Chromogranin B gene ablation reduces the catecholamine cargo and decelerates exocytosis in chromaffin secretory vesicles

Abstract

Chromogranins/secretogranins (Cgs) are the major soluble proteins of large dense-core secretory vesicles (LDCVs). We have recently reported that the absence of chromogranin A (CgA) caused important changes in the accumulation and in the exocytosis of catecholamines (CAs) using a CgA-knock-out (CgA-KO) mouse. Here, we have analyzed a CgB-KO mouse strain that can be maintained in homozygosis. These mice have 36% less adrenomedullary epinephrine when compared to Chgb(+/+) [wild type (WT)], whereas the norepinephrine content was similar. The total evoked release of CA was 33% lower than WT mice. This decrease was not due to a lower frequency of exocytotic events but to less secretion per quantum (approximately 30%) measured by amperometry; amperometric spikes exhibited a slower ascending but a normal decaying phase. Cell incubation with L-DOPA increased the vesicle CA content of WT but not of the CgB-KO cells. Intracellular electrochemistry, using patch amperometry, showed that L-DOPA overload produced a significantly larger increase in cytosolic CAs in cells from the KO animals than chromaffin cells from the WT. These data indicate that the mechanisms for vesicular accumulation of CAs in the CgB-KO cells were saturated, while there was ample capacity for further accumulation in WT cells. Protein analysis of LDCVs showed the overexpression of CgA as well as other proteins apparently unrelated to the secretory process. We conclude that CgB, like CgA, is a highly efficient system directly involved in monoamine accumulation and in the kinetics of exocytosis from LDCVs.

Figure 1.

The secretory profile of chromaffin cells lacking CgB. A, A typical amperometric trace obtained from a CgB-KO cell after a 10 s pressure application of BaCl₂ (5 mm); left black calibration bar is for the amperometry trace (in picoamperes) and the right gray bar for the integrated signal (in picocoulombs). B, Quantification of the cumulated release after 2 min (means ± SEM) expressed in picocoulombs. The number of cells used is in brackets. *p = 0.053 (U Mann–Whitney). Stimuli were applied at 0 s time. C, Total amount of spikes (means ± SEM) counted for 2 min after stimulation. D, Distribution of spikes expressed in hertz. Each point is the average of counting spikes at 1 s intervals.

Figure 2.

Effects of l-DOPA overload on free cytosolic catechols. Cells were incubated with l-DOPA as described in Table 2; all bathing media contained 10 μm pargyline. A, Individual cells were observed with patch amperometry in the whole-cell configuration mode, and the cytosol was put in direct contact with the carbon fiber electrode by aspiration. B, Typical 40 s amperometric trace observed once the cell membrane was broken. The oxidation current decayed rapidly, and many secretory spikes can be observed; a locally written macro for Igor Pro was used to eliminate the contribution of spikes to the net free cytosolic amines (inset). The integration plot (in picocoulombs) is superimposed on the graph. C, Pooled data from 120 s integration of the cytosolic amines obtained under basal conditions (C) and after l-DOPA treatment (L-D) from cells of wild-type and CgB-KO mice. The number of cells is indicated in brackets. *p = 0.011, ^†p = 0.004, ^¥p = 0.008 (global comparison Kruskal–Wallis p = 0.001 with Bonferroni correction, α = 0.012).

Figure 3.

Adrenal CgA is overexpressed in CgB-KO mice. A, Typical Western blots from adrenal medullary tissues confirming the lack of CgB and the overexpression of CgA. α-Tubulin was used as internal loading control. B, Pooled data from three different experiments. Neither SgII nor tyrosine hydroxylase (TH) levels were affected. *p = 0.03, Mann–Whitney test.

Figure 4.

Two-dimensional gel electrophoresis of chromaffin secretory vesicles from WT and CgB-KO mice. Fractions 3 to 5 from Optiprep gradients were used to perform the 2D SDS-PAGE. Panels show original gels stained with colloidal Coomassie blue. Proteins were identified by MALDI-TOF (see Table 3).

Figure 5.

Kinetics profiles of amperometric spikes from CgA- or CgB-KO chromaffin cells. A, Traces illustrate the kinetics changes observed in the exocytosis from cells lacking in CgA or CgB. Spikes were constructed by averaging spikes; their corresponding values of I_max, t_1/2, m, Ft₁, and Ft₂ coincide with the values given in Table 1 of a previous paper (Montesinos et al., 2008) and from Table 1 of this paper. Discontinuous lines show the ascending slopes (m) obtained by linear fit of 25–75% segment of the ascending portion of spikes. B, Spike amplitude versus quantal size of secretory spikes from CgB-KO (CgB^−/−) and WT mice. All spikes (from WT and CgB-KO) were pooled regardless of whether they were from WT or KO cells and then distributed into 10 intervals of increasing charge containing the same number of spikes. The spikes were then split into WT and KO, and their I_max (mean ± SEM) was analyzed. Note that, at similar quantal size, the spikes from CgB-KO have a smaller I_max than WT. U Mann–Whitney with the correction of Bonferroni, **p < 0.001. Data are averaged from the spikes of Table 1.