Controlling the excitability of IPANs: a possible route to therapeutics.

Recent studies have shown that intrinsic primary afferent neurons (IPANs) express a much larger range of ionic currents than non-sensory neurons of the enteric nervous system. These ionic currents can be modulated by neurotransmitters that are synaptically released onto the soma (unlike cranial and spinal sensory neurons). The membrane receptors and ionic channels that are involved in the sensory transduction processes of IPANS are beginning to be defined. IPANS can move between a large range of excitability states that are influenced by neurotransmitters and hormones. An additional cause of variability in excitability is the actions of inflammatory mediators. It is becoming apparent that the variation in excitability of IPANS might play a critical role in determining the physiological state of the intestine.

Sensitization of enteric reflexes in the rat colon in vitro.

We have investigated sensitization of reflexes in the isolated rat colon in order to develop a model that might prove useful for investigating how the sensitivity of enteric reflexes can be altered by prior stimulation. Records were taken of circular muscle tension, 7-10 mm oral and anal to radial distension exerted by a hook passed through the wall of the colon. A test stimulus of 1.5 g produced consistent contractions both oral and anal to the distension. A conditioning protocol, consisting of repeated application of 3 g for 30 s with 30 s between the stimuli for 30 min, doubled the amplitudes of reflex contractions that were evoked by the test stimuli but did not change the sensitivity of the muscle to the direct action of carbachol. The enhanced responses persisted for at least 40 min. The enhancement of reflexes was not reduced by antagonists of tachykinin NK3 receptors or of 5-HT3 receptors, but the reflex oral to stimulation was reduced by NK1 and NK3 antagonists added together. Sensitization was abolished by the cyclo-oxygenase and thromboxane synthase inhibitor, indomethacin. We conclude that sensitization can be reliably induced in vitro and that the model described in the present work can be used to investigate drugs that interfere with the sensitization process.

Ionic conductances in sustentacular cells of the mouse olfactory epithelium.

The electrical properties of sustentacular cells (SCs) in the olfactory epithelium (OE) were investigated in tissue slices taken from neonatal mice (P0-P4). Conventional whole-cell recordings were obtained from SCs and also from olfactory receptor neurones (ORNs) in situ. SCs had a larger apparent cell capacitance (C(cell)) (18.6 +/- 0.5 pF) than ORNs (4.4 +/- 0.4 pF) and a lower apparent membrane resistance (R(m)) (160 +/- 11 MOmega versus 664 +/- 195 MOmega, respectively). When corrected for a seal resistance of 1 GOmega, these mean R(m) values were increased to 190 MOmega and 2 GOmega in SCs and ORNs, respectively. SCs generated a TTX (1 microm)-resistant voltage-activated Na(+) current (I(Na)) that had a peak density at -38 mV of -44 pA pF(-1) and supported action potential firing. Peak current density of I(Na) in neurones was 510 +/- 96 pA pF(-1). The outward K(+) current in SCs was composed (> 70%) of a TEA (2 mm)-sensitive component that was mediated by the opening of large-conductance (237 +/- 10 pS; BK) channels. The resting leak conductance (g(L)) of SCs was permeable to monovalent cations and anions and was largely inhibited by substitution of external Na(+) with NMDG and by internal F(-) with gluconate. g(L) deactivated up to 50% at potentials negative of -70 mV and was inhibited by 18beta-glycyrrhetinic acid (20 mum). SCs were identified using fluorescent dyes (Lucifer Yellow and Alexa Fluor 488) in the whole-cell patch pipette-filling solution. Our findings indicate that SCs in the OE of neonates are electrically excitable and are distinguishable from neurones by a having a resting g(L).

Electrical coupling in sustentacular cells of the mouse olfactory epithelium.

Sustentacular cells (SCs) line the apical surface of the olfactory epithelium (OE) and provide trophic, metabolic, and mechanical support for olfactory receptor neurons. Morphological studies have suggested that SCs possess gap junctions, although physiological evidence for gap junctional communication in mammalian SCs is lacking. In the present study we investigated whether coupling exists between SCs situated in tissue slices of OE from neonatal (P0-P4) mice. Using whole cell and cell-attached patch recordings from SCs, we demonstrate that SCs are electrically coupled by junctional resistances on the order of 300 M(omega). Under whole cell recording conditions, Alexa 488 added to the pipette solution failed to reveal dye coupling between SCs. Electrical coupling was deduced from the biexponential decay of capacitive currents recorded from SCs and from the bell-shaped voltage dependency of a P2Y-receptor-activated current, both of which were abolished by 18beta-glycyrrhetinic acid (20-50 microM), a blocker of gap junctions. These data provide strong evidence for functional coupling between SCs, the physiological importance of which is discussed.

Altered excitability of intestinal neurons in primary culture caused by acute oxidative stress.

Neurons were isolated from the intestine of guinea pigs and grown in primary culture for < or =15 days. Using conventional whole cell recording techniques, we demonstrated that the majority of neurons express a prolonged poststimulus afterhyperpolarization (slow AHP). These neurons also had large-amplitude (approximately 100 mV), broad-duration (approximately 2 ms) action potentials and generated a hyperpolarization activated inward current (Ih). Application of H2O2 (0.22-8.8 mM) hyperpolarized these neurons but not those lacking slow AHPs. The H2O2-induced hyperpolarization was followed by irreversible depolarization at higher concentrations (more than approximately 1 mM) of H2O2 while it was maintained after washout of submillimolar H2O2. The ionic mechanisms underlying the hyperpolarization included the suppression of Ih and the activation of an inwardly rectifying outward current, which was blocked by glybenclamide (25-50 microM) and TEA (30 mM). In addition, H2O2 suppressed the slow AHP and its underlying current. Internal perfusion of catalase and glutathione opposed the H2O2-mediated decrease in IsAHP. Our results indicate that acute oxidative stress has neuron- and conductance-specific actions in intestinal neurons that may underlie pathophysiological conditions.

SK channels and the varieties of slow after-hyperpolarizations in neurons.

Action potentials and associated Ca2+ influx can be followed by slow after-hyperpolarizations (sAHPs) caused by a voltage-insensitive, Ca2+-dependent K+ current. Slow AHPs are a widespread phenomenon in mammalian (including human) neurons and are present in both peripheral and central nervous systems. Although, the molecular identity of ion channels responsible for common membrane potential mechanisms has been largely determined, the nature of the channels that underlie the sAHPs in neurons, both in the brain and in the periphery, remains unresolved. This short review discusses why there is no clear molecular candidate for sAHPs.

PKA-mediated inhibition of a novel K+ channel underlies the slow after-hyperpolarization in enteric AH neurons.

Postspike after-hyperpolarizations (AHPs) control the excitability of neurons and are important in shaping firing patterns. The duration of some of these events extends to tens of seconds and they can render neurons inexcitable for much of their time course. While consensus is strong that the medium duration (< 1 s AHPs are mediated by the opening of small conductance Ca2+-activated K+ channels, the K+ channels mediating slow AHPs (> 5 s in a subset of enteric (AH) neurons) have an intermediate unit conductance (IKCa). Using whole-cell and excised-patch recording, we have demonstrated that the cAMP-protein kinase A (PKA) pathway regulates the activity of these channels. In whole-cell mode, forskolin (0.003-1 microM) inhibited the current underlying the slow AHP (IsAHP) by 90 %, and this was partially sensitive to inhibition of PKA with internal Rp-cAMPS (500 microM). Rp-cAMPS alone increased the current following break-in and caused a 20 mV hyperpolarization, suggesting that PKA maintains slow AHP channels in the closed state. Internal perfusion of the inhibitory peptide PKI5-24 slightly increased the IsAHP and opposed the inhibitory action of forskolin. Internal perfusion of the catalytic subunit of PKA (PKAcat) suppressed the IsAHP by 50 % without affecting membrane potential or action potential configuration. In inside-out patches containing IKCa-like channels, PKAcat decreased the open probability of IKCa-like channels while alkaline phosphatase activated them. These results suggest that the IKCa-like channels that underlie the slow AHP in myenteric AH neurons are subject to inhibition by PKA-dependent phosphorylation and that PKA plays an integral role in their gating.

TEA- and apamin-resistant K(Ca) channels in guinea-pig myenteric neurons: slow AHP channels.

The patch-clamp technique was used to record from intact ganglia of the guinea-pig duodenum in order to characterize the K(+) channels that underlie the slow afterhyperpolarization (slow AHP) of myenteric neurons. Cell-attached patch recordings from slow AHP-generating (AH) neurons revealed an increased open probability (P(o)) of TEA-resistant K(+) channels following action potentials. The P(o) increased from < 0.06 before action potentials to 0.33 in the 2 s following action potential firing. The ensemble averaged current had a similar time course to the current underlying the slow AHP. TEA- and apamin-resistant Ca(2+)-activated K(+) (K(Ca)) channels were present in inside-out patches excised from AH neurons. The P(o) of these channels increased from < 0.03 to approximately 0.5 upon increasing cytoplasmic [Ca(2+)] from < 10 nM to either 500 nM or 10 microM. P(o) was insensitive to changes in transpatch potential. The unitary conductance of these TEA- and apamin-resistant K(Ca) channels measured approximately 60 pS under symmetric K(+) concentrations between -60 mV and +60 mV, but decreased outside this range. Under asymmetrical [K(+)], the open channel current showed outward rectification and had a limiting slope conductance of about 40 pS. Activation of the TEA- and apamin-resistant K(Ca) channels by internal Ca(2+) in excised patches was not reversed by washing out the Ca(2+)-containing solution and replacing it with nominally Ca(2+)-free physiological solution. Kinetic analysis of the single channel recordings of the TEA- and apamin-resistant K(Ca) channels was consistent with their having a single open state of about 2 ms (open dwell time distribution was fitted with a single exponential) and at least two closed states (two exponential functions were required to adequately fit the closed dwell time distribution). The Ca(2+) dependence of the activation of TEA- and apamin-resistant K(Ca) channels resides in the long-lived closed state which decreased from > 100 ms in the absence of Ca(2+) to about 7 ms in the presence of submicromolar cytoplasmic Ca(2+). The Ca(2+)-insensitive closed dwell time had a time constant of about 1 ms. We propose that these small-to-intermediate conductance TEA- and apamin-resistant Ca(2+)-activated K(+) channels are the channels that are primarily responsible for the slow AHP in myenteric AH neurons.

Regulation of K+ channels underlying the slow afterhyperpolarization in enteric afterhyperpolarization-generating myenteric neurons: role of calcium and phosphorylation.

1. Myenteric afterhyperpolarization-generating myenteric (AH) neurons serve as intrinsic primary afferent neurons of the enteric nervous system and generate prolonged or slow afterhyperpolarizing potentials (slow AHP). The slow AHP is generated by an increase in a Ca2+-activated K+ conductance (gK-Ca) and is inhibited by enteric neurotransmitters leading to increased excitability. 2. Using cell-attached patch-clamp recordings from AH neurons, we have shown that K+ channels with an intermediate unitary conductance (IK channels) open following action potential firing. 3. In excised patches from AH neurons, we have identified an IK-like channel that can be activated by submicromolar levels of cytoplasmic Ca2+ and is not voltage dependent. 4. Application of the catalytic subunit of cAMP-dependent protein kinase to the cytoplasmic surface of inside-out patches inhibits the opening of IK-like channels previously activated by Ca2+. 5. The IK-like channels are resistant to external tetraethylammonium (5 mmol/L) and apamin (0.3-1 micro mol/L), but are inhibited by clotrimazole (10 micro mol/L). 6. Our present data support the idea that an increase in the open probability of IK-like channels in AH neurons following an increase in cytoplasmic [Ca2+] is responsible for the slow AHP and their opening is modulated by kinases.