흰쥐 이하선 선세포에서 Arachidonic acid 및 Myristic acid에 의한 Carbachol-induced K+ -release의 변화

Abstract

[한글]

타액선 선세포막에는 세포막에 있는 여러 종류의 수용체 (muscarinic, α 및 β-adrenergic, 그리고 peptidergic 수용체 등)가 있으며, 이들 수용체가 자극을 받으면 선세포막에 있는 K**+ 및 Cl**- 통로 (channel)가 열리게 되고 이어 Na**+과 물이 따라 나가 일차 타액을 형성하게 된다. 실제 외부 자극을 받아 타액이 분비될 때, 세포내 신호전환 (signal transduction) 과정을 거쳐 이차전령체 (secondary messenger인 Ca**2+ 이나 혹은 cAMP의 증가가 수반되기 때문에 (이를 각각 Ca**2+ 경로, CAMP 경로라고 부른다), 새포내 Ca**2+ 이나 CAMP의 농도 변화를 타액분비의 중요한 과정으로 인식하고 있다. 이와 같이 Ca**2+ 과 cAMP는 수용체를 자극할 수 있는 각각의 표현제 (agonist)에 의하여 그 농도가 변화되지만, 이외에 지방산인 arachidonic acid (AA)나 myristic acid (MA)도 Ca**2+ 의존성 phospholipase A2, phnspholipase C와 diacylglycerol을 복합적으로 활성화시키는 것으로 보아, 이들 지방산을 inositol trisphosphate (IP3)와 연계된 Ca**2+ 동원에 관여하는 조절인자로 보고 있으나, AA나 MA가 이하선 선세포막의 K**+ 통로에 어떤 영향을 미치는지에 대해서는 아직 명백히 밝혀진 바 없다.

따라서 본 실험에서는 흰쥐 (Sprgue Dawley)외 이하선을 분리하여 미세조직 절편을 만들고 이 조직절편을 perifusion chamber에 넣고 Krebs-Rinser bicarbonate (KRB) 용액을 관류시키면서 선세포를 자극한 후 이로부터 분비되는 K**+ -release를 K**+ 에 민감한 유리전극(K**+ -sensitive electrode)으로 계측함으로써, 1) AA나 MA에 의한 K**+ -release의 변화와, 2) 다수 불포화지방산인 AA와 MA가 carbacbol-induced K**+ -release (CCh-induced K**+ -release)에 미치는 영향을 관찰하였다. 실험결과 AA (100 μM/L)와 MA (100 μM/L) 그 자체는 모두 흰쥐 이하선 조직절편에서 K**+ 을 유리시켰으나, 이는 CGh-induced K**+ -release외 4.8-11.25% 정도로 적었다. 이와 같이 AA와 MA는 이하선 선세포로부터 K**+ -release를 증가시켰는데도 AA나 MA를 CCh과 동시에 투여한 경우 CCh-induced K*

*+ -release를 억제하여 이들 지방산과 CCh간에는 서로 교차반응 (cross interaction)이 있는 것으로 생각된다. 특히 세포외부에 Ca**2+ 이 없는 조건에서, AA 및 MA는 carbachol-induced K**+ -release를 각각 31.6%, 34.8% 감소시켜, 이들 지방산이 carbachol-induced K**+ -release를 억제하는 효과는 같은 정도였다. 그러나 Ca**2+ 이 존재하는 경우 AA와 MA가 carbachol-induced K**+ -release를 감소시키는 효과는 각각 27.3%, 7.5%로 다르게 나타났다. 이는 AA 및 MA에 의한 carbochol-induced K**+ -release의 억제 기전이 서로 다르다는 사실을 암시하는 것으로 해석되는데, 즉 myristic acid는 Ca**2+ entry와 Ca**2+ mobilization을 동시에 감소시켜 carbochol-induced K**+ -release를 줄이는 것으로 보이나, 이와는 달리 AA는 Ca**2+ entry와 무관한 세포내부의 Ca**2+ mobilization만을 차단하여 carbachol-induced K**+ -release를 억제하는 것으로 생각된다.

[영문]

In rat salivary glands, many hormones and transmitter receptors (muscarinic, adrenergic, and peptidergic receptor etc.) transduce signals in a wide variety of cells though the activation of phospholipases and the production of second messenger molecules derived from phospholipids. Particularly, the stimulation of

muscarinic receptor in rat parotid acini results in the increase in the intracellular calcium (Ca**2+) level via inositol trisphosphate (IP3). Thus, a rise of Ca**2+ activates K**+ channels in the basolateral membrane and Cl**- channels in the apical membrane, which in turn evokes the salivary secretion Cytosolic Ca**2+ modulating K**+ channel is mobilized via two different routes : 1) Ca**2+ entry though the Ca**2+ channel, 2) Ca**2+ mobilization released from the intracellular Ca**2+ pool. In regard to Ca**2+ mobilization the existence of IP3-insensitive

Ca**2+ pool (IICP) has bean reported. Furthermore, close intercommunication between two Ca**2+ pool has been proposed to account for the mechanism of intracellular Ca**2+ mobilization.

In the rat carotid gland, two main signal transduction processes have actually been described ; the cAMP pathway involving adenylyl cyclase and Ca**2+ /phosphlipid pathway involving a phospholipase C. In addition to these two signal

pathways, fatty acids such as arachidonic acid and myristic acid might regulate the signal pathways and directly the function of ionic channel in pancreatic acini. Therefore, the aims of the present study are to examine 1) the effect of arachidonic acid and myristic acid ctrl K**+ -release from the rat parotid acini,

2) the changes in CCh-induced K**+ -release in response to arachidonic acid and myriltic acid, and 3) alterations in CCh-induced K**+ -release by the exposure arachidonic acid and myristic acid in the absence of extracellular Ca**2+ .

Rat (Sprague Dawley) carotid gland was dissected out and the tissue minced with scissor. The minced tissue was placed in the perifusion chamber with the K**+ sensitive electrode(Orion Research), followed by perifusing the parotid acini with Krebs-Ringer bicarbonate(KRB) solution. To stimulate the parotid acini, CCh was perifused with KRB solution. Then K**+ -release from parotid acini was measured using K**+ -sensitive electrode and interpreted in terms of intracellular Ca**2+ concentrate. In the results of these study, the small increase in K**+ -release

from the rat parotid was detected in response to the stimulation with arachidonic acid and myristic acid. The amount of K**+ -release increased by the stimulation with arachidonic acid find myristic acid was 4.8-11.25% as compared to that increased by stimulation with carbachol omly. However, the simutaneous

administration of myristic acid and arachidonic acid attenuated the K**+ -release induced by carbachol only, which is interpreted as some cross interactions between these fatty acids and CCh. The attenuation effects by AA and MA were by 31.6% and 34.8% respectively, under the condition of the absence of extracellular Ca**2+ .

But these responses were different under the environment of calcium solution, resulting in the attenuation by 27.3% and 7,5% each, which can be interpreted as a possibility of the difference in the maimer of reducing ability of CCh-induced K**+ -release by each fatty acid.

Taken all together, arachidonic acid and myristic acid itself might act to open the K**+ -channel via the increases in intracellular Ca**2+ concentration. However, it may be suggested that the atenuation effect of K**+ -release in response to simultaneous administration of fatty acids and CCh was different. In another words, Myristic acid seemed to reduce the carbachol-induced K**+ -release by interfering with both Ca**2+ entry and Ca**2+ mobilization, but AA is thought to be effective only in interfering with the process of Ca**2+ mobilization.