e.burcher@unsw.edu.au

Neurochemicals and their receptors : functions in human health and disease

Bladder dysfunction. Urinary incontinence is a “taboo subject”. This debilitating chronic disease is responsible for loss of social dignity, with major socio-economic costs. This disorder occurs in 4% of otherwise healthy 20 year olds, and increases with age. The accepted dogma has been that urge incontinence arises from involuntary contractions of the detrusor muscle, which cause leakage of urine. Most treatments have focused on the use of muscarinic receptor antagonists to inhibit such involuntary spasms. This approach rarely achieves long term cure. For the first time, we have recently shown the presence of mRNA and protein for muscarinic receptors on the human urothelium, which lines the bladder. Such receptors may in fact be the unintended target of these antagonists. Our ongoing studies also show expression of purinergic, vanilloid and tachykinin receptors on human urothelium. Thus the urothelium is no longer regarded as an inert barrier but a metabolically active organ responsible for signalling the sensation of “urgency”. The projects outlined below form part of our ongoing studies in the human urinary bladder.

Our specific aims are: (a) To identify and localize muscarinic, vanilloid, purinergic and tachykinin receptors on the human urothelium and detrusor, using (i) RT-PCR, (ii) western blotting, (iii) immunohistochemistry and (iv) in situ hybridisation; (b) To determine the functional roles of these key receptors, in relation to the urothelium and detrusor.

Project 1. Sensory receptor changes in bladder contractility disorders.

Contact: e.burcher@unsw.edu.au, 9385 2562

Availability for 1 student.

The aim of this project is to determine the expression of receptors for the tachykinin neuropeptides (Nk1 and NK2 receptors) in the smooth muscle and mucosa of the human urinary bladder, in biopsy samples obtained from patients with disorders of micturition involving excessive / uncontrollable contractility, leading to urinary incontinence. These disorders are relatively common, particularly in the elderly, but are not commonly discussed due to a perception that such disorders are shameful and also that there is no cure. We have well-established collaborations with clinicians to provide samples from both patients and age- and gender-matched “control” patients.

Techniques: RT-PCR, quantitative competitive RT-PCR, in situ hybridisation.

Selected References (Available on request)

• Mansfield, K.J., Liu, L., Mitchelson, F.J., Moore, K.H., Millard, R.J. & Burcher, E. (2005) Muscarinic receptor subtypes in human bladder detrusor and mucosa, studied by radioligand binding and quantitative competitive RT-PCR: Changes in ageing. Br. J. Pharmacol. 144, 1089−1099.

• Werkstrom, V., Lam, D.S.H., Farnsworth, R.H., Awad, N., Burcher, E. & Moore, K.H. (2000) In vitro contractile responses of detrusor to carbachol and neurokinin A, in children with recurrent urinary tract infection or day wetting. BJU Int. 85, 319-325.

• Burcher, E., Zeng, X.-P., Strigas, J., Shang, F., Millard, R.J. & Moore, K.H. (2000) Autoradiographic localization of tachykinin and calcitonin gene-related peptide receptors in adult urinary bladder. J. Urol. 163, 331-337.

• De Groat WC (2004) The urothelium in overactive bladder: passive bystander or active participant? Urology, 64 (suppl 6A): 7-11 (Review)

• Millard R.J, Moore K.H (1996) Urinary incontinence: the Cinderella subject. Med J Australia 165: 124-5.

Project 2. Cellular signalling mechanisms in human smooth muscle

Co-supervisor: Dr Lu Liu, email: lu.liu@unsw.edu.au, 9385 8762

 Availability for 1 student

Tachykinins are peptides conserved throughout evolution, with many actions including contraction of smooth muscle of the gut, secretion, neuronal activation, etc. Tachykinin receptors belong to the superfamily of guanine nucleotide binding protein-coupled receptors (GPCR). When the tachykinin peptides bind to the receptor, conformational changes occur: the C-terminal tail of the receptor will couple to different G-proteins and activate intracellular pathways which then trigger a variety of cellular responses. Our previous binding and functional studies in human tissues have shown that tachykinins are potent smooth muscle contractors and that tachykinin receptors (particular NK2 receptors) are densely localized to intestinal smooth muscle.

The aim of this study is to establish signalling pathways utilised by tachykinin receptors in human colon. Functional studies will be performed in control human colonic muscle strip. Tachykinins (substance P and neurokinin A) induced contraction will be recorded in the presence of nifedipine, an L-type Ca²⁺ channel blocker, and ryanodine, an inhibitor of Ca²⁺ release from the sarcoplasmic reticulum to determine whether tachykinins is dependent on calcium influx through L-type Ca²⁺ channels and/or calcium release from intracellular calcium stores. In addition, the effects of U73122, a phospholipase C (PLC) inhibitor; xestospongin-C, an inositol 1,4,5-triphosphate (IP₃) receptor antagonist; GF109203X, a protein kinase C (PKC) inhibitor; SQ 22,536, an adenylyl cyclase inhibitor will also be examined. In second messenger studies, IP3 and cAMP formation will be measured by radioimmunoassay.

Techniques: Isolated organ bath, radioimmunoassay

Selected References (Available on request)

• Warner, F.J., Liu, L., Lubowski, D.Z. & Burcher, E. (2000) Circular muscle contraction, messenger signalling and localisation of binding sites for neurokinin A in human sigmoid colon. Clin. Exp. Pharmacol. Physiol. 27, 928-933.

• Liu, L., Shang, F., Markus, I. & Burcher, E. (2002)  Role of substance P receptors in human colon circular muscle: alterations in diverticular disease. J. Pharmacol. Exp. Ther. 302, 627-635.