Sigma Receptors Inhibit High-Voltage–Activated Calcium Channels in Rat Sympathetic and Parasympathetic Neurons

2002 ◽  
Vol 87 (6) ◽  
pp. 2867-2879 ◽  
Author(s):  
Hongling Zhang ◽  
Javier Cuevas
Keyword(s):  
Calcium Channels ◽  
Calcium Channel ◽  
Receptor Activation ◽  
Neonatal Rat ◽  
Sigma Receptor ◽  
Sigma Receptors ◽  
Receptor Agonists ◽  
Parasympathetic Neurons ◽  
Ganglion Neurons ◽  
Channel Currents

Studies on the expression and cellular function of sigma receptors in autonomic neurons were conducted in neonatal rat intracardiac and superior cervical (SCG) ganglia. Individual neurons from SCG and intracardiac ganglia were shown to express transcripts encoding the sigma-1 receptor using single-cell RT-PCR techniques. The relationship between sigma receptors and calcium channels was studied in isolated neurons of these ganglia under voltage-clamp mode using the perforated-patch configuration of the whole cell patch-clamp recording technique. Bath application of sigma receptor agonists was shown to rapidly depress peak calcium channel currents in a reversible manner in both SCG and intracardiac ganglion neurons. The inhibition of barium ( I Ba) currents was dose-dependent, and half-maximal inhibitory concentration (IC50) values for haloperidol, ibogaine, (+)-pentazocine, and 1,3-Di- O-tolylguanidin (DTG) were 6, 31, 61, and 133 μM, respectively. The rank order potency of haloperidol > ibogaine > (+)-pentazocine > DTG is consistent with the effects on calcium channels being mediated by a sigma-2 receptor. Preincubation of neurons with the irreversible sigma receptor antagonist, metaphit, blocked DTG-mediated inhibition of Ca2+ channel currents. Maximum inhibition of calcium channel currents was ≥95%, suggesting that sigma receptors block all calcium channel subtypes found on the cell body of these neurons, which includes N-, L-, P/Q-, and R-type calcium channels. In addition to depressing peak Ca2+ channel current, sigma receptors altered the biophysical properties of these channels. Following sigma receptor activation, Ca2+ channel inactivation rate was accelerated, and the voltage dependence of both steady-state inactivation and activation shifted toward more negative potentials. Experiments on the signal transduction cascade coupling sigma receptors and Ca2+ channels demonstrated that neither cell dialysis nor intracellular application of 100 μM guanosine 5′-O-(2-thiodiphosphate) trilithium salt (GDP-β-S) abolished the modulation of I Ba by sigma receptor agonists. These data suggest that neither a diffusible cytosolic second messenger nor a G protein is involved in this pathway. Activation of sigma receptors on sympathetic and parasympathetic neurons is likely to modulate cell-to-cell signaling in autonomic ganglia and thus the regulation of cardiac function by the peripheral nervous system.

Opioid Receptor-Mediated Inhibition of ω-Conotoxin GVIA-Sensitive Calcium Channel Currents in Rat Intracardiac Neurons

1998 ◽  
Vol 79 (2) ◽  
pp. 753-762 ◽  
Author(s):  
David J. Adams ◽  
Carlo Trequattrini
Keyword(s):  
Calcium Channel ◽  
Opioid Receptor ◽  
Voltage Dependence ◽  
Neonatal Rat ◽  
Patch Clamp Technique ◽  
Receptor Agonists ◽  
Channel Current ◽  
Opioid Receptor Agonists ◽  
Channel Currents ◽  
Intracardiac Neurons

Adams, David J. and Carlo Trequattrini. Opioid receptor-mediated inhibition of ω-conotoxin GVIA-sensitive calcium channel currents in rat intracardiac neurons. J. Neurophysiol. 79: 753–762, 1998. Modulation of depolarization-activated ionic conductances by opioid receptor agonists was investigated in isolated parasympathetic neurons from neonatal rat intracardiac ganglia by using the whole cell perforated patch clamp technique. Met-enkephalin (10 μM) altered the action potential waveform, reducing the maximum amplitude and slowing the rate of rise and repolarization but the afterhyperpolarization was not appreciably altered. Under voltage clamp, 10 μM Met-enkephalin selectively and reversibly inhibited the peak amplitude of high-voltage–activated Ca2+ channel currents elicited at 0 mV by ∼52% and increased three- to fourfold the time to peak. Met-enkephalin had no effect on the voltage dependence of steady-state inactivation but shifted the voltage dependence of activation to more positive membrane potentials whereby stronger depolarization was required to open Ca2+ channels. Half-maximal inhibition of Ba2+ current ( I Ba) amplitude was obtained with 270 nM Met-enkephalin or Leu-enkephalin. The opioid receptor subtype selective agonists, DAMGO and DADLE, but not DPDPE, inhibited I Ba and were antagonized by the opioid receptor antagonists, naloxone and naltrindole with IC50s of 84 nM and 1 μM, respectively. The κ-opioid receptor agonists, bremazocine and dynorphin A, did not affect Ca2+ channel current amplitude or kinetics. Taken together, these data suggest that enkephalin-induced inhibition of Ca2+ channels in rat intracardiac neurons is mediated primarily by the μ-opioid receptor type. Addition of Met-enkephalin after exposure to 300 nM ω-conotoxin GVIA, which blocked ∼75% of the total Ca2+ channel current, failed to cause a further decrease of the residual current. Met-enkephalin inhibited the ω-conotoxin GVIA-sensitive but not the ω-conotoxin-insensitive I Ba in rat intracardiac neurons. Dialysis of the cell with a GTP-free intracellular solution or preincubation of the neurons in Pertussis toxin (PTX) abolished the attenuation of I Ba by Met-enkephalin, suggesting the involvement of a PTX-sensitive Gprotein in the signal transduction pathway. The activation of μ-opioid receptors and subsequent inhibition of N-type Ca2+ channels in the soma and terminals of postganglionic intracardiac neurons is likely to inhibit the release of ACh and thereby regulate vagal transmission to the mammalian heart.

Effects of Serotonin on Caudal Raphe Neurons: Inhibition of N- and P/Q-Type Calcium Channels and the Afterhyperpolarization

1997 ◽  
Vol 77 (3) ◽  
pp. 1362-1374 ◽  
Author(s):  
Douglas A. Bayliss ◽  
Yu-Wen Li ◽  
Edmund M. Talley
Keyword(s):  
Calcium Channels ◽  
Calcium Channel ◽  
Calcium Current ◽  
Tryptophan Hydroxylase ◽  
Residual Current ◽  
Neonatal Rat ◽  
Repetitive Firing ◽  
Firing Behavior ◽  
Channel Currents ◽  
Raphe Neurons

Bayliss, Douglas A., Yu-Wen Li, and Edmund M. Talley. Effects of serotonin on caudal raphe neurons: inhibition of N- and P/Q-type calcium channels and the afterhyperpolarization. J. Neurophysiol. 77: 1362–1374, 1997. We characterized whole cell barium currents through calcium channels and investigated the effects of serotonin (5-HT) on calcium channel currents and firing behavior in visualized caudal raphe neurons of the neonatal rat in brain stem slices ( n = 201). A subpopulation of recorded neurons was recovered after staining for tryptophan hydroxylase (TPH), the 5-HT synthesizing enzyme ( n = 21); of those cells, 86% were TPH immunoreactive, suggesting that the majority of recorded neurons was serotonergic. Calcium channel currents began to activate at about −40 mV in caudal raphe neurons and showed a peak amplitude of 952.2 ± 144.2 (SE) pA at −10 mV. A small low-voltage-activated current was also observed (∼22 pA). Calcium channel currents were potently inhibited by bath-applied 5-HT in most cells tested (∼90%). The EC50 for inhibition of calcium current by 5-HT was 0.1 μM; a saturating concentration (1.0 μM) blocked ∼49% of the current evoked at 0 mV from a holding potential of −70 mV ( n = 101). Current inhibition was associated with a slowing of activation kinetics and a shift in the peak of the current-voltage relationship, and was partially relieved by strong depolarizations. Current inhibition by 5-HT was mimicked by 8-OH-DPAT, a specific 5-HT1A agonist, and blocked by the 5-HT1A antagonists NAN 190 and (+)WAY 100135, but was unaffected by ketanserin, a 5-HT2A/C antagonist. ω-Conotoxin GVIA (ω-CgTx)-sensitive N-type channels and ω-agatoxin IVA (ω-AgaIVA)-sensitive P/Q-type channels together accounted for most of the calcium current (36 and 37%, respectively). Nimodipine had no effect on calcium current, indicating that caudal raphe neurons do not express dihydropyridine-sensitive L-type currents. A substantial residual current (27%) remained after application of ω-CgTx, ω-AgaIVA, and nimodipine. Most of the 5-HT-sensitive calcium current was blocked by ω-CgTx and ω-AgaIVA; 5-HT had little effect on the residual current. Inhibition of calcium current by 5-HT was irreversible when GTPγS, a nonhydrolyzable guanosine 5′-triphosphate (GTP) analogue, was substituted for GTP in the pipette. In addition, the effects of 5-HT were blocked by pretreating slices with pertussis toxin (PTX). Together these data indicate that inhibition of N- and P/Q-type calcium current in serotonergic caudal raphe neurons is mediated by a 5-HT1A receptor via PTX-sensitive G proteins. Under current clamp, calcium channel toxins (ω-CgTx and ω-AgaIVA) and 5-HT each caused a decrease in the spike afterhyperpolarization and enhanced the repetitive firing response to injected current. The similar effects of 5-HT and the calcium channel toxins on firing behavior suggest that those effects of 5-HT were secondary to inhibition of N- and P/Q-type calcium channels.

scholarly journals M4 Muscarinic Receptor Activation Modulates Calcium Channel Currents in Rat Intracardiac Neurons

1997 ◽  
Vol 78 (4) ◽  
pp. 1903-1912 ◽  
Author(s):  
J. Cuevas ◽  
D. J. Adams
Keyword(s):  
Calcium Channel ◽  
Muscarinic Receptor ◽  
High Voltage ◽  
Receptor Activation ◽  
Muscarinic Agonist ◽  
Muscarinic Agonists ◽  
Maximal Inhibition ◽  
Cell Recording ◽  
Channel Currents ◽  
Intracardiac Neurons

Cuevas, J. and Adams, D. J. M4 muscarinic receptor activation modulates calcium channel currents in rat intracardiac neurons. J. Neurophysiol. 78: 1903–1912, 1997. Modulation of high-voltage–activated Ca2+ channels by muscarinic receptor agonists was investigated in isolated parasympathetic neurons of neonatal rat intracardiac ganglia using the amphotericin B perforated-patch whole cell recording configuration of the patch-clamp technique. Focal application of the muscarinic agonists acetylcholine (ACh), muscarine, and oxotremorine-M to the voltage-clamped soma membrane reversibly depressed peak Ca2+ channel current amplitude. The dose-reponse relationship obtained for ACh-induced inhibition of Ba2+ current ( I Ba) exhibited a half-maximal inhibition at 6 nM. Maximal inhibition of I Ba amplitude obtained with 100 μM ACh was ∼75% compared with control at +10 mV. Muscarinic agonist-induced attenuation of Ca2+ channel currents was inhibited by the muscarinic receptor antagonists pirenzepine (≤300 nM) and m4-toxin (≤100 nM), but not by AF-DX 116 (300 nM) or m1-toxin (60 nM). The dose-response relationship obtained for antagonism of muscarine-induced inhibition of I Ba by m4-toxin gave an IC50 of 11 nM. These results suggest that muscarinic agonist-induced inhibition of high-voltage–activated Ca2+ channels in rat intracardiac neurons is mediated by the M4 muscarinic receptor. M4 receptor activation shifted the voltage dependence and depressed maximal activation of Ca2+ channels but had no effect on the steady-state inactivation of Ca2+ channels. Peak Ca2+ channel tail current amplitude was reduced ≥30% at +90 mV in the presence of ACh, indicating a voltage-independent component to the muscarinicreceptor-mediated inhibition. Both dihydropyridine- and ω-conotoxin GVIA–sensitive and -insensitive Ca2+ channels were inhibited by ACh, suggesting that the M4 muscarinic receptor is coupled to multiple Ca2+ channel subtypes in these neurons. Inhibition of I Ba amplitude by muscarinic agonists was also observed after cell dialysis using the conventional whole cell recording configuration. However, internal perfusion of the cell with 100 μM guanosine 5′-O-(2-thiodiphosphate) trilithium salt (GDP-β-S) or incubation of the neurons in Pertussis toxin (PTX) abolished the modulation of I Ba by muscarinic receptor agonists, suggesting the involvement of a PTX-sensitive G-protein in the signal transduction pathway. Given that ACh is the principal neurotransmitter mediating vagal innervation of the heart, the presence of this inhibitory mechanism in postganglionic intracardiac neurons suggests that it may serve for negative feedback regulation.

P2 receptor-mediated afferent arteriolar vasoconstriction during calcium blockade

2002 ◽  
Vol 282 (2) ◽  
pp. F245-F255 ◽  
Author(s):  
Edward W. Inscho ◽  
Anthony K. Cook
Keyword(s):  
Calcium Channels ◽  
Calcium Channel ◽  
Calcium Release ◽  
Calcium Influx ◽  
Receptor Activation ◽  
P2 Receptor ◽  
Channel Blockade ◽  
Calcium Channel Blockade ◽  
Vasoconstrictor Response ◽  
Arteriolar Diameter

Experiments were performed to determine the role of L-type calcium channels on the afferent arteriolar vasoconstrictor response to ATP and UTP. With the use of the blood-perfused juxtamedullary nephron technique, kidneys were perfused at 110 mmHg and the responses of arterioles to α,β-methylene ATP, ATP, and UTP were determined before and during calcium channel blockade with diltiazem. α,β-Methylene ATP (1.0 μM) decreased arteriolar diameter by 8 ± 1% under control conditions. This response was abolished during calcium channel blockade. In contrast, 10 μM UTP reduced afferent arteriolar diameter to a similar degree before (20 ± 4%) and during (14 ± 4%) diltiazem treatment. Additionally, diltiazem completely prevented the vasoconstriction normally observed with ATP concentrations below 10 μM and attenuated the response obtained with 10 μM ATP. These data demonstrate that L-type calcium channels play a significant role in the vasoconstrictor influences of α,β-methylene ATP and ATP but not UTP. The data also suggest that other calcium influx pathways may participate in the vasoconstrictor response evoked by P2 receptor activation. These observations support previous findings that UTP-mediated elevation of intracellular calcium concentration in preglomerular vascular smooth muscle cells relies primarily on calcium release from intracellular pools, whereas ATP-mediated responses involve both voltage-dependent calcium influx, through L-type calcium channels, and the release of calcium from intracellular stores. These results support the argument that P2X and P2Y receptors influence the diameter of afferent arterioles through activation of disparate signal transduction mechanisms.

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