Search

To determine the mechanism of endothelium-dependent relaxation by bradykinin, we simultaneously measured changes in cytosolic calcium concentrations ([Ca2+]i) and force of fura-2-loaded strips of porcine coronary artery. We also examined effects of captopril, an angiotensin converting enzyme inhibitor, on bradykinin-induced relaxation. Bradykinin, in a concentration-dependent manner (10(-10) to 10(-7) M), decreased both [Ca2+]i and force to resting levels, during 10(-5) M prostaglandin F2 alpha-induced contractions, only when endothelium was intact. Treatment with 10(-5) M captopril enhanced the bradykinin-induced decreases in [Ca2+]i and force and shifted the concentration-response curve to the left. During 118 mM K+ depolarization, bradykinin induced a greater relaxation than that expected from the reduction in [Ca2+]i. Captopril had no effects on the relationship between reduction in [Ca2+]i and relaxation induced by bradykinin. Bradykinin relaxes porcine coronary artery in an endothelium-dependent manner, by decreasing [Ca2+]i and also by controlling the Ca2+ sensitivity of the contractile apparatus of smooth muscle. Captopril enhanced the bradykinin-induced relaxation, with no apparent direct effect on Ca2+ sensitivity of the contractile apparatus.

Cerebral vasospasm determines the prognosis of subarachnoid hemorrhage (SAH). The increased vascular reactiveness has an important role in the development of cerebral vasospasm. This study analyzed the roles of the receptor-mediated signaling and the myofilament Ca2+ sensitivity in the increased vascular reactiveness in SAH, using the basilar artery of a rabbit SAH model. Endothelin-1, thrombin, and phenylephrine induced transient increases in [Ca2+]i, myosin light chain phosphorylation, and contraction in the controls. All these responses were not only enhanced but also became sustained in SAH. In the sequential stimulation of thrombin receptor or α1-adrenoceptor, the second response was substantially attenuated in the controls, whereas it was maintained in SAH. The thrombin-induced contraction in SAH irreversibly persisted even after terminating the thrombin stimulation. This contraction was completely reversed by trypsin and a Gαq inhibitor YM254890, thus suggesting the sustained receptor activity during the sustained contraction. YM254890 also inhibited the endothelin-1- and phenylephrine-induced sustained contraction. Furthermore, the GTPγS-induced transient contraction in the control α-toxin-permeabilized strips was converted to a sustained contraction in SAH. The results provide the first evidence that the feedback inactivation of the receptor activity and the myofilament Ca2+ sensitivity was impaired in SAH, thus contributing to the increased vascular reactiveness.

Thrombus formation is commonly associated with pulmonary arterial hypertension (PAH). Thrombin may thus play an important role in the pathogenesis and pathophysiology of PAH. Hence, we investigated the contractile effects of thrombin and its mechanism in pulmonary artery.The cytosolic Ca(2+) concentrations ([Ca(2+)](i)), 20 kDa myosin light chain (MLC20) phosphorylation and tension development were evaluated using the isolated porcine pulmonary artery.Thrombin induced a sustained contraction in endothelium-denuded strips obtained from different sites of a pulmonary artery, ranging from the main pulmonary artery to the intrapulmonary artery. In the presence of endothelium, thrombin induced a transient relaxation. The contractile effect of thrombin was abolished by either a protease inhibitor or a proteinase-activated receptor 1 (PAR(1)) antagonist, while it was mimicked by PAR(1)-activating peptide (PAR(1)AP), but not PAR(4)AP. The thrombin-induced contraction was associated with a small elevation of [Ca(2+)](i) and an increase in MLC20 phosphorylation. Thrombin and PAR(1)AP induced a greater increase in tension for a given [Ca(2+)](i) elevation than that obtained with high K(+)-depolarization. They also induced a contraction at a fixed Ca(2+) concentration in alpha-toxin-permeabilized preparations.The present study revealed a unique property of the pulmonary artery. In contrast to normal arteries of the systemic circulation, thrombin induces a sustained contraction in the normal pulmonary artery, by activating PAR(1) and thereby increasing the sensitivity of the myofilament to Ca(2+). This responsiveness of the pulmonary artery to thrombin may therefore contribute to the pathogenesis and pathophysiology of PAH.

1. Using front-surface fluorimetry and Fura-2, we determined the effects of caffeine on cytosolic calcium concentration ([Ca2+]i) and on tension of strips of the rat thoracic aorta. We also determined the effects of caffeine on 45Ca2+ influx into the strips. The objective was to elucidate the mechanism of contraction and relaxation in vascular smooth muscle, as induced by caffeine. 2. In normal physiological salt solution (PSS), caffeine induced a transient tension development, while it induced a biphasic change in [Ca2+]i. The initial transient peak in [Ca2+]i which coincided with tension development was followed by a sustained increase. Thus, changes in tension did not follow changes in [Ca2+]i. In Ca(2+)-free PSS, both the caffeine-induced contraction and the increase in [Ca2+]i were transient. It was suggested that in both the presence and absence of extracellular Ca2+, the transient increase in [Ca2+]i was due to the release of Ca2+ from the intracellular store. Although the sustained increase in [Ca2+]i depended on extracellular Ca2+, it was not affected by diltiazem, a Ca2+ antagonist. 3. Caffeine inhibited the increase in [Ca2+]i and tension development during 118 mM-K+ depolarization, in a concentration-dependent manner. The extent of reduction in tension (relaxation) was greater than that expected from the reduction in [Ca2+]i based on the [Ca2+]i-tension relationship observed with K+ depolarization. Pretreatment of the strips with ryanodine did not alter the inhibitory effects of caffeine. 4. Caffeine inhibited the increased [Ca2+]i and developed tension during stimulation by 10(-5) M-noradrenaline, in a concentration-dependent manner. 5. Dibutyryl cAMP (10(-4) M) inhibited both high K(+)-induced and noradrenaline-induced tension development. Inhibition of an increase in [Ca2+]i in relation to the inhibition of tension during noradrenaline stimulation was much greater than that in 118 mM-K+ depolarization. 6. Although caffeine per se had no effect on 45Ca2+ influx in the strips in normal PSS, caffeine did inhibit the increase in 45Ca2+ influx stimulated by 118 mM-K+ or by 10(-5) M-noradrenaline, to a similar extent and with similar IC50 values. 7. The characteristic features of the effects of caffeine on vascular smooth muscle, i.e. the transient nature of contraction and the relaxation of precontracted strips could be explained as follows: caffeine is able to reduce [Ca2+]i after releasing Ca2+ from intracellular stores; however, this may play a minor role. Independent of the [Ca2+]i reduction, the second messenger, cAMP, might directly influence the [Ca2+]i-tension relationship, and if so, would play a major role.

Chest CT on admission of a 58-year-old woman with bloody sputum showed a mass shadow at the hilum of the right lung suggesting invasion to the mediastinum, and contralateral mediastinal lymph node (#6) metastasis. Bronchial brush cytology yielded a diagnosis of small cell lung cancer (SCLC). The clinical stage was T4N3M0, stage IIIB, limited disease (LD). On admission, her platelet count was only 40 x 10(3)/microl. Blood biochemistry and bone marrow puncture revealed immune thrombocytopenic purpura (ITP). We speculated that she had secondary ITP (ITP-like syndrome) associated with cancer. Only 11 cases of lung cancer with secondary ITP have ever been reported, 4 cases of which attained complete response of cancer and complete remission of ITP by anti-cancer therapy. Therapeutic procedures employed were surgery in 3 cases of adenocarcinoma and a high dose chemotherapy (HDC) with autologous peripheral blood stem cell transplantation (APBSCT) in 1 case of SCLC. In the present case, concurrent chemoradiotherapy (four cycles of cisplatin/etoposide (PE) combined with 45Gy of thoracic radiotherapy) was performed, which resulted in a complete response of SCLC and a complete remission of the secondary ITP. This is apparently the first report of successful treatment of SCLC with secondary ITP by standard chemoradiotherapy. In a SCLC patient with ITP-like symptoms, treatment for SCLC may simultaneously resolve the ITP-like symptoms.

A 63-year-old man who had general malaise and dyspnea on effort, was admitted to our hospital. Chest X-ray film on admission showed left pleural effusion. Chest and abdominal CT after left chest drainage revealed left pleural thickening, mediastinal lymph node swelling, multiple lung nodules, osteolytic change of the left 4th rib, and multiple liver tumors. Right pleural effusion and ascites was also recognized. Cytology of the left pleural effusion suggested malignant mesothelioma. He had a skin tumor on his anterior chest. Biopsy revealed metastasis of malignant epithelioid mesothelioma. Upper gastrointestinal endoscopy showed a duodenal tumor and colonoscopy showed a cecal erosion. Endoscopic biopsy revealed metastases of malignant mesothelioma identical to the skin tumor. Because of the left pleural thickening, the primary site was considered to be in the left pleura. Here we report a case of malignant pleural mesothelioma (MPM) with multiple distant metastases to the duodenum, cecum, skin, lung, liver, and rib. Gastrointestinal metastases of MPM detected by endoscopic biopsy are very rare. Only one case of cecal metastasis has ever been reported.

1. We examined temporal changes in the relationship between cytosolic calcium concentrations ([Ca2+]i) and developed tension during histamine-induced contractions of strips of the coronary artery of the pig, by making use of simultaneous measurements of fura-2 fluorescence and force. 2. The relationship between [Ca2+]i and developed tension observed with cumulative applications of extracellular Ca2+ ([Ca2+]o), ranging from 0 mM to 10 mM, during 118 mM K(+)-depolarization was similar to that observed in chemically skinned strips of the porcine coronary artery, as noted by other investigators. [Ca2+]i at 0 mM [Ca2+]o, at 50% of maximum, and at maximum tension development were 76 nM, 424 nM, and 3050 nM, respectively. 3. Cumulative applications of histamine induced dose-dependent increases in [Ca2+]i and tension and the extent of tension for a given change in [Ca2+]i increased, i.e. greater effectiveness of [Ca2+]i-tension relationship, than seen with K(+)-depolarization. 4. When histamine 10(-5) M was applied, [Ca2+]i abruptly rose and reached the first peak within several seconds. After a slight dip at 30 s, [Ca2+]i reached a second peak at 3 min, and then gradually declined. On the other hand, tension developed rapidly reached a maximum at 4 min, then gradually declined. The relation between [Ca2+]i and tension in the early, rising phase of contraction was similar to that obtained during depolarization. At the time of maximum tension development, the relation was greater than that observed during depolarization, which persisted in the phase of declining tension. 5. To examine the role of protein kinase C in the increased effectiveness of [Ca2+]i-tension relation of histamine-induced contractions, we used 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7), as an inhibitor. Exposure of strips to H-7, i0-' M, reduced the effectiveness of [Ca2+]i-tension relation toward the level observed during depolarization, while it had no effect on the [Ca2 ]i-tension relation in the early phase of contraction either in the presence or absence of extracellular Ca2 + 6. In intact smooth muscle of the coronary artery of the pig, the relationship between [Ca2+]i and developed tension varies during contraction. Histamine, a receptor-mediated stimulus, induced a greater tension for a given change in [Ca2+]i during the steady state of contraction than did depolarization. The greater effectiveness of contraction, particularly in the later phase, seems to be mediated by activation of an H-7-sensitive mechanisms.

Background and Purpose— The level of platelet-derived growth factor (PDGF) in cerebrospinal fluid is elevated in subarachnoid hemorrhage (SAH). Therefore, the contractile effect of PDGF on the basilar artery was examined in SAH. Methods and Results— A rabbit double-hemorrhage SAH model was used. In the medial layers of the control basilar artery, PDGF had no effect on contraction up to 1 nmol/L, whereas 3 nmol/L PDGF induced slight contraction. In SAH, PDGF induced an enhanced contraction with an increase in [Ca 2+ ] i at 1 nmol/L and higher concentrations. The levels of [Ca 2+ ] i and tension induced by 1 nmol/L PDGF in SAH were 17% and 20%, respectively, of those obtained with 118 mmol/L K + depolarization. The PDGF-induced elevation of [Ca 2+ ] i and contraction seen in SAH were abolished in the absence of extracellular Ca 2+ . In α-toxin–permeabilized strips of SAH animals, PDGF induced no further development of tension during contraction induced by 300 nmol/L Ca 2+ , suggesting no direct effect on myofilament Ca 2+ sensitivity. Genistein at 10 μmol/L completely inhibited the tension induced by 1 nmol/L PDGF. The level of myosin light-chain phosphorylation was significantly increased by 1 nmol/L PDGF. Conclusions— These results show that the contractile response to PDGF of the basilar artery was enhanced in SAH. The PDGF-induced contraction depended mostly on tyrosine phosphorylation and Ca 2+ -dependent myosin light-chain phosphorylation. The enhancement of the responsiveness to PDGF may therefore contribute to the development of cerebral vasospasm after SAH.

Background and Purpose— Cerebral vasospasm is one of the major complications of subarachnoid hemorrhage (SAH). Its pathogenesis still remains elusive, and effective therapeutic strategies are yet to be established. We investigated the role of proteinase-activated receptor-1 (PAR1) in the hypercontractile state in SAH. Methods— Rabbit double hemorrhage model was used as a model of SAH. The contractile response to thrombin and the PAR1 expression were evaluated in the isolated rings of basilar artery. Results— Thrombin exhibited only a minor contractile effect in the control, whereas it induced augmented contractions in SAH. The expression of PAR1 was upregulated in SAH. Intracisternal injection of PAR1 antagonist E5555 prevented the enhancement of the contractile responses to thrombin in SAH. The maximal prevention was obtained with 2 μg/kg weight/injection. The contractile responses to K + depolarization or endothelin-1 remained unaffected. The upregulation of PAR1 was also prevented by E5555 (2 μg/kg weight/injection) to a level similar to that seen in the control. Ex vivo treatment with E5555 (1 μmol/L) inhibited the contraction induced by thrombin, whereas it had little effect on the contraction induced by K + depolarization or endothelin-1, in the basilar artery of SAH. E5555 also inhibited the [Ca 2+ ] i elevation induced by thrombin, but not trypsin, in cultured smooth muscle cells. Conclusions— PAR1 plays a critical role in upregulating PAR1 itself, thereby enhancing the contractile response to thrombin in SAH. PAR1 could thus be a therapeutic target. However, the usefulness of PAR1 antagonist remains to be investigated in vivo.

Proteinase-activated receptors 1 and 4 (PAR(1) and PAR(4)) are the major receptors mediating thrombin-induced NO production in endothelial cells. The intracellular signaling following their activation still remains to be elucidated. The present study provides the first evidence for the distinct Ca(2+) requirement for the NO production between PAR(1) and PAR(4). The activation of PAR(1) by the activating peptide (PAR(1)-AP) elevated cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and activated NO production in porcine aortic and human umbilical vein endothelial cells, whereas it had little effect on bovine aortic endothelial cells. PAR(4) activation by PAR(4)-AP consistently induced NO production without an appreciable [Ca(2+)](i) elevation in three types of endothelial cells. The PAR(1)-mediated NO production was significantly inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), whereas the PAR(4)-mediated NO production was resistant. NO production following the PAR(1) and PAR(4) activation was significantly inhibited by pertussis toxin, but it was resistant to a Galpha(q/11) inhibitor, YM254890 [(1R)-1-[(3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-[(1R)-1-methoxyethyl]-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl]-2-methylpropyl rel-(2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate]. However, YM254890 abrogated the PAR(1)-mediated Ca(2+) signal. PAR(4)-mediated NO production was substantially inhibited by the inhibitors of phosphotidylinositol-3 kinase (PI3K) and Akt, as well as by the dominant negative mutant of Akt. The PAR(1)-mediated NO production was relatively resistant to inhibitors of PI3K. An immunoblot analysis revealed a transient increase in the phosphorylation of Akt and endothelial NO synthase following the PAR(4) stimulation. In conclusion, PAR(1) and PAR(4) engage distinct signal transduction mechanisms to activate NO production in vascular endothelial cells. PAR(4) preferably activates Galpha(i/o) and induced NO production in a manner mostly independent of Ca(2+) but dependent on the PI3K/Akt pathway, whereas PAR(1) activates both the Ca(2+)-dependent and -independent mechanisms.

The mechanism for the transcriptional up-regulation of p27 Kip1 due to the formation of the cell–cell contact was investigated in vascular endothelial cells. The induction of the cell–cell contact by adding an extra number of endothelial cells activated Rac1, up-regulated p27 Kip1 mRNA and protein, and also facilitated the cell cycle arrest. Transduction of the Rac1 inhibitor protein using the cell-penetrating peptide or treatment with a Rac1 inhibitor NSC23766 inhibited the p27 Kip1 up-regulation and delayed the cell cycle arrest. Rac1 was therefore suggested to mediate the contact-induced transcriptional up-regulation of p27 Kip1 . The role of Rac1 in the regulation of the p27 Kip1 promoter activity was next examined with a luciferase reporter assay. The promoter activity was increased by inducing the cell–cell contact, which was significantly inhibited by the Rac1 inhibitory protein and NSC23766. The evaluation of various truncated promoter regions determined region − 620 to − 573 nucleotides from the initiation codon to be responsible for the contact-induced, Rac1-dependent activation of the p27 Kip1 promoter. The present study thus demonstrated for the first time that the activation of Rac1 due to the cell–cell contact plays a critical role in the transcriptional up-regulation of p27 Kip1 in vascular endothelial cells.

PURPOSE To determine the mechanisms underlying prostaglandin (PG) F(2)alpha-(,) carbachol (CCh)-, or latanoprost (a PGF(2)alpha analogue)-induced contraction of the pig iris sphincter muscle. METHODS Effects of these agents on myofilament Ca(2+) sensitivity were evaluated and compared with the use of receptor-coupled permeabilized preparations by alpha-toxin. The effects of PGF(2)alpha and CCh on the phosphorylation of myosin light chain (MLC) were also analyzed. RESULTS In the intact strips, all three of these agents induced contractions. In permeabilized strips, PGF(2)alpha and CCh, but not latanoprost, caused an additional tension development at a fixed intracellular Ca(2+) concentration ([Ca(2+)](i)) and also shifted the [Ca(2+)](i)-tension curve to the left, thus indicating that PGF(2)alpha and CCh, but not latanoprost, induced increases in Ca(2+) sensitivity (Ca(2+) sensitization). This Ca(2+) sensitization could have been inhibited by Y27632, a rho kinase inhibitor, but not by GF109203X, a protein kinase C (PKC) inhibitor or by PD98059, a mitogen-activated protein (MAP) kinase inhibitor. PGF(2)alpha increased the level of MLC phosphorylation at a constant [Ca(2+)](i). CONCLUSIONS PGF(2)alpha, but not latanoprost, induced Ca(2+) sensitization of the pig iris sphincter muscle in an MLC phosphorylation-dependent manner through the rho-rho kinase pathway. The effect of latanoprost on the Ca(2+) sensitization mechanism was different from that of PGF(2)alpha and was thought to play a beneficial role in glaucoma treatment.

Thrombin and other proteinases exert vascular effects by activating the proteinase-activated receptors (PARs). The expression of PARs has been shown to be upregulated after balloon injury and in human arteriosclerosis. However, the relationship between the receptor upregulation and the alteration of vasomotor function remains to be elucidated. We herein demonstrated that the contractile responses to the PAR-1 and PAR-2 agonist were markedly enhanced in the rabbit femoral arteries after balloon injury. Neointimal thickening was established 4 wk after the injury. No histological change was observed in the sham operation, where the saphenous artery was ligated without any balloon injury. The contractile response to K+depolarization was significantly attenuated 1 wk after the injury and then partly recovered after 4 wk. Thrombin, PAR-1-activating peptide, trypsin, and PAR-2-activating peptide induced no significant contraction in the control. All these stimulants induced enhanced responses 1 wk after balloon injury. Such enhanced responses were seen 4 wk after the injury, except for thrombin. There was no change in the Ca2+sensitivity of the contractile apparatus as evaluated in the permeabilized preparations. PAR-1-activating peptide (100 μmol/l), but no other stimulants, induced an enhanced contraction in the sham operation. The expression of PAR-1 and PAR-2 slightly increased after the sham operation, whereas it markedly and significantly increased after balloon injury. Our observations suggest that balloon injury induced the receptor upregulation, thereby enhancing the contractile response before the establishment of vascular lesions. The local inflammation associated with the sham operation may also contribute to the receptor upregulation.

We investigated the involvement of G i/o protein in NO production following the activation of proteinase-activated receptor-4 (PAR-4) in cultured bovine aortic endothelial cells. AYPGKF-NH 2 (PAR-4 activating peptide), thrombin, and ionomycin induced a concentration-dependent NO production, with the maximal production seen at 30 μM, 0.1 U/ml, and 1 μM, respectively. Ionomycin elevated [Ca 2+ ] i in a concentration-dependent manner. However, AYPGKF-NH 2 and thrombin induced no [Ca 2+ ] i elevation. The loading of cells with BAPTA almost completely inhibited both the NO production and [Ca 2+ ] i elevation induced by 1 μM ionomycin, while it had no significant effect on the AYPGKF-NH 2 -induced NO production. Treatment with pertussis toxin inhibited the AYPGKF-NH 2 -induced NO production, while it had no effect on the ionomycin-induced NO production. Our findings thus demonstrate, for the first time, that PAR-4 activation induced NO production in a manner mostly independent of the Ca 2+ signal and also that G i/o is involved in such NO production in vascular endothelial cells.

We reported previously that various radiocontrast media cause apoptosis in porcine proximal tubular (LLC-PK 1 ) cells, in which reduction in B-cell lymphoma (Bcl)-2 expression and caspase-3 activation are implicated. In the present study, we investigated a role for ceramide in radiocontrast media-induced apoptosis in renal tubular cells. LLC-PK 1 cells were exposed to radiocontrast media for 30 min, followed by incubation for 24 h in normal medium. Cell viability was assessed by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt assay, while apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling stain. Immunofluorescent stains were performed using antibodies against phosphorylated Akt (pAkt) and cAMP response element binding protein (CREB) (pCREB), and ceramide. The mRNA expression and protein content of Bcl-2 were determined by reverse transcriptase-polymerase chain reaction and enzyme immunoassay, respectively. In vivo model of contrast-induced renal injury was induced in mice with unilateral renal occlusion. The cell injury induced by the nonionic radiocontrast medium ioversol was reversed by inhibiting de novo ceramide synthesis with fumonisin B 1 (FB 1 ) and L-cycloserine, but not by suppressing sphingomyelin breakdown with D609. FB 1 reversed ioversol-induced decrease in the immunoreactivities of pAkt and pCREB, reduction in Bcl-2 expression and caspase-3 activation. Like ioversol, C2 ceramide and the Akt inhibitor Src homology-6 induced apoptosis by reducing pAkt and pCREB-like immunoreactivities, lowering Bcl-2 expression and enhancing caspase-3 activity. Indeed, various radiocontrast media, excluding iodixanol which showed the least nephrotoxicity, enhanced ceramide-like immunoreactivity. The role for de novo ceramide synthesis was also shown in the in vivo model of radiocontrast nephropathy. We demonstrated here for the first time that the enhancement of de novo ceramide synthesis contributes to radiocontrast nephropathy.

Background Rho kinase plays an important role in vascular smooth muscle cell (VSMC) contraction and other cellular functions, such as proliferation, migration, and apoptosis. Recent studies have demonstrated that long-term inhibition of Rho kinase suppresses coronary artery spasm and vascular lesion formation after arterial injury. In the cardiovascular surgery field, intimal thickening in vein grafts is the major cause of late graft failure, for which no effective treatment has yet been developed. In this study, we examined whether long-term inhibition of Rho kinase suppresses intimal thickening in autologous vein grafts in rabbits. Methods Male rabbits were randomly divided into two groups and received normal chow (control group) or a special chow containing 0.09% fasudil (fasudil group). After oral administration, fasudil is metabolized to a specific Rho kinase inhibitor, hydroxyfasudil. Each group underwent reversed autologous vein graft surgery with the internal jugular vein into the left common carotid artery. At 1, 2, and 4 weeks after the operation, we examined the extent of intimal thickening of the graft and VSMC proliferation and apoptosis. Results The intimal thickening was significantly suppressed in the fasudil group compared with the control group at 2 and 4 weeks after the operation. In the fasudil group, VSMC proliferation was suppressed at 1 and 2 weeks after the operation, whereas VSMC apoptosis was enhanced at 2 weeks after the procedure. Conclusions These results indicate that Rho kinase is substantially involved in the pathogenesis of intimal thickening of vein grafts and that it is an important therapeutic target for the prevention of graft failure.

We have previously reported that the contractile response to thrombin and trypsin was enhanced in the pregnant rat myometrium. We herein determined whether or not sex hormones contribute to this enhancement and the expression of protease-activated receptors (PARs). The nonpregnant rats received daily injections of either 17beta-estradiol or progesterone, and then the contractile response of the myometrium was examined ex vivo. Treatment with either 17beta-estradiol or progesterone had almost no significant enhancing effect on the high K(+)- or oxytocin-induced contraction. On the other hand, both 17beta-estradiol and progesterone dose-dependently enhanced the contractile response to trypsin. A maximal enhancement was obtained at 25 and 40 mg kg weight(-1) day(-1) for 17beta-estradiol and progesterone, respectively. The extent of the enhancement of the trypsin-induced contraction seen in the sex hormone-treated rats in the present study was comparable to that reported in the pregnant rats. However, the contractile response to thrombin and PAR1/PAR2-AP, SFLLRNP was not enhanced either by progesterone or 17beta-estradiol. PAR2-AP and PAR4-AP failed to induce contraction under any conditions. PAR1 mRNA was scarcely detected in the control myometrium by an RT-PCR analysis, while it slightly increased only in the progesterone-treated rats. Neither PAR2 nor PAR4 mRNA was detected. We thus conclude that the responsiveness to trypsin, but not thrombin, is controlled by sex hormones. A novel type of receptor, other than PAR1, PAR2 or PAR4, is suggested to mediate the trypsin-induced contraction as in the case of the pregnant rat myometrium.

The contractile activity of prostatic stromal cells contributes to symptoms of benign prostatic hyperplasia (BPH). However, the mechanisms for this contraction have not yet been fully elucidated. In this study, we investigated the role of protein kinase C (PKC) in prostatic contraction by measuring the isometric tension development of cultured human prostatic stromal cells (CHPSCs) derived from BPH patients. Fresh human BPH tissue was used only in a Western blot analysis. A ring preparation made of CHPSCs and collagen gel could develop an isometric tension during activation with various agonists. Phorbol 12,13 dibutyrate (PDBu), a PKC activator, induced a relaxation. A Western blot analysis revealed the expression of PKC-potentiated protein phosphatase-1 inhibitory protein (CPI-17) in both CHPSCs and fresh human BPH tissue to be much lower than that in the rabbit aorta. When CPI-17 was over-expressed, PDBu induced a large contraction, but the agonist-induced contraction did not become larger than expected. In alpha-toxin permeabilized preparations, PDBu induced a relaxation in control CHPSCs, while it induced a contraction at a constant [Ca2+]i in CPI-17 over-expressing CHPSCs. These results indicated that the activation of PKC in CHPSCs induces a relaxation probably due to low expression level of CPI-17 and also that the PKC-CPI-17 pathway does not appear to play a major role in the agonist-induced contraction even when CPI-17 was over-expressed.

1 The contractile mechanism of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) was investigated in the guinea-pig Taenia coli, by simultaneously monitoring the changes in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) and force. 2 fMLP induced a significant elevation of [Ca(2+)](i) and force at concentrations higher than 10 nM. The maximal response was obtained at a concentration of higher than 1 microM. 3 fMLP (10 microM) augmented the force development induced by a stepwise increment of the extracellular Ca(2+) concentration during 60 mM K(+) depolarization, while it had no effect on the [Ca(2+)](i) elevation, and thus produced a greater force for a given elevation of [Ca(2+)](i) than 60 mM K(+) depolarization. 4 The removal of extracellular Ca(2+) completely abolished the fMLP-induced contraction. The fMLP-induced [Ca(2+)](i) elevation was inhibited substantially but not completely by 10 microM diltiazem, partly by 10 microM SKF 96365, and completely by their combination. 5 Y27632, a specific inhibitor of rho-kinase, had no significant effect on the fMLP-induced [Ca(2+)](i) elevation and force development. 6 Chenodeoxycholic acid, a formyl peptide receptor antagonist, specifically abolished the fMLP-induced contraction but not high K(+)- or carbachol-induced contractions. 7 A dual lipoxygenase/cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, a nonselective leukotriene receptor antagonist, and a selective type 1 cysteinyl-containing leukotriene receptor antagonist specifically reduced the fMLP-induced contraction. 8 We suggest that the low-affinity-type fMLP receptor and lipoxygenase metabolites of arachidonic acid are involved in the fMLP-induced contraction in the guinea-pig T. coli. This contraction mainly depends on the [Ca(2+)](i) elevation due to Ca(2+) influx and the enhancement of Ca(2+) sensitivity in the contractile apparatus.

The Ca2+-dependent, reversible phosphorylation of the 20 kDa regulatory myosin light chain (MLC) plays a primary role in regulating the contraction of smooth muscle. However, it is well known that the Ca2+ signal is not the only factor which regulates such contraction, however, the alteration of the Ca2+ sensitivity in the contractile apparatus is also known to play an important role. The degree of MLC phosphorylation is determined by the balance of the activity between phosphorylation and dephosphorylation. Either the Ca2+-independent activation of MLC phosphorylation or the inhibition of MLC dephosphorylation causes a greater MLC phosphorylation for a given level of Ca2+ signal and thereby potentiates the myofilament Ca2+ sensitivity. The smooth muscle myosin light chain phosphatase (MLCP) consisting of three subunits was first isolated and cloned in the early '90s. The intensive investigation thereafter has uncovered the biochemical basis for regulating the activity of MLCP. The regulation of the MLCP activity is now considered to play a critical role in regulating the myofilament Ca2+ sensitivity. There are three major mechanisms in the regulation of MLCP; (1) the phosphorylation of a 110 kDa regulatory subunit of MLCP (2) the conformational change of the trimeric structure, and (3) the inhibition by a smooth muscle specific inhibitor protein, CPI-17. Furthermore, some kinases have been found to phosphorylate the MLC and activate the contraction of smooth muscle in a Ca2+-independent manner. Numerous protein kinases have been found to be involved in the regulation of MLC phosphorylation, and rho-kinase is one of the most frequently investigated kinases. The smooth muscle physiology is now asked to integrate the current understanding of the biochemical mechanisms and to clarify which kinases and/or proteins in the contractile apparatus play a physiological role in regulating the myofilament Ca2+ sensitivity and how such extracellular contractile stimulation modulates these mechanisms.

Estrogen has been reported to inhibit apoptosis in vascular endothelial cells. However, its precise mechanism still remains to be elucidated. Here we determined the role of Akt in the anti-apoptotic effect of estrogen. 17β-Estradiol prevented the apoptosis induced by TNF-α in bovine aortic endothelial cells, as evaluated by double staining with fluorescein isothiocyanate-conjugated annexin V and propidium iodide. Introducing a dominant negative mutant of Akt by using a cell-penetrating peptide of Tat protein inhibited the anti-apoptotic effect of estrogen in a concentration-dependent manner, and resulted in the complete inhibition of the anti-apoptotic effect of 17β-estradiol at 1 nM and higher concentrations. The dominant negative mutant without the cell-penetrating peptide and Tat peptide-conjugated protein A had no effect. The intracellular protein transduction was confirmed by immunoblot analysis. Our observations thus provide first direct evidence that Akt plays a central role in the anti-apoptotic effect of estrogen in vascular endothelial cells.

Estrogen suppresses contractile response and increases vasodilator response, partly by modulating endothelial function. However, the effect of estrogen on the contractility of vascular smooth muscle remains to be elucidated. We investigated the effect of a long-term estrogen deficiency on vascular contractility and the Ca(2+) sensitivity of the contractile apparatus in arterial smooth muscle.Female rabbits were divided into the following three groups: control group, an ovariectomized group (OVX), and a group supplemented with 17beta-estradiol after ovariectomy (OVX+E2). Twelve weeks later, the mesenteric artery was isolated, and the vascular contractility was evaluated.In OVX, the contractile responses to phenylephrine and 118 mM potassium were enhanced, and the basal release of nitric oxide decreased in the strips with endothelium compared with either OVX+E2 or control. An enhancement of contraction was also observed in the strips without endothelium. However, the extent of enhancement was smaller than that observed in the presence of endothelium. The simultaneous measurement of calcium ([Ca(2+)](i)) and tension revealed no significant difference in the [Ca(2+)](i) elevations induced by phenylephrine among the three groups. In the alpha-toxin permeabilized strips, the Ca(2+)-tension relationships obtained both with and without phenylephrine and guanosine triphosphate were similar among the three groups. No difference in the myosin expression and the histology of vascular tissue was observed among the three groups.Long-term estrogen deficiency increased the vascular tone mainly by enhancing smooth muscle contractility. Endothelial dysfunction is considered to play a minor role in the augmentation of vascular tone.

Oxidative stress plays a critical role in the pathogenesis of vasospasm after a subarachnoid hemorrhage. We demonstrate that 24-h incubation of the isolated bovine middle cerebral arteries in the serum-free media at 37 degrees C converted the response to bradykinin from relaxation to contraction, in a manner sensitive to free radical scavengers. In the freshly prepared strips, bradykinin induced an endothelium-dependent relaxation, while having no direct effect on the smooth muscle. However, in the strips treated in serum-free media, bradykinin failed to induce endothelium-dependent relaxation, but did demonstrate a direct contractile effect on smooth muscle. The addition of superoxide dismutase and ascorbic acid or 5% serum during the 24-h incubation in the serum-free media prevented the loss of endothelium-dependent relaxation and the development of a contractile response to bradykinin. SB203580 (4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole), a p38 mitogen-activated protein kinase inhibitor, and genistein (4',5,7-Trihydroxyisoflavone), a tyrosine kinase inhibitor, also demonstrated a similar preventive effect. In conclusion, serum-deprivation induced endothelial dysfunction and the responsiveness of smooth muscle to bradykinin due to failure of eliminating oxidative stress. p38 mitogen-activated protein kinase and tyrosine kinase were suggested to play a critical role in this endothelial dysfunction.

Objective— The region of the 110 kDa regulatory subunit (MYPT1) of smooth muscle myosin phosphatase involved in the regulation of contraction was determined under physiological conditions. Methods and Results— Using HIV Tat protein-mediated protein transduction, the N-terminal fragments of MYPT1 were introduced to the intact porcine coronary arterial strips. Pre-incubation with 3 μmol/L TAT-MYPT1 1–374 , a construct containing the Tat peptide and the residues 1 to 374 of MYPT1, for 15 minutes augmented (2.4-fold) the subsequent contraction induced by adding 1.25 mmol/L of extracellular Ca 2+ under 118 mmol/L K + depolarization, with no augmentation of the [Ca 2+ ] i elevation. The deletion of the Tat peptide, MYPT1 1–374 , abolished the augmenting effect. TAT-MYPT1 1–296 demonstrated a weaker but significant augmentation (1.7-fold). However, TAT-MYPT1 1–171 , TAT-MYPT1 39–374 , TAT-MYPT1 39–296 , and TAT-MYPT1 297–374 had no augmenting activity. The myosin light chain phosphorylation level as a function of extracellular Ca 2+ concentrations was shifted to the left in the strips pretreated with TAT-MYPT1 1–374 compared with the control. Conclusions— Region 1 to 296 was the minimal region involved in the enhancement of contraction, and region 297 to 374 played a supplemental role. These results suggested that the interaction mainly between catalytic subunit and MYPT1 play a critical role in the regulation of the endogenous myosin phosphatase in intact smooth muscle.

Theophylline, a nonselective phosphodiesterase inhibitor, has long been regarded as a major bronchodilator in the treatment of human asthma. Using front-surface fluorometry with fura-2 and alpha-toxin permeabilization, the effects of theophylline on intracellular Ca2+ concentration ([Ca2+]i), tension development and Ca2+ sensitivity of the contractile apparatus were investigated in porcine tracheal smooth muscle strips. Application of theophylline induced a relaxation without a significant decrease in [Ca2+]i when strips were precontracted by 40 mm K+ depolarization, while theophylline significantly decreased both [Ca2+]i and tension induced by carbachol. The effects of theophylline on the increases in [Ca2+]i and tension induced by carbachol were significantly inhibited by iberiotoxin, an inhibitor of large-conductance Ca2+-activated K+ channels. In the absence of extracellular Ca2+, theophylline significantly attenuated carbachol-induced transient increases in tension development, while it did not affect carbachol-induced transient increase in [Ca2+]i. The [Ca2+]i-force relationship, which was determined by cumulative applications of extracellular Ca2+ (0-5 mm) during 40 mm K+ depolarization, was significantly shifted to the right by theophylline. In alpha-toxin permeabilized strips, theophylline significantly increased the EC50 value of [Ca2+]i for contraction and enhanced the effect of cAMP, but not of cGMP. These results indicate that theophylline induces relaxation of the porcine tracheal smooth muscle through an activation of BK channels, and a resultant decrease in [Ca2+]i and an attenuation of Ca2+ sensitivity, presumably through the action of cAMP.

G A A b st ra ct s significant difference was observed for age (mean age 52 years in both groups, p=0.922), gender (50% men vs 34%, p=0.097) and body mass index (median 25 kg/m2 vs 24.7 kg/ m2, p=0.569). Patients with typical symptoms were non-responders in 86%, those with atypical symptoms alone in 74% and those with atypical symptoms dominant in 92%. The results of pH-impedance tests were not significantly different between PPI-responders and non-responders. The prevalence of PDS was significantly higher in PPI-non responders, while the prevalence of EPS and IBS almost reach statistical significance (Table). Conclusion This study confirms that pH-impedance monitoring does not help to predict clinical response or non-response to PPI treatment. Functional digestive disorders (EBS, PDS or IBS) appear more frequent in PPI-non responders. Visceral hypersensitivity may be a predictive factor of PPI-non-response in patients with typical or atypical GERD symptoms. References 1. Zerbib et al. Aliment Pharmacol Ther 2005 2. Zerbib et al. Clin Gastroenterol Hepatol 2013 prevalence of functional digestive disorders according to PPI response

1. To investigate the mechanism underlying the trypsin-induced endothelium-dependent relaxation, cytosolic Ca(2+) concentration ([Ca(2+)](i)) and tension development of smooth muscle were simultaneously monitored in the porcine coronary artery, and [Ca(2+)](i) of in situ endothelial cells were monitored in the porcine aortic valvular strips, using fura-2 fluorometry. 2. During the contraction induced by 30 nM U46619, a thromboxane A(2) analogue, 100 nM trypsin induced a rapid transient significant decrease in both [Ca(2+)](i) (from 67.9+/-5.1 to 15.7+/-4.4%) and tension (from 97.5+/-9.2 to 16.8+/-3.5%) of smooth muscle only in the presence of endothelium (100% level was assigned to the level obtained with the 118 mM K(+)-induced contraction). [Ca(2+)](i) and the tension thus returned to the levels prior to the application of trypsin by 5 and 10 min, respectively. 3. The initial phase of this relaxation was partly inhibited by 100 microM N(omega)-nitro-L-arginine (L-NOARG), and was completely inhibited by L-NOARG plus 40 mM K(+) or L-NOARG plus 100 nM charybdotoxin and 100 nM apamin, while the late phase of the relaxation was inhibited by L-NOARG alone. 4. Trypsin induced a transient [Ca(2+)](i) elevation in the endothelial cells mainly due to the Ca(2+) release from the intracellular stores, at the concentrations (1 - 100 nM) similar to those required to induce relaxation. 5. In conclusion, trypsin induced an elevation in [Ca(2+)](i) mainly due to Ca(2+) release in endothelial cells, and thereby caused endothelium-dependent relaxation. The early phase of relaxation was due to nitric oxide and hyperpolarizing factors, while the late phase was mainly due to nitric oxide in the porcine coronary artery.

p27Kip1 is an inhibitor of cyclin-dependent kinases. It has been implicated as having a role in the induction of growth arrest at the G1 phase of the cell cycle in response to anti-mitogenic signals such as cell contact and serum starvation. Proteasome-mediated degradation plays an important role in the rapid inactivation of p27Kip1, causing quiescent cells to re-enter the cell cycle. Although the existence of a second isoform has been suggested, no such isoform was isolated. Through screening of a cDNA library derived from growth-arrested confluent porcine endothelial cells, we obtained clones for a novel isoform of p27Kip1 in addition to the original isoform. The novel isoform differed from the original isoform at the C-terminus. The tissue-specific expression of the original and novel isoforms was demonstrated at the mRNA and protein levels. An in vitro degradation assay demonstrated this novel isoform to be resistant to proteasome-mediated destruction. The expression as a fusion protein with green fluorescent protein revealed this isoform to be targeted to the nucleus by a bipartite nuclear-localization signal with a C-terminal part different from that of the original isoform. The expression of the novel isoform caused the growth arrest of HeLa cells and an accumulation of cells in the G0/G1 phase, and this effect was similar to that seen with the original isoform. The present study suggests that the novel isoform functions as a negative regulator of the cell cycle, and may play a distinct role. The novel isoform was named p27Kip1R because of its resistance to degradation.

Background —Peroxisome proliferator-activated receptor γ (PPARγ) activators, such as troglitazone (Tro), not only improve insulin resistance but also suppress the neointimal formation after balloon injury. However, the precise mechanisms have not been determined. Angiotensin II (Ang II) plays crucial roles in the pathogenesis of atherosclerosis, hypertension, and neointimal formation after angioplasty. We examined the effect of PPARγ activators on the expression of Ang II type 1 receptor (AT 1 -R) in cultured vascular smooth muscle cells (VSMCs). Methods and Results —AT 1 -R mRNA and AT 1 -R protein levels were determined by Northern blot analysis and radioligand binding assay, respectively. Natural PPARγ ligand 15-deoxy-Δ 12,14 -prostaglandin J 2 , as well as Tro, reduced the AT 1 -R mRNA expression and the AT 1 -R protein level. The PPARγ activators also reduced the calcium response of VSMCs to Ang II. PPARγ activators suppressed the AT 1 -R promoter activity measured by luciferase assay but did not affect the AT 1 -R mRNA stability, suggesting that the suppression occurs at the transcriptional level. Conclusions —PPARγ activators reduced the AT 1 -R expression and calcium response to Ang II in VSMCs. Downregulation of AT 1 -R may contribute to the inhibition of neointimal formation by PPARγ activators.

Correlation between the expression of the α-tropomyosin isoforms and cell growth was investigated in rat aortic smooth muscle cells. The levels of exon 1a, exons 1a+2a (smooth muscle type) and exons 1a+2b (non-smooth muscle type) were determined by reverse transcription-polymerase chain reaction (RT–PCR). When the cells were cultured, the level of exons 1a+2b transiently increased while reaching a maximum at 3–5 days. When the serum-deprived confluent cells were stimulated with 3–20% serum for 1.5 h, the level of exons 1a+2b increased by about twofold. The 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) but not 2-[1-(3-dimethylaminopropyl)-1 H -indol-3-yl]-3-(1 H -indol-3-yl)-maleimide (GF 109203X) inhibited this up-regulation. Phorbol-12,13-dibutyrate (PDB) mimicked the effect of serum. The DNA synthesis as determined by the incorporation of 5-bromo-2′-deoxy-uridine (BrdU) was not enhanced by the 1.5 h stimulation with serum or phorbol ester. The up-regulation of non-smooth muscle isoform of α-tropomyosin occurred during G 0 /G 1 transition before entering S phase. Protein phosphorylation is suggested to be involved in the up-regulation. However, the responsible kinase(s) remain to be elucidated.

p27(Kip1) is a cyclin-dependent kinase inhibitor, and its nuclear localization is a prerequisite for it to function as a cell cycle regulator. In the present study, the minimal requirement for the nuclear localization signal (NLS) of p27(Kip1) was determined by analyzing the localization of various mutants of p27(Kip1) tagged with green fluorescent protein (GFP) in HeLa cells and porcine aortic endothelial cells. Wild-type p27(Kip1) exclusively localized into nucleus, while GFP alone localized in both cytosol and nucleus. A comparison of various truncation mutants revealed residues 153-166 to be the minimal region necessary for nuclear localization. However, a fusion of this region to GFP showed cytoplasmic retention in addition to nuclear localization, thus suggesting that some extension flanking this region is required to achieve a full function of NLS. The site-directed mutation of the full-length p27(Kip1) therefore showed that four basic residues (K153, R154, K165, R166), especially R166, play a critical role in the nuclear localization of p27(Kip1).

1. Using fura-PE3 fluorimetry and alpha-toxin permeabilization, the characteristics of the contractile responses to neurokinin A (NKA) were determined in the pregnant rat myometrium. 2. NKA induced contractions in rat myometrium in a concentration-dependent manner. There were no significant differences in the maximum contractions and EC(50) values between the pregnant and non-pregnant myometrium, however, the contraction of only the former was greatly enhanced in the presence of phosphoramidon (PPAD), an endopeptidase inhibitor. 3. In the pregnant myometrium, NKA induced sustained increases in [Ca(2+)](i) and tension in normal physiological saline solution, while only small transient increases in [Ca(2+)](i) and tension were observed in Ca(2+)-free solution. 4. Both diltiazem (10 microM) and SK-F 96365 (10 microM) significantly inhibited the NKA-induced elevations of [Ca(2+)](i) and tension. The effects were additive when these drugs were used together. 5. NKA induced a significant leftward shift of the [Ca(2+)](i)-tension curve obtained by changing the external Ca(2+) (0 - 2.5 mM) during depolarization with high K(+) solution. This Ca(2+)-sensitizing effect by NKA was also observed in the alpha-toxin permeabilized myometrium. 5. These results indicated that in the pregnant rat myometrium: (1) the responsiveness to NKA increased, although it was masked by the increase in the endopeptidase activity; (2) NKA induced contractions of the myometrium by increasing both [Ca(2+)](i) and the myofilament Ca(2+) sensitivity and (3) The NKA-induced [Ca(2+)](i) elevation was partly due to the intracellular Ca(2+) release and mainly due to the Ca(2+) influx, which was thought to be through both voltage dependent calcium channels and non-specification channels.

The correlation between the intracellular Ca(2+) signal and the tube formation in collagen gels induced by vascular endothelial cell growth factor (VEGF) was investigated using cultured bovine aortic endothelial cells. The VEGF-induced sustained elevation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) was similarly inhibited by 10 microM 1-¿beta-[3-(4-methoxyphenyl)propyl]-4-methoxyphenethyl¿-1H-imidazole hydrochloride (SKF 96365) and 10 microM troglitazone. However, 10 microM diltiazem had no effect. The basal tube formation obtained with 1% serum was augmented twofold by 100 ng/ml VEGF. SKF 96365 (0. 1-10 microM) inhibited the VEGF-induced and basal tube formation, while 10 microM troglitazone or 10 microM diltiazem had no effect. The proliferation of endothelial cells was markedly inhibited by SKF 96365 but only slightly by troglitazone and diltiazem. The inhibition of tube formation by three Ca(2+) entry blockers thus correlated with the inhibition of cell proliferation. The [Ca(2+)](i) elevation is thus not a prerequisite for VEGF to induce tube formation.

To investigate the effects of troglitazone on the capacitative Ca 2+ entry, we monitored changes in cytosolic Ca 2+ concentrations ([Ca 2+ ] i ) induced by thapsigargin in fura-2-loaded porcine endothelial cells in situ and in primary culture. In aortic valve endothelial cells in situ, thapsigargin induced sustained elevation of [Ca 2+ ] i . Both troglitazone and SKF 96365 inhibited the steady state increase in [Ca 2+ ] i in a concentration-dependent manner. At 30 μM, troglitazone and SKF 96365 inhibited the [Ca 2+ ] i elevation to 19.4±3.6% and 43.9±4.5%, respectively. In aortic endothelial cells in primary culture, both troglitazone (10 μM) and SKF 96365 (100 μM) completely inhibited the thapsigargin-induced [Ca 2+ ] i increase. The EC 50 value of troglitazone (1.4±0.1 μM) was lower than that of SKF 96365 (10.0±3.3 μM). We suggest that troglitazone would be a useful tool to investigate the capacitative Ca 2+ entry.

The effects of the small noncatalytic subunit of myosin light chain phosphatase (MLCPsr) on the Ca2+-induced contraction of smooth muscle were investigated in the Triton X-100-permeabilized porcine renal artery. The full-length recombinant chicken MLCPsr obtained by the bacterial expression system induced an additional contraction at a constant [Ca2+]i and shifted the [Ca2+]i-force relation curve to the left. A deletion mutant containing the N-terminal 78 amino acids of MLCPsr retained the full action, compared with the full-length MLCPsr, while the deletion of this region completely abolished its effect. The process of relaxation was also delayed by the fragment containing the N-terminal 78 amino acids. These results indicated that MLCPsr increases the Ca2+ sensitivity of the contractile apparatus while the N-terminal 78 amino acids are responsible for this effect in vascular smooth muscle.

A yeast two-hybrid screen of a chicken gizzard cDNA library detected the interaction of the catalytic subunit of protein phosphatase type 1 with a novel subunit. Subsequent characterization established similarity (58%) to the rat liver glycogen-binding subunit. Northern analyses showed expression in a wide range of tissues.

Myosin II light chains (MLC20) are phosphorylated by a Ca2+/calmodulin-activated kinase and dephosphorylated by a phosphatase that has been purified as a trimer containing the delta isoform of type 1 catalytic subunit (PP1C delta), a myosin-binding 130-kDa subunit (M130) and a 20-kDa subunit. The distribution of M130 and PP1C as well as myosin II was examined in smooth muscle cells and fibroblasts by immunofluorescence microscopy and immunoblotting after differential extraction. Myosin and M130 colocalized with actin stress fibers in permeabilized cells. However, in nonpermeabilized cells the staining for myosin and M130 was different, with myosin mostly at the periphery of the cell and the M130 appearing diffusely throughout the cytoplasm. Accordingly, most M130 was recovered in a soluble fraction during permeabilization of cells, but the conditions used affected the solubility of both M130 and myosin. The PP1C alpha isoform colocalized with M130 and also was in the nucleus, whereas the PP1C delta isoform was localized prominently in the nucleus and in focal adhesions. In migrating cells, M130 concentrated in the tailing edge and was depleted from the leading half of the cell, where double staining showed myosin II was present. Because the tailing edge of migrating cells is known to contain phosphorylated myosin, inhibition of myosin LC20 phosphatase, probably by phosphorylation of the M130 subunit, may be required for cell migration.

Interactions of the type 1 phosphatase catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MBP) were compared using affinity columns. In the absence of ATP, MBP bound to dephosphorylated myosin, heavy meromyosin (HMM), and subfragment 1. In contrast, PP1c was not bound. In the presence of ATP, the binding of MBP occurred only with phosphorylated protein. The interaction of MBP with phosphorylated proteins also was demonstrated using thiophosphorylated proteins as competitive inhibitors. Kinetics parameters were determined. With phosphorylated light chains (P-LC20), the major difference between PP1c and MBP was a lower K(m) for the latter. With myosin, MBP showed a marked increase in kcat, compared to PP1c. ATP did not affect these parameters. To investigate the role of the large phosphatase subunit, two recombinant proteins representing the N-terminal two-thirds of the molecule were expressed. These activated PP1c, and activation was maximum at approximately an equimolar ratio. The equimolar mixture of recombinant fragment and PP1c exhibited K(m) values similar to MBP and increased kcat values, compared to PP1c alone. An affinity column was prepared using the recombinant fragment. Phosphorylated HMM and P-LC20 were bound in the presence and absence of ATP. The interaction of P-LC20 was not ATP-dependent. Dephosphorylated HMM did not bind in the presence of ATP. The N-terminal fragment of the large subunit also contained a binding site for PP1c. These results indicate that the N-terminal portion of the large subunit of MBP contained binding sites for P-LC20 and PP1c.

The two-hybrid system was used to screen for binding proteins of type 1 protein phosphatase. Two plasmids were constructed, one containing the cDNA of the delta isoform of the type 1 catalytic subunit and the other containing a chicken gizzard cDNA library. Yeast (Y190) were transformed with the plasmids and screened for interacting species. 35 positive clones were categorized into 19 gene groups. Most of these were not identified. One clone, however, contained a sequence identical to the C-terminal portion of the chicken ribosomal protein L5 and corresponded to nucleotide residues 606-975. L5 was isolated from rat liver ribosomes as the L5.5 S RNA complex. This activated phosphatase activity of a myosin-bound phosphatase and the isolated type 1 catalytic subunit using phosphorylated myosin light chains and phosphorylase alpha as substrates. In addition, it was found that phosphatase sedimented with ribosomal subunits containing L5 but did not sediment with those deficient in L5. These data indicate that L5 binds to the catalytic subunit of the type 1 protein phosphatase and may act as a target molecule for phosphatase in ribosomal function or other cell mechanisms.

Bacterial endotoxic reactions can cause osteonecrosis in humans by disseminated intravascular coagulation. The authors first used a combination of the Shwartzman reaction and corticoid injections in rabbits to develop a new animal model of osteonecrosis. This model showed a significantly higher incidence and wider area of osteonecrosis in the femur and humerus than that found in rabbits with either Shwartzman reaction or steroid injection alone. Osteonecrosis was observed in several foci that were distributed from the diaphysis to the epiphysis in both bones. Histologically, the bone marrow cells underwent necrosis, whereas the bone trabeculae demonstrated either empty lacunae or pycnotic nuclei of osteocytes. Exogenous steroids appeared to potentiate the Shwartzman reaction and the magnitude of osteonecrosis, perhaps by increasing endothelial damage and hypercoagulability of those intraosseous and extraosseous vessels that subsequently thrombosed. This model may not only be useful in clarifying the etiology and early pathogenesis of human osteonecrosis after corticoid therapy, but also in designing pharmaceuticals for prevention and early treatment.