Nerve endings communicate with their receiving cells by the secretion of primary neurotransmitter substances, and, may also regulate their own activity by the co-release of presynaptic neuromodulatory substances. Adenosine derivatives are such modulatory substances. Indeed, most synapses in the vertebrate nervous system are responsive to physiological levels of extracellular adenosine derivatives. The main interests of my laboratory are studying the presynaptic and postsynaptic mechanisms underlying chemical synaptic transmission with emphasis on the processes which co-released adenosine derivatives modulate excitatory synaptic transmission.
1) Presynaptic processes
With respect to presynaptic events, earlier results from this laboratory have demonstrated: (i) the release of adenosine derivatives from motor nerve endings, (ii); the presence of specific adenosine receptors on motor nerve endings and (iii) the blockade of Ca2+-dependent secretion by adenosine. In the past few years, we have been able to measure nerve terminal Ca2+ currents simultaneously with the electrophysiological correlates of evoked acetylcholine (ACh) release. Adenosine does not affect the Ca2+ current that mediates ACh release in the frog and inhibits ACh release by methods that bypass active calcium channels with a very brief latency. These recent results confirm our published suggestions beginning in 1981 that adenosine and other neuromodulators may inhibit neurotransmitter release downstream of Ca2+ entry, and, that G proteins can inhibit transmitter release apart from changes in soluble second messenger substances.
This effect of adenosine has considerable physiological significance as we have recently shown that all of the neuromuscular depression that occurs during repetitive nerve stimulation at normal levels of ACh release in the frog is due to the neurally-evoked release of ATP, which after degradation to adenosine acts as a negative feedback modulator of ACh release. We have even found that ATP is released quantally from synaptic vesicles in motor nerve terminals using the outside-out patches of membrane containing ATP receptors as biosensors for ATP release. These results are now being confirmed at mammalian neuromuscular junctions. The clinical implications of these results are intriguing. Specifically, could specific adenosine receptor antagonists (e.g., 8-cyclopentyl-theophylline derivatives) maintain a high safety factor at the neuromuscular junction by preventing the normal depression of ACh release and thus alleviate the symptom of patients in disease states where skeletal muscle is readily fatigued (e.g. myasthenia gravis)? Indeed, it is our intent to extend the work originally conducted at normal motor nerve endings to other synaptic junctions (both normal and diseased) in subsequent years.
With respect to the mechanisms of neurotransmitter release, apart from considerations of adenosine, we have found that it is possible to deliver hydrophilic membrane-impermeant molecules (ions, nucleotides, etc.) to the cytoplasm of the small motor nerve ending using lipid vesicles (liposomes) as vehicles and simultaneously study the electrophysiological correlates of evoked ACh. Our results suggest that physiological ACh release by nerve impulses can occur when Ca2+ channels are bypassed. Briefly, Ca2+ delivery to the nerve terminal cytoplasm via liposomes appears sufficient to promote evoked ACh release without the participation of active Ca2+ channels. This result is consistent with our proposed model for Ca2+-dependent ACh release which suggests that a Ca2+ binding protein at the secretory apparatus is the main determinant of ion selectivity and of the magnitude of evoked ACh release. We are currently improving the yield of liposomes for encapsulation and delivery of synapse-specific antibodies to the cytoplasm of frog and mammalian nerve endings in vitro and determine which if any of these molecules are required for normal synaptic function. We will also combine the liposome technique with studies of gene knockout mice in which specific presynaptic proteins have been deleted to determine the importance of these presynaptic proteins for normal neuromuscular function and for the action of adenosine.
2) Postsynaptic processes
With respect to postjunctional events, we employed patch clamp techniques to be one of the first to demonstrate that ATP mediates fast synaptic excitation between adult mammalian neurons (sympathetic ganglion cells of the guinea-pig celiac ganglion). Excised membrane patches with the outside surface of the membrane facing the bathing solution (outside-out patches) demonstrate that these ATP receptors incorporate an ionic channel. These studies also demonstrated a remarkable interaction between P2X ATP receptors and nicotinic receptors for ACh. Indeed, activation of these two types of apparently independent ligand-gated channels are mutually occlusive, even at low concentrations of agonists that do not activate ion channels. Specifically, nicotine inhibits the non-selective cationic current generated by ATP, and, ATP receptor agonists inhibit the cation current through nicotinic receptors. We will be studying these interactions further in adult neurons and in cloned human nicotinic and P2X ATP receptors expressed in tandem.
