Engineered protein systems have been created to impart new functions, or “re-program” mammalian cells for applications including cancer and HIV/AIDS therapies. The successful development of mammalian cells for re-programming will depend on having well-defined, modular systems. Migration is a particularly important cell function that will determine the efficiency and efficacy of many re-programming applications in vivo, and Rho proteins are responsible for regulation of cell migration natively. While there have been several reports of photo-activated Rho proteins, no strategy has been developed such that Rho proteins and cell migration can be controlled by a variety of extracellular stimuli that may be compatible with signaling in large organisms. Here, several methods are described for engineering Ca2+-sensitive Rho proteins so that the large, natural toolbox of Ca2+-mobilizing proteins can use the Ca2+ intermediate to activate Rho proteins in response to a variety of exogenous stimuli, including chemicals, growth factors, and light. First, an unreported calmodulin binding site was identified in RhoA. This knowledge was used to create a tandem fusion of RhoA and calmodulin that mediated Ca2+-sensitive bleb retraction in response to a variety of Ca2+-elevating chemicals. Ca2+-mobilizing modules including channelrhodopsin-2 and nicotinic acetylcholine receptor α4 were used for light- and acetylcholine-dependent bleb retraction. Second, a more robust morphology switch was created by embedding a calmodulin binding site into RhoA to enable Ca2+-responsive bleb formation. A wider range of Ca2+-mobilizing modules were also used here including LOVS1K/Orai1 and vascular endothelial growth factor 2. Combining Ca2+-mobilizing and Ca2+-responsive modules increased amoeboid-like cell migration in wound closure and transwell assays. Finally, the embedded peptide design was applied to Rac1 and Cdc42 to enable control of new morphologies and migration modes. The modular Ca2+ control over Rho proteins developed here is an important contribution to cell re-programming because it shows that control over cell migration can be rewired in a way that is flexible and tunable.

No description available.

Purinergic receptors are membrane ion channels that are activated by extracellular ATP. In vertebrates, seven genes encode subunits of P2X receptors. The subunits, designated P2X1-7, are 40 - 50% identical in amino acid sequences. P2X receptors are composed of three subunits and are found as homo- and heterotrimers in tissues of vertebrates. P2X receptors have a wide distribution in the organism, functional receptors are found in neurons, glial cells, muscle cells and also in nonexcitable tissues as epithelial, endothelial, and in hemopoietic tissue. Purinergic signalling plays an important role in pain transmission, at CNS injury and immune processes. P2X receptor subunit consists of two transmembrane domains, extracellular domain and intracellular N-and C-termini. Each transmembrane domain contains two amino acids conserved across all P2X subunits. In the first transmembrane domain receptor P2X2 are that Gly30 and Tyr43. In previous experiments performed on P2X2 receptor, electrophysiological measurements demonstrated that substitution of conserved Tyr43 in the first transmembrane domain with alanine prolongs the deactivation time of ion channel after agonist wash out. This work is focused on clarifying the role of conserved tyrosine in the process of opening and closing of ion channel of P2X...

Angiotensin II (Ang II) plays a very important role in regulating cardiac and vascular contraction and proliferation/hypertrophy via stimulation of AT1 receptors. A few studies have demonstrated that 5-lipoxygenase (5-LO) derived cysteinyl leukotrienes (CysLT) contribute to Ang II evoked tension responses in rat aortic rings. Whether CysLT would contribute to Ang II evoked Ca2+ mobilization in neonatal rat cardiomyocytes (NRC) and rat aortic smooth muscle cells (ASMC) has not been investigated. In the present study, using primary cultures of NRC and minimally passaged cultures of rat ASMC, an effort was made to address this key issue. The agonists evoked increase in cytosolic free calcium ([Ca2+]i) level was determined by fura-2 fluorescence measurement in NRC and ASMC. Total CysLT levels in the culture medium were determined using an ELISA kit. CysLT1/CysLT2 receptor mRNA levels of NRC and ASMC were quantified by Northern blot analysis. In NRC, the AT1 but not the AT2 selective antagonist, attenuated the elevations in [Ca2+]i and CysLT levels evoked by Ang II. Vasopressin (AVP) and endothelin-1 (ET-1) increased [Ca2+]i but not CysLT levels. The 5-LO inhibitor, AA861, and the CysLT1 selective antagonist, MK-571, reduced the maximal [Ca2+]i responses (Emax) to Ang II but not to AVP and ET-1. While CysLT1 antagonist reduced the Emax to leukotriene D4, (LTD4), the dual CysLT1/CysLT2 antagonist, BAY u9773, completely blocked the [Ca2+]i elevation to both LTD4 and leukotriene C4 (LTC4). Both CysLT1 and CysLT2 mRNA were detected in NRC. The inositol 1,4,5 triphosphate (InsP3) antagonist, 2-aminoethoxyphenyl borate (2-APB), attenuated the [Ca2+]i responses to Ang II and LTD4. In ASMC, Ang II, ET-1 and AVP evoked [Ca2+]i responses were significantly higher in the cultured ASMC isolated from spontaneously hypertensive rats (SHR) compared to ASMC derived from age-matched normotensive Wistar-Kyoto (WKY) strain. Addition of either MK571 or BAY u9773, reduced the Emax values to Ang II (but not to ET-1and AVP) in both strains. While BAY u9773 abolished the [Ca2+]i responses evoked by both LTD4 and LTC4, MK571, the CysLT1 antagonist reduced the responses evoked by LTD4 but not LTC4. The basal CysLT levels were higher in the ASMC of SHR. Ang II but not ET-1 and AVP evoked time and concentration dependent increases in CysLT levels in ASMC of both WKY and SHR strains. The AT1 selective antagonist, losartan, but not the AT2 antagonist, PD123319, attenuated the increases in [Ca2+]i and CysLT levels evoked by Ang II. The InsP3 antagonist, attenuated the [Ca2+]i responses to Ang II, LTD4 and LTC4. Both CysLT1 and CysLT2 mRNA were detected in the ASMC of either strain; but they were significantly higher in SHR. These data suggest that AT1 mediated CysLT production contributes to Ang II evoked Ca2+ mobilization in NRC and that elevated CysLT production along with increased expression of both CysLT1/CysLT2 receptors may account for the exaggerated [Ca2+]i responses to Ang II in ASMC of SHR due to enhanced mobilization of Ca2+ from InsP3 sensitive intracellular Ca2+ stores.

Th The kinetics of exocytotic secretion depend not only on the spatial relationship between calcium channels and the exocytotic apparatus, but also on the total amount of Ca2+ influx through Ca2+ channels, the free Ca2+ around the release site and the filling state of the release-ready vesicles. These factors may differ between neurons and endocrine cells. Bovine chromaffin cells (BCCs) are neuroendocrine cells responsible for catecholamine release from the adrenal glands. Ca2+ imaging experiments have shown that localized zones of Ca2+ influx exist on BCC membranes, but how different Ca2+ channel subtypes are distributed, and the mechanisms by which they are targeted, remain to be elucidated. CASK (calcium, calmodulin associated serine kinase) and Mint1 (Munc-18-interacting protein 1), which are modular adaptor proteins involved in synaptic targeting, have recently been found to function in targeting of á1B Ca2+ channels in hippocampal neurons. These data led to the proposal that Ca2+ channels are clustered in BCCs and that CASK and Mint1 play important roles in targeting and/or anchoring channels to their proper location. p*Using RT-PCR and Western blotting, CASK is demonstrated present in isolated BCCs. Mint1 is shown to be present by Western blotting as well. Immunocytochemical experiments and experiments in which BCCs were transfected with plasmids expressing á1A, á1B, and á1C subunits labeled with green fluorescent protein, have shown that á1A and á1B subunits are clustered on the plasma membranes of BCCs, while the á1C subunit is distributed in diffuse patches. With immunoprecipitation, it was determined that CASK interacts biochemically with á1A and á1B Ca2+ channels. Transfection of BCCs with NC3-GFP, which codes for the sequence of the á1B Ca2+ channel that interacts with CASK and Mint1, results in a punctate pattern of fluorescence, which is consistent with the binding of GFP labeled peptide to complexes of CASK and Mint1 at sites of release. Furthermore, immunocytochemical analysis of cells transfected with NC3-GFP showed that á1B Ca2+ channels have a dispersed distribution suggesting that they have been displaced from the binding sites. These data suggest that CASK and Mint1 are important in clustering and targeting Ca2+ channels in the BCC plasma membrane. This study is the first to show the existence and function of CASK and Mint1 in BCCs, and may contribute to our understanding of the exocytotic process in neuroendocrine cells

The purpose of this study was to investigate whether skeletal muscle sarcoplasmic reticulum (SR) Ca2+ transport efficiency and expression levels of major SR Ca2+ regulatory proteins are associated with resting metabolic rate (RMR) in humans. Twenty five healthy and weight stable participants with mean age, height and weight of 22±3.6 years, 174.6±8.0 cm and 72.8±21 kg respectively, were recruited for the study. RMR was calculated using the Weir equation based upon measures of VO2 and VCO2, which were collected using the Vmax breath by breath indirect calorimetry system. Ca2+-ATPase activity, Ca2+ uptake and Ca2+ leak analyses were performed in vitro on homogenates that were prepared from vastus lateralis muscle biopsies. Ionophore (IONO) ratio was assessed by measuring Ca2+-ATPase activity in the presence and absence of Ca2+ Ionophore. The coupling ratio, a measure of SR Ca2+ transport efficiency, was calculated by taking the ratio of Ca2+ uptake to Ca2+-ATPase activity. Expression levels of the major SR Ca2+ regulatory proteins, including SERCA1a, SERCA2a, phospholamban (PLN), and calsequestrin (CSQ) were assessed using Western blotting techniques. Pearson correlation coefficient analysis demonstrated a weak but significant negative correlation between coupling ratio and RMR (r2= 0.2108, p =0.0240). Content of the SR Ca2+ regulatory proteins, IONO ratio and Ca2+ leak were not found to be significantly related to either RMR or coupling ratio, with the exception of the ratio of SERCA1a to SERCA2a, which showed a weak but significant positive relationship with RMR (r2=0.1781, p=0.0400). Thus, the relationship between coupling ratio and RMR is not influenced by Ca2+ leak, SERCA pump efficiency or the SR Ca2+ regulatory proteins. Overall, these results suggest that the efficiency of SR Ca2+ transport is weakly related to whole body RMR. Further analysis is needed to assess this relationship, and to determine which SR Ca2+ handling properties are influencing the relationship between coupling ratio and RMR.

Angiotensin II (Ang II) plays a very important role in regulating cardiac and vascular contraction and proliferation/hypertrophy via stimulation of AT1 receptors. A few studies have demonstrated that 5-lipoxygenase (5-LO) derived cysteinyl leukotrienes (CysLT) contribute to Ang II evoked tension responses in rat aortic rings. Whether CysLT would contribute to Ang II evoked Ca2+ mobilization in neonatal rat cardiomyocytes (NRC) and rat aortic smooth muscle cells (ASMC) has not been investigated. In the present study, using primary cultures of NRC and minimally passaged cultures of rat ASMC, an effort was made to address this key issue. The agonists evoked increase in cytosolic free calcium ([Ca2+]i) level was determined by fura-2 fluorescence measurement in NRC and ASMC. Total CysLT levels in the culture medium were determined using an ELISA kit. CysLT1/CysLT2 receptor mRNA levels of NRC and ASMC were quantified by Northern blot analysis. In NRC, the AT1 but not the AT2 selective antagonist, attenuated the elevations in [Ca2+]i and CysLT levels evoked by Ang II. Vasopressin (AVP) and endothelin-1 (ET-1) increased [Ca2+]i but not CysLT levels. The 5-LO inhibitor, AA861, and the CysLT1 selective antagonist, MK-571, reduced the maximal [Ca2+]i responses (Emax) to Ang II but not to AVP and ET-1. While CysLT1 antagonist reduced the Emax to leukotriene D4, (LTD4), the dual CysLT1/CysLT2 antagonist, BAY u9773, completely blocked the [Ca2+]i elevation to both LTD4 and leukotriene C4 (LTC4). Both CysLT1 and CysLT2 mRNA were detected in NRC. The inositol 1,4,5 triphosphate (InsP3) antagonist, 2-aminoethoxyphenyl borate (2-APB), attenuated the [Ca2+]i responses to Ang II and LTD4. In ASMC, Ang II, ET-1 and AVP evoked [Ca2+]i responses were significantly higher in the cultured ASMC isolated from spontaneously hypertensive rats (SHR) compared to ASMC derived from age-matched normotensive Wistar-Kyoto (WKY) strain. Addition of either MK571 or BAY u9773, reduced the Emax values to Ang II (but not to ET-1and AVP) in both strains. While BAY u9773 abolished the [Ca2+]i responses evoked by both LTD4 and LTC4, MK571, the CysLT1 antagonist reduced the responses evoked by LTD4 but not LTC4. The basal CysLT levels were higher in the ASMC of SHR. Ang II but not ET-1 and AVP evoked time and concentration dependent increases in CysLT levels in ASMC of both WKY and SHR strains. The AT1 selective antagonist, losartan, but not the AT2 antagonist, PD123319, attenuated the increases in [Ca2+]i and CysLT levels evoked by Ang II. The InsP3 antagonist, attenuated the [Ca2+]i responses to Ang II, LTD4 and LTC4. Both CysLT1 and CysLT2 mRNA were detected in the ASMC of either strain; but they were significantly higher in SHR. These data suggest that AT1 mediated CysLT production contributes to Ang II evoked Ca2+ mobilization in NRC and that elevated CysLT production along with increased expression of both CysLT1/CysLT2 receptors may account for the exaggerated [Ca2+]i responses to Ang II in ASMC of SHR due to enhanced mobilization of Ca2+ from InsP3 sensitive intracellular Ca2+ stores.

Th The kinetics of exocytotic secretion depend not only on the spatial relationship between calcium channels and the exocytotic apparatus, but also on the total amount of Ca2+ influx through Ca2+ channels, the free Ca2+ around the release site and the filling state of the release-ready vesicles. These factors may differ between neurons and endocrine cells. Bovine chromaffin cells (BCCs) are neuroendocrine cells responsible for catecholamine release from the adrenal glands. Ca2+ imaging experiments have shown that localized zones of Ca2+ influx exist on BCC membranes, but how different Ca2+ channel subtypes are distributed, and the mechanisms by which they are targeted, remain to be elucidated. CASK (calcium, calmodulin associated serine kinase) and Mint1 (Munc-18-interacting protein 1), which are modular adaptor proteins involved in synaptic targeting, have recently been found to function in targeting of á1B Ca2+ channels in hippocampal neurons. These data led to the proposal that Ca2+ channels are clustered in BCCs and that CASK and Mint1 play important roles in targeting and/or anchoring channels to their proper location. p*Using RT-PCR and Western blotting, CASK is demonstrated present in isolated BCCs. Mint1 is shown to be present by Western blotting as well. Immunocytochemical experiments and experiments in which BCCs were transfected with plasmids expressing á1A, á1B, and á1C subunits labeled with green fluorescent protein, have shown that á1A and á1B subunits are clustered on the plasma membranes of BCCs, while the á1C subunit is distributed in diffuse patches. With immunoprecipitation, it was determined that CASK interacts biochemically with á1A and á1B Ca2+ channels. Transfection of BCCs with NC3-GFP, which codes for the sequence of the á1B Ca2+ channel that interacts with CASK and Mint1, results in a punctate pattern of fluorescence, which is consistent with the binding of GFP labeled peptide to complexes of CASK and Mint1 at sites of release. Furthermore, immunocytochemical analysis of cells transfected with NC3-GFP showed that á1B Ca2+ channels have a dispersed distribution suggesting that they have been displaced from the binding sites. These data suggest that CASK and Mint1 are important in clustering and targeting Ca2+ channels in the BCC plasma membrane. This study is the first to show the existence and function of CASK and Mint1 in BCCs, and may contribute to our understanding of the exocytotic process in neuroendocrine cells

Depressive disorder is one of the most important diseases influencing human health in the 21st century. Antidepressants can improve some depressive symptoms and signs of depressive disorder in patients. It is thought that neurotransmitters (especially serotonin and/or norepinephrine and/or dopamine) have important roles in antidepression effects, but their pharmacological effects on the intracellular signal transduction pathway remain unclear. The aim of this thesis is to explore the effect of the antidepressants paroxetine (a selective serotonin reuptake inhibitor) and nortriptyline (a tricyclic antidepressant) on the intracellular Ca2+-related signal transduction and apoptosis of human prostate cancer PC3 cells. By using the fura-2 method, in PC3 cell, we found paroxetine (at concentrations between 10-150 £gM) and nortriptyline (at concentrations between 50-500 £gM) increased [Ca2+]i of PC3 cells in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+ indicating that Ca2+ entry and release both contributed to the [Ca2+]i rise. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor nearly abolished paroxetine and nortriptyline-induced Ca2+ release. Conversely, pretreatment with nortriptyline greatly reduced the inhibitor-induced [Ca2+]i rise, suggesting that antidepressants released Ca2+ from the endoplasmic reticulum. Inhibition of phospholipase C with U73122 inhibited paroxetine-induced [Ca2+]i rise by 80%, but did not change nortriptyline-induced [Ca2+]i rise. Paroxetine-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and protein kinase C modulators. Nortriptyline-induced Ca2+ influx was inhibited by activation of protein kinase C. Nortriptyline at a concentration of 10 £gM increased the viability of the PC3 cells. At 50 £gM, nortriptyline killed 45% of the cells, and induced significant apoptosis, as measured by propidium iodide staining. Collectively, in PC3 cells, paroxetine induced [Ca2+]i rise is caused by phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels in a manner regulated by protein kinase C and phospholipase A2. Nortriptyline increased [Ca2+]i via phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx from the protein kinase C-sensitive pathway. Nortriptyline also induced apoptosis at a higher level. The results of this thesis may be helpful for understanding the effects of antidepressants on the intracellular signaling of cultured cells, and might illustrate a new possible mechanism concerning antidepressants¡¦ therapeutic effects or clinical side effects.

The effect of capsazepine and nonylphenol on cytosolic free Ca2+ concentrations ([Ca2+]i) in MDCK renal tubular cells is unclear. This study explored whether capsazepine and nonylphenol changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+-selective fluorescent dye. Capsazepine at concentrations between 10 and 200 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partially by 40% after removing extracellular Ca2+. Capsazepine induced Mn2+ quench of fura-2 fluorescence, indirectly implicating Ca2+ entry. Capsazepine-induced Ca2+ influx was not changed by L-type Ca2+ entry inhibitors and protein kinase C modulators [phorbol 12-myristate 13-acetate (PMA) and GF109203X]. In Ca2+-free medium, 100microM capsazepine-induced Ca2+ release was substantially suppressed by pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Pretreatment with capsazepine nearly abolished thapsigargin-induced Ca2+ release. Nonylphenol also increased [Ca2+]i in a concentration- dependent manner like capsazepine does. Similar response in [Ca2+]i rise can be found by inhibition of phospholipase C and using thapsigargin. Different from capasazpine, the [Ca2+]i rise was inhibited by PMA. At concentrations between 5 and 100microM, nonylphenol killed cells in a concentration-dependent manner. Collectively, in MDCK cells, capsazepine induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via non-L-type Ca2+ channels. Nonylphenol induced [Ca2+]i increase in MDCK cells via evoking Ca2+ entry through protein kinase C-regulated Ca2+ channels, and releasing Ca2+ from endoplasmic reticulum and other cellular storage in a phospholipase C-independent manner.