Expression, function and modulation of nicotinic ACh receptors and P2- purinoceptors in rat parasympathetic neurons

Liu, Dongmei. (2002). Expression, function and modulation of nicotinic ACh receptors and P2- purinoceptors in rat parasympathetic neurons PhD Thesis, School of Biomedical Sciences, The University of Queensland.

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Author Liu, Dongmei.
Thesis Title Expression, function and modulation of nicotinic ACh receptors and P2- purinoceptors in rat parasympathetic neurons
School, Centre or Institute School of Biomedical Sciences
Institution The University of Queensland
Publication date 2002
Thesis type PhD Thesis
Supervisor David Adams
Total pages 190
Language eng
Subjects 06 Biological Sciences
Formatted abstract
In the mammalian parasympathetic nervous system, ACh is a primary neurotransmitter, however, other non-cholinergic, non-adrenergic neurotransmitters are also found, for example vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase activation peptide (PACAP) and adenosine 5-triphosphate (ATP).

VIP has been shown to cause an increase in heart rate and cardiac output. In present study, the effects of VIP and PACAP on dissociated parasympathetic neurons of rat intracardiac and submandibular ganglia were examined using the voltage clamp technique. VIP and PACAP selectively and reversibly potentiate nicotinic ACh-evoked currents in a concentration-dependent manner without affecting other ionic conductances. The potentiation was inhibited reversibly by bath application of VIP receptor antagonists or the neuronal nicotinic receptor antagonist, mecamylamine. The signal transduction pathway mediating VIP-induced potentiation of ACh-evoked currents involves the activation of both a specific cell-surface receptor and a pertussis toxin-sensitive G-protein but not cyclic AMP. The potentiation of nicotinic ACh-evoked currents by VIP and PACAP is likely to contribute to the altered neuronal activity observed in the mammalian intracardiac cardiac ganglia in vivo and consequent changes in heart rate and cardiac contractility.

ATP has been suggested as neurotransmitter in both peripheral and central nervous systems. However, no direct evidence has been presented to show that ATP mediates synaptic transmission in the parasympathetic nervous systems. In acutely dissociated submandibular neurons, focal application of ATP evoked an excitatory response and firing, whereas in intact preparations no response was observed in postganglionic neurons. These were explained by the differential expression of P2 receptors in intact and dissociated neurons. The permeability properties of native P2X receptor-channels were examined in dissociated neurons by measuring the reversal potential in presence of different monovalent and divalent inorganic cations and monovalent organic cations. The P2X receptor-channel exhibited weak selectivity among the alkali metals with a selectivity sequence of Li+ > Na+ > Cs+ > Rb+ > K+, and permeability ratios relative to Cs+ (Px/Pcs) ranging from 1.42 to 0.86. The selectivity for the divalent alkaline earth cations was also weak with the sequence Ca2+ > Sr2+ > Ba2+ > Mn2+ > Mg2+ The permeability sequence obtained for the saturated organic cations is inversely correlated with the size of the cation. The unsaturated organic cations, guanidinium, imidazole and acetamidine have a higher permeability than that predicted by molecular size. Acidification potentiated the ATP-evoked current, which is similar to cloned P2X2 and P2X2/3 receptor subtypes in submandibular neurons. P2X receptor antibodies were used to further identify the functional subtypes of P2X receptor. Cell dialysis with either anti- P2X2 or anti-P2X4 but not P2X1 antibodies attenuated the ATP-evoked currents. Single channel properties were also examined and at least four conductances were observed: 18 pS, 27 pS, 36 pS and 56 pS.

The mobilization of Ca2+ by purinoceptor activation and the relative contributions of intra- and extracellular sources of Ca^* were also investigated using microfluorimetric measurements of fura-2 loaded rat intracardiac neurons. Brief application of either ATP or UTP caused transient increases in ICa2+]i. Removal of external Ca2+ did not significantly reduce the ATP- or UTP-induced [Ca2+]i responses. The agonist potency profile for [Ca2+]i increases was ATP = UTP > 2- MeSATP > ADP » adenosine. ATP- and UTP-induced rise of [Ca2+]i were completely and reversibly blocked by 10 µM PPADS and partially inhibited by 100 µM suramin. In the presence of cyclopiazonic acid (10 µM) in Ca2+-free media, the [Ca2+]i responses evoked by ATP were progressively decreased and abolished. ATP- and UTP-induced [Ca2+]i rises were insensitive to pertussis toxin, caffeine and ryanodine. However, U-73122, a phospholipase C (PLC) inhibitor, significantly reduced the ATP- and UTP-induced [Ca2+]i responses. Thus, the signalling pathway appears to involve a PTX-insensltive G protein coupled to PLC generation of IP3, which triggers the release of Ca2+ from a ryanodine-insensitive Ca2+ store(s). ATP and UTP also evoked slow outward currents at -60 mV. concomitant with the rise in [Ca2+]i, in approximately one third of rat intracardiac neurons. This Ca2+-activated outward current may underlie the slow prolonged hyperpolarization observed in a subpopulation of intracardiac neurons.
Keyword Nicotinic receptors
Parasympathetic nervous system
Acetylcholine -- Receptors

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
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Created: Thu, 05 May 2011, 11:31:46 EST by Talha Alam on behalf of The University of Queensland Library