Intraluminal pressure triggers myogenic response via activation of calcium spark and calcium-activated chloride channel in rat renal afferent arteriole.

Myogenic contraction of renal arterioles is an important regulatory mechanism for renal blood flow autoregulation. We have previously demonstrated that integrin-mediated mechanical force increases the occurrence of Ca2+ sparks in freshly isolated renal vascular smooth muscle cells (VSMCs). To further test whether the generation of Ca2+ sparks is a downstream signal of mechanotransduction in pressure-induced myogenic constriction, the relationship between Ca2+ sparks and transmural perfusion pressure was investigated in intact VSMCs of pressurized rat afferent arterioles. Spontaneous Ca2+ sparks were found in VSMCs when afferent arterioles were perfused at 80 mmHg. The spark frequency was significantly increased when perfusion pressure was increased to 120 mmHg. A similar increase of spark frequency was also observed in arterioles stimulated with ß1-integrin-activating antibody. Moreover, spark frequency was significantly higher in arterioles of spontaneous hypertensive rats at 80 and 120 mmHg. Spontaneous membrane current recorded using whole cell perforated patch in renal VSMCs showed predominant activity of spontaneous transient inward currents instead of spontaneous transient outward currents when holding potential was set close to physiological resting membrane potential. Real-time PCR and immunohistochemistry confirmed the expression of Ca2+-activated Cl- channel (ClCa) TMEM16A in renal VSMCs. Inhibition of TMEM16A with T16Ainh-A01 impaired the pressure-induced myogenic contraction in perfused afferent arterioles. Our study, for the first time to our knowledge, detected Ca2+ sparks in VSMCs of intact afferent arterioles, and their frequencies were positively modulated by the perfusion pressure. Our results suggest that Ca2+ sparks may couple to ClCa channels and trigger pressure-induced myogenic constriction via membrane depolarization.

Calcium-dependent FAK/CREB/TNNC1 signalling mediates the effect of stromal MFAP5 on ovarian cancer metastatic potential.

Ovarian cancer is the most lethal gynaecologic 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 are yet to be characterized. Here we identify a stromal gene signature for advanced high-grade serous ovarian cancer using microdissected stromal ovarian tumour 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 signalling 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 tumour growth and metastasis in vivo, suggesting that it may be a new modality of ovarian cancer treatment.

Remanent cell traction force in renal vascular smooth muscle cells induced by integrin-mediated mechanotransduction.

It was previously demonstrated in isolated renal vascular smooth muscle cells (VSMCs) that integrin-mediated mechanotransduction triggers intracellular Ca(2+) mobilization, which is the hallmark of myogenic response in VSMCs. To test directly whether integrin-mediated mechanotransduction results in the myogenic response-like behavior in renal VSMCs, cell traction force microscopy was used to monitor cell traction force when the cells were pulled with fibronectin-coated or low density lipoprotein (LDL)-coated paramagnetic beads. LDL-coated beads were used as a control for nonintegrin-mediated mechanotransduction. Pulling with LDL-coated beads increased the cell traction force by 61 ± 12% (9 cells), which returned to the prepull level after the pulling process was terminated. Pulling with noncoated beads had a minimal increase in the cell traction force (12 ± 9%, 8 cells). Pulling with fibronectin-coated beads increased the cell traction force by 56 ± 20% (7 cells). However, the cell traction force was still elevated by 23 ± 14% after the pulling process was terminated. This behavior is analogous to the changes of vascular resistance in pressure-induced myogenic response, in which vascular resistance remains elevated after myogenic constriction. Fibronectin is a native ligand for α(5)ß(1)-integrins in VSMCs. Similar remanent cell traction force was found when cells were pulled with beads coated with ß(1)-integrin antibody (Ha2/5). Activation of ß(1)-integrin with soluble antibody also triggered variations of cell traction force and Ca(2+) mobilization, which were abolished by the Src inhibitor. In conclusion, mechanical force transduced by α(5)ß(1)-integrins triggered a myogenic response-like behavior in isolated renal VSMCs.

Cyanobacteria cultivation in industrial wastewaters and biodiesel production from their biomass: a review.

As an alternative fuel biodiesel has become increasingly important due to diminishing petroleum reserves and adverse environmental consequences of exhaust gases from petroleum-fueled engines. Recently, research interest has focused on the production of biofuel from microalgae. Cyanobacteria appeared to be suitable candidates for cultivation in wastes and wastewaters because they produce biomass in satisfactory quantity and can be harvested relatively easily due to their size and structure. In addition, their biomass composition can be manipulated by several environmental and operational factors to produce biomass with concrete characteristics. Herein, we review the culture of cyanobacteria in wastewaters and also the potential resources that can be transformed into biodiesel successfully for meeting the ever-increasing demand for biodiesel production.

Impedance analysis of renal vascular smooth muscle cells.

Impedance of renal vascular smooth muscle cells (VSMCs) cultured on microelectrodes was measured by electric cell-substrate impedance sensing. Changes in measured impedance as a function of frequency were compared with the calculated values obtained from an extended cell-electrode model to estimate the junctional resistance, distance between the ventral cell surface and the substratum, and apical and basolateral membrane capacitances of renal VSMCs. This cell-electrode model was derived to accommodate the slender and rectangular shape of VSMCs. The calculated changes in impedance (Z(cal)) based on the model agreed well with the experimental measurement (Z(exp)), and the percentage error defined as |(Z(cal)-Z(exp))/Z(exp)| was 1.0%. To test the sensitivity of the new model for capturing changes in cell-cell and cell-substrate interactions induced by changes in cellular environment, we then applied this model to analyze impedance changes induced by an integrin binding peptide in renal VSMCs. Our result demonstrates that integrin binding peptide decreases junctional resistance between cells, increases the distance between the basolateral cell surface and substratum, and increases the apical membrane capacitance, whereas the basolateral membrane capacitance stays relatively stable. This model provides a generic approach for impedance analysis of cell layers composed of slender, rectangular cells.

Calcium signaling in vasopressin-induced aquaporin-2 trafficking.

It has been the general consensus that cAMP-mediated PKA-dependent phosphorylation of aquaporin-2 is the primary mechanism of vasopressin to regulate osmotic water permeability in kidney collecting duct. By using laser scanning confocal microscopy to monitor [Ca2+]i and apical exocytosis in individual cells of inner medullary collecting duct, we have demonstrated that vasopressin also triggers intracellular Ca2+ mobilization, which is coupled to apical exocytotic insertion of aquaporin-2. Vasopressin-induced Ca2+ mobilization is in the form of oscillations, which involves both intracellular Ca2+ release from ryanodine-gated Ca2+ stores and extracellular Ca2+ influx via capacitative calcium entry. Each individual cell operates as an independent calcium oscillator with time variance in frequency and amplitude. Vasopressin-induced Ca2+ mobilization is mediated by cAMP, but is independent of PKA. Exogenous cAMP analog (8-pCPT-2'-O-Me-cAMP), which activates Epac (exchange protein directly activated by cAMP), but not PKA, triggers Ca2+ mobilization and apical exocytosis. These observations suggest that activation of Epac by cAMP may also contribute to the action of vasopressin in regulating osmotic water permeability. There are multiple plausible candidates for downstream effectors of vasopressin-induced Ca2+ signal including calmodulin, myosin light chain kinase, calmodulin kinase II, and calcineurin. All of them have been implicated in the regulation of aquaporin-2 trafficking and/or water permeability.

Integrin-mediated mechanotransduction in renal vascular smooth muscle cells: activation of calcium sparks.

Integrins are transmembrane heterodimeric proteins that link extracellular matrix (ECM) to cytoskeleton and have been shown to function as mechanotransducers in nonmuscle cells. Synthetic integrin-binding peptide triggers Ca(2+) mobilization and contraction in vascular smooth muscle cells (VSMCs) of rat afferent arteriole, indicating that interactions between the ECM and integrins modulate vascular tone. To examine whether integrins transduce extracellular mechanical stress into intracellular Ca(2+) signaling events in VSMCs, unidirectional mechanical force was applied to freshly isolated renal VSMCs through paramagnetic beads coated with fibronectin (natural ligand of alpha(5)beta(1)-integrin in VSMCs). Pulling of fibronectin-coated beads with an electromagnet triggered Ca(2+) sparks, followed by global Ca(2+) mobilization. Paramagnetic beads coated with low-density lipoprotein, whose receptors are not linked to cytoskeleton, were minimally effective in triggering Ca(2+) sparks and global Ca(2+) mobilization. Preincubation with ryanodine, cytochalasin-D, or colchicine substantially reduced the occurrence of Ca(2+) sparks triggered by fibronectin-coated beads. Binding of VSMCs with antibodies specific to the extracellular domains of alpha(5-) and beta(1)-integrins triggered Ca(2+) sparks simulating the effects of fibronectin-coated beads. Preincubation of microperfused afferent arterioles with ryanodine or integrin-specific binding peptide inhibited pressure-induced myogenic constriction. In conclusion, integrins transduce mechanical force into intracellular Ca(2+) signaling events in renal VSMCs. Integrin-mediated mechanotransduction is probably involved in myogenic response of afferent arterioles.