Tony D. Gover

Inhibition of mechanical activation of guinea-pig airway afferent neurons by amiloride analogues

The aim of this study was to investigate a role for Epithelial Sodium Channels (ENaCs) in the mechanical activation of low‐threshold vagal afferent nerve terminals in the guinea‐pig trachea/bronchus. Using extracellular single‐unit recording techniques, we found that the ENaC blocker amiloride, and its analogues dimethylamiloride and benzamil caused a reduction in the mechanical activation of guinea‐pig airway afferent fibres. Amiloride and it analogues also reduced the sensitivity of afferent fibres to electrical stimulation such that greater stimulation voltages were required to induce action potentials from their peripheral terminals within the trachea/bronchus. The relative potencies of these compounds for inhibiting electrical excitability of afferent nerves were similar to that observed for inhibition of mechanical stimulation (dimethylamiloride∼#38;benzamil>amiloride). This rank order of potency is incompatible with the known rank order of potency for blockade of ENaCs (benzamil>amiloride>>dimethylamiloride). As voltage‐gated sodium channels play an important role in determining the electrical excitability of neurons, we used whole‐cell patch recordings of nodose neuron cell bodies to investigate the possibility that amiloride analogues caused blockade of these channels. At the concentration required to inhibit mechanical activation of vagal nodose afferent fibres (100 μM), benzamil caused significant inhibition of voltage‐gated sodium currents in neuronal cell bodies acutely isolated from guinea‐pig nodose ganglia. Combined, our findings suggest that amiloride and its analogues did not selectively block mechanotransduction in airway afferent neurons, but rather they reduced neuronal excitability, possibly by inhibiting voltage‐gated sodium currents.

Calcium and calcium-activated currents in vagotomized rat primary vagal afferent neurons.

Adult inferior vagal ganglion neurons (nodose ganglion neurons, NGNs) were acutely isolated 4–6 days after section of their peripheral axons (vagotomy) and examined with the whole‐cell patch‐clamp technique. A subset (∼25 %) of vagotomized NGNs displayed depolarizing after‐potentials (DAPs), not present in control NGNs. DAPs were inhibited by niflumic acid (125 μm) or cadmium (100 μm), and had a reversal potential near ECl, indicating that they were due to Ca2+‐activated chloride current (ICl(Ca)). N‐type, L‐type, T‐/R‐ and other types of voltage‐dependent Ca2+ channels provided about 43, 2, 16 and 40 % of the trigger Ca2+ for DAP generation, respectively. Intracellular Ca2+ concentration ([Ca2+]i) was estimated using fura‐2 fluorescence. Resting [Ca2+]i and peak [Ca2+]i elevation induced by activating Ca2+‐induced Ca2+ release (CICR) stores with 10 mm caffeine were not significantly different among control NGNs, vagotomized NGNs with DAPs and vagotomized NGNs without DAPs, averaging 54 ± 7.9 (n= 19; P= 0.49) and 2022 ± 1059 nm (n= 19; P= 0.44), respectively. Blocking CICR with 10 μm ryanodine reduced DAP amplitude by ∼37 %. Ca2+ influx induced by action potential waveforms was increased by over 250 % in vagotomized NGNs with DAPs (19.0 ± 2.1 pC) compared to control NGNs (5.0 ± 0.8 pC) or vagotomized NGNs without DAPs (7.0 ± 0.8 pC). L‐type, N‐type, T‐/R‐type and other types of Ca2+ influx were increased proportionately in vagotomized NGNs with DAPs. In conclusion, a subset of vagotomized NGNs have increased Ca2+ currents and express ICl(Ca). These NGNs respond electrically to increases in [Ca2+]i during regeneration.

Role of Calcium in Regulating Primary Sensory Neuronal Excitability

The fundamental role of calcium ions (Ca(2+)) in an excitable tissue, the frog heart, was first demonstrated in a series of classical reports by Sydney Ringer in the latter part of the nineteenth century (1882a, b; 1893a, b). Even so, nearly a century elapsed before it was proven that Ca(2+) regulated the excitability of primary sensory neurons. In this chapter we review the sites and mechanisms whereby internal and external Ca(2+) can directly or indirectly alter the excitability of primary sensory neurons: excitability changes being manifested typically by variations in shape of the action potential or the pattern of its discharge.

Calcium regulation in individual peripheral sensory nerve terminals of the rat

Ca2+ is vital for release of neurotransmitters and trophic factors from peripheral sensory nerve terminals (PSNTs), yet Ca2+ regulation in PSNTs remains unexplored. To elucidate the Ca2+ regulatory mechanisms in PSNTs, we determined the effects of a panel of pharmacological agents on electrically evoked Ca2+ transients in rat corneal nerve terminals (CNTs) in vitro that had been loaded with the fluorescent Ca2+ indicator, Oregon Green 488 BAPTA‐1 dextran or fura‐2 dextran in vivo. Inhibition of the sarco(endo)plasmic reticulum Ca2+‐ATPase, disruption of mitochondrial Ca2+ uptake, or inhibition of the Na+–Ca2+ exchanger did not measurably alter the amplitude or decay kinetics of the electrically evoked Ca2+ transients in CNTs. By contrast, inhibition of the plasma membrane Ca2+‐ATPase (PMCA) by increasing the pH slowed the decay of the Ca2+ transient by 2‐fold. Surprisingly, the energy for ion transport across the plasma membrane of CNTs is predominantly from glycolysis rather than mitochondrial respiration, as evidenced by the observation that Ca2+ transients were suppressed by iodoacetate but unaffected by mitochondrial inhibitors. These observations indicate that, following electrical activity, the PMCA is the predominant mechanism of Ca2+ clearance from the cytosol of CNTs and glycolysis is the predominant source of energy.

Electrophysiological properties and chemosensitivity of acutely dissociated trigeminal somata innervating the cornea.

Adult rat sensory trigeminal ganglion neurons innervating the cornea (cTGNs) were isolated and identified following retrograde dye labeling with FM1-43. Using standard whole-cell patch clamp recording techniques, cTGNs could be subdivided by their action potential (AP) duration. Fast cTGNs had AP durations <1 ms (40%) while slow cTGNs had AP durations >1 ms and an inflection on the repolarization phase of the AP. With the exception of membrane input resistance, the passive membrane properties of fast cTGNs were different from those of slow cTGNs (capacitance: 61+/-4.5 pF vs. 42+/-2.6 pF, resting membrane potential: -59+/-0.7 mV vs. -53+/-0.9 mV, for fast and slow cTGNs respectively). Active membrane properties also differed between fast and slow cTGNs. Slow cTGNs had a higher AP threshold (-25+/-1.6 mV vs. -38+/-0.8 mV), a larger rheobase (14+/-1.9 pA/pF vs. 6.8+/-1.0 pA/pF), and a smaller AP undershoot (-56+/-1.7 mV vs. -67+/-2.5 mV). The AP overshoot, however was similar between the two types of neurons (46+/-3.1 mV vs. 48+/-4 mV). Slow cTGNs were depolarized by capsaicin (1 microM, 80%) and 60% of their APs were blocked by tetrodotoxin (TTX) (100 nM). Fast cTGNs were unaffected by capsaicin and 100% of their APs were blocked by TTX. Similarly, cTGNs were also heterogeneous with respect to their responses to exogenous ATP and 5-HT. The current work shows that cTGNs have distinctive electrophysiological properties and chemosensitivity profiles. These characteristics may mirror the distinct properties of corneal sensory nerve terminals. The availability of isolated identified cTGNs constitutes a tractable model system to investigate the biophysical and pharmacological properties of corneal sensory nerve terminals.