Hisakazu Kimura

Impaired expression of Gsα protein mRNA in rat ventricular myocardium with aging

We have recently reported that the responsiveness of adrenoceptors is decreased with aging in rat ventricular myocardium. Thus, the current study determined aging-dependent changes in: (a) characteristics of myocardial G proteins as determined by Western blot analysis; (b) steady-state levels of G protein mRNA as determined by Northern blot analysis; and (c) the intropic response to isoproterenol, a beta-adrenoceptor agonist. Cardiac preparations were isolated from male Wistar rats of 6 (adult) and 24 (old) months old. Compared with adults, aging decreased the combined level of the three Gs alpha subunits (45, 47 and 52 kDa) by a total of 23% in ventricular membrane preparations. In contrast, levels of Gi alpha (40/41 kDa), Gq alpha (42 kDa), Go alpha (39 kDa) and G common beta (35/36 kDa) immunoreactivity were not affected by aging in the same membrane preparations. In ventricular myocardium, steady-state levels of Gs alpha mRNA (1.9 kb) decreased by 20-28% between 6 and 24 months of age with no change in Gi alpha mRNA (2.4 kb). An aging-associated decline in beta-adrenergic stimulation was observed in the maximum positive inotropic effect elicited by isoproterenol in the presence of prazosin in left papillary muscles, with no change in ED50 values. These results suggest that age-related changes in cardiac excitation and contraction coupling following beta-adrenoceptor stimulation are mediated, at least in part, by Gs alpha protein dysfunction.

Down-regulation of β-adrenoceptors and loss of Gsα subunit levels in ventricular myocardium of rats treated with isoproterenol

Abstract We investigated the influence of chronic β-adrenergic stimulation on the β-adrenoceptor-G protein-adenylyl cyclase system in rat ventricular myocardium. The rats received twice-daily injections of 4 mg/kg isoproterenol (ISO) alone or with 8 mg/kg propranolol (PROP) for 4 days. The ISO (10 μM)-induced increase in tissue cAMP production was lower (50%) after chronic ISO treatment than in control myocardium. The β-adrenoceptor density decreased by 43% in ventricular membranes from ISO-treated rats. Immunoblotting techniques using specific antibodies against G proteins revealed that ventricular myocardium contains three Gsα subunit isoforms of 45, 47 and 52kDa. ISO treatment decreased levels of the three Gsα subunits by a total of 40%, while no change in Giα (40/41kDa) and Gcommonβ (35/36kDa) levels were found in the same membrane preparations. The antagonist PROP almost totally blocked the effects of ISO treatment on cAMP, β-adrenoceptors and Gsα subunits. These results suggest that chronic β-adrenergic stimulation causes not only down-regulation of β-adcenoceptors, but also loss of Gsα subunit levels in rat ventricular myocardium.

Role of α1-adrenoceptor subtypes which mediate positive chronotropy in neonatal rat cardiac myocytes

We investigated the involvement of alpha 1-adrenoceptor subtypes in the positive chronotropic response to norepinephrine (NE) in neonatal rat cardiac myocytes at day 3 of culture. The cardiac myocytes at day 3 of culture exhibited a dose-dependent positive chronotropic response to NE in the presence of propranolol, a beta-adrenoceptor antagonist. The positive chronotropic responses to NE were completely antagonized by the alpha 1-adrenoceptor antagonist prazosin. The NE-induced positive chronotropic response was inhibited 68% by the alpha 1B-adrenoceptor antagonist, chloroethylclonidine (CEC), but partially (41%) so by the alpha 1A-adrenoceptor antagonist, WB4101. In the membrane fraction derived from cardiac myocytes at day 3 of culture, pretreatment with CEC decreased the Bmax of the alpha 1-adrenoceptor to 22% of the control value. The NE-induced positive chronotropic response was inhibited 62 and 77% by the voltage-gated Ca2+ channel blocker such as nifedipine and verapamil, respectively. These findings indicate (1) that cultured neonatal rat cardiac myocytes possess both alpha 1-adrenoceptor subtypes, i.e., alpha 1A and alpha 1B, (2) that the predominant alpha 1-adrenoceptor subtypes mediating NE-induced positive chronotropy in neonatal rat cardiac myocytes at day 3 of culture are alpha 1B-subtypes, and (3) that NE-induced positive chronotropy may be caused via voltage-gated Ca2+ channel activation.

Relation between spontaneous contraction and sarcoplasmic reticulum function in cultured neonatal rat cardiac myocytes.

The relation between spontaneous contraction, Ca2+ oscillations, and sarcoplasmic reticulum (SR) function was studied in cultured neonatal rat cardiac myocytes. Spontaneous contraction and Ca2+ oscillations were irregular at day 2 of culture but became regular at day 6 of culture in neonatal rat cardiac myocytes. The rate of spontaneous contraction and the frequency of Ca2+ oscillations were decreased by verapamil and were abolished in the absence of extracellular Ca2+ at both day 2 and day 6 of culture. Ryanodine and thapsigargin increased the rate of contraction and the frequency of Ca2+ oscillations at day 2 of culture but did not affect contractions and Ca2+ oscillations at day 6 of culture. Ultrastructural observation showed that the structure of SR developed less at day 6 of culture. The present results suggest that spontaneous contraction and Ca2+ oscillations are due mainly to extracellular Ca2+ influx but not to Ca2+ release from SR in neonatal rat cardiac myocytes.

α1-adrenoceptors in neonatal rat cardiac myocytes: Hypoxia alters the responsiveness of α1A and α1B subtypes

Abstract We investigated the contribution of α 1 -adrenoceptor subtypes to the chronotropic response to norepinephrine (NE) in cultured neonatal rat cardiac myocytes under normoxia and hypoxia. A dose-dependent negative chronotropic response was induced by NE in the presence of propranolol. Hypoxic exposure inverted the negative chronotropic response to NE to a positive one. All of these chronotropic responses were completely antagonized by prazosin. In normoxic conditions, the NE-induced negative chronotropic response was completely antagonized by WB4101 but only partially (55%) so by chloroethylclonidine (CEC). After hypoxic exposure, WB4101 partially antagonized the positive chronotropic response to NE (54%), while CEC completely suppressed the action of NE. Hypoxic exposure did not alter the number of α 1A - and α 1B -adrenoceptor subtypes as measured by [ 3 H]prazosin binding following CEC treatment. These results indicate (1) that cultured neonatal rat cardiac myocytes contain both α 1 -adrenoceptor subtypes, i.e., α 1A and α 1B , and (2) that the predominant α 1 -adrenoceptor subtypes mediating NE-induced chronotropy were altered by hypoxia.

Characterization of α1-adrenoceptors which mediate chronotropy in neonatal rat cardiac myocytes

Abstract 1. In the present study, we investigated the effect of culture on α 1 -adrenoceptors that mediate chronotropy and on α 1 -adrenergic signal transduction in neonatal rat cardiac myocytes. 2. The spontaneous beating rate of neonatal rat myocytes after 3 or 7 days in culture was 37.4 ± 4.2 or 102.0 ± 4.3 beats min − , respectively. The α 1 -adrenoceptor-mediated chronotropic effect of norepinephrine was positive at day 3 of culture. In contrast to day 3 of culture, the neonatal myocytes exhibited a negative chronotropic response to norepinephrine on day 7 of culture. Both of these effects of norepinephrine were completely abolished by prazosin. 3. The affinity ( K d ) and/or density ( B max ) of α 1 -adrenoceptors labeled with [ 3 H]prazosin in membranes from cultured myocytes were not significantly different between day 3 and day 7 of culture. 4. The expression of G s , G i , G q and G o , α-subunits in membranes from cultured myocytes was found to be significantly increased with the passage of culture time by immunoblot analysis. In contrast, no significant differences in G β -subunit expression were observed between day 3 and day 7 of culture. 5. Norepinephrine-stimulated inositol 1,4,5-trisphosphate production by radio-binding protein in neonatal myocytes after 7 days of culture was significantly higher than that of the day 3 counterpart. 6. No significant changes in phospholipid and cholesterol contents in membranes from neonatal myocytes were observed with longer culture times. 7. These results suggest that changes in the responsiveness to α 1 -adrenergic stimulation from positive to negative chronotropy during culture of cardiac myocytes are mediated, at least in part, by functional alterations in the α 1 -adrenergic signal transduction systems, including both G-protein expression and inositol 1,4,5-trisphosphate production.

Hypoxia inverts the negative chronotropic response to norepinephrine in normoxia in cultured neonatal rat cardiac myocytes: role of the α1 adrenergic signal transduction system

In the present study, we investigated the effect of hypoxia on the chronotropic response to norepinephrine (NE) of cultured neonatal rat ventricular myocytes. We measured beating of myocytes with the Fotonic sensor, using a newly developed method for a noncontact displacement measurement. The beating rate counted with the sensor had a high correlation coefficient with that counted visually under a microscope (r = 0.997, P < 0.01). NE concentrations of 10(-8) - 10(-4) M caused negative chronotropy dose dependently in the presence of 5 x 10(-7) M propranolol. NE-induced chronotropy was completely antagonized by 10(-6) M prazosin. Three hours hypoxia decreased the spontaneous beating rate 40% (P < 0.01). Negative chronotropy induced by 10(-4) M NE in normoxia was inverted to positive and was antagonized by prazosin. Hypoxia increased the basal level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) to 190% (P < 0.01), while NE-stimulated Ins(1,4,5)P3 production was significantly suppressed. Immunoblotting analysis of G protein subunits demonstrated no quantitative changes in Gi alpha, Gq alpha, Go alpha and G beta common subunits in hypoxia. In a saturation binding assay with [3H]prazosin, Kd values were increased to 152% by hypoxia (P < 0.05) without significant change in Bmax. Basal activity of low Km-GTPase was increased to 122% by hypoxia (P < 0.05). These results suggest that the hypoxia-induced increase in low-Km GTPase activity, which could stimulate phospholipase C by an activated alpha GTP subunit of G protein and consequently induce receptor-independent increase in Ins(1,4,5)P3, may be responsible for the inversion of the NE-induced negative chronotropic response in normoxia.

The direct effect of halothane on myocardial contraction in rat myocytes with poorly developed gap junctional intercellular communication

Background: The gap junction channel plays an important role in synchronous beating in the heart, and the reduction in the amount of gap junctional intercellular communication (GJIC) is thought to be the main arrhythmogenic factor in diseased heart. However, the effect of halothane on myocardial contraction in heart tissue with less GJIC is not well known. The purpose of the present study is to examine the direct effect of halothane on myocardium with poorly expressed GJIC.

Ca2+ channel modulation alters halothane-induced depression of ventricular myocytes

PurposeThis study examined the direct myocardial depressant effect of halothane and determined whether an L-type Ca2+ channel agonist and antagonists altered the myocardial depression induced by halothane in cultured rat ventricular myocytes.MethodsVentricular myocytes were obtained from neonatal rats by enzymatic digestion with collagenase and then cultured for 6 to 7 days. The myocytes were stabilized in a serum-free medium, and the spontaneous beating rate and amplitude were measured. To assess the halothane-induced conformational changes in L-type Ca2+ channel, receptor binding study was performed using a dihydropyridine derivative, [3H] PN 200-110, in cardiac membrane preparation.ResultsHalothane (1%, 2%, 3%, 4%) decreased the beating rate and amplitude in a concentration-dependent manner (P < 0.05). The myocardial depressant effects of halothane were potentiated by nifedipine or verapamil (P < 0.05). Bay K 8644, an L-type Ca2+ channel agonist, completely prevented the halothane-induced depression in amplitude (P < 0.05), but affected the beating rate less. Adding halothane (2%) decreased (P < 0.05) the maximum binding site density for [3H] PN 200-110 (from 198.6 ± 23.7 fmol·mg−1 protein to 115.3 ± 21.6 fmol·mg−1 protein) but did not affect binding affinity (from 0.461 ± 0.077 nM to 0.307 ± 0.055 nM).ConclusionThe reduction of Ca2+ current via sarcolemmal L-type Ca2+ channel, probably due to conformational changes in dihydropyridine binding sites, plays an important role in halothane-induced myocardial depression in living heart cells.RésuméObjectifLétude actuelle a examiné l’effet dépresseur myocardique direct de l’halothane et déterminé si un agoniste ou un antagoniste des canaux calciques de type L modifie la dépression myocardique induite par l’halothane sur des myocytes ventriculaires de rats, en culture.MéthodesLes myocytes ventriculaires ont été obtenus à partir de rats nouveau-nés, par digestion enzymatique avec collagénase, et puis mis en culture pendant 6 ou 7 jours. Les myocytes ont été stabilisés dans un milieu sans sérum et le rythme et l’amplitude des battements spontanés ont été mesurés. Pour évaluer les changements de conformation induits par l’halothane dans le canal calcique de type L, l’étude de la liaison aux récepteurs a été réalisée en utilisant un dérivé dihydropyridine, [3H] PN 200-110, d’une préparation de membranes cardiaques.RésultatsL’halothane (1 %, 2 %, 3 %, 4%) a diminué le rythme des battements et leur amplitude en fonction de la concentration (P < 0,05). Les effets dépresseurs myocardiques de l’halothane ont été accentués par la nifédipine ou le vérapamil. Le Bay K 8644, un agoniste des canaux calciques de type L, a complètement empêché la dépression d’amplitude induite par l’halothane (P < 0,05), mais a eu moins d’effet sur le rythme des battements. L’accroissement de l’halothane (2 %) a diminué (P < 0,05) la densité maximale au site de liaison pour le [3H] PN 200-110 (de 198,6 ± 23,7 fmol·mg−1 de protéine à 115,3 ± 21,6 fmol·mg−1 de protéine), mais n’a pas affecté l’affinité de la liaison (de 0,461 ± 0,077 nM à 0,307 ± 0,055 nM).ConclusionLa réduction de l’entrée de calcium dans le canal calcique sarcolemmique de type L joue un rôle important dans la dépression myocardique induite par l’halothane dans des cellules cardiaques en culture à cause, probablement, des changements de conformation des sites de liaison de la dihydropyridine.

Involvement of Connexin43 Localization and Gap Junctional Intercellular Communication in the Establishment of a Synchronized Contraction of Cultured Neonatal Rat Cardiac Myocytes

Abstract We have established a method for the long-term maintenance of functional cardiac myocytes that are cultured confluently. In our culture system, spontaneous but irregular contraction was observed even for cells 18 hours after seeding. The contractile rate of the myocytes increased with the passage of time, and a synchronized contraction was gradually formed. At day 7 of culture, the confluent myocytes exhibited synchronous contractile activity with a relatively constant rate (145.6±16.6 beats/min). With an immunohistochemical study, the localization of connexin43 on cell membranes in contact became clearer with culture time. The permeability of gap junctions investigated by the microinjection-dye transfer method indicated that the cell-cell communication increased with time, that is, cultured cardiac myocytes exhibiting synchronous contractile activity had excellent gap junctional intercellular communication. These results suggest that the establishment of a synchronized contraction in cultured neonatal rat cardiac myocytes has a close relation to gap junctional intercellular communication.