Met-enkephalin-induced mobilization of intracellular Ca2+ in rat intracardiac ganglion neurones

Abstract

The effects of Met-enkephalin on Ca 2+-dependent K + channel activity were investigated using the cell-attached patch recording technique on isolated parasympathetic neurones of rat intracardiac ganglia. Large-conductance, Ca 2+ -dependent K + channels (BK Ca ) were examined as an assay of agonist-induced changes in the intracellular free calcium ion concentration ([Ca 2+ ] i ). These BK Ca channels had a conductance of ˜200 pS and were charybdotoxin-and voltage-sensitive. Caffeine (5 mM), used as a control, evoked a large increase in BK Ca channel activity, which was inhibited by 10 μM ryanodine. Met-enkephalin (10 μM) evoked a similar increase in BK Ca channel activity, which was dependent on the presence of extracellular Ca 2+ and inhibited by either ryanodine (10 μM) or naloxone (1 μM). In Fura-2-loaded intracardiac neurones, Met-enkephalin evoked a transient increase in [Ca 2+ ] i . Metenkephalin-induced mobilization of intracellular Ca 2+ may play a role in neuronal excitability and firing behaviour in mammalian intracardiac ganglia.Keywords: Parasympathetic ganglia; Intracellular Ca 2+ ; Caffeine; Ryanodine; Opioid receptor; CaIndex Terms: heart innervation Endogenous opioid peptides have been found in the extrinsic and intrinsic innervation of the rat heart [7] and vagal transmission to the heart has been shown to be inhibited by exogenous opioid substances [11,18] channels via a Pertussis toxin-sensitive G-protein [1]. However, the action potential after-hyperpolarization due to a Ca 2+ -dependent K + conductance(s) in these neurones was unaltered in the presence of Met-enkephalin. In the present study, the actions of Met-enkephalin and caffeine, a known intracellular Ca 2+ mobilizing agent, on intracellular Ca 2+ mobilization and activation of the large conductance Ca 2+ -dependent K + channels (BK Ca ) were investigated.Parasympathetic neurones from neonatal rat intracardiac ganglia were isolated as previously described [19]. The dissociated neurones were plated onto glass coverslips coated with laminin, incubated at 37°C in 95% O 2 -5% CO 2 atmosphere and used for experiments within 36-72 h. At the time of experiments, the neuroneplated glass coverslips were transferred to a recording chamber (0.5 ml volume) and mounted on the stage of an inverted phase contrast microscope. Unitary current recordings were obtained using the cell-attached patch recording configuration of the patch clamp technique [6]. In the cell-attached configuration, K + currents in the membrane patch follow the relationship i K =γ(V m −V h −E K ). When the cells were bathed in a depolarizing external solution (V m ˜0 mV) and V h was held at 0 mV, the membrane potential in the patch was close to 0 mV. Unitary currents were recorded using an Axopatch 200B patch clamp amplifier (Axon Instruments, Foster City, CA), filtered at 5 kHz, and stored on digital tape using a DAT digital recorder (DTR-1204, Biologic Science Instruments, Claix, France). Records were transferred to a Pentium computer using an analogue-to-digit...

VPAC Receptor Modulation of Neuroexcitability in Intracardiac Neurons

“…Thapsigargin also eliminated the rise in [Ca 2ϩ ] i elicited by bath application of 5 mM caffeine (Fig. 3D), which has been shown to evoke release of Ca 2ϩ from intracellular stores in these cells (49 3 ) and a second that is sensitive to ryanodine and caffeine (39,49). The PACAP/VIP receptors have been shown to mobilize Ca 2ϩ from both IP 3 -and ryanodine/caffeine-sensitive stores in other cell types (44,46).…”

“…Microfluorometric Measurements-The calcium-sensitive dye fura-2/AM was used for measuring intracellular free calcium concentrations in intracardiac neurons, as previously described (39). Cells plated on coverslips were incubated for 1 h at room temperature in physiological saline solution (PSS) consisting of 140 mM NaCl, 3 mM KCl, 2.5 mM CaCl 2 , 1.2 mM MgCl 2 , 7.7 mM glucose, and 10 mM HEPES (pH to 7.2 with NaOH), which also contained 1 M fura-2/AM and 0.1% Me 2 SO.…”

Muscarinic and Nicotinic ACh Receptor Activation Differentially Mobilize Ca2+in Rat Intracardiac Ganglion Neurons

“…On the other hand, ryanodine, in micromolar concentrations is an inhibitor of caffeine-induced Ca 2ϩ release from intracellular Ca 2ϩ stores in rat autonomic neurons (Smith and Adams 1999;Thayer et al 1988). Focal application of caffeine (10 mM) evoked a transient [Ca 2ϩ ] i increase (114 Ϯ 19 nM, n ϭ 4) that was inhibited by bath application of 10 M ryanodine (14 Ϯ 5% of control, n ϭ 4, P Ͻ 0.005) as shown in Fig.…”

Opioid Receptor Trafficking and Signaling: What Happens After Opioid Receptor Activation?

“…Furthermore, Met-enkephalin-induced increase of intracellular Ca 2? in rat intracardiac neurons was found to be inhibited by ryanodine (Smith and Adams 1999). All these findings indicated that Ca 2?…”

“…or cyclic ADP ribose (Zalk et al 2007). Although all three isoforms of the RyRs (RyR1-3) have been found in the central nervous system, RyR2 is the most prevalent (Smith and Adams 1999). DOR activation has been reported to increase the release of Ca 2?…”