Project 22

Rho-Kinase and synaptic transmission in the autonomic nervous system

Dr Lynette Fernandez (Pharmacology) and A/Prof. Alan Everett (Physiology)

Activation of the Rho-kinase pathway promotes phosphorylation of myosin light chain kinase and increases [Ca²⁺]_i, causing smooth muscle contraction. A component of elevated bronchial obstruction in asthma is driven by cholinergic nerves which provide the dominant neural input in the airways. I have recently shown that inhibition of Rho-kinase activity by Y-27632 suppresses cholinergic nerve-mediated contractile activity in murine airways. In addition, Y-26732 alters acetylcholine release from murine tracheal preparations. The mechanism by which Rho-kinase modulates cholinergic nerve activity is yet to be determined. However, myosin light chain kinase, which is regulated by Ca²⁺/calmodulin, has been shown to modulate presynaptic function in the hippocampus (Ryan, 1999, J. Neurosci., 14, 1317-1323).

This study will significantly increase our understanding of the involvement of Rho-kinase in cholinergic nerve function and will be of considerable value in assessing the utility of Rho-kinase inhibitors in airway diseases such as asthma. Completion of this project will provide critical data to be included in future grant applications.

General Hypothesis

The Rho-kinase pathway controls the phosphorylation of myosin light chain kinase in synaptic boutons of autonomic nerves and is a prime determinant of release probability.

General Method

Synaptic function will be measured by optical procedures that employ a styryl dye (FM1-43) that fluorescently labels synaptic vesicle membrane when vesicles fuse with the presynaptic membrane. Subsequent recycling of the membrane leads to activity-dependent loading of terminals with dye and provides a quantitative measure of synaptic function. Studies will be conducted in tracheal preparations obtained from CBA/CaH mice (AEC approval No. 03/100/274).

Experiment 1: To measure the effect of Rho-kinase on synaptic bouton recruitment

Rho-kinase may convert boutons from “silent” to “active” by recruiting vesicles to active zones of silent (very low probability) boutons. Murine tracheal smooth muscle preparations will be dual-labelled with synaptic vesicle protein 2 (SV2, a ubiquitous marker for synaptic vesicles) and the fluorescent dye FM1-43. SV2 labels vesicular proteins and, therefore, the total synaptic vesicle pool within nerve terminals. FM1-43 labelling occurs when synaptic vesicles exocytose and undergo recycling in response to electrical field stimulation. The ratio of FM1-43:SV2 labelling will indicate the proportion of boutons recycling vesicles and presumably releasing transmitter. The experiment will investigate the extent of bouton recruitment in the absence and presence of the Rho-kinase inhibitor Y-27632.

Experiment 2: To measure the effect of Rho-kinase on synaptic vesicle turnover

Rho-kinase may stimulate vesicle pool turnover without recruiting new boutons. We will investigate this possibility by monitoring the loss of dye from prelabelled boutons – the dye is lost from the vesicle membrane during exocytosis conducted in the absence of dye in the extracellular medium. Murine tracheal smooth muscle preparations will be electrically stimulated in the presence of FM1-43 to saturate the total recycling pool. This will be followed by another period of electrical stimulation during which vesicles release FM1-43. FM1-43 release during this unloading phase will be assessed in the absence and presence of Y-27632. This involves quantitative imaging of boutons during application of electrical stimulation. These data will indicate the effect of Rho-kinase on the release of acetylcholine from the total recycling pool of individual boutons