Water-soluble jack-knife prawn extract inhibits 5-hydroxytryptamine-induced vasoconstriction and platelet aggregation in humans.

Coronary artery spasm plays an important role in the pathogenesis of various ischemic heart diseases or serious arrhythmia. The aim of this study is to look for functional foods which have physiologically active substances preventing 5-hydroxytryptamine (5-HT)-related vasospastic diseases including peri- and postoperative ischemic complications of coronary artery bypass grafting (CABG) from ocean resources in Japanese coastal waters. First, we evaluated the effect of water-soluble ocean resource extracts on the response to 5-HT in HEK293 cells which have forcibly expressed cyan fluorescent protein-fused 5-HT2A receptors (5-HT2A-CFP). Among 5 different water-soluble extracts of ocean resources, the crude water-soluble jack-knife prawn extract (WJPE) significantly reduced maximal Ca(2+) influx induced by 0.1 µM 5-HT in a concentration-dependent manner. The Crude WJPE significantly inhibited, in a concentration-dependent manner, 5-HT-induced constriction of human saphenous vein. 5-HT released from activated platelets plays a crucial roles in the constriction of coronary artery. Next the WJPE was purified for applying the experiment of 5-HT-induced human platelet aggregation. The purified WJPE significantly inhibited 5-HT-induced human platelet aggregation also in a concentration-dependent manner. Based on our findings, jack-knife prawn could be one of a functional food with health-promoting benefits for most people with vasospastic diseases including patients who have gone CABG.

Endothelin-1-induced down-regulation of NaV1.7 expression in adrenal chromaffin cells: attenuation of catecholamine secretion and tau dephosphorylation.

Endothelin-1 and voltage-dependent sodium channels are involved in control and suppression of neuropathological factors, which contribute to sculpting the neuronal network. We previously demonstrated that veratridine-induced NaV1.7 sodium channel activation caused intracellular calcium elevation, catecholamine secretion and tau dephosphorylation in adrenal chromaffin cells. The aim of this study was to examine whether endothelin-1 could modulate NaV1.7. Our results indicated that endothelin-1 decreased the protein level of NaV1.7 and the veratridine-induced increase in intracellular calcium. In addition, it also abolished the veratridine-induced dephosphorylation of tau and the phosphorylation of glycogen synthase kinase-3ß and extracellular signal-regulated kinase. These findings suggest that the endothelin-1-induced down-regulation of NaV1.7 diminishes NaV1.7-related catecholamine secretion and dephosphorylation of tau.

Insulin induces internalization of the plasma membrane 5-hydroxytryptamine2A (5-HT2A) receptor in the isolated human endothelium-denuded saphenous vein via the phosphatidylinositol 3-kinase pathway.

The aim of this study was to investigate the relaxant effect of insulin on the 5-hydroxytryptamine (5-HT)-induced constriction of the human endothelium-denuded saphenous vein (SV) and its signal transduction pathway. During the 5-HT-induced sustained constriction of vessels, insulin induced vasorelaxation in a concentration-dependent manner. This insulin-induced vasorelaxation was partially attenuated by L-NAME, a nitric oxide synthase (NOS) inhibitor, and was abolished by wortmannin, a phosphatidylinositol 3-kinase (PI3-K) inhibitor. Insulin increased the Ser(473) phosphorylation of Akt. Endothelial NOS and inducible NOS protein expressions were observed in SV smooth muscle when insulin induced relaxation of SV vessels preconstricted with 5-HT. Although insulin did not affect the total protein level of 5-HT(2A) receptors, it decreased the particulate protein level and reciprocally increased the soluble protein level of 5-HT(2A) receptors in a concentration-dependent manner. These results demonstrate that insulin can induce the internalization of 5-HT(2A) receptors from the plasma membrane to the cytoplasm. The insulin-induced internalization of 5-HT(2A) receptors was abolished by wortmannin but was not affected by L-NAME. These results suggest that the relaxant effect of insulin on 5-HT-induced vasoconstriction is mediated in part by the internalization of plasma membrane 5-HT(2A) receptors and the production of nitric oxide via the PI3-K/Akt pathway.

The defective protein level of myosin light chain phosphatase (MLCP) in the isolated saphenous vein, as a vascular conduit in coronary artery bypass grafting (CABG), harvested from patients with diabetes mellitus (DM).

We examined the contractile reactivity to 5-hydroxytryptamine (5-HT) in isolated human saphenous vein (SV), as a vascular conduit in coronary artery bypass grafting (CABG), harvested from patients with diabetes mellitus (DM) and non-DM (NDM). Vascular rings of endothelium-denuded SV were used for functional and biochemical experiments. The vasoconstrictions caused by 5-HT were significantly greater (hyperreactivity) in the DM group than in the NDM group. RhoA/ROCK pathway is activated by various G-protein-coupled receptor agonists and consequently induces phosphorylation of myosin phosphatase target subunit 1 (MYPT1), a subunit of myosin light chain phosphatase (MLCP), which inhibits MLCP activity. In the resting state of the vessels, total tissue protein levels of 5-HT(2A) receptor, 5-HT(1B) receptor, RhoA, ROCK1, and ROCK2 did not differ between NDM and DM groups. However, the total protein level of MYPT1 was significantly lower in the DM group than in the NDM group. Furthermore, the ratio of P(Thr(696))-MYPT1 to total MYPT1 was significantly higher in the DM group than in the NDM group. These results suggest that the hyperreactivity to 5-HT in the SV smooth muscle of patients with DM is due to not only enhanced phosphorylation of MLCP but also defective protein level of MLCP. Thus, we reveal for the first time that the defective protein level of MLCP in the DM group can partially explain the poor patency of SV graft harvested from patients with DM.

Insulin-induced neurite-like process outgrowth: acceleration of tau protein synthesis via a phosphoinositide 3-kinase~mammalian target of rapamycin pathway.

Both insulin and tau, promoting neuronal differentiation (neurite outgrowth, neuronal polarity, and myelination) and cell survival, are associated with neurodegenerative disease (e.g., Alzheimer's disease). The aim of this study was to explore relation between insulin-induced activation of insulin signal and expression of tau protein on neurite-like process outgrowth in adrenal chromaffin cells. Primary cultured bovine adrenal chromaffin cells were incubated with insulin to determine whether stimulant of insulin signal could affect tau expression and neurite-like process outgrowth. Chronic treatment with insulin (⩾6h) led neurite-like process outgrowth as well as increased tau protein level by ∼99% in a concentration (EC(50) 5.5nM)- and time-dependent manner, without changing Ser(396)-phosphorylated tau level. The insulin-induced increase of tau protein level was abolished by LY294002 [an inhibitor of phosphoinositide 3-kinase (PI3K)] and rapamycin [an inhibitor of mammalian target of rapamycin (mTOR)], but not by PD98059 and U0126 [two inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK)]. Additionally, insulin-induced increase of tau was blocked by cyclohexamide (an inhibitor of protein synthesis), but not by actinomycin D (an inhibitor of gene transcription). Pulse-label followed by polyacrylamide gel electrophoresis revealed that insulin accelerated tau protein synthesis rate (t(1/2)) from 2.6 to 1.9h. Insulin did not change tau mRNA level. Taken together, these results suggest that insulin-induced activation of PI3K∼mTOR pathway up-regulated tau protein via acceleration of protein synthesis, on which insulin promoted neurite-like process outgrowth.

Dexmedetomidine and clonidine inhibit the function of Na(v)1.7 independent of α(2)-adrenoceptor in adrenal chromaffin cells.

PURPOSE: Besides being administered systemically for sedation and analgesia, α(2)-agonists such as dexmedetomidine and clonidine have been administered with intrathecal, epidural, or perineural injections, leading to an antinociceptive effect at the spinal cord or peripheral nerve level. However, the mechanism for this remains unclear. In the present study, we examined whether dexmedetomidine and clonidine could inhibit the function of tetrodotoxin-sensitive Na(+) channels, which play important roles in the generation of pain. METHODS: Cultured bovine adrenal chromaffin cells expressing the tetrodotoxin-sensitive Na(v)1.7 Na(+) channel isoform were incubated in KRP buffer containing 2 µCi (22)NaCl for 5 min without or with dexmedetomidine or clonidine in the absence or presence of veratridine, α-scorpion venom, ß-scorpion venom, Ptychodiscus brevis toxin-3 or ouabain. Cells were then washed and counted radioactively. RESULTS: Dexmedetomidine and clonidine reduced veratridine-induced (22)Na(+) influx via Na(v)1.7 in a concentration-dependent manner (EC(50) = 50 µM and 530 µM), even in the presence of ouabain, an inhibitor of Na(+), K(+)-ATPase. Dexmedetomidine and clonidine shifted the concentration-response curve of veratridine for (22)Na(+) influx downward without altering the EC(50) of veratridine. Atipamezole and yohimbine, α(2)-antagonists, did not prevent the inhibition of veratridine-induced (22)Na(+) influx by dexmedetomidine. Dexmedetomidine and clonidine combined with lidocaine induced more inhibition of veratridine-induced (22)Na(+) influx than each drug did individually. Atipamezole and yohimbine did not prevent the lidocaine-enhancing effect of dexmedetomidine and clonidine. CONCLUSION: Dexmedetomidine and clonidine inhibit the function of Na(v)1.7 independent of α(2)-adrenoceptor. These results may lead to a deeper understanding of the peripheral antinociceptive effects of α (2)-agonists.

Relative contributions of 5-hydroxytryptamine (5-HT) receptor subtypes in 5-HT-induced vasoconstriction of the distended human saphenous vein as a coronary artery bypass graft.

It is established that the segment of saphenous vein (SV) that is widely used as a conduit vessel in coronary artery bypass graft (CABG) surgery is distended with high pressure to check for leaks and to increase the patency before implantation into coronary arterial circulation. The aim of the present study was to elucidate the relative contributions of 5-hydroxytryptamine (5-HT) receptor subtypes responsible for 5-HT-induced vasoconstriction of the distended human SV. Whereas about half of the 5-HT-induced vasoconstriction still remained in the presence of supramaximum concentration of sarpogrelate or of SB224289 (5-HT(2A) and 5-HT(1B) receptor antagonists, respectively), simultaneous treatment with sarpogrelate and SB224289 almost completely inhibited the 5-HT-induced vasoconstriction. Immunopositive staining for 5-HT(2A) and 5-HT(1B) receptors was detected in smooth muscle cells of the distended human SV and there was no significant difference between the immunopositive areas of 5-HT(2A) and 5-HT(1B) receptors. These results demonstrate that 5-HT(2A) and 5-HT(1B) receptors similarly contribute to 5-HT-induced vasoconstriction in human distended SV. Thus, when the SV is used as a CABG conduit, a combination of 5-HT(2A) and 5-HT(1B) receptor antagonists would appear to be most useful to prevent 5-HT-induced spasm.

Nav1.7-Ca2+ influx-induced increased phosphorylations of extracellular signal-regulated kinase (ERK) and p38 attenuate tau phosphorylation via glycogen synthase kinase-3beta: priming of Nav1.7 gating by ERK and p38.

In cultured bovine adrenal chromaffin cells expressing Nav1.7 sodium channel isoform, we previously showed that veratridine-induced Na+ influx via Nav1.7 and the subsequent Ca2+ influx via voltage-dependent calcium channels activated protein kinase C-alpha and Akt, which converged on increasing inhibitory Ser9-phosphorylation of glycogen synthase kinase-3beta, decreasing constitutive Ser396-phosphorylation of tau. Here, veratridine increased constitutive Tyr204-phosphorylation of extracellular signal-regulated kinase-1/-2 (ERK1/ERK2) and constitutive Thr180/Tyr182-dual phosphorylation of p38 by approximately 118% (EC50=2.8 microM). Veratridine-induced increased phosphorylation levels of ERK1/ERK2 and p38 were abolished by tetrodotoxin, extracellular Ca2+ removal, or Gö6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole;Go6976] (protein kinase C-alpha inhibitor). PD98059 (2'-amino-3'-methoxyflavone) (ERK1/ERK2 inhibitor) or SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] (p38 inhibitor) attenuated veratridine-induced increased phosphorylation of glycogen synthase kinase-3beta and decreased phosphorylation of tau by approximately 54% and approximately 56%, as partial blockade by Gö6976. Additionally, basal constitutive phosphorylation levels of ERK1/ERK2 and p38 were decreased by PD98059 or SB203580, but not by SB216763 [3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indolo-3-yl)-1H-pyrrole-2,5-dione] (glycogen synthase kinase-3beta inhibitor) or extracellular Ca2+ removal. In this condition, PD98059 or SB203580 (but not SB216763 or extracellular Ca2+ removal) inhibited veratridine-induced 22Na+ influx and 45Ca2+ influx, without changing nicotine-induced 22Na+ influx via nicotinic receptor-associated cation channels and nicotine-induced 45Ca2+ influx via voltage-dependent calcium channels. These results suggest that Nav1.7-Ca2+ influx-protein kinase C-alpha pathway activated ERK1/ERK2 and p38, which increased phosphorylation of glycogen synthase kinase-3beta, decreasing tau phosphorylation. In veratridine-nontreated cells, basal constitutive activities of ERK1/ERK2 and p38 primed Nav1.7 to increase 22Na+ influx.

Homologous posttranscriptional regulation of insulin-like growth factor-I receptor level via glycogen synthase kinase-3beta and mammalian target of rapamycin in adrenal chromaffin cells: effect on tau phosphorylation.

In cultured bovine adrenal chromaffin cells, approximately 24 h-treatment with insulin-like growth factor-I (IGF-I) decreased cell surface (125)I-IGF-I binding capacity and IGF-I receptor protein level by approximately 64% (EC(50) = 5.0 nM; t(1/2) = approximately 7 h). IGF-I-induced IGF-I receptor decrease was abolished by LY294002 (phosphoinositide 3-kinase inhibitor) and partially attenuated by rapamycin (an inhibitor of mammalian target of rapamycin [mTOR]). SB216763 (an inhibitor of glycogen synthase kinase-3 [GSK-3]) down-regulated IGF-I receptor, which was further decreased by IGF-I. IGF-I increased inhibitory Ser(9)-phosphorylation of GSK-3beta and stimulatory Ser(2448)-phosphorylation of mTOR. l-leucine increased phosphorylation of mTOR (but not GSK-3beta), and down-regulated IGF-I receptor, both events being abolished by rapamycin. IGF-I-induced IGF-I receptor decrease was not prevented by proteolysis inhibitors. Pulse-label with [(35)S]methionine/cysteine followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that SB216763 or L-leucine retarded synthesis of IGF-I receptor and its precursor molecule. SB216763 (but not l-leucine) destabilized IGF-I receptor mRNA and decreased its level, without changing IGF-I receptor gene transcription. In SB216763-treated cells, IGF-I-induced Tyr-autophosphorylation of IGF-I receptor was decreased by 36%, compared to nontreated cells. IGF-I attenuated constitutive Ser(396)-phosphorylation of tau by 30% in nontreated cells, but not in SB216763-treated cells. IGF-I-induced down-regulations of (125)I-IGF-I binding and IGF-I receptor, as well as IGF-I-induced phosphorylations of GSK-3beta and mTOR were restored to the control levels of nontreated cells after washout of IGF-I (10 nM for 12 h)-treated cells. Thus, IGF-I down-regulated functional IGF-I receptor via GSK-3beta inhibition and mTOR activation; constitutive activity of GSK-3beta maintained IGF-I receptor level in nonstimulated cells.

Chronic lithium treatment up-regulates cell surface Na(V)1.7 sodium channels via inhibition of glycogen synthase kinase-3 in adrenal chromaffin cells: enhancement of Na(+) influx, Ca(2+) influx and catecholamine secretion after lithium withdrawal.

In cultured bovine adrenal chromaffin cells expressing Na(V)1.7 isoform of voltage-dependent Na(+) channels, we have previously reported that lithium chloride (LiCl) inhibits function of Na(+) channels independent of glycogen synthase kinase-3 (GSK-3) (Yanagita et al., 2007). Here, we further examined the effects of chronic lithium treatment on Na(+) channels. LiCl treatment (1-30 mM, > or = 12 h) increased cell surface [(3)H]saxitoxin ([(3)H]STX) binding by approximately 32% without altering the affinity of [(3)H]STX binding. This increase was prevented by cycloheximide and actinomycin D. SB216763 and SB415286 (GSK-3 inhibitors) also increased cell surface [(3)H]STX binding by approximately 31%. Simultaneous treatment with LiCl and SB216763 or SB415286 did not produce an increased effect on [(3)H]STX binding compared with either treatment alone. LiCl increased Na(+) channel alpha-subunit mRNA level by 32% at 24 h. LiCl accelerated alpha-subunit gene transcription by 35% without altering alpha-subunit mRNA stability. In LiCl-treated cells, LiCl inhibited veratridine-induced (22)Na(+) influx as in untreated cells. However, washout of LiCl after chronic treatment enhanced veratridine-induced (22)Na(+) influx, (45)Ca(2+) influx and catecholamine secretion by approximately 30%. Washout of LiCl after 24 h treatment shifted concentration-response curve of veratridine upon (22)Na(+) influx upward, without altering its EC(50) value. Ptychodiscus brevis toxin-3 allosterically enhanced veratridine-induced (22)Na(+) influx by two-fold in untreated and LiCl-treated cells. Whole-cell patch-clamp analysis indicated that I-V curve and steady-state inactivation/activation curves were comparable between untreated and LiCl-treated cells. Thus, GSK-3 inhibition by LiCl up-regulated cell surface Na(V)1.7 via acceleration of alpha-subunit gene transcription, enhancing veratridine-induced Na(+) influx, Ca(2+) influx and catecholamine secretion.

Nav1.7 sodium channel-induced Ca2+ influx decreases tau phosphorylation via glycogen synthase kinase-3beta in adrenal chromaffin cells.

In cultured bovine adrenal chromaffin cells expressing Na(v)1.7 sodium channel isoform, veratridine increased Ser(473)-phosphorylation of Akt and Ser(9)-phosphorylation of glycogen synthase kinase-3beta by approximately 217 and approximately 195%, while decreasing Ser(396)-phosphorylation of tau by approximately 36% in a concentration (EC(50)=2.1 microM)- and time (t(1/2)=2.7 min)-dependent manner. These effects of veratridine were abolished by tetrodotoxin or extracellular Ca(2+) removal. Veratridine (10 microM for 5 min) increased translocation of Ca(2+)-dependent conventional protein kinase C-alpha from cytoplasm to membranes by 47%; it was abolished by tetrodotoxin, extracellular Ca(2+) removal, or Gö6976 (an inhibitor of protein kinase C-alpha), and partially attenuated by LY294002 (an inhibitor of phosphatidylinositol 3-kinase). LY294002 (but not Gö6976) abrogated veratridine-induced Akt phosphorylation. In contrast, either LY294002 or Gö6976 alone attenuated veratridine-induced glycogen synthase kinase-3beta phosphorylation by 65 or 42%; however, LY294002 plus Gö6976 completely blocked it. Veratridine (10 microM for 5 min)-induced decrease of tau phosphorylation was partially attenuated by LY294002 or Gö6976, but completely blocked by LY294002 plus Gö6976; okadaic acid or cyclosporin A (inhibitors of protein phosphatases 1, 2A, and 2B) failed to alter tau phosphorylation. These results suggest that Na(+) influx via Na(v)1.7 sodium channel and the subsequent Ca(2+) influx via voltage-dependent calcium channel activated (1) Ca(2+)/protein kinase C-alpha pathway, as well as (2) Ca(2+)/phosphatidylinositol 3-kinase/Akt and (3) Ca(2+)/phosphatidylinositol 3-kinase/protein kinase C-alpha pathways; these parallel pathways converged on inhibitory phosphorylation of glycogen synthase kinase-3beta, decreasing tau phosphorylation.

Drug development targeting the glycogen synthase kinase-3beta (GSK-3beta)-mediated signal transduction pathway: the role of GSK-3beta in the maintenance of steady-state levels of insulin receptor signaling molecules and Na(v)1.7 sodium channel in adrenal chromaffin cells.

Glycogen synthase kinase-3 (GSK-3) is constitutively active in nonstimulated cells, where the majority of its substrates undergo inactivation/proteolysis by phosphorylation. Extracellular stimuli (e.g., insulin) catalyze inhibitory Ser(9)-phosphorylation of GSK-3beta, turning on signaling and causing other biological consequences otherwise constitutively suppressed by GSK-3beta. Regulated and dysregulated activities of GSK-3beta are pivotal to health, disease, and therapeutics (e.g., insulin resistance, neurodegeneration, tumorigenesis, inflammation); however, the underlying mechanisms of multifunctional GSK-3beta remain elusive. In cultured bovine adrenal chromaffin cells, 1) constitutive and negatively-regulated activities of GSK-3beta up- and down-regulated insulin receptor, insulin receptor substrate-1 (IRS-1), IRS-2, and Akt levels via controlling proteasomal degradation and protein synthesis; 2) nicotinic receptor/protein kinase C-alpha (PKC-alpha)/extracellular signal-regulated kinase (ERK) pathway up-regulated IRS-1 and IRS-2 levels, enhancing insulin-induced the phosphoinositide 3-kinase (PI3K)/Akt/GSK-3beta pathway; 3) inhibition of calcineurin by cyclosporin A or FK506 down-regulated IRS-2 level, attenuating insulin-like growth factor-I (IGF-I)-induced ERK and GSK-3beta pathways; and 4) insulin, IGF-I or therapeutics (e.g., lithium) up-regulated the voltage-dependent Na(v)1.7 sodium channel.

Regulation of Akt mRNA and protein levels by glycogen synthase kinase-3beta in adrenal chromaffin cells: effects of LiCl and SB216763.

In cultured bovine adrenal chromaffin cells, where Akt1 is the predominant isoform over Akt2 and Akt3, chronic (> or =12 h) treatment with 1-20 mM LiCl, an inhibitor of glycogen synthase kinase-3, decreased Akt1 level by approximately 52% (EC50=3.7 mM; t1/2=l2 h); it was associated with LiCl-induced increased levels of Ser9-phosphorylated glycogen synthase kinase-3beta (approximately 37%) and beta-catenin (approximately 59%), two hallmarks of glycogen synthase kinase-3beta inhibition. The same LiCl treatment did not change phosphoinositide 3-kinase, phosphoinositide-dependent kinase 1, and extracellular signal-regulated kinase-1/2 levels. Treatment with SB216763 [3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione], a selective inhibitor of glycogen synthase kinase-3, lowered Akt1 level by approximately 67% (EC50=2 microM; t1/2=l2 h), when SB216763 caused concentration- and time-dependent increase of beta-catenin level by approximately 76%. LiCl- or SB216763-induced Akt1 decrease, as well as increases of Ser9-phosphorylated glycogen synthase kinase-3beta and beta-catenin were restored to the control levels of nontreated cells after the washout of LiCl (20 mM for 24 h)- or SB216763 (30 microM for 24 h)-treated cells. LiCl-induced Akt1 reduction was not prevented by beta-lactone, lactacystin (two inhibitors of proteasome), calpastatin (an inhibitor of calpain), or leupeptin (an inhibitor of lysosome). LiCl decreased Akt1 mRNA level by 20% at 6 h, with no effect on Akt1 mRNA stability. These results suggest that glycogen synthase kinase-3beta inhibition caused down-regulation of Akt1 mRNA and Akt1 protein levels; conversely, constitutive activity of glycogen synthase kinase-3beta maintains steady-state level of Akt1 in quiescent adrenal chromaffin cells.