Investigation of PKC isoform-specific translocation and targeting of the current of the late afterhyperpolarizing potential of myenteric AH neurons (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.
Autonomic Neuroscience, 2004
We have investigated the effects of protein kinase inhibitors on the sustained slow postsynaptic excitation (SSPE) that is evoked by prolonged stimulation of synaptic inputs to intrinsic primary afferent neurons (IPANs) in the small intestines of guinea pigs. Stimulation of synaptic inputs to the IPANs caused depolarisation, increased input resistance, and increased excitation that continued after the cessation of stimulation. The excitation was substantially reduced by the broad-spectrum kinase inhibitor staurosporine (1 AM), PKC inhibitors Ro 31-8220 (3.3 AM) and calphostin C (1 AM), but not by the PKA inhibitor H89 (1 AM). At a higher concentration, 10 AM Ro 31-8220 reduced the excitability of axons to electrical stimulation. Phorbol dibutyrate (1 AM) caused excitability increases, membrane depolarisation, and increased input resistance that mimicked the SSPE. We conclude that the generation of the SSPE requires a phosphorylation step that is mediated by protein kinase C. D 2004 Elsevier B.V. All rights reserved.
Pathway-specific expression of PKC and PKA in sympathetic neurons
NeuroReport, 1999
WE used multiple-labelling immuno¯uorescence, intracellular dye injection, electrophysiological recording and confocal microscopy to examine the expression of immunoreactivity to protein kinase C (PKC) and protein kinase A (PKA) in sympathetic ganglia of guinea-pigs. PKCá and PKCã were widespread in vasoconstrictor and pilomotor neurons. High levels of PKA RIIá and RIIâ were restricted to neurons that lacked signi®cant expression of PKC, including somatostatin-containing neurons projecting to the gut, and non-noradrenergic vasodilator neurons. In coeliac ganglia, most neurons with PKC contained neuropeptide Y and displayed phasic patterns of action potential ®ring, often with a long after-hyperpolarization. Tonically ®ring neurons lacked both neuropeptide Y and PKC. These results show remarkably pathway-speci®c expression of protein kinases in functionally identi®ed populations of sympathetic neurons. NeuroReport 10: 979±979 # 1999 Lippincott Williams & Wilkins.
Brain Research, 1995
Alterations in the parameters of action potentials upon changes in protein kinase C (PKC) activity were studied on neurons of the visceral ganglion of Aplysia californica. The amplitude and maximum speed of the up-and downstroke of the action potentials were measured. Intracellulady injected PKC and intra-and extracellularly applied phorbol-12,13-diacetate (PDAc) had similar effects on the Aplysia neurons, the most prominent being an increase of the upstroke speed of the AP in every neuron. The non-PKC-activating 4a-phorbol didecanoate had no effect, and the effects of the PKC blocker H-7 were opposite to those of PDAc. It was concluded that the changes of the AP evoked by PDAc are mediated through PKC activation.
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...
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...
Autonomic Neuroscience, 2006
Ca 2+ -activated K + channels play an important role in the control of neuronal excitability via the generation of the afterhyperpolarization. While both small and large conductance Ca 2+ -activated K + channels underlie afterhyperpolarizations in different neuron types, the role of intermediate conductance Ca 2+ -activated K + channels (IK Ca ) in the generation of afterhyperpolarizations remains unclear. The effects of blockade of IK Ca on guinea pig coeliac and ileal myenteric neurons were studied using single microelectrode current and voltage clamp. In coeliac neurons, TRAM-39, a selective blocker of IK Ca , depressed the amplitude of the prolonged conductance underlying the slow afterhyperpolarization, (gKCa2) by 57%. In contrast, the conductance underlying the prolonged afterhyperpolarization in AH-type myenteric neurons was unaffected by TRAM-39, although it has been suggested that this AHP is mediated by IK Ca . In both types of neurons, TRAM-39 did not alter the resting cell properties or the properties of the action potential. TRAM-39 had no effect on the amplitude of the fast component of the afterhyperpolarization present in sympathetic LAH neurons. The results of this study suggest that in sympathetic LAH neurons, activation of IK Ca underlies at least part of the prolonged afterhyperpolarization while the nature of the channel underlying the AHP in enteric neurons remains unclear. D
Proceedings of the National Academy of Sciences, 1987
Recent studies suggest that phosphorylation may regulate the rate of desensitization of nicotinic acetylcholine (AcCho) receptors (AcChoR) in vertebrate muscle and Torpedo. It is not known if phosphorylation is involved in regulation of the neuronal AcChoR, however. In this study we examine the possibility that protein kinase C might regulate nicotinic AcChoR function in neurons. Several activators of protein kinase C (1-oleyol-2-acetylglycerol, phorbol 12,13diacetate, and phorbol 12,13-dibutyrate) were tested for their ability to modulate AcChoR function in embryonic chicken sympathetic ganglion neurons. Neurons were voltage-clamped at the resting potential, and the response to AcCho was tested before and after treatment with activators of protein kinase C. We find that all of these agents enhance the rate of decay of AcCho-induced current without affecting peak current amplitude or cellular input resistance. The drugs were ineffective if applied concurrently with AcCho: significant effects could be detected after 60 sec of pretreatment. A phorbol that does not increase protein kinase C activity (48-phorbol) was ineffective in enhancing the decay of AcCho-induced current. Thus, the effects of these agents on AcChoR function are likely to be mediated by their interaction with C kinase, rather than by direct interaction with the AcChoR channel. Our data suggest that kinase C may regulate agonist-induced desensitization of the neuronal AcChoR channel.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1996
alpha2-Adrenoceptors regulate the efficacy at the sympathoeffector junction by means of a feedback inhibition of transmitter release. In chick sympathetic neurons, the mechanism involves an inhibition of N-type calcium channels, and we now present evidence that this effect involves an atypical, phorbol ester-insensitive protein kinase C (PKC). The inhibition of voltage-gated Ca2+ currents by the specific alpha2-adrenergic agonist UK 14,304 was significantly attenuated when the PKC inhibitors PKC(19-36), staurosporine, or calphostin C were included in the internal solution used to fill the patch pipettes, or if staurosporine or calphostin C were applied extracellularly; however, phorbol esters as classical activators of PKC or oleoylacetylglycerol did not mimic the effect of UK 14,304, and chronic exposure to 4-beta-phorbol dibutyrate (PDBu) did not attenuate it, ever though PKCalpha and -epsilon isozymes were translocated to plasma membranes by PDBu. The atypical isozyme PKCzeta was...
Journal of neurophysiology, 2000
The types of Ca(2+)-dependent K(+) channel involved in the prolonged afterhyperpolarization (AHP) in a subgroup of sympathetic neurons have been investigated in guinea pig celiac ganglia in vitro. The conductance underlying the prolonged AHP (gKCa2) was reduced to a variable extent in 100 nM apamin, an antagonist of SK-type Ca(2+)-dependent K(+) channels, and by about 55% in 20 nM iberiotoxin, an antagonist of BK-type Ca(2+)-dependent K(+) channels. The reductions in gKCa2 amplitude by apamin and iberiotoxin were not additive, and a resistant component with an amplitude of nearly 50% of control remained. These data imply that, as well as apamin- and iberiotoxin-sensitive channels, other unknown Ca(2+)-dependent K(+) channels participate in gKCa2. The resistant component of gKCa2 was not abolished by 0.5-10 mM tetraethylammonium, 1 mM 4-aminopyridine, or 5 mM glibenclamide. We also investigated which voltage-gated channels admitted Ca(2+) for the generation of gKCa2. Blockade of Ca(2...