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.

Breast cancer has the highest incidence rate in women, accounting for more than 22% of all cancers and possessing a strong disposition to metastasize to bone. These skeletal metastases become a significant cause of morbidity and mortality in patients with the primary symptom being pain. Pain is a major concern in determining a patient’s quality of life and there have been many attempts to understand and control bone pain with little success. Previous studies have shown that glutamate plays a role in bone cancer pain, with an excess in free glutamate able to cause pain either directly through excitotoxic pathways or indirectly though the dysregulation of osteoclasts and osteoblasts, causing bone dysregulation. TRPV-1 receptors have also has been implicated in the mechanisms of bone cancer pain, as osteoclasts release protons during bone remodeling which can elicit a TRPV-1-related nociceptive response from neurons in the surrounding periosteum. Capsazepine was identified during a high throughput screen of 30,000 compounds to be a potent inhibitor of breast cancer cell-mediated glutamate release, a neurotransmitter with known associations in neural signaling, bone homeostasis, and pain. Capsazepine also has antagonistic effects on transient receptor potential vanilloid type 1 (TRPV-1) receptors which act as key players in both heat and vanilloid-induced nociception. These findings suggest that Capsazepine may provide a multi-site effect for the treatment of cancer-induced bone pain. An animal model of breast cancer-induced bone pain involved intrafemorally injecting MDA-MB-231 cancer cells to measure pain. Behavioural tests are then performed measuring dynamic weight bearing and paw withdrawal thresholds. These measurements are used to demonstrate both movement-evoked and spontaneous pain-related behaviour of the affected limb. Using Capsazepine, we demonstrate a dose-dependent attenuation of pain behaviour in vivo, while confirming tumour presence using immunohistochemistry (IHC). We show that TRPV-1 and glutamate play an important role in the onset and severity of bone cancer pain and blocking these pain pathways provide relief from pain commonly associated with cancer in the bone. / Thesis / Master of Health Sciences (MSc)

Background and aim: The calcium ion (Ca2+) is an important ion involved in intracellular signalling. An increase in the free intracellular calcium concentration ([Ca2+]i) is essential for triggering insulin secretion from pancreatic beta cells. Beta cell death or disturbed insulin secretion are key factors in the pathogenesis of type 1 and type 2 diabetes respectively. A number of Ca2+ channels located on the plasma membrane or on the endoplasmic reticulum (ER) mediate Ca2+ increase in beta cells. Among the plasma membrane Ca2+ channels, members of the Transient Receptor Potential (TRP) family are currently of great interest. Transient Receptor Potential Vanilloid subtype 1 (TRPV1) is one of the 28 members of the TRP family. This ion channel is activated by heat and pungent chemicals like capsaicin. The main aim of this study was to investigate if functional TRPV1 channels are present in insulin secreting cells. Further more we examined if TRP channels could be studied by using microfluorometry in single cells. A third objective was to investigate if members of the TRP family could be identified by western blot. Methods: We used S5 cells, a highly differentiated rat insulinoma cell line, as a model of beta cells. A ratiometric fluorescence technique was used for measurement of [Ca2+]i concentration from single Fura-2 loaded cells. [Ca2+]i was measured continuously using microscope based fluorometry with the time resolution of 1 Hz. For western blot we used proteins extracted from S5 cells and human islets. The blots were probed with antibodies directed against both the N-terminal and the C-terminal end of the protein. Results: Capsaicin, an activator of TRPV1, increased [Ca2+]i in a dose-dependent manner with a half maximal effective concentration (EC50) ~ 100 nM. In nominally Ca2+ free buffer the capsaicin-induced [Ca2+]i increase was completely lost, while the intracellular depots of Ca2+ were not emptied as shown by administration of carbachol. The capsaicin-induced [Ca2+]i increase was completely blocked by capsazepine, an antagonist of TRPV1. An increase in temperature in the range of 43 – 49 °C increased [Ca2+]i, whereas temperatures < 42 °C did not. In nominally Ca2+ free medium the response to heat was reduced. Subsequent administration of carbachol showed that intracellular depots of Ca2+ were not emptied. Ruthenium red, an antagonist of TRPV1, also reduced the heat induced [Ca2+]i response. Another heat-sensitive, Ca2+ permeable protein Transient Receptor Potential Melastatin-like subtype 2 (TRPM2) was detected in S5 cells and human islets by western blot. The 171 kDa band represents the full length TRPM2 and is clearly visible in human islets, while the 95 KDa band represents the truncated form of TRPM2 and is more prominent in S5 cells. Interpretation and conclusions: Microscope based fluorometry is a powerful method for studying ion channels of the TRP family in single living cells. We found that pancreatic beta cells express functional TRPV1 channels that were activated by capsaicin and heat. TRPV1 channels of beta cells are located on the plasma membrane and not on the ER. TRP channel proteins can also be detected by the western blot technique. The ease of studying TRP channels by microfluorometry and our demonstration of functionalTRPV1 channels in beta cells paves the way for studying the role of these channels in insulin secretion and in the pathogenesis of diabetes.

<p>Background and aim: The calcium ion (Ca2+) is an important ion involved in intracellular signalling. An increase in the free intracellular calcium concentration ([Ca2+]i) is essential for triggering insulin secretion from pancreatic beta cells. Beta cell death or disturbed insulin secretion are key factors in the pathogenesis of type 1 and type 2 diabetes respectively. A number of Ca2+ channels located on the plasma membrane or on the endoplasmic reticulum (ER) mediate Ca2+ increase in beta cells. Among the plasma membrane Ca2+ channels, members of the Transient Receptor Potential (TRP) family are currently of great interest. Transient Receptor Potential Vanilloid subtype 1 (TRPV1) is one of the 28 members of the TRP family. This ion channel is activated by heat and pungent chemicals like capsaicin. The main aim of this study was to investigate if functional TRPV1 channels are present in insulin secreting cells. Further more we examined if TRP channels could be studied by using microfluorometry in single cells. A third objective was to investigate if members of the TRP family could be identified by western blot.</p><p>Methods: We used S5 cells, a highly differentiated rat insulinoma cell line, as a model of beta cells. A ratiometric fluorescence technique was used for measurement of [Ca2+]i concentration from single Fura-2 loaded cells. [Ca2+]i was measured continuously using microscope based fluorometry with the time resolution of 1 Hz. For western blot we used proteins extracted from S5 cells and human islets. The blots were probed with antibodies directed against both the N-terminal and the C-terminal end of the protein.</p><p>Results: Capsaicin, an activator of TRPV1, increased [Ca2+]i in a dose-dependent manner with a half maximal effective concentration (EC50) ~ 100 nM. In nominally Ca2+ free buffer the capsaicin-induced [Ca2+]i increase was completely lost, while the intracellular depots of Ca2+ were not emptied as shown by administration of carbachol. The capsaicin-induced [Ca2+]i increase was completely blocked by capsazepine, an antagonist of TRPV1. An increase in temperature in the range of 43 – 49 °C increased [Ca2+]i, whereas temperatures < 42 °C did not. In nominally Ca2+ free medium the response to heat was reduced. Subsequent administration of carbachol showed that intracellular depots of Ca2+ were not emptied. Ruthenium red, an antagonist of TRPV1, also reduced the heat induced [Ca2+]i response. Another heat-sensitive, Ca2+ permeable protein Transient Receptor Potential Melastatin-like subtype 2 (TRPM2) was detected in S5 cells and human islets by western blot. The 171 kDa band represents the full length TRPM2 and is clearly visible in human islets, while the 95 KDa band represents the truncated form of TRPM2 and is more prominent in S5 cells.</p><p>Interpretation and conclusions: Microscope based fluorometry is a powerful method for studying ion channels of the TRP family in single living cells. We found that pancreatic beta cells express functional TRPV1 channels that were activated by capsaicin and heat. TRPV1 channels of beta cells are located on the plasma membrane and not on the ER. TRP channel proteins can also be detected by the western blot technique. The ease of studying TRP channels by microfluorometry and our demonstration of functionalTRPV1 channels in beta cells paves the way for studying the role of these channels in insulin secretion and in the pathogenesis of diabetes.</p>