In the present study, we show that the eicosanoid compound, 20-hydroxyeicosatetraenoic acid (20-HETE), an important arachidonic acid metabolite, activates mouse TRPC6 in a stable, overexpressing HEK293 cell line, Hek-t6.11. Application of 20-HETE rapidly induced an inward, non-selective current in whole-cell recordings, which was inhibited by N-methyl-D-glucamine, 1.8 mM Ca 2؉ , and 100 M Gd 3؉ but remained unaffected by flufenamate and indomethacin. The current-voltage relationship obtained at low concentrations of 20-HETE (1-10 M) demonstrated slight inward rectification, whereas the highest concentration of 20-HETE tested (30 M) showed outward rectification, as shown previously for these channels using 100 M 1-oleoyl-2-acetylsn-glycerol. Dose-response curves indicate that 20-HETE activated TRPC6 channels with an EC 50 ‫؍‬ 0.8 M. Single channel analysis using inside-out patches revealed that 20-HETE increased open probability of mouse TRPC6 channels ϳ3-fold, and this was in a membrane-delimited fashion. Interestingly, 20-HETE did not provoke changes in intracellular Ca 2؉ concentrations. Thus, we have identified an arachidonic acid metabolite, 20-HETE, as a novel activator for a TRP family member, TRPC6.Cationic channels thought to be responsible for capacitative Ca 2ϩ entry, as originally defined by Putney (1, 2), have been identified in the plasmalemmal membrane of a spectrum of cells (3, 4). Capacitative Ca 2ϩ entry, through store-operated channels (SOCs) 1 is triggered by the emptying of intracellular Ca 2ϩ stores by a variety of maneuvers (4 -6). SOCs differ in their ionic selectivity, conductance, and sensitivity to inorganic and organic blockers. The best characterized SOC is the calcium release-activated calcium channel, a highly selective Ca 2ϩ ion channel first described in mast cells (7) and T lymphocytes (8), whereas non-selective cationic channels activated by store depletion have also been described (9). The molecular identity of store-operated channels has not yet been firmly established despite accumulating evidence that they could be members of the TRPC family (10 -12). The transient receptor potential (dTRP) channel, found in Drosophila eye, is a storeoperated Ca 2ϩ channel, whereas the trp-like (dTRPL) protein functions as a constitutively activated, non-selective cation channel (13-15). In mammals, seven dTRP homologs, TRPC1-TRPC7 (16 -18), have been found. Overexpression studies have demonstrated that these channels show important differences in a number of properties including their mode of activation, unitary conductance, and selectivity, which may or may not be a result of the expression system used (for reviews, see Refs. 4, 17, and 19). Moreover, the formation of heteromeric TRPC channels has been reported, whose characteristics were quite distinct from those recorded for the individual, homomeric channels; such behavior was shown for coassembly of TRPC1/ TRPC3 (20) and TRPC1/TRPC5 (21). A number of studies have reported that some members of the TRPC family can be activated by produ...

The present report details the role of Ca 2 ϩ in the early events of ACTH action in human adrenal glomerulosa cells.

Previous studies have shown that human fetal adrenal gland from 17-to 20-week-old fetuses expressed pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, which were localized on chromaffin cells. Pituitary adenylate cyclase-activating polypeptide is a 38-residue ␣-amidated neuropeptide (PACAP-38) 1 originally isolated from the ovine hypothalamus for its ability to stimulate cAMP formation in rat anterior pituitary cells. Processing of PACAP-38 can generate a 27-amino acid amidated peptide (PACAP-27) that exhibits 68% sequence identity with vasoactive intestinal polypeptide (VIP), thus identifying PACAP as a member of the VIP/secretin/glucagon superfamily of regulatory peptides (1, 2).The effects of PACAP are mediated through interaction with two types of high affinity receptors: type I receptors are selectively activated by PACAP, whereas type II receptors bind PACAP and VIP with similar affinity (3). Three isoforms of PACAP receptors have now been cloned and designated as PACAP-specific receptor I (PAC 1 -R) (4, 5) and VIP/PACAP mutual receptors 1 and 2 (VPAC 1 -R and VPAC 2 -R) (6, 7). Both PAC 1 -R (type 1 receptors) and VPAC 1 -R/VPAC 2 -R (type 2 receptors) belong to the seven-transmembrane domain, G-protein-coupled receptor family, and are all positively coupled to adenylyl cyclase (2). Eight isoforms of PAC 1 -R, resulting from alternative splicing, have been characterized to date. These variants display differential signal transduction properties with regard to adenylyl cyclase and phospholipase C (PLC) stimulation (1, 2). In addition to these classical signaling pathways, PACAP has been found to stimulate a Ca 2ϩ -calmodulin nitric oxide synthase (8) and mitogen-activated protein kinase activity (9). These various transduction mechanisms are involved in the neurotrophic activities exerted by PACAP (i.e. inhibition of apoptosis and stimulation of neurite outgrowth) during development (9 -11).PACAP and its receptors are actively expressed in the adrenal medulla (12)(13)(14). In particular, we have previously demonstrated the occurrence of PACAP-38 (15) and PACAP binding sites (16) in chromaffin cells from 16-to 20-week-old fetal human adrenal glands. Activation of these receptors by PACAP-38 causes stimulation of cAMP production and induces a modest increase in inositol 1,4,5-triphosphate (IP 3 ) formation (16), suggesting a role for the neuropeptide in the developing

Ionic currents of primary cultured glomerulosa cells from human adrenal glands were studied with the patch-clamp technique. Two types of outward K+ currents and two types of inward Ca2+ currents were described. The transient outward K+ current activated at potential positive to -40 mV and demonstrated a marked time-dependent inactivation. It was blocked by 4-aminopyridine but not tetraethylammonium. A second type of outward current activated rapidly at the depolarization onset and then increased slowly with no time-dependent inactivation. The transient inward T-type Ca2+ current was activated for potential positive to -60 mV with a maximal current amplitude at -30 mV and zero current voltage at +40 mV; it was completely inactivated for membrane potential positive to -40 mV. The pharmacological studies of the T-type channel showed that Ni2+ was a potent blocker but that the channel was not sensitive to dihydropyridine. The long-lasting inward Ca2+ current was activated for potentials positive to -20 mV with a maximum current amplitude at +70 mV. This current was increased by the agonist Bay K 8644 and blocked by the antagonist nifedipine; in addition, it was blocked by Cd2+ but less sensitive to Ni2+. This study revealed that glomerulosa cells from human adrenal demonstrated the presence of K+ and Ca2+ currents similar to those found in rat and bovine cells. Moreover, the main stimuli of aldosterone secretion, ACTH and angiotensin II, induce an increase in aldosterone secretion which is inhibited in a Ca(2+)-free external medium.

Angiotensin II (Ang II) has been reported to induce migration in neuronal cell types. Using time-lapse microscopy, we show here that Ang II induces acceleration in NG108-15 cell migration. This effect was antagonized by PD123319, a selective AT2 receptor antagonist, but not by DUP753, a selective AT1 receptor antagonist, and was mimicked by the specific AT2 receptor agonist CGP42112. This Ang II-induced acceleration was not sensitive to the inhibition of previously described signaling pathways of the AT2 receptor, guanylyl cyclase/cyclic GMP or p42/p44 mapk cascades, but was abolished by pertussis toxin treatment and involved PP2A activation. Immunofluorescence studies indicate that Ang II or CGP42112 decreased the amount of filamentous actin at the leading edge of the cells. This decrease was accompanied by a concomitant increase in globular actin levels. Regulation of actin turnover in actin-based motile systems is known to be mainly under the control of the actin depolymerizing factor and cofilin. Basal migration speed decreased by 77.2% in cofilin-1 small interfering RNA-transfected NG108-15 cells, along with suppression of the effect of Ang II. In addition, the Ang II-induced increase in cell velocity was abrogated in serum-free medium as well as by genistein or okadaic acid treatment in a serum-containing medium. Such results indicate that the AT2 receptor increases the migration speed of NG108-15 cells and involves a tyrosine kinase activity, followed by phosphatase activation, which may be of the PP2A type. Therefore, the present study identifies actin depolymerization and cofilin as new targets of AT2 receptor action, in the context of cellular migration.