Keiji Yanagisawa

Taurine and β-alanine act on both GABA and glycine receptors in Xenopus oocyte injected with mouse brain messenger RNA

The responding pathway (process from agonist binding to channel opening) of taurine and beta-alanine was investigated in Xenopus oocytes injected with mouse brain poly(A)+ RNA. Responses to gamma-aminobutyric acid (GABA), glycine, taurine and beta-alanine were induced in oocytes injected with poly(A)+ RNA extracted from 3 regions, cerebrum, cerebellum and brainstem of the mouse brain. From comparison, responses to these 4 inhibitory amino acids in each regional poly(A)+ RNA-injected oocytes were categorized into at least 3 groups: (1) GABA, (2) glycine, and (3) taurine and beta-alanine. No cross-desensitization was observed between GABA response and glycine response, but taurine and beta-alanine responses cross-desensitized both the GABA and glycine responses. Taurine and beta-alanine responses were partially inhibited by the GABA antagonist, bicuculline, and also by the glycine antagonist, strychnine. The results suggest that the taurine or the beta-alanine response in the brain is caused through both the GABA receptor and the glycine receptor.

Quantitative changes in the mRNA for contractile proteins and metabolic enzymes in masseter muscle of bite-opened rats.

To study the effects of bite opening on the fibre phenotypes of rat masseter, the mRNAs of four predominant myosin heavy-chain isoforms (MHC I, IIa, IId/x and IIb) and two alkali light-chain isoforms (LC1f and 3f) as well as those of two metabolic enzymes, carbonic anhydrase III (CAIII, oxidative enzyme) and glucose-phosphate isomerase (GPI, glycolytic enzyme), were measured in relation to the total RNA of masseter muscle by competitive, reverse transcriptase-polymerase chain reaction in control and bite-opened rats. Bite opening (2.8 mm increase in the vertical dimension for 1 week) significantly (P<0.05) increased the amount of MHC IIa mRNA but decreased (P<0.001) the amount of MHC IIb mRNA without changing the amount of MHC IId/x mRNA. No MHC I mRNA was found in any masseter studied. A significant (P<0.01) increase in the mRNA of LC1f associated with a decrease (P<0.05) in that of LC3f was observed after the bite opening. The CAIII mRNA increased significantly (P<0.001), while the GPI mRNA decreased (P<0.05) in association with the bite opening. These results strongly suggest that in 1 week of bite opening changes the rat masseter muscle from a glycolytic, MHC IIb-LC3f-dominant fibre to an oxidative, MHC IIa-LC1f-dominant fibre.

Adrenaline increases the rate of cross-bridge cycling in rat cardiac muscle

To characterize the myocardial cross-bridge dynamics in catecholamine-induced positive inotropic state, we studied the effects of adrenaline (6 X 10(-6) M) on the transient central segment length (SL) response to step decrease in tension in rat right ventricular papillary muscle in barium contracture. The time course of this response is thought to reflect the kinetics of actin-myosin interaction. The muscle was released stepwise from the steady contracture tension (Tc) to new steady tension levels (Tr) of varying magnitudes at 22 degrees C. When the tension decrease was less than 0.7 Tc, the SL transient responses comprised, in most cases, four phases. The first phase was a rapid and minute shortening during tension reduction; the second was a slow further shortening; the third, a slow lengthening; and the fourth, an extremely slow shortening toward a new steady length under the new tension. Adrenaline showed almost no effect on Tc and the amplitude of SL transients, but markedly reduced the duration of the second (D2) and third (D3) phases of SL transient regardless of the amplitude of tension reduction. The reduction of duration was 14 +/- 3% in D2 and 26 +/- 5% in D3 at Tr/Tc of 0.84 +/- 0.03 on the average (mean +/- S.D.) in nine preparations. The velocity measured from the quasi-steady SL shortening in the second phase increased with the addition of adrenaline, regardless of the amplitude of tension reduction. The increase in the shortening velocity was 16 +/- 6% (mean +/- S.D., n = 9) at Tr/Tc of 0.18 +/- 0.04. These results suggest that adrenaline increases the rate of cross-bridge cycling in cardiac muscle independent of activation level.

Mechanical properties and ATPase activity in glycerinated cardiac muscle of hyperthyroid rabbit

Isometric tension, tension transients in response to rapid step stretches in length and ATPase activity were measured at constant levels of various Ca2+ activations in glycerinated right ventricular papillary muscle ofl-thyroxine-treated (14 daily injections of 0.2 mg/kg) and control rabbits. The isometric tension increased sigmoidally as Ca2+ was varied from slightly below pCa 7 to about pCa 6 both in thyroxine-treated and control preparations. The maximum isometric tension in thyroxine-treated preparations, however, was only about 66% of that in control. The tension transients were characterized by clear three distinct phases; the first phase of an immediate tension increase coincident with the stretch, the second phase of a rapid quasi-exponential tension decrease and the third phase of a delayed quasi-exponential tension rise. In thyroxine-treated preparations, relative to controls, the time for 63% tension reduction in the second phase decreased from 39.3±2.8 ms (mean ± SD,n=5) to 20.2±2.0 ms (P<0.001) and the time for 63% tension rise in the third phase decreased from 483.9±14.3 ms to 298±15.9 ms (p<0.001). The ATPase activity increased in a sigmoid fashion with increasing Ca2+ from slightly above pCa 7 to slightly below pCa 6 both in thyroxine-treated and control preparations. However, the tension cost (ATPase activity/tension) was about two times greater in the tyyroxine-treated preparations than in controls.

Adaptation of Guinea-pig superficial masseter muscle to an increase in occlusal vertical dimension

To study the effect of increased occlusal vertical dimension on the fibre phenotypes of the superficial masseter muscle, the composition of myosin heavy-chains (MHC), myosin light-chains (MLC) and tropomyosin was investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis in conjunction with densitometric analysis in normal (control) and bite-opened (5.7 mm increase in the vertical dimension for 1 week) guinea-pigs. The superficial masseter contained two fast-type MHC isoforms, II-1 and II-2, in both the bite-opened and control groups; their relative content (mean+/-SD, n = 7) was 47.8+/-2.9% and 52.2+/-2.9%, in the bite-opened and 44.4+/-3.0% and 55.6+3.0% in control preparations, indicating no significant (p>0.05) changes in MHC composition in association with the bite opening. On the other hand, significant differences in MLC and tropomyosin composition were found between the two preparations. Although the MLC consisted of three components, LC1f, LC2f and LC3f, in both preparations, their relative content (mean+/-SD, n = 7) was 37.1+/-2.4%, 49.6+/-1.6% and 13.2+/-3.2%, respectively, in the bite-opened and 28.1+/-3.1%, 50.9+/-1.6% and 21.0+/-3.5% in the control preparations, indicating that the bite opening induced a significant (p < 0.0001) increase in the relative content of LC1f at the expense of that of LC3f. Although the tropomyosin consisted of two components, TM-alpha and TM-beta, in both preparations, their relative content (mean+/-SD, n = 7) was 91.8%+/-1.9% and 8.2+/-1.9%, respectively, in the bite-opened and 95.9+/-0.7% and 4.1+/-0.7% in the control preparations, showing a significant (p < 0.001) increase in the relative content of TM-beta in relation to the bite opening. These results indicate that in guinea-pigs an increase in occlusal vertical dimension for 1 week changes the composition of MLC and tropomyosin, with no significant change in MHC, in the masseter muscle. These changes might be required to meet altered functional demands.

Monoclonal antibody analysis of phosphatidylserine and protein kinase C localizations in developing rat cerebellum.

Abstract: Understanding the topographical relationships between phosphatidylserine (PS) and protein kinase C (PKC) within neurons can provide clues about the mechanism of translocation and activation of PKC. For this purpose we applied monoclonal antibodies (Abs) of PS and PKC to sections of developing rat cerebellum. The anti‐PKC Ab immunohistochemical pattern showed homogeneous staining of Purkinje cells over various postnatal ages, whereas the anti‐PS Ab staining showed a heterogeneous localization over these ages. Purkinje cells did not stain well between postnatal day 14 (PND 14) and PND 21, suggesting that the PS was lost from the membrane during preparation of the sections during this period. These data imply that interactions between PS and PKC vary in Purkinje cells during postnatal development.

ATPase activity and tension development in mechanically-skinned feline jaw muscle

At a muscle length L0 (just taut), isometric tension and ATPase activity were measured at constant levels of various Ca2+ activations in muscle fibres (2-3 mm long and 34-66 microns in diameter) isolated from temporal, masseter and digastric (anterior belly) muscles. The isometric tension increased in a sigmoid fashion with increasing Ca2+ concentration from about pCa 6.60 to pCa 5.00 in the temporalis and from about pCa 6.37 to pCa 5.00 in both the masseter and digastric. The maximum isometric tension from four preparations of each muscle averaged 44.4 +/- 6.6 g/mm2 in the temporalis, 31.9 +/- 4.0 g/mm2 in the masseter, and 23.9 +/- 5.5 g/mm2 in the digastric. The ATPase activity also increased sigmoidally with increasing Ca2+ concentration from slightly below pCa 7.0 to pCa 5.0 in the temporalis, and from slightly above pCa 6.0 to pCa 5.0 in both the masseter and digastric. The maximum ATPase activity obtained from four preparations of each muscle averaged 0.35 +/- 0.06 mumol/mg protein per min in the temporalis, 0.29 +/- 0.06 mumol/mg protein per min in the masseter, and 0.18 +/- 0.04 mumol/mg protein per min in the digastric. The tension cost (ATPase activity/tension) estimated from these results was lower in the digastric than in the temporalis or masseter, indicating more economical ATP consumption in the digastric.

Ca2+ sensitivities and transient tension responses to step-length stretches in feline mechanically-stripped single-fibre jaw-muscle preparations

At a muscle length, Lo (just taut), isometric tension at constant levels of various Ca2+ activations and transient tension responses to rapid length stretches (less than 1 per cent of Lo within 2 ms) at maximal Ca2+ activation level were measured in temporal, masseter and digastric (anterior belly) muscles (2-3 mm long and 24-48 micron in diameter). Steady isometric tension increased in a sigmoid fashion with increasing Ca2+ concentration from about pCa 7.28 to 4.49 in temporalis, from about pCa 6.18 to 4.40 in masseter and from about pCa 5.82 to 4.40 in digastric. The maximum tension was 75.5 +/- 10.2 g/mm2 in temporalis, 44.7 +/- 14.1 g/mm2 in masseter, and 46.1 +/- 20.1 g/mm2 in digastric. In the resting state, the sarcomere length at Lo was 2.34 +/- 0.06 micron in temporalis, 2.20 +/- 0.08 micron in masseter, and 2.20 +/- 0.00 micron in digastric. When the sarcomere length was stretched from 2.20 to 2.34 micron (the sarcomere length of temporalis at Lo) in the masseter and digastric, the Ca2+ sensitivity increased without significant change of the maximum tension in either muscle. The transient tension responses in all three muscles showed two distinct phases; an immediate tension increase coincident with the length stretch followed by an exponential tension decrease. The mean value of the time constant in the second phase was 58.5 +/- 19.7 ms in temporalis, 58.5 +/- 12.6 ms in masseter, and 362.6 +/- 16.8 ms in digastric. Thus temporalis showed a higher Ca2+ sensitivity at Lo and a greater maximum tension-producing capability than the other muscles and the cross-bridge turnover rate appears to be slower in digastric than in the others.

Increase in vertical dimension alters mechanical properties and isometric ATPase activity in guinea pig masseter.

To study the changes in mechanical and metabolic properties associated with an increase in the vertical dimension of the face, isometric tension, isometric ATPase activity, unloaded shortening velocity (Vmax), and the tension transients in response to step stretches in length were measured at constant levels of various Ca2+ activations in glycerinated masseter muscles (75 to 150 microns in diameter and about 3 mm long) from normal and bite opened (6 mm increase in the vertical dimension, period of 1 week) guinea pigs. The isometric tension increased sigmoidally with an increase in Ca2+ concentration in both preparations. However, the bite opening shifted the pCa-relative tension relationship in the direction of increasing Ca2+ required for activation, and the pCa at Km (Ca2+ concentration required to develop half maximum tension) was 6.40 +/- 0.08 in control and 6.27 +/- 0.05 in the bite opened preparations. The maximum isometric tension at the saturating Ca2+ concentration was greater in the bite opened preparation. The ATPase activity changed almost in parallel with the tension (i.e., Ca2+ concentration) in both preparations. However, the tension cost (ATPase activity/tension) was significantly (p < 0.05) lower in the bite opened preparation, compared with the control. The Vmax at the saturating Ca2+ concentration was lower in the bite opened preparations. The tension transients in response to step stretch in length were characterized by four distinct phases; the first phase of an immediate tension increase coincident with the stretch, the second phase of a rapid quasi-exponential tension reduction, the third phase of a minute tension increase and the fourth phase of a slow tension reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Ba2+ on the mechanical properties of glycerinated heart muscle

At a muscle length, L0 (just taut), isometric tension and tension transients in response to rapid step stretches in length (mostly less than 1.2% of L0 within 2 ms) were measured at constant levels of Ba2+ activation of varying magnitude in glycerinated cat right-ventricular papillary muscles (2–3 mm long, 130–200 μm in diameter). The majority of the experiments were carried out at room temperature (26–27°C) and at a pH of 6.8 The steady isometric tension increased as Ba2+ was varied from slightly below pBa 6 to about pBa 4. The concentration range of Ba2+-activated muscle was roughly 10 times higher than that of Ca2+-activated muscle. Maximum Ba2+-activated isometric tension was 79.0±4.2% (mean±SD,n=8) of that activated with Ca2+. The tension transients in Ba2+-activated muscle were characterized by at least three distinct phases; an immediate tension increase coincident with the stretch, a rapid exponential tension decrease (time constant, τ1 = 7.3 ± 1.0 ms,n = 5) and a delayed exponential tension rise (τ2 = 104 ± 5.7 ms,n=5). The profile of tension response was quite similar to that of Ca2+-activated muscle. These results suggest that in Ba2+-activated glycerinated heart muscle the cross-bridge turnover is taking place as in the Ca2+-activated muscle, but the number of active cross-bridges at maximally activated state is smaller than that of Ca2+-activated muscle.