Hiroki Yokoo

Nitric oxide and endothelial cellular senescence

Cellular senescence is characterized by permanent exit from the cell cycle and the appearance of distinct morphological and functional changes associated with an impairment of cellular homeostasis. Many studies support the occurrence of vascular endothelial cell senescence in vivo, and the senescent phenotype of endothelial cells can be transformed from anti-atherosclerotic to pro-atherosclerotic. Thus, endothelial cell senescence promotes endothelial dysfunction and may contribute to the pathogenesis of age-associated vascular disorders. Emerging evidence suggests that increasing nitric oxide (NO) bioavailability or endothelial NO synthase (eNOS) activity activates telomerase and delays endothelial cell senescence. In this review, we discuss the potential mechanisms underlying the ability of NO to prevent endothelial cell senescence and describe the possible changes in the NO-mediated anti-senescence effect under pathophysiological conditions, including oxidative stress and hyperglycemia. Further understanding of the mechanisms underlying the anti-senescence effect of NO in endothelial cells will provide insights into the potential of eNOS-based anti-senescence therapy for age-associated vascular disorders.

Aquaporin subtypes in rat cerebral microvessels

We investigated the expression of aquaporin (AQP) subtypes in the rat cerebral microvessels by reverse transcription-polymerase chain reaction, immunoblotting and immunohistochemistry. mRNA for AQP4, but not for AQP1, 2, 3 or 5, was detected in the microvessels. Immunoblot analysis showed that AQP4 protein was detected as a 30 kDa band with higher molecular weight bands. Immunohistochemical staining showed that AQP4 was located on cell surface of the cerebral microvessels. These results suggest that AQP4 in the cerebral microvessels is involved in the regulation of water transport between blood and brain.

Up-regulation of sodium channel subunit mRNAs and their cell surface expression by antiepileptic valproic acid: activation of calcium channel and catecholamine secretion in adrenal chromaffin cells.

Abstract: Treatment of cultured bovine adrenal chromaffin cells with a therapeutic concentration (0.6 mM) of valproic acid (VPA) for >24 h caused a time‐dependent (t1/2 = 74 h) increase in [3H]saxitoxin binding up to 1.4‐fold without altering the KD value; it was prevented by the simultaneous treatment with cycloheximide (an inhibitor of protein synthesis). VPA also raised Na+ channel α‐ and β1‐subunit mRNA levels 1.4‐ and 1.7‐fold at 24 h, and 1.6‐ and 1.8‐fold at 72 h, respectively. Chronic (but not acute) exposure to VPA enhanced 22Na+ influx caused by various concentrations of veratridine 1.4–2.1‐fold, even when assayed in the presence of Na+,K+‐ATPase inhibitor, but did not change the EC50 value of veratridine. Ptychodiscus brevis toxin‐3 allosterically potentiated veratridine‐induced 22Na+ influx by ∼2‐fold in VPA‐treated cells as in nontreated cells. Long‐term treatment with VPA augmented veratridine‐induced 45Ca2+ influx via voltage‐dependent Ca2+ channels and catecholamine secretion, but had no effect on 45Ca2+ influx and catecholamine secretion caused by high K+ (a direct activation of voltage‐dependent Ca2+ channels). Chronic treatment with VPA also enhanced nicotine‐induced 22Na+ influx via the nicotinic receptor‐ion channel complex 1.2–1.4‐fold with little change in the EC50 value of nicotine, thereby increasing the nicotine‐induced 45Ca2+ influx via voltage‐dependent Ca2+ channels and catecholamine secretion. These results suggest that chronic treatment with VPA up‐regulates cell surface expression of Na+ channels via the transcription/translation‐dependent mechanisms, and probably of nicotinic receptors, thereby resulting in the enhancement of Ca2+ channel gating and catecholamine secretion.

Up-Regulation of Functional Voltage-Dependent Sodium Channels by Insulin in Cultured Bovine Adrenal Chromaffin Cells

Abstract: Treatment of cultured bovine adrenal chromaffin cells with 100 nM insulin raised [3H]saxitoxin ([3H]STX) binding in a time‐dependent manner (t1/2 = 26 h). Insulin (100 nM for 4 days) increased the Bmax of [3H]STX binding by 49% without changing the KD value and also augmented the maximal influx of 22Na+ due to 560 µM veratridine by 39% without altering the EC50 value of veratridine. The stimulatory effect of insulin on 22Na+ influx was concentration‐dependent with an EC50 of 3 nM, whereas insulin‐like growth factor (IGF)‐I had little effect at 1 nM. Ptychodiscus brevis toxin‐3 allosterically potentiated veratridine (100 µM)‐induced 22Na+ influx by approximately twofold in both insulin‐treated cells and untreated cells. Veratridine‐induced 45Ca2+ influx via voltage‐dependent Ca2+ channels and catecholamine secretion were also enhanced by insulin treatment, whereas insulin did not alter nicotine‐induced 22Na+ influx via the nicotinic receptor‐ion channel complex and high‐K+ (direct activation of voltage‐dependent Ca2+ channels)‐induced 45Ca2+ influx. Stimulatory effects of insulin on [3H]STX binding and veratridine‐induced 22Na+ influx were nullified by simultaneous treatment with either 5,6‐dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, or cycloheximide, an inhibitor of protein synthesis, whereas insulin treatment did not appreciably increase the level of mRNA encoding the Na+ channel α‐subunit. These results suggest that the binding of insulin to insulin (but not IGF‐I) receptors mediates the up‐regulation of functional Na+ channel expression at plasma membranes; this up‐regulation may be due, at least in part, to the de novo synthesis of an as yet unidentified protein(s).

Protein Kinase C-α and -ε Down-Regulate Cell Surface Sodium Channels via Differential Mechanisms in Adrenal Chromaffin Cells

Abstract: In cultured bovine adrenal chromaffin cells, our [3H]saxitoxin ([3H]STX) binding, immunoblot, and northern blot analyses specified protein kinase C (PKC) isoform‐specific posttranscriptional and posttranslational mechanisms that direct down‐regulation of cell surface Na channels. Immunoblot analysis showed that among 11 PKC isoforms, adrenal chromaffin cells contained only conventional (c)PKC‐α, novel (n)PKC‐ε, and atypical (a)PKC‐ζ. Treatment of adrenal chromaffin cells with 100 nM 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) or 100 nM phorbol 12,13‐dibutyrate (PDBu) caused a rapid (< 15 min) and sustained (> 15 h) translocation of PKC‐α and ‐ε (but not ‐ζ) from cytosol to membranes, whereas a biologically inactive 4α‐TPA had no effect. Thymeleatoxin (TMX), an activator of cPKC, produced similar membrane association of only PKC‐α at 100 nM, with the potency of TMX being comparable with those of TPA and PDBu. Treatment with either 100 nM TPA or 100 nM TMX reduced cell surface [3H]STX binding to a comparable extent at 3, 6, and 12 h, whereas TPA lowered the binding to a greater extent than TMX at 15, 18, and 24 h; at 15 h, Gö6976, a specific inhibitor of cPKC, completely blocked TMX‐induced decrease of [3H]STX binding while preventing by merely 57% TPA‐induced decrease of [3H]STX binding. Treatment with 100 nM TPA lowered the Na channel α‐subunit mRNA level between 3 and 12 h, with its maximum 52% fall at 6 h, and it was accompanied by a subsequent 61% rise of the β1‐subunit mRNA level at 24 h. Gö6976 failed to prevent TPA‐induced reduction of the α‐subunit mRNA level; TMX did not change the α‐and β1‐subunit mRNA levels throughout the 24‐h treatment. Brefeldin A, an inhibitor of vesicular exit from the trans‐Golgi network, augmented TPA‐ and TMX‐induced decrease of [3H]STX binding at 1 and 3 h. Our previous and present studies suggest that PKC down‐regulates cell surface Na channels without altering the allosteric gating of Na channels via PKC isoform‐specific mechanisms; cPKC‐α promotes Na channel internalization, whereas nPKC‐ε decreases the α‐subunit mRNA level by shortening the half‐life of α‐subunit mRNA without changing its gene transcription.

Adrenomedullin inhibits spontaneous and bradykinin‐induced but not oxytocin‐ or prostaglandin F2α‐induced periodic contraction of rat uterus

In isolated rat uterine strips, adrenomedullin (AM) inhibited the spontaneous periodic contraction in a concentration‐dependent manner (IC50=22.3±0.7 nM). The inhibitory effect of AM was prevented by either AM22–52, a putative antagonist for AM receptors, or calcitonin gene‐related peptide (CGRP)8–37, a putative antagonist for CGRP receptors. AM also attenuated bradykinin (BK)‐induced periodic uterine contraction, which was blocked by AM22–52 or CGRP8–37, whereas AM had no effect on the periodic contraction caused by oxytocin or prostaglandin F2α (PGF2α). RT–PCR analysis showed that mRNAs for calcitonin receptor‐like receptor (CRLR), receptor‐activity‐modifying protein (RAMP)1, RAMP2 and RAMP3 were expressed in the rat uterus. These results demonstrate that AM selectively inhibits spontaneous and BK‐induced periodic contraction via activating receptors for AM and CGRP.

Modulation of glucocorticoid receptor expression, inflammation, and cell apoptosis in septic guinea-pig lungs using methylprednisolone

The use of glucocorticoids for treatment of sepsis has waxed and waned during the past several decades, and recent randomized controlled trials have evoked a reassessment of this therapy. Most glucocorticoid actions are mediated by its specific intracellular receptors (GRs). Thus we initially evaluated whether sepsis and high-dose corticosteroid therapy can regulate guinea pig pulmonary expression of GRs: active receptor, GRalpha, and dominant negative receptor, GRbeta. Sepsis induction by LPS injection (300 mug/kg ip) decreased mRNA and protein levels of GRalpha and increased protein expression of GRbeta in lungs. High-dose methylprednisolone (40 mg/kg ip), administered simultaneously with LPS, markedly potentiated the decrease in GRalpha expression but slightly affected the increase in GRbeta expression. Consequently, this led to a significant reduction in GRalpha nuclear translocation. Nevertheless, methylprednisolone treatment strongly eliminated LPS induction of NF-kappaB activity, as determined by NF-kappaB nuclear translocation and by gel mobility shift assays. Furthermore, the LPS-induced increase in inflammatory cells in bronchoalveolar lavage fluid was blunted by administration of the corticosteroid. On the other hand, immunofluorescent staining for cleaved caspase-3 showed a marked increase in this proapoptotic marker in lung sections, and terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) represented an enhanced appearance of cell apoptosis in lungs and spleen when methylprednisolone was given together with LPS. Cell apoptosis is now considered to play a role in the pathogenesis of septic syndrome. We thus suggest that the action of glucocorticoids at high doses to accelerate sepsis-induced cell apoptosis may overwhelm their therapeutic advantages in septic shock.

REGULATION OF CELL SURFACE EXPRESSION OF VOLTAGE-DEPENDENT Nav1.7 SODIUM CHANNELS: mRNA STABILITY AND POSTTRANSCRIPTIONAL CONTROL IN ADRENAL CHROMAFFIN CELLS

Regulated expression of Na+ channels is indispensable to physiological events, whereas dysregulated expression of otherwise silent or even normal Na+ channel isoforms causes Na+ channelopathies; however, the regulatory mechanisms remain unknown. In quiescent cultured bovine adrenal chromaffin cells, constitutive phosphorylation/activation of extracellular signal-regulated kinase-1 (ERK1) and ERK2 destabilized Nav l.7 Na+ channel alpha-subunit mRNA and decreased its level without altering alpha-subunit gene transcription, thus negatively regulating steady-state level of Na+ channels. Activation of protein kinase C (PKC) down-regulated Na+ channels via PKC isoform-specific mechanisms; conventional PKC-alpha promoted endocytic internalization of Na+ channels, whereas novel PKC-epsilon destabilized alpha-subunit mRNA without altering its gene transcription. Long-lasting (but not short-term) increase of cytoplasmic Ca2+ down-regulated Na+ channels; a slowly-developing moderate increase of Ca2+ activated PKC-alpha and calpain, promoting internalization of Na+ channels, whereas an immediate monophasic and salient plateau increase of Ca2+ lowered alpha- and beta1-subunit mRNA levels. Calcineurin, or FK506 binding protein- and rapamycin-associated protein (FRAP), a serine/threonine protein kinase, down-regulated, whereas insulin receptor tyrosine kinase or protein kinase A (PKA) up-regulated, Na+ channels via modulating Na+ channel internalization, and/or Na+ channel externalization from the trans-Golgi network. Neuroprotective, antiepiletic, antipsychotic, and local anesthetic drugs up-regulated Na+ channels via transcriptional/translational events.

Successful Treatment of Acute Lung Injury with Pitavastatin in Septic Mice: Potential Role of Glucocorticoid Receptor Expression in Alveolar Macrophages

There is growing evidence that the HMG-CoA reductase inhibitors (statins) provide some of the beneficial effects that are independent of their lipid-lowering effects. Recent animal experiments and clinical trials suggest that statin use may limit the development of sepsis and associated systemic inflammation. The aim of this study was to explore the potential role of statins in the prevention treatment of sepsis-induced acute lung injury (ALI). Mice were rendered septic by cecal ligation and puncture (CLP). An intraperitoneal injection of 3 mg/kg per day of pitavastatin was initiated 4 days before surgery and was maintained for life support afterward, which significantly improved the survival of CLP mice. Treatment with pitavastatin prevented the ALI development in CLP mice, as indicated by the findings that severe hypoxemia, increased pulmonary vascular permeability, and histological lung damage, including inflammatory cell infiltrate, were greatly remedied. This was associated with down-regulation of increased activity of nuclear factor-κB (NF-κB) in septic lungs. Although plasma cortisol showed a sharp rise, glucocorticoid receptor (GCR) expression in the lungs was strikingly reduced after the onset of CLP-induced sepsis. It is noteworthy that pitavastatin increased GCR expression with an increase in alveolar macrophages in which GCRs are localized, without modifying the sepsis-associated rise in plasma cortisol. These results confirm significant protection by pitavastatin on septic ALI and demonstrate that down-regulated NF-κB activation associated with the GCR expression increase consequent to the increased number of alveolar macrophages may explain, in part, the mechanisms responsible for favorable effects of statins on the ALI management.

Enhancement of insulin-induced PI3K/Akt/GSK-3β and ERK signaling by neuronal nicotinic receptor/PKC-α/ERK pathway: up-regulation of IRS-1/-2 mRNA and protein in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells treated with nicotine (10 µm for 24 h), phosphorylation of Akt, glycogen synthase kinase‐3β (GSK‐3β) and extracellular signal‐regulated kinase (ERK)1/2 induced by insulin (100 nm for 10 min) was enhanced by ∼ 62%, without altering levels of these protein kinases. Nicotine produced time (> 12 h)‐ and concentration (EC50 3.6 and 13 µm)‐dependent increases in insulin receptor substrate (IRS)‐1 and IRS‐2 levels by ∼ 125 and 105%, without altering cell surface density of insulin receptors. In these cells, insulin‐induced tyrosine phosphorylation of IRS‐1/IRS‐2 and recruitment of phosphoinositide 3‐kinase (PI3K) to IRS‐1/IRS‐2 were augmented by ∼ 63%. The increase in IRS‐1/IRS‐2 levels induced by nicotine was prevented by nicotinic acetylcholine receptor (nAChR) antagonists, the Ca2+ chelator 1,2‐bis(2‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetra‐acetic acid tetrakis‐acetoxymethyl ester, cycloheximide or actinomycin D. Nicotine increased IRS‐1 and IRS‐2 mRNA levels by ∼ 57 and ∼ 50%, and this was prevented by conventional protein kinase C (cPKC) inhibitor Gö6976, or ERK kinase inhibitors PD98059 and U0126. Nicotine phosphorylated cPKC‐α, thereby increasing phosphorylation of ERK1/ERK2, as demonstrated by using Gö6976, PD98059 or U0126. Selective activation of cPKC‐α by thymeleatoxin mimicked these effects of nicotine. Thus, stimulation of nAChRs up‐regulated expression of IRS‐1/IRS‐2 via Ca2+‐dependent sequential activation of cPKC‐α and ERK, and enhanced insulin‐induced PI3K/Akt/GSK‐3β and ERK signaling pathways.