NHERF1 regulates parathyroid hormone receptor membrane retention without affecting recycling.

Na/H exchange regulatory factor-1 (NHERF1) is a PDZ protein that regulates trafficking of several G protein-coupled receptors. The phenotype of NHERF1-null mice suggests that the parathyroid hormone (PTH) receptor (PTH1R) is the principal GPCR interacting with NHERF1. The effect of NHERF1 on receptor recycling is unknown. Here, we characterized NHERF1 effects on PTH1R membrane tethering and recycling by radio-ligand binding and recovery after maximal receptor endocytosis. Using Chinese hamster ovary cells expressing the PTH1R, where NHERF1 expression could be induced by tetracycline, NHERF1 inhibited PTH1R endocytosis and delayed PTH1R recycling. NHERF1 also inhibited PTH-induced receptor internalization in MC4 osteoblast cells. Reducing constitutive NHERF1 levels in HEK-293 cells with short hairpin RNA directed against NHERF1 augmented PTH1R endocytosis in response to PTH. Mutagenesis of the PDZ-binding domains or deletion of the MERM domain of NHERF1 demonstrated that both are required for inhibition of endocytosis and recycling. Likewise, an intact COOH-terminal PDZ recognition motif in PTH1R is needed. The effect of NHERF1 on receptor internalization and recycling was not associated with altered receptor expression or binding, activation, or phosphorylation but involved beta-arrestin and dynamin. We conclude that NHERF1 inhibits endocytosis without affecting PTH1R recycling in MC4 and PTH1R-expressing HEK-293 cells. Such an effect may protect against PTH resistance or PTH1R down-regulation in certain cells harboring NHERF1.

D5 dopamine receptor regulation of reactive oxygen species production, NADPH oxidase, and blood pressure.

Activation of D1-like receptors (D1 and/or D5) induces antioxidant responses; however, the mechanism(s) involved in their antioxidant actions are not known. We hypothesized that stimulation of the D5 receptor inhibits NADPH oxidase activity, and thus the production of reactive oxygen species (ROS). We investigated this issue in D5 receptor-deficient (D5-/-) and wild-type (D5+/+) mice. NADPH oxidase protein expression (gp91(phox), p47(phox), and Nox 4) and activity in kidney and brain, as well as plasma thiobarbituric acid-reactive substances (TBARS) were higher in D5-/- than in D5+/+ mice. Furthermore, apocynin, an NADPH oxidase inhibitor, normalized blood pressure, renal NADPH oxidase activity, and plasma TBARS in D5-/- mice. In HEK-293 cells that heterologously expressed human D5 receptor, its agonist fenoldopam decreased NADPH oxidase activity, expression of one of its subunits (gp91(phox)), and ROS production. The inhibitory effect of the D5 receptor activation on NADPH oxidase activity was independent of cAMP/PKA but was partially dependent on phospholipase D2. The ability of D5 receptor stimulation to decrease ROS production may explain, in part, the antihypertensive action of D5 receptor activation.

Enhanced activation of RhoA by angiotensin II in SHR preglomerular microvascular smooth muscle cells.

Angiotensin II causes a greater renal vasoconstriction in spontaneously hypertensive rats (SHR) than in normotensive Wistar Kyoto rats (WKY), and alpha2-adrenoceptor agonists potentiate angiotensin II-induced renal vasoconstriction more in SHR. Because angiotensin II activates RhoA, and RhoA contributes to vasoconstriction, we tested the hypothesis that the ability of angiotensin II to stimulate RhoA in preglomerular vascular smooth muscle cells and the ability of alpha2-adrenoceptor activation to potentiate this response are augmented in cells from SHR. In SHR preglomerular vascular smooth muscle cells, angiotensin II (1 micromol/L) greatly stimulated RhoA activity, and this effect was markedly potentiated by UK 14,304 (1 micromol/L; alpha2-adrenoceptor agonist) (fold increase from vehicle-treated cells: 9.0 +/- 2, 0.8 +/- 0.2, and 13.6 +/- 3.2 in cells treated with angiotensin II, UK 14,304, and angiotensin II + UK 14,304, respectively). In contrast, in WKY cells, angiotensin II only mildly activated RhoA (2.0 +/- 0.50), and this response was not enhanced by UK 14,304. The expression of Galpha12 and Galpha13, G-proteins thought to link G-protein-coupled receptors to RhoA, was not increased in SHR cells. We conclude that angiotensin II-induced activation of RhoA is much more robust in the preglomerular microcirculation of SHR compared with WKY and that this may contribute to the etiology of genetic hypertension.

AT2 receptors cross talk with AT1 receptors through a nitric oxide- and RhoA-dependent mechanism resulting in decreased phospholipase D activity.

ANG II activation of phospholipase D (PLD) is required for ERK and NAD(P)H oxidase activation, both of which are involved in hypertension. Previous findings demonstrate that ANG II stimulates PLD activity through AT(1) receptors in a RhoA-dependent mechanism. Additionally, endogenous AT(2) receptors in preglomerular smooth muscle cells attenuate ANG II-mediated PLD activity. In the present study, we examined the signal transduction mechanisms used by endogenous AT(2) receptors to modulate ANG II-induced PLD activity through either PLA(2) generation of lysophosphatidylethanolamine or Galpha(i)-mediated generation of nitric oxide (NO) and interaction with RhoA. Blockade of AT(2) receptors, Galpha(i) and NO synthase, but not PLA(2), enhanced ANG II-mediated PLD activity in cells rich in, but not poor in, AT(2) receptors. Moreover, NO donors, a direct activator of guanylyl cyclase and a cGMP analog, but not lysophosphatidylethanolamine, inhibited ANG II-mediated PLD activity, whereas an inhibitor of guanylyl cyclase augmented ANG II-induced PLD activity. AT(2) receptor- and NO-mediated attenuation of ANG II-induced PLD activity was completely lost in cells transfected with S188A RhoA, which cannot be phosphorylated on serine 188. Therefore, our data indicate that AT(2) receptors activate Galpha(i), subsequently stimulating NO synthase and leading to increased soluble guanylyl cyclase activity, generation of cGMP, and activation of a protein kinase, resulting in phosphorylation of RhoA on serine 188. Furthermore, because AT(2) receptors inhibit AT(1) receptor signaling to PLD via modulating RhoA activity, AT(2) receptor signaling can potentially regulate multiple vasoconstrictive signaling systems through inactivating RhoA.

AT2 receptors attenuate AT1 receptor-induced phospholipase D activation in vascular smooth muscle cells.

Previous studies indicate that angiotensin (AT)(1) receptor-induced activation of phospholipase D (PLD) may importantly contribute to vascular hypertrophy, injury, and contraction. However, the role of AT(2) receptors in regulating AT(1) receptor-induced PLD activation is unknown. In this study, we identified angiotensin II receptors on cultured preglomerular vascular smooth muscle cells (PGSMCs) from spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) by reverse transcription-polymerase chain reaction (RT-PCR) and binding assays and examined their functional effects on angiotensin II-mediated PLD activity. Both RT-PCR and binding indicated that cultured SHR and WKY PGSMCs expressed AT(1) and AT(2) receptors, and the combined total of AT(1) and AT(2) receptors was similar between the strains. However, the number of AT(1) and AT(2) receptors differed between SHR and WKY PGSMCs in so much as the ratio of AT(1) to AT(2) receptors was approximately 1 to 1 and 3 to 1 in WKY and SHR PGSMCs, respectively. As previously reported, angiotensin II more potently activated PLD in SHR PGSMCs (SHR EC(50) = 4 nM; WKY EC(50) = 47 nM). Addition of an AT(2) receptor-specific antagonist or agonist shifted the angiotensin II-mediated PLD concentration-response curve of WKY PGSMCs in a manner consistent with AT(2) receptors producing an inhibitory signal. In contrast, in SHR little change was observed. Our findings indicate that the ratio of AT(1) to AT(2) receptors in vascular smooth muscle cells may be a determinant of the net effects of angiotensin II on PLD activity due to AT(2)-dependent inhibition of AT(1)-mediated PLD activity. Furthermore, cultured WKY PGSMCs provide an excellent model system to study endogenous AT(2) receptor signal transduction.

Role of EGFR transactivation in angiotensin II signaling to extracellular regulated kinase in preglomerular smooth muscle cells.

Angiotensin (Ang) II promotes the phosphorylation of extracellular regulated kinase (ERK); however, the mechanisms leading to Ang II-induced ERK phosphorylation are debated. The currently accepted theory involves transactivation of epidermal growth factor receptor (EGFR). We have shown that generation of phosphatidic acid (PA) is required for the recruitment of Raf to membranes and the activation of ERK by multiple agonists, including Ang II. In the present report, we confirm that phospholipase D-dependent generation of PA is required for Ang II-mediated phosphorylation of ERK in Wistar-Kyoto and spontaneously hypertensive rat preglomerular smooth muscle cells (PGSMCs). However, EGF stimulation does not activate phospholipase D or generate PA. These observations indicate that EGF recruits Raf to membranes via a mechanism that does not involve PA, and thus, Ang II-mediated phosphorylation of ERK is partially independent of EGFR-mediated signaling cascades. We hypothesized that phosphoinositide-3-kinase (PI3K) can also act to recruit Raf to membranes; therefore, inhibition of PI3K should inhibit EGF signaling to ERK. Wortmannin, a PI3K inhibitor, inhibited EGF-mediated phosphorylation of ERK (IC50, approximately 14 nmol/L). To examine the role of the EGFR in Ang II-mediated phosphorylation of ERK we utilized 100 nmol/L wortmannin to inhibit EGFR signaling to ERK and T19N RhoA to block Ang II-mediated ERK phosphorylation. Wortmannin treatment inhibited EGF-mediated but not Ang II-mediated phosphorylation of ERK. Furthermore, T19N RhoA inhibited Ang II-mediated ERK phosphorylation, whereas T19N RhoA had significantly less effect on EGF-mediated ERK phosphorylation. We conclude that transactivation of the EGFR is not primarily responsible for Ang II-mediated activation of ERK in PGSMCs.