Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 135-153).

No description available.

The mechanism of action of notexin in the facilitation of spontaneous transmitter release at neuromuscular synapse was investigated in Xenopus cell culture by using whole-cell patch clamp recording. Exposure of the culture to notexin dose-dependently enhanced the frequency of spontaneous synaptic currents (SSCs). Either buffering of intracellular Ca2+ rise by BAPTA or replacing culture medium with Ca2+-free Ringer¡¦s solution effectively hampered notexin effect, suggesting Ca2+ influx is requisite for this facilitation. Pretreatment of the cultures with a TRP channel inhibitor SKF96365, instead of voltage-dependent L-type Ca2+ channel blockers nifedipine or verapamil, significantly abolished the SSC facilitating effect of notexin. Furthermore, knockdown the expression of TRPC channels by TRPC-specific morpholino abruptly abolished notexin effect, suggesting that TRPC channel is the major entranceway of extracellular Ca2+. The notexin-enhanced SSC frequency was also obviously reduced under intracellular Ca2+ store depletion by pretreatment of the cultures with pharmacological Ca2+-ATPase inhibitor thapsigargin or CPA. Bath application of membrane-permeable inositol 1,4,5-triphosphate (IP3) inhibitor, XeC, effectively occluded the facilitation of SSC frequency elicited by notexin. Furthermore, notexin-induced SSC frequency facilitation was blocked in the presence of phospholipase C (PLC) inhibitor, U73122. Taken collectively, these results suggest that notexin elicits a Ca2+ release from the IP3-sensitive intracellular Ca2+ store which resulted in further store depletion-operated Ca2+ influx through membrane TRPC channels of the presynaptic nerve terminal. This is done via PLC signaling cascade, leading to an enhancement of spontaneous transmitter release. Notexin-induced synaptic facilitation is potentially reduced while structural modification with phenylglyoxal. In addition, bath application of PLA2 inhibitor either aristolochic acid or glycyhirrzin effectively abolished notexin effect, suggesting the PLA2 activity is involved in notexin-induced SSC frequency facilitation. Previously it has been suggested local accumulation of PLA2-induced lipid metabolites myristoyl lysophosphatidylcholine (mLPC) and oleic acid (OA) promotes the fusion of the hemifused synaptic vesicles with plasma membrane, hence facilitating the neurotransmitter release in cultured cerebella granular neurons and thus resulted in bulges formation along the neurite. Our real-time morphometric analysis and synaptic activity assays showed that bulges formation along the neurite appeared significantly earlier and was induced at lower notexin concentrations than synaptic activity facilitation. Bath application of either mLPC, OA alone or their mixtures failed to mimic the notexin-induced facilitation in spontaneous transmitter release. Attenuation of PLA2 activity by chemical modification (notexin-80) resulted in correlated decrease of notexin-induced synaptic facilitation but not the degenerative morphological sign. Moreover, PLP-notexin, a site-specific modification of notexin with full intact PLA2 activity, shows significant loss the ability of notexin-induced neurite degeneration. Overall, results from our studies suggest the morphologic changes and synaptic facilitating effect induced by notexin are resulted from different cellular mechanisms.

Transient receptor potential (TRP) channels play important roles in cellular physiology and biology. The present PhD project investigated the functional expression of TRPV and TRPM channels in human preadipocytes and cardiac c-kit+ progenitor cells and their roles in regulating cell proliferation, adipogenic differentiation or migration. In addition, the role of store-operated Ca2+ entry (SOCE) channels in regulating cell proliferation and migration was also studied in human cardiac c-kit+ progenitor cells using multiple approaches including whole-cell patch voltage-clamp, confocal microscope, molecular biology, etc. We found that TRPV2, TRPV4 and TRPM7 channels were abundantly expressed in human preadipocytes. Activation of TRPV2 channels by probenecid caused a long-lasting intracellular Ca2+ transient, while activation of TRPV4 channels by 4-PDD induced Ca2+ oscillations. TRPM7 current was recorded with a Mg2+-free pipette solution, and inhibited by 2-aminoethyl diphenyl borate (2-APB). Silence of TRPV2 or TRPM7, but not TRPV4, with the specific shRNA, reduced cell proliferation via inhibiting cyclin D1, cyclin E, and p-ERK1/2. Individually silencing these three channels decreased adipogenic differentiation by reducing p-Akt kinase. The results indicate that TRPV2, TRPV4 and TRPM7 are involved in adipogenesis, while TRPV2 and TRPM7, but not TRPV4, regulate cell proliferation in human preadipocytes. In second part of the thesis, abundant expression of TRPV2, TRPV4, and TRPM7 channels was demonstrated in human cardiac c-kit+ progenitor cells. Similar to human preadipocytes, probenecid and 4-PDD activated Ca2+ signaling, and TRPM7 current recorded with a Mg2+-free pipette solution was inhibited by 2-APB. Silencing TRPV2 or TRPM7, but not TRPV4, inhibited cell proliferation by arresting cells at G0/G1 phase with a reduced cyclin D, cyclin E, and p-ERK1/2. Cell migration was decreased with silence of TRPV2, TRV4 or TRPM7 via inhibiting p-Akt kinase. The results show that TRPV2, TRPV4 and TRPM7 mediate cell migration, while TRPV2 and TRPM7, but not TRPV4 channels, participate in regulating cell proliferation. In third part of the thesis, we demonstrated that SOCE channels were composed of TRPC1, STIM1 and Orai1 by protein-protein interaction. Silence of TRPC1, STIM1, or Orai1 with specific siRNA reduced Ca2+ influx through SOCE channels, decreased cell proliferation by inhibiting cyclin D1 and cyclin E, and slowed down cell migration via reducing p-Akt kinase. These results suggest that TRPC1, STIM1 and Orai1 are the major components of SOCE channels in human cardiac c-kit+ cells. SOCE channels play an essential role in regulating cell proliferation and migration. Collectively, this PhD project has demonstrated for the first time that 1) TRPV2, TRPV4, and TRPM7 are abundantly expressed in human preadipocytes and cardiac c-kit+ progenitor cells. 2) These TRP channels regulate adipogenic differentiation in preadipocytes and migration in cardiac c-kit+ progenitor cells. 3) TRPV2 and TRPM7, but not TRPV4, are involved in cell proliferation of human preadipocytes and cardiac c-kit+ progenitor cells. 4) TRPC1, STIM1 and Orai1 are interacted to form SOCE channels and regulate cell proliferation and migration in human cardiac c-kit+ cells. 5) All the above physiological roles of TRPV2, TRPV4, TRPM7, and SOCE channels are mediated by cyclin D1, cyclin E, p-ERK1/2, and/or p-Akt. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Autophagy is a major catabolic pathway for maintaining cell homeostasis through degradation and recycle of macromolecules and organelles. Autophagy can be activated under environmental stress conditions, including reactive oxygen species (ROS). TRPM2, a non-selective trans-membrane calcium channel, can be activated by ROS that, in turn, leads to intracellular 〖Ca〗^(2+) increase through 〖Ca〗^(2+) influx. It is well known that ROS regulates autophagy, and vice versa. Yet, the molecular mechanisms underlying the interplay between ROS and autophagy remain elusive. Here we studied the role of TRPM2-mediated 〖Ca〗^(2+) influx in interplay between ROS and autophagy. From our study, we found that ROS activated TRPM2 for 〖Ca〗^(2+) influx via ADPR to inhibit early autophagy induction, which ultimately led to apoptosis in TRPM2 expressing cancer cell lines. On the other hand, ROS induced autophagy, not apoptosis, for cell survival in cancer cell lines which do not express TRPM2, and autophagy inhibition, either by ATG5 knockdown or by treating cells with bafilomycin A1 (an autophagy inhibitor), converted cells to apoptosis upon ROS treatment. In addition, ROS dramatically changed mitochondrial morphology, increased mitochondrial 〖Ca〗^(2+) content, and abolished mitochondrial membrane potential in TRPM2 expressing cells. Moreover, we found that ROS-induced Ca2+ influx via TRPM2 actually activated calmodulin-dependent protein kinase II (CaMKII) to phosphorylate Ser295 on Beclin1. Phosphorylated Beclin1, in turn, decreased the association between Beclin1 and VPS34, but induced the binding between Beclin1 and BCL-2. In summary, our data demonstrated that the TRPM2/〖Ca〗^(2+)/CaMKII/ Beclin1 cascade is the molecular switch between autophagy and apoptosis in response to ROS. Since dysregulation of ROS and autophagy has been associated with a variety of human diseases, e.g. cancer, neurological disorders, heart diseases, and liver diseases, manipulating the TRPM2/〖Ca〗^(2+)/CaMKII/ Beclin1 cascade should provide novel treatment option for these diseases. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

No description available.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2005. / Includes bibliographical references (leaves 102-113).

Thesis (M.S.)--Villanova University, 2006. / Chemistry Dept. Includes bibliographical references.

Le cancer de la prostate (CaP) représente la deuxième cause de mortalité par cancer dans les pays développés. L'invasion des tissus environnants et l'angiogenèse tumorale promeut la métastase de CaP vers des organes éloignés. L’expression de plusieurs canaux TRP (Transient Receptor Potential) est dérégulée dans les cellules cancéreuses et les cellules endothéliales (CE) dérivées de tumeurs. Ils ont donc été proposés comme marqueurs pour la progression du cancer ainsi que comme cibles potentielles pour une thérapie pharmaceutique. Afin d'étudier les canaux TRP dans la vascularisation du CaP, nous avons isolé et caractérisé trois lignées de CE derivées du CaP (PTEC). Nous avons testé sur les PTEC l'effet de deux molécules anti-angiogénique en combinaison avec des médicaments anti-androgèniques. Les résultats démontrent un comportement résistant des PTEC à des médicaments anti-angiogéniques par rapport à des CE normales. Nous avons criblé l'expression de tous les canaux TRP dans les CE saines et celles dérivées de trois types tumoraux (prostate, sein, rein). Nous avons identifié cinq candidats ‘spécifiques’ du CaP dérégulés seulement dans les PTEC qui ont été caractérisés au niveau fonctionnel et leur rôle potentiel en tant que modulateurs de l'angiogenèse in vitro a été testé. En outre, nous avons étudié le rôle inhibiteur de TRPM8 dans la migration des cellules cancéreuses prostatiques CaP et nous avons également détecté TRPM8 dans les CE dans lesquelles nous avons observé aussi un rôle anti migratoire de TRPM8. Pris dans leur ensemble, nos résultats mettent en lumière de nouveaux acteurs moléculaires pour cibler sélectivement la progression du CaP et son angiogenèse. / Prostate cancer (PCa) is the second most lethal male tumor in developed countries. Metastasis to distant organs is mainly mediated by tissue invasion and angiogenesis, which are indeed two of the main cancer hallmarks. Several Transient Receptor Potential (TRP) proteins are deregulated in cancer cells and angiogenesis and have been suggested as valuable markers in predicting cancer progress and as potential targets for pharmaceutical therapy. In order to screen and study TRP channels in PCa vasculature, we isolated and characterized three lines of human endothelial cells (ECs) from PCa patients (PTEC). We tested the effect of two anti-angiogenic in combination with anti-androgen drugs. The results clearly demonstrate a resistant behavior of endothelial cells isolated from prostate cancer to specific anti-angiogenic drugs compared to normal endothelial cells. We fully profiled the expression of TRP channels in tumor (prostate, breast and renal) and healthy ECs, with particular interest for prostate tumor EC. We identified five ‘prostate specific’ candidates deregulated in PTEC compared to endothelial derived from healthy prostate. ‘Prostate specific’ TRP candidates were functionally characterized and their potential role as in vitro angiogenesis modulators investigated. Our laboratory has already extensively studied the role of TRPM8 in PCa progression and migration. For this reason, we further investigated the molecular mechanism underling this effect in PCa cells as well as in ECs. Taken together, our results bring to light TRP channels as novel molecular players to selectively target prostate tumor progression and angiogenesis.

The first sensory synapse is the site where sensory afferent fibers make synaptic connections with second order neurons. Somatic and craniofacial afferents terminate in spinal cord dorsal horn (SDH) and caudal spinal trigeminal nucleus (CSTN). Neurotransmitter release from first order nerve terminals regulates ascending sensory transmission. Several lines of evidence indicate that plasticity in the spinal cord dorsal horn underlies secondary hyperalgesia. The sensory receptors, Transient Receptor Potential (TRP) channels, are expressed not only at peripheral terminals, but also at the central terminals of sensory neurons. While the role of these channels at the periphery is detecting environmental stimuli, their function at central terminals is not fully understood. Furthermore, TRP channel expression has been shown in CNS nuclei like hippocampus that are not tightly linked to somatosensation. In this study, I first determined the functionality of TRP channels at the first sensory synapse and hippocampus using pharmacological activators. I then determined if putatively endogenous TRP channel activators modulate synaptic transmission at the first sensory synapse. Lastly, I determined if recordings that respond to capsaicin demonstrate synaptic plasticity in either hippocampus or spinal cord, in an attempt to attribute synaptic plasticity mechanisms to TRPV1 activity at glutamatergic terminals. I have used slice patch-clamp technique to record miniature, spontaneous and evoked currents in lamina II neurons of spinal cord dorsal horn, CSTN and hippocampus. In lamina II neurons of SDH and CSTN, capsaicin, a TRPV1 agonist, robustly increased the frequency of mEPSCs and sEPSCs in a dose dependant manner. Although capsaicin increased m/sEPSC frequency, eEPSC amplitude, which reflects synchronous action potential propagation at glutamatergic terminals, was markedly depressed by capsaicin. Our studies indicate capsaicin inhibits action potential dependant transmission at central terminals. Resiniferatoxin (RTX) is a TRPV1 agonist that displays higher potency (>100 fold) compared to capsaicin, and deactivation with this agonist is minimal. RTX also depressed eEPSC amplitude in lamina II neurons of SDH and CSTN; unexpectedly, RTX increased m/sEPSC frequency to lesser extent compared to capsaicin. The TRPA1 agonist, N-methyl maleimide (NMM), increased s/mEPSC frequency in lamina II neurons; however, NMM did not depress eEPSC amplitude like capsaicin and RTX. It is possible that inhibition of nerve terminal firing is a unique property of TRPV1 agonists compared to other noxious chemicals. To justify a physiological relevance for nociceptive TRP channel expression at the first sensory synapse, I studied the effect of endogenous TRP channel agonists on synaptic transmission at the first sensory synapse. Anandamide (AEA) is an agonist of CB1/CB2 and TRPV1 receptors; it is less potent at TRPV1 receptors than capsaicin. AEA increased sEPSC frequency in 70% of neurons, whereas the remainder of neurons showed a decrease in sEPSC frequency. Unlike capsaicin and RTX, anandamide did not dramatically depress eEPSC amplitude. Methyl glyoxal (MG) is a putative TRPA1 agonist produced during conditions of hyperglycemia. MG increased the frequency of sEPSCs in SDH lamina II neurons. I next used high frequency synaptic stimulation (HFS-100 Hz, 1s) to model synaptic activity during pain transmission. HFS induced a modest increase in sEPSC frequency and minimally changed eEPSC amplitude; patches that showed HFS modulation also responded to capsaicin. In studying the role of TRP channels in modulating synaptic transmission at central synapses, I finally performed experiments in hippocampus with 2 objectives; 1) to determine extent of capsaicin responsiveness as an indicator of TRPV1 functionality, and 2) to evaluate synaptic plasticity in response to HFS. Capsaicin effect on sEPSC frequency in CA1 and CA3 neurons was minimal in comparison to its effect in dorsal horn neurons. HFS at schaffer collateral region caused LTP in CA1 neurons that was more pronounced than for spinal cord. In conclusion, TRP channels are expressed at central terminals of nociceptors where they modulate glutamatergic transmission. Studying their role at the first sensory synapse enhances our understanding of nociceptive transmission, and this study suggests this receptor for a target for intervening in pathological pain transmission at the level of spinal cord.