Kazuo Nagai

Modification of dopamine release by nociceptin in conscious rat striatum

Nociceptin (NOC), an endogenous ligand for the orphan receptor ORL1, has recently been recognized as a neuropeptide. We used brain microdialysis and on-line high performance liquid chromatography to examine the effect of NOC on the basal outflow of dopamine (DA) and its metabolite in the freely moving rat striatum in vivo. The percent change of DA release induced by NOC at concentrations of 10-6 and 10-5 M were 383% and 398%, respectively. This effect of NOC was attenuated by naloxone, suggesting that NOC activates classic (micro, delta, kappa) receptors in a very little way. These data indicates that NOC may act as a neuropeptide which enhances DA release from the striatum of rat brain via an opioid receptor.

Phosphatidyl inositol-phospholipase C in squid photoreceptor membrane is activated by stable metarhodopsin via GTP-binding protein, Gq

Phosphatidyl inositol-phospholipase C (PI-PLC) in squid retina was studied by immunoblotting and its activities were determined using [3H]phosphatidyl inositol bisphosphate ([3H]PIP2) as substrate. PI-PLC activity was found mostly in soluble fraction when the retina homogenate was treated with 400 mM KCl, but was associated with rhabdomal membranes under low salt conditions (20 mM Hepes). A protein with apparent molecular mass of 130kD was recognized by an antibody against PLC beta 4/norp A in both 400 mM KCl soluble and rhabdomal membrane fractions. A 42 kD protein recognized by antibody against the C-terminus of Gq alpha was also present in these two fractions. GTP gamma S stimulated only the PI-PLC activity associated with membrane and was magnesium dependent. PI-PLC activity was found to be (i) highly dependent upon calcium concentrations, (ii) enhanced by GTP but not by other nucleotides, and (iii) significantly stimulated by light at lower concentrations of GTP gamma S. The stimulation by light was still observed when irradiated membrane was incubated at 10 degrees C for 10 min and then mixed with GTP gamma S. These results suggest that stable metarhodopsin stimulates a PLC beta 4/norp A-like enzyme via a G-protein, Gq.

Squid photoreceptor phospholipase C is stimulated by membrane Gqα but not by soluble Gqα

Phospholipase C (PLC) was purified from squid retina. Soluble Gqα, membrane Gqα and Gβγ were isolated from GTPγS‐treated and light‐illuminated photoreceptor membranes. The membrane Gqα stimulated phosphatidyl inositoi‐phospholipase C (PI‐PLC) activity in a dose‐dependent manner. Soluble Gqα and membrane Gβγ showed no stimulating effects on PLC. GTPγS‐binding was found exclusively in membrane fraction, with very little present in the KCl‐soluble fraction which contained soluble Gqα. These results indicate that light‐activated rhodopsin activates PLC through membrane‐bound Gqα and suggest that the rhodopsin/Gq/PLC cascade might be the pathway of phototransduction in squid photoreceptors.

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.

Modification of acetylcholine release by nociceptin in conscious rat striatum.

Nociceptin (NOC), an endogenous ligand for the orphan opioid receptor ORL1 (ORL1), has recently been recognized as a neuropeptide. We used brain microdialysis and on-line high performance liquid chromatography (HPLC) to examine the effect of NOC on the basal outflow of acetylcholine (ACh) in the freely moving rat striatum in vivo. ACh release was reduced by nociceptin at a concentration of 10(-5) M to 79% of control release. This effect of NOC was attenuated by [Phe1Psi(CH2-NH)Gly2]nociceptin-(1-13)-NH2 (PhePsi), suggesting that NOC activates the ORL1 receptor and (PhePsi) acts as an antagonist on ORL1 in rat striatum in vivo. These findings indicate that NOC may act as a neuropeptide which inhibits ACh release in the striatum via ORL1.

Endothelin-3 modification of dopamine releases in anaesthetised rat striatum; An in vivo microdialysis study

Endothelin-3 (ET-3), a member of the vasoconstrictive peptide family, has recently been recognized as a neuropeptide. We used brain microdialysis and on-line HPLC to examine the effect of ET-3 on the basal outflow of monoamines and their metabolites in the ketamine-anaesthetised rat striatum in vivo. Although intrastriatal infusion of ET-3 (40 pmol/rat) did not change basal dopamine (DA) release, after perfusion of DA releasing agent (5 x 10(-5) M ouabain or 120 mM KCl), ET-3 could increase the DA level. Further, these effects of ET-3 were attenuated by calcium-free Ringer. These data indicated that ET-3 may act by modifying the exocytosis from the striatum of rat brain to enhance DA release after depolarization induced by an agent such as KCl or ouabain.

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.

The isolation of the green rod pigment of the frog, rana catesbeiana

Green rods account for 8% of the total rod population in a frog retina (Rana temporaria) and absorb the blue light [ 11. The h,, of green rod pigment was determined to be 430-440 nm by the two steps partial bleaching analysis of the extract from outer segments with digitonin [2-41. The protein of green. rod pigment may be like the cone pigment because green rod pigment regenerates more quickly than red rod pigment, rhodopsin, and is destroyed by hydroxylamine [3-51. However, the properties of green rod pigment was not studied sufficiently because the extract of outer segments contained green rod pigment below 10%. Here, we report the separation of green rod pigment of bullfrog and some properties of this pigment.

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.