Nitric Oxide Inhibits L-Type Ca²⁺Current in Glomus Cells of the Rabbit Carotid Body Via a cGMP-Independent Mechanism

Abstract

Previous studies have shown that nitric oxide (NO) inhibits carotid body sensory activity. To begin to understand the cellular mechanisms associated with the actions of NO in the carotid body, we monitored the effects of NO donors on the macroscopic Ca2+ current in glomus cells isolated from rabbit carotid bodies. Experiments were performed on freshly dissociated glomus cells from adult rabbit carotid bodies using the whole cell configuration of the patch-clamp technique. The NO donors sodium nitroprusside (SNP; 600 microM, n = 7) and spermine nitric oxide (SNO; 100 microM, n = 7) inhibited the Ca2+ current in glomus cells in a voltage-independent manner. These effects of NO donors were rapid in onset and peaked within 1 or 2 min. In contrast, the outward K+ current was unaffected by SNP (600 microM, n = 6), indicating that the inhibition by SNP was not a nonspecific membrane effect. 2-(4-carboxyphenyl)-4,4,5, 5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxy-PTIO; 500 microM), an NO scavenger, prevented inhibition of the Ca2+ current by SNP (n = 7), whereas neither superoxide dismutase (SOD; 2,000 U/ml, n = 4), a superoxide scavenger, nor sodium hydrosulfite (SHS; 1 mM, n = 7), a reducing agent, prevented inhibition of the Ca2+ current by SNP. However, SNP inhibition of the Ca2+ current was reversible in the presence of either SOD or SHS. These results suggest that NO itself inhibits Ca2+ current in a reversible manner and that subsequent formation of peroxynitrites results in irreversible inhibition. SNP inhibition of the Ca2+ current was not affected by 30 microM LY 83, 583 (n = 7) nor was it mimicked by 600 microM 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP; n = 6), suggesting that the effects of NO on the Ca2+ current are mediated, in part, via a cGMP-independent mechanism. N-ethylmaleimide (NEM; 2.5 mM, n = 6) prevented the inhibition of the Ca2+ current by SNP, indicating that SNP is acting via a modification of sulfhydryl groups on Ca2+ channel proteins. Norepinephrine (NE; 10 microM) further inhibited the Ca2+ current in the presence of NEM (n = 7), implying that NEM did not nonspecifically eliminate Ca2+ current modulation. Nisoldipine, an L-type Ca2+ channel blocker (2 microM, n = 6), prevented the inhibition of Ca2+ current by SNP, whereas omega-conotoxin GVIA, an N-type Ca2+ channel blocker (1 microM, n = 9), did not prevent the inhibition of Ca2+ current by SNP. These results demonstrate that NO inhibits L-type Ca2+ channels in adult rabbit glomus cells, in part, due to a modification of calcium channel proteins. The inhibition might provide one plausible mechanism for efferent inhibition of carotid body activity by NO.

cGMP/Protein Kinase G-Dependent Inhibition of N-Type Ca2+Channels Induced by Nitric Oxide in Human Neuroblastoma IMR32 Cells

“…The findings that emerge from the present study also fit nicely with the NO-induced inhibition of L-type Ca 2ϩ channels that has been observed in various experimental models, although the mechanisms responsible for these effects may be different. NO has been reported to inhibit Ca 2ϩ channels either through a direct action consisting in S-nitrosylation of the channel protein (Hu et al, 1997;Summers et al, 1999) or by activating guanylate cyclase. The increase in cGMP levels can produce channel inhibition through activation of phosphodiesterase, with consequent downregulation of the cAMP/PKA-activating pathway or through protein kinase G [see references in Carabelli et al (2002)].…”

Carotid body function in heart failure

“…To date however, very little is known of the function of these channels in the CB in the setting of CHF. Indirectly, it is known that NO inhibits L-type Ca 2+ channels in rabbit glomus cells via a cGMP independent mechanism (Summers et al 1999). The role of Ang II on L-type Ca 2+ channel activity in the CB has not been addressed.…”

Inhibitory effects of NO on carotid body: contribution of neural and endothelial nitric oxide synthase isoforms

“…33 for review). NO has been proposed to produce vasodilatation in the CB (5,8,28,38), retrograde inhibition of the glomus cell's excitability (39), inhibition of Ca 2ϩ channels in glomus cells (36), modulation of petrosal ganglion neuron's excitability (1), and inhibition of mitochondrial metabolism (22,28). At least three isoforms of NOS have been isolated (32): neuronal (nNOS), endothelial (eNOS), and inducible.…”