| Summary: | 碩士 === 國立陽明大學 === 生物化學研究所 === 87 === In our previous research， we found that ET-1 alone could stimulate 2-deoxyglucose uptake and the synergism between ET-1 and cAMP on glucose transport in 3T3-L1 adipocytes. We can make a conclusion that expression of the endothelin receptor in 3T3-L1 adipocytes. In addition, Fura-2 analysis reveal that the initial and transient rise of cytosolic Ca2+ evoked by ET-1 in 3T3-L1 adipocytes . If the 3T3-L1 adipocytes were pretreated with BQ788, we found the effect on ET-1`s induction of intracellular calcium release was inhibited by 50%. We can make a conclusion that expression of the endothelin receptor in 3T3-L1 adipocytes. In this report, we further characterize what kind of the endothelin receptor subtypes in 3T3-L1 adipocytes.
At first, we align the sequences of the endothelin receptors updated in GCG database for designing a pair of primer. We get the RT-PCR product that is about 0.6kb large on DNA agarose gel. Through the TA cloning , we can pick up a clone and its sequence has 94% similarity to updated partial mouse- ETA receptor. We labeled the fragment as probe for Northern blot analysis and a dominant band was located on 4kb. If the partial sequence of rat-ETB receptor were used as probe, two weak bands could be seen. One was located on 3kb, another was located on 1.7kb. At next step, we analyzed the tissue specific distribution of the ETAR. We found that there are high amount expression of ETA R in Heart and lung and weakly expression in liver、kidney、skeletal muscle and testis. If 3T3-L1 adipocytes were treated with ET-1、cAMP or ET-1 and cAMP for 8h , expression of ETAR were enhanced. If 3T3-L1 adipocytes were pre treated with PD98059 for 2h, action of ET-1 on the expression of ETAR is cancellated. If adipocytes were pretreated with staurosporine for 30 mins, action of ET-1 or cAMP on the expression of ETAR are reduced.
We use binding study to characterize expression of endothelin receptor subtypes in 3T3-L1 adipocytes. The number of ET-1 binding sites was determined in saturation binding studies. 3T3-L1 adipocytes have two endothelin binding sites. KD value of picomolar range binding site is 545pM and Bmax is1.786x105 sites/cell. KD value of nanomolar range binding site is 6.5nM and Bmax is 1.547x106 sites/cell。 BQ123, an ETAR-selective antagonist, displaced 125I-ET-1 binding from 3T3-L1 adipocytes with an IC50 value of 4pM. In addition, BQ788, an ETBR-selective antagonist, displaced 125I-ET-1 binding from 3T3-L1 adipocytes with an IC50 value of 300pM.
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