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COVID-19, which originated in Wuhan, China, 2 years ago, became a pandemic, that continues to this day. The Journal of Disaster Research (JDR) edited a special issue, “Special Issue on COVID-19 and Historical Pandemics” at the end of 2020 and an electronic version of the issue was published in January 2021 (JDR Vol.16, No.1, pp. 1-117). Even though one year has passed, life has not yet returned to normal due to the continued spread of COVID-19. Although the percentage of the general public that has been vaccinated continues to increase and various drugs and treatments have been developed, the number of COVID-19 cases also continues to increase. A huge number of COVID-19 cases have been reported, especially in Europe and the Americas. Accordingly, we planned to publish Special Issue Part 2 and called for papers. This Special Issue Part 2 includes 18 manuscripts, which deal with general epidemiology, effects on socioeconomic or educational areas, and clinical medicine, including vaccines and medical treatments. The virus spike protein plays an important role in its intrusion into human cells and the onset of COVID-19 infection. Although SARS-CoV-2 (the pathogenic virus of COVID-19) originated in Wuhan, China, various variant strains with mutation in gene coding in the spike protein have appeared in multiple countries. The WHO classifies these variants into the categories of VOC (variant of concern), VOI (variant of interest), or VUM (variant under monitoring). The definitions of VOC and VOI are as follows. VOC: “A SARS-CoV-2 variant that meets the definition of a VOI and, through a comparative assessment, has been demonstrated to be associated with one or more changes in the degree of global public health significance.” VOI: “Increase in transmissibility or detrimental change in COVID-19 epidemiology, increase in virulence or change in clinical disease presentation, or decrease in effectiveness of public health and social measures or available diagnostics, vaccines and therapeutics.” The WHO uses Greek letters designating the variants. Accordingly as they have appeared, variants have been designated as alpha (α: the UK variant), beta (β: the South Africa variant), and delta (δ: the Indian variant). After the deadline for the manuscript of this special issue, a new variant, omicron (o) originated in South Africa and Botswana, and it immediately began spreading worldwide. Unfortunately, it was impossible to include the manuscript on the omicron variant in this Special Issue Part 2, as the deadline had already passed. As COVID-19 continues to spread, the JDR will plan to edit Part 3 of this COVID-19 special issue.

Pectenotoxin-2 (PCTX-2) is one of the polyether macrolide toxins isolated from scallops involved in diarrheic shellfish poisoning via actin depolymerization. In the present study, we examined the bioactive mechanism of PCTX-2 in smooth muscle cells and clarify mode of action of the PCTX-2-induced actin depolymerization using purified skeletal actin. PCTX-2 (300 nM-3 µM) non-selectively inhibited vascular smooth muscle contractions elicited by high K+ or phenylephrine in a dose-dependent manner. However, elevated cytosolic Ca2+ and myosin light chain phosphorylation stimulated by high K+ were only slightly inhibited by PCTX-2. By monitoring the fluorescent intensity of pyrenyl-actin, PCTX-2 was found to inhibit both the velocity and degree of actin polymerization. The critical concentration of G-actin was linearly increased in accordance with the concentration of PCTX-2, indicating sequestration of G-actin with 1 to 1 ratio. The kinetics of F-actin depolymerization by dilution assay indicated that PCTX-2 does not sever F-actin. Transmission electron microscopic and confocal microscopic observations demonstrated that PCTX-2 selectively depolymerized filamentous actin without affecting tublin. In conclusion, PCTX-2 is a potent natural actin depolymerizer which sequesters G-actin without severing F-actin.

COVID-2019 was first identified in Wuhan, China, at the end of 2019, and from there it spread worldwide. Due to this worldwide distribution of COVID-19 cases, the WHO declared a COVID-19 pandemic. The pathogen of COVID-19, a novel corona virus, resembles SARS-CoV, the pathogen of SARS (severe acute respiratory syndrome, a pandemic in 2003), so the International Committee on Taxonomy Virology named it SARS-CoV-2. However, COVID-19 is a different disease from SARS, and should be controlled to the extent possible with the effective vaccines and therapeutic medicines. Although one year has passed since the first appearance of the disease, the number of COVID-19 cases continues to increase, and the pandemic is now in its third large wave. It is thought that it will be difficult to eradicate the disease completely, because SARS-CoV-2 is possible to invade and live in various host animals in addition to humans. During this pandemic, the JDR has put together the special issue “COVID-19 and Historical Pandemics.” Because there have been many pandemics that have transformed society in various ways, the special issue includes historical pandemics in addition to COVID-19. The manuscripts in this issue include various subjects related to COVID-19, including methods of analyzing the pandemic, suggestions for countermeasures against it, methods of prevention and epidemiological reviews, among others. The WHO has released a large volume of pandemic information on an ongoing basis, including its “COVID-19 Weekly Epidemiological Update.” In the weekly edition of December 27, it reported the cumulative number of cases and percentages of the global total from around the world: the Americas 34,403,371 (43%), Europe 25,271 (31%), Africa 1,831,227 (2%), and the Western Pacific 1.059,751 (1%). It is notable that 74% of cumulative global total number of cases have been reported in the Americas and Europe, where most developed countries are included, whereas the numbers in Africa, which includes many developing countries, and the Western Pacific region, which includes Thailand, the Philippines, Vietnam, and Malaysia in addition to Japan and China, are 2% and 1%, respectively. This indicates that the COVID-19 pandemic is a different from previous epidemics caused by other infectious diseases. As the pandemic spreads, the JDR plans to edit Part 2 of this COVID-19 special issue.

In 2011, the radionuclide released from the nuclear power plant in Fukushima contaminated 3.4% of agricultural and marine products to a level exceeding the standard. After measures were introduced to inhibit radionuclide absorption and to decontaminate farmland, contamination exceeding regulatory limits fell to 0.002% in 2014. The 0.002% of contamination remaining involves fungi and wild edible plants from forested regions and some fish caught in rivers where decontamination has not been done. Restrictions were placed on shipments of food exceeding regulatory limits, so such food was never distributed. The allowable dose, which is the basis of regulatory limits of radionuclide concentrations in food in Japan is 1 mSv per year based on the guideline set by the Codex Alimentarius Commission which is responsible for establishing international standards for food. The issue of radioactive food contamination was thus resolved in a short time. According to a survey conducted 2014, the estimated radiation dose a person would consume over 1 year from radioactive cesium contained in food distributed for consumption ranged from 0.0007 to 0.0022 mSv a year. This amounts to less than 1% of the yearly permissible dose of 1 mSv.

The first BSE case in Japan was found in 2001. The BSE risk in Japan was small and the measures taken by the government successfully prevented the spread of BSE. However, because consumers did not have accurate information, they did not trust the government and refused to consume beef. Based on the lessons learned, the government enacted the Food Safety Basic Act in 2003, and risk communication in the food field was started. In 2003, the first BSE case was found in the U.S. that were supplying nearly one third of the beef consumed in Japan, and the government banned beef import from the U.S. The BSE risk in the U.S. was also small and it was possible to resume imports of beef after the appropriate measures. Despite the government efforts of risk communication, consumers rejected the resumption of imports. In 2011, food was contaminated with radioactive substances discharged from the Fukushima Daiichi nuclear power plant. Although government eliminated the contaminated food from the market, some consumers rejected all of the agricultural products of the Fukushima region, again a failure of risk communication. Here, the current situation and problems of the risk communication in Japan will be described.

In 1986, after bovine spongiform encephalopathy (BSE) was discovered in the United Kingdom, over 183,000 cases have been confirmed to date. In 1996, the UK government announced that BSE may be transmitted to humans, causing variant Creutzfeldt-Jakob disease (vCJD). Retrospectively, the measures taken by the UK government were appropriate and the number of new cases of both BSE and vCJD decreased. Because of the long incubation period between infection and the appearance of clinical symptoms in both BSE and vCJD, a long time was needed to determine the effects of these measures. The inappropriate risk communication, however, caused people to lose trust in the UK government, and fear spread. In Japan, a cow infected with BSE was found in 2001. Although no cattle showing BSE symptoms were found and the risk of BSE infection was low, fear again spread due to inadequate risk communication. To allay consumer anxieties, the government began testing all cattle at slaughter facilities. This, in turn, generated the "blanket testing myth" - the misunderstanding that BSE testing was the most important measure needed to maintain the safety of beef consumption.

知識人の趣味であった科学が，19世紀以後，社会の問題解決という目的を持つようになり，研究者の数が増えて科学者集団が生まれ，集団内の成功が社会における成功につながった．これは必然的に科学の不正につながり，その社会的影響が無視できなくなった．これに加えて，国立大学の法人化と産学連携の推進により，これまでは主に企業研究者の問題であった利益相反も多くの研究者の問題になりつつある．不正の発見はピアレビューと追試による検証で行われるが，ピアレビューは性善説に準拠するために，意図的な不正を見抜く力はない．また検証はその結果が出るまでには長い時間がかかり，その間に他の研究者や社会が損失を被ることがある．内部告発も有効ではあるが，慎重な取り扱いが必要である．科学の不正は，一般社会における不正と同じく，いつでも起こりうる．不正を防止するためには科学者集団が科学の品質を保証するシステムを構築するとともに，研究者が功利主義ではなく義務論に基づいて行動し，一般社会に通用する透明性と説明責任を果たすことが何より重要であり，そのために一般道徳に加えて科学者倫理の教育を強化することが必要である．

Objective— We investigated the effects of fluvastatin on hypoxia-induced (1 to 3 weeks, 10% O 2 ) pulmonary hypertension with focus on endothelial nitric oxide synthase (eNOS) activity. Methods and Results— Oral fluvastatin treatment (1 mg/kg daily) prevented the causing and progression of pulmonary hypertension as determined by the right ventricular pressure, right ventricular hypertrophy, and muscularization of pulmonary artery. We also revealed that fluvastatin treatments prevented the hypoxia-induced decrease in cGMP production in the rat lung and restored the endothelium-dependent relaxation in the pulmonary artery. We revealed that this beneficial effect was not dependent on the increase in eNOS mRNA or protein expression, but was dependent on the inhibition of the eNOS-tight coupling with caveolin-1, the eNOS dissociation from heat shock protein 90, and the decrease in eNOS Ser 1177 –phosphorylation induced by hypoxia. Furthermore, in a whole-mount immunostaining the hypoxia-induced eNOS protein condensation with caveolin-1 of pulmonary endothelial cells was restored by the fluvastatin-treatment. Conclusion— These results suggest that the fluvastatin exerts beneficial effects on chronic hypoxia-induced pulmonary hypertension by protecting against the eNOS activity at the post-transcriptional level.

Contractions of vascular and other types of smooth muscle are regulated mainly by changes in the concentration of free cytosolic Ca 2+ {[Ca 2+ ] c }. Because abnormal contraction of smooth muscle results in various diseases, including hypertension, stroke and asthma, it is important to identify changes in [Ca 2+ ] c at the subcellular level. In this article, a brief history of the techniques used to estimate [Ca 2+ ] c in smooth muscle is presented, beginning in the 1960s with the use of radioactive 45 Ca 2+ . Difficulties associated with measuring the very small [Ca 2+ ] c required to regulate contraction in the presence of higher levels of Ca 2+ bound to the membrane surface and intracellular organelles resulted, in the early 1970s, in the development of a method to selectively replace membrane-bound 45 Ca 2+ with La 3+ . However, Ca 2+ detected using this method also included a large amount of Ca 2+ accumulated in mitochondria. Eventually, in the late 1980s, only cytosolic free Ca 2+ was detected using fluorescent Ca 2+ indicators. Further improvements in the spatial and temporal resolution of the method of detecting Ca 2+ might help elucidate cellular regulation mechanisms and help develop pharmaceuticals, including vasodilators and anti-asthmatic drugs.

In our previous general screening experiments, we found that NSU-242, a dibenzothiepine derivative (1-10 mg/kg), inhibited antigen-induced immediate asthmatic response in actively sensitized guinea pigs in a dose-dependent manner. The purpose of the present study was to assess the mechanism of the relaxing effect of NSU-242 on smooth muscle contractions in isolated smooth muscle tissues of the porcine trachea and rat aorta. NSU-242 administration resulted in a concentration-dependent inhibition of the tracheal-tissue contractions induced by carbachol and high K(+) and the aortic-tissue contractions induced by norepinephrine and high K(+). The IC(50) values of these inhibitions were 1-10 microM, and there was no selectivity for the type of stimulation. In tracheal tissue, the relaxations were accompanied by neither changes in cAMP nor changes in cGMP. Carbachol (1 microM) and high K(+) (59.2 mM) increased myosin light chain (MLC) phosphorylation in the trachea, and NSU-242 (3-30 microM) had no effect on the level of MLC phosphorylation. Furthermore, NSU-242 (300 microM) had no effect on contractions in membrane-permeabilized tracheal tissue. FITC-phalloidin staining of the actin fiber in cultured vascular smooth muscle cells (A7r5) indicated that NSU-242 (10-100 microM) altered the configuration of actin stress fiber in the cytosol. However, unlike cytochalasin D, NSU-242 did not inhibit actin polymerization as assessed by in vitro assay. These results suggest that NSU-242 inhibits smooth muscle contractions without any effect on the Ca(2+)-dependent MLC phosphorylation. NSU-242 may uncouple the force generated by the activated actomyosin interaction, possibly by modifying the actin assembly in smooth muscle cells without a direct effect on actin molecules.

Urotensin II induced sustained contraction with an EC50 value of 2.29 ± 0.12 nM in rat aorta. Urotensin II (100 nM) transiently increased cytosolic Ca2+ level ([Ca2+]i), followed by a small sustained phase superimposed with rhythmic oscillatory change. In the presence of verapamil and La3+, the [Ca2+]i oscillation was completely inhibited, although a small transient increase in [Ca2+]i remained. The urotensin II-induced contraction was also partially inhibited by verapamil and La3+. Combined application of verapamil, La3+, and thapsigargin completely inhibited the increase in [Ca2+]i with only partial inhibition of the contraction elicited by urotensin II. Urotensin II increased myosin light chain (MLC) phosphorylation to a level greater than that induced by 72.7 mM KCl (high K+). Pretreatment with Go6983 (PKC inhibitor), U0126 (MEK inhibitor), or SB203580 (p38MARK inhibitor) partially inhibited the urotensin II-induced contraction with no effects on the high K+-induced contractions. Wortmannin (MLC kinase inhibitor) only partially inhibited urotensin II-induced contraction, although it completely inhibited the high K+-induced contraction. These results suggest that urotensin II-induced contraction is mediated by the Ca2+/calmodulin/MLC kinase system and modulated by the Ca2+ sensitization mechanisms to increase MLC phosphorylation. In addition, activations of PKC, p38MAPK, and ERK1/2 modulate the contractility mediated by urotensin II in rat aorta. Keywords:: urotensin II, rat aorta, Ca2+, myosin phosphorylation, Ca2+ sensitization

Interleukin-1beta (IL-1beta) is a proinflammatory cytokine that plays a central role in inflammatory bowel disease (IBD). In order to elucidate the mechanism of motility disorders frequently observed in IBD, we investigated the long term effects of IL-1beta on rat ileal smooth muscle contractility by using an organ culture system. When ileal smooth muscle strips were cultured with IL-1beta (10 ng/ml), contractions elicited by high K+ and carbachol were inhibited in a time-dependent manner. IL-1beta more strongly inhibited the carbachol-induced contractions than high K+ with decreasing myosin light chain phosphorylation. In the alpha-toxin-permeabilized ileal muscle, carbachol with GTP or guanosine 5'-3-O-(thio)triphosphate increased the Ca2+ sensitivity of contractile elements, and this G protein-coupled Ca2+ sensitization was significantly reduced in the IL-1beta-treated ileum. Among the functional proteins involved in the smooth muscle Ca2+ sensitization, CPI-17 expression was significantly reduced after the culture with IL-1beta, whereas the expressions of RhoA, ROCK-I, ROCK-II, MYPT-1, myosin light chain kinase, and myosin phosphatase (PP1) were unchanged. The phosphorylation level of CPI-17 by carbachol was low in accordance with the decrease in CPI-17 expression due to IL-1beta treatment. In contrast, constitutively phosphorylated MYPT-1 was also decreased in the IL-1beta-treated muscles. These results suggest that long term treatment with IL-1beta decreases either CPI-17 expression or MYPT-1 phosphorylation, which may result in an increase in myosin phosphatase activity to reduce force generation. Based on these findings, we consider IL-1beta to be an important mediator of gastrointestinal motility disorders in IBD, and CPI-17 and MYPT-1 are key molecules in the decreased smooth muscle contractility due to IL-1beta.

Activation of protein kinase C (PKC) by phorbol 12,13-dibutylate (PDBu, 1 μM) induced sustained contractions with no increase in [Ca2+]i in nonpregnant and pregnant human myometria. The contractile effects of PDBu in pregnant myometrium were much greater than those in nonpregnant myometrium, and the contractions in pregnant myometrium were accompanied by an increase in myosin light chain (MLC) phosphorylation at Ser19. The contraction induced by PDBu in pregnant myometrium was inhibited by the inhibitors of conventional PKC isoforms, bisindolylmaleimides and indolocarbazole, such as Go6976, Go6983, and Go6850 (1 μM). LY333531 (1 μM), a specific inhibitor of PKCβ, also inhibited the PDBu-induced contraction in the pregnant myometrium. In the pregnant myometrium permeabilized with α-toxin, PDBu increased the contractions induced at fixed Ca2+ concentration (0.3 μM) both in nonpregnant and pregnant myometria, indicating Ca2+ sensitization of contractile elements. Western immunoblot analysis indicated that pregnant myometrium contained PKC isozymes such as conventional PKC (α, β, γ), novel PKC (δ, ɛ, θ), and atypical PKC (ζ but not ι and λ). RT-PCR and real-time RT-PCR analysis indicated that, among the conventional PKC, the levels of mRNA of β isoform in pregnant human myometrium were greater than those in nonpregnant myometrium. CPI-17 is a substrate for PKC, and the phosphorylated CPI-17 is considered to inhibit myosin phosphatase. The levels of CPI-17 mRNA and protein expression were also greater in the pregnant myometrium. These results suggest that the PKC-mediated contractile mechanism is augmented in human myometrium after gestation, and that this augmentation may be attributable to the increased activity of the β PKC isoform and CPI-17. British Journal of Pharmacology (2003) 140, 1303–1312. doi:10.1038/sj.bjp.0705552

We investigated the mechanisms of dysmotility of the colonic circular muscle of the Crohn's disease rat model. Contractions induced by KCl, carbachol, and Bay K 8644 were decreased in circular smooth muscles isolated from 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat colon. However, the absolute force and Ca2+ sensitivity of contractile proteins were not affected as assessed in α-toxin permeabilized smooth muscle. The current density of the L-type Ca2+ channel in circular smooth muscle cells was significantly decreased in the TNBS-treated colonic cells. However, expressions of the L-type Ca2+ channel mRNA and protein did not differ between control and TNBS-treated preparations. Pretreatment with the NF-κB inhibitors pyrrolidinedithiocarbamate and sulfasalazine partially recovered the decreased contractility and current density of the L-type Ca2+ channel by TNBS treatment. These results suggest that the decrease in the contraction of circular smooth muscle isolated from TNBS-induced colitis rat colon, which may be related to gut dysmotility in Crohn's disease, is attributable to the decreased activity of the L-type Ca2+ channel. The dysfunction of the L-type Ca2+ channel may be mediated by NF-κB-dependent pathways.

In neonatal mouse right ventricles, endothelin-1 (ET-1, 1-300 nM) induced a dose-dependent increase in twitch contractions and the dose-response curve was shifted to the right by BQ-123 (10 microM), an endothelin ET(A) receptor antagonist. The ET-1 (100 nM)-induced positive inotropy was accompanied by an increase in [Ca(2+)](i) transients without any change in the [Ca(2+)](i)-force relationship. Ryanodine (1 microM) partially decreased the [Ca(2+)](i) transients and contractile force, but did not affect the ET-1 (100 nM)-induced positive inotropy. Reduction of [Na(+)](o) elicited an increase in contractile force, and this effect was significantly inhibited by KB-R7943 (30 microM), an inhibitor of the Na(+)-Ca(2+) exchanger. KB-R7943 (30 microM) almost completely suppressed the positive inotropic effect of ET-1. Activation of protein kinase C (PKC) by phorbol 12,13-dibutylate (100 nM) decreased the contractile force, an effect which was suppressed by bisindolylmaleimide I (3 microM). On the other hand, the ET-1-induced positive inotropic effect was unaffected by bisindolylmaleimide I (3 microM). These results suggest that the positive inotropic effect of ET-1 in neonatal mouse right ventricles is caused by the increase in [Ca(2+)](i) transients through activation of the endothelin ET(A) receptor and the increase in Ca(2+) influx via the Na(+)-Ca(2+) exchanger during an action potential. Furthermore, the ET-1-induced positive inotropy is independent of the effects of PKC, which makes it distinct from the ET-1-mediated pathways reported for cardiac tissues in other species.

1 Phorbol ester decreases muscle tension in the rat myometrium, and the effect is more potent in latepregnant myometrium than in nonpregnant myometrium. In the present study, we have examined the contribution of protein kinase C (PKC) isoforms to the phorbol ester-induced inhibition of tension in rat uterine smooth muscle. 2 Thymeleatoxin (THX), a selective activator of conventional-type PKC (cPKC), and 12deoxyphorbol 13-isobutyrate (DPB), an activator of pan PKC, inhibited the tension induced by high K + , and inhibitions were significantly increased in pregnant myometrium compared to nonpregnant myometrium. The inhibition by DPB and THX of high K + -induced tension was significantly attenuated when PKC was downregulated by long-term pretreatment with THX and inhibited by Go6976, a cPKC inhibitor. 3 Of the cPKCs, PKCa is predominantly expressed in the rat myometrium, as detected by Western blot analysis. The expression of PKCa gradually increases from the beginning of gestation, reaching a maximum at day 21 of pregnancy. Treatment with DPB induced PKCa to translocate from the cytosol to the membrane in the pregnant myometrium. PKCe and PKCz, other dominant PKC isoforms in the rat myometrium, decrease during gestation, reaching a minimum in late pregnancy. 4 These results suggest that cPKC may be at least partly involved in the PKC-mediated inhibition of muscle tension in the rat myometrium. British Journal of Pharmacology (2003) 139, 408 – 414. doi:10.1038/sj.bjp.0705237

1 Xestospongin-C isolated from a marine sponge, Xestospongia sp., has recently been shown to be a membrane-permeable IP3 receptor inhibitor. In this study we examined the eAects of this compound on smooth muscle from guinea-pig ileum. 2 In guinea-pig ileum permeabilized with a-toxin, xestospongin-C (3 mM) inhibited contractions induced by Ca 2+ mobilized from sarcoplasmic reticulum (SR) with IP3 or carbachol with GTP, but not with caAeine. 3 In intact smooth muscle tissue, xestospongin-C (3‐10 mM) inhibited carbachol- and high-K + induced increases in [Ca 2+ ]i and contractions at sustained phase. 4 It also inhibited voltage-dependent inward Ba 2+ currents in a concentration-dependent manner with an IC50 of 0.63 mM. Xestospongin-C (3‐10 mM) had no eAect on carbachol-induced inward Ca 2+ currents via non-selective cation channels; but it did reduce voltage-dependent K + currents in a concentration-dependent manner with an IC50 of 0.13 mM. 5 These results suggest that xestospongin-C inhibits the IP3 receptor but not the ryanodine receptor in smooth muscle SR membrane. In intact smooth muscle cells, however, xestospongin-C appears to inhibit voltage-dependent Ca 2+ and K + currents at a concentration range similar to that at which it inhibits the IP3 receptor. Xestospongin-C is a selective blocker of the IP3 receptor in permeabilised

1. We evaluated the role of the cross-linking of Fc epsilon RI-mediated inositol 1,4,5-triphosphate (IP(3)) in the increase in cytosolic Ca(2+) level ([Ca(2+)](i)) using xestospongin C, a selective membrane permeable blocker of IP(3) receptor, in RBL-2H3 mast cells. 2. In the cells sensitized with anti-dinitrophenol (DNP) IgE, DNP-human serum albumin (DNP-HSA) and thapsigargin induced degranulation of beta-hexosaminidase and a sustained increase in [Ca(2+)](i). Xestospongin C (3 - 10 microM) inhibited both of these changes that were induced by DNP-HSA without changing those induced by thapsigargin. 3. In the absence of external Ca(2+), DNP-HSA induced a transient increase in [Ca(2+)](i). Xestospongin C (3 - 10 microM) inhibited this increase in [Ca(2+)](i). 4. In the cells permeabilized with beta-escin, the application of IP(3) decreased Ca(2+) in the endoplasmic reticulum (ER) as evaluated by mag-fura-2. Xestospongin C (3 - 10 microM) inhibited the effect of IP(3). 5. After the depletion of Ca(2+) stores due to stimulation with DNP-HSA or thapsigargin, the addition of Ca(2+) induced capacitative calcium entry (CCE). Xestospongin C (3 - 10 microM) inhibited the DNP-HSA-induced CCE, whereas it did not affect the thapsigargin-induced CCE. 6. These results suggest that Fc epsilon RI-mediated generation of IP(3) contributes to Ca(2+) release not only in the initial phase but also in the sustained phase of the increase in [Ca(2+)](i), resulting in prolonged Ca(2+) depletion in the ER. The ER Ca(2+) depletion may subsequently activate CCE to achieve a continuous [Ca(2+)](i) increase, which is necessary for degranulation in the RBL-2H3 mast cells. Xestospongin C may inhibit Ca(2+) release and consequently may attenuate degranulation.

The benefit of organ culture is to retain the original structural relationship between various cell species and their interactions and enable us to study the long-term effects of exogenous stimuli. Organ culture methods have been used especially in the studies of the proliferative vascular diseases, such as atherosclerosis and restenosis. We describe here that organ culture is a useful in vitro method to study the biology of vascular and other smooth muscle organs.

We investigated the chronic effects of doxorubicin (DXR) on morphological and functional changes in the rabbit mesenteric artery using an organ culture system. In arteries cultured with 0.3 μM DXR for 7 days, the contractions induced by noradrenaline, but not those induced by endothelin-1 or high K+, were strongly inhibited. This reaction was followed by a decrease in the induction of the α1A-adrenoceptor without any change in the mRNA level. Inhibition of noradrenaline-induced contractions by DXR was attenuated by superoxide dismutase, and α1A-adrenoceptor protein expression recovered. In the arteries cultured with 1 μM DXR for 7 days, contractions induced by endothelin-1 or high K+ and absolute force in the permeabilized muscles were also inhibited. Morphological examinations revealed the existence of concentrated nuclei and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL)-positive smooth muscle cells, and internucleosomal DNA fragmentation was also detected, indicating the induction of apoptosis. In the arteries cultured with 10 μM DXR for 7 days, nuclear swelling, karyolysis and random DNA fragmentation indicative of necrosis were observed, and muscle contractility was abolished. These results suggest that 0.3 μM DXR selectively down-regulates the α1A-adrenoceptor protein expression, resulting in a decrease in the noradrenaline-induced contraction. This down-regulation may be at least partly due to the production of a superoxide radical. DXR also caused a decrease in muscle contractility followed by apoptotic changes at 1 μM and necrotic changes at 10 μM. These changes might be responsible for the disturbance of the circulatory system that is often observed during the course of repetitive chemotherapy. British Journal of Pharmacology (2001) 132, 1365–1373; doi:10.1038/sj.bjp.0703959

Although beta-adrenergic stimulation has been shown in many studies to decrease myofilament Ca2+ sensitivity in various types of cardiac muscle such as rat and rabbit ventricles, other studies disagree with this conclusion. In the present study, we aimed to explain these contradictory findings. We examined the effect of beta-adrenoceptor stimulation on Ca2+ sensitivity using guinea pig and rat ventricles. We performed the experiment at two different temperatures and compared the results. In guinea pig ventricles, isoproterenol and forskolin did not alter the relationship between [Ca2+]i and muscle force during the relaxation phase of tetanic contraction at either 24 degrees C or 30 degrees C. In rat ventricles, in contrast, isoproterenol shifted the [Ca2+]i-force curve to the right at 24 degrees C, but not at 30 degrees C. In guinea pig ventricles permeabilized by alpha-toxin, in which the cAMP/PK-A system is intact, the addition of cAMP did not decrease Ca2+ sensitivity. These results suggest that there are species- and temperature-dependent differences in the regulation of myofilament Ca2+ sensitivity by beta-adrenergic stimulation.

We have attempted to determine whether muscarinic stimulation induces RhoA/ROCK-mediated Ca2+ sensitization of contractions in chicken gizzard smooth muscles. rhoA is a small GTP-binding protein, and ROCK is a rhoA-associated coiled coil-forming serine/threonine kinase. The relationship between the cytosolic Ca2+ level ([Ca2+]i) and muscle force in the presence of a high K+ concentration was not different from that in the presence of carbachol. Verapamil inhibited muscle force in proportion to the decrease in [Ca2+]i in both the muscle stimulated with high K+ and that stimulated with carbachol. In addition, Y-27632 (10 microM), a ROCKs inhibitor, had no effect on the contractions. In the alpha-toxin-permeabilized muscles, Ca2+ induced a greater contraction in the presence of guanosine 5'-O-(3-thiotriphosphapte) (GTP[gamma-S]), whereas carbachol with GTP was not effective. The GTP[gamma-S]-induced Ca2+ sensitization was completely inhibited by Clostridium botulinum exoenzyme C3. Western blot analysis revealed both rhoA and ROCKII in the muscle extract. In addition, reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed the expression of both ROCKI and ROCKII mRNAs. These results suggest that Ca2+ sensitization in the chicken gizzard is elicited via a rhoA/ROCKs pathway, and that this pathway may be responsible for the augmentation of contraction by GTP[gamma-S] in the permeabilized muscles. If such a pathway does exist, however, carbachol-induced contraction may not be coupled to it, which explains the absence of Ca2+ sensitization in the intact chicken gizzard stimulated by carbachol.

1 Recent evidence has indicated that the mitogen platelet-derived growth factor (PDGF) can act acutely to regulate arterial tone. In this study we demonstrate that the BB isoform of PDGF (PDGF-BB) can cause endothelium-independent relaxation of rabbit isolated mesenteric arteries. 2 In endothelium-denuded arteries, PDGF-BB (40 pM‐8.0 nM) caused concentration-dependent relaxation of noradrenaline-induced tone. The relaxation to PDGF-BB was abolished by a cyclooxygenase inhibitor, indomethacin (10 mM) and by the PDGF receptor-associated tyrosine kinase inhibitor, tyrphostin AG1295 (50 mM), but not by N G -monomethyl-L-arginine (L-NMMA, 200 mM), an inhibitor of nitric oxide (NO) synthase. 3 PDGF-BB (4.0 nM) significantly increased the release of prostacyclin (PGI2) in endotheliumdenuded arteries. Exogenously applied iloprost (1 mM), a stable analogue of PGI2, relaxed the endothelium-denuded artery. PDGF-BB (4.0 nM) significantly increased the cyclic AMP content. 4 In the absence of Ca 2+ , PDGF-BB (4.0 nM) failed to relax the artery, and the release of PGI2 was almost completely suppressed. In addition, the release of PGI2 by PDGF-BB (4.0 nM) was significantly reduced in the presence of endothelium. The eAect of endothelium was eliminated by LNMMA (200 mM) and PGI2 release increased. 5 These data indicate that in rabbit mesenteric arteries, PDGF-BB can evoke endotheliumindependent relaxation by stimulating the synthesis of PGI2. The PDGF-BB-induced prostaglandin synthesis is dependent on both Ca 2+ and tyrosine phosphorylation of the PDGF receptor, and is attenuated by endothelium-derived NO. British Journal of Pharmacology (2000) 131, 1546‐1552

To elucidate the function of calponin in intact contracting smooth muscle cells in vivo, we generated mice with a mutated basic calponin (h1) locus (Yoshikawa et al., Genes Cells 3, 685–695, 1998). Crossbridge cycling rates were estimated in aortic smooth muscle by the force redevelopment following an isometric step shortening as a function of time after K+ depolarization. Evidence is presented that calponin is involved in the inhibition of shortening velocity in the tonic phase of contraction. The phosphorylation levels of myosin regulatory light chain and cytosolic calcium concentrations were not significantly different in paired comparisons between calponin-deficient (−/−) and wild-type (+/+) muscles at any time point after stimulation. The force-velocity relationships in vas deferens smooth muscle showed that the maximum shortening velocity of −/− muscle was significantly faster than that of +/+ muscle. There was no change in the length-force relationships in both −/− and +/+ muscles of aorta and vas deferens. The results suggest that calponin plays a role in regulation of the crossbridge cycling and that it may be responsible for reduced shortening velocity during a maintained contraction of mammalian smooth muscle.

Acidic calponin is an actin binding protein expressed in smooth muscle and brain. Although the role of smooth muscle calponin (basic calponin) has been well studied, few studies have been performed on acidic calponin. In the present study, we demonstrated that acidic calponin binds to filamentous actin, but not monomeric actin. A co-sedimentation assay indicated that acidic calponin binds to actin with an apparent binding constant of 4 x 10(5) M(-1). In the presence of an excess amount of calmodulin, the binding of acidic calponin to actin was inhibited. The binding of acidic calponin to calmodulin was Ca(2+)-dependent with K(d) of 31 microM. We next investigated whether or not acidic calponin could be a substrate for mu-calpain in vitro, since it has been shown that basic calponin is cleaved by mu-calpain. The results showed that acidic calponin was also cleaved by mu-calpain. Neither the proteolytic pattern nor velocity of acidic calponin was different in the absence or presence of calmodulin. When acidic calponin had bound to actin, however, the susceptibility of the acidic calponin to mu-calpain was significantly reduced, which was reversed by the addition of calmodulin. Our results suggest that acidic calponin might be involved in the mu-calpain-regulated actin cytoskeleton.

Vasoinhibitory effects of (-)-(S)-2-[3,5-bis(1, 1-dimethylethyl)-4-hydroxyphenyl]-3-[3-[N-methyl-N-[2-(3, 4-methylenedioxyphenoxy)ethyl]amino]propyl]-1,3-thiazolidin- 4-one hydrogen fumarate (CP-060S), a synthesized cardioprotective agent, were examined. In the rat aortic rings, the contractile responses to cumulative application of angiotensin II, [Arg(8)]-vasopressin (vasopressin), or prostaglandin F(2alpha) were inhibited by CP-060S in a concentration-dependent manner. The Ca(2+)-induced contractions in the presence of vasopressin or prostaglandin F(2alpha) were also inhibited by CP-060S in a concentration-dependent manner. The inhibitory effect of 10(-5) M CP-060S on phenylephrine-induced contraction was as potent as that of 10(-6) M nifedipine, and the combined addition of 10(-6) M nifedipine and 10(-5) M CP-060S showed the effect similar to that of 10(-5) M CP-060S alone. In rat aorta loaded with a Ca(2+) indicator, fura-PE3, 10(-5) M CP-060S completely inhibited the high K(+)-induced increase in cytosolic Ca(2+) level ([Ca(2+)](i)) and contraction. In contrast, 10(-5) M CP-060S only partially inhibited the increase in [Ca(2+)](i) and contraction due to phenylephrine or prostaglandin F(2alpha). In the presence of 10(-6) M nifedipine, 10(-5) M CP-060S did not inhibit the increase in [Ca(2+)](i) and contraction induced by prostaglandin F(2alpha). In a Ca(2+)-free medium, the phasic increases in contraction and [Ca(2+)](i) induced by phenylephrine were not affected by 10(-5) M CP-060S. These results suggest that the vasoinhibitory effect of CP-060S in rat aortic rings is due mainly to the inhibition of L-type voltage-dependent Ca(2+)-channels.

We evaluated the role of the inositol 1,4,5-triphosphate (IP(3)) receptor-mediated Ca(2+) release on the positive inotropic effects of alpha-adrenergic stimulation using a novel, potent, selective membrane-permeable blocker of IP(3) receptor, xestospongin C. Guinea-pig papillary muscle permeabilized with saponin exhibited spontaneous oscillatory contractions in solution buffered with pCa(2+) 6.5 by a low concentration of EGTA. The oscillatory activity was increased by adding 100 microM IP(3) and abolished by 1 microM ryanodine or 30 microM cyclopiazonic acid. Xestospongin C (3 microM) inhibited the IP(3)-induced increase in the oscillatory contractions without affecting basal oscillations. In intact papillary muscle, xestospongin C (3 microM) inhibited the positive inotropic effects of phenylephrine, resulting in a rightward and downward shift of the concentration-response curve for phenylephrine. On the contrary, xestospongin C did not affect the concentration-response curve for phenylephrine obtained in the presence of ryanodine (1 microM). On the other hand, xestospongin C affected neither basal contractions nor the positive inotropic effects of a high extracellular Ca(2+) concentration (3.2 mM) or that of isoprenaline (1 and 10 nM). These results suggest that the IP(3)-mediated increase in Ca(2+) release is involved in the positive inotropic effects of alpha-adrenergic stimulation in the guinea-pig cardiac muscle.

It has been reported that endothelium-dependent relaxation is impaired in pulmonary hypertensive vessels. The underlying mechanisms for this phenomenon, however, have not yet been identified. In this study, the mechanisms responsible for decreased endothelium-dependent relaxation in the pulmonary artery isolated from monocrotaline (MCT)-induced pulmonary hypertensive rat (MCT rat) were examined. MCT (60 mg kg−1), or its vehicle was administered by a single subcutaneous injection to 6-week-old male Sprague Dawley rats. Endothelium-dependent relaxation induced by carbachol or ionomycin in the MCT rat artery was significantly smaller than that in vehicle-treated rat (control rat) artery. Cyclic GMP levels, measured by enzyme-immunoassay, under resting or stimulation with carbachol or ionomycin were also smaller in the MCT rat artery. However, sodium nitroprusside-induced cyclic GMP accumulation in the endothelium-denuded artery was similar in control and MCT rats. These results suggest that MCT treatment decreases endothelial nitric oxide (NO) production. Resting endothelial Ca2+ levels ([Ca2+]i) in the fura-PE3-loaded MCT rat artery, were not different from those in the control rat. However, the increase in endothelial [Ca2+]i elicited by carbachol was attenuated in the MCT rat. In quantitative RT–PCR analysis, the expression of mRNA encoding endothelial NO synthase was rather increased in the MCT rat artery, suggesting an up-regulation of eNOS expression. These results provide evidence that impaired NO-mediated arterial relaxation in the MCT rat is due to dissociation between eNOS expression and NO production. This dissociation may be derived from an inhibition of receptor-mediated Ca2+ metabolism and also from the apparent decrease in Ca2+ sensitivity of eNOS. British Journal of Pharmacology (1999) 128, 1098–1104; doi:10.1038/sj.bjp.0702878

Platelet-derived growth factor (PDGF) has been shown to act chronically on blood vessels to regulate not only proliferation but also vascular tone. These effects may be at least partly due to the chronic effect of PDGF on vascular endothelium. To evaluate this possibility, we examined the effects of PDGF on the endothelium-dependent relaxation (EDR) and total RNA for endothelial nitric oxide (NO) synthase (eNOS) using an organ culture system. In rabbit mesenteric arteries cultured in a serum-free medium for 1 wk, amplitude of the substance P-induced EDR did not change, whereas dependency of the EDR on NO (∼60.0% vs. 18.9%) and the total amounts of recoverable eNOS mRNA estimated by RT-PCR were increased compared with those in freshly isolated arteries. Culture with PDGF for 1 wk decreased the relaxant effect of substance P and ionomycin ( P < 0.01 compared with the arteries without PDGF), NO production estimated by bioassay ( P < 0.01), and eNOS mRNA level, whereas the sodium nitroprusside-induced relaxation did not change. These results suggest that PDGF has a chronic effect on vascular endothelium to decrease eNOS mRNA and NO production and to impair NO-dependent EDR.

Treatment with acetylcholine (ACh) of a beta-escin-permeabilized intrapulmonary bronchial smooth muscle of the rat induced force when the Ca2+ concentration was clamped at 1 microM. The ACh-induced Ca2+ sensitization of myofilaments was significantly greater in antigen-induced airway hyperresponsive rats than in control rats. The ACh-induced Ca2+ sensitization was completely blocked by treatment with Clostridium botulinum C3 exoenzyme, an inactivator of Rho family of proteins. Moreover, the protein level of RhoA in the intrapulmonary bronchi was significantly increased in the airway hyperresponsive rats. Thus, increased airway smooth muscle contractility observed in asthmatics may be related to augmented agonist-induced, Rho-mediated Ca2+ sensitization of myofilaments.

It is evident that smooth muscle contraction is regulated not only by the Ca2+/calmodulin/myosin light chain kinase system but also by modulation of Ca2+ sensitivity. Changes in free calmodulin concentrations, myosin light chain phosphorylation elicited by rho/rho-kinase, regulation of myosin phosphatase activity and thin filament-linked mechanisms are the possible mechanisms for regulation of Ca2+ sensitivity.

The mechanism by which cicletanine (3-(4-chlorophenyl)-1,3-dihydro-7-hydroxy-6-methylfuro-[3,4-c]pyri dine) induces vasodilatation was examined in isolated vascular smooth muscle. Cicletanine inhibited the contraction induced by high K+, norepinephrine (NE) and prostaglandin F2alpha in a concentration-dependent manner in rat aorta. High K+ (15.8-72.7 mM) elicited elevation of cytosolic Ca2+ level ([Ca2+]i) and contraction in a concentration-dependent manner. Cicletanine (300 microM) inhibited the high K+-induced contractions without changing the [Ca2+]i/tension relationship. NE (3-300 nM) elicited greater contractions than high K+ at a given [Ca2+]i, suggesting that NE increased Ca2+ sensitivity of the contractile elements. Cicletanine inhibited the NE-induced contractions without changing the slope of the [Ca2+]i/tension relationship. Cicletanine inhibited the transient increases in both [Ca2+]i and muscle tension elicited by NE but not the transient increase in [Ca2+]i elicited by caffeine in Ca2+-free solution. Cicletanine did not inhibit contraction induced by Ca2+ in the permeabilized rabbit mesenteric artery with alpha-toxin. These results suggest that cicletanine inhibits vascular smooth muscle contraction by multiple mechanisms: 1) inhibition of Ca2+ influx via voltage-dependent Ca2+ channel and 2) inhibition of Ca2+ release mediated by the alpha-adrenoceptors, but not by caffeine.

In vitro effects of mycalolide-B (MB), isolated from marine sponge, were investigated with regard to the activation of rat platelets. Collagen-induced platelet aggregation in platelet-rich plasma (PRP) was slightly but significantly potentiated by lower concentrations of MB (0.3 and 1 microM) but was inhibited by higher concentrations (3 and 10 microM). ADP-induced platelet aggregation in PRP was also significantly prevented by MB (1-10 microM). Potentiation of ADP-induced aggregation by MB (0.3 microM) was hardly observed. G-actin contents, determined by DNase I inhibition assay, were increased in resting washed platelets incubated with MB (3 microM). In contrast, cytochalasin-D (CD) at 3 microM slightly reduced G-actin contents in resting platelets. After platelet aggregation with collagen (3 microg/ml) or ADP (10 microM), G-actin contents in platelets were reduced, indicating de novo actin polymerization. MB (3 microM) and CD (3 microM) abolished both ADP (10 microM)- and collagen (3 microg/ml)-induced platelet aggregation and actin polymerization in washed platelets. MB (1-10 microM) had no effects on intracellular Ca2+ concentrations in ADP (10 microM)-stimulated platelets. [125I]-fibrinogen binding to activated platelets with ADP (10 microM)(was inhibited by MB (0.3-3 microM) in a concentration-dependent manner. Thrombin-induced platelet-fibrin clot retraction was inhibited by MB (1 and 10 microM). These results suggest that MB inhibits platelet activation by interfering with actin polymerization through a different mechanism of action from CD. MB may be a useful tool for studying the role of actin polymerization in various cells.

Stellettamide A (ST-A), a novel marine toxin isolated from a marine sponge, inhibited high K+(72.7 mM)-induced contraction in the smooth muscle of guinea-pig taenia coli with an IC50 of 88 μM. In the taenia permeabilized with Triton X-100, ST-A inhibited Ca2+ (3 and 10 μM)-induced contractions with an IC50 of 46 μM for 3 μM Ca2+ and 105 μM for 10 μM Ca2+. In the permeabilized taenia, calyculin-A (300 nM), a potent inhibitor of type-1 and type-2A phosphatases, induced sustained contraction in the absence of Ca2+. ST-A had no effect on this contraction. ST-A inhibited Mg2+-ATPase activity in native actomyosin prepared from chicken gizzard with an IC50 of 25 μM. In a reconstituted smooth muscle contractile system containing calmodulin, myosin light chain (MLC) and MLC kinase, ST-A inhibited MLC phosphorylation with an IC50 of 152 μM. The inhibitory effect of ST-A was antagonized by increasing the concentration of calmodulin. ST-A inhibited calmodulin activity, assessed by Ca2+/calmodulin-dependent enzymes, (Ca2+-Mg2+)-ATPase of erythrocyte membrane, with an IC50 of 100 μM and phosphodiesterase prepared from bovine cardiac muscle with an IC50 of 52 μM. The inhibitory effect on phosphodiesterase activity was antagonized by increasing the calmodulin concentration. Interaction between ST-A and calmodulin was demonstrated by instantaneous quenching of the intrinsic tyrosine fluorescence of calmodulin by ST-A (3–300 μM). Similar results were obtained in the presence or absence of Ca2+ suggesting that ST-A binds to calmodulin and that Ca2+ is not essential for the binding of ST-A to calmodulin. These results suggest that ST-A, isolated from marine metabolites, is a novel inhibitor of calmodulin.

Palytoxin (PTX) is one of the most potent toxins isolated from marine coelenterates of the genus Palythoa. It induces depolarization in various types of cells by increasing the permeability for monovalent cations. It has been reported that PTX induces endothelium-dependent relaxation of vascular smooth muscle. In this study, we examined the effect of PTX on the cytosolic Ca2+ concentration ([Ca2+]i) in the endothelium of rabbit aortic valves loaded with fluorescent Ca2+ indicators, fura-PE3 or fluo-3. PTX (10 pM-300 nM) irreversibly increased endothelial [Ca2+]i in a concentration-dependent manner. ATP and thapsigargin also increased [Ca2+]i. Imaging of [Ca2+]i with a confocal microscope revealed that PTX increased [Ca2+]i in all endothelial cells studied (n = 13). An inorganic Ca2+ entry blocker, La3+ (30 microM), had no effect on the increase in [Ca2+]i induced by PTX whereas it inhibited the sustained phase of the increase in [Ca2+]i induced by ATP or thapsigargin. The PTX-induced increase in [Ca2+]i was partially inhibited by ouabain and was abolished by removal of external Ca2+ although decrease of Na+ concentration in the incubation medium was ineffective. Activation of protein kinase C by 1 microM 12-deoxyphorbol 13-isobutyrate or inhibition of phosphatase by 10 nM calyculin-A had no effect on the increase in [Ca2+]i induced by PTX, whereas both agents inhibited the sustained phase of the increase in [Ca2+]i induced by ATP or thapsigargin. Mn2+ influx, measured by the quenching of fura-PE3 fluorescence, was accelerated by ATP or thapsigargin, but not by PTX. These results suggest that PTX increases [Ca2+]i in the endothelium of the rabbit aortic valve by increasing Ca2+ influx through a pathway which is different from that activated by ATP or thapsigargin.