We determined the relationship between the localization of rhoA and Ca 2؉ sensitization of force in smooth muscle. In ␣-toxin-permeabilized rabbit portal vein at pCa 6.5, the particulate hydrophobic fraction of rhoA (10 ؎ 1.6% of the total) was significantly increased by phenylephrine to 18 ؎ 5.5% at 5 min, by AlF 4 ؊ to 26 ؎ 8.4% at 20 min, and dose-dependently up to 62 ؎ 9.5% by guanosine 5-O-(3-thiotriphosphate) (GTP␥S; 0.3-50 M). Translocation of rhoA was selective (Rac1 and Cdc42 were not translocated) and was quantitatively correlated (up to ϳ50%; r ‫؍‬ 0.91, p < 0.05) with Ca 2؉ sensitization; high GTP␥S concentrations ( 10 M) further increased translocation without increasing force. The initial recruitment of rhoA to the membrane paralleled the time course of contraction, but sensitization could be reversed without a decrease in particulate rhoA. High [Ca 2؉] (pCa 4.5) also increased particulate rhoA to 31 ؎ 5.8%. Membrane-associated rhoA in unstimulated portal vein was a good substrate for in vitro ADP-ribosylation, whereas the large amount translocated by GTP␥S was not. We conclude that 1) translocation of rhoA plays a causal role in Ca 2؉ sensitization, and 2) membrane-bound rhoA can exist in two or more states.Phosphorylation of the regulatory light chain of myosin (MLC 20 ) 1 by a calcium/calmodulin-dependent protein kinase is the primary determinant of force developed by smooth muscle. However, this phosphorylation can also be increased ("Ca 2ϩ sensitization") at constant [Ca 2ϩ ] by a G-protein-coupled mechanism (1-4) that inhibits the trimeric phosphatase (SMPP-1M) (5-7) that dephosphorylates MLC 20 . The Ca 2ϩ -sensitizing effect of recombinant p21rhoA and the inhibition of agonist-induced Ca 2ϩ sensitization by selective ADP-ribosylation of p21 rhoA (either recombinant or endogenous) have implicated this monomeric G-protein in Ca 2ϩ sensitization (8 -11). Because bacterially expressed p21rhoA that lacks the prenylated C terminus required for membrane association (12) did not show significant Ca 2ϩ -sensitizing activity and even active (geranylgeranylated) p21rhoA failed to Ca 2ϩ -sensitize preparations heavily permeabilized with Triton X-100 (11), we suggested that association of p21rhoA with the plasma membrane may be required for its Ca 2ϩ -sensitizing effect (11). Recruitment of cytosolic proteins to the membrane is an important component of several other signaling systems, such as the Raf-Ras pathway (13) and protein kinase C cascades (conventional and novel) (14). The purpose of this study was to determine whether p21 rhoA signaling of Ca 2ϩ sensitization also involves its translocation to the cell membrane in vivo. We now show that GTP␥S-induced translocation of p21 rhoA is quantitatively and kinetically associated with Ca 2ϩ sensitization of smooth muscle and provide evidence of more than one conformational state of membrane-associated p21 rhoA . MATERIALS AND METHODSIsometric Tension Measurement-Small strips (200 m wide and 3 mm long) of rabbit portal vein and ileum longitud...

The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) MgCl2/1 mM dithiothreitol/1 mM phenylmethylsulfonyl fluoride/20 ,tM GTP (buffer A), disrupted in a glass homogenizer, and centrifuged at 640 x g for 15 min, and the supernatant was decanted. The residue was twice resuspended in 70 ml of buffer A and recentrifuged. The combined supernatants containing the cell membrane fragments were centrifuged at 186,000 x g for 90 min, and the pellet was resuspended in 50 ml of buffer A containing 2% octyl glucoside, homogenized, and gently shaken for 45 min. The solution was centrifuged at 142,000 x g for 90 min, and the supematant was filtered through a 0.45-,tm Minisart cellulose acetate filter (Sartorius). The solution (-0.4 mg of protein per ml) was loaded into a 10-ml MonoS column equili-

Adipose tissue expresses components of the renin-angiotensin system (RAS). Angiotensin converting enzyme (ACE2), a new component of the RAS, catabolizes the vasoconstrictor peptide ANG II to form the vasodilator angiotensin 1-7 [ANG-(1-7)]. We examined whether adipocytes express ACE2 and its regulation by manipulation of the RAS and by high-fat (HF) feeding. ACE2 mRNA expression increased (threefold) during differentiation of 3T3-L1 adipocytes and was not regulated by manipulation of the RAS. Male C57BL/6 mice were fed low-(LF) or high-fat (HF) diets for 1 wk or 4 mo. At 1 wk of HF feeding, adipose expression of angiotensinogen (twofold) and ACE2 (threefold) increased, but systemic angiotensin peptide concentrations and blood pressure were not altered. At 4 mo of HF feeding, adipose mRNA expression of angiotensinogen (twofold) and ACE2 (threefold) continued to be elevated, and liver angiotensinogen expression increased (twofold). However, adipose tissue from HF mice did not exhibit elevated ACE2 protein or activity. Increased expression of ADAM17, a protease responsible for ACE2 shedding, coincided with reductions in ACE2 activity in 3T3-L1 adipocytes, and an ADAM17 inhibitor decreased media ACE2 activity. Moreover, ADAM17 mRNA expression was increased in adipose tissue from 4-mo HF-fed mice, and plasma ACE2 activity increased. However, HF mice exhibited marked increases in plasma angiotensin peptide concentrations (LF: 2,141 Ϯ 253; HF: 6,829 Ϯ 1,075 pg/ml) and elevated blood pressure. These results demonstrate that adipocytes express ACE2 that is dysregulated in HF-fed mice with elevated blood pressure compared with LF controls.

The ovulatory process is activated by a surge of LH, a pituitary gonadotropin, which initiates a cohort of dramatic changes in biochemical, physical, and gene expression in the ovary, leading to follicle rupture and oocyte release. Here we report the identification of endothelin-2 (EDN2) as a last moment-trigger of follicle rupture. In the ovary, EDN2 is exclusively and transiently expressed in the granulosa cells immediately before ovulation. Administration of EDN2 to the ovarian tissue induced rapid contraction, whereas addition of tezosentan, an endothelin receptor antagonist, diminishes the EDN2 effect. In vivo, treatment of tezosentan before ovulation substantially decreases gonadotropin-induced superovulation. As a target tissue of EDN2 action, we identified a layer of smooth muscle cells in the follicular wall of each follicle. Taken together, our data indicate that EDN2 induces follicular rupture by constricting periovulatory follicles.

The effects of the Rho-kinase inhibitor, Y-27632 [1] on Ca P+ -sensitization of force induced by arachidonic acid (AA), phorbol 12,13-dibutyrate (PDBu), GTPQ QS, and by the stable thromboxane analog, 9,11-dideoxy-9K K,11K K-methanoepoxy-PGF PK (U-46619), were determined in K K-toxin-permeabilized smooth muscles. Y-27632 relaxed (up to 99%) Ca P+ -sensitization by GTPQ QS (10 W WM) and U-46619 (1 W WM), but not by PDBu (20 W WM), and reduced GTPQ QS-induced myosin light chain (MLC PH ) phosphorylation from 28% to 17% (P = 0.002). GTPQ QS-induced force sensitization was inhibited by Y-27632 more potently when the inhibitor was added during the plateau of force than prior to stimulation. In K K-toxin-permeabilized smooth muscle, Y-27632 inhibited AA (50 W WM)-induced Ca P+ -sensitization of force (by 66 þ 1.3%) and reduced MLC PH phosphorylation. In contrast, Y-27632 did not relax force Ca P+ -sensitized by AA in smooth muscle permeabilized with Triton X-100. We conclude that (i) AA induces Ca P+ -sensitization through dual mechanisms, one mediated by Rho-kinase (or a related kinase), and (ii) Rho-kinase is not required for phorbol ester-induced Ca P+ -sensitization.z 1998 Federation of European Biochemical Societies.