Stimulation of voltage-dependent Ca²⁺channels by NO at rat myenteric neurons

Abstract

The aim of the present study was to characterize the action of the neurotransmitter NO on rat myenteric neurons. A NO donor such as GEA 3162 (10(-4) mol/l) induced an increase in the intracellular Ca2+ concentration as indicated by an increase in the fura 2 ratio in ganglia loaded with this Ca2+-sensitive fluorescent dye. The effect of GEA 3162 was strongly reduced in the absence of extracellular Ca2+, suggesting an influx of Ca2+ from the extracellular space evoked by NO. A similar nearly complete inhibition was observed in the presence of Ca2+ channel blockers such as Ni2+ (5 x 10(-4) mol/l) or nifedipine (10(-6) mol/l). Whole cell patch-clamp recordings confirmed the activation of voltage-dependent Ca2+ channels, measured as inward current carried by Ba2+, by the NO donor. The peak Ba2+-carried inward current increased from -100 +/- 19 to -185 +/- 34 pA in the presence of sodium nitroprusside (10(-4) mol/l). The consequence was a hyperpolarization of the membrane, which was blocked by intracellular Cs+ and thus most probably reflects the activation of Ca2+-dependent K+ channels. Furthermore, at least two subtypes of NO synthases, NOS-1 (neuronal form) and NOS-3 (endothelial form), were found as transcripts in mRNA isolated from the rat myenteric ganglia. The expression of these NO synthases was confirmed immunohistochemically. These observations suggest that NO, released from nitrergic neurons within the enteric nervous system, not only affects target organs such as smooth muscle cells in the gut but has in addition profound effects on the enteric neurons themselves, the key players in the regulation of many gastrointestinal functions.

Diet‐induced obesity has neuroprotective effects in murine gastric enteric nervous system: involvement of leptin and glial cell line‐derived neurotrophic factor

“…This increase in neuronal density and in particular of NOS‐IR neurons could be responsible for the larger Ca 2+ concentration induced by fibre tract nerve stimulation in the ENS in WD as compared to ND mice. Indeed, NO has been shown to increase intracellular calcium concentration in myenteric neurons (Sitmo et al 2007).…”

Galanin inhibition of voltage‐dependent Ca2+ influx in rat cultured myenteric neurons is mediated by galanin receptor 1

“…Primary antisera were diluted in this blocking solution, and preparations were incubated overnight. For these experiments, we used a specific antibody raised against GalR1 (rabbit GalR1 Y225–238 antiserum [1:1,000]) that has been previously characterized (Pham et al, 2002); a chicken antibody raised against microtubule‐associated proteins 2 (MAP2) (1:3,000; Neuromics, Edina, MN) as a neuronal marker (Scheuermann et al, 1991); a monoclonal antibody raised against human smooth muscle actin (clone 1A4) (1:100; Dako, Carpinteria, CA) as smooth muscle marker (Ennes et al, 1997); or a mouse monoclonal antibody against glial fibrillary acidic protein (GFAP) (1:1,000; Neuromics, Edina, MN) as marker for glial cells (Sitmo et al, 2007). After incubation in the primary antibody, coverslips were rinsed with 0.1 M PB solution and incubated for 1 hr at room temperature in affinity purified goat anti‐rabbit IgG Alexa 568 (1:1,000), goat anti‐mouse IgG Alexa 568 (1:1,000), or goat anti‐chicken IgG Alexa 488 (1:1,000) (Invitrogen).…”

The Chemistry of Bioactive Mesoionic Heterocycles

“…It is suggested that NO stimulates the voltagedependent Ca 2+ channels in rat myenteric neurons [74]. (42) has been one of the most studied compounds.…”