Characterization of cultured chemoreceptor cells dissociated from adult rabbit carotid body

Abstract

Short-term cell cultures were obtained from enzymatically dissociated carotid bodies from adult rabbits, and morphological and functional characterization of the cultured chemoreceptor cells were carried out. Under phase contrast, freshly isolated type I cells are round, bright, and 10-14 microns in diameter and exhibit strong fluorescence when stained with the glyoxylic acid technique. The content of endogenous dopamine in the cultures increased from 80 pmol/10(5) cells 2 h after plating the cells to 200 pmol/10(5) cells on the 3rd day, and the rate of synthesis and storage of [3H]dopamine from the precursor [3H]tyrosine increased from 1.7 pmol.10(5) cells-1.h-1 in 1-day cultures to 4 pmol.10(5) cells-1.h-1 on the 3rd day; the later values represent 80-85% of the expected values for the intact carotid body. After labeling with [3H]tyrosine, cultured chemoreceptor cells release [3H]dopamine when challenged by hypoxia, high external K+, or the protonophore dinitrophenol, the pattern of response being similar to that of the intact carotid body. When studied by whole cell clamp recording, individual chemoreceptor cells exhibit a marked variability in the properties of some ionic currents; the data, however, do not support the existence of distinct subpopulations of type I cells.

Viral Gene Transfer of Dominant-Negative Kv4 Construct Suppresses an O2-Sensitive K+Current in Chemoreceptor Cells

“…After tracheostomy, the carotid artery bif urcations were removed, and the animals were killed by an intracardiac bolus injection of pentobarbital sodium. The CBs were cleaned of surrounding connective tissue and enzymatically dispersed as described elsewhere (Pérez-García et al, 1992;L ópez-L ópez et al, 1997). Dispersed cells were plated onto poly-L-lysine-coated coverslips placed in 6-well dishes with 1 ml of DM EM:F-12 (1:1) with 5% FBS and maintained in culture for up to 96 hr.…”

“…In the case of the rabbit CB chemoreceptors, three different voltage-dependent K ϩ channels have been described in singlechannel studies (Ganfornina and López Barneo, 1992a), and a detailed electrophysiological characterization of these currents, particularly of the O 2 -sensitive K ϩ current, has been provided by several studies López-Barneo, 1991, 1992a,b;Pérez-García et al, 1992;López-López et al, 1993); however, no attempts have been made to establish the molecular identity of the different channels. Indeed, the minute size of the organ, together with its structural complexity, has delayed its characterization with conventional molecular biology techniques.…”

“…Nevertheless, low pO 2 inhibition represents only a small fraction of the amplitude of the transient outward K ϩ current of chemoreceptor cells (López-López et al, 1993), making conceivable the hypothesis that Kv1.4 could contribute to the component of the transient outward K ϩ current that is inhibited by hypoxia, so that its blockade by the Kv1.xDN construct could be unperceived when looking at the transient current amplitude caused by the cell to cell variability (Pérez-García et al, 1992). To explore this possibility, we have studied if hypoxic inhibition of the transient K ϩ current is still present in Kv1.xDN cells.…”

Effect of p47phoxgene deletion on ROS production and oxygen sensing in mouse carotid body chemoreceptor cells

“…In rat type I cells, the current-voltage characteristics of maxiK channels suggest that they do not open until E m is more positive than Ϫ20 mV. Multiple studies have reported that specific maxiK blockers fail to activate isolated type I cells, which has supported the notion that these channels modulate cell activity and facilitate repolarization of the membrane (6,17,42). However, in thin slices of rat carotid body, Pardal and López-Barneo (37) demonstrated that the specific maxiK blocker iberiotoxin evokes catecholamine release from type I cells.…”

Inhibition of [3H]catecholamine release and Ca2+ currents by prostaglandin E2 in rabbit carotid body chemoreceptor cells.

“…Chemoreceptor cell culture and electrophysiological recordings Isolation and culture of chemoreceptor cells from adult rabbit CBs was done following protocols described previously (PerezGarcia, Obeso, Lopez-Lopez, Herreros & Gonzalez, 1992). In brief, CBs were incubated for 20 min at 37°C in 2 ml of nominally Ca2`-and Mg2+-free Tyrode solution (mM: NaCl, 140; KCl, 5; N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (Hepes), 10; glucose, 5; pH 7 2) containing trypsin (2 mg ml-', type II; Sigma, Alcobendas, Madrid, Spain) collagenase (2 mg ml-', type IV; Sigma), and deoxyribonuclease (DNase; 0 5 mg ml-', type II; Sigma).…”