In vitro studies suggest that the G protein-coupled receptor (GPR) 30 is a functional estrogen receptor. However, the physiological role of GPR30 in vivo is unknown, and it remains to be determined whether GPR30 is an estrogen receptor also in vivo. To this end, we studied the effects of disrupting the GPR30 gene in female and male mice. Female GPR30((-/-)) mice had hyperglycemia and impaired glucose tolerance, reduced body growth, increased blood pressure, and reduced serum IGF-I levels. The reduced growth correlated with a proportional decrease in skeletal development. The elevated blood pressure was associated with an increased vascular resistance manifested as an increased media to lumen ratio of the resistance arteries. The hyperglycemia and impaired glucose tolerance in vivo were associated with decreased insulin expression and release in vivo and in vitro in isolated pancreatic islets. GPR30 is expressed in islets, and GPR30 deletion abolished estradiol-stimulated insulin release both in vivo in ovariectomized adult mice and in vitro in isolated islets. Our findings show that GPR30 is important for several metabolic functions in female mice, including estradiol-stimulated insulin release.

Abstract-The reactivity of the vascular wall to endothelin-1 (ET-1) is influenced by cholesterol, which is of possible importance for the progression of atherosclerosis. To elucidate signaling steps affected, the cholesterol acceptor methyl-␤-cyclodextrin (m␤cd, 10 mmol/L) was used to manipulate membrane cholesterol and disrupt caveolae in intact rat arteries. In endothelium-denuded caudal artery, contractile responsiveness to 10 nmol/L ET-1 (mediated by the ET A receptor) was reduced by m␤cd and increased by cholesterol. Neither ligand binding nor colocalization of ET A and caveolin-1 was affected by m␤cd. Ca 2ϩ inflow via store-operated channels after depletion of intracellular Ca 2ϩ stores was reduced in m␤cd-treated caudal arteries, as shown by Mn 2ϩ quench rate and intracellular [Ca 2ϩ ] response. Expression of TRPC1, 3, and 6 was detected by reverse transcriptase-polymerase chain reaction, and colocalization of TRPC1 with caveolin-1 was reduced by m␤cd, as seen by immunofluorescence. Part of the contractile response to ET-1 was inhibited by Ni 2ϩ (0.5 mmol/L) and by a TRPC1 blocking antibody. In the basilar artery, exhibiting less store-operated channel activity than the caudal artery, ET-1-induced contractions were insensitive to the TRPC1 blocking antibody and to m␤cd. Increased store-operated channel activity in basilar arteries after organ culture correlated with increased sensitivity of ET-1 contraction to m␤cd. These results suggest that cholesterol influences vascular reactivity to ET-1 by affecting the caveolar localization of TRPC1. Key Words: arterial smooth muscle Ⅲ methyl-␤-cyclodextrin Ⅲ caveolae Ⅲ endothelin Ⅲ store-operated Ca 2ϩ channels H ypercholesterolemia increases reactivity to endothelin-1 (ET-1) in experimental animals and humans. [1][2][3][4] This has been pointed out as one possible factor in the progression of atherosclerosis. [5][6][7] The mechanism of action has not been elucidated, although both endothelial dysfunction and altered smooth muscle reactivity have been proposed. 5 Lipoprotein particles may directly influence endothelial membrane-associated endothelial NO synthase activity by interfering with cholesterol-rich domains referred to as caveolae. 8 Although these effects modulate the endothelial influence on vascular tone, less is known regarding direct effects of cholesterol on vascular smooth muscle functions.Caveolae are 50-to 100-nm membrane invaginations that integrate many cellular receptor functions. 9 For instance, ET A receptors expressed in COS cells colocalize with the caveolae-associated protein caveolin. 10,11 The caveolar structure is disrupted after depletion of cholesterol with cyclodextrins, 12 and this correlates with a decreased contractility to ET-1, but not to depolarization or ␣ 1 -receptor stimulation, in endothelium-denuded rat caudal arteries. 13 Cholesterol might thus modulate the strength of caveolae-associated signaling, providing a basis for altered contractility in response to ET-1.Activation of the ET A receptor stimulates Ca 2ϩ inflow ov...

Objective-This study assessed the role of cholesterol-rich membrane regions, including caveolae, in the regulation of arterial contractility. Methods and Results-Rat tail artery devoid of endothelium was treated with the cholesterol acceptor methyl-␤-cyclodextrin, and the effects on force and Ca 2ϩ handling were evaluated. In cholesterol-depleted preparations, the force responses to ␣ 1 -adrenergic receptors, membrane depolarization, inhibition of myosin light chain phosphatase, and activation of G proteins with a mixture of 20 mmol/L NaF and 60 mol/L AlCl 3 were unaffected. In contrast, responses to 5-hydroxytryptamine (5-HT), vasopressin, and endothelin were reduced by Ͼ50%. The rise in global intracellular free Ca 2ϩ concentration in response to 5-HT was attenuated, as was the generation of Ca 2ϩ waves at the cellular level. By electron microscopy, cholesterol depletion was found to disrupt caveolae. The 5-HT response could be restored by exogenous cholesterol, which also restored caveolae. Western blots showed that the levels of 5-HT 2A receptor and of caveolin-1 were unaffected by cholesterol extraction. Sucrose gradient centrifugation showed enrichment of 5-HT 2A receptors, but not ␣ 1 -adrenergic receptors, in the caveolin-1-containing fractions, suggesting localization of the former to caveolae. Conclusions-These results show that a subset of signaling pathways that regulate smooth muscle contraction depends specifically on cholesterol. Furthermore, the cholesterol-dependent step in serotonergic signaling occurs early in the pathway and depends on the integrity of caveolae. Key Words: smooth muscle Ⅲ caveolae Ⅲ 5-hydroxytryptamine Ⅲ endothelin Ⅲ intracellular calcium C ellular cholesterol, of which most (up to 90%) 1 resides in the plasma membrane, is crucial for normal membrane permeability and fluidity and also plays a role in cellular signaling, via several proposed mechanisms that fall into at least 4 categories. First, cellular cholesterol may influence gene transcription in the nucleus through sterol regulatory element binding proteins. 2 Second, the activity of membrane receptors, ion channels, and transporters may depend on the membrane fluidity, per se. 3 Third, membrane protein function may be regulated through specific cholesterol-protein interactions. 3,4 Fourth, cholesterol stabilizes the structure of caveolae and lipid rafts.Caveolae, which are 50-to 100-nm membrane invaginations that are abundant in vascular endothelium and smooth muscle cells, are defined by their characteristic morphology and contents of caveolin and cav-p60. 5,6 No definitive definition of rafts has appeared because they do not exhibit a characteristic structure, but the term is used for planar aggregations of specific lipids and proteins. Caveolae and lipid rafts are envisaged to serve as platforms for a dynamic association of signaling proteins and for the initiation or modulation of signaling. 5,7,8 Some agonists causing contraction of vascular smooth muscle act on receptors that are believed to be located in caveo...