Biomedical subjects

Y Imaizumi

Publications and source records attributed to Y Imaizumi.

At least 19 recordsLinked to original sources

PubMed2001

Local Ca(2+) transients and distribution of BK channels and ryanodine receptors in smooth muscle cells of guinea-pig vas deferens and urinary bladder.

1. The relationship between Ca(2+) sparks spontaneously occurring at rest and local Ca(2+) transients elicited by depolarization was analysed using two-dimensional confocal Ca(2+) images of single smooth muscle cells isolated from guinea-pig vas deferens and urinary bladder. The current activation by these Ca(2+) events was also recorded simultaneously under whole-cell voltage clamp. 2. Spontaneous transient outward currents (STOCs) and Ca(2+) sparks were simultaneously detected at -40 mV in approximately 50 % of myocytes of either type. Ca(2+) sparks and corresponding STOCs occurred repetitively in several discrete sites in the subplasmalemmal area. Large conductance Ca(2+)-dependent K(+) (BK) channel density in the plasmalemma near the Ca(2+) spark sites generating STOCs was calculated to be 21 channels microm(-2). 3. When myocytes were depolarized from -60 to 0 mV, several local Ca(2+) transients were elicited within 20 ms in exactly the same peripheral sites where sparks occurred at rest. The local Ca(2+) transients often lasted over 300 ms and spread into other areas. The appearance of local Ca(2+) transients occurred synchronously with the activation of Ca(2+)-dependent K(+) current (I(K,Ca)). 4. Immunofluorescence staining of the BK channel alpha-subunit (BKalpha) revealed a spot-like pattern on the plasmalemma, in contrast to the uniform staining of voltage-dependent Ca(2+) channel alpha1C subunits along the plasmalemma. Ryanodine receptor (RyR) immunostaining also suggested punctate localization predominantly in the periphery. Double staining of BKalpha and RyRs revealed spot-like co-localization on/beneath the plasmalemma. 5. Using pipettes of relatively low resistance, inside-out patches that included both clustered BK channels at a density of over 20 channels microm(-2) and functional Ca(2+) storage sites were obtained at a low probability of approximately 5%. The averaged BK channel density was 3-4 channels microm(-2) in both types of myocyte. 6. These results support the idea that a limited number of discrete sarcoplasmic reticulum (SR) fragments in the subplasmalemmal area play key roles in the control of BK channel activity in two ways: (i) by generating Ca(2+) sparks at rest to activate STOCs and (ii) by generating Ca(2+) transients presumably triggered by sparks during an action potential to activate a large I(K,Ca) and also induce a contraction. BK channels and RyRs may co-localize densely at the junctional areas of plasmalemma and SR fragments, where Ca(2+) sparks occur to elicit STOCs.

Aniline Compounds↗

PubMed2001

Molecular cloning and expression of the novel splice variants of K(+) channel-interacting protein 2.

Two cDNAs encoding the splice variants of K(+) channel-interacting protein 2 (KChIP2) recently reported as human KChIP2 have been identified from rat, mouse, and human heart by RT-PCR. A longer variant, KChIP2L encodes a protein of 270 amino acids, which has a 50-amino-acid insertion in N-terminus in comparison with a shorter one, KChIP2S. Interestingly, both KChIP2S and KChIP2L (KChIP2S/L) but not the original KChIP2 were expressed in human heart and umbilical vein endothelial cells (HUVECs). KChIP2S transcripts but not KChIP2L were predominantly expressed in rat, mouse, and human heart and HUVECs, whereas both transcripts were expressed at low levels in other tissues such as brain, aorta, and kidney. Using chimeric proteins of green fluorescence protein (GFP) fused to the N-terminus of KChIP2S/L, the interactions between Kv4.3 and KChIP2S/L were analyzed in native and Kv4.3-expressed HEK293 cells. Specific localization of GFP-fused KChIP2S/L proteins on or near cell membrane was observed only in Kv4.3-expressed HEK293 cells.

Amino Acid Sequence↗

PubMed2001

Regional expression of the splice variants of Kv4.3 in rat brain and effects of C-terminus deletion on expressed K+ currents.

In situ hybridization and RT-PCR analyses have revealed that, among three Kv4.3 splice variants (a, b, and c) with distinct C-terminal cytoplasmic domains, the mRNA for Kv4.3a is abundant in cerebral cortex, cerebellum, olfactory bulb, and medulla oblongata, whereas the mRNA for Kv4.3c is localized mainly to hippocampus. Three new distinct splice variants of Kv4.3 (Kv4.3d, e and f), which consist of 601, 635, and 628 amino acids, respectively, and have distinct C-terminal cytoplasmic domains, were isolated from rat brain by RT-PCR. Kv4.3b, d, e and f were expressed at much lower levels in brain. Mutagenesis which removed 149 amino acids in C-terminal domain of Kv4.3a significantly slowed its rate of recovery from inactivation as measured in heterologous expression in HEK293 cells. Surprisingly, however, neither the rate of inactivation nor voltage dependence of the activation and inactivation were changed.

Amino Acid Sequence↗

PubMed2001

Comparative study of the molecular and functional expression of L-type Ca2+ channels and large-conductance, Ca2+-activated K+ channels in rabbit aorta and vas deferens smooth muscle.

The relationship between the density of ionic currents through major two channels, voltage-dependent L-type Ca2+ channels (L-type VDCC) and large-conductance, Ca2+-activated K+ channels (BKC), and the mRNA expression levels of alpha1C subunit of L-type VDCC (alpha1C) and alpha/beta subunits of BKC (alphaBK/betaBK) were compared in smooth muscle cells (SMC) of rabbit aorta and vas deferens using whole cell-voltage clamp and reverse transcription-polymerase chain reaction (RT-PCR) analyses. The density of the currents through VDCC (ICa) and BKC (IK,Ca) at +10 mV in aortic SMC was approximately one-seventh and one-sixth respectively of that in vas deferens. Whilst application of the Ca2+ channel agonist Bay K 8644 increased ICa by 75-90% in these SMC, the increase in IK,Ca was far greater in aorta than in vas deferens. The expression of the alpha1C transcript in vas deferens was approximately 3.5 times higher than that in aorta. In contrast, expression of alphaBK/betaBK was almost identical in both tissues, indicating the dissociation of IK,Ca density from the expression levels of BKC transcripts in aorta. The results were supported by Western blot and immunocytochemical analyses using subunit-specific antibodies. The lower Ca2+ influx through VDCC in aorta activates only a very limited fraction of BKC compared with that in vas deferens. The greater expression of BKC than of VDCC in aortic SMC contributes to a strong negative feed-back mechanism that minimizes membrane depolarization and acts as a safety margin to maintain low membrane excitability.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2001

BK channel activation by NS-1619 is partially mediated by intracellular Ca2+ release in smooth muscle cells of porcine coronary artery.

1. Effects of NS-1619, an opener of large conductance Ca2+-activated K+ (BK) channel, on intracellular Ca2+ concentration ([Ca2+]i) and membrane potential were examined in single myocytes freshly isolated from porcine coronary artery. 2. Under current clamp mode, the application of 1-30 microM NS-1619 hyperpolarized the membrane in concentration-dependent manner. The NS-1619-induced hyperpolarization was abolished by the presence of 100 nM iberiotoxin. 3. Application of 1-10 microM NS-1619 hyperpolarized the membrane by approximately 6 mV or less but did not change significantly the [Ca2+]i. When membrane hyperpolarization of 12 mV or so was caused by 30 microM NS-1619, [Ca2+]i was unexpectedly increased by approximately 200 nM. This increase in [Ca2+]i and the concomitant outward current activation were also observed under voltage-clamp at holding potential of -40 mV. 4. The increase in [Ca2+]i by 30 microM NS-1619 occurred mainly in peripheral regions than in the centre of the myocytes. The removal of extracellular Ca2+ affected neither the membrane hyperpolarization nor the increase in [Ca2+]i. 5. In the presence of 10 mM caffeine and 10 microM ryanodine, the increase in [Ca2+]i by 30 microM NS-1619 was not observed and the membrane hyperpolarization was reduced to approximately 67% of the control. 6. These results indicate that the opening of BK channels by NS-1619 at 30 microM, which is the most frequently used concentration of this agent, is partly due to Ca2+ release from caffeine/ryanodine-sensitive intracellular storage sites but is mainly due to the direct activation of the channels.

Animals↗

PubMed2001

Effects of KRN2391 on ionic currents in rabbit femoral arterial myocytes.

The effects of KRN2391, an ATP-sensitive K+ channel opener (KCO) which also acts as a nitrate, on ionic membrane currents in rabbit femoral arterial myocytes were examined. Under whole-cell clamp conditions where cells were superfused with physiological salts solution containing 5.9 mM K+, KRN2391 elicited an outward current at a holding potential of -30 mV. KRN2391-induced current had a reversal potential of -78 mV and was abolished by glibenclamide (glib). KRN2391 was approximately 25 times more potent than nicorandil to activate an ATP-sensitive K+ current (I:(KATP)). On the other hand, 10 microM KRN2391 did not affect either voltage-dependent Ca(2+) or delayed rectifier K+ channel currents. In the inside-out patch configuration, KRN2391 activated 47 pS K+ channels in the presence of nucleotide diphosphates (NDPs) under the symmetrical 140 mM K+ conditions. Glib and intracellular ATP reversibly inhibited the activity of the 47 pS K+ channels. The 47 pS K+ channels activated by KRN2391 are similar in their conductance and other properties to NDP-sensitive K+ channels (K(NDP) channels) described in other smooth muscles and the cloned channels. KRN2391 is a potent activator of the 47 pS K+ channels and the activation can contribute to the KRN2391-induced vasodilation in arterial muscles.

Adenosine Diphosphate↗

PubMed2001

Perinatal mortality in twins and factors influencing mortality in Japan, 1980--98.

The perinatal mortality rate (PMR) in twins was analysed using Japanese vital statistics during the period 1980-98. There was a total of 1992 twin perinatal deaths. The PMR in twins significantly decreased from 91 per 1000 births in 1980 to 31 per 1000 in 1998. The PMR was higher in males than females, but the differences between the sexes decreased over time. It was significantly higher in the second-born than the first-born of the twins. The fetal death rate was 2.8 times higher in monozygotic than dizygotic twins. The PMR decreased to one-sixth for the maternal age group > or =40 years, one-fifth for <20 years, and between one-third and a half for 20--39 years during the period 1980--98. It was lowest for birthweights (BW) between 2500 g and 2999 g during the entire period; the PMR decreased with gestational age (GA) up to 38--39 weeks and increased thereafter. The effect of BW on the PMR was greater than that of GA except in the shorter GA group. The proportion of twin perinatal deaths with extremely low BW (<1000 g) increased from 50% in 1980 to 68% in 1998. Thus, the declining PMR was unlikely to be due to the improvement in BW in twins. It is likely that it is related to the improved medical management of twins during the perinatal period and the first week of life.

Algorithms↗

PubMed2001

Imaging of Ca2+ release by caffeine and 9-methyl-7-bromoeudistomin D and the associated activation of large conductance Ca2+-dependent K+ channels in urinary bladder smooth muscle cells of the guinea pig.

Ca2+ release by caffeine and 9-methyl-7-bromoeudistomin D (MBED) and the concomitant activation of large conductance Ca2+-dependent K+ (BK) channels were analyzed using confocal Ca2+ imaging and whole cell voltage-clamp methods in guinea pig urinary bladder smooth muscle cells. Puff application of 3 or 10 mM caffeine for several seconds (2 - 5 s) elicited a large increase in intracellular Ca2+ concentration ([Ca2+]i) and induced a phasic outward current at a holding potential of -40 mV. The phasic outward current was the summation of spontaneous transient outward currents (STOCs) due to marked activation of BK channels and was followed by a short cessation of STOCs. Although the increase in superficial [Ca2+]i by caffeine was faster than that in global [Ca2+]i, the peak [Ca2+]i was identical in these areas. Puff application of 100 microM MBED also markedly enhanced STOCs for a few seconds. This response to MBED was not observed when stored Ca2+ was depleted by caffeine. The increase in [Ca2+]i by MBED occurred mainly in superficial areas. Longer application of 100 microM MBED for 2 min did not induce significant global [Ca2+]i increase but decreased the amount of Ca2+ release and cell shortening during the subsequent application of 10 mM caffeine. These results indicate that short application of MBED releases Ca2+ preferentially from superficial storage sites, presumably due to its slow approach to deeper sites. MBED may be a good pharmacological tool to manipulate selectively the superficial Ca2+ stores related to STOCs.

Animals↗

PubMed2001

Ca2+-images of smooth muscle cells and endothelial cells in one confocal plane in femoral artery segments of the rat.

Simultaneous recording of Ca2+-images in one confocal plane from vascular smooth muscle cells (SMCs) and endothelial cells (ECs) of an intact rat femoral artery segment was performed using indo-1 and a confocal microscope. During application of 10 microM acetylcholine (ACh), elevation and oscillation of intracellular Ca2+ concentration ([Ca2+]i) were observed in ECs but not in SMCs. Sequential conduction of Ca2+ oscillation from an EC to the neighboring ECs in one longitudinal direction was often observed in the presence of ACh. On the other hand, the activation of voltage-dependent Ca2+ channels by external 30 mM K+ resulted in the elevation of [Ca2+]i only in SMCs. When 10 microM ACh was added in the presence of 30 mM K+, it was observed in one confocal plane that [Ca2+]i in ECs and SMCs was almost simultaneously increased and decreased, respectively. The simultaneous recording method in this intact preparation will provide a line of valuable information about the interactions between SMCs and ECs, based on spatio-temporal analyses of absolute values of [Ca2+]i in individual cells.

Acetylcholine↗

PubMed2001

Usefulness and limitation of DiBAC4(3), a voltage-sensitive fluorescent dye, for the measurement of membrane potentials regulated by recombinant large conductance Ca2+-activated K+ channels in HEK293 cells.

The usefulness of bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC4(3)), a voltage-sensitive fluorescent dye, for the measurement of membrane potentials (MPs) was evaluated in HEK293 cells, where alpha or alpha plus beta1 subunits of large conductance Ca2+-activated K+ (BK) channels were expressed (HEKBK alpha and HEKBK alphabeta). The fluorescent intensity of DiBAC4(3) was measured at various potentials under voltage-clamp for calibration to estimate the absolute MP semi-quantitatively. The resting MPs measured with DiBAC4(3) were roughly comparable to those recorded with a microelectrode; the MP in HEKBK alphabeta was 10-20 mV more negative than that in native HEK. In HEKBK alpha, the membrane hyperpolarization induced by 10 microM Evans blue, a BK channel opener, was detected with DiBAC4(3). NS-1619, another BK channel opener, induced gradual but substantial change in F/F(K) even in native HEK, while the BK channel opening effect was detected. Oscillatory membrane hyperpolarization was induced in HEKBK alphabeta by application of 10 microM acetylcholine via increase in intracellular Ca2+ concentration. The oscillatory hyperpolarization was, however, detected only as a slow hyperpolarization with DiBAC4(3). It can be concluded that relatively slow effects of BK channel modulators can be semi-quantitatively measured by use of DiBAC4(3) in HEKBK, while the limited temporal resolution and possible artifacts should be taken into account.

Barbiturates↗

PubMed2000

Nifedipine and nisoldipine modulate membrane potential of vascular endothelium via a myo-endothelial pathway.

Effects of nifedipine (Nif) and nisoldipine (Nis), dihydropyridine Ca2+ channel blockers (DHPs) on membrane potential and currents of endothelial cells, which are enzymatically dispersed (dis-ECs) from or exist in arterial segments (seg-ECs) of rabbit and rat aorta, were examined. Outward currents induced by 1-10 microM acetylcholine (ACh) in dis-ECs were neither affected by a receptor operated Ca2+ channel blocker, SK&F 96365 (SKF), nor DHPs. ACh hyperpolarized dis-ECs and seg-ECs by 15-20 mV, whereas phenylephrine (Phe) elicited oscillatory depolarization in seg-ECs but not in dis-ECs. The Phe-induced response in seg-ECs was significantly inhibited by treatment with 18beta-glycyrrhetinic acid, a disrupter of gap junctions. Application of 0.3 microM Nif or Nis effectively inhibited the Phe-induced oscillatory depolarization, while these DHPs did not affect ACh-induced hyperpolarization in seg-ECs. The lack of effect on dis-ECs indicates that DHPs have little effect on dis-ECs themselves, nevertheless DHPs inhibit the Phe-induced endothelial potential oscillation which is conducted from smooth muscle cells via a myo-endothelial pathway.

Animals↗

PubMed2000

Inhibition of smooth-muscle myosin-light-chain phosphatase by Ruthenium Red.

Ruthenium Red (RuR) is widely used as an inhibitor of ryanodine receptor Ca(2+) release channels, but has additional effects such as the induction of Ca(2+) sensitization of contraction of permeabilized smooth muscles. To address the mechanism underlying this process, we examined the effects of RuR on contractility in permeabilized guinea-pig ileum and on the activity of myosin-light-chain phosphatase (MP). RuR increased the force at submaximal [Ca(2+)] (pCa 6.3) approx. 4-fold. This effect was not observed after thiophosphorylation of MP. RuR also seemed capable of preventing the thiophosphorylation of MP, suggesting a direct interaction of RuR with MP. Consistent with this possibility, smooth-muscle MP was inhibited by RuR in a concentration-dependent manner (IC(50) 23 microM). Exogenous calmodulin significantly increased RuR-induced contraction at pCa 6.3 but had little effect on contraction induced by microcystin at this [Ca(2+)]. Ca(2+)-independent contraction was induced by RuR (EC(50) 843 microM) and by microcystin (EC(50) 59 nM) but the maximal force induced by RuR was smaller than that induced by microcystin. The addition of 300 microM RuR enhanced the contraction induced by 30 nM microcystin but markedly decreased that induced by 1 microM microcystin. Such a dual action of RuR on microcystin-induced effects was not observed in experiments using purified MP. We conclude that the RuR-induced Ca(2+) sensitization of smooth-muscle contraction is due to the direct inhibition of MP by RuR.

Adenosine Triphosphate↗

PubMed2000

Cromakalim-induced membrane current in guinea-pig tracheal smooth muscle cells.

The characteristics of the cromakalim-induced membrane current were examined in single tracheal myocytes of the guinea-pig under voltage-clamp conditions. When K(+) concentrations in the pipette and bathing solutions were approximately 140 mM, cromakalim activated a membrane current (I(crom)) which was inward at -60 mV and reversed at -2 mV. I(crom) was blocked by 10 microM glibenclamide and potentiated when the ATP concentration in the pipette solution was decreased. The K(d) and Hill coefficient of glibenclamide for I(crom) block were 200 nM and 1.05, respectively. Application of the tyrosine kinase inhibitors, genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamid (ST638), reduced I(crom) in a concentration-dependent manner. Daidzein, which does not inhibit tyrosine kinase, was about 10 times less effective than genistein. Herbimycin A had no effect on I(crom). Internal application of these inhibitors from the pipette did not affect I(crom). In conclusion, cromakalim is a potent activator of the ATP-sensitive K(+) channel (K(ATP) channel) in guinea-pig tracheal myocytes. The inhibition of I(crom) by genistein and ST638 may be due to the direct block of the channel from outside.

Adenosine Triphosphate↗

PubMed2000

Deaths from cerebrovascular diseases correlated to month of birth: elevated risk of death from subarachnoid hemorrhage among summer-born.

It has been suggested that maternal nutrition, and fetal and infant growth have an important effect on the risk of cardiovascular disease in adult life. We investigated the population-based distribution of deaths from cerebrovascular diseases (ICD9 codes 430, 431, or 434) in Japan in 1986-1994 as a function of birth month, by examining death-certificate records. For a total of 853 981 people born in the years 1900-1959, the distribution of the number of deaths according to the month of birth was compared with the distribution expected from the monthly numbers of all births for each sex and for the corresponding birth decade. For those born between 1920 and 1949, there were significant discrepancies between the actual numbers of deaths from subarachnoid hemorrhage (ICD9 430) and the numbers expected, and these differences were related to the month of birth. Those born in summer, June-September, consistently had an elevated risk of death, particularly men, where the excess risk was 8%-23%. This tendency was also observed, less distinctly but significantly, for deaths from intracerebral hemorrhage (ICD9 431), but was not observed for those dying from occlusion of the cerebral arteries (ICD9 434). The observation that the risk of dying from subarachnoid hemorrhage was more than 10% higher among those born in the summer implies that at least one in ten deaths from subarachnoid hemorrhage has its origin at a perinatal stage. Although variations in hypertension in later life, which could possibly be "programmed" during the intra-uterine stages, could be an explanation for this observation, the disease-specific nature of the observation suggests the involvement of aneurysm formation, which is a predominant cause of subarachnoid hemorrhage.

Aged↗

PubMed2000

Conserved smooth muscle contractility and blood pressure increase in response to high-salt diet in mice lacking the beta3 subunit of the voltage-dependent calcium channel.

Voltage-dependent calcium channels are crucially important for calcium influx and the following smooth muscle contraction. Beta subunits of these channels are known to modify calcium currents through pore-forming alpha subunits. Among the four reported independent beta subunits, the beta3 subunit is expressed in smooth muscle cells and thought to compose L-type calcium channels in the tissue. To determine the role of the beta3 subunit in the cardiovascular system, we have analyzed beta3-null mice. Electrophysiological examinations proved the existence of dihydropyridine (DHP)-sensitive. L-type calcium channels in the smooth muscle cells. Beta3-null mice show no apparent changes in smooth muscle contraction and sensitivity to DHP, and normal blood pressure when they are raised on a normal diet, but the 13 subunit deficient mice show elevated blood pressure in response to a high-salt diet, with significant reductions in plasma catecholamine concentrations. Our finding strongly suggests a close relationship between voltage-dependent channels and high blood pressure.

Animals↗

PubMed2000

Protective effect of benidipine against the abnormal electrical activity in single ventricular myocytes of the guinea pig under simulated ischemic conditions and reperfusion.

Induction of electrical abnormalities (EAs) under simulated ischemic conditions and after reperfusion was measured from single cardiac myocytes isolated from guinea pig ventricle using whole-cell voltage or current clamp with perforated patch variation. Conditions of simulated ischemia were produced by the exchange of medium from the standard one oxygenated with 95% O2-5% CO2 gas (pH 7.4) to the modified one, which contained no glucose, 8 mM K+ and 30 mM sodium-D,L-lactate and was gassed with 90% argon-10% CO2 (pH 6.6). Under the simulated ischemia for 20 min, EAs such as delayed afterdepolarization, early afterdepolarization, automatic activity or transient inward current were observed in about 37% of myocytes driven electrically at 1 Hz. Irreversible hypercontracture occurred in myocytes of 10% or less. Upon reperfusion with the standard solution, EAs and hypercontracture were observed in about 43% and 22% of cells, respectively. Glibenclamide-sensitive current was detected during ischemia, but tended to be enhanced during reperfusion. Amplitude of Ca2+ current and ATP-sensitive K+ current after reperfusion varied widely with time and from cell to cell. When myocytes were pretreated for 10 min with 10 nM benidipine, a 1,4-dihydropyridine derivative Ca2+ blocker, the incidence of EAs and hypercontracture was markedly reduced, suggesting the protective effect of benidipine against cardiac cell injury during ischemia and reperfusion.

Action Potentials↗

PubMed2000

SK4 encodes intermediate conductance Ca2+-activated K+ channels in mouse urinary bladder smooth muscle cells.

The single channel current of intermediate conductance Ca2 +-activated K+ channel (IK channel) was measured in mouse urinary bladder myocytes (MBM), and the molecular basis of the channel was suggested to be the SK4 subtype by RT-PCR. Among Ca2+-activated K+ channel subtypes (SK2, SK3, SK4 and BK), the mRNAs of SK4 and BK were predominantly expressed in MBM. IK channel currents recorded from MBM showed: 38.7 pS slope conductance under symmetrical 140 mM K+ conditions, Ca2+-dependent activation, and blockade by charybdotoxin and econazole. These results strongly suggest that SK4 encodes IK channels in MBM.

Animals↗

PubMed2000

Molecular analysis of non-specific supersensitivity induced by AF64A in rat iris smooth muscle.

Characteristics of supersensitivity induced by the pretreatment with AF64A, an inhibitor of choline uptake at parasympathetic nerve endings, were examined in rat iris sphincter. In preparations isolated and skinned by beta-escin after the micro injection of AF64A to eyes in vivo, the amplitude of maximum contraction in pCa 4.5 solution was increased by 180% of the control from the contralateral eyes. The Ca2+ sensitivity of the contractile system was slightly but significantly increased by AF64A injection; the half maximum contraction was obtained at pCa 5.87 and 6.05 in the control and AF64A-injected eyes, respectively. The increase in maximum contraction in AF64A injected ones was neither affected by the addition of calmodulin, GTPgammaS nor H-7. The increase in Ca2+ sensitivity by AF64A injection was not affected by calmodulin, enhanced by GTPgammaS and abolished by H-7. AF64A injection increased the total protein content only by 30% of the control. The contents of contractile proteins per iris were quantified using Western blotting with monoclonal antibodies. The contents of actin and calponin were increased by AF64A, whereas those of myosin, calmodulin and caldesmon were not affected. The results indicate that AF64A-induced enhancement of the maximum contraction is not mainly due to the increase in the contents of major contractile proteins and that the increase in Ca2+ sensitivity could be due to the mechanism in which changes in protein kinase C and/or GTP binding protein activity are involved.

Animals↗