Suppressing the activation of protein kinase A as a DNA damage-independent mechanistic lead for dihydromethysticin prophylaxis of NNK-induced lung carcinogenesis.

Our earlier work demonstrated varying potency of dihydromethysticin (DHM) as the active kava phytochemical for prophylaxis of tobacco carcinogen nicotine-derived nitrosamine ketone (NNK)-induced mouse lung carcinogenesis. Efficacy was dependent on timing of DHM gavage ahead of NNK insult. In addition to DNA adducts in the lung tissues mitigated by DHM in a time-dependent manner, our in vivo data strongly implicated the existence of DNA damage-independent mechanism(s) in NNK-induced lung carcinogenesis targeted by DHM to fully exert its anti-initiation efficacy. In the present work, RNA seq transcriptomic profiling of NNK-exposed (2 h) lung tissues with/without a DHM (8 h) pretreatment revealed a snap shot of canonical acute phase tissue damage and stress response signaling pathways as well as an activation of protein kinase A (PKA) pathway induced by NNK and the restraining effects of DHM. The activation of the PKA pathway by NNK active metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) at a concentration incapable of promoting DNA adduct was confirmed in a lung cancer cell culture model, potentially through NNAL binding to and activation of the ß-adrenergic receptor. Our in vitro and in vivo data overall support the hypothesis that DHM suppresses PKA activation as a key DNA damage-independent mechanistic lead, contributing to its effective prophylaxis of NNK-induced lung carcinogenesis. Systems biology approaches with a detailed temporal dissection of timing of DHM intake versus NNK exposure are warranted to fill the knowledge gaps concerning the DNA damage-driven mechanisms and DNA damage-independent mechanisms to optimize the implementation strategy for DHM to achieve maximal lung cancer chemoprevention.

Myofibroblast ß2 adrenergic signaling amplifies cardiac hypertrophy in mice.

Abnormal ß-adrenergic signaling plays a central role in human heart failure. In mice, chronic ß-adrenergic receptor (ßAR) stimulation elicits cardiac hypertrophy. It has been reported that cultured cardiac fibroblasts express ßAR; however, the functional in vivo requirement of ßAR signaling in cardiac fibroblasts during the development of cardiac hypertrophy remains elusive. ß2AR null mice exhibited attenuated hypertrophic responses to chronic ßAR stimulation upon continuous infusion of an agonist, isoprenaline (ISO), compared to those in wildtype controls, suggesting that ß2AR activation in the heart induces pro-hypertrophic effects in mice. Since ß2AR signaling is protective in cardiomyocytes, we focused on ß2AR signaling in cardiac myofibroblasts. To determine whether ß2AR signaling in myofibroblasts affects cardiac hypertrophy, we generated myofibroblast-specific transgenic mice (TG) with the catalytic subunit of protein kinase A (PKAcα) using Cre-loxP system. Myofibroblast-specific PKAcα overexpression resulted in enhanced heart weight normalized to body weight ratio, associated with an enlargement of cardiomyocytes at 12 weeks of age, indicating that myofibroblast-specific activation of PKA mediates cardiac hypertrophy in mice. Neonatal rat cardiomyocytes stimulated with conditioned media from TG cardiac fibroblasts likewise exhibited significantly more growth than those from controls. Thus, ß2AR signaling in myofibroblasts plays a substantial role in ISO-induced cardiac hypertrophy, possibly due to a paracrine effect. ß2AR signaling in cardiac myofibroblasts may represent a promising target for development of novel therapies for cardiac hypertrophy.

Stimulation of alpha-(methylamino) isobutyric acid uptake by interleukin-1 in human synovial cells. Involvement of a cAMP dependent pathway.

After one hour incubation with interleukin-1 beta (IL-1 beta), the uptake of alpha-(methylamino) isobutyric acid (MeAIB) by human osteoarthritic synovial cells appeared significantly increased. This effect, observed with 0.1 to 5 ng/ml of cytokine, was inhibited by cycloheximide, indicating that protein synthesis is involved. In addition, this effect seems mediated by a pertussis toxin-sensitive G protein. Finally, intracellular cAMP concentration measurements, the use of a phorbol ester, protein kinase inhibitors and forskolin+3-isobutyl-1-methylxantine (IBMX) provided evidence that a cAMP-dependent protein kinase is associated with interleukin-1 beta-mediated alpha-(methylamino) isobutyric acid uptake.

Effects of isoquinolinesulfonamide H-8 on Fundulus heteroclitus ovarian follicles: role of cyclic nucleotide-dependent protein kinases on steroidogenesis and oocyte maturation in vitro.

The possible role of cyclic nucleotide-dependent protein kinases in mediating the stimulatory actions of Fundulus heteroclitus pituitary extract (FPE) during ovarian steroidogenesis and oocyte maturation in vitro was investigated. Follicle-enclosed oocytes were cultured in the presence of FPE and/or N-[2-Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), a compound that inhibits protein kinase A (PKA) and cGMP-dependent protein kinase. H-8 alone (0.1-1 mM) promoted oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner. However, the process of GVBD initiated by H-8 was much slower that that triggered by 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 betaP), the natural inducer of oocyte maturation in F. heteroclitus. Treatment with H-8 also increased 17,20 betaP production by the follicles and the accumulation of this steroid in the media was much slower than that initiated by FPE. However, in contrast to the FPE action on the oocyte, which is mediated by 17,20 betaP, the stimulatory action of H-8 on GVBD appears to be independent of follicular steroid production, since aminoglutethimide (AGI), an inhibitor of steroidogenesis, did not-block H-8-induced GVBD while inhibiting H-8 induced 17, 20 betaP production. Moreover, addition of H-8 to FPE-treated follicles significantly reduced 17,20 betaP secretion and the percentage of GVBD. These results provide further support for the involvement of PKA in the mechanism by which FPE stimulates ovarian steroidogenesis in F. heteroclitus. Furthermore, the fact that H-8 alone increased 17,20 betaP levels may imply that basal follicular production of this steroid could be induced by inactivation of cyclic nucleotide-dependent protein kinases. Data also indicate that inhibition of PKA and/or c-GMP-dependent protein kinase in the oocyte may be involved in the mechanism leading to resumption of meiosis in this species.

The inhibition of aortic smooth muscle cell proliferation by the intravenous anesthetic ketamine.

Smooth muscle cell (SMC) proliferation has been recognized as central to the pathology of both major forms of vascular disease, atherosclerosis and hypertension. Recently, we reported that ketamine inhibits rat mesangial cell proliferation, suggesting that ketamine inhibits cell growth. Although the IV anesthetic ketamine has been widely used clinically, the exact effects of ketamine on vascular SMC proliferation have not been studied. In this study, we investigated the effects of ketamine on vascular SMC proliferation. Ketamine inhibited [(3)H]thymidine incorporation and decreased the number of SMCs in a concentration-dependent manner (10-200 microM); neither propofol nor fentanyl inhibited [(3)H]thymidine incorporation into human aortic SMCs. The protein kinase C (PKC) inhibitor GF109203x abolished the ketamine-induced inhibition of [(3)H]thymidine incorporation into SMC, but the inhibition was not affected by either the protein kinase A inhibitor H-89 or the protein kinase G inhibitor KT5823. A histological analysis demonstrated the inhibitory effect of ketamine on the intimal thickening of the balloon-injured rat aorta. Based on these results, ketamine inhibits SMCs at clinical concentrations via the PKC pathway. Our results indicate that ketamine might prevent the proliferation of SMCs clinically.