects of beta-adrenoceptor agonists and other cAMP-elevating agents were significantly blocked by the cAMP-dependent protein kinase inhibitor, H-89 (N-[2-(p-(bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; 10(-7) M). The mitogenic effect of insulin upon hepatocytes was almost completely suppressed by genistein (5 x 10(-6) M), wortmannin (10(-7) M) and by rapamycin (10 ng/ml). These results show that insulin rapidly induced the proliferation of adult rat hepatocytes in primary culture. The mitogenic effects of insulin were potentiated by beta-adrenoceptor agonists and cAMP-elevating agents. The effects of beta-adrenoceptor agonists and cAMP-elevating agents may be mediated through cAMP-dependent protein kinase. In addition, the activation of receptor tyrosine kinase, phosphoinositide 3-kinase and p70 ribosomal protein S6 kinase may be involved in the insulin signal transduction pathway.

Analysis of simultaneous transport and metabolism of ethyl nicotinate in hairless rat skin

AbstractPurpose. Simultaneous skin transport and metabolism of ethyl nicotinate (EN), a model drug, were measured and theoretically analyzed. Methods. Several permeation studies of EN or its metabolite nicotinic acid (NA) were done on full-thickness skin or stripped skin with and without an esterase inhibitor. Permeation parameters such as partition coefficient of EN from the donor solution to the stratum corneum and diffusion coefficients of EN and NA in the stratum corneum and the viable epidermis and dermis were determined by these studies. Enzymatic parameters (Michaelis constant Km and maximum metabolism rate Vmax were obtained from the production rate of NA from different concentrations of EN in the skin homogenate. Obtained permeation data were then analyzed by numerical method based on differential equations showing Ficks second law of diffusion in the stratum corneum and the law with Michaelis-Menten metabolism in the viable epidermis and dermis. Results. Fairly good steady-state fluxes of EN and NA through the skin were obtained after a short lag time for all the concentrations of EN applied. These steady-state fluxes were not proportional to the initial donor concentration of EN: EN and NA curves were concave and convex, respectively, which suggests that metabolic saturation from EN to NA takes place in the viable skin at higher EN application. The steady-state fluxes of EN and NA calculated by the differential equations with resulting permeation and enzymatic parameters were very close to the obtained data. Conclusions. The present method is a useful tool to analyze simultaneous transport and metabolism of many drugs and prodrugs, especially those showing Michaelis-Menten type-metabolic saturation in skin.

Change in the electrochemical properties of skin and the lipid packing in stratum corneum by ultrasonic irradiation

Effect of ultrasound on the skin permeation of benzoate anion (BA), a model compound, through excised hairless rat skin was investigated using electrochemical techniques. When the skin surface was sonicated at 150 kHz frequency and 111 mW/cm2 intensity, skin impedance measured by alternative current with 10 Hz frequency was decreased and skin permeation rate (flux) of BA and deuterium oxide was correspondingly increased. A constant current iontophoresis with 0.1 mA/cm2 after pretreatment of skin by the ultrasound for 60 min significantly increased the BA flux through the skin compared to that without the pretreatment. In contrast, electric potential difference across the skin during iontophoresis with the ultrasonic pretreatment was one-third lower than that without the pretreatment. Analysis of these results using the Nernst-Planck equation suggests that the ultrasound increased aqueous region in the stratum corneum (s.c.) as well as effective diffusivity of BA in the skin as a result of a structural disorder in the stratum corneum lipids. The ultrasound significantly increased leaching of sterols and ceramides, typical lipids, from the skin when 0.1% Tween 20 was used as a donor solution, and thus disordered the lipid packing of s.c. to lower the skin impedance and to increase the diffusivity in the s.c. We concluded that ultrasound acts on the s.c. lipids and increases the diffusivity of polar molecules in the skin barrier.

Fundamental investigation of a novel drug delivery system, a transdermal delivery system with jet injection

Abstract A new drug delivery system simultaneously using jet injection and a transdermal delivery system was proposed. After pretreatment of hairless rat skin by a jet injector containing physiological saline without any drug to make a pore in the skin, aqueous solution containing gentamycin sulfate, nicardipine hydrochloride or theophylline was applied on the pore. Absorption clearance (volume flow rate) through the skin (the product of permeability coefficient and application area) was almost the same (0.4 μl/h) with any type of drug and regardless of properties and concentrations. Plasma concentration of gentamycin when the distance between the injector and skin surface was 5 mm was twice that when there was no space between the two. It was found by morphological observation that injection from 5 mm away made a larger pore in the stratum corneum (about 0.3 mm 2 ) and a clear saline reservoir in the viable epidermis and dermis. Mathematical analysis showed that this larger pore greatly increased the skin delivery (absorption) rate, whereas the saline reservoir increased it little. This high delivery rate continued for over 1 week with theophylline.

Tumor necrosis factor (TNF) receptor-2-mediated DNA synthesis and proliferation in primary cultures of adult rat hepatocytes: The involvement of endogenous transforming growth factor-α

We investigated the effects of tumor necrosis factor (TNF)-alpha on DNA synthesis and proliferation, and its signal transduction pathways in primary cultures of adult rat hepatocytes. TNF-alpha induced time- and dose-dependent increases in hepatocyte DNA synthesis and proliferation. The hepatocyte proliferation stimulated by 30 ng/ml TNF-alpha was significantly inhibited by anti-TNF receptor 2 antibody, but not by anti-TNF receptor 1 antibody. TNF-alpha-induced hepatocyte DNA synthesis and proliferation were blocked by AG1478 (10(-7) M), PD98059 (10(-6) M), LY 294002 (10(-7) M), and rapamycin (100 ng/ml). TNF-alpha at 30 ng/ml significantly increased phosphorylation of receptor tyrosine kinase (175 kDa) and p42 mitogen-activated protein (MAP) kinase. This data suggests that the proliferative signal for primary cultured hepatocytes induced by TNF-alpha is mediated by TNF receptor 2 and the receptor tyrosine kinase/MAP kinase pathway. In addition, TNF-alpha-induced hepatocyte mitogenesis was significantly blocked by somatostatin (10(-6) M), adenylate cyclase inhibitor dideoxyadenosine (10(-7) M), protein kinase A inhibitor H-89 (10(-7) M), and neutralizing antibody to transforming growth factor (TGF)-alpha in culture. Indeed, 30 ng/ml TNF-alpha was found to rapidly stimulate secretion of TGF-alpha, and this secretion was also blocked by anti-TNF receptor 2 antibody. Moreover, TGF-alpha secretion induced by TNF-alpha was suppressed by dideoxyadenosine, H-89, and somatostatin. Together, these results indicate that stimulation of TNF receptor 2 by 30 ng/ml TNF-alpha induces autocrine secretion of TGF-alpha via the adenylate cyclase/protein kinase A pathway, after which TGF-alpha induces hepatocyte DNA synthesis and proliferation through the TGF-alpha receptor-linked tyrosine kinase (175 kDa)/MAP kinase signaling system.

Combined effect of ultrasound and chemical enhancers on the skin permeation of aminopyrine

Abstract The combined effect of 150 kHz ultrasound with 111 mW/cm 2 intensity and chemical enhancers on the skin permeation of aminopyrine (AMP) was investigated using excised hairless rat skin. Monoterpenes ( l -menthol, l -calvone and D-limonene), laurocapram (Azone®), glycerol monocaprylate (Sefsol-318®), isopropyl myristate and ethanol were selected as enhancers. Combined application of ultrasound and enhancers increased the skin permeation rate (flux) of AMP compared with ultrasound or enhancers alone. Better effects were obtained by the combination with monoterpenes. The influence of detailed conditions of ultrasound and enhancer applications on the AMP flux was further investigated using l -menthol. The enhancement effect by this combination was increased with an increase in ultrasonic application duration and l -menthol concentration, suggesting that these conditions might be used to achieve the controlled drug delivery. A pretreatment experiment with ultrasound or l -menthol was carried out, and l -menthol content in the skin and the skin permeation of deuterium oxide (D 2 O), used as a donor vehicle, were measured to understand the role of ultrasound in the combined effect. Application of ultrasound to the l -menthol-pretreated skin increased the AMP flux, while the effect of l -menthol on ultrasonic-pretreated skin was similar to that of l -menthol alone. The ultrasound increased the l -menthol content in the skin as well as the skin permeation of D 2 0 from a vehicle with l -menthol. These results suggested that simultaneous application of ultrasound and enhancers is essential to obtain the pronounced effect. Ultrasound application also strongly assisted migration of l -menthol into skin, which increases the enhancing action on the skin permeation for a drug.