Diabetic cystopathy is a chronic and common complication of diabetes with a classical triad of symptoms; decreased bladder sensation, increased bladder capacity and impaired detrusor muscle contractility (Hunter and Moore, 2003). In animal models of diabetes such as streptozotocin-induced diabetes in the rat, abnormalities of bladder function have been reported (Longhurst and Belis, 1986). The prototypic TRPV channel, TRPV1, is activated by capsaicin, which has been shown to cause contraction of the rat bladder (Saitoh et al., 2007), and this is reduced in STZ-diabetic rat bladder (Pinna et al., 1994). Therefore we hypothesize that TRPV1 function will be reduced in the diabetic bladder. The aim of this study are the following: Firstly, to investigate the effect of the streptozotocin (STZ) model of diabetes on a range of TRP channel functions in the urinary bladder smooth muscle preparation using TRP channel agonists and antagonists and to study the neurotransmitters involved in the contractile or relaxant responses. Some studies were also performed on colon tissues. Secondly, to explore the involvement of cholesterol modudation in TRP channel signalling. Thirdly, to study the change in TRP channel response with time following the treatment with streptozotocin. The results showed that the contractile responses to the TRPV1 agonist capsaicin, TRPV4 agonist 4-α-PDD, and TRPA1 agonist allyl isothiocyanate were significantly reduced in diabetic bladder. The selective TRPV1 antagonist, SB-366791, inhibited the contractile responses to capsaicin confirming the involvement of TRPV1 channels. The effect of diabetes is unlikely to be at the level of contractile machinery since the contractile responses to muscarinic receptor agonist carbachol were not significantly reduced in diabetic tissues. It is reported for the first time that the combination of neurokinin 1 and 2 antagonists GR-205171 and SB-207164 inhibited the contractile responses to capsaicin suggesting that a neurokinin may be the neurotransmitter involved in the capsaicin responses. In addition, the reduction of the responses to capsaicin in STZ-induced diabetic tissues occurred not only in urinary bladder but also in colon. Cholesterol-PEG significantly lowered the maximal contractile responses to capsaicin of rat bladder strips. Methyl-β-cyclodextrin, α-cyclodextrin and β-cyclodextrin at the same concentrations enhanced the contractile responses to capsaicin in the control and diabetic rat bladder strips. These effects of cyclodextrin are specific to capsaicin activated contractions and not seen with TRPA1 activation, suggesting that the effects are not mediated downstream of channel activation. Since α-cyclodextrin does not sequester cholesterol, the enhanced responses to cyclodextrins may not be due to the cholesterol modulations. Instead, theses novel findings may possibly occur by changing the local membrane lipid environment of the TRPV1 channel. As early as 36 hours after induction of diabetes by STZ, the contractile responses to capsaicin were significantly reduced in comparison to those of the controls and this reduction persisted until the eight weeks time point. In contrast, responses to the TRPA1 agonist allyl isothiocyanate were not affected at early time points but were reduced one week after STZ treatment. This detailed time course analysis suggests that there are novel mechanisms of modulation of the TRPV1 channels in this STZ model. In conclusion, in the rat urinary bladder or colon preparations, diabetes mellitus using STZ animal model caused 1) the impairment of a number of TRP channel subfamily functions, TRPV1, TRPV4 and TRPA1 but not TRPM8. The combination of NK1 and NK2 antagonists significantly inhibited the responses to capsaicin. This may suggest the involvement of neurokinin in postsynaptic transmission in rat bladder following the activation of TRPV1 channel, 2) the impairment caused by STZ-induced diabetes occurred very early (within 36 hours after diabetes induction) in TRPV1 channel but not TRPA1 channel. There are specific early effects of STZ treatment on TRPV1 channel function at a time when other afferent nerve terminal channels (TRPA1) are functioning normally, suggesting that early onset of dysfunction in TRPV1 signalling may not merely be the consequence of nerve damage, 3) the mechanism of this impairment may not be the effect of neuropathy on neurotransmitter release or nerve damage. Improving the responsiveness of nerves of bladder in diabetic patients might be of therapeutic benefit. The present studies suggest that it is possible to enhance function using indirect modulators such as bradykinin which potentiated the TRPV1 channel function in diabetic rat bladders.

Thesis (Ph. D.)--Univeristy of Nevada, Reno, 2002. / Includes bibliographical references. Online version available on the World Wide Web.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2010. / Title from PDF title page (viewed on June 25, 2010). Includes bibliographical references.

Intracellular calcium and oxidative stress dyshomeostasis, which can be highly interactive, occur in bipolar disorder (BD), but the pathogenesis of these disturbances is unknown. The transient receptor potential (TRP) melastatin subtype 2 (TRPM2) and canonical subtype 3 (TRPC3) calcium-permeable non-selective ion channels, already implicated in BD, are involved in calcium and oxidative stress signalling. Thus, I sought to determine whether the expression and function of these channels are modulated by oxidative stress exposure in rat cortical neurons, astrocytes, and in human B lymphoblast cell lines (BLCLs), a cell model that reports diagnostically relevant abnormalities in BD. This thesis work demonstrated that TRPC3 expression and function are decreased after chronic, but not acute oxidative stress exposure in both human and rat cell models. TRPM2 expression, on the other hand, was increased after both acute and chronic stressor treatments in rat cortical neurons. In BLCLs, TRPM2-mediated calcium entry was blunted although no difference in TRPM2 mRNA expression was detected. Moreover, BLCLs from BD-I patients exhibited greater susceptibility to cell death and a differential sensitivity of TRPM2-mediated calcium influx to acute oxidative stress compared with healthy subjects, further supporting reduced cellular resilience in the pathophysiology of BD-I. I also demonstrated that TRPC3 protein is expressed in human brain from 8 days to 83 years old supporting an ongoing role in the developing and adult human brain. These findings support an important role for TRPM2 and TRPC3 in sensing and responding to oxidative stress, and in transducing oxidative stress signalling to intracellular calcium homeostatic and cellular stress responses, which have been implicated in the pathophysiology of BD. Finally, this work has highlighted an inherent difference in TRPM2 channel functionality in BD type I subjects compared with controls, adding functional evidence to the genetic and differential expression findings implicating TRPM2 dysfunction in BD.

Intracellular calcium and oxidative stress dyshomeostasis, which can be highly interactive, occur in bipolar disorder (BD), but the pathogenesis of these disturbances is unknown. The transient receptor potential (TRP) melastatin subtype 2 (TRPM2) and canonical subtype 3 (TRPC3) calcium-permeable non-selective ion channels, already implicated in BD, are involved in calcium and oxidative stress signalling. Thus, I sought to determine whether the expression and function of these channels are modulated by oxidative stress exposure in rat cortical neurons, astrocytes, and in human B lymphoblast cell lines (BLCLs), a cell model that reports diagnostically relevant abnormalities in BD. This thesis work demonstrated that TRPC3 expression and function are decreased after chronic, but not acute oxidative stress exposure in both human and rat cell models. TRPM2 expression, on the other hand, was increased after both acute and chronic stressor treatments in rat cortical neurons. In BLCLs, TRPM2-mediated calcium entry was blunted although no difference in TRPM2 mRNA expression was detected. Moreover, BLCLs from BD-I patients exhibited greater susceptibility to cell death and a differential sensitivity of TRPM2-mediated calcium influx to acute oxidative stress compared with healthy subjects, further supporting reduced cellular resilience in the pathophysiology of BD-I. I also demonstrated that TRPC3 protein is expressed in human brain from 8 days to 83 years old supporting an ongoing role in the developing and adult human brain. These findings support an important role for TRPM2 and TRPC3 in sensing and responding to oxidative stress, and in transducing oxidative stress signalling to intracellular calcium homeostatic and cellular stress responses, which have been implicated in the pathophysiology of BD. Finally, this work has highlighted an inherent difference in TRPM2 channel functionality in BD type I subjects compared with controls, adding functional evidence to the genetic and differential expression findings implicating TRPM2 dysfunction in BD.

Investigations of structural and functional relationships of rat transient receptor potential cation channel, subfamily V, member 1 (TRPV1), also known as the capsaicin receptor, and human transient receptor potential cation channel, subfamily A, member 1, also known as TRPA1, are presented. Capsaicin induced Ca2+ -dependent desensitization of rat TRPV1 channel is studied and lead to the identification of key amino acid residues in the C- terminal domain of TRPV1 interacting with the membrane phospholipid PIP2 and an intradomain interaction that controls the open and desensitized state of the TRPV1 channel. Further the molecular basis of agonist AITC- and voltage-dependent gating on TRPA1 is explained. Hereby, residue P949 located near the center of the sixth transmembrane spanning helix (S6) is structurally required for normal functioning of the receptor and the distal bi-glycine G958XXXG962 motif controls its activation/deactivation properties. Furthermore, the gating region is extended towards the cytoplasmic part of the channel, putatively located near the inner mouth of the channel pore. A following series of experiments lead to the identification of a limited number of residues that appear important for allosteric regulation of the channel by chemical and voltage stimuli (K969, R975, K989, K1009, K1046, K1071, K1092 and K1099). In addition, three charge-neutralizing `gain-of- function{\crq} mutants (R975A, K988A, and K989A) which exhibited higher sensitivity to depolarizing voltages were characterized, indicating that these residues are directly involved in voltage-dependent modulation of TRPA1.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Sept. 16, 2008). Includes bibliographical references.

Ca2+-activated potassium channels (KCa) and canonical transient receptor potential (TRPC) channels are essential to endothelial function. In ischemic heart disease, or in cardiac surgery, coronary endothelium is subjected to ischemia-reperfusion (I-R) / hypoxia-reoxygenation (H-R) injury. Hyperkalemic cardioplegic or organ preservation solutions used in cardiac surgery including heart transplantation also impair endothelial function. The present study was designed to mainly investigate whether endothelial dysfunction occurring in H-R or in hyperkalemic exposure is attributable to alterations of intermediate- and small-conductance KCa (IKCa and SKCa) channels, or TRPC channels, in particular, the TRPC3 channel. / Exposure to 60-min hypoxia followed by reoxygenation inhibited the vasorelaxant response of coronary arteries to IKCa / SKCa activator 1-EBIO. H-R reduced endothelial IKCa and SKCa currents and downregulated IKCa expression in PCECs. 1-EBIO enhanced endothelial K+ current that was blunted by H-R. / Exposure to hyperkalemic solutions decreased Ca2+ influx via TRPC3 in PCECs. The reduced Ca2+ influx in PCECs and the attenuated EDHF-mediated vasorelaxation in porcine coronary arteries, which were caused by hyperkalemic or cardioplegic / organ preservation solutions, were restored by OAG. / In PCECs, hypoxia for 60-min with reoxygenation reduced TRPC3 current and Ca2+ influx via TRPC3, which was accompanied by decreased NO release and endothelium-dependent vasorelaxation of porcine coronary arteries. The compromised endothelial function was restored by OAG. The translocation of TRPC3 to endothelial membrane was inhibited by H-R. / In TRPC3-overexpressing HEK293 cells, followed by reoxygenation, short-time hypoxia (10-min) enhanced, whereas prolonged hypoxia (60-min) reduced the current induced by TRPC3/6/7 activator OAG. / Our results indicate that: (1) Endothelial IKCa, SKCa and TRPC3 play an important role in regulating vascular tone; TRPC3 contributes to NO release from endothelial cells and is also involved in the function of EDHF. (2) H-R (60-30 min) reduces endothelial IKCa and SKCa currents with downregulation ofthe protein expression of IKCa. (3) H-R has dual effect on TRPC3 with short-time hypoxia (lO-min) enhancing whereas prolonged hypoxia (60-min) decreasing the electrophysiological activity of this channel. H-R (60-30 min) inhibits the translocation of TRPC3 to endothelial membrane. Furthermore, H-R inhibits Ca2+ influx via TRPC3 and such inhibition is associated with a decrease of NO production. (4) The activator of IKCa / SKCa or TRPC protects coronary endothelium against H-R injury. In coronary endothelium exposed to hyperkalemic or cardioplegic / organ preservation solutions, TRPC activator also exhibits protective effect. / The above findings are likely to have significant implications in ischemic heart disease and in modem cardiopulmonary surgery. / Whole-cell membrane currents of IKCa, SKCa, or TRPC3 were recorded by patch-clamp in primary cultured porcine coronary endothelial cells (PCECs). TRPC3 current was also studied in human embryonic kidney cells (HEK293 cells) transiently overexpressed with TRPC3 gene. Protein or mRNA expression of these channels was detected by Western blot or RT-PCR. Intracellular Ca2+ concentration was measured by Ca2+ imaging technique. Isometric force study was performed in a wire myograph and endothelial nitric oxide (NO) release was measured electrochemically by using a NO-specific microsensor in porcine coronary small arteries. / Huang, Junhao. / "December 2010." / Adviser: Qin Yang. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 138-165). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

No

Diabetes mellitus is a complex disease characterised by chronic hyperglycaemia due to compromised insulin synthesis and secretion, or decreased tissue sensitivity to insulin, if not all three conditions. Endothelial dysfunction is a common complication in diabetes in which endothelium-dependent vasodilation is impaired. The aim of this study was to examine the involvement of TRPV4 in diabetes endothelial dysfunction. Male Charles River Wistar rats (350-450 g) were injected with 65mg/kg streptozotocin (STZ) intraperitoneally. STZ-injected rats were compared with naïve rats (not injected with STZ) or control rats (injected with 10ml/kg of 20mM citrate buffer, pH 4.0-4.5), if not both. Rats with blood glucose concentrations greater than 16mmol/L were considered to be diabetic. As the results revealed, STZ-diabetic rats showed significant endothelial dysfunction characterised by impaired muscarinic-induced vasodilation, as well as significant impairment in TRPV4-induced vasodilation in aortic rings and mesenteric arteries. Furthermore, STZ-diabetic primary aortic endothelial cells (ECs) showed a significant reduction in TRPV4-induced intracellular calcium ([Ca2+]i) elevation. TRPV4, endothelial nitric oxide synthase (eNOS), and caveolin-1 (CAV-1) were also significantly downregulated in STZ-diabetic primary aortic ECs and were later significantly restored by in vitro insulin treatment. Methylglyoxal (MGO) was significantly elevated in STZ-diabetic rat serum, and nondiabetic aortic rings incubated with MGO (100μM) for 12 hours showed significant endothelial dysfunction. Moreover, nondiabetic primary aortic ECs treated with MGO (100μM) for 5 days showed significant TRPV4 downregulation and significant suppression of 4-α-PDD-induced [Ca2+]i elevation, which was later restored by L-arginine (100μM) co-incubation. Incubating nondiabetic aortic rings with MGO (100μM) for 2 hours induced a spontaneous loss of noradrenaline-induced contractility persistence. Moreover, MGO induced significant [Ca2+]i elevation in Chinese hamster ovary cells expressing rat TRPM8 channels (rTRPM8), which was significantly inhibited by AMTB (1-5μM). Taken together, TRPV4, CAV-1, and eNOS can form a functional complex that is downregulated in STZ-diabetic aortic ECs and restored by insulin treatment. MGO elevation might furthermore contribute to diabetes endothelial dysfunction and TRPV4 downregulation. By contrast, MGO induced the loss of contractility persistence, possibly due to MGO's acting as a TRPM8 agonist.