The crucial role of chromogranins in storage and exocytosis revealed using chromaffin cells from chromogranin A null mouse

Abstract

Chromogranins (Cgs) are the major soluble proteins of dense-core secretory vesicles. Chromaffin cells from Chga null mice [chromogranin A knock-out (CgA-KO)] exhibited approximately 30% reduction in the content and in the release of catecholamines compared with wild type. This was because of a lower secretion per single exocytotic event, rather than to a lower frequency of exocytotic events. Cell incubation with L-DOPA produced an increase in the vesicular amine content of wild-type, but not CgA-KO vesicles. In contrast, intracellular electrochemistry showed that L-DOPA produced a significantly larger increase in cytosolic amines in CgA-KO cells than in the wild type. These data indicate that the mechanisms for vesicular accumulation in CgA-KO cells were fully saturated. Patch-amperometry recordings showed a delayed initiation of the amperometric signal after vesicle fusion, whereas no changes were observed in vesicle size or fusion pore kinetics despite the smaller amine content. We conclude that intravesicular proteins are highly efficient systems directly implicated in transmitter accumulation and in the control of neurosecretion.

Figure 1.

Immunohistochemical characterization of CgA-KO mice. Adrenal gland sections were compared with their control strains (isogenic background, WT). Primary polyclonal rabbit antibodies against CgA or monoclonal anti-mouse against TH were used. As a secondary antibody we used an anti-rabbit or anti-mouse IgG labeled with horseradish peroxidase. The figure shows sections of the mouse adrenal gland immunostained with anti-CgA or anti-TH. Scale bars: (in A) A, B, E, F, 300 μm; (in C) C, D, G, H, 50 μm.

Figure 2.

Changes in the expression of granins observed in CgA-KO (−/−) versus WT (+/+) mice. A, Western blots were performed on CgA-KO adrenal medullary homogenates using anti-α-tubulin as an internal control. B, Results (mean ± SEM) from six different analyses were pooled. *p < 0.05, Student's t test.

Figure 3.

Secretory responses of CgA-KO chromaffin cells studied with amperometry. A, Representative trace (gray) showing the amperometric recording from an isolated cell stimulated for 5 s with 5 mm BaCl₂. Each spike represents the CA released from a single vesicle. The black outline shows the cumulative secretory response obtained by integration of the amperometric charge after baseline subtraction. B, Total secretion (mean ± SEM) comparing WT with CgA-KO cells; the cumulative charges were measured for 2 min after the stimulus and expressed in picocoulombs. The number of cells from each group is expressed in parentheses. *p < 0.05, Student's t test.

Figure 4.

Quantal secretion of catecholamines from adrenal chromaffin cells. Probability distribution of secretory events during a 2 min recording period. Secretion was elicited by pressure injection of 5 mm Ba²⁺ for 5 s from a micropipette placed 40 μm away from the cell. Each circle represents the average of spike frequencies recorded within 1 s bins from control cells (n = 10). The solid line is the curve fitting. The distribution of probability reaches a maximum at 20 s after stimulation by BaCl₂.

Figure 5.

Spike amplitude versus quantal size of secretory spikes from CgA-KO and WT mice. All spikes (from WT and CgA-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) analyzed. Note that, at similar quantal size, the spikes from CgA-KO have a larger I _max than WT. *p < 0.005; **p < 0.001, Mann–Whitney U test with Bonferroni correction. Data are averaged from the spikes accounted in Table 1.

Figure 6.

Effects of l-DOPA incubation on the secretory spikes from WT and CgA-KO cells. Cells attached to coverslips were incubated in a culture medium containing 0 (C) or 100 μm l-DOPA (L–D) for 90 min at 37°C. The coverslips were then washed twice with Krebs-HEPES buffer and used for conventional amperometry. A–C, The effects of l-DOPA on spike height (A), quantal size (B), and t_1/2 (C). Numbers in parentheses indicate the number of cells used. *p < 0.05 and **p < 0.01 (Student's t test) when comparing effects of l-DOPA incubation against the corresponding control; ^# p < 0.05 when comparison was done between untreated (C) cells from WT- and CgA-KO-animals.

Figure 7.

Effects of l-DOPA overload on the relation between catecholamine content of vesicles (Q) and the maximal concentration reaching the carbon fiber electrode (I _max). Plots show the relation of spike amplitude versus quantal size in the absence (control) and after cell incubation with 100 μm l-DOPA for 90 min in WT cells (n = 1483 spikes from 15 cells; left) and CgA-KO cells (n = 1127 spikes from 13 cells; right). The description of the method to perform this analysis is in the legend of Figure 5. Note that the tight relation between I _max and Q in the spikes recorded from WT cells did not occur in the CgA-KO. In these cells, l-DOPA treatment promoted the increase in the I _max from vesicles of similar quantal size. *p < 0.005; **p < 0.001, Mann–Whitney U test with Bonferroni correction.

Figure 8.

L-DOPA overload suggests a lack of accumulation of newly synthesized CA (after overload) in vesicles from the CgA-KO mouse. A, Diagram showing the experimental approach using patch amperometry in the whole-cell configuration. The plasma membrane was ruptured by suction and a whole-cell configuration was achieved. This allows secretory vesicles, CA, and other soluble molecules to diffuse from the cytosol into the pipette. B, The capacitance jump indicates the opening of the plasma membrane to the whole cell configuration. The cytosolic oxidable molecules are monitored as a rapid increase in current followed by a slow decaying wave by the amperometry microelectrode. Observe the presence of amperometric spikes in the zoomed portion of the decaying slope. After spike area subtraction, the total area of this oxidation current wave (gray) was used as a measure of the amount of cytosolic free CA depicted as the smooth ascending line. C, Averaged time course of free CA levels in cells from WT mice not exposed to l-DOPA (thick trace, n = 8) and from other cells after l-DOPA + pargyline treatment (thin trace, n = 7). D, As in C but in CgA-KO cells (n = 6 and 7 cells). E, Average values from integration of the curves from C and D. Numbers in parentheses indicate the number of cells used. *p < 0.05; **p < 0.01, Student's t test.

Figure 9.

Fusion pore kinetics are not involved in the descending portion of spikes. Figure shows the four traces obtained using cell-attached patch amperometry. Top trace shows the conductance of the fusion pore (Gp), the second trace shows the real projection directly taken from the lock-in amplifier (Re), the third trace shows the capacitance trace (Im), and the bottom trace corresponds to the amperometric signal. Vertical dashed lines indicate the starting point of the fusion (i), the beginning of secretory spike (ii), and the point on the descending slope of the amperometric spike that equals the value of Ft₁ (iii). The time interval between i and ii defines Δt (Table 2). A, Time course of a complete fusion event. B, Horizontal expansion of traces from A.