Evidence for protein kinase involvement in long-term postsynaptic excitation of intrinsic primary afferent neurons in the intestine (original) (raw)
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Pfl�gers Archiv European Journal of Physiology, 2003
Low-frequency stimulation of synaptic inputs to after-hyperpolarising (AH) neurons in the guinea-pig small intestine causes sustained increases in excitability that far outlast the stimulus period. This excitation has been called sustained, slow, post-synaptic excitation (SSPE). Intracellular microelectrodes were used to record the effects of the protein kinase C (PKC) stimulant, phorbol dibutyrate (PDBu), and compare these with changes seen during the SSPE, in AH neurons of the small intestine of the guinea-pig. PDBu (1 nM-1 µM) increased excitability, depolarised the membrane and increased input resistance concentration dependently, mimicking the effects of low-frequency stimulation of pre-synaptic inputs. These changes developed slowly after the start of infusion and were only slowly reversible after wash out. PDBu suppressed a late after-hyperpolarising potential (AHP) that depends on Ca 2+ entry via voltagegated Ca 2+ channels during the action potential. The effects of PDBu (10 nM) on the late AHP were indistinguishable from those observed during the SSPE. PDBu, at a concentration that inhibited the AHP, had no effect on the action potential half-width or the slope of its first repolarisation phase (the early phase of repolarisation is slowed by the Ca 2+ influx of the action potential). Thus PDBu inhibited K + channel opening underlying the late AHP, but did not suppress Ca 2+ entry during the action potential. The hyperpolarisation-activated cation current (I h ) in intrinsic primary afferent neurons (IPANs) was not affected by PDBu. We conclude that PDBu mimics the sustained excitation caused by low-frequency stimulation of synaptic inputs to IPANs by closing IK channels responsible for the AHP or restricting their opening by Ca 2+ and by reducing the current carried by K + channels that are active at rest. IK channels, the opening of which results in the AHP, have consensus sites for PKC and are likely targets for phosphorylation during the SSPE.
The European journal of neuroscience, 2005
AH neurons in the enteric nervous system play an essential role in initiating intestinal reflexes and factors that control AH neuron excitability therefore influence the state of the digestive system. Prominent afterhyperpolarizations that follow action potentials in these neurons strongly affect their excitability. In the present work, we have investigated the regulation of the afterhyperpolarizing current (I AHP ) by protein kinase C (PKC). Electrophysiological responses and protein translocation were investigated in AH neurons of freshly dissected preparations of myenteric ganglia from the guinea-pig ileum. The activator of conventional and novel PKCs, phorbol dibutyrate, but not the activator of novel PKCs, ingenol, blocked the I AHP . Phorbol dibutyrate had no effect on the hyperpolarization-activated current (I h ) or on the A current (I A ). Stimulation of synaptic inputs to the neurons also reduced the I AHP , and had no effect on I h or I A . Phorbol dibutyrate also reduced a background outward current that was present after the I AHP current had been blocked by clotrimazole. Both phorbol dibutyrate and ingenol caused translocation of the novel PKC, PKCe, in these neurons. Only phorbol dibutyrate caused translocation of PKCc, a conventional PKC. The studies thus indicate that the activation of PKC by phorbol esters and by nerve stimulation affects AH neurons in a similar way, and that PKC activation targets both the I AHP and another background K + current. The I AHP is targeted by a conventional PKC, suggested to be PKCc, as this is the only conventional PKC that is prominent in AH neurons.
Neuroscience, 2004
Recent studies have shown that tachykinins mediate slow synaptic transmission to myenteric AH (afterhyperpolarising) neurons via neurokinin-3 receptors (NK 3 R). This study investigated a similar role for neurokinin-1 receptors (NK 1 R) and compared the effect of selective receptor antagonists on non-cholinergic slow excitatory post-synaptic potentials (EPSPs) recorded in myenteric AH neurons of the guinea-pig ileum. Slow EPSPs evoked by electrical stimulation of circumferentially oriented presynaptic nerves were mimicked by application of senktide, an NK 3 R agonist. [Sar 9 ,Met(O 2) 11 ]-substance P, an NK 1 R agonist, depolarised a smaller number of neurons. SR142801, a selective NK 3 R antagonist (100 nM), inhibited slow EPSPs and responses to senktide, but had no effect on depolarisations evoked by forskolin, an activator of adenylate cyclase. SR140333, a selective NK 1 R antagonist, inhibited slow EPSPs in a subset of neurons and blocked responses to [Sar 9 ,Met(O 2) 11 ]substance P, but not to senktide or forskolin. Slow EPSPs that were predominantly mediated by NK 1 R had significantly shorter latencies than those due to activation of NK 3 R. After blockade of slow EPSPs, slow hyperpolarizing responses to presynaptic nerve stimulation were revealed in one-third of neurons. These events, which were associated with a decrease in input resistance and blocked by tetrodotoxin, were equated with slow inhibitory postsynaptic potentials. They were abolished by the 5-hydroxytryptamine 1A receptor antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine (NAN-190), but unaffected by phentolamine, an ␣adrenoceptor antagonist. In conclusion, these results provide the first direct evidence that NK 1 R mediate some slow excitatory synaptic input to myenteric AH neurons, and suggest that NK 1 R and NK 3 R activate distinct signal transduction pathways. These results also demonstrate that slow inhibitory synaptic transmission, which may be mediated by 5-hydroxytryptamine, is more prevalent in the myenteric plexus than previously indicated.
2004
Synaptic transmission between neurones intrinsic to the wall of the intestine involves multiple neurotransmitters. This study aimed to identify neurotransmitters responsible for noncholinergic excitatory synaptic transmission in the submucous plexus of the guinea pig ileum. Intracellular recordings were made from secretomotor and vasodilator neurones. A single electrical stimulus to a fibre tract evoked excitatory postsynaptic potentials (EPSPs) with three different time courses -fast, slow and an EPSP with an intermediate time course (latency 96 ms, duration 1.2 s). In all neurones, blocking nicotinic receptors reduced fast EPSPs, but they were abolished in only 57 of 78 neurones. Fast EPSPs were also reduced by P2 purinoceptor blockade (5
Mediation by protein kinases C and A of Go-linked slow responses of enteric neurons to 5-HT
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1997
5-HT activates the peristaltic reflex and is the neurotransmitter of a subset of myenteric interneurons. Hyperpolarizing afterpotential (AH)/type 2 neurons respond to 5-HT with a long-lived depolarization that is caused by the inhibition of a Ca(2+)-activated K+ conductance (gKCa). This effect is mediated by a G-protein-coupled receptor, 5-HT1P. 5-HT1P agonists specifically activate G alpha o, the immunoreactivity of which was found to be highly abundant and membrane-associated in almost all enteric neurons. Responses of hyperpolarizing AH/type 2 neurons to 5-HT were inhibited by intracellular injection of GDP beta S or anti-G alpha o Fab fragments but were potentiated and prolonged by intracellular GTP gamma S. Responses to 5-HT were antagonized by pertussis toxin, downregulation of protein kinase C (PKC) and inhibitors of phosphatidylcholine phospholipase C (PC-PLC), PKC (including pseudosubstrate peptides, chelerythrine, and the alpha/beta isoform-specific inhibitor Go 6976), pro...
2003
5-hydroxytryptamine (HT) 4 receptor agonists stimulate gastrointestinal motility partly by facilitating acetylcholine release from myenteric neurones. However, the signalling mechanisms that couple 5-HT 4 receptor activation to increased transmitter release in the myenteric plexus are unknown. We used conventional intracellular electrophysiological methods to record fast excitatory postsynaptic potentials (fEPSPs) from neurones in the guinea-pig ileum myenteric plexus preparation. The substituted benzamide, renzapride, acted at 5-HT 4 receptors to facilitate fEPSPs. This response was mimicked by forskolin, an activator of adenylate cyclase. Facilitation of fEPSPs by renzapride and forskolin was not blocked by treating tissues with pertussis toxin (PTX) (2 h, 2 lg mL )1 ). Facilitation of fEPSPs caused by renzapride was blocked by the non-selective protein kinase inhibitors, staurosporine (1 lmol L )1 ) and H-8 (30 lmol L )1 ) and by the selective protein kinase A (PKA) inhibitor, H-89 (10 lmol L )1 ). These data indicate that 5-HT 4 receptors act via a PTX-resistant mechanism to activate PKA. Protein kinase A activation leads to an increase in transmitter release from myenteric nerve terminals and a facilitation of fast excitatory synaptic transmission.
Journal of Neurophysiology, 1994
1. In the pleural mechanoafferent sensory neurons of Aplysia, serotonin (5-HT)-induced spike broadening consists of at least two components: a cAMP and protein kinase A (PKA)-dependent, rapidly developing component and a protein kinase C (PKC)-dependent, slowly developing component. Voltage-clamp experiments were conducted to identify currents that are modulated by PKC and thus may contribute to the slowly developing component of 5-HT-induced spike broadening. 2. We compared the effects of phorbol esters, activators of PKC, on membrane currents with those of 5-HT. Bath application of 5-HT had complex modulatory effects on currents elicited by voltage-clamp pulses to potentials > 0 mV. The kinetics of both activation and inactivation of the membrane currents were slowed by 5-HT. This led to a decrease in an outward current at the beginning of the voltage-clamp pulse and an increase at the end of the pulse. Previous work has shown that these effects represent, in part, the modulati...