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

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Calcium Channel Currents in Acutely Dissociated Intracardiac Neurons From Adult Rats

Journal of Neurophysiology ◽

10.1152/jn.1997.77.4.1769 ◽

1997 ◽

Vol 77 (4) ◽

pp. 1769-1778 ◽

Cited By ~ 36

Author(s):

Seong-Woo Jeong ◽

Robert D. Wurster

Keyword(s):

Calcium Channel ◽

Fold Change ◽

Dependent Manner ◽

High Concentration ◽

Patch Clamp Technique ◽

Adult Rats ◽

Voltage Dependent ◽

Steady State Inactivation ◽

Channel Currents ◽

Intracardiac Neurons

Jeong, Seong-Woo and Robert D. Wurster. Calcium channel currents in acutely dissociated intracardiac neurons from adult rats. J. Neurophysiol. 77: 1769–1778, 1997. With the use of the whole cell patch-clamp technique, multiple subtypes of voltage-activated calcium channels, as indicated by measuring Ba2+ currents, were pharmacologically identified in acutely dissociated intracardiac neurons from adult rats. All tested neurons that were held at −80 mV displayed only high-voltage-activated (HVA) Ca2+ channel currents that were completely blocked by 100 μM CdCl2. The current density of HVA Ca2+ currents was dependent on the external Ca2+ concentration. The Ba2+ (5 mM) currents were half-activated at −16.3 mV with a slope of 5.6 mV per e-fold change. The steady-state inactivation was also voltage dependent with half-inactivation at −33.7 mV and a slope of −12.1 mV per e-fold change. The most effective L-type channel activator, FPL 64176 (2 μM), enhanced the Ba2+ current in a voltage-dependent manner. When cells were held at −80 mV, the saturating concentration (10 μM) of nifedipine blocked ∼11% of the control Ba2+ current. The major component of the Ca2+ channels was N type (63%), which was blocked by a saturating concentration (1 μM) of ω-conotoxin GVIA. Approximately 19% of the control Ba2+ current was sensitive to ω-conotoxin MVIIC (5 μM) but insensitive to low concentrations (30 and 100 nM) of ω-agatoxin IVA (ω-Aga IVA). In addition, a high concentration (1 μM) of ω-Aga IVA occluded the effect of ω-conotoxin MVIIC. Taken together, these results indicate that the ω-conotoxin MVIIC-sensitive current represents only the Q type of Ca2+ channels. The current that was insensitive to nifedipine and various toxins represents the R-type current (7%), which was sensitive to 100 μM NiCl2. In conclusion, the intracardiac neurons from adult rats express at least four different subtypes (L, N, Q, and R) of HVA Ca2+ channels. This information is essential for understanding the regulation of synaptic transmission and excitability of intracardiac neurons by different neurotransmitters and neural regulation of cardiac functions.

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

Journal of Neurophysiology ◽

10.1152/jn.2002.87.6.2867 ◽

2002 ◽

Vol 87 (6) ◽

pp. 2867-2879 ◽

Cited By ~ 112

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.

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