Kazuhiko Oishi

Oxytocin contracts rat uterine smooth muscle in Ca2(+)-free medium without any phosphorylation of myosin light chain.

Contraction of rat uterine smooth muscle related to phosphorylation state of myosin light chain under various conditions was investigated. In the Ca2(+)-containing medium, both high K+ and oxytocin induced marked contraction of the muscle accompanied by pronounced phosphorylation of myosin light chain. In the Ca2(+)-free medium, although both vanadate and oxytocin induced slight contraction, phosphorylation of myosin light chain was only evident for vanadate but not for oxytocin. It was suggested that another mechanism distinct from myosin light chain phosphorylation might be involved in Ca2(+)-independent contraction of uterine smooth muscle elicited by oxytocin.

Role of the short isoform of myosin light chain kinase in the contraction of cultured smooth muscle cells as examined by its down-regulation

GbaSM-4 cells, smooth muscle cells derived from brain basilar artery, which express both 210-kDa long and 130-kDa short isoforms of myosin light chain kinase (MLCK), were infected with an adenovirus vector carrying a 1.4-kb catalytic portion of MLCK–cDNA in an antisense orientation. Western blot analysis showed that the expression of short MLCK was depressed without affecting long MLCK expression. The contraction of the down-regulated cells was measured by the cell-populated collagen-fiber method. The tension development after stimulation with norepinephrine or A23187 was depressed. The additional infection of the down-regulated cells with the adenovirus construct containing the same insert in a sense direction rescued not only the short MLCK expression but also contraction, confirming the physiological role of short MLCK in the contraction. To examine the role of long MLCK in the residual contraction persisting in the short MLCK-deficient cells, long MLCK was further down-regulated by increasing the multiplicity of infection of the antisense construct. The additional down-regulation of long MLCK expression, however, did not alter the residual contraction, ruling out the involvement of long MLCK in the contractile activity. Further, in the cells where short MLCK was down-regulated specifically, the extent of phosphorylation of 20-kDa myosin light chain (MLC20) after the agonist stimulation was not affected. This finding suggests that there are additional factors to MLC20 phosphorylation that contribute to regulate smooth muscle contraction.

Involvement of protein kinase C in Ca2+-independent contraction of rat uterine smooth muscle

Abstract The contribution of protein kinase C to the contraction by oxytocin of rat uterine longitudinal smooth muscle in Ca 2+ -free solution was investigated. Immunological analysis revealed that type II (β) and III (α) protein kinase C subspecies were present in rat uterine smooth muscle. The pretreatment of a diacylglycerol kinase inhibitor R59022 to accumulate diacylglycerol potentiated the Ca 2+ -independent contraction. The contractile activity was diminished with the depletion of protein kinase C, when the contraction was evoked repeatedly by oxytocin during the prolonged exposure to a tumor-promoting phorbol ester 12- O -tetradecanoylphorbol 13-acetate. These results suggested the involvement of protein kinase C in oxytocin-induced contraction in Ca 2+ -free solution.

Protein kinase C-independent sensitization of contractile proteins to Ca2+ in α-toxin-permeabilized smooth muscle cells from the guinea-pig stomach

Involvement of protein kinase C in receptor‐operated Ca2+ sensitization of cell shortening was investigated by use of α‐toxin‐permeabilized smooth muscle cells from the fundus of the guinea‐pig. Most of the isolated cells responded to 0.6 μm Ca2+ with a maximal shortening to approximately 65% of the resting cell length. Addition of acetylcholine (ACh) at a maximal concentration (10 μm) resulted in a marked decrease in the concentration of Ca2+ required to trigger a threshold response from 0.6 μm to 0.2 μm. The augmentation of Ca2+ sensitivity by ACh was not inhibited by specific protein kinase C inhibitors, calphostin C and K‐252b at a concentration of 1 μm. These findings suggest that protein kinase C is not involved in the muscarinic receptor‐operated augmentation of Ca2+ sensitivity.

Receptor‐coupled shortening of α‐toxin‐permeabilized single smooth muscle cells from the guinea‐pig stomach

1 Isolated single smooth muscle cells from the fundus of the guinea‐pig stomach were permeabilized by use of Staphylococcus aureus α‐toxin. Receptor‐coupled shortening of individual cells was monitored under phase contrast microscopy. 2 Most of the isolated cells responded to 0.6 μm Ca2+, but not to 0.3 μm Ca2+, with a resulting maximal shortening to approximately 65% of the resting cell length. The contractile activity of these permeabilized cells lasted for several hours and repeated shortening was readily achieved after washing out. 3 Addition of acetylcholine (ACh) at a maximal concentration (10 μm) resulted in a marked decrease in the concentration of Ca2+ required to trigger a threshold response from 0.6 μm to 0.2 μm, and 1 mm guanosine 5′‐diphosphate (GDP) blocked this decrease. Moreover, treatment with 100 μm guanosine 5′‐triphosphate (GTP) mimicked the action of ACh. 4 Addition of 100 μm inositol 1,4,5‐trisphosphate (InsP3) with 0.2 μm Ca2+ did not cause cell shortening, whereas 10 μm ACh with 0.2 μm Ca2+ did, suggesting that InsP3‐induced Ca2+ release is not involved in ACh‐operated cell shortening. 5 The present study demonstrates an α‐toxin‐permeabilized single smooth muscle cell preparation which retains its receptor function and also provides an insight into mechanisms leading to augmentation of Ca2+ sensitivity by stimulation of muscarinic receptors or GTP‐binding proteins.

Impaired neural differentiation of induced pluripotent stem cells generated from a mouse model of Sandhoff disease.

Sandhoff disease (SD) is a glycosphingolipid storage disease that arises from mutations in the Hexb gene and the resultant deficiency in β-hexosaminidase activity. This deficiency results in aberrant lysosomal accumulation of the ganglioside GM2 and related glycolipids, and progressive deterioration of the central nervous system. Dysfunctional glycolipid storage causes severe neurodegeneration through a poorly understood pathogenic mechanism. Induced pluripotent stem cell (iPSC) technology offers new opportunities for both elucidation of the pathogenesis of diseases and the development of stem cell-based therapies. Here, we report the generation of disease-specific iPSCs from a mouse model of SD. These mouse model-derived iPSCs (SD-iPSCs) exhibited pluripotent stem cell properties and significant accumulation of GM2 ganglioside. In lineage-directed differentiation studies using the stromal cell-derived inducing activity method, SD-iPSCs showed an impaired ability to differentiate into early stage neural precursors. Moreover, fewer neurons differentiated from neural precursors in SD-iPSCs than in the case of the wild type. Recovery of the Hexb gene in SD-iPSCs improved this impairment of neuronal differentiation. These results provide new insights as to understanding the complex pathogenic mechanisms of SD.

Carbachol-induced desensitization of rat basophilic leukemia (RBL-2H3) cells transfected with human m3 muscarinic acetylcholine receptors

1. Carbachol-induced homologous desensitization of the secretory response was investigated by transfecting RBL-2H3 cells with cDNA encoding the human m3 muscarinic acetylcholine receptor (RBL-m3). 2. Exposure of RBL-m3 cells to 100 microM carbachol for 30 min in Ca2+-free medium inhibited the secretion induced by the subsequent addition of 10 microM carbachol plus Ca2+. 3. Desensitized cells bound [3H]quinuclidinyl benzilate with a similar Bmax and Kd to those of control cells. 4. The carbachol-induced transient increase in levels of inositol 1,4,5-trisphosphate was not changed by desensitization. 5. Homologous desensitization persisted when desensitized cells were permeabilized with Staphylococcal alpha-toxin.

Protein kinase c promotes spontaneous relaxation of streptolysin-o-permeabilized smooth muscle cells from the guinea-pig stomach

Isolated single smooth muscle cells from the fundus of a guinea-pig stomach were permeabilized by use of streptolysin-O (0.5 U/ml). Most of the permeabilized cells responded to 0.6 microM Ca2+, but not to 0.2 microM Ca2+, with a resulting maximal cell shortening to approximately 71% of the resting cell length. These cells were relaxed again by washing with the Ca2+-free solution (2.5 nM free Ca2+) for 3-5 min. Addition of 10 microM acetylcholine (ACh) resulted in both a marked decrease in the concentration of Ca2+ required to trigger a threshold response and an increase in the maximal cell shortening, indicating that the cells retained the muscarinic receptor function. When the cell treated with a protein kinase C (PKC) inhibitor, K-252b (1 microM), for 3 min was exposed to 10 microM ACh in the presence of K-252b, the cell shortened within 2 min with a maximal cell shortening. When the cell shortening was induced by 10 microM ACh plus 1 microM Ca2+ in the presence of K-252b (1 microM) or more selective PKC inhibitors, such as calphostin C (1 microM) or PKC pseudosubstrate peptide (100 microM), the extension of the shortened cells, by washing with the Ca2+-free solution, was significantly inhibited. In contrast, K-252b (1 microM) did not inhibit the relaxation of Ca2+-induced shortened cells. These results suggest that the receptor-mediated activation of PKC in the process of ACh-induced cell shortening plays a role in the subsequent relaxation of the shortened cells.

FcεRI-stimulated Ca2+-dependent secretion from rat basophilic leukemia (RBL-2H3) cells permeabilized with Staphylococcal α-toxin: FcεRI-operated signals are not mimicked by the actions of GTPγS

Abstract 1. RBL-2H3 cells permeabilized with α-toxin responded to dinitrophenol (30–40 mol/mol)-conjugated human serum albumin, as antigen, to secrete [ 14 C]serotonin in the micromolar range of free Ca 2+ . 2. Calcium ion alone did not cause substantial secretion. 3. Guanosine 5′- O -(3-thiotriphosphate) (GTP γ S) (100 μM) in combination with Ca 2+ produced only negligible [ 14 C]serotonin secretion. 4. GTP γ S, in the presence of cytochalasin D, caused optimal secretion of [ 14 C]serotonin in a Ca 2+ -dependent manner.

Actin-severing and Ca2+-induced reversal of smooth muscle contraction that is independent of Ca2+

1. Intracellular actin filament organization of gastric smooth muscle cells of the guinea pig in primary culture was examined with rhodamine-labelled phalloidin using a confocal laser fluorescence microscope. 2. The resting cells, both in the presence and absence of Ca2+, showed an even distribution of microfilamentous actin fibers. 3. The characteristic image of the stimulated cells with 10 microM acetylcholine in the presence of 1.8 mM Ca2+ was that the actin filaments were located only on the periphery of the cell. 4. The characteristic image of the cells stimulated as above, but in the absence of Ca2+, was that the actin filaments were unevenly distributed in the cell. 5. The characteristic image of the cells stimulated in the presence of 1 microM Ca2+, which inhibits the above contraction, was pultaceous with the actin filaments absent, indicating severing of actin filaments by a Ca(2+)-activated system, such as gelsolin.