血管增壓素（Vasopressin），又稱抗利尿激素（Antidiuretics），在人體脫水時產生。它的主要功能是促進水份之再吸收作用及血管收縮。而前列腺激素E2（Prostaglandin E2），可抗拮血管增壓素之作用，促進血管平滑肌之擴張，使血壓下降。此外，前列腺激素E2可以藉由抑制腎小管之再吸收作用而引發腎臟利尿作用（Diuresis）。血管增壓素及前列腺激素E2如何互相作用及抗衡，以維持腎臟正常的水份再吸收功能，曾被廣泛研究。環腺甘酸（cAMP），此細胞之重要第二訊息傳導物質，以被證實牽涉其中。但許多文獻報告之結果並不一致，主要原因是腎臟內部構造相當複雜且在組織準備方法上有所不同所致。腎臟集尿管（Collecting ducts）是腎臟中，發生水份再吸收作用相當頻繁的一段區域。在這段區域中，血管增壓素及前列腺激素E2如何互相傳達彼此訊息，及其與環腺甘酸（cAMP）之間如何互動，並不是很清楚，值得加以研究。血管增壓素及前列腺激素E2都是經由結合其在細胞膜上之受體，來引發一連串訊息傳導。此二者之受體都很複雜且有不同亞型（subtypes）。V2 receptors及EP3 receptors可能分別是血管增壓素及前列腺激素E2在腎臟集尿管中最主要之受體亞型。此二種亞型之受體，都是屬於G蛋白質連結之受體。V2受體已被證實連結到Gs蛋白質，而EP3受體已被證實連結到Gi蛋白質。藉由所連結之G蛋白質的不同，可以調節細胞內環腺甘酸之含量。大部分的研究都著重在此二受體所結合之G蛋白質上，但忽略了腺甘酸環化脢（Adenylyl Cyclases），此催化環腺甘酸形成之酵素，對於細胞內環腺甘酸之含量，亦伴演了舉足輕重之調控角色。腺甘酸環化脢在近年來，陸續有不少isozymes被發現，各種isozymes分布在不同組織，且受到細胞內其他因子，如蛋白質激脢（protein kinase C），鈣離子（Ca+2）之調控。我對於腎臟中，存在何種腺甘酸環化脢及此酵素如何調節由血管增壓素及前列腺激素E2引發之訊息傳導（Signal Transduction）感到興趣。利用RT-PCR，我們偵測到腺甘酸環化脢第六亞型是老鼠腎臟及腎臟集尿管細胞株（M-1 cell line）中，最主要之腺甘酸環化脢。而第二亞型血管增壓素受體，則是分布在腎臟集尿管細胞株（M-1 cell line）中，最主要的血管增壓素受體。至於前列腺激素E2受體之分布情形，更為複雜。以我們目前的結果顯示，EP3 beta form是老鼠腎臟中最主要之EP3 receptor。為了進一步瞭解腺甘酸環化脢，血管增壓素及前列腺激素E2之間的互動關係，我們將利用in situ PCR的技術，來偵測腺甘酸環化脢，血管增壓素受體及前列腺激素E2受體在腎臟集尿管的分布情形。此外，將利用co-transfection之方法，將V2 receptors及EP3 receptors此二種不同受體分別與不同腺甘酸環化脢isozymes送入真核細胞表現系統，以瞭解此二種受體與何種腺甘酸環化脢isozymes結合，來調控細胞內環腺甘酸之含量。藉由瞭解血管增壓素及前列腺激素E2之間的訊息傳導，及此二者與腺甘酸環化脢之間的相互關係，腺甘酸環化脢在腎臟生理功能上扮演之角色，將可得到進一步瞭解。
Vasopressin (AVP), also known as antidiuretic hormone (ADH), acts on the kidney to promote water retention and cause vasoconstriction. On the other hand, PGE2, the most important PG in the kidney, is well known as a potent antagonist of the AVP and causes vasodilation. It also inhibits water reabsorption from kidney cells into the blood and causes diuresis. The water reabsorption mechanism in the kidney is regulated by AVP via a cAMP-dependent pathway. The effect of AVP and PGE2 on cell cAMP content has been a subject of intensive investigation. However, the observations are contradictory and no definitive mechanisms have been proved yet. These different results may be due to the complex nephron heterogeneity and difference in tissue preparation. Both AVP and PGE2 exert their physiological functions through binding to their receptors on the cell membranes. Different subtypes of AVP and PGE2 receptors have been reported. The collecting ducts are the major segments in the kidney which undergo water reabsorption. V2 receptors and EP3 receptors are the major receptors found in the collecting ducts. According to previous reports, both V2 receptors and EP3 receptors are G protein coupled receptors. V2 receptors are linked to Gs protein while the EP3 receptors are linked to Gi protein. A lot of attention has paid to the G protein in modulating the cAMP level inside the cell. However, not much work has been done in investigating which adenylyl cyclase (AC) isozymes are coupled to these receptors. ACs catalyze the formation of cAMP from ATP. In the recent years, the control of cAMP content in the cell has become more complex by the finding of several types of ACs with different regulatory properties. Using RT-PCR, we have investigated the distribution of AC isozymes, V2 receptors and EP3 receptors in the rat kidney and M-1 cells. AC VI was the major AC isozyme found in both rat kidney and M-1 cells. V2 receptor was detected as the major AVP receptor in M-1 cells. Further in situ PCR will be used to investigate the co-localization of V2 receptor and AC VI in the collecting duct. Co-transfection of V2 receptor and AC VI into CHO-K1 cells will provide the coupling efficiency between V2 receptor and this specific AC. For EP3 receptors, EP3 beta form seems to be the major isoform in the rat kidney. Whether this isoform is the major EP3 receptor in M-1 cell and the collecting duct will be verified by RT-PCR. The co-localization of EP3 receptors with AC isozymes and V2 receptor in the collecting duct will be tested by in situ PCR. By understanding the cellular signaling of AVP and PGE2 and their coupling efficiency to ACs, the relevance of ACs to the renal physiological functions, such as water reabsorption in collecting ducts, will be clarified.
