Sadao Miyata

A simple procedure for the extraction of the native chromophore of visual pigments : The formaldehyde method

The chromophore of visual pigments was efficiently extracted in its original isomeric conformation by dichloromethane/hexane in the presence of excess formaldehyde. This new method of extraction (the formaldehyde method) in combination with HPLC is useful for the determination of the chromophore composition of visual pigments. The chromophore isolated by this method is biologically active and can be used for regeneration experiments.

Effects of H-7 (protein kinase inhibitor) and phorbol ester on aortic strips from spontaneously hypertensive rats

We investigated the vascular responsiveness to vasoactive agents and the inhibition by H-7 (1-(5-isoquinoline-sulfonyl)-2-methylpiperazine), which inhibits cyclic nucleotide-dependent protein kinases and protein kinase C(PKC) equally potently in helically cut strips of thoracic aortas from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The susceptibility of norepinephrine (NE)- and angiotensin II (Ang II)-induced contractions to H-7 was significantly higher in the aortas from SHR than in those from WKY. H-7 decreased the contractile responses to KCl to a similar extent in both strains without affecting the high K(+)-stimulated Ca2+ influx. H-7 produced a shift to the right of the dose-response curve for the PKC activator, 12-o-tetradecanoylphorbol-13-acetate (TPA) in the case of SHR aortas, while no such shift was noted in tissues from WKY. Functional alterations in the PKC branch of the Ca2+ messenger system in vascular smooth muscle may play an important role in SHR during the sustained contraction.

Competition between retinal and 3-dehydroretinal for opsin in the regeneration of visual pigment

Rhodopsin regenerated faster than porphyropsin in all preparations of bullfrog opsin, bullfrog rod outer segment membrane and cattle opsin. When opsin was incubated with excess amount of an equimolar mixture of 11-cis-retinal and 11-cis-3-dehydroretinal, the composition of the regenerated pigment was simply dependent on the ratio of regeneration rates of rhodopsin and porphyropsin. This result can provide a mechanism to account for the discrepancy in vitamin A1/A2 composition between the retina and the pigment epithelium. The property of opsin preferring retinal to 3-dehydroretinal may be one of the basic factors affecting vitamin A1/A2 visual pigment systems.

Spatial Distribution of Visual Pigment and Dopamine in the Bullfrog Retina.

Visual pigments and a neurotransmitter, dopamine, were quantitatively investigated in the retina of adult bullfrog, Rana catesbeiana. The adult bullfrog (body length 15-16 cm, body weight 375 +/- 52 g, n = 10) had 21.4 +/- 4.2 nmol visual pigment and 209 +/- 28 pmol dopamine in retinal areas of 266 +/- 27 mm2. Greater pigment densities were recorded in a semicircular band around the optic disc, extending to the nasal and temporal peripheries of the ventral retina. The area with the highest concentration of visual pigment was found in the middle of the dorsal retina, 3-4 mm dorsal to the optic disc. A high concentration of vitamin A2-based pigment was found in the dorsal quarter of the retina (porphyropsin zone); the zone extended up to the most ventral part along peripheral regions of the retina. There was also a band with higher dopamine concentrations although it was not so prominent as that of the visual pigment; the highest concentration of dopamine was found in the area immediately dorsotemporal to the optic disc. Fluorescence micrography indicated that the distribution pattern of catecholamine-containing amacrine cells paralleled that of the dopamine content. The topographic map of dopamine was slightly different from that of visual pigment in the bullfrog retina.

Calcium dependence of ouabain-induced contraction in aortas from spontaneously hypertensive rats

The calcium sensitivity of ouabain-induced contractions of aortic strips from spontaneously hypertensive rat (SHR) was examined using several drugs which affect Na+ and Ca2+ movements across the cell membrane, and the results were compared with those obtained with age-matched Wistar-Kyoto rat (WKY). The Ca2+ concentration-response curves (10(-3) M ouabain-treated preparations) made with aortic strips from SHR lay to the left of those made with aortic strips from WKY (Ca EC50 values: SHR, 0.51 +/- 0.16 mM, n = 6; WKY, 1.23 +/- 0.41 mM, n = 7; P less than 0.05). Amiloride (a Na+ entry blocker) and nifedipine (a Ca2+ entry blocker) attenuated the sensitivity to Ca2+ of SHR and WKY aortic strips. With 2 x 10(-4) M amiloride, WKY vessels showed a 1.3-fold increase in the Ca EC50 value and SHR a 2.1-fold increase. With 10(-6) M nifedipine. WKY vessels showed a 1.1-fold increase in the Ca EC50 value and SHR a 1.5-fold increase. Addition of monensin (Na ionophore) produced a dose-dependent potentiation in ouabain-treated aorta from WKY, but not in ouabain-treated aorta from SHR. Addition of 1.5 x 10(-5) M A23187 (Ca ionophore) eliminated the difference between the Ca2(+)-induced contractions in aortas from SHR and WKY. These results suggest that enhancement of Ca2+ influx by Na(+)-Ca2+ exchange and/or voltage-dependent Ca2+ channels in vascular smooth muscle cell membranes may be an important factor in the difference between ouabain-induced contractions in aorta from SHR and WKY.

Ouabain-induced contraction of vascular smooth muscle in spontaneously hypertensive rats and the effect of hydralazine

The effect of ouabain (10(-3) M) on contractile responses of SHR (spontaneously hypertensive rat) and WKY (Wistar-Kyoto rat) aortas and mesenteric arteries was studied. Ouabain addition caused a rapid contraction of aortic strips with a steeper rate of rise and a larger maximal force development in strips from SHR than WKY. This difference in contractile response is known to occur in the prehypertensive period of SHR (4-week-old). Phentolamine (10(-6) M) pretreatment had no effect on the ouabain-induced contraction but partially suppressed it in both SHR and WKY aortas when diltiazem (10(-5) M) was also added. The difference in the ouabain-induced contractions of SHR and WKY aortas was more apparent in the residual contraction during suppression by diltiazem. The 45Ca uptake in the presence of ouabain was significantly larger in the early period of incubation in SHR aorta than in WKY aorta. The ouabain-induced contraction of hydralazine-treated SHR aorta from the prehypertensive period was very similar to that of non-treated WKY aorta. These results suggested that the abnormality of the ouabain-induced contraction in SHR arterial smooth muscle could have arisen from an increased Ca2+ movement due to Ca2+ leakage when ouabain inhibited the Na+-pump in the membrane. This abnormality seems to start during the prehypertensive period and continue in the hypertensive stage.

3-hydroxyretinol in the rhodopsin-porphyropsin retina of an invertebrate (Procambarus clarkii)

Abstract 1. 1. A polar retinoid was found in the crayfish retina which has the rhodopsin-porphyropsin visual pigment system. Analyses with high-performance liquid chromatography and spectroscopy indicated that this compound was 11- cis 3-hydroxyretinol. 2. 2. The 3-hydroxyretinol existed in esterified forms and accounted for 2–9% of the total retinols stored in the retina. The 3-hydroxyretinol is not the chromophore precursor of visual pigment because no 3-hydroxyretinal (aldehyde) was present in the retina. 3. 3. The 3-hydroxyretinol appeared in the retina when the animal was kept at low temperature (10°C) and disappeared at high temperature (25°C). 4. 4. These results suggest that the 3-hydroxyretinol is an intermediate in the conversion of retinol to dehydroretinol.

Substitution of Porphyropsin for Rhodopsin in Mouse Retina

Weanling, male albino mice were placed on a vitamin A-free diet for three months to deplete their vitamin A stores. The vitamin A-deficient mice were injected intraperitoneally with all-trans 3-dehydroretinol. 3-dehydroretinol was rapidly incorporated into the liver as a fatty acid ester. The chromophore of visual pigment increased gradually and reached a normal level 13 days after the injection. 3-Dehydroretinal accounted for 95% of the total chromophore in the retina. The high proportion of 3-dehydroretinal was observed also in the long-term experiment which was continued for six weeks with the injection of 3-dehydroretinol once a week. When the animal was injected with a mixture of 3-dehydroretinol and retinol, the ratio of dehydroretinal/retinal in the retina was far lower than the ratio of dehydroretinol/retinol in the liver. These results indicate that 3-dehydroretinol is not converted to retinol in mouse and is used less efficiently than retinol for the chromophore of visual pigment. The synthesis of visual pigment was observed even when the animal was kept in complete darkness after the injection of all-trans 3-dehydroretinol. This fact indicates that light is not required for the production of 11-cis chromophore of visual pigment.

Rearing and Learning Behaviors of Rats After Long-Term Administration of Nicotine

The effects of long-term oral administration of nicotine (N; 0.01%) on the rearing response and the complex T-maze learning of WKY rats were investigated. N slightly increased the locomotion, and increased the rearing response more than the control (C) rats in the night. During daily 6-trial T-maze learning for 4 days, N enhanced the learning in both the number of errors and the latency measures. Scopolamine (40mg/kg) administered on the 5th session reduced the T-maze performance in the C rats, but not in the N animals.