Up-regulation of tetrodotoxin-sensitive sodium currents by prostaglandin E2 in type-4 rat dorsal root ganglion cells.

Mechanisms were studied by which prostaglandin E(2) (PGE(2)) up-regulates Na(+) currents (INa) in medium diameter dorsal root ganglion (DRG) cells that express large T-type Ca(2+) currents (type-4 DRG cells). PGE(2) or the adenylyl cyclase (AC) activator forskolin (10 µM) up-regulated peak INa evoked by test potentials (TP) to -10 mV by an average of 13.5% and 21.8%, respectively. The PGE(2) and forskolin induced up-regulation of INa, evoked with TPs to -10 mV, began approximately 15-20 s after initiation of drug exposure and continued gradually over the course of 2-3 min. Both PGE(2) and forskolin significantly increased peak conductance without significantly shifting the voltage at which INa was ½ activated (V(a)) or ½ steady state inactivated. However, although V(a) was not significantly shifted, both PGE(2) and forskolin induced a proportionally greater percent increase in conductance at weak TPs to around -30 mV compared to stronger TPs to around 10 mV. The PGE(2)-induced up-regulation of INa was occluded by prior up-regulation with forskolin, and the up-regulation of INa by both PGE(2) and forskolin was blocked by Rp-cAMPs and 50 nM tetrodotoxin (TTX). In the presence of Rp-cAMPs, both PGE(2) and forskolin induced decreases in INa that peaked around 25 s following initiation of PGE(2)/forskolin application. The decrease induced by PGE(2) averaged 8.5%, which was significantly greater than the average 3.5% decrease induced by forskolin. Estimation of kinetic rate constants by fitting INa with a Markov channel state model, suggested that both PGE(2) and forskolin up-regulated INa by changing channel gating rather than by increasing channel number or unitary conductance. The data suggest that application of PGE(2) may initially induce a relatively rapid down-regulation of TTX-sensitive INa (signaling pathway uncharacterized), followed by a gradual up-regulation of INa via activation of an AC/PKA-dependent signaling pathway. The up-regulation of INa in sensory neurons with type-4 cell bodies may increase excitability and strengthen signaling, and may play some role in the allodynia and hyperalgesia associated with injury to nerves and peripheral tissues.

Analysis of the variation in use-dependent inactivation of high-threshold tetrodotoxin-resistant sodium currents recorded from rat sensory neurons.

This study addressed variation in the use-dependent inactivation (UDI) of high-threshold tetrodotoxin-resistant Na+ currents (TTX-R currents) and action potential firing behavior among acutely isolated rat dorsal root ganglion (DRG) cells. UDI was quantified as the percent decrease in current amplitude caused by increasing the current activation rate from 0.1-1.0 Hz for 20 s. TTX-R current UDI varied from 6% to 66% among 122 DRG cells examined, suggesting the existence of two or more levels of UDI. The voltage-dependency of the TTX-R currents was consistent with Na(V)1.8, regardless of UDI. However, TTX-R currents with more UDI had a more negative voltage-dependency of inactivation, a greater tendency to enter slow inactivation, and a slower recovery rate from slow inactivation, compared with those with less UDI. TTX-R currents with more UDI ran down faster than those with less UDI. However, UDI itself changed little over time, regardless of the initial UDI level observed in a particular DRG cell. Together, these two observations suggest that individual DRG cells did not express mixtures of TTX-R channels that varied regarding UDI. TTX-R current UDI was correlated with expression of a low-threshold A-current and whole-cell capacitance, suggesting that it varied among different nociceptor types. Whole-cell inward currents (WCI-currents), recorded without channel blockers, also exhibited UDI. WCI-current UDI varied similarly to TTX-R current UDI in magnitude, and relative to whole-cell capacitance and A-current expression, suggesting that the WCI-currents were carried predominantly by TTX-R channels. DRG cells with more WCI-current UDI exhibited a greater decrease in action potential amplitude and number, and a greater increase in action potential threshold over seven ramp depolarizations, compared with DRG cells with less WCI-current UDI. Variation in UDI of Na(V)1.8 channels expressed by different nociceptor types could contribute to shaping their individual firing patterns in response to noxious stimuli.

Low-threshold L-type calcium channels in rat dopamine neurons.

Ca(2+) channel subtypes expressed by dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) were studied using whole cell patch-clamp recordings and blockers selective for different channel types (L, N, and P/Q). Nimodipine (Nim, 2 microM), omega-conotoxin GVIA (Ctx, 1 microM), or omega-agatoxin IVA (Atx, 50 nM) blocked 27, 36, and 37% of peak whole cell Ca(2+) channel current, respectively, indicating the presence of L-, N-, and P-type channels. Nim blocked approximately twice as much Ca(2+) channel current near activation threshold compared with Ctx or Atx, suggesting that small depolarizations preferentially opened L-type versus N- or P-type Ca(2+) channels. N- and L-channels in DA neurons opened over a significantly more negative voltage range than those in rat dorsal root ganglion cells, recorded from using identical conditions. These data provide an explanation as to why Ca(2+)-dependent spontaneous oscillatory potentials and rhythmic firing in DA neurons are blocked by L-channel but not N-channel antagonists and suggest that pharmacologically similar Ca(2+) channels may exhibit different thresholds for activation in different types of neurons.

Muscarine reduces calcium-dependent electrical activity in substantia nigra dopaminergic neurons.

The effect of muscarine on Ca2+ dependent electrical activity was studied in dopamine (DA) neurons located in the substantia nigra pars compacta (SNc) in brain slices from young rats, using sharp electrodes. In most DA neurons tested, muscarine (50 microM) reduced the amplitude of spontaneous oscillatory potentials and evoked Ca2+-dependent potentials recorded in the presence of TTX. Muscarine also reduced the amplitude of the slow afterhyperpolarization (sAHP) following action potentials in most DA neurons. These data suggest that muscarine reduces Ca2+ entry in SNc DA neurons. The reduction of the amplitude of the sAHP by muscarine in DA neurons may facilitate bursting initiated by glutamatergic input by increasing the frequency at which DA neurons can fire. The reduction of the sAHP via activation of muscarinic receptors in vivo may provide a mechanism whereby cholinergic inputs to DA neurons from the tegmental peduncular pontine nucleus could modulate dopamine release at dopaminergic targets in the brain.

5HT increases excitability of nociceptor-like rat dorsal root ganglion neurons via cAMP-coupled TTX-resistant Na(+) channels.

The physiological effects of 5HT receptor coupling to TTX-resistant Na(+) current, and the signaling pathway involved, was studied in a nociceptor-like subpopulation of rat dorsal root ganglion (DRG) cells (type 2), which can be identified by expression of a low-threshold, slowly inactivating A-current. The 5HT-mediated increase in TTX-resistant Na(+) current in type 2 DRG cells was mimicked and occluded by 10 microM forskolin. Superfusion of type 2 DRG cells on the outside with 1 mM 8-bromo-cAMP or chlorophenylthio-cAMP (CPT-cAMP) increased the Na(+) current, but less than 5HT itself. However, perfusion of the cells inside with 2 mM CPT-cAMP strongly increased the amplitude of control Na(+) currents and completely occluded the effect of 5HT. Thus it appears that the signaling pathway includes cAMP. The phosphodiesterase inhibitor 3-isobutyl-L-methylxanthine (200 microM) also mimicked the effect of 5HT on Na(+) current, suggesting tonic adenylyl cyclase activity. 5HT reduced the amount of current required to evoke action potentials in type 2 DRG cells, suggesting that 5HT may lower the threshold for activation of nociceptor peripheral receptors. The above data suggest that serotonergic modulation of TTX-resistant Na(+) channels through a cAMP-dependent signaling pathway in nociceptors may participate in the generation of hyperalgesia.

Serotonergic modulation of hyperpolarization-activated current in acutely isolated rat dorsal root ganglion neurons.

1. The effect of serotonin (5-HT) on the hyperpolarization-activated cation current (IH) was studied in small-, medium- and large-diameter acutely isolated rat dorsal root ganglion (DRG) cells, including cells categorized as type 1, 2, 3 and 4 based on membrane properties. 5-HT increased IH in 91 % of medium-diameter DRG cells (including type 4) and in 67 % of large-diameter DRG cells, but not other DRG cell types. 2. The increase of IH by 5-HT was antagonized by spiperone but not cyanopindolol, and was mimicked by 5-carboxyamidotryptamine, but not (+)-8-hydroxydipropylaminotetralin (8-OH-DPAT) or cyanopindolol. These data suggested the involvement of 5-HT7 receptors, which were shown to be expressed by medium-diameter DRG cells using RT-PCR analysis. 3. 5-HT shifted the conductance-voltage relationship of IH by +6 mV without changing peak conductance. The effects of 5-HT on IH were mimicked and occluded by forskolin, but not by inactive 1,9-dideoxy forskolin. 4. At holding potentials negative to -50 mV, 5-HT increased steady-state inward current and instantaneous membrane conductance (fast current). The 5-HT-induced inward current and fast current were blocked by Cs+ but not Ba2+ and reversed at -23 mV, consistent with the properties of tonically activated IH. 5. In medium-diameter neurons recorded from in the current clamp mode, 5-HT depolarized the resting membrane potential, decreased input resistance and facilitated action potential generation by anode-break excitation. 6. The above data suggest that in distinct subpopulations of DRG neurons, 5-HT increases cAMP levels via activation of 5-HT7 receptors, which shifts the voltage dependence of IH to more depolarized potentials and increases neuronal excitability.

5HT4 receptors couple positively to tetrodotoxin-insensitive sodium channels in a subpopulation of capsaicin-sensitive rat sensory neurons.

The distribution of tetrodotoxin (TTX)-sensitive and -insensitive Na+ currents and their modulation by serotonin (5HT) and prostaglandin E2 (PGE2) was studied in four different types of dorsal root ganglion (DRG) cell bodies (types 1, 2, 3, and 4), which were previously identified on the basis of differences in membrane properties (). Types 1 and 2 DRG cells expressed TTX-insensitive Na+ currents, whereas types 3 and 4 DRG cells exclusively expressed TTX-sensitive Na+ currents. Application of 5HT (1-10 microM) increased TTX-insensitive Na+ currents in type 2 DRG cells but did not affect Na+ currents in type 1, 3, or 4 DRG cells. The 5HT receptor involved resembled the 5HT4 subtype. It was activated by 5-methoxy-N,N-dimethyltryptamine (10 microM) but not by 5-carboxyamidotryptamine (1 microM), (+)-8-hydroxydipropylaminotetralin (10 microM), or 2-methyl-5HT (10 microM), and was blocked by ICS 205-930 with an EC50 of approximately 2 microM but not by ketanserin (1 microM). PGE2 (4 or 10 microM) also increased Na+ currents in varying portions of cells in all four groups. The effect of 5HT and PGE2 on Na+ currents was delayed for 20-30 sec after exposure to 5HT, suggesting the involvement of a cytosolic diffusible component in the signaling pathway. The agonist-mediated increase in Na+ current, however, was not mimicked by 8-chlorophenylthio-cAMP (200 microM), suggesting the possibility that cAMP was not involved. The data suggest that the 5HT- and PGE2-mediated increase in Na+ current may be involved in hyperesthesia in different but overlapping subpopulations of nociceptors.

Two parallel signaling pathways couple 5HT1A receptors to N- and L-type calcium channels in C-like rat dorsal root ganglion cells.

The coupling of serotonin receptors to Ca2+ channels was studied in a subpopulation of acutely isolated rat dorsal root ganglion (DRG) cell bodies (type 1 DRG cells), which have membrane properties similar to C-type nociceptive sensory neurons. In these cells, serotonin (5HT) inhibited high-threshold Ca2+ channel current and decreased action potential duration. The inhibitory effects of 5HT and the 5HT1A agonist 8-OH-DPAT were shown to be antagonized by the 5HT1A antagonists spiperone and pindolol, respectively, indicating involvement of a 5HT1A receptor. Several observations suggest that 5HT1A receptors couple to N- and L-type Ca2+ channels by two different signaling pathways in type 1 DRG cells. The inhibition of Ca2+ channel currents produced by 10 microM 5HT occurred in two phases, an initial slowing of current activation rate (kinetic slowing), which was complete within 10 s, and a simultaneous reduction in steady state current amplitude (steady state inhibition), which peaked in approximately 1 min. The kinetic slowing, but not steady state inhibition, was reversed by a positive prepulse to +70 mV (prepulse). Blockade of N-type Ca2+ channels selectively reduced the kinetic slowing and its reversal by prepulses. Chelation of intracellular Ca2+ or blockade of L-type Ca2+ channels selectively reduced the steady state inhibition. Recordings using the cell-attached patch configuration suggest that steady state inhibition required a component that was diffusible in the cytosol, while kinetic slowing occurred via a membrane delimited pathway. The application of 5HT to the cell body outside the patch pipette reduced macroscopic Ca2+ channel currents in 33% of small-diameter DRG cells tested, indicating the participation of a cytosolic diffusible component. Application of 5HT (a membrane impermeant compound) outside the patch pipette produced steady state inhibition only, whereas similar application of membrane permeant 5HT1A agonists, 8-OH-DPAT or 5-methoxy-N,N-dimethyl-tryptamine, produced kinetic slowing and steady state inhibition. Together these data suggest that 5HT1A receptors couple negatively to Ca2+ channels via two pathways: a membrane-delimited pathway that couples to N-channels and actuates voltage-sensitive kinetic slowing and a pathway dependent on a cytosolic diffusible component and free intracellular Ca2+, which couples to L channels and actuates steady state inhibition.

Capsaicin preferentially affects small-diameter acutely isolated rat dorsal root ganglion cell bodies.

The effects of capsaicin were tested on 221 acutely isolated dorsal root ganglion neurons of the rat, which ranged in diameter from 15 to 55 microns. In a sub-population of these cells, ranging in diameter from 17.5 to 33 microns (n = 117), capsaicin (1 microM) produced an inward shift in holding current that was associated with an increase in membrane conductance in most cells (114 of 117). These effects of capsaicin were reversible upon washout of the drug. Other cells ranging in diameter from 15 to 52.5 microns (n = 104) were unaffected in this manner by the 1 micron concentration of capsaicin. Capsaicin-sensitive cells had, on average, significantly longer duration action potentials and expressed significantly less IH than capsaicin-insensitive cells. The relatively long duration action potentials and/or small cell body diameter and paucity of IH observed in most of the capsaicin-sensitive cells is consistent with their representing C- or A delta-type sensory neurons.

Variation in serotonergic inhibition of calcium channel currents in four types of rat sensory neurons differentiated by membrane properties.

1. Rat dorsal root ganglion (DRG) cell bodies were screened according to action potential (AP) duration, capsaicin sensitivity, expression of IH, IA, and N-, L-, and T-type Ca2+ channel currents. AP duration was measured at half of total amplitude at a membrane potential of -60 mV. Sensitivity to capsaicin was defined as production of an inward current at a holding potential (HP) of -60 mV by 1 microM capsaicin. IH was evoked by a 787-ms hyperpolarization to -110 mV from an HP of -60 mV. IA was evoked by repolarization to -60 mV after a 787-ms hyperpolarization to -110 mV. High-threshold Ca2+ channel current was evoked by a depolarization to -10 or 0 mV from an HP of -60 mV, and L- and N-type Ca2+ channel current was fractionated using selective Ca2+ channel blockers (nimodipine and omega-conotoxin GVIA). T-type Ca2+ channel current was evoked by a depolarization to -40 mV from an HP of -90 mV. Ninety-seven of the 116 DRG cells studied fit closely into one of four categories based on expression of the above characteristics. These four categories, referred to as types 1-4, are described below. 2. Type 1 DRG cells (soma diameter 24.6 +/- 0.5 microns, mean +/- SE; n = 34) had long-duration APs (average = 9.8 ms) with a prominent shoulder on the falling limb and were capsaicin sensitive. Significant IH or IA was not expressed. High-threshold Ca2+ channel current was on average 28% omega-conotoxin GVIA sensitive (N-type) and 46% nimodipine sensitive (L-type); 26% was resistant to both blockers (resistant). T-type Ca2+ channel currents averaged 245 pA. 3. Type 2 DRG cells (soma diameter 25.2 +/- 0.9 microns, n = 19) had short-duration APs (average = 2.9 ms) with a small shoulder on the falling limb and were capsaicin sensitive. IH was negligible but IA averaged 184 pA. High-threshold Ca2+ channel current averaged 42% N-type, 23% L-type, and 35% resistant. T-type Ca2+ channel currents averaged 47 pA. 4. Type 3 DRG cells (soma diameter 18.6 +/- 0.8 microns, n = 21) had short-duration APs (average = 1.8 ms) and were insensitive to capsaicin. IA was not expressed but IH averaged 147 pA. High-threshold Ca2+ channel current averaged 27% N-type, 44% L-type, and 29% resistant. T-type Ca2+ channel currents averaged 306 pA. 5. Type 4 DRG cells (soma diameter 33.9 +/- 0.4 microns, n = 23) had short-duration APs (average = 1.1 ms) and were capsaicin insensitive. IA was not expressed but IH averaged 810 pA. High-threshold Ca2+ channel current was 16% N-type, 4% L-type, and 80% resistant. T-type Ca2+ channel currents averaged 4,031 pA. 6. There was a large variation in the inhibition of high-threshold Ca2+ channel currents by serotonin (5-HT) and (+)8-OH-DPAT in type 1 DRG cells versus types 2-4. On average, 5-HT (10 microM) inhibited high-threshold Ca2+ channel current by an average of 42% in type 1 DRG cells, compared with 15%, 18%, and 7% inhibition in types 2-4, respectively. Similarly, (+)8-OH-DPAT (1 microM) inhibited high-threshold Ca2+ channel current by an average of 35% in type 1 DRG cells, compared with 5%, 8%, and 3% inhibition in types 2-4, respectively. 7. It is possible that DRG cells that vary in their expression of membrane properties may represent sensory neurons that transmit different types of sensory information. Thus the variation in inhibition of Ca2+ channel current by 5-HT and (+)8-OH-DPAT in the above categories of DRG cells may indicate that 5-HT1A receptor activation inhibits Ca2+ entry into some types of DRG sensory neurons more than others.

Serotonin inhibits high-threshold Ca2+ channel currents in capsaicin-sensitive acutely isolated adult rat DRG neurons.

1. The effect of serotonin (5HT) was studied on high-threshold Ca2+ channel currents in a subpopulation of acutely isolated rat dorsal root ganglion cell bodies that had long-duration action potentials, lacked IH current, were capsaicin-sensitive, and thus resembled C-type nociceptors. 2. In these neurons, 10 microM 5HT inhibited peak high-threshold Ca2+ channel currents by 61.5 +/- 6.9% (mean +/- SE), (n = 7). The effects of 5HT were mimicked by 1 microM (+)8-hydroxy-2-(di-n-propylamino)-tetralin HBr [(+)8-OH-DPAT] in five neurons tested, and the effects of 1 microM (+)8-OH-DPAT were antagonized by 100 nM 1-(2-methoxyphenyl)-4-[4-(2-phthalimmido)butyl]piperazine HBr (NAN-190) in six neurons tested. 3. The above data leads us to hypothesize that 5HT, released into the spinal cord by descending systems, may produce antinociception by inhibiting Ca2+ entry into afferent terminals of nociceptors via activation of 5HT1A receptors.

Determination of cardiac output and ejection fraction with the dual cardiac probe.

Cardiac output and left ventricular ejection fraction were determined noninvasively at the bedside in 26 patients by using a dual scintillation probe. The probe is a nonimaging detector that records a high frequency time-activity curve of the passage of an intravenously injected radioactive bolus through the heart. Results were correlated with ejection fraction measured by biplane cineangiography (r = 0.80) and cardiac output determined by green dye dilution (R = 0.86). It is concluded that the dual probe provides an accurate noninvasive means of measuring these parameters, and that it may be particularly applicable to serial measurements in patients in the intensive care unit.