PubMed · 4386319
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V P Whittaker. 1968. Synaptic transmission.. https://doi.org/10.1073/pnas.60.4.1081
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1. Intracellular recording techniques were used to examine the voltage-activated calcium channels mediating neurotransmitter release from nerve terminals of extrinsic, sympathetic origin and intrinsic (enteric) origin innervating submucosal neurones of the guinea-pig caecum. 2. The noradrenergic slow inhibitory postsynaptic potential (IPSP) was abolished by superfusion of omega-conotoxin (omega-CTX) GVIA (3-300 nM), with an apparent IC50 of 8.6 nM. Superfusion of omega-CTX MVIIC (500 nM) also suppressed the amplitude of slow IPSPs, but both omega-agatoxin IVA (100 nM) and nicardipine (1-10 microM) were ineffective. The hyperpolarization induced by exogenous noradrenaline was not affected by omega-CTX GVIA (100 nM). 3. In contrast to the slow IPSP, the amplitude of the cholinergic fast excitatory postsynaptic potential (EPSP) was partially inhibited, but not abolished, by omega-CTX GVIA (0.1-1 microM). Furthermore, omega-agatoxin IVA (0.1-1 microM) or omega-CTX MVIIC (0.1-1 microM) also affected the fast EPSP, but nicardipine (1-10 microM) was ineffective. In combination, omega-CTX GVIA (100 nM) and omega-agatoxin IVA (100 nM) inhibited the fast EPSP by 74 +/- 6 %; the residual fast EPSP was not affected by omega-CTX MVIIC (100 nM). The fast EPSP was completely abolished by low Ca2+, high Mg2+ Krebs solution or Krebs solution containing Co2+ (2 mM) and Cd2+ (400 microM). The depolarization induced by exogenous acetylcholine was not affected by either omega-CTX GVIA (100 nM), omega-agatoxin IVA (100 nM) or omega-CTX MVIIC (100 nM). 4. Taken together, these results suggest that, in the submucosal plexus of the guinea-pig caecum, release of noradrenaline from extrinsic nerve terminals is regulated by N-type calcium channels, whereas release of acetylcholine from intrinsic nerve terminals involves several types of calcium channel.
1. Acetylcholine (ACh) is important as the transmitter responsible for neuromuscular transmission. Here we report the non-quantal release of ACh from embryonic myocytes. 2. Co-cultures of spinal neurons and myotomal muscle cells were prepared from 1-day-old Xenopus embryos. Single channel currents were recorded in the non-innervated myocytes. When the patch pipette was filled with Ringer solution alone, spontaneous single channel currents occurred, which were inhibited by d-tubocurarine (d-Tc). 3. The channel conductance appearing in Ringer solution (37.3 pS) was similar to that of an embryonic-type ACh channel (36.9 pS), indicating that ACh is probably released from myocytes in normal Ringer solution. 4. When the patch pipette was filled with anticholinesterase alone to prevent hydrolysis of ACh released from myocytes, both physostigmine and neostigmine in a concentration-dependent manner increased channel open probability; it was reduced by d-Tc or alpha-bungarotoxin. 5. Vesamicol and quinacrine, vesicular transporter inhibitors, reduced the channel open probability caused by ACh released from myocytes in the presence of neostigmine or physostigmine. 6. Intracellular alkalinization with NH4Cl inhibited the ACh release from myocytes, whereas, extracellular alkalinization, brought about by replacing normal Ringer solution, with pH 8.6 Ringer solution enhanced ACh release. 7. The immunocytochemistry of choline acetyltransferase (ChAT) showed that ChAT exists in both myocytes and neuronal cells but not in fibroblasts. 8. These results suggest that embryonic myocytes are capable of synthesizing and releasing ACh in a non-quantal manner. Extracellular alkalinization enhanced and intracellular alkalinization inhibited ACh release from myocytes.
A common feature of neuronal nicotinic acetylcholine receptors (nAChRs) is that they conduct inward current at negative membrane potentials but little outward current at positive membrane potentials, a property referred to as inward rectification. Physiologically, inward rectification serves important functions, and the main goal of our study was to investigate the mechanisms underlying the rectification of these receptors. We examined recombinant alpha3beta4 and alpha4beta2 neuronal nAChR subtypes expressed in Xenopus oocytes and native nAChRs expressed on superior cervical ganglion (SCG) neurons. Whole-cell ACh-evoked currents recorded from these receptors exhibited strong inward rectification. In contrast, we showed that single-channel currents from these neuronal nAChRs measured in outside-out patches outwardly rectify. On the basis of recent findings that spermine, a ubiquitous intracellular polyamine, confers rectification to glutamate receptors and inwardly rectifying potassium channels, we investigated whether spermine causes neuronal nAChRs to inwardly rectify. When spermine was added to the patch electrode in outside-out recordings, it caused a concentration- and voltage-dependent block of ACh-evoked single-channel currents. Using these single-channel data and physiological concentrations of intracellular spermine, we could account for the inward rectification of macroscopic whole-cell ACh-evoked conductance-voltage relationships. Therefore, we conclude that the voltage-dependent block by intracellular spermine underlies inward rectification of neuronal nAChRs. We also found that extracellular spermine blocks both alpha3beta4 and alpha4beta2 receptors; this finding points to a mechanism whereby increases in extracellular spermine, perhaps during pathological conditions, could selectively block these receptors.