Effects of caveolin-1 on the 17beta-estradiol-mediated inhibition of VSMC proliferation induced by vascular injury

Estrogen has a protective effect on the cardiovascular system. Yet the mechanism of how estrogen inhibits vascular smooth muscle cell (VSMC) proliferation after vascular injury and the role of caveolin-1 in this process are not clear. To understand the protection effect of estrogen and caveolin-1, we employed a vascular balloon-injury model. Sixteen New Zealand White rabbits with or without estrogen were tested. 17beta-estradiol is able to inhibit VSMC proliferation in a range from 10(-10)-10(-5) mol/L, with an optimal concentration of 10(-8) mol/L. Estrogen exerted its effect through suppressing the activity of p42/44 MAPK, which can be blocked by tamoxifen. Moreover, in estrogen pretreated cells as well as in common carotid arteries of the balloon injury model, expression of caveolin-1 is enhanced compared to the estrogen-deficient group, as assessed by both western blotting and RT-PCR and morphological studies. Our results showed that the inhibition effect of estrogen in VSMCs is mediated by p42/44 MAPK. Caveolin-1 plays an important role in this protective process.     

Caveolin-1蛋白在171β-雌二醇抑制内皮素-1诱导血管平滑肌细胞增殖中的作用

本工作旨在研究Caveolin-1在17β-雌二醇（17β-estradiol,E2)抑制内皮素-1（endothelin-1,ET-1)诱导血管平滑肌细胞（vascular smooth muscle cells,VSMCs前后ET-1对DNA合成和caveolin-1蛋白表达的影响。结果显示,ET-1可以刺激VSMCs增殖。E2作用24h后,可明显抑制ET-1的上述作用。免疫荧光证实,在VSMCs上有cavellin-1分布,ET-1刺激VSMCs增殖的过程中,VSMCs上caveolin-1蛋白荧光强度下,而事称给予E2可逆转这种下降。Western bolt证实,ET-1可抑制caveolin-1蛋白的表达而E2则可增加caveolin-1蛋白的表达。以上结果表明E2抑制ET-1诱导的VSMCs增殖可能与其增加caveolin-1蛋白表达有关。     

Caveolin-1 in cytokine-induced enhancement of intracellular Ca(2+) in human airway smooth muscle

Diseases such as asthma are characterized by airway hyperresponsiveness. Enhanced airway smooth muscle (ASM) intracellular Ca(2+) ([Ca(2+)](i)) response to agonist stimulation leading to increased airway constriction has been suggested to contribute to airway hyperresponsiveness. Caveolae are flask-shaped plasma membrane invaginations that express the scaffolding protein caveolin and contain multiple proteins important in [Ca(2+)](i) signaling (e.g., agonist receptors, ion channels). We recently demonstrated that caveolae and caveolin-1 are important in [Ca(2+)](i) regulation in human ASM. Proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-13 modulate [Ca(2+)](i) in ASM. We hypothesized that cytokine upregulation of caveolar signaling in ASM contributes to enhanced agonist-induced [Ca(2+)](i) in inflammation. Enzymatically dissociated human ASM cells were exposed to medium (control), 20 ng/ml TNF-α, or 50 ng/ml IL-13 for 24 h. Caveolae-enriched membrane fractions displayed substantial increase in caveolin-1 and -2 expressions by TNF-α and IL-13. Transfection with caveolin-1-mRed DNA substantially accelerated and increased plasma membrane caveolin-1 expression by TNF-α and to a lesser extent by IL-13. Caveolin-1 enhancement was inhibited by nuclear factor-κB and mitogen-activated protein kinase inhibitors. In fura 2-loaded ASM cells, [Ca(2+)](i) responses to 1 μM ACh, 10 μM histamine, or 10 nM bradykinin were all exaggerated by TNF-α as well as IL-13 exposure. However, disruption of caveolae using caveolin-1 suppression via small-interfering RNA resulted in significant blunting of agonist-induced [Ca(2+)](i) responses of vehicle and TNF-α-exposed cells. These functional data were correlated to the presence of TNFR(1) receptor (but not the IL-4/IL-13 receptor) within caveolae. Overall, these results indicate that caveolin-1 plays an important role in airway inflammation by modulating the effect of specific cytokines on [Ca(2+)](i).     

Hemodynamic effects of acute and repeated exposure to raloxifene in ovariectomized sheep

We hypothesize that administration of acute and daily doses of raloxifene will have significant effects on ovine coronary and uterine hemodynamics and that these changes are estrogen receptor dependent. Eleven ovariectomized sheep were instrumented to measure mean arterial pressure, heart rate (HR), cardiac output (CO), and coronary (CBF) and uterine artery blood flows (UBF). A dose-response curve was generated for raloxifene (1, 3, and 10 microg/kg) and compared with a standard dose of estradiol-17beta (1 microg/kg) given intravenously. In a second group of animals, raloxifene (10 microg.kg-1.day-1) was administered intravenously for 14 consecutive days, and cardiovascular responses were compared with a group of animals administered estradiol-17beta (10 microg/kg) daily for the same period. To determine whether raloxifene-related vascular responses were estrogen receptor (ER) mediated, the animals were pretreated with estrogen antagonist ICI-182,780 given intravenously. Finally, RT-PCR was preformed to determine the presence of ERalpha and ERbeta mRNA in ovine coronary and uterine vessels. Raloxifene increased CBF and UBF dose dependently with a parallel decrease in the associated vascular resistances. Acute cardiovascular responses to daily doses of raloxifene and estradiol-17beta were sustainable. In contrast to estradiol-17beta, which significantly increases CO by increasing HR but not stroke volume, raloxifene significantly increased stroke volume without a significant parallel increase in HR. ICI-182,780 abolished raloxifene-induced hemodynamic responses, and ERalpha and ERbeta mRNA are present in both ovine coronary and uterine vessels. Hence, the hemodynamic effects of raloxifene are dose dependent, sustainable, and estrogen receptor mediated.     

Induction of endothelial nitric-oxide synthase phosphorylation by the raloxifene analog LY117018 is differentially mediated by Akt and extracellular signal-regulated protein kinase in vascular endothelial cells

Raloxifene is a tissue-selective estrogen receptor modulator. The effect of estrogen on cardiovascular disease is mainly dependent on direct actions on the vascular wall involving activation of endothelial nitric oxide synthase (eNOS) via Akt and extracellular signal-regulated protein kinase (ERK) cascades. Although raloxifene is also known to activate eNOS in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), the raloxifene analog LY117018 caused acute phosphorylation of eNOS that was unaffected by actinomycin D and was blocked by the pure estrogen receptor antagonist ICI182,780. Activation of Akt by raloxifene reached a plateau at 15-30 min and declined thereafter, a similar time frame to that of Akt activation by 17beta-estradiol. On the other hand, both activation and phosphorylation of ERK by raloxifene showed a biphasic pattern (peaks at 5 min and 1 h), whereas ERK activation and phosphorylation by 17beta-estradiol reached a plateau at 5 min and declined thereafter. A MEK inhibitor, PD98059, had no effect on the raloxifene-induced Akt activity, suggesting an absence of cross-talk between the ERK and Akt cascades. Either exogenous expression of a dominant-negative Akt or pretreatment of TRLECs with PD98059 decreased the raloxifene-induced eNOS phosphorylation. Moreover, raloxifene stimulated the activation of Akt, ERK, and eNOS in Chinese hamster ovary cells expressing estrogen receptor alpha but not Chinese hamster ovary cells expressing estrogen receptor beta. Our findings suggest that raloxifene-induced eNOS phosphorylation is mediated by estrogen receptor alpha via a nongenomic mechanism and is differentially mediated by Akt- and ERK-dependent cascades.     

Comparative study of the short-term effects of a novel selective estrogen receptor modulator, ospemifene, and raloxifene and tamoxifen on rat uterus

To investigate the differential short-term effects of selective estrogen receptor (ER) modulators (SERMs) on uterus, we treated adult ovariectomized rats with a novel SERM, ospemifene (Osp), two previously established SERMs (tamoxifen and raloxifene (Ral)) and estradiol. The expression of two estrogen-regulated early response genes c-fos and vascular endothelial growth factor (VEGF), and DNA synthesis were analysed at 1-24 h after treatment of ovariectomized rats. Induction of c-fos mRNA by each of the SERMs showed a biphasic pattern with peaks at 3 and 20 h, respectively. The maximum level of VEGF mRNA was observed at 1 h after raloxifene and 6 h after tamoxifen or ospemifene treatment. Maximum levels of the c-fos and VEGF mRNA after raloxifene treatment were higher than those seen after treatments with E2 or a corresponding dose of tamoxifen or ospemifene. DNA synthesis was significantly increased by ospemifene, tamoxifen and raloxifene both in luminal and glandular epithelium. The stimulation was transient, peaking at 16 h. In comparison, the maximum level observed at 16 h after E2 treatment sustained at least until 24 h. DNA synthesis in stromal cells was increased by the SERMs but not by E2 at 24 h. When treated together with E2, the SERMs were able to antagonise E2-stimulated DNA synthesis at 16 h. Our results demonstrate that the initial response of uterus to ospemifene, raloxifene and tamoxifen includes activation of early response genes and even transient stimulation of DNA synthesis in spite of their different long-term effects. However, the early stimulatory events may be mediated by different mechanisms leading to diverging pathways in various tissue compartments and development of differential SERM-specific long-term responses of uterus.     

Caveolin-1 is required for vascular endothelial insulin uptake

As insulin's movement from plasma to muscle interstitium is rate limiting for its metabolic action, defining the regulation of this movement is critical. Here, we address whether caveolin-1 is required for the first step of insulin's transendothelial transport, its uptake by vascular endothelial cells (ECs), and whether IL-6 and TNFα affect insulin uptake or caveolin-1 expression. Uptake of FITC-labeled insulin was measured using confocal microscopy in control bovine aortic ECs (bAECs), in bAECs in which caveolin-1 was either knocked down or overexpressed, in murine ECs from caveolin-1(-/-) mice and in bAECs exposed to inflammatory cytokines. Knockdown of caveolin-1 expression in bAECs using specific caveolin-1 siRNA reduced caveolin-1 mRNA and protein expression by ～ 70%, and reduced FITC-insulin uptake by 67% (P < 0.05 for each). Over-expression of caveolin-1 increased insulin uptake (P < 0.05). Caveolin-1-null mouse aortic ECs did not take up insulin and re-expression of caveolin-1 by transfecting these cells with FLAG-tagged caveolin-1 DNA rescued FITC-insulin uptake. Knockdown of caveolin-1 significantly reduced both insulin receptor protein level and insulin-stimulated Akt1 phosphorylation. Knockdown of caveolin-1 also inhibited insulin-induced caveolin-1 and IGF-1 receptor translocation to the plasma membrane. Compared with controls, IL-6 or TNFα (20 ng/ml for 24 h) inhibited FITC-insulin uptake as well as the expression of caveolin-1 mRNA and protein (P < 0.05 for each). IL-6 or TNFα also significantly reduced plasma membrane-associated caveolin-1. Thus, we conclude that insulin uptake by ECs requires expression of caveolin-1 supporting a role for caveolae mediating insulin uptake. Proinflammatory cytokines may inhibit insulin uptake, at least in part, by inhibiting caveolin-1 expression.     

Caveolin-1 regulates BMPRII localization and signaling in vascular smooth muscle cells

Recent studies demonstrate the interaction of BMPRII and caveolin-1 in various cell types. In this study we test the hypothesis that caveolin-1 interacts with and regulates BMPRII-dependent signaling in vascular smooth muscle cells. We demonstrate that BMPRII localizes to caveolae and directly interacts with caveolin-1 in mouse aortic smooth muscle cells. We demonstrate that this interaction is mediated by the caveolin-1 scaffolding domain and is regulated by caveolin-1 phosphorylation. Downregulation of caveolin-1 via siRNA resulted in a loss of BMP-dependent SMAD phosphorylation and gene regulation. Further studies revealed that loss of caveolin-1 results in decreased BMPRII membrane localization and decreased association of BMPRII with the type I BMP receptor BMPRIa. Dominant negative caveolin-1 decreased BMPRII membrane localization suggesting a role for caveolin-1 in BMPRII trafficking. Taken together, our findings establish caveolin-1 as an important regulator of downstream signaling and membrane targeting of BMPRII in vascular smooth muscle cells.     

Nerve growth factor-enhanced airway responsiveness involves substance P in ferret intrinsic airway neurons

Nerve growth factor (NGF), a member of the neurotrophin family, enhances synthesis of neuropeptides in sensory and sympathetic neurons. The aim of this study was to examine the effect of NGF on airway responsiveness and determine whether these effects are mediated through synthesis and release of substance P (SP) from the intrinsic airway neurons. Ferrets were instilled intratracheally with NGF or saline. Tracheal smooth muscle contractility to methacholine and electrical field stimulation (EFS) was assessed in vitro. Contractions of isolated tracheal smooth muscle to EFS at 10 and 30 Hz were significantly increased in the NGF treatment group (10 Hz: 33.57 +/- 2.44%; 30 Hz: 40.12 +/- 2.78%) compared with the control group (10 Hz: 27.24 +/- 2.14%; 30 Hz: 33.33 +/- 2.31%). However, constrictive response to cholinergic agonist was not significantly altered between the NGF treatment group and the control group. The NGF-induced modulation of airway smooth muscle to EFS was maintained in tracheal segments cultured for 24 h, a procedure that causes a significant anatomic and functional loss of SP-containing sensory fibers while maintaining viability of intrinsic airway neurons. The number of SP-containing neurons in longitudinal trunk and superficial muscular plexus and SP nerve fiber density in tracheal smooth muscle all increased significantly in cultured trachea treated with NGF. Pretreatment with CP-99994, an antagonist of neurokinin 1 receptor, attenuated the NGF-induced increased contraction to EFS in cultured segments but had no effect in saline controls. These results show that the NGF-enhanced airway smooth muscle contractile responses to EFS are mediated by the actions of SP released from intrinsic airway neurons.     

Pharmacological evidence for both neuronal and smooth muscular PAC1 receptors and a VIP-specific receptor in rat colon

The receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) were characterised in vitro on rat colon longitudinal smooth muscle with adherent myenteric ganglia. VIP, PACAP-38 and PACAP-27 all caused concentration-dependent relaxations. PACAP-27 and PACAP-38 were equipotent, while VIP was less potent. Tetrodotoxin (10(-6) M), L-NAME (10(-4) M), 7-NINA (10(-5) M) and ODQ (3 x 10(-6) M) reduced the amplitude of the relaxatory responses to PACAP-38 but did not affect relaxations induced by VIP or PACAP-27. Apamin (10(-6) M) almost totally abolished the PACAP-27- and PACAP-38-induced relaxations, while VIP-induced relaxations were only slightly reduced. Tetraethylammonium (TEA) reduced VIP- but not PACAP-27-induced relaxations, while charybdotoxin was ineffective. Cross-desensitisation between PACAP-27, PACAP-38 and VIP could be revealed to some extent. In conclusion: VIP, PACAP-27 and PACAP-38 are effective relaxants in rat colon longitudinal muscle. The receptors involved are classified as: (1) a neuronal PAC1 receptor localised on NO-synthesising neurones, the preferred ligand being PACAP-38. Activation of this receptor leads to an increased NO production. (2) A smooth muscle PAC1 receptor, the preferred ligand being PACAP-27. However, also PACAP-38 and, to a less extent, VIP activate this receptor. The relaxatory responses elicited by both PACAP-27 and PACAP-38 are abolished by apamin and thus mediated through small conductance Ca2+-activated K+ channels. (3) A VIP-specific receptor localised on smooth muscle cells. The mechanisms whereby this receptor elicits a relaxatory response involve, at least to some extent, TEA-sensitive K+ channels.     

Estrogen receptor beta dependent attenuation of cytokine-induced cyclooxygenase-2 by androgens in human brain vascular smooth muscle cells and rat mesenteric arteries

Androgens may provide protective effects in the vasculature under pathophysiological conditions. Our past studies have shown that dihydrotestosterone (DHT) decreases expression of cyclooxygenase-2 (COX-2) during cytokine, endotoxin, or hypoxic stimulation in human vascular smooth muscle cells, in an androgen receptor (AR)-independent fashion. Classically DHT is regarded as a pure AR agonist; however, it can be endogenously metabolized to 5α-androstane-3β, 17β-diol (3β-diol), which has recently been shown to be a selective estrogen receptor (ERβ) agonist. Therefore, we hypothesized that DHT's anti-inflammatory properties following cytokine stimulation are mediated through ERβ. Using primary human brain vascular smooth muscle cells (HBVSMC), we tested whether DHT's effect on IL-1β induced COX-2 expression was mediated via AR or ERβ. The metabolism of DHT to 3β-diol is a viable pathway in HBVSMC since mRNA for enzymes necessary for the synthesis and metabolism of 3β-diol [3alpha-hydroxysteroid dehydrogenase (HSD), 3β-HSD, 17β-HSD, CYP7B1] was detected. In addition, the expression of AR, ERα, and ERβ mRNA was detected. When applied to HBVSMC, DHT (10nM; 18 h) attenuated IL-1β-induced increases in COX-2 protein expression. The AR antagonist bicalutamide did not block DHT's ability to reduce COX-2. Both the non-selective estrogen receptor antagonist ICI 182,780 (1 μM) and the selective ERβ antagonist PHTPP (1 μM) inhibited the effect of DHT, suggesting that DHT actions are ERβ-mediated. In HBVSMC and in rat mesenteric arteries, 3β-diol, similar to DHT, reduced cytokine-induced COX-2 levels. In conclusion, DHT appears to be protective against the progression of vascular inflammation through metabolism to 3β-diol and activation of ERβ.     

Mechanisms of angiotensin II-induced expression of B2 kinin receptors

Although the primary roles of the kallikreinkinin system and the renin-angiotensin system are quite divergent, they are often intertwined under pathophysiological conditions. We examined the effect of ANG II on regulation of B(2) kinin receptors (B2KR) in vascular cells. Vascular smooth muscle cells (VSMC) were treated with ANG II in a concentration (10(-9)-10(-6) M)- and time (0-24 h)-dependent manner, and B2KR protein and mRNA levels were measured by Western blots and PCR, respectively. A threefold increase in B2KR protein levels was observed as early as 6 h, with a peak response at 10(-7) M. ANG II (10(-7) M) also increased B2KR mRNA levels twofold 4 h after stimulation. Actinomycin D suppressed the increase in B2KR mRNA and protein levels induced by ANG II. To elucidate the receptor subtype involved in mediating this regulation, VSMC were pretreated with losartan (AT(1) receptor antagonist) and/or PD-123319 (AT(2) receptor antagonist) at 10 microM for 30 min, followed by ANG II (10(-7) M) stimulation. Losartan completely blocked the ANG II-induced B2KR increase, whereas PD-123319 had no effect. In addition, expression of B2KR mRNA levels was decreased in AT(1A) receptor knockout mice. Finally, to determine whether ANG II stimulates B2KR expression via activation of the MAPK pathway, VSMC were pretreated with an inhibitor of p42/p44(mapk) (PD-98059) and/or an inhibitor of p38(mapk) (SB-202190), followed by ANG II (10(-7) M) for 24 h. Selective inhibition of the p42/p44(mapk) pathway significantly blocked the ANG II-induced increase in B2KR expression. These findings demonstrate that ANG II regulates expression of B2KR in VSMC and provide a rationale for studying the interaction between ANG II and bradykinin in the pathogenesis of vascular dysfunction.     

Alterations in lipid peroxidation and antioxidant status in pregnancy with preeclampsia

Soy consumption is associated with a lower risk of atherosclerotic disease in the oriental population. Genistein is a soy isoflavone bearing estrogenic properties. Previous experiments in our laboratory demonstrated the potentiation of endothelium-independent relaxation of coronary artery by both estrogen and genistein. The potentiating effects of both estrogen and genistein were mediated through the cAMP-signaling pathway. We hypothesize that genistein could enhance protein kinase A (PKA) activity in porcine coronary artery smooth muscle, thereby offering a mechanism for the potentiation of vascular relaxation by genistein. In our study, a high concentration of genistein (10^−4.5 M) significantly increased PKA activity in porcine coronary artery rings. While genistein at 10^−5.5 M and forskolin at 10⁻⁷ M had no effect on PKA activity, the combination of the two compounds at the prescribed concentrations caused a significant increase in PKA activity. The increase in PKA activity by genistein was abolished by SQ 22536 (adenylate cyclase blocker), but not by NF 449 (Gs protein blocker) or ICI 182780 (estrogen receptor antagonist). Our results suggest that the action of genistein is mediated via adenylate cyclase, but does not appear to involve Gs protein or ICI 182780-sensitive estrogen receptor.     

Caveolin-1 is required for contractile phenotype expression by airway smooth muscle cells

Airway smooth muscle cells exhibit phenotype plasticity that underpins their ability to contribute both to acute bronchospasm and to the features of airway remodelling in chronic asthma. A feature of mature, contractile smooth muscle cells is the presence of abundant caveolae, plasma membrane invaginations that develop from the association of lipid rafts with caveolin-1, but the functional role of caveolae and caveolin-1 in smooth muscle phenotype plasticity is unknown. Here, we report a key role for caveolin-1 in promoting phenotype maturation of differentiated airway smooth muscle induced by transforming growth factor (TGF)-β(1). As assessed by Western analysis and laser scanning cytometry, caveolin-1 protein expression was selectively enriched in contractile phenotype airway myocytes. Treatment with TGF-β(1) induced profound increases in the contractile phenotype markers sm-α-actin and calponin in cells that also accumulated abundant caveolin-1; however, siRNA or shRNAi inhibition of caveolin-1 expression largely prevented the induction of these contractile phenotype marker proteins by TGF-β(1). The failure by TGF-β(1) to adequately induce the expression of these smooth muscle specific proteins was accompanied by a strongly impaired induction of eukaryotic initiation factor-4E binding protein(4E-BP)1 phosphorylation with caveolin-1 knockdown, indicating that caveolin-1 expression promotes TGF-β(1) signalling associated with myocyte maturation and hypertrophy. Furthermore, we observed increased expression of caveolin-1 within the airway smooth muscle bundle of guinea pigs repeatedly challenged with allergen, which was associated with increased contractile protein expression, thus providing in vivo evidence linking caveolin-1 expression with accumulation of contractile phenotype myocytes. Collectively, we identify a new function for caveolin-1 in controlling smooth muscle phenotype; this mechanism could contribute to allergic asthma.     

Raloxifene,a mixed estrogen agonist/antagonist,induces apoptosis through cleavage of BAD in TSU-PR1 human cancer cells

Selective estrogen receptor modulator is a proven agent for chemoprevention and chemotherapy of cancer. Raloxifene, a mixed estrogen agonist/antagonist, was developed to prevent osteoporosis and potentially reduce the risk of breast cancer. In this study, we examined the effect of raloxifene on the TSU-PR1 cell line. This cell line was originally reported to be a prostate cancer cell line, but recently it has been shown to be a human bladder transitional cell carcinoma cell line. The TSU-PR1 cell line contains high levels of estrogen receptor beta. Following treatment with raloxifene, evidence of apoptosis, including change in nuclear morphology, DNA fragmentation, and cytochrome c release, was observed in a dose-dependent manner in the TSU-PR1 cells (10(-9) to 10(-6) m range). We observed no detectable change in the steady-state levels of Bax, Bcl-2, and Bcl-X(L) following raloxifene treatment. However, raloxifene induced caspase-dependent cleavage of BAD to generate a 15-kDa truncated protein. Overexpression of a double mutant BAD resistant to caspase 3 cleavage blocked raloxifene-induced apoptosis. These results demonstrate that raloxifene induces apoptosis through the cleavage of BAD in TSU-PR1 cells. This molecular mechanism of apoptosis suggests that raloxifene may be a therapeutic agent for human bladder cancer.     

Human esophageal smooth muscle cells express muscarinic receptor subtypes M(1) through M(5)

Receptor characterization in human esophageal smooth muscle is limited by tissue availability. We used human esophageal smooth muscle cells in culture to examine the expression and function of muscarinic receptors. Primary cultures were established using cells isolated by enzymatic digestion of longitudinal muscle (LM) and circular muscle (CM) obtained from patients undergoing esophagectomy for cancer. Cultured cells grew to confluence after 10-14 days in medium containing 10% fetal bovine serum and stained positively for anti-smooth muscle specific alpha-actin. mRNA encoding muscarinic receptor subtypes M(1)-M(5) was identified by RT-PCR. The expression of corresponding protein for all five subtypes was confirmed by immunoblotting and immunocytochemistry. Functional responses were assessed by measuring free intracellular Ca(2+) concentration ([Ca(2+)](i)) using fura 2 fluorescence. Basal [Ca(2+)](i), which was 135 +/- 22 nM, increased transiently to 543 +/- 29 nM in response to 10 microM ACh in CM cells (n = 8). This response was decreased <95% by 0.01 microM 4-diphenylacetoxy-N-methylpiperidine, a M(1)/M(3)-selective antagonist, whereas 0.1 microM methoctramine, a M(2)/M(4)-selective antagonist, and 0.1 microM pirenzepine, a M(1)-selective antagonist, had more modest effects. LM and CM cells showed similar results. We conclude that human smooth muscle cells in primary culture express five muscarinic receptor subtypes and respond to ACh with a rise in [Ca(2+)](i) mediated primarily by the M(3) receptor and involving release of Ca(2+) from intracellular stores. This culture model provides a useful tool for further study of esophageal physiology.     

Differential effects of 17beta-estradiol and raloxifene on VSMC phenotype and expression of osteoblast-associated proteins

Several studies demonstrate an association between osteoporosis and arterial calcific disease, both of which being common in elderly women. Estradiol and raloxifene, a selective estrogen receptor modulator, prevent bone loss in postmenopausal women. Little is known regarding how these agents affect arterial calcification. The aim of this study was to determine whether or not 17beta-estradiol and raloxifene reduced vascular smooth muscle cell (VSMC) differentiation and expression of bone-associated proteins during phosphate-induced calcification in vitro. Aortic VSMC were cultured from adult, gonadally intact, and ovariectomized (OVX) female pigs. Calcifying medium was added, and cells were treated with solvent (control), 17beta-estradiol (E(2)), or raloxifene. Extent of calcification and phenotypic expression of bone-associated proteins [matrix gla protein (MGP), osteoprotegerin (OPG), and bone sialoprotein (BSP)] were examined at 3-day intervals over 2 wk. Calcium content increased in all groups but was greater in VSMC derived from intact compared with OVX animals. E(2) reduced calcification and preserved a contractile phenotype. Expression of OPG significantly decreased with time; this decrease was significantly greater in VSMC derived from OVX compared with gonadally intact pigs. E(2) and raloxifene preserved expression of OPG only in VSMC from intact pigs. Expression of MGP increased significantly with time and was not affected by E(2) or raloxifene treatments. E(2) treatment significantly inhibited synthesis of BSP in cells from both groups. In conclusion, E(2) slows differentiation of VSMC induced by excess phosphate. Effectiveness of raloxifene to preserve expression of bone cell-associated proteins depends on the hormonal status of the tissue donor.     

Modulation of gonadotropin-releasing hormone receptor concentration in cultured female rat pituitary cells by estradiol treatment

Short-term (0.5-4 h) treatment of rat pituitary cells in culture with estradiol (E2) results in a significant decrease of Gonadotropin-Releasing Hormone (GnRH) induced LH-release. We studied whether changes in the concentrations of GnRH-receptors (GnRH-R) might account for this phenomenon: pituitary cells from adult female rats were incubated for 4 or 24 h in the presence or absence of 10(-9) M E2. Then saturation curves of D-Ala6-des-Gly10-GnRH ethylamide binding were obtained. In addition, binding studies were carried out in cultures incubated for 0.5, 1, 2 or 4 h with or without 10(-9) M E2 using a near saturating concentration of GnRH-analog. No changes of GnRH-R affinity occurred (4 h experiments: Ka in vehicle treated cells: 0.94 +/- 0.2 x 10(9) M-1, Ka in E2 treated cells: 1.06 +/- 0.3 x 10(9) M-1; 24 h experiments: Ka vehicle: 0.95 +/- 0.2 x 10(9) M-1, Ka E2: 0.82 +/- 0.3 x 10(9) M-1). The GnRH-R concentrations, however, were significantly reduced (44 +/- 3%; P less than 0.001) by 4 h E2 treatment and increased (by 68 +/- 8%; P less than 0.01) by 24 h of E2 treatment. The GnRH induced LH-release in aliquots of the same cell preparations was significantly reduced after 4 h and markedly increased after 24 h of E2 treatment. The experiments on the time-course of the reduction of D-Ala6-GnRH-binding by E2 treatment showed that the number of GnRH-R was significantly decreased (24 +/- 1%; P less than 0.05) already after 0.5 h of exposure to the estrogen. This is also the time period after which the negative E2-effect on GnRH-induced LH-release becomes significant. These data provide first evidence that the short-term negative E2-effect on GnRH induced LH-release by rat pituitary cells in culture could be mediated via a reduction of available GnRH-R.