Ikuo Maruyama

Improvement of a procedure for preparing single smooth muscle cells from guinea pig tenia coli by purified collagenase and papain

Procedures for isolating single smooth muscle cells from tenia coli of guinea pigs have been developed. The cells were isolated with a combination of highly purified collagenase and a small amount of strong peptidase, papain, in the presence of Ca. This combination resulted in approximately a 100-fold yield of the single cells as compared with the ordinary method in which only collagenase preparations contaminated with various peptidases were used. More than 95% of the single cells were viable when examined by the trypan blue exclusion technique. In addition, this combination greatly increased the responsiveness to agonists such as carbachol (CCh). A concentration of CCh for the half maximum response for this preparation was about one five-hundredth that of cells prepared by the ordinary method. This value was approximately one-quarter that of intact tissue. This preparation can be used to investigate the physiology, pharmacology, and biochemistry of smooth muscle.

Conflicting effects of noradrenaline on ciliary movement of frog palatine mucosa

The effects of noradrenaline on ciliary movement of frog palatine mucosa were investigated using the particle transport method. Prostaglandins and cyclic AMP concentrations in the mucosa were determined by radioimmunoassay. Concentrations of noradrenaline below 10(-7) M suppressed ciliary movement by about 40% as compared to the control level. The changeover from inhibition to acceleration of ciliary movement occurred at 10(-7) M noradrenaline. Ciliary movement was markedly accelerated by concentrations of noradrenaline over 2 X 10(-6) M. This acceleration was eliminated in the presence of inhibitors of prostaglandin biosynthesis such as indomethacin (14 microM) and aspirin (25 microM). The acceleration was not eliminated by an inhibitor of thromboxane A2 biosynthesis (OKY-1555). On the other hand, E type prostaglandin release was increased about 8 fold of control by treatment with 5 X 10(-5) M noradrenaline, while F type prostaglandin release was not affected by the treatment with 5 X 10(-5) M noradrenaline. Ciliary movement was markedly accelerated when prostaglandin E1 was administered. The concentration of cyclic AMP was increased about 2.1 fold by treatment with 5 X 10(-5) M noradrenaline. The possible mechanism of the effects of noradrenaline on ciliary movement is discussed in the light of these results.

The role of serotonin in mucociliary transport system in the ciliated epithelium of frog palatine mucosa

The effects of serotonin (5-hydroxytryptamine, 5-HT) on mucociliary transport and mucus secretion were investigated pharmacologically. The presence of 5-HT in the ciliated epithelium of frog palatine mucosa was examined chromatographically. Mucociliary transport was accelerated by the application of 10(-6) M 5-HT. Mucus secretion was also stimulated significantly by 5-HT (30 mg/kg). 5-HT in the mucosa was detected by a chromatographic technique when a large amount of 5-HT precursor, 5-hydroxytryptophan (5-HTP), was given previously. Phenylhydrazine was used as MAO inhibitor. When phenylhydrazine (20 mg/kg) was given before 5-HTP, 5-HT was markedly concentrated in the mucosa. Radioactive 5-HT was also detected in the mucosa after incubation with [14C]5-HTP. One hour after systemic administration of [3H]5-HTP, a large number of silver grains appeared in the autoradiograms of some epithelial cells. These results suggest the possibility that epithelial cells with the ability to synthesize 5-HT from 5-HTP exist in ciliated epithelium. A possible functional relationship is proposed between the regulation of mucociliary transport with mucus secretion and 5-HT-containing cells.

Dopaminergic innervation and inhibition of ciliary movement in the ciliated epithelium of frog palatine mucosa

The effect of dopamine on ciliary movement and the existence of dopamine-containing cells in the ciliated epithelium of frog palatine mucosa were investigated pharmacologically and by means of electron and fluorescence microscopy, respectively. Ciliary movement was suppressed markedly when the perfusion medium contained 10(-6) M dopamine, thereby indicating that dopamine has a suppressive effect on ciliary movement. Electron microscopy revealed at least two types of granule-containing (GC) cells, type 1 cells containing small spherical granular vesicles (about 100-150 nm in diameter) and type 2 cells containing elongated dense granules (about 150 X 250 nm in diameter). One hour after systemic administration of dihydroxyphenylalanine-[3H(G)] [( 3H]DOPA), a large number of silver grains appeared only in type 2 GC cells, thus indicating that type 2 cells are catecholamine-storing. Blue-green fluorescent cells were detected in the ciliated epithelium. On microspectrofluorometry, catecholamine fluorescence in the cells showed a main excitation and emission maxima at 415 and 480 nm, respectively. When the fluorescent cells were exposed to HCl vapor for several s, the excitation peak shifted to 380 nm and this peak was unchanged after treatment for an additional 5 min. It may be considered that these fluorescent cells contain dopamine and correspond to type 2 GC cells. A possible functional relationship between regulation of ciliary movement and dopamine-containing cells was also discussed.

Effect of DFP on loading of fura 2/AM and quin 2/AM into single smooth muscle cells prepared from guinea pig taenia coli

The effect of diisopropyl fluorophosphate (DFP), a potent cholinesterase (ChE) inhibitor, on loading quin 2 acetoxymethyl ester (quin 2/AM) and fura 2/AM into smooth muscle cells isolated from guinea pig taenia coli was investigated spectrofluorometrically. The presence of DFP during the loading permitted the incorporation of quin 2 into the cells, so that it became possible to measure intracellular Ca2+ concentrations using the ester of this dye. Also, DFP significantly enhanced the incorporation of fura 2 into the cells. These results indicate that loading of quin 2/AM and fura 2/AM into the smooth muscle cells may depend on the suppression of ChE or various serine protease activities outside cells.

Photochemical activities of chloroplasts treated with diazonium-1,2,4-triazole

Chloroplast membranes are composed of two major chlorophyll particles, one enriched in chlorophyll a and the other in chlorophyll b. Many works on the chlorophyll particles have shown that the particles enriched in c~orophyll Q and b are correlated with phot~hemical system I and II, respectively [l-6] . We have previously shown that diazonium1,2,4-triazole reacted preferentially with the particle enriched in chlorophyll a and inferred that the particles responsible for the photochemical system I may be located on the external surface of the grana membrane and more reactive with the diazoni~ compound [7--93. The same conclusion was reached by Dilley et al. f lo] who treated chloroplast with a non-penetrating 3s S-p(diazonium)benzene sulfonic acid and found that photochemical system I but not system II was readily labeled with this reagent. Since diazonium compound reacts with a number of amino acid residues such as histidine, tyrosine and lysine in proteins, the coupling reaction may result in the inactivation of the modified protein molecule. Thus it is to be expected that the photochemical activities of system I are decreased by the treatment of chloroplasts with diazonium-i ,2,4-triazole. The present study was undertaken to examine this possibility.

[Digestant effects of a new digestive enzyme capsule, Excelase, on jejunectomized and pancreato-jejunectomized Beagle dogs (author's transl)].

The digestant effects of a new digestive capsule, excelase containing sanactase, proctase, meicelase, olipase-2S and pancreatic digestive enzyme TA, were investigated in vivo. Jejunectomy and pancreato-jejunectomy were performed to cause an artificial disturbance of gastro-intestinal digestion and absorption in Beagle dogs. Absorption of protein and fat was measured using chromic oxide as an indicator. Excelase (3 capsules/dog/day) was given orally to Beagle dogs 1 week after each operation for 7 weeks. Changes in body weight and absorption of protein and fat were observed during the administration. The decrease in body weight of dogs treated with excelase fully recovered, however, that of controls remained even 8 weeks after the surgery. Absorption of protein and fat in the groups of dogs treated with excelase was greatly improved as compared with controls. The digestant effects of excelase on percent absorption of protein and fat were more manifest in pancreato-jejunectomized dogs than in jejunectomized dogs. These results indicate that excelase is effective for gastro-intestinal disturbances of digestion and absorption. The digestant effects of excelase on starch, protein and cellulose were also investigated in vitro using a gastro-intestinal model.