Monitoring the Secretory Behavior of the Rat Adrenal Medulla by High-Performance Liquid Chromatography-Based Catecholamine Assay from Slice Supernatants

Abstract

Catecholamine (CA) secretion from the adrenal medullary tissue is a key step of the adaptive response triggered by an organism to cope with stress. Whereas molecular and cellular secretory processes have been extensively studied at the single chromaffin cell level, data available for the whole gland level are much scarcer. We tackled this issue in rat by developing an easy to implement experimental strategy combining the adrenal acute slice supernatant collection with a high-performance liquid chromatography-based epinephrine and norepinephrine (NE) assay. This technique affords a convenient method for measuring basal and stimulated CA release from single acute slices, allowing thus to individually address the secretory function of the left and right glands. Our data point that the two glands are equally competent to secrete epinephrine and NE, exhibiting an equivalent epinephrine:NE ratio, both at rest and in response to a cholinergic stimulation. Nicotine is, however, more efficient than acetylcholine to evoke NE release. A pharmacological challenge with hexamethonium, an α3-containing nicotinic acetylcholine receptor antagonist, disclosed that epinephrine- and NE-secreting chromaffin cells distinctly expressed α3 nicotinic receptors, with a dominant contribution in NE cells. As such, beyond the novelty of CA assays from acute slice supernatants, our study contributes at refining the secretory behavior of the rat adrenal medullary tissue, and opens new perspectives for monitoring the release of other hormones and transmitters, especially those involved in the stress response.

Keywords: acetylcholine; acute adrenal slice; catecholamine release; fluorescence derivatization; hexamethonium; high-performance liquid chromatography; medullary tissue; stimulus-secretion coupling.

Figure 1

Schematic illustration of the experimental protocol leading to the high-performance liquid chromatography (HPLC)-based measurement of catecholamines [E and norepinephrine (NE)] in rat acute adrenal slice supernatants. The protocol can be divided into four main steps, including (i) the gland slicing using a vibratome, (ii) the challenge of individual slices with drugs of interest, (iii) the calculation of the medulla volume of each challenged slices after a hematoxylin staining, and (iv) the HPLC-based measurement of the amounts of E and NE released by each slice. C, cortex; M, medulla.

Figure 2

Validation of the use of high-performance liquid chromatography coupled with fluorescence detection for the determination of basal and stimulated catecholamine (CA) release in slice supernatants. (A) Chemical reaction leading to the production of the UV-fluorescent benzoxazole derivative. [(B), a] Representative chromatograms of basal CAs released from six successive 150 μm-thick slices (#1 to #6, assay in 20 µl supernatant). [(B), b] Plots of the correlation between hormone secretion [measured by the calculation of the area under the curve of E- and norepinephrine (NE)-related chromatographic peaks] and slice medulla volume {same slices than in panel [(B), a]}. Although not statistically significant in the example illustrated in the figure, the Spearman’s rank correlation coefficient ρ indicates a positive correlation between the medulla volume and hormone chromatographic peaks. [(C), a] Superimposed chromatograms illustrating the increased catecholamine (CA) (E and NE) content in a 10 µM acetylcholine chloride (Ach)-stimulated slice supernatant. [(C), b] Calculation of the areas under the curve of NE- and E-related peaks allowing the quantification of CAs released before (basal) and after ACh stimulation.

Figure 3

Comparison of the adrenal medulla volume between the left and the right glands. (A) Pictures illustrating the medulla surface (dashed line) in six serial slices (hematoxylin labeling) of the same adrenal gland. As expected from the anatomy of the rat adrenal gland and the central location of the medullary tissue, the medulla is smaller in the first cut slices (#1 and #2) and gradually increases in the deeper slices (#4, #5, and #6). Scale bar: 1 mm. [(B), a] Calculation of the medulla volume in the left and right glands of 12 rats. The statistical analysis of the paired data indicates that the left medullary tissue is significantly greater than the right one (non-parametrical Wilcoxon’s matched pair test, *p < 0.05). [(B), b] No significant correlation between the medullary tissue volume and the body weight (Spearman’s rank correlation coefficient ρ = −0.189 for the left gland, p > 0.05 and ρ = 0.175 for the right gland, p > 0.05).

Figure 4

Basal catecholamine (CA) secretion from the left and the right gland. (A) CAs amounts [E, left panels and norepinephrine (NE), right panels] assayed in 20 µl supernatant collected from eight consecutive slices, and comparison to the medulla volume of associated slices. Pooled data from three to six left glands [(A), a] and two to seven right glands [(A), b]. The Spearman’s rank correlation coefficient ρ between the secreted hormone amount and the slice medulla volume is shown in insets. For E, larger is the medullary tissue volume in a slice, greater is the secreted hormone amount (ρ = 0.786 and 0.881 for the left and the right glands, respectively). For NE, no significant correlation is found between the secreted amount and the medulla volume. (B–E) E and NE are secreted equally by the left and the right glands. (B) Data expressed as a function of slice serial position [same slices as in panel (A)]. (C) Pooled data of all slices. (D–E) E:NE ratios calculated for the left and the right gland. Raw results in panel (D), showing a greater secretion of E versus NE, for both the left and the right adrenal (each dot illustrates a single slice). (E) No significant difference in the E:NE ratio between the two glands.

Figure 5

Acetylcholine-evoked catecholamine secretion from the left and the right glands. (A) Slices were challenged with a 5-min bath application of acetylcholine chloride (Ach) (10, upper graph or 100 µM, lower graph). Regardless of ACh concentration and of the left or right gland, E is main hormone secreted by individual slices. E:norepinephrine (NE) ratio is dose dependent but does not differ between the left and the right gland (B).

Figure 6

No meaningful difference in the amounts of hormone released from slices containing chromaffin cells mostly located close to the cortex or in the medulla center. Due to the round-shaped morphology of the adrenal glands, the first and last cut slices (white bars) contain numerous chromaffin cells that are in the vicinity of the cortical region. By contrast, the intermediate slices (gray bars) mostly contain chromaffin cells that are distant from the cortex. When comparing the amounts of E and norepinephrine (NE) released from first/last slices to hormones released from intermediate slices, no significant difference is observed, whether for basal secretion (A), acetylcholine chloride (Ach)-evoked (B), and nicotine-evoked secretion (C). (D) Histogram illustrating the ratio peripheral/central catecholamines (CAs) release under basal, ACh-evoked, and nicotine-evoked hormone secretion. Note the bias toward a shift from central to peripheral E and NE release upon ACh challenges.

Figure 7

Dose-dependent acetylcholine chloride (Ach)- and nicotine-evoked catecholamine secretion and blockade by hexamethonium. (A) Individual slices were stimulated for 5 min by either ACh (10 and 100 µM) or nicotine (1, 3, 10, and 100 µM). Both E [(A), a] and norepinephrine (NE) [(A), b] release dose-dependently increases with agonist concentration. Note that for a same agonist concentration, nicotine is more efficient to evoke E and NE secretion [(A), c]. (B) Effect of the α3-containing nAChR antagonist hexamethonium. Slices were incubated for 5 min with hexamethonium (200 µM) before challenging with 10 µM nicotine (or saline for control experiments). While basal E and NE secretion is not affected by hexamethonium, nicotine-triggered E [(B), a] and NE [(B), b] release is significantly reduced. [(B), c] Pooled data illustrating a strongest ability for hexamethonium to reduce NE secretion compared to E secretion (88 versus 57% decrease). *p < 0.05, **p < 0.01, ***p < 0.001.