Behavior of Junction Channels Between Rat Glomus Cells During Normoxia and Hypoxia

Abstract

The activity of gap junction channels between cultured and clustered carotid body glomus cells of the rat was studied with dual voltage clamping during normoxia (PO(2) 300 Torr) and hypoxia induced by sodium dithionite (Na(2)S(2)O(4)) or 100% N(2). Na(2)S(2)O(4) reduced the saline PO(2) to approximately 10 Torr, whereas 100% N(2) reduced ambient O(2) to approximately 60 Torr. The following observations were made. 1) In normoxia, the intercellular macroconductance (G(j) = 3.0 +/- 1.01 ns, mean +/- SE) was changed unevenly (increased and decreased) under hypoxic conditions by either agent, although N(2) produced the largest changes. 2) The intercellular microconductances of the channels (g(j) = 104.44 +/- 10.16 pS under normoxic conditions) significantly decreased in 100% N(2) but showed depressions and enhancements in Na(2)S(2)O(4). 3) The conductance of single-junction channels (SChs), calculated as g(j) variance/mean g(j), yielded a mean of approximately 17.6 pS. Larger values were obtained with manual measurements of the data (approximately 34 pS). Hypoxic hypoxia (induced by 100% N(2)) significantly depressed the conductance of SChs when calculated from digitized records or from manual measurements. Hypoxia induced by Na(2)S(2)O(4) did not significantly change junctional conductance. 4) The number of intercellular channels, calculated as g(j)/SCh g(j), had a mean of approximately 452 (range 1 to 2,471). During N(2)-induced hypoxia, this number significantly decreased to approximately 84 but remained unchanged during Na(2)S(2)O(4) hypoxia. 5) The mean open time of junction channels varied from 4 to 30 ms in different experiments, having an overall mean of mu = 11.33 +/- 0.33 ms. This value was significantly reduced by 100% N(2) but was not changed by Na(2)S(2)O(4). 6) Intracellular calcium ([Ca(2+)](i)), 46.2 +/- 4.84 nM under normoxia, significantly increased to 77.32 +/- 11.27 nM with Na(2)S(2)O(4) and to 66.39 +/- 11.64 nM with 100% N(2). It is concluded that 100% N(2) uncouples glomus cells by significantly reducing intercellular macro- and microconductances. Hypoxia induced by Na(2)S(2)O(4) had variable effects. The coupling effects of hypoxia may depend on, or be aided by, increases in [Ca(2+)](i) and/or intracellular pH changes. However, secreted transmitters and ATP plus the effects of hypoxia on second messengers and other cytoplasmic components may also play an important role in this phenomenon.

Role of K2P channels in stimulus-secretion coupling

“…Presence of spontaneous oscillation in [Ca 2+ ] i has also been reported earlier [28]. Because glomus cells in the carotid body are believed to be electrically-coupled by gap junctions [2], it is possible that spontaneous [Ca 2+ ] i oscillations occur under normoxic conditions for all glomus cells in vivo, but not in dispersed cells. Such [Ca 2+ ] i oscillations could account for the basal release of catecholamines in normoxia unrelated to any effect on TASK activity.…”

Real-time dynamics of methyl-CpG-binding domain protein 3 and its role in DNA demethylation by fluorescence correlation spectroscopy

“…For efficient and acute hypoxia induction, 1 mM sodium dithionite (Na 2 S 2 O 4 , Sigma, 157953) was used to chemically consume the dissolved oxygen in the culture medium. [74][75][76] It is reported that Na 2 S 2 O 4 can lower pO 2 to 10 Torr compared with 30 Torr pO 2 induced by 100% N 2 . 77 After adding Na 2 S 2 O 4 , the pH of the DMEM/F-12 medium was adjusted to 7.4 and maintained by HEPES buffer considering the acidification potential of Na 2 S 2 O 4 .…”

An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells

“…Several authors have shown that, in hypoxic chambers, between 3 and 24 hours are required for the oxygen concentrations in the liquid phase to reach equilibrium with the gas mixture in the chamber (Allen et al, 2001; Mueller et al, 2009), making the clinically relevant rapid induction of hypoxic conditions impossible in these models. Hypoxic conditions can also be induced by the addition of cobalt chloride (Cheng et al, 2013; Garayoa et al, 2000; Gotoh et al, 2012) or sodium dithionite (Abudara et al, 2002; Mancarella et al, 2011; Yu et al, 2007), or by the application of the hypoxia-inducing enzymes glucose oxidase and catalase (Askoxylakis et al, 2011; Baumann et al, 2008; Mueller et al, 2009). Although the last-mentioned systems are easy to handle and rapidly induce hypoxic conditions, one of their major disadvantages is that the cells are in direct contact with the hypoxia-inducing agents.…”