AC6 Expression Research Articles

Calmodulin Is Required for Vasopressin-stimulated Increase in Cyclic AMP Production in Inner Medullary Collecting Duct

Calmodulin plays a critical role in regulation of renal collecting duct water permeability by vasopressin. However, specific targets for calmodulin action have not been thoroughly addressed. In the present study, we investigated whether Ca2+/calmodulin regulates adenylyl cyclase activity in the renal inner medullary collecting duct. Rat inner medullary collecting duct suspensions were incubated in the presence or absence of 0.1 nM vasopressin and the calmodulin inhibitors, monodansylcadaverine, W-7, and trifluoperazine, followed by measurement of cAMP. Vasopressin-stimulated cAMP elevation was significantly attenuated in the presence of calmodulin inhibitors. Analysis of transglutaminase 2 knock-out mice confirmed that these compounds were not acting through inhibition of transglutaminase 2 activity. Calmodulin inhibitors also blocked both cholera toxin- and forskolin-stimulated cAMP accumulation. In isolated perfused tubules, W-7 reversibly blocked vasopressin-stimulated urea permeability, a process that requires a rise in intracellular cAMP but does not appear to involve protein trafficking to the apical plasma membrane. These results suggest that calmodulin is required for vasopressin-stimulated adenylyl cyclase activity in the intact inner medullary collecting duct. Reverse transcription-PCR, immunoblotting, and immunohistochemistry revealed the presence of the calmodulin-sensitive adenylyl cyclase type 3 in the rat collecting duct, an isoform previously not known to be expressed in the collecting duct. Long-term treatment of Brattleboro rats with a vasopressin analog markedly decreased adenylyl cyclase type 3 protein abundance, providing an explanation for long-term down-regulation of vasopressin response in the collecting duct. These studies demonstrate the importance of calmodulin in the regulation of collecting duct adenylyl cyclase activity and transport function.

Analysis of Oxygen-Sensitive Human Cardiac L-Type Ca2+ Channel α1C Subunit (hHT Isoform)

Publisher Summary This chapter discusses the analysis of oxygen-sensitive human cardiac L-type Ca 2+ channel α 1C subunit (hHT isoform). A study is described, which demonstrated the hypoxic inhibition of a recombinant human cardiac L-type Ca 2+ channel α1C subunit (the hHT splice variant). Inhibition was observed following stable α1C expression in HEK293 cells, in the absence of auxiliary subunits, and the effects of hypoxia appeared to mimic perfectly the effects of hypoxia on native L-type Ca 2+ channel activity in smooth muscle cells. In this chapter, the techniques used to generate mutant L-type Ca 2+ channels and to record their responses to hypoxia following expression in HEK293 cells are discussed. Experimental methodology for the construction of mutant L-type Ca 2+ channels, swapping C-tail segments between hHT and rHT cDNA clones, experimental procedures for blue–white colony screening (colorimetric assay) and purification and concentration of DNA are also described.

Neuropeptide Y Modulates L-Type Ca 2+ Current During Heart Development

An interesting and potentially very important study1 in this issue of Circulation Research reports that during development of the mammalian heart, neuropeptide Y can significantly increase L-type Ca2+ current, thereby enhancing heart rate and strength of contraction. In this study, a combination of targeted deletion of the NPY gene, electrophysiological recordings, and immunoblot analysis of Ca2+ channel α1C expression is used in an experimental design that involves study of hearts from pre- and postnatal animals at predetermined stages.1 These findings can perhaps be put in context by the following brief review of NPY physiology and pharmacology in mammalian tissue. Neuropeptide Y (NPY) was identified by Tatemoto and Mutt in 1982 through use of a novel chemical assay that detected the amidated C-terminal tyrosine residue of this 36-amino acid peptide.2,3 Isolated first from brain, it was soon discovered to be widely distributed throughout the body, primarily in postganglionic sympathetic nerves and often colocalized with norepinephrine.4 Indeed, NPY is the most widely distributed and abundant neuropeptide in the nervous system. NPY shares structural and functional homology with two other 36-amino acid peptides, pancreatic polypeptide (PP) and peptide YY (PYY).4 All …

Localization of Adenylyl Cyclase Isoforms and G Protein-Coupled Receptors in Vascular Smooth Muscle Cells: Expression in Caveolin-Rich and Noncaveolin Domains

A number of different agonists activate G protein-coupled receptors to stimulate adenylyl cyclase (AC), increase cAMP formation, and promote relaxation in vascular smooth muscle. To more fully understand this stimulation of AC, we assessed the expression, regulation, and compartmentation of AC isoforms in rat aortic smooth muscle cells (RASMC). Reverse transcription-polymerase chain reaction detected expression of AC3, AC5, and AC6 mRNA, whereas immunoblot analysis indicated expression of AC3 and AC5/6 protein primarily in caveolin-rich membrane (cav) fractions relative to noncaveolin (noncav) fractions. Beta(1)-adrenergic receptors (AR), beta(2)AR, and G(s) were detected in both cav and noncav fractions, whereas the prostanoid receptors EP(2)R and EP(4)R were excluded from cav fractions. We used an adenoviral construct to increase AC6 expression. Overexpressed AC6 localized only in noncav fractions. Two-fold overexpression of AC6 caused enhancement of forskolin-, isoproterenol- and prostaglandin E(2)-stimulated cAMP formation but no changes in basal levels of cAMP. At higher levels of AC6 overexpression, basal and adenosine receptor-stimulated cAMP levels were increased. Stimulation of cAMP levels by agents that increase Ca(2+) in native cells was consistent with the expression of AC3, but overexpression of AC6, which is inhibited by Ca(2+), blunted the Ca(2+)-stimulable cAMP response. These data indicate that: 1) RASMC express multiple AC isoforms that localize in both caveolin-rich and noncaveolin domains, 2) expression of AC6 in non-caveolin-rich membranes can increase basal levels of cAMP and response to several stimulatory agonists, and 3) Ca(2+)-mediated regulation of cAMP formation depends upon expression of different AC isoforms in RASMC. Compartmentation of GPCRs and AC is different in cardiomyocytes than in RASMC, indicating that targeting of these components to caveolin-rich membranes can be cell-specific. Moreover, our results imply that the colocalization of GPCRs and the AC isoforms they activate need not occur in caveolin-rich fractions.

496 Adenylyl cyclase mRNA expression and effects of adenylyl cyclase cascade on contractility in unilateral pregnant rat myometrium

496 ADENYLYL CYCLASE MRNA EXPRESSION AND EFFECTS OF ADENYLYL CYCLASE CASCADE ON CONTRACTILITY IN UNILATERAL PREGNANT RAT MYOMETRIUM KIMISATO ASANO 1, TOSHIAKI OKAWA t, HIDENORI TAKAHASHI 1, SATOSHI HASHIMOTO 1, HIROSHI ANBE t, AKIRA SATO I, YURI VEDERNIKOV~, GEORGE SAADE 2, ROBERT GARFIELD 2, YOSUKE SUZUKI t , ISAO MATSUOKA3; ]Fukushima Medical University, Obstetrics and Gynecology, Fukushima; 2University of Texas Medical Branch, Obstetrics and Gynecology, Galveston, TX; 3Fukushima Medical University, Pharmacology, Fukushima OBJECTIVE: To determine the effect of gestation on adenylyl cyclase (AC) mRNA expression, and on the response to protein kinase C (PKC) and a2 adrenergic agonists in gravid and non-gravid uterine horns of pregnant rats. STUDY DESIGN: Non-pregnan t and day 17 p regnan t Wistar rats were used (n = 6 per group) . In the p regnan t animals, one of the oviducts was ligated before mating in order to result in pregnancy in only one uterine horn. mRNA levels of AC2, AC4, AC6 ad AC9 were determined in the uterine horns with (fetus+) and without (fetus-) fetuses by semi-quantitive reverse transcription-polynaerase chain react ion (RT-PCR). In addit ion, strips of myometr ium fi-om the fetus+ and fetussides were p repa red for isometric tension recording in F-a'ebs-Ringer solution (37°C). The strips were treated with PK-C st inmlant (phorbol myristate acetat: PMA; 1 ~tM) or an a 2 adrenerg ic agonis t (UK-14304; 1 IxM). The changes of contracti l i ty were determined as %change from basal contractile activity. RESULTS: mRNA levels of AC4, AC6 and AC9 were no t significantly different between fetus+ and fetussides on day 47 of gestation. AC2's mRNA levels on the fetusside was significantly lower than the fetus+ side, and were not significantly different than the mRNA levels in the uteri of non-pregnant animals. Uterine contractil i ty was significantly decreased by PMA and UK14304 on the fetus+ side, but was not affected on the fetusside. CONCLUSION: Pregnancy increases AC4, AC6 and AC9 mRNA expression in the uterus by systenfic factors, but the gestational increase in AC2 is dependent on the local presence of pups. AC2 inhibits uterine contractility via a PKC and an ~2 adrenergic receptor in preglmnt myometrium. O f the various adenylyl cyclases, AC2 appears to play the d o m i n a n t role in mainta ining uter ine quiescence in pregnancy. Local factors affecting AC2's expression or function may initiate term or pre term labor. SMFM Abstracts S217