Chromogranin A deficiency in transgenic mice leads to aberrant chromaffin granule biogenesis

Abstract

The biogenesis of dense-core secretory granules (DCGs), organelles responsible for the storage and secretion of neurotransmitters and neuropeptides in chromaffin cells, is poorly understood. Chromogranin A (CgA), which binds catecholamines for storage in the lumen of chromaffin granules, has been shown to be involved in DCG biogenesis in neuroendocrine PC12 cells. Here, we report that downregulation of CgA expression in vivo by expressing antisense RNA against CgA in transgenic mice led to a significant reduction in DCG formation in adrenal chromaffin cells. The number of DCGs formed in CgA antisense transgenic mice was directly correlated with the amount of CgA present in adrenal medulla. In addition, DCGs showed an increase in size, with enlargement in the volume around the dense core, a phenomenon that occurs to maintain constant "free" catecholamine concentration in the lumen of these granules. The extent of DCG swelling was inversely correlated with the number of DCGs formed, as well as the amount of CgA present in the adrenal glands of CgA antisense transgenic mice. These data indicate an essential role of CgA in regulating chromaffin DCG biogenesis and catecholamine storage in vivo.

Figure 1.

Reduction of DCG formation in CgA antisense transgenic mice. A, Diagram of CgA antisense transgene plasmid. A transgenic vector (pCAS2C) containing mouse CgA antisense sequence under the control of the endogenous mouse CgA promoter for the tissue-specific expression was generated previously (Liu et al., 2001). PCR amplification using a specific primer set (C1 and P1) with genomic DNAs from WT, transgenic animals, and a plasmid (pCAS3C) is shown. Only TG and a plasmid amplification showed PCR product at ∼520 bp. B, Western blotting analysis representing the level of CgA, CgB, and CPE in WT and transgenic animals. C, Bar graph represents the level of CgA in adrenal glands (open bars) and the number of DCGs (filled bars). The level of CgA was reduced to 68% (TG-4) and 31% (TG-5) compared with WT-2 (100%). C, The level of CgA in TG-1 was reduced to 51% compared with WT-1. The number of DCGs are 7.3 ± 0.9 (WT-1; n = 11 EM images), 6.7 ± 0.3 (WT-2; n = 5), 4.6 ± 0.5 (TG-1; n = 12; ^**p < 0.01 vs WT-1; ^*p < 0.05 vs WT-2), 5.3 ± 0.2 (TG-4; n = 15; ^**p < 0.01 vs WT-2), and 3.8 ± 0.2 (TG-5; n = 9; ^***p < 0.001 vs WT-2) granules per square micrometer of cytoplasm (±SEM; two-tailed Student's t test).

Figure 2.

Morphological changes of DCGs in CgA antisense transgenic mice. EM micrographs represent cytoplasmic images from adrenal chromaffin cells in WT-2, TG-4, and TG-5. Two images per animal are shown. Scale bar, 300 nm.

Figure 3.

Increased size of DCGs in CgA antisense transgenic mice. A, EM morphometric analysis showing the percentage of DCGs with different diameters. The arrow indicates DCG population with a diameter >500 nm in TG-5. B, Average diameters of DCGs (filled bars) and dense cores (open bars) are shown. Average sizes of DCGs are 219.2 ± 1.4 nm (±SEM; n = 2062), 263.9 ± 1.5 nm (±SEM; n = 3648; ^***p < 0.0001 vs WT-2), and 302.9 ± 2.6 nm (±SEM; n = 2757; ^***p < 0.0001 vs WT-2) for WT-2, TG-4, and TG-5, respectively. Average diameters of the dense cores are 170.2 ± 1.4 nm (±SEM; n = 1256), 163.2 ± 1.4 nm (n = 1017; ^***p < 0.0001 vs WT-2), and 157.9 ± 1.2 nm (n = 1116; ^**p < 0.01 vs WT-2) for WT-2, TG-4, and TG-5, respectively (two-tailed Student's t test).