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PhD Thesis: Kirk Pak

Dissertation Abstract:
The Role of the M2 Muscarinic Receptor in Maintaining Urinary Bladder Function in a Streptozotocin Model of Diabetes

By Kirk Pak, PhD
Doctor of Philosophy in Pharmacology and Toxicology
University of California, Irvine, 2010
Professor Frederick J. Ehlert, Chair

Urinary bladder function depends on autonomic innervation and a complex neural control system consisting of the brain and spinal cord. Its reliance on central nervous control distinguishes the bladder from other visceral structures such as those found in the cardiovascular and gastrointestinal systems. The efficient regulation by afferent and efferent activity in the urinary bladder is critical in its unusual task of both storage and intermittent voiding. And the appropriate postjunctional response to upstream neural regulation is imperative to mediate normal urinary voiding function. A major neurotransmitter influencing bladder contractile function is acetylcholine, which acts via M2 and M3 muscarinic receptors in smooth muscle. The M3 receptor mediates direct contraction, whereas the M2 receptor has been shown to enhance M3-mediated function and to inhibit relaxation mediated by adenylate cyclase.

Diabetes is the most common cause of autonomic neuropathy in the developed world. Accordingly, visceral structures such as the urinary bladder are negatively affected and inadequate and aberrant control leads to dysfunction. In addition to neuropathy, diabetic bladders are also associated with distension. These abnormalities are reproduced in animals treated with streptozotocin (STZ), a pancreatic ß-cell toxin. Through circumstantial evidence showing substantial enlargement in bladder from STZ-treated M2 muscarinic receptor knockout mouse (M2 KO), we speculated that bladder distension might impose excessive physical strain on surrounding nerves already under hyperglycemic stress. An upregulation of M2 receptor expression and an increase in muscarinic receptor function in diabetic bladder has been reported as well as the enhanced functional profile of the M2 receptor when bladders are distended.

We hypothesize, therefore, that the M2 receptor may have a protective role and may sustain function in the impaired diabetic bladder by opposing neuropathy secondary to excessive distension of bladder. We postulate that M2-mediated postjunctional compensatory mechanisms may also contribute to maintaining bladder function.

We test these hypotheses in the following sections.

In Chapter 2, we confirm that known postjunctional M2 receptor-mediated mechanisms in bladder can be replicated in an electrical field stimulation experiment, which acts as a reporter assay for the activation of the M2 receptor by neurotransmitter release.

Chapter 3 investigates the differential postjunctional roles of M2 and M3 receptors in STZ-induced diabetes. Bladders were isolated from wild type and M2 KO mice treated with vehicle or STZ. Contractions elicited to the bath-applied muscarinic agonist oxotremorine-M were measured in the absence and presence of the adenylate cyclase stimulator forskolin. We found that M2-mediated responses sustain bladder contraction when M3-mediated function is impaired in STZ-induced diabetes. And the ability of the M2 receptor to inhibit the relaxant effects of forskolin is increased in this model.

Chapter 4 investigates whether bladder lacking the M2 receptor is pre-disposed to excessive enlargement during the course of STZ-induced diabetes. We hypothesize that an over-distended bladder phenotype may impinge surrounding nerves and, thereby, expedite the progression of diabetic neuropathy.

Chapter 5 studies whether the contractile role of the M2 receptor in gastric fundus is similar to that in bladder during the course of diabetes, since the fundus shares with the bladder the quality of being able to distend. The ileum does not share this quality to the same extent and so is not expected to exhibit a similar role for the M2 receptor. Atrial contraction is also examined to determine muscarinic receptor involvement in another important system affected by diabetes.

In Chapter 6, we discuss various possibilities, both theoretical and empirically explored by my research, by which the M2 receptor may mediate its protective role during the course of diabetes.