Kazuhito Ichikawa

Inhibitory Phosphorylation Site for Rho-associated Kinase on Smooth Muscle Myosin Phosphatase

It is clear from several studies that myosin phosphatase (MP) can be inhibited via a pathway that involves RhoA. However, the mechanism of inhibition is not established. These studies were carried out to test the hypothesis that Rho-kinase (Rho-associated kinase) via phosphorylation of the myosin phosphatase target subunit 1 (MYPT1) inhibited MP activity and to identify relevant sites of phosphorylation. Phosphorylation by Rho-kinase inhibited MP activity and this reflected a decrease in V max. Activity of MP with different substrates also was inhibited by phosphorylation. Two major sites of phosphorylation on MYPT1 were Thr695 and Thr850. Various point mutations were designed for these phosphorylation sites. Following thiophosphorylation by Rho-kinase and assays of phosphatase activity it was determined that Thr695 was responsible for inhibition. A site- and phosphorylation-specific antibody was developed for the sequence flanking Thr695 and this recognized only phosphorylated Thr695 in both native and recombinant MYPT1. Using this antibody it was shown that stimulation of serum-starved Swiss 3T3 cells by lysophosphatidic acid, thought to activate RhoA pathways, induced an increase in Thr695 phosphorylation on MYPT1 and this effect was blocked by a Rho-kinase inhibitor, Y-27632. In summary, these results offer strong support for a physiological role of Rho-kinase in regulation of MP activity.

Rho-associated Kinase of Chicken Gizzard Smooth Muscle

Rho-associated kinase (Rho-kinase) from chicken gizzard smooth muscle was purified to apparent homogeneity (160 kDa on SDS-polyacrylamide gel electrophoresis) and identified as the ROKα isoform. Several substrates were phosphorylated. Rates with myosin phosphatase target subunit 1 (MYPT1), myosin, and the 20-kDa myosin light chain were higher than other substrates. Thiophosphorylation of MYPT1 inhibited myosin phosphatase activity. Phosphorylation of myosin at serine 19 increased actin-activated Mg+-ATPase activity, i.e. similar to myosin light chain kinase. Myosin phosphorylation was increased at higher ionic strengths, possibly by formation of 6 S myosin. Phosphorylation of the isolated light chain and myosin phosphatase was decreased by increasing ionic strength. Rho-kinase was stimulated 1.5–2-fold by guanosine 5′-O-3-(thio)triphosphate·RhoA, whereas limited tryptic hydrolysis caused a 5–6-fold activation, independent of RhoA. Several kinase inhibitors were screened and most effective were Y-27632, staurosporine, and H-89. Several lipids caused slight activation of Rho-kinase, but arachidonic acid (30–50 μm) induced a 5–6-fold activation, independent of RhoA. These results suggest that Rho-kinase of smooth muscle may be involved in the contractile process via phosphorylation of MYPT1 and myosin. Activation by arachidonic acid presents a possible regulatory mechanism for Rho-kinase.

Effects of the Phosphorylation of Myosin Phosphatase by Cyclic GMP-dependent Protein Kinase

Cyclic GMP-dependent protein kinase (PKG) phosphorylated, in vitro, the large (MYPT1) and small (M20) regulatory subunits of myosin phosphatase (MP) with maximum stoichiometries of 1.8 and 0.6 mol of phosphate/mol subunit, respectively. The phosphorylation of these subunits by PKG did not affect the phosphatase activity towards the 20 kDa myosin light chain. However, phosphorylation of the MP holoenzyme decreased the binding of MP to phospholipid. The phosphorylation of the serine residue of the C-terminal part of MYPT1 was crucial for these interactions. These results suggest that the phosphorylation of MP by PKG is not a direct mechanism in activating MP activity, and that other indirect mechanisms, including the interaction between MP and phospholipids, might be candidates for Ca2+ desensitization via cGMP in smooth muscle.

Isolated Congenital Left Ventricular Diverticulum in an Adult: A Case Report

A sixty-three-year-old man presented himself with atrial flutter and congestive heart failure. Cardiac catheterization revealed that left ventricular diverticulum was located on the anterobasal wall with narrow connection to the left ventricular cavity. Coronary angiography revealed normal coronary arteries. The patient had been asymptomatic until adult life with no other thoracoabdominal or cardiac anomalies. This is an extremely rare finding in the adult population.

IDENTIFICATION AND CHARACTERIZATION OF ISOENZYMES OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE IN HUMAN KIDNEY AND HEART, AND THE EFFECTS OF NEW CARDIOTONIC AGENTS ON THESE ISOENZYMES

The present study was done to identify and characterize the isoenzymes of cyclic nucleotide phosphodiesterase (PDE) and to determine their intracellular distribution in human kidney and heart. The in vitro effects of new cardiotonic agents, namely, NSP-805 (4,5-dihydro-5-methyl-6-[4-[(2-methyl-3-oxo-1-cyclopentenyl)amino] phenyl]-3(2H)-pyridazinone), TZC-5665 (6-[4-[2-[3-(5-chloro-2-cyanophenoxy)-2-hydroxypropylamino]-2-methylpropylamino]phenyl]-5-methyl-4,5-dihydro-3(2 H)-pyridazinone) and its metabolites, OPC-18790 ((±)-6-[3-(3,4-dimethoxybenzylamino)-2-hydroxypropoxy]-2-(1H)-quinolinone), MS-857 (4-acetyl-l-methyl-7-(4-pyridyl)-5,6,7,8-tetrahydro-3(2H)-isoquinolinone) and E-1020 (1,2-dihydro-6-methyl-2-oxo-5-(imidazo[1,2-a]pyridin-6yl)-3-pyridine carbonitrile hydrochloride monohydrate), on these human PDE isoenzymes were also investigated.PDE isoenzymes were separated from cytosolic and particulate fractions of homogenates of human kidney and heart by DEAE-Sepharose chromatography. PDE isoenzymes were identified by their elution characteristics, substrate specificities, sensitivities to regulation by effectors and by the use of isoenzyme-specific inhibitors.In a cytosolic fraction from kidney, Ca2+/calmodulin-dependent PDE (CaM-PDE), cyclic GMP-stimulated PDE (cGS-PDE), cyclic GMP-inhibited PDE (cGI-PDE) and two forms of cyclic AMP-specific PDE (cAMP-PDE) were resolved. One form of cAMP-PDE (cAMP-PDEα), which was eluted at a lower ionic strength than cGI-PDE during DEAE-Sepharose chromatography, was newly recognized in human tissues, though the other form (cAMP-PDEβ), which eluted later than cGI-PDE, had been previously isolated. Both of these cAMP-specific PDEs had similar properties in that they predominantly hydrolyzed cAMP with similar Km values for cAMP and were inhibited to almost equal extents by 3-isobutyl-l-methylxanthine (IBMX) but were hardly inhibited by cGMP. However, cAMP-PDEα was inhibited about 10 times more weakly (on the basis of IC50 values) by rolipram (4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone) and Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone than was cAMP-PDEβ. In a cytosolic fraction from heart ventricle, four distinct PDE isoenzymes, CaM-PDE, cGS-PDE, cGI-PDE and cAMP-PDEβ, were recognized. cAMP-PDEβ was the major component of the cAMP-hydrolyzing activity in the cytosolic fraction from human kidney, while CaM-PDE and cGI-PDE represented more than 90% of the total cAMP phosphodiesterase activity in the cytosolic fraction from human heart. In the particulate fractions from human kidney and heart, three activities, those of cGI-PDE and of two forms of cAMP-PDE, were identified. In kidney, cAMP/PDEα was the main cAMP-hydrolyzing PDE, while in heart cGI-PDE accounted for most of the activity. Furthermore, we evaluated the inhibitory effects on these human PDE isoenzymes of newly synthesized compounds with inotropic effects, namely, NSP-805, metabolites of TZC-5665 referred to as M-1 (6-(4-aminophenyl)-5-methyl-4,5-dihydro-3(2H)-pyridazinone) and M-2 (6-(4-acetyl-aminophenyl)-5-methyl-4,5-dihydro-3(2H)-pyridazinone), OPC-18790, MS-857 and E-1020. These drugs potently inhibited the activity of cGI-PDE and very weakly inhibited the activities of CaM-PDE and cGS-PDE. With respect to inhibitory effects on cardiac cAMP-PDEβ, there were some differences between the pyridazinone derivatives, for example NSP-805 and M-2, and the nonpyridazinone derivatives OPC-18790, MS-857 and E-1020. From the IC50 values, it was clear that the pyridazinone derivatives inhibited the activity of cGI-PDE at concentrations that were two to four orders of magnitude lower than that required for the inhibition of the activity of cAMP-PDEβ, while for the nonpyridazinone derivatives the difference was about one order of magnitude. The inhibition of the activity of human cardiac cGI-PDE by NSP-805, M2, OPC-18790, MS-857 and E-1020 was competitive with respect to cAMP with Ki values of 0.012, 0.32, 0.42, 1.3 and 0.15 μmol/l, respectively.These results suggest that there may be two isoforms of cAMP-PDE, which exist not only in the particulate fraction but also in the cytosolic fraction of human tissues, and that PDE inhibitors, which exert their cardiotonic effects by inhibiting cGI-PDE, have different selectivities with respect to the inhibition of the other human PDE isoenzymes.

Smooth Muscle Myosin Phosphatase

The endogenous phosphatase of chicken gizzard actomyosin contains two subunits of 58 and 38kDa, respectively, as we showed previously. This phosphatase was active with both isolated myosin light chain and intact myosin that had been phosphorylated by myosin light chain kinase as substrates, suggesting that phosphorylated myosin might be a substrate in situ. The 38-kDa subunit was identified as a catalytic subunit of a type lδ protein phosphatase (PP1δc), and the 58-kDa subunit was revealed to play a regulatory role in the binding of myosin. Studies with a monoclonal antibody (MoAb) to the 58-kDa subunit revealed that the 58-kDa protein was a product of proteolytic degradation of a protein of 130–133 kDa. The distribution of the 130-kDa component in chicken tissues was examined with the MoAb. An immunoreactive band of appropriate mobility was detected in analyses of all tissues except liver and skeletal muscle, and higher concentrations of the 130-kDa component were found in samples of smooth muscle. Intact myosin phosphatase was purified from chicken gizzard with the MoAb as probe. The purified holoenzyme was a heterotrimer that consisted of PP1bc (38kDa) and regulatory subunits of 130/133 kDa and 20 kDa. cDNA clones encoding the 130/133-kDa subunits were isolated from chicken gizzard cDNA libraries. The isolation of overlapping clones suggested the presence of the two isoforms. There were open reading frames of 2889 and 3012 bases that encoded proteins of 963 and 1004 amino acids with masses of 106.7 and 111.6 kDa, respectively. The deduced sequence of the larger isoform was 123 nucleotides longer in its central coding region than the other isoform. The characteristic N-terminal region was almost entirely composed of eight tandem repeats of 33 amino acids, which have been called the cdcl0/SWI6, or ankyrin, repeat. The 58-kDa fragment with the ability to bind to both myosin and the catalytic subunit was revealed to be located in the N-terminal half of the molecule. A cDNA clone (1338 bp) encoding chicken gizzard PPlδc was also isolated and sequenced. This clone contained an open reading frame that encoded a protein of 327 amino acids with a calculated molecular mass of 37kDa. Its amino acid sequence was identical to the analogous isoform from rats. These results suggest that smooth muscle myosin phosphatase is a novel holoenzyme composed of a type 1δ protein phosphatase and unique regulatory subunits.