Abstract-Chronic hypoxic pulmonary hypertension is associated with profound vascular remodeling and alterations in Ca 2ϩ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Recent studies show that transient receptor potential (TRPC) genes, which encode store-operated and receptor-operated cation channels, play important roles in Ca 2ϩ regulation and cell proliferation. However, the influence of chronic hypoxia on TRPC channels has not been determined. Here we compared TRPC expression, and store-and receptor-operated Ca 2ϩ entries in PASMCs of normoxic and chronic hypoxic rats. Reverse-transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining showed consistently that TRPC1, TRPC3, and TRPC6 were expressed in intralobar pulmonary arteries (PAs) and PASMCs. Application of 1-oleoyl-2-acetyl-sn-glycerol (OAG) to directly activate receptor-operated channels, or thapsigargin to deplete Ca 2ϩ stores, caused dramatic increase in cation entry measured by Mn 2ϩ quenching of fura-2 and by Ca 2ϩ transients. OAG-induced responses were Ϸ700-fold more resistant to La 3ϩ inhibition than thapsigargin-induced responses. siRNA knockdown of TRPC1 and TRPC6 specifically attenuated thapsigargin-and OAG-induced cation entries, respectively, indicating that TRPC1 mediates store-operated entry and TRPC6 mediates receptor-operated entry. In hypoxic PAs, there were 2-to 3-fold increases in TRPC1 and TRPC6 expression. They were accompanied by significant increases in basal, OAG-induced, and thapsigargin-induced cation entries in hypoxic PASMCs. Moreover, removal of Ca 2ϩ or inhibition of store-operated Ca 2ϩ entry with La 3ϩ and SK&F-96365 reversed the elevated basal [Ca 2ϩ ] i in PASMCs and vascular tone in PAs of chronic hypoxic animals, but nifedipine had minimal effects. Our results for the first time to our knowledge show that both store-and receptor-operated channels of PASMCs are upregulated by chronic hypoxia and contribute to the enhanced vascular tone in hypoxic pulmonary hypertension. Key Words: pulmonary hypertension Ⅲ transient receptor potential channels Ⅲ store-operated Ca 2ϩ channels Ⅲ receptor-operated Ca 2ϩ channels P rolonged exposure to alveolar hypoxia causes pulmonary hypertension with profound vascular remodeling and increase in vasomotor tone. The increase in vascular tone is in part attributable to alterations in vasoconstricting and vasorelaxing influences imposed by the endothelially derived and circulating factors. 1 Recent evidence indicates that chronic hypoxia also causes intrinsic changes in ionic balance and Ca 2ϩ homeostasis in pulmonary arterial smooth muscle cells (PASMCs), including membrane depolarization, elevation in resting [Ca 2ϩ ] i , and changes in electrophysiological and Ca 2ϩ responses to vasoconstrictors and vasodilators. [2][3][4][5][6] The mechanism for alteration in Ca 2ϩ homeostasis in hypoxic PASMCs is controversial. Previous studies found significant suppression of voltage-gated K ϩ (K V ) currents and K V channel expression in PASMCs isolated fr...

Ovarian cancer is the most lethal gynecological malignancy. It is usually diagnosed at a late stage, with a 5-yr survival rate of <30%. The majority of ovarian cancer cases are diagnosed after tumors have widely spread within the peritoneal cavity, limiting the effectiveness of debulking surgery and chemotherapy. Owing to a substantially lower survival rate at late stages of disease than at earlier stages, the major cause of ovarian cancer deaths is believed to be therapy-resistant metastasis. Although metastasis plays a crucial role in promoting ovarian tumor progression and decreasing patient survival rates, the underlying mechanisms of ovarian cancer spread have yet to be thoroughly explored. For many years, researchers have believed that ovarian cancer metastasizes via a passive mechanism by which ovarian cancer cells are shed from the primary tumor and carried by the physiological movement of peritoneal fluid to the peritoneum and omentum. However, the recent discovery of hematogenous metastasis of ovarian cancer to the omentum via circulating tumor cells instigated rethinking of the mode of ovarian cancer metastasis and the importance of the "seed-and-soil" hypothesis for ovarian cancer metastasis. In this review we discuss the possible mechanisms by which ovarian cancer cells metastasize from the primary tumor to the omentum, the cross-talk signaling events between ovarian cancer cells and various stromal cells that play crucial roles in ovarian cancer metastasis, and the possible clinical implications of these findings in the management of this deadly, highly metastatic disease.

In the renal collecting duct, vasopressin increases osmotic water permeability (P f ) by triggering trafficking of aquaporin-2 vesicles to the apical plasma membrane. We investigated the role of vasopressin-induced intracellular Ca 2؉ mobilization in this process. In isolated inner medullary collecting ducts (IMCDs) Arginine vasopressin (AVP) 1 regulates water transport across the epithelium of the renal collecting duct, allowing precise control of water excretion. Water transport across the collecting duct is mediated by molecular water channels, the aquaporins (1, 2). Aquaporin-2 provides the water transport pathway across the apical plasma membrane of the collecting duct principal cells, whereas aquaporins-3 and -4 facilitate water transport across the basolateral plasma membrane. AVP increases the osmotic water permeability (P f ) of the collecting duct cells by triggering translocation of intracellular vesicles containing aquaporin-2 to the apical plasma membrane (3), thus increasing the number of water channels in the ratelimiting barrier for transepithelial water transport. This response depends on the binding of AVP to V 2 vasopressin receptors in the basolateral plasma membrane. These receptors couple to the heterotrimeric G protein, G s , which activates the effector enzyme adenylyl cyclase type VI (4) and increases cyclic AMP levels in the cells. Vasopressin, acting via the V 2 receptor, also causes a transient increase in intracellular Ca 2ϩ (5-8). Little is known about the mechanism of the vasopressininduced increase in intracellular Ca 2ϩ , although previous studies establish that it occurs in the absence of activation of the phosphoinositide signaling pathway (9). Little is known also about the physiological role of the vasopressin-induced increase in intracellular Ca 2ϩ in the regulation of aquaporin-2 trafficking. However, studies of a wide variety of vesicular-trafficking processes have pointed to a key role for localized increases in intracellular Ca 2ϩ in triggering the fusion of vesicles with their target membranes (10), raising the possibility that the same could be true for aquaporin-2 vesicle trafficking. One calciumdependent mediator that has been suggested to play a role in water permeability regulation in the vasopressin-responsive toad bladder epithelium is calmodulin (11). Based on recent studies of homotypic fusion of yeast vacuoles, Peters and Mayer conclude that a critical final step in the process of vesicle fusion is dependent on calmodulin (12), and calmodulin actions can be postulated at other steps involved in vasopressin signaling or aquaporin-2 trafficking. In the present study, we investigate the role of intracellular Ca 2ϩ and calmodulin in the AVPmediated regulation of aquaporin-2 trafficking, assessed through the measurement of osmotic water permeability (P f ) in isolated perfused inner medullary collecting duct (IMCD) segments and through immunofluorescence localization of aquaporin-2 in cultured IMCD cells. The results support the view that stimulation of aquaporin-2...

SUMMARY Advanced stage papillary serous tumors of the ovary are responsible for the majority of ovarian cancer deaths, yet the molecular determinants modulating patient survival are poorly characterized. Here, we identify and validate a prognostic gene expression signature correlating with survival in a series of microdissected serous ovarian tumors. Independent evaluation confirmed the association of a prognostic gene microfibril-associated glycoprotein 2 (MAGP2) with poor prognosis, whereas in vitro mechanistic analyses demonstrated its ability to prolong tumor cell survival and stimulate endothelial cell motility and survival via the αVβ3 integrin receptor. Increased MAGP2 expression correlated with microvessel density suggesting a proangiogenic role in vivo. Thus, MAGP2 may serve as a survival-associated target.

Ovarian cancer is the most lethal gynecologic malignancy in the United States, and advanced serous ovarian adenocarcinoma is responsible for most ovarian cancer deaths. However, the stroma-derived molecular determinants that modulate patient survival have yet to be characterized. Here we identify a stromal gene signature for advanced high-grade serous ovarian cancer using microdissected stromal ovarian tumor samples and find that stromal microfibrillar-associated protein 5 (MFAP5) is a prognostic marker for poor survival. Further functional studies reveal that FAK/CREB/TNNC1 signaling pathways mediate the effect of MFAP5 on ovarian cancer cell motility and invasion potential. Targeting stromal MFAP5 using MFAP5 specific siRNA encapsulated in chitosan nanoparticles significantly decreases ovarian tumor growth and metastasis in vivo, suggesting that it may be a new modality of ovarian cancer treatment.

Arginine vasopressin (AVP) regulates the osmotic water permeability (P f ) of renal collecting duct, conferring the precise control of renal excretion of water. AVP increases P f in collecting ducts by triggering translocation and insertion of aquaporin-2 (AQP2) to the apical membrane of principal cells in the collecting duct (Wade et al. 1981; Nielsen et al. 1995a). These responses depend on the binding of AVP to basolateral V 2 vasopressin receptors in collecting duct cells, which leads to the activation of adenylyl cyclase and an increase of cyclic AMP level in the cells (Chabardes et al. 1996). Cyclic AMP-dependent protein kinase A is then activated, which subsequently phosphorylates AQP2 at Ser 256 (Nishimoto et al. 1999). However, the steps that lead to the eventual insertion of AQP2 into apical membrane are not known. A number of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, which are known to regulate exocytosis of synaptic vesicles, have been identified in renal collecting duct (Sollner et al. 1993 (Burgoyne & Morgan, 1998). This raises the possibility that the exocytotic insertion of AQP2 is also Ca 2+ dependent. Studies from other investigators showed that 10 n AVP was required to induce a detectable increase of [Ca 2+ ] i in perfused inner medullary collecting duct (IMCD) (Star et al. 1988). However, 0.01-0.1 n AVP is sufficient to increase cyclic AMP production and to induce a submaximal increase of P f in IMCD (Star et al. 1988). Detection of Ca 2+ mobilization from a perfused IMCD based on conventional fluorescence imaging might be compromised by out-of-focus fluorescence and asynchronous [Ca 2+ ] i oscillations in individual cells. Conventional methods for measuring changes in P f of a perfused IMCD also do not provide sufficient spatial and temporal resolution to capture the dynamics of AVPinduced exocytosis in individual IMCD cells. Coupling of vasopressin-induced intracellular Ca

Ca+ sparks originating from ryanodine receptors (RyRs) are known to cause membrane hyperpolarization and vasorelaxation in systemic arterial myocytes. By contrast, we have found that Ca2+ sparks of pulmonary arterial smooth muscle cells (PASMCs) are associated with membrane depolarization and activated by endothelin-1 (ET-1), a potent vasoconstrictor that mediates/modulates acute and chronic hypoxic pulmonary vasoconstriction. In this study, we characterized the effects of ET-1 on the physical properties of Ca2+ sparks and probed the signal transduction mechanism for spark activation in rat intralobar PASMCs. Application of ET-1 at 0.1-10 nM caused concentration-dependent increases in frequency, duration, and amplitude of Ca2+ sparks. The ET-1-induced increase in spark frequency was inhibited by BQ-123, an ETA-receptor antagonist; by U-73122, a PLC inhibitor; and by xestospongin C and 2-aminoethyl diphenylborate, antagonists of inositol trisphosphate (IP3) receptors (IP3Rs). However, it was unrelated to sarcoplasmic reticulum Ca2+ content, activation of L-type Ca2+ channels, PKC, or cADP ribose. Photorelease of caged-IP3 indicated that Ca2+ release from IP3R could cross-activate RyRs to generate Ca2+ sparks. Immunocytochemistry showed that the distributions of IP3Rs and RyRs were similar in PASMCs. Moreover, inhibition of Ca2+ sparks with ryanodine caused a significant rightward shift in the ET-1 concentration-tension relationship in pulmonary arteries. These results suggest that ET-1 activation of Ca2+ sparks is mediated via the ETA receptor-PLC-IP3 pathway and local Ca2+ cross-signaling between IP3Rs and RyRs; in addition, this novel signaling mechanism contributes significantly to the ET-1-induced vasoconstriction in pulmonary arteries.

Hydrostatic pressure and flow in renal proximal tubules oscillate at 30-40 mHz in normotensive rats anesthetized with halothane. The oscillations originate in tubuloglomerular feedback, a mechanism that provides local blood flow regulation. Instead of oscillations, spontaneously hypertensive rats (SHR) have aperiodic tubular pressure fluctuations; the pattern is suggestive of deterministic chaos. Normal rats made hypertensive by clipping one renal artery had similar aperiodic tubular pressure fluctuations in the unclipped kidney, and the fraction of rats with irregular fluctuations increased with time after the application of the renal artery clip. Statistical measures of deterministic chaos were applied to tubular pressure data. The correlation dimension, a measure of the dimension of the phase space attractor generating the time series, indicated the presence of a low-dimension strange attractor, and the largest Lyapunov exponent, a measure of the rate of divergence in phase space, was positive, indicating sensitivity to initial conditions. These time series therefore satisfy two criteria of deterministic chaos. The measures were the same in SHR as in rats with renovascular hypertension. Since two different models of hypertension displayed similar dynamics, we suggest that chaotic behavior is a common feature of renal vascular control in the natural history of the disease.