Cellular and subcellular localization of tritiated gentamicin in the guinea pig cochlea following combined treatment with ethacrynic acid.

Guinea pigs (GPs) receiving one intra-muscular injection of gentamicin (GM) (150 mg/kg) in which 2 mg of tritiated GM (2 mCi) were incorporated, followed 1.5 h later by an intra-cardiac injection of ethacrynic acid (EA) (30 mg/kg) were sacrificed 25 min, 1, 4 and 24 h after the EA injection. Other GPs were treated with one injection of GM or EA alone and sacrificed 24 h later. Cochlear function was monitored by recording VIIIth nerve compound action potential (CAP) responses to clicks at 70 dB peak-equivalent Sound Pressure Level (pe SPL) and CAP audiograms. At 24 h thresholds were significantly elevated for high frequencies only in GPs treated with the GM/EA combination. GM was revealed in the cochlea and kidney by autoradiography using light and electron microscopy. In the kidney GM was already detected in the proximal tubule cells at 25 min and at 24 h. In the cochlea GM was systematically not observed at 25 min. At 1 h a weak labelling was detected in vessels of the stria vascularis and in sensory cells at the base of the cochlea. At 4 h the labelling disappeared in stria vascularis but increased in the hair cells. At 24 h GM labelling was found exclusively in hair cells, particularly outer hair cells, with a gradient from base to apex and from first to 3rd row, this distribution pattern correlating well with the pattern of threshold changes prominent at high frequencies.(ABSTRACT TRUNCATED AT 250 WORDS)

Norepinephrine-containing glomus cells in the rabbit carotid body. I. Autoradiographic and morphometric study after tritiated norepinephrine uptake.

Rabbit carotid bodies were investigated by autoradiography at both the light and electron microscope levels following tritiated norepinephrine administration either in vivo or in vitro. Two kinds of labelled structures were found: nerve fibres (absent in sympathectomized carotid bodies) and some type I glomus cells. Desipramine (a specific norepinephrine uptake inhibitor) prevented labelling. Most of the labelled cells differed from unlabelled ones by the presence of (i) large dense-cored vesicles characterized by a large halo between the membrane and an eccentric dense core; (ii) a nucleus showing a more electron dense chromatin and a more irregular shape; and (iii) relatively abundant glycogen particles. A new weakly-labelled cells were characterized by a pyknotic nucleus and very swollen dense-cored vesicles, and were presumed to be degenerating. Dense core diameters of dense-cored vesicles were distributed according to a unimodal distribution in labelled cells as in unlabelled ones but with an extension towards both large and very small diameters in labelled cells. The mean diameter was higher in labelled cells than in unlabelled ones (127 nm versus 113 nm, P less than 0.01). The labelling intensity (as estimated by the number of silver grains per unit of cytoplasmic area) was maximum in cells having dense-cored vesicles whose mean diameter was between 130 and 170 nm, but decreased for cells with mean diameter of dense cores smaller than 130 nm, or larger than 170 nm. Thus, in the rabbit carotid body, some glomus cells differ from others by their ability to take up tritiated norepinephrine and by the presence of larger dense-cored vesicles. However, this distinction is not clearcut and there are many intermediates. The observations suggest a phenomenon of evolution deriving from a unique cell type and typified by both metabolic norepinephrine uptake ability, glycogen accumulation) and morphologic changes (increase in diameter of dense-cored vesicles). It seems, therefore, more appropriate to consider these results in terms of different functional states rather than different types of glomus cells.

Norepinephrine-containing glomus cells in the rabbit carotid body. II. Immunocytochemical evidence of dopamine-beta-hydroxylase and norepinephrine.

The presence of noradrenergic glomus cells in the rabbit carotid body was investigated at the light and electron microscope levels, using dopamine-beta-hydroxylase and norepinephrine immunocytochemistry as well as the chromaffin reaction. Frozen and semi-thin plastic sections showed some dopamine-beta-hydroxylase immunoreactive glomus cells either isolated in the connective tissue or, more frequently, mixed with unreactive cells. At the ultrastructural level immunopositive cells differed from immunonegative ones by the larger size of most of their dense-cored vesicles. Similar observations were made after using anti-norepinephrine antibodies. Immunoreactive cells to anti-dopamine-beta-hydroxylase and anti-norepinephrine antibodies were relatively few although their number varied from carotid body to carotid body. The immunolabelling intensity was very variable from cell to cell. Consecutive frozen sections processed for norepinephrine- and dopamine-immunocytochemistry showed many cell clusters containing both norepinephrine and dopamine-immunoreactive glomus cells. Some chromaffin glomus cells were clearly identifiable by the very strong electron opacity of their dense-cored vesicles; most of these vesicles were characterized by their large size, as the dense-cored vesicles observed in dopamine-beta-hydroxylase- and norepinephrine-immunopositive cells. These results demonstrated that dopamine-beta-hydroxylase and norepinephrine-immunopositive, as well as chromaffin cells, were identical to the cells which take up exogenous norepinephrine, described in part I of this study. However, many intermediate levels were found between norepinephrine-immunonegative and strongly norepinephrine-immunopositive glomus cells, suggesting that the distinction between these two kinds of cells is not clearcut.

Long-term hypoxia increases the number of norepinephrine-containing glomus cells in the rat carotid body: a correlative immunocytochemical and biochemical study.

A group of adult rats was divided into two subgroups: one was submitted to long-term normobaric hypoxia (10% O2, two weeks) while the other (control group) was kept in the same room, breathing air. Animals from each subgroup were used to study either the norepinephrine content of the carotid body by high pressure liquid chromatography or to localize norepinephrine-containing structures using an immunocytochemical procedure (peroxidase-labelled antibodies on cryostat sections). The biochemical study showed, as expected, a large increase in carotid body norepinephrine content (19-fold) and turnover (ten-fold) in hypoxic rats. The immunocytochemical study revealed only a few norepinephrine-immunopositive glomus cells in sections through the carotid body of normoxic rats, whereas the carotid body of hypoxic rats showed a very large number of norepinephrine-positive glomus cells. This increase was quantified, using an image analyser, and it was found to constitute a 61-fold increase in the number of immunopositive profiles per section and a 29-fold increase in the immunopositive profile area/section area ratio. It is concluded that long-term hypoxia increases rat carotid body norepinephrine content by inducing norepinephrine synthesis in glomus cells in which this amine was not detectable previously, before hypoxia.

Localization of dopamine D2 receptor mRNA in glomus cells of the rabbit carotid body by in situ hybridization.

The localization of mRNA coding for the dopamine D2 receptor was studied in the rabbit carotid body using in situ hybridization with synthetic 35S-labelled oligodeoxynucleotides. Using autoradiography on cryostat or semi-thin sections, labelling was observed over the cytoplasm of glomus cells, but not over sustentacular cells. A quantitative study showed that labelling intensity (silver grain density) was increased by haloperidol treatment. These results suggest that glomus cells express the dopamine D2 receptor gene and that this expression is regulated.