by Ai-Dong Qi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 219-256). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

by So Siu Cheung, Eddie. / "September 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2000. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Muscle contraction and relaxation are controlled by the intracellular free Ca²⁺ concentration. The sarcoplasmic reticulum (SR) is an intracellular membrane system which regulates this internal free Ca²⁺ concentration. Responding to an electrical excitation of the cell surface membrane, the SR releases Ca²⁺ through a specific Ca²⁺ release channel, thus elevating the Ca²⁺ concentration inside muscle cell and causing the muscle to contract. Subsequent sequestration of Ca²⁺ by the SR Ca²⁺ pumps restores the resting state of the muscle cell. This research focuses on the Ca²⁺ release channel from skeletal muscle SR. The planar lipid bilayer technique was used to study the channel at the single channel level. The SR Ca²⁺ release channel was identified and isolated via its interaction with specific sulfhydryl oxidizing agents. This protein of a molecular mass of 106 kDa was then incorporated into a planar lipid bilayer membrane (BLM). In an asymmetrical Ca²⁺ solution, the channel protein demonstrates a single channel conductance of 107 ± 13 pS and a permeability ratio of Ca²⁺ versus Tris⁺ of 7.4 ± 3.3. In a symmetrical 250 mM NaCl solution, the channel protein displays a large single channel conductance of 400 ± 20 pS, and a weak voltage-dependence. The channel is activated by millimolar ATP and inhibited by micromolar ruthenium red. Nanomolar concentrations of ryanodine modify the channel by changing it from a rapidly gating full conductance state to a long-lived subconductance state. These results demonstrate that the isolated 106 kDa protein channel has properties similar to those observed following fusion of SR vesicles to a BLM. The bilayer system was also used to examine the effect of Ag⁺ on the SR Ca²⁺ release channel. Ag⁺ (0.2-1. 0 μM ) activates the SR Ca²⁺ release channel. Activation by Ag⁺ does not require the presence of Ca²⁺, Mg²⁺, or ATP. Ag⁺ activates the channel by increasing the open probability Po. Ag⁺ activation is always followed by a spontaneous inactivation. The channel is still sensitive to ruthenium red inhibition after exposure to Ag⁺. Isolated SR vesicles were fused to a BLM to study the effect of the photooxidizing dye, rose bengal, on the gating characteristics of the reconstituted SR Ca²⁺ release channel. Rose bengal activates the Ca²⁺ release channel in the presence of light by increasing the channel open probability and leaving the single channel conductance unchanged. This photoactivation is independent of the myoplasmic Ca²⁺ concentration, and can be achieved from either side of the membrane. In addition, the effect is inhibited by addition of 10-20 μM ruthenium red. When modified to its subconducting state by ryanodine, subsequent addition of rose Bengal reactivates the channel to a rapidly fluctuating full conducting state. These studies carried out at the single channel level utilizing the planar lipid bilayer technique have not only enhanced our understanding of the Ca²⁺ release mechanism of skeletal muscle SR, but also provided information about the toxic effects on biological membrane systems caused by heavy metals and oxidizing agents.

Calcium ions are important mediators in the mechanism of contraction and relaxation of muscle fibers. Depolarization of sarcolemma and transverse tubule causes an increase of myoplasmic ca2+ concentration which induces contraction of the myofibrils. In skeletal muscle fibers, the intracellular Ca2+ concentraton is regulated by an extensive membrane system, the sarcoplasmic reticulum (SR). Ca2+-release from SR is initiated by depolarization of the transverse tubule via a process referred to as excitation-contraction coupling. The Ca2+ - release channel located in the junctional SR plays an important role in this mechanism.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 213-239). Also available in electronic version.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus are responsible for the synthesis and secretion of vasopressin (VP), which is important for fluid homeostasis, and oxytocin (OT), which is responsible for uterine contraction during parturition and milk let-down during lactation. VP-ergic MNCs undergo a number of structural and functional changes during dehydration, including the adoption of a bursting pattern of firing, the retraction of glial processes from MNC somata and terminals, the translocation of kappa-opioid receptors from internal stores to the plasma membrane, and the somatodendritic release of VP and OT. Since voltage-gated Ca2+ channels have been found on intracellular granules, and since an increase in Ca2+ current could regulate firing patterns and neuropeptide release, the surface expression of Ca2+ channel subtypes in MNCs was tested to determine if it would be altered by 16-24 hours of water deprivation. Using radioligand binding of antagonists of N-type and L-type Ca2+ channels, channel density was measured in the supraoptic nucleus (SON), which is largely composed of MNC somata, and in the neurohypophysis (NH), which is largely composed of MNC terminals. Dehydration caused an increase in the density of L-type channels in the SON, while causing no significant change in the N-type density. No change in density of either channel type was observed in the NH. Electrophysiological measurements in isolated MNC somata showed no change in total Ca2+ current, but a significant increase in the nifedipine-sensitive current following dehydration. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated no increase in messenger RNA levels for L-type channels, suggesting that the increase in channel density is not a consequence of de novo synthesis. These results suggest that L-type Ca2+ channels may be translocated from internal stores to the plasma membrane of MNCs in response to dehydration. Such a process may be important in maximizing secretion of VP when the physiological need is high.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus are responsible for the synthesis and secretion of vasopressin (VP), which is important for fluid homeostasis, and oxytocin (OT), which is responsible for uterine contraction during parturition and milk let-down during lactation. VP-ergic MNCs undergo a number of structural and functional changes during dehydration, including the adoption of a bursting pattern of firing, the retraction of glial processes from MNC somata and terminals, the translocation of kappa-opioid receptors from internal stores to the plasma membrane, and the somatodendritic release of VP and OT. Since voltage-gated Ca2+ channels have been found on intracellular granules, and since an increase in Ca2+ current could regulate firing patterns and neuropeptide release, the surface expression of Ca2+ channel subtypes in MNCs was tested to determine if it would be altered by 16-24 hours of water deprivation. Using radioligand binding of antagonists of N-type and L-type Ca2+ channels, channel density was measured in the supraoptic nucleus (SON), which is largely composed of MNC somata, and in the neurohypophysis (NH), which is largely composed of MNC terminals. Dehydration caused an increase in the density of L-type channels in the SON, while causing no significant change in the N-type density. No change in density of either channel type was observed in the NH. Electrophysiological measurements in isolated MNC somata showed no change in total Ca2+ current, but a significant increase in the nifedipine-sensitive current following dehydration. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated no increase in messenger RNA levels for L-type channels, suggesting that the increase in channel density is not a consequence of de novo synthesis. These results suggest that L-type Ca2+ channels may be translocated from internal stores to the plasma membrane of MNCs in response to dehydration. Such a process may be important in maximizing secretion of VP when the physiological need is high.

Pain is the most common and debilitating medical problem for which patients seek medical care. Opioids remain the gold standard in the treatment of pain but are limited by poor side effect profiles such as emesis, constipation, dependence/addiction and respiratory depression. Despite a myriad of analgesic compounds on the market, tri-cyclic antidepressants, opioids, anticonvulsants, non-steroidal anti-inflammatory agents and combinations thereof, nearly two thirds of the chronic pain patients report inadequate pain relief; therefore, a new approach in the development of pain management is necessary. In recent years, the N-Type voltage gated calcium channel (CaV2.2) has become an attractive target in the treatment of chronic pain. Ziconotide, a selective CaV2.2 blocker, has been FDA approved in the United States for the treatment of severe chronic pain that is refractory to other treatments, but due to its profound side effect profile (nausea/vomiting, somnolence, vertigo, muscle spasms, myalgia, insomnia, anxiety, tremor, memory impairment and induced psychiatric disorders), the use of Ziconotide is severely limited. Mapping of the CaV2.2 interactome led to the identification of novel regulatory proteins, including collapsin response mediator protein 2 (CRMP2). Initially identified as an intracellular protein in the specification of axon/dendrite fate and axonal outgrowth, it is now known that this protein can regulate the activity of CaV2.2 and hence may be a critical regulatory node in pain modulation. Here, I describe a novel peptide aptamer derived from CRMP2, designated CaV2.2 binding domain 3 (CBD3), which when fused with the HIV transactivator of transcription protein (TAT), created tat-CBD3, which was able to significantly reverse thermal and mechanical hypersensitivity induced by the surgical incision on the plantar surface of the left hind paw in rats, a pre-clinical model of post-operative pain. Additionally, tat-CBD3 significantly attenuated thermal hypersensitivity induced via intraplantar injection of carrageenan, a model of acute inflammatory pain. Furthermore, the administration of tat-CBD3 did not produce any rewarding behaviors as measured by the conditioned placed preference (CPP) paradigm, nor did the administration of tat-CBD3 produced any motor coordination deficits measured using the rotarod performance test. Moreover, the addition of a 14-carbon myristate (myr) group to the parent peptide, myr-tat-CBD3, had increased efficacy in the attenuation of paw incision and carrageenan induced thermal/mechanical hypersensitivities when compared to the parent peptide (tat-CBD3). These types of novel compounds that lack unwanted side effects and addiction propensities are urgently needed to relieve individuals suffering from chronic pain.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 116-121).

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 133-169). Also available in electronic version.