Thesis (Ph.D.)--Biology, Georgia Institute of Technology, 2008. / Committee Chair: Dr. Nael A. McCarty; Committee Co-Chair: Dr. Darrell Jackson; Committee Member: Dr. Alfred H. Merrill; Committee Member: Dr. Barbara D. Boyan; Committee Member: Dr. Harish Radhakrishna; Committee Member: Dr. Marion B. Sewer

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xviii, 178 p.; also includes graphics. Includes bibliographical references (p. 163-178). Available online via OhioLINK's ETD Center

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.

No description available.

Caveolae are integral part of the smooth muscle membrane and caveolins, the defining proteins of caveolae, act as scaffolding proteins for several G protein-coupled receptor signaling molecules and regulate cellular signaling through direct and indirect interactions with signaling proteins. Caveolin-1 is the predominant isoform in the smooth muscle and drives the formation of caveolae. However, little is known about the role of caveolin-1 in the regulation of excitation-contraction and excitation-transcription coupling in gastrointestinal smooth muscle. In the present study we have characterized muscarinic m2 and m3 receptor signaling in gastric smooth muscle and tested the hypothesis that caveolin-1 positively regulates muscarinic receptor signaling and contractile protein expression in smooth muscle. The role of caveolae/caveolin-1 in the regulation of muscarinic signaling was examined using complementary approaches: a) methyl b-cyclodextrin (MbCD) to deplete cholesterol in dispersed muscle cells, b) caveolin-1 siRNA to suppress caveolin-1 expression in cultured muscle cells, and c) caveolin-1 knockout (KO) mice. RT-PCR, western blot and radioligand binding studies demonstrated the selective expression of m2 and m3 receptor in gastric smooth muscle cells. Carbachol (CCh), acting via m3 receptors caused stimulation of phosphoinositide (PI) hydrolysis, Rho kinase and ZIP kinase activity, and induced phosphorylation of MYPT1 (at Thr696) and MLC20 (at Ser19), and muscle contraction, and acting via m2 receptors caused inhibition of forskolin stimulated cAMP formation. Stimulation of PI hydrolysis, Rho kinase and ZIP kinase activities, phosphorylation of MYPT1 and MLC20 phosphorylation and muscle contraction in response to CCh was attenuated in dispersed cells treated with MbCD or in cultured cells transfected with caveolin-1 siRNA. Similar inhibition of all responses was obtained in gastric muscle cells from caveolin-1 KO mice compared to gastric muscle cells to WT mice. Although, caveolin-1 had no effect on m2 receptor signaling, agonist-induced internalization of m2, but not m3 receptors was blocked in dispersed cells treated with MbCD or in cultured cells transfected with caveolin-1 siRNA. These results suggest that caveolin-1 selectively and positively regulates Gq/13-coupled m3 receptor signaling, Gi-coupled m2 receptor internalization. The expression of contractile proteins, g-actin and caldesmone and the transcription factors SRF and myocardin that regulate the expression of contractile proteins are down regulated, whereas EGF-stimulated EGF receptor phosphorylation and ERK1/2 activity are up-regulated in cells transfected with caveolin-1 siRNA. These results suggest using pharmacological, molecular and genetic approaches provide conclusive evidence that caveolae and caveolin-1 play an important role in orchestrating G protein coupled receptor signaling to have dual pro- excitation-contraction and excitation-transcription coupling, and anti-proliferative role in gastric smooth muscle.

Muscarinic acetylcholine receptors (mAChR) belong to the family of G-protein coupled receptors. There are five subtypes of mAChR denoted M1 to M5 that are widely and differentially distributed in both the central nervous system and periphery and play an important role in many specific physiological functions. Impairment of muscarinic neurotransmission occurs in serious disorders such as Alzheimer's disease, schizophrenia or Parkinson's disease that are accompanied by cognitive decline mainly due to the disruption of M1 receptor signaling in the brain. Unfortunately, the high degree homology of the orthosteric binding site among muscarinic receptor subtypes makes it very difficult to obtain subtype- selective agonists. One of the few known selective agonists is xanomeline that preferentially activates the M1 and M4 subtypes. Xanomeline exerts unique interactions with muscarinic receptors comprising reversible binding to the orthosteric domain, and wash-resistant allosteric interaction with a secondary binding site. The basis of xanomeline functional selectivity remains largely unknown. In an attempt to probe into such mechanisms we investigated the immediate and long-term effects of xanomeline on activation of muscarinic receptors, using intact Chinese hamster ovary (CHO) cells expressing individual...

Evidence from previous studies on the multiple memory systems model suggests that specific brain regions cooperate and compete to mediate the navigational strategies used to locate a goal in a spatial environment. Specifically, the cholinergic system within these discrete brain regions plays a key role in balancing this mediation such that acetylcholine release, genomic changes, and receptor regulation at cholinergic synapses are altered following learning and subsequent memory consolidation. Based on previous findings, we proposed to test learning-induced changes in muscarinic receptor binding expression in adult male rats following training on a water maze task guided either by a cue proximal to the escape platform (stimulus-response strategy), by cues surrounding the maze (place strategy) or by alternating between the two strategies (strategy-switching). The primary findings of the current study demonstrate that adult male rats that navigated to an escape platform guided by cues surrounding a water maze (place-trained) learned the task at a significantly slower rate than males that were guided by a cue proximal to the platform (stimulus-response-trained) or males that were required to switch strategies on alternating days. Additionally, males that were required to switch strategies over alternating days expressed higher ratios of muscarinic binding in the hippocampus relative to the striatum compared to place-trained rats, stimulus-response-trained rats, and swim-only controls. These results indicate that the use of a place learning strategy slows acquisition of a water maze task while the requirement to switch strategies as the demands of the task change over days engages the cholinergic system in the hippocampus most heavily. Taken together, the results from the current study further confirm the involvement of cholinergic function in regulating the balance between multiple memory systems. / acase@tulane.edu

Thesis (Ph.D.)--Tufts University, 1998. / Adviser: Barry A. Trimmer. Submitted to the Dept. of Biology. Includes bibliographical references (leaves 92-105). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Muscarinic ligand binding sites in fragments of rat hypothalamus and pituitary were studied using [3H]quinuclidinyl benzilate (QNB). In the hypothalamus, the highest amount of specific QNB binding was to n. paraventricularis and n. dorsomedialis. Specific QNB binding in other hypothalamic regions varied within a relatively narrow range. Fragments of whole pituitary also bound QNB but to a much smaller degree than brain. Pituitary binding of QNB was blocked by atropine but not by hexamethonium or d-tubocurarine. Within the pituitary, specific QNB binding to posterior pituitary was three times greater than to anterior pituitary. These findings are consistent with the operation of cholinergic mechanisms in hypothalamic and pituitary function.