Developmental Changes in Gap Junction Expression in Rat Adrenal Medullary Chromaffin Cells

Abstract

Cell-to-cell communications are desirable for efficient functioning in endocrine cells. Gap junctions and paracrine factors are major mechanisms by which neighboring endocrine cells communicate with each other. The current experiment was undertaken to morphologically examine gap junction expression and developmental changes in rat adrenal medullary chromaffin (AMC) cells. The expression of connexin 43 (Cx43) was conspicuous in the rat adrenal cortex, but not detected immunohistochemically in neonatal or adult AMC cells. Consistent with the morphological findings, the phosphorylated and non-phosphorylated forms of Cx43 were predominantly and faintly detected by immunoblotting in the adrenal cortical and medullary homogenates, respectively. In contrast to Cx43, Cx36-like immunoreactive (IR) material was detected in neonatal AMC cells, a fraction of which were in the process of migration to the center of the adrenal gland, but this was not seen in adult AMC cells. The current results raise the possibility that the mechanism for cell-to-cell communication changes in a developmental manner in rat AMC cells.

Keywords: chromaffin cell; chromogranin A; connexin; development; gap junction.

Fig. 1.

Developmental change in immunostaining for chromogranin A in rat adrenal glands. (A) Immunohistochemical staining for chromogranin A in adrenal gland sections. Right, enlargement of the square shown in left. Thin slices of adrenal glands of indicated ages (D: day; W: week) were subjected to immunohistochemistry for chromogranin A. Immunoreaction was visualized as DAB reaction. The sections were counterstained with hematoxylin. (B and C) Summaries of fractions (%) of Adrenal medullary chromaffin (AMC) cells in the adrenal gland and areas of the adrenal gland, respectively. AMC cells were immunohistochemically identified as chromogranin A-positive cells. Data represent means ± SEM of 4 slices for each newborn and adult rat. Newborn rats include 1- and 5-day old rats; adult rats represent 8 to 12-week-old rats.

Fig. 2.

Immunostaining and immunoblotting for connexin 43. (A) Immunostaining for connexin 43 (Cx43) of adrenal slices of 1- and 7-day old rats. Image in the 2nd row represents the enlargement of a square in the 1st row. (B) Immunostaining for Cx43 of an adrenal slice of the adult rat. The immunoreaction was visualized as DAB reaction. (C) Immunoblots of rat adult heart (H), adrenal cortex (AC) and adrenal medulla (AM) homogenates for Cx43 and actin. The same amount of proteins (5 μg) was loaded in each lane. (D) Immunoblot for Cx43 of heart homogenates treated (AP(+)) or not treated (AP(−)) with alkaline phosphatase. AP(+) denotes the preparations treated with alkaline phosphatase for the indicated periods (min) at 36°C, whereas AP(−) denotes the preparation treated for 50 min at 36°C without alkaline phosphatase. Arrows indicate three forms of Cx43.

Fig. 3.

Immunostaining for Cx36 of the rat adrenal gland. (A) Immunostaining of MIN6 β cells for Cx36. Left, confocal image of Cx36-like immunoreactive (IR) material; right, merge of confocal and differential interference contrast (DIC) images. Immunoreaction was visible as rhodamine-like fluorescence. (B) Immunostaining for Cx36 of an adrenal gland slice of 1-day old rat with the indirect immunoperoxidase method. a, control slice, where the treatment with an anti-Cx36 Ab was omitted; b, immunostaining with the anti-Cx36 Ab; c, enlargement of a square in b. The arrow and the arrowhead indicate the area inside the uttermost layer and the core with significant DAB reaction, respectively. (C) Line scan of DAB reaction intensity along the straight line in Bb. peri and core represent the area inside the uttermost layer (arrow in Bb) and that in the center of the adrenal gland (arrowhead). Intensity is expressed in arbitrary unit (au). (D) Double immunostaining of an adrenal gland slice of a 1-day-old rat for Cx36 and chromogranin A. a, confocal image of merge of Cx36- and chromogranin A-like fluorescence; b, enlargement of a square in a; c and d, confocal Cx36- and chromogranin A-like fluorescence images, respectively. Cx36- and chromogranin A-like IR material were visible as FITC- and rhodamine-like fluorescence, respectively. (E) Immunostaining for Cx36 of an adult rat adrenal gland slice with the indirect immunoperoxidase method. The slice was counterstained with hematoxylin. a, control slice, where the treatment with the anti-Cx36 Ab was omitted; b, immunostaining with the anti-Cx36 Ab.