Br-DIF-1 accelerates dimethyl sulphoxide-induced differentiation of P19CL6 embryonic carcinoma cells into cardiomyocytes.

BACKGROUND AND PURPOSE: Stem cell transplantation therapy is a promising option for treatment of severe ischaemic heart disease. Dimethyl sulphoxide (DMSO) differentiates P19CL6 embryonic carcinoma cells into cardiomyocyte-like cells, but with low differentiation capacity. To improve the degree of this differentiation, we have assessed several derivatives of the differentiation-inducing factor-1 (DIF-1), originally found in the cellular slime mould Dictyostelium discoideum, on P19CL6 cells. EXPERIMENTAL APPROACH: P19CL6 cells were cultured with each derivative and 1% DMSO for up to 16 days. Differentiation was assessed by measuring the number of beating and non-beating aggregates, and the expression of genes relevant to cardiac tissue. The mechanism of action was investigated using a T-type Ca(2+) channel blocker. KEY RESULTS: Of all the DIF-1 derivatives tested only Br-DIF-1 showed any effects on cardiomyocyte differentiation. In the presence of 1% DMSO, Br-DIF-1 (0.3-3 µM) significantly and dose-dependently increased the number of spontaneously beating aggregates compared with 1% DMSO alone, by day 16. Expression of mRNA for T-type calcium channels was significantly increased by Br-DIF-1 + 1% DMSO compared with 1% DMSO alone. Mibefradil (a T-type Ca(2+) channel blocker; 100 nM) and a small interfering RNA for the T-type Ca(2+) channel both significantly decreased the beating rate of aggregates induced by Br-DIF-1 + 1% DMSO. CONCLUSIONS AND IMPLICATIONS: Br-DIF-1 accelerated the differentiation, induced by 1% DMSO, of P19CL6 cells into spontaneously beating cardiomyocyte-like cells, partly by enhancing the expression of the T-type Ca(2+) channel gene.

Pathophysiological role of endothelin in ectopic ossification of human spinal ligaments induced by mechanical stress.

Ossification of the posterior longitudinal ligament (OPLL) of the spine is characterized by progressive ectopic bone formation in the spinal ligament. To identify the genes related to ossification affected by mechanical stress during OPLL, analyses using cDNA microarray were carried out using cultured human spinal ligament cells that had been subjected to uniaxial cyclic stretching. Samples were obtained from a total of 14 patients: seven cervical or thoracic OPLL patients and seven control patients. Spinal ligament cells derived from tissues of OPLL (OPLL cells) and control (non-OPLL cells) patients were subjected to uniaxial sinusoidal cyclic stretching (0.5 Hz, 20% stretch) for various time periods (0-9 hours). cDNA microarrays revealed that ranges of distribution of both up- and downregulated genes evoked by cyclic stretching were significantly wider in OPLL cells than in non-OPLL cells. Increases in the mRNA expression of endothelin-1 (ET-1) as well as various marker genes related to ossification were also observed. mRNA expression of ET-1 and alkaline phosphatase was increased by mechanical stress in a time-dependent manner, while addition of ET-1 to static cultures of OPLL cells increased mRNA expression of alkaline phosphatase in a dose-dependent manner. During 9 hours of cyclic stretching, ET-1 release increased to about sixfold the amount observed in nonstretched cells. In non-OPLL cells, neither cyclic stretching nor ET-1 induced any increase in alkaline phosphatase expression. These results suggest that mechanical stress promotes the progression of ossification in OPLL cells through autocrine and/or paracrine mechanisms of ET-1.

Uni-axial cyclic stretch induces Cbfa1 expression in spinal ligament cells derived from patients with ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments. Mechanical stress, which acts on the posterior ligaments, is thought to be an important factor in the progression of OPLL. To clarify this mechanism, we investigated the effects of in vitro cyclic stretch (120% peak to peak, at 0.5 Hz) on cultured spinal ligament cells derived from OPLL (OPLL cells) and non-OPLL (non-OPLL cells) patients. The mRNA expressions of Cbfa1 (an osteoblast-specific transcription factor), type I collagen, alkaline phosphatase (ALP), osteocalcin and integrin beta1 (a mechanotransducer) were increased by cyclic stretch in OPLL cells, whereas no change was observed in non-OPLL cells. The effects of cyclic stretch on the spinal ligament tissues derived from OPLL and non-OPLL patients were also analyzed by immunohistochemistry using an antibody against Cbfa1. The expression of Cbfa1 was increased by cyclic stretch at the center of the spinal ligament tissues of OPLL patients, whereas no change was observed in the tissues of non-OPLL patients. Furthermore, U0126, a specific inhibitor of MAPK kinase (MEK), suppressed the stretch-induced mRNA expressions of Cbfa1, ALP and type I collagen in OPLL cells. These results suggest that in OPLL cells, mechanical stress is converted by integrin beta1 into intracellular signaling and that Cbfa1 is activated through the MAP kinase pathway. Therefore, we propose that mechanical stress plays a key role in the progression of OPLL through an increase in Cbfa1 expression.

Uniaxial cyclic stretch induces osteogenic differentiation and synthesis of bone morphogenetic proteins of spinal ligament cells derived from patients with ossification of the posterior longitudinal ligaments.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments. Mechanical stress, which acts on the posterior ligaments, is thought to be an important factor in the progression of OPLL. To elucidate this mechanism, we investigated the effects of in vitro sinusoidal cyclic stretch (120% peak to peak, at 1 Hz) on cultured spinal ligament cells derived from OPLL and non-OPLL patients. The mRNA expressions of alkaline phosphatase (ALP), osteopontin, bone morphogenetic protein (BMP)-2, BMP-4, and BMP receptors as well as ALP activity in cell layers and production of BMPs into the conditioned medium were significantly increased by cyclic stretch in OPLL cells, whereas no change was observed in non-OPLL cells. A stretch-activated Ca(2+) channel blocker, Gd(3+), the voltage-dependent L-type Ca(2+) channel blockers diltiazem and nifedipine, and Ca(2+)-free medium suppressed stretch-induced ALP activity, which suggests a role of Ca(2+) influx in the signal transduction of mechanical stress to the osteogenic response of OPLL cells. Our study provides first evidences that mechanical stress plays a key role in the progression of OPLL through the induction of osteogenic differentiation in spinal ligament cells and the promotion of the autocrine/paracrine mechanism of BMPs in this lesion.

Coronary vasodilation and positive inotropism by urocortin in the isolated rat heart.

Corticotropin-releasing factor (CRF) has a coronary vasodilator effect and a positive inotropic effect on the isolated rat heart. Recently, expression of CRF receptor type 2 (CRF-R2) has been demonstrated in the heart. In addition, urocortin (Ucn), a new member of the CRF family, has been reported to have much greater affinity for CRF-R2 than CRF. It is suggested that the cardiac effects of Ucn may be more potent than those of CRF. We compared the effect of Ucn with that of CRF on isolated rat heart. The effects of Ucn were then analyzed to determine whether these effects were mediated by CRF receptors and/or any other mediators under the following conditions: perfusion buffer containing (1) alpha-helical CRF 9-41, (2) indomethacin, (3) N(G)-nitro-l -arginine methylester and (4) propranolol. Ucn exhibited a greater effect with a longer duration of action than CRF. Indomethacin significantly attenuated the vasodilator effects of Ucn (P<0.05). CRF receptor antagonist diminished both coronary vasodilation and the positive inotropic effects of Ucn (P<0.05). These results suggest that the cardiac effects of Ucn may be mediated by a CRF receptor, and prostaglandins may be involved in the vasodilator effect.

Novel acyl alpha-pyronoids, dictyopyrone A, B, and C, from Dictyostelium cellular slime molds.

For the elucidation of the diversity of secondary metabolites of Dictyostelium cellular slime molds, we investigate the constituent of three species of slime molds. From the methanol extract of their fruit bodies, we obtained three novel compounds, dictyopyrone A (1) and B (2) from D. discoideum and D. rhizoposium and dictyopyrone C (3) from D. longosporum. They possess a unique alpha-pyrone moiety with a side chain at the C-3 position. Their relative structures were elucidated by spectral means, and the absolute configuration was confirmed by asymmetric synthesis of 1. Since these compounds were obtained from different species of Dictyostelium slime molds, they may be a type of compound common to this genus.

Modulation of actomyosin ATPase by thiotetromycin is mediated through conformational change of actin.

Thiotetromycin isolated from the culture broth of Streptomyces sp. strain OM-674 slightly enhanced the superprecipitation and the ATPase activity of myosin B from skeletal muscle. The ATPase activity of troponin-tropomyosin-free myosin B was inhibited by thiotetromycin. The inhibitory effect of thiotetromycin was significantly attenuated by troponin-tropomyosin complex. The ATPase activity of actomyosin reconstituted from actin and myosin was inhibited by pretreatment of actin with thiotetromycin. Thiotetromycin induced a concentration-dependent decrease in the fluorescence intensity of actin and pyrenyl-F-actin. By using surface plasmon resonance (SPR), it was proved that thiotetromycin bound to actin. Thiotetromycin caused a concentration-dependent decrease in sedimentation of F-actin by hard centrifugation. This was a cross-correlation among the concentration-inhibition curves for thiotetromycin in the activity of actomyosin ATPase and the fluorescence intensity. These results suggest that thiotetromycin binds to actin to cause a conformational change, resulting in modulation of the interaction between actin and myosin, and in depolymerization of F-actin.

A fluorometric assay for cyclic guanosine 3',5'-monophosphate incorporating a Sep-Pak cartridge and enzymatic cycling.

We developed a sensitive and nonradioactive fluorometric assay for cyclic guanosine 3',5'-monophosphate (cGMP). Guanine nucleotides except cGMP were enzymatically phosphorylated to GTP. cGMP, absorbed into a Sep-Pak amino propyl cartridge, was eluted separately from GTP. Purified cGMP was enzymatically converted to GTP, which was applied to the GTP-GDP cycle using succinic thiokinase and pyruvate kinase. When pyruvic acid produced by the GTP-GDP cycle was reduced by lactate dehydrogenase, a reduced form of nicotinamide adenine dinucleotide (NADH) was equivalently oxidized to NAD(+). NAD(+) was further converted into fluorescent compound, which was excited at 370 nm and emitted fluorescence at 460 nm, by a strong alkali. When 20 nmol NADH was used for this assay, the calibration curve over 50 to 500 fmol cGMP became sufficiently linear. The detection limit for cGMP was ca. 5 fmol (signal to noise ratio >3). Using this assay, we confirmed that the cGMP content in the left atrial strip of dog was changed from 11.4 +/- 3.8 to 19.3 +/- 2.6 fmol/mg wet wt of tissue (mean +/- SE, n = 6) by electrical driving at 1 Hz. Carbachol (1 microM) further increased the cGMP to 45.6 +/- 9.2 fmol/mg wet wt of tissue. From these results, it is suggested that this novel assay for cGMP is highly sensitive and can be applied to various biological samples.

Gamma-mangostin, a novel type of 5-hydroxytryptamine 2A receptor antagonist.

Gamma-mangostin, purified from the fruit hull of the medicinal plant Garcinia mangostana caused a parallel rightwards shift of the concentration/response curve for the contraction elicited by 5-hydroxytryptamine (5-HT) in the rabbit aorta (pA2 = 8.2) without affecting the contractile responses to KCl, phenylephrine (alpha1) or histamine (H1). The perfusion pressure response of rat coronary artery to 5-HT (5-HT2A) was reduced concentration dependently by gamma-mangostin (IC50 = 0.32 microM). 5-HT amplified, ADP-induced aggregation of rabbit platelets (5-HT2A) was inhibited by gamma-mangostin (IC50 = 0.29 microM), whereas that induced by thrombin was not affected, nor did gamma-mangostin affect 5-HT-induced contraction of the guinea-pig ileum (5-HT3)in the presence of 5-HT1, 5-HT2 and 5-HT4 receptor antagonists. Furthermore, 5-HT-induced contraction of the rat fundus (5-HT2B) and 5-HT-induced relaxation of the rabbit aorta in the presence of ketanserin (5-HT1) and carbachol-induced contraction of the guinea-pig ileum (muscarinic M3) were not affected by gamma-mangostin (5 microM). Gamma-mangostin inhibited [3H]spiperone binding to cultured rat aortic myocytes (IC50 = 3.5 nM). The Kd for [3H]spiperone binding was increased by gamma-mangostin (3 nM) from 11.7 to 27.4 nM without affecting Bmax. These results suggest that gamma-mangostin is a novel competitive antagonist, free from a nitrogen atom, for the 5-HT2A receptors in vascular smooth muscles and platelets.

The mechanism of rabbit platelet aggregation induced by 2,5-di-(tert-butyl)-1,4-benzohydroquinone, an inhibitor of endoplasmic reticulum Ca2+-ATPase.

2,5-Di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), an inhibitor of endoplasmic reticulum Ca2+-ATPase, caused aggregation accompanied by a marked increase in intracellular Ca2+ concentrations ([Ca2+]i) in washed rabbit platelets. Treatment of platelets with SK&F96365 or removal of extracellular Ca2+ inhibited both Ca2+ influx and platelet aggregation induced by tBuBHQ. These responses of platelets to tBuBHQ were also inhibited by genistein. Western blots using antibody against phosphorylated amino acid residues revealed that tBuBHQ activated tyrosine kinase independently of extracellular Ca2+. These results suggest that tBuBHQ induces Ca2+ influx into platelets probably through activation of tyrosine kinase, resulting in platelet aggregation.

[Ca2+ -ATPase].

The importance of the Ca2+ -ATPases of the surface and endoplasmic reticulum membranes in regulating the intracellular Ca2+ concentrations in living cells has been recognized as a result of the development of biophysical techniques and finding of useful pharmacological tool drugs. The number of possible isoforms of these Ca2+ -ATPases are increased by a complex pattern of alternative splicing and the knowledge of the functional difference and tissue distribution of these isoforms has been accumulated. In this review, we discuss recent progress in our understanding of the roles of the Ca2+ -ATPases in physiological and pathophysiological conditions.

Maxi K+ channels are stimulated by cyclic guanosine monophosphate-dependent protein kinase in canine coronary artery smooth muscle cells.

By using a patch clamp technique, we examined the effect of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (G kinase) on Ca(2+)-activated maxi K+ channels in canine coronary artery smooth muscle cells. Maxi K+ channels (274 +/- 4 pS in symmetrical 140 mM KCl at 24-26 degrees C) were activated by cytoplasmic Ca2+ and were completely blocked by 100 nM charybdotoxin (CTX). G kinase (300 U/ml) added to the cytoplasmic face of the membrane patch shifted the voltage dependence of these channels by about 25 mV in the negative direction in the presence of 1 microM Ca2+, 50 microM cGMP and 1 mM magnesium adenosine triphosphate. At -50 mV and 1 microM Ca2+, G kinase treatment increased the mean number of open channels 4.5-fold compared with the control. alpha-Human atrial natriuretic peptide (ANP, 100 nM) reduced the isometric tension of coronary arterial rings elicited by 14 or 24 mM KCl, but failed to relax the artery contracted by 34 mM KCl. Addition of 100 nM CTX augmented tension development elicited by 24 mM KCl and totally prevented ANP from relaxing the arterial rings. These results indicate that G kinase-dependent protein phosphorylation activates maxi K+ channels in canine coronary smooth muscle, and further suggest that the G kinase-induced activation of maxi K+ channels may cause hyperpolarization and relaxation of coronary artery.

The conventional and saturation transfer electron paramagnetic resonance of spin-labeled myosin subfragment-1 in the presence of F-actin and nucleotides.

SH-1 thiol of S-1 was modified with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetoamide spin label (IASL). The extent of dissociation, alpha, of spin-labeled myosin subfragment-1 (IASL-S-1) from acto-IASL-S-1 by a nucleotide was measured by an ultracentrifugal separation method, a light-scattering method, and a saturation transfer EPR method. The alpha values obtained by these three methods were the same within the limits of the experimental errors. The dependence of alpha on the concentrations of AMPPNP, [S], and F-actin, [A], could be described by the equation: alpha-1 = 1 + (1 + Ks/[S])[A]/KA. The Ks and KA values were 0.65-1.2 mM and 1.7-2.7 mg/ml, respectively, in 0.5 M KCl and 4 mM MgCl2 at pH 7.0 and 20 degrees C. The height of the weakly immobilized peak of the conventional EPR spectrum of IASL-S-1, W, increased linearly with increase in the ATP or AMPPNP concentration, and became saturated at 1 mol nucleotide/mol IASL-S-1. No change in W was observed upon the binding of IASL-S-1 with F-actin. The dependence of the extent of change in W, delta W, on [A] and [S] was given by delta W-1 = 1 + Ks/[S], where Ks = Ks/(1 + KA/[A]). This finding indicates that the delta W value is proportional to the amount of a nucleotide bound to IASL-S-1 and independent of the binding of F-actin to IASL-S-1.