• The Korean Journal of Physiology and Pharmacology
• /
• 제2권6호
• /
• pp.725-732
• /
• 1998

In order to elucidate the molecular mechanism of the intracellular $Ca^{2+}$ overload frequently reported from diabetic heart, diabetic rats were induced by the administration of streptozotocin, the membrane vesicles of junctional SR (heavy SR, HSR) were isolated from the ventricular myocytes, and SR $Ca^{2+}$ uptake and SR $Ca^{2+}$ release were measured. The activity of SR $Ca^{2+}-ATPase$ was $562{\pm}14$ nmol/min/mg protein in control heart. The activity was decreased to $413{\pm}30$ nmol/min/mg protein in diabetic heart and it was partially recovered to $485{\pm}18$ nmol/min/mg protein in insulin-treated diabetic heart. A similar pattern was observed in SR $^{45}Ca^{2+}$ uptakes; the specific uptake was the highest in control heart and it was the lowest in diabetic heart. In SR $^{45}Ca^{2+}$ release experiment, the highest release, 45% of SR $^{45}Ca^{2+}$, was observed in control heart. The release of diabetic heart was 20% and it was 30% in insulin-treated diabetic heart. Our results showed that the activities of both SR $Ca^{2+}-ATPase$ and SR $Ca^{2+}$ release channel were decreased in diabetic heart. In order to evaluate how these two factors contribute to SR $Ca^{2+}$ storage, the activity of SR $Ca^{2+}-ATPase$ was measured in the uncoupled leaky vesicles. The uncoupling effect which is able to increase the activity of SR $Ca^{2+}-ATPase$ was observed in control heart; however, no significant increments of SR $Ca^{2+}-ATPase$ activities were measured in both diabetic and insulin-treated diabetic rats. These results represent that the $Ca^{2+}$ storage in SR is significantly depressed and, therefore, $Ca^{2+}-sequestering$ activity of SR may be also depressed in diabetic heart.

• BMB Reports
• /
• 제29권1호
• /
• pp.22-26
• /
• 1996

Agents that activate or inhibit the $Ca^{2+}$ release channel in cardiac sarcoplasmic reticulum (SR) were tested for their abilities to affect the activity of the SR $Ca^{2+}$-ATPase. Vesicles of junctional SR (heavy SR, HSR) from terminal cisternae were prepared from porcine cardiac muscle by density gradient centrifugation. The steady-state activity of $Ca^{2+}$-ATPases in intact HSR vesicles was/$347{\pm}5\;nmol/min{\cdot}mg$ protein (${\pm}$ SD). When the HSR vesicles were made leaky, the activity was increased to $415{\pm}5\;nmol/min{\cdot}mg$ protein. This increase is probably due to the uncoupling of HSR vesicles. Caffeine (10 mM), an agonist of the SR $Ca^{2+}$ release channel, increased $Ca^{2+}$-ATPase activity in the intact HSR vesicle preparation to $394{\pm}30\;nmol/min{\cdot}mg$ protein. However, caffeine had no significant effect in the leaky vesicle preparation and in the purified $Ca^{2+}$-ATPase preparation. The effect of caffeine on SR $Ca^{2+}$-ATPase was investigated at various concentrations of $Ca^{2+}$. Caffeine increased the pump activity over the whole range of $Ca^{2+}$ concentrations, from $1\;{\mu}M$ to $250\;{\mu}M$, in the intact HSR vesicles. When the SR $Ca^{2+}$-ATPase was inhibited by thapsigargin, no caffeine effect was observed. These results imply that the caffeine effect requires the intact vesicles and that the increase in $Ca^{2+}$-ATPase activity is not due to a direct interaction of caffeine with the enzyme. We propose that the activity of SR $Ca^{2+}$-ATPase is linked indirectly to the activity of the $Ca^{2+}$ release channel (ryanodine receptor) and may depend upon the amount of $Ca^{2+}$ released by the channels.

• The Korean Journal of Physiology and Pharmacology
• /
• 제3권2호
• /
• pp.175-182
• /
• 1999

In the present study, the underlying mechanisms for diabetic functional derangement and insulin effect on diabetic cardiomyopathy were investigated with respect to sarcoplasmic reticulum (SR) $Ca^{2+}-ATPase$ and phospholamban at the transcriptional and translational levels. The maximal $Ca^{2+}$ uptake and the affinity of $Ca^{2+}-ATPase$ for $Ca^{2+}$ were decreased in streptozotocin-induced diabetic rat cardiac SR, however, even minimal amount of insulin could reverse both parameters. Levels of both mRNA and protein of phospholamban were significantly increased in diabetic rat hearts, whereas the mRNA and protein levels of SR $Ca^{2+}-ATPase$ were significantly decreased. In case of phospholamban, insulin treatment reverses these parameters to normal levels. Minimal amount of insulin could reverse the protein levels; however, it could not reverse the mRNA level of SR $Ca^{2+}-ATPase$ at all. Thus, the decreased SR $Ca^{2+}$ uptake appear to be largely attributed to the decreased SR $Ca^{2+}-ATPase$ level, which is further impaired due to the inhibition by the increased level of phospholamban. These results indicate that insulin is involved in the control of intracellular $Ca^{2+}$ in the cardiomyocyte through multiple target proteins via multiple mechanisms for the decrease in the mRNA for both SR $Ca^{2+}-ATPase$ and phospholamban which are unknown and needs further study.

• The Korean Journal of Physiology and Pharmacology
• /
• 제3권2호
• /
• pp.223-230
• /
• 1999

Alterations of cardiovascular function associated with various thyroid states have been studied. In hyperthyroidism left ventricular contractility and relaxation velocity were increased, whereas these parameters were decreased in hypothyroidism. The mechanisms for these changes have been suggested to include alterations in the expression and/or activity levels of various proteins; ${\alpha}-myosin$ heavy chain, ${\beta}-myosin$ heavy chain, ${\beta}-receptors,$ the guanine nucleotide-binding regulatory protein, and the sarcolemmal $Ca^{2+}-ATPase.$ All these cellular alterations may be associated with changes in the intracellular $Ca^{2+}$ concentration. The most important regulator of intracellular $Ca^{2+}$ concentration is the sarcoplasmic reticulum (SR), which serves as a $Ca^{2+}$ sink during relaxation and as a $Ca^{2+}$ source during contraction. The $Ca^{2+}-ATPase$ and phospholamban are the most important proteins in the SR membrane for muscle relaxation. The dephosphorylated phospholamban inhibits the SR $Ca^{2+}-ATPase$ through a direct interaction, and phosphorylation of phospholamban relieves the inhibition. In the present study, quantitative changes of $Ca^{2+}-ATPase$ and phospholamban expression and the functional consequences of these changes in various thyroid states were investigated. The effects of thyroid hormones on (1) SR $Ca^{2+}$ uptake, (2) phosphorylation levels of phospholamban, (3) SR $Ca^{2+}-ATPase$ and phospholamban protein levels, (4) phospholamban mRNA levels were examined. Our findings indicate that hyperthyroidism is associated with increases in $Ca^{2+}-ATPase$ and decreases in phospholamban levels whereas opposite changes in these proteins occur in hypothyroidism.

• The Korean Journal of Physiology and Pharmacology
• /
• 제6권2호
• /
• pp.101-107
• /
• 2002

In the present study, the postnatal developmental changes in the expressional levels of cardiac sarcoplasmic reticulum (SR) $Ca^{2+}$ regulatory proteins, i.e. $Ca^{2+}-ATPase,$ phospholamban, and $Ca^{2+}$ release channel, were investigated. Both SR $Ca^{2+}-ATPase$ and phospholamban mRNA levels were about 35% of adult levels at birth and gradually increased to adult levels. Protein levels of both SR $Ca^{2+}-ATPase$ and phospholamban, which were measured by quantitative immunoblotting, were closely correlated with the mRNA levels. The initial rates of $Ca^{2+}$ uptake at birth were about 40% of adult rates and also increased gradually during the myocardial development. Consequently, the relative phospholamban/$Ca^{2+}-ATPase$ ratio was 1 in developmental hearts. $Ca^{2+}$ release channel (ryanodine receptor) mRNA was about $50{\sim}60%$ at birth and increased gradually to adult level throughout the postnatal rat heart development. $^3[H]ryanodine$ binding increased gradually during postnatal myocardial development, which was closely correlated with ryanodine mRNA expression levels during the development except the ryanodine mRNA level at birth. These findings indicate that cardiac SR $Ca^{2+}-ATPase,$ phospholamban, and $Ca^{2+}$ release channel are expressed coordinately, which may be necessary for intracellular $Ca^{2+}$ regulation during the rat heart development.

• 대한약리학회지
• /
• 제30권1호
• /
• pp.117-124
• /
• 1994

Phospholamban은 심근 근장그물 $Ca^{2+}-ATPase$ 조절단백이다. 조절작용기전은 탈인산화된 phospholamban에 의해 $Ca^{2+}-ATPase$가 억제됨으로 나타나며, 이 phospholamban이 인산화됨으로 $Ca^{2+}-ATPase$에 대한 억제가 반전됨을 보인다. 최근에 phospholamban의 cytoplasmic domain만으로 $Ca^{2+}-ATPase$를 억제하기에는 불충분하다는 보고가 있어 본 실험을 계획하였다. $Ca^{2+}-ATPase$의 활성을 억제하는 phospholamban domain을 밝히기 위하여 합성한 phospholamban 펩타이드(아미노산 1-25)의 $Ca^{2+}$ uptake에 대한 효과를 살펴보았다. 골격근 근장그물에서 $Ca^{2+}-ATPase$를 분리한 후 phosphatidylcholine이나 phosphatidylcholine과 phosphatidylserine을 포함한 liposome에 재조합시켰다. Phospholamban 펩타이드는 phosphatidylcholine을 이용하여 재조합된 vesicles의 초기 $Ca^{2+}$ uptake rate를 억제하고, cAMP 의존성 protein kinase의 catalytic subunit로 인산화시킨 phospholamban 펩타이드는 이 억제를 반전시킴을 보여 주었다. Phosphatidylcholine과 phosphatidylserine을 포함한 제조합 vesicles에서도 같은 양상을 보였다. 이상의 결과로 미루어 볼 때 인산화 sites를 포함하고 있는 phospholamban의 cytoplasmic domain은 그 자체만으로도 근장그물 칼슘펌프를 억제하기에 충분하다고 결론지을 수 있다.

• The Korean Journal of Physiology
• /
• 제22권2호
• /
• pp.163-177
• /
• 1988

세포막을 구성하고 있는 지질의 물리학적 성상이 단백질의 세포막 속으로의 삽입과정 및 단백질의 기능에 미치는 영향을 관찰하기 위하여 골격근의 근세망(SR)으로부터 $Ca^{2+}-ATPase$ 단백질을 분리한 후 이를 세포막의 주 구성성분인 포스파티딜콜린(PC)과 포스파티딜에타노라민(PE)의 혼합지질과 재조합(reconstitution)시켰다. 이와같이 인공적으로 재조합된 구조물에서 $Ca^{2+}-ATPase$의 기능을 측정하기 위하여 칼슘지시색소인 아르세나죠III(AIII)를 이용한 분광방법과 방사선동위원소를 이용한 여과법으로 칼슘흡수율을 측정하였고 또한 ATP 가수분해 능력을 측정하였다. 실험결과 칼슘의 흡수율은 포스파티딜코린의 함량이 많은 혼합지질과 재조합시킬 때에 증가하였고, ATP 가수분해 능력은 포스파티딜함량이 25%까지는 포스타피딜코린의 양에 비례하여 증가하였으나 50%이상에서는 약간 감소하는 경향을 보였다. 한편 지질세포막속으로 단백질이 삽입되는 양은 포스파티딜 함량이 25%일 때 최고의 값을 보였으며 함량이 그 이하 또는 이상일 때는 감소하였다. 이상의 실험결과로보아 단백질의 기능은 세포막이 "bilayer" 구조를 갖출때에 증가하고 세포막속으로 단백질이 삽입되는 양은 세포막이 "non-bilayer" 구조를 형성할 때에 증가함을 알 수 있다.

• 한국생물물리학회:학술대회논문집
• /
• 한국생물물리학회 2001년도 학술 발표회 진행표 및 논문초록
• /
• pp.66-66
• /
• 2001

Diabetic cardiomyopathy has been suggested to be caused by abnormal intracellular $Ca^{2＋}$ homeostasis in the myocardium, which is partly due to a defect in calcium transport by the cardiac sarcoplasmic reticulum (SR). In the present study, the underlying mechanism for this functional derangement was investigated with respect to SR $Ca^{2＋}$-ATPase and phospholamban (PLB, the inhibitor of SR $Ca^{2+}$-ATPase).(omitted)d)

• 한국생물물리학회:학술대회논문집
• /
• 한국생물물리학회 1998년도 학술발표회
• /
• pp.29-29
• /
• 1998

Previously, we reported two types of vanadate-sensitive ATPases in the micro somes of tracheal epithelial cells, a high-affinity one and a low-affinity one. The low affinity vanadate-sensitive (LAVS) ATPase was sensitive to thapsigargin and cyclopiazonic acid, specific antagonists of ER-type Ca$\^$2＋/-ATPase, and mediated microsomal $\^$45/Ca$\^$2+/ uptake, implying that the LAVS-ATPase is an ER/SR-type Ca$\^$2+/-ATPase.(omitted)

• 한국생물물리학회:학술대회논문집
• /
• 한국생물물리학회 1999년도 학술발표회 진행표 및 논문초록
• /
• pp.48-48
• /
• 1999

Two types of vanadate-sensitive $Ca^{2＋}$-ATPases have been characterized in the microsomes of tracheal epithelial cells, a high affinity vanadate-sensitive (HA VS) and a low affinity vanadate-sensitive (LAVS) $Ca^{2＋}$-ATPases. The LA VS $Ca^{2＋}$-ATPase was sensitive to thapsigargin, implying that it is an ER/SR-type $Ca^{2+}$-ATPase.(omitted)d)