Masaatsu K. Uchida

Inhibition by calcium antagonists of histamine release and calcium influx of rat mast cells: difference between induction of histamine release by concanavalin A and compound 48/80.

The relation between calcium influx and histamine release from rat mast cells was investigated. When purified mast cells pretreated with a calcium antagonist (MnCl2 or methoxyverapamil (D-600)) were exposed to concanavalin A or compound 48/80 in Tyrode solution (pH 7.4) at 37 degrees C, the calcium antagonists inhibited the extracellular calcium-dependent component of concanavalin A-induced histamine release. MnCl2 also inhibited the extracellular calcium-dependent component of compound 48/80-induced histamine release, whereas D-600 did not inhibit the release. D-600 inhibited the 45Ca uptake induced by concanavalin A, but did not inhibit the 45Ca uptake induced by compound 48/80. It was found that the inhibitory action of calcium antagonists depended on the uptake of extracellular calcium. These observations suggest that concanavalin A and compound 48/80 stimulate different mechanisms of calcium influx. Studies on inactivation of the mechanisms of calcium influx showed that calcium influx into cells activated by concanavalin A stopped when concanavalin A was washed out, whereas the influx activated by compound 48/80 was still operative after compound 48/80 had been washed out.

Oxytocin-induced Ca-free contraction of rat uterine smooth muscle: effects of preincubation with EGTA and drugs.

1. The effect of the time of preincubation with 3 mM EGTA on oxytocin-induced Ca-free contraction of rat uterus was investigated. It was found 10(-2) unit/ml oxytocin induced sustained Ca-free contraction only after preincubation for 50 min or more. 2. The tension developed showed a biphasic relation to the time of preincubation with a minimum at 25 min. 3. Sustained Ca-free contraction could be repeated with only slight reduction in magnitude. 4. Oxytocin-induced Ca-free contraction was relaxed by isoproterenol. The effect of isoproterenol was inhibited by 3 X 10(-4) M 1,1-diphenyl-3-piperazino-1-butanol hydrochloride.

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.

Identification of NDRG1 as an early inducible gene during in vitro maturation of cultured mast cells

Coculture of mouse bone marrow-derived mast cells (BMMC) with fibroblasts in the presence of stem cell factor (SCF) facilitates morphological and functional maturation toward a connective tissue mast cell (CTMC)-like phenotype. By means of cDNA subtraction, we identified several inducible genes during this mast cell maturation process. Of approximately 100 sequenced clones induced, nearly 50% were chromosome 14-associated serine proteases. Approximately 14% encoded NDRG1, a 43-kDa cytosolic protein that has been implicated in cell differentiation. NDRG1 was distributed in the cytosol of cultured mast cells and CTMC in rat skin. Overexpression of NDRG1 in RBL-2H3 cells resulted in enhanced degranulation in response to various stimuli. Thus, NDRG1 may be a mast cell maturation-associated inducible protein that allows the cells to be susceptible to extracellular stimuli leading to degranulation. Additionally, several unique maturation-associated inducible genes were identified, molecular and functional characterization of which will provide new insights into mast cell biology.

Effects of bergenin on experimental ulcers—Prevention of stress induced ulcers in rats

Abstract 1. 1. The effects of bergenin on various experimental ulcers were tested. Bergenin is a constituent of folk medicines for gastritis and gastric ulcers. 2. 2. It was effective in preventing stress-induced gastric ulcers (30 mg/kg i.v.). 3. 3. One of the mechanisms of its effectiveness may be inhibition of acetylcholine release, which induces gastric acid secretion and enhances gastric motility, both being well known ulcerogenic factors.

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.

Manganese-induced contraction in K+-depolarized rat uterine smooth muscle

Abstract 1. 1. Manganese induced biphasic contraction of K+-depolarized myometrium. The first phase, evoked by lower concentrations of 10−3–10−2 M, was affected by treatments causing calcium-loading or calcium-depletion of the cells. The second phase, evoked by a higher concentration of 3 × 10−2 M, was not affected by these procedures. 2. 2. A difference in the manganese requirements of the two phases of contraction was demonstrated using a calcium antagonist, D-600.

Influence of pH on the inhibitory effects of local anesthetics on histamine release induced from rat mast cells by concanavalin A and compound 48/80.

The study concerned the effects of lidocaine and mepivacaine on rat mast cell histamine release. At low concentrations these drugs inhibited the histamine release induced by concanavalin A and compound 48/80 although, at high concentrations, they caused cell lysis. The mechanism of their inhibition of histamine release was studied by examining the pH dependence of the inhibitory action. In the absence of drugs, the release of histamine was not affected by the pH of the medium, but the inhibitory effects of the drugs increased with increase in pH. The percent inhibition of histamine release by these drugs at each pH was plotted against the calculated concentration of the nonionized form of the drug, according to the Henderson-Hasselbalch equation. The inhibitory action was correlated with the concentration of nonionized molecules, which readily penetrate the cell membrane. From the interaction of lidocaine with the phospholipid bilayer, it was concluded that this drug penetrates the lipid bilayer and that this effect increases with increasing pH of the medium. Thus, it seems that it is the nonionized form of local anesthetics that inhibits histamine release and that it is nonionized molecules that penetrate the mast cell membrane.

Effects of calmodulin inhibitor on histamine release from rat peritoneal mast cells induced by concanavalin A and ionophore A23187

The involvement of calmodulin in stimulus-secretion coupling of histamine release from rat mast cells was examined using a specific calmodulin inhibitor, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide]. W-7 at low concentrations inhibited histamine release induced by either concanavalin A or ionophore A23187 and at high concentrations enhanced the release. But its congener, W-5 [N-(6-aminohexyl)-1-naphthalenesulfonamide], which has virtually no calmodulin-inhibiting activity, did not affect the release. It is concluded that W-7 modulates histamine release by inhibiting some functions of calmodulin in the cells.