The granin VGF promotes genesis of secretory vesicles, and regulates circulating catecholamine levels and blood pressure

Abstract

Secretion of proteins and neurotransmitters from large dense core vesicles (LDCVs) is a highly regulated process. Adrenal LDCV formation involves the granin proteins chromogranin A (CgA) and chromogranin B (CgB); CgA- and CgB-derived peptides regulate catecholamine levels and blood pressure. We investigated function of the granin VGF (nonacronymic) in LDCV formation and the regulation of catecholamine levels and blood pressure. Expression of exogenous VGF in nonendocrine NIH 3T3 fibroblasts resulted in the formation of LDCV-like structures and depolarization-induced VGF secretion. Analysis of germline VGF-knockout mouse adrenal medulla revealed decreased LDCV size in noradrenergic chromaffin cells, increased adrenal norepinephrine and epinephrine content and circulating plasma epinephrine, and decreased adrenal CgB. These neurochemical changes in VGF-knockout mice were associated with hypertension. Germline knock-in of human VGF1-615 into the mouse Vgf locus rescued the hypertensive knockout phenotype, while knock-in of a truncated human VGF1-524 that lacks several C-terminal peptides, including TLQP-21, resulted in a small but significant increase in systolic blood pressure compared to hVGF1-615 mice. Finally, acute and chronic administration of the VGF-derived peptide TLQP-21 to rodents decreased blood pressure. Our studies establish a role for VGF in adrenal LDCV formation and the regulation of catecholamine levels and blood pressure.

Keywords: CG; LDCV; adrenal; chromaffin granule; large dense core vesicle; norepinephrine.

Figure 1.

Expression of exogenous VGF in nonendocrine NIH 3T3 cells results in the formation of LDCV-like vesicles, visualized by CLEM, and regulated secretion of VGF. A–D) NIH 3T3 cells were cotransfected with plasmids encoding GFP and either full-length rat VGF or empty vector, and identified GFP-positive cells were processed for transmission EM as detailed in Materials and Methods. Representative transmission electron micrographs of 3T3 cells transfected with pEGFPN1 alone (A), and those cotransfected with pEGFPN1 and empty pcDNA3 vector (B), or pEGFPN1 and rat VGF_1–617 expression plasmids (C, D), are shown. Arrowheads indicate LDCV-like structures. E–H) NIH 3T3 cells were similarly transfected with plasmid encoding VGF_1–65:GFP:VGF_452–617 (VGF:GFP:VGF), and LDCV-like structures were visualized in GFP-positive, green cells (E) by CLEM (F–H). Arrowheads indicate LDCV-like structures. I) To measure secretion, NIH 3T3 cells were transfected with expression plasmids encoding rat VGF_1–617, VGF_1–65:GFP:VGF_452–617 (VGF:GFP:VGF), or constitutively secreted Igκ:GFP (26). After 48–72 h, cells were rinsed, incubated for 30 min in serum-free medium (basal release), rinsed, and incubated for 30 min in serum-free medim supplemented with 2 mM BaCl₂ (stimulated release). In a subset of experiments, cells were pretreated for 2 h with CHX (1 μg/ml), which blocks constitutive but has less effect on regulated secretion, or with vehicle, and basal or stimulated release was assayed as described above, in the presence of CHX or vehicle. Secreted VGF, VGF:GFP:VGF, or Igκ:GFP proteins were quantified by Western blot analysis and ImageJ, as described in Materials and Methods. Normalized to basal release (1±0.2), VGF secretion was stimulated 2.8 ± 0.1-fold by BaCl₂ (P=0.004, 2-tailed Student's t test). VGF:GFP:VGF secretion under basal conditions (1±0.3) was stimulated 18.6 ± 1.3-fold by BaCl₂ (P<0.001, ANOVA with Tukey's post hoc comparison, n=3), while basal + CHX release (2±0.4) was stimulated 12.2 ± 0.7-fold in BaCl₂ + CHX-treated cells (P<0.001, ANOVA with Tukey's post hoc comparison, n=3) (all values normalized to VGF:GFP:VGF secretion under basal conditions). Igκ:GFP secretion under basal conditions (1±0.12) was not stimulated by BaCl₂ treatment but rather was significantly reduced (0.4±0.14; P<0.05, ANOVA with Tukey's post hoc comparison, n=3). Secretion of Igκ:GFP in the presence of CHX (0.5±0.07) was not stimulated by BaCl₂ + CHX (0.2±0.03; nonsignificant compared to CHX). Igκ:GFP secretion in BaCl₂ + CHX-treated cells was significantly reduced compared to basal secretion (P=0.0025) (all values normalized to Igκ:GFP secretion under basal conditions). Results are representative of ≥3 experiments.

Figure 2.

DCG (LDCV) area but not number is decreased in adrenal medullary chromaffin cells from VGF-knockout (Vgf^−/−) compared to wild-type (Vgf^+/+) mice. A–D) Representative EM micrographs of Vgf^+/+ (A, C) and Vgf^−/− (B, D) adrenal medullary chromaffin cells, taken at ×10,000 (A, B) and at ×30,000 (C, D). Scale bars = 2 μm (A, B); 500 nm (C, D). E–G) Tissues were prepared for EM, and ×30,000 images were analyzed using MetaMorph to determine mean LDCV core area in square nanometers (E), LDCV number per square micrometer of cytoplasm (F), and the distribution of LDCVs having specified square nanometer areas (G). Values are means ± sem. LDCV core area in Vgf^+/+ chromaffin cells was significantly different from that in Vgf^−/− cells (n=3228 Vgf^+/+ DCVs; n=2551 Vgf^−/− DCVs). ***P < 0.0001; 2-tailed Student's t test. H–K) As shown (H), the area around the dense core (J) and the area of the membrane-bound LDCV (K) were both significantly decreased in Vgf^−/− chromaffin cells (I). *P < 0.05, **P < 0.005; unpaired 2-tailed Student's t test.

Figure 3.

Adrenal CgB protein levels are reduced in Vgf^+/− and Vgf^−/− mice in comparison to wild-type Vgf^+/+ mice. Adrenal glands were isolated from wild-type Vgf^+/+, heterozygous-knockout Vgf^+/−, and homozygous-knockout Vgf^−/− mice and extracted in RIPA buffer, and Western blot analysis for CgA, CgB, SgII, and SgIII was carried out. Levels of CgA (A), CgB (B), SgII (C), and SgIII (D) were determined by densitometry of scanned films (E) and normalized to their respective actin loading controls, and are expressed relative to the level of the respective wild-type granin (means±sem; n=2–5 mice/group). *P < 0.05, **P < 0.005; ANOVA with Tukey's post hoc comparison.

Figure 4.

Coexpression of CgB and VGF in adrenal chromaffin and PC12 pheochromocytoma cells. A, B) Coimmunoprecipitation analysis was used to determine whether CgB and VGF associate in PC12 pheochromocytoma cells. PC12 cell lysates were immunoprecipitated with rabbit anti-VGF or normal rabbit IgG (A), and with goat anti-CgB or normal goat IgG (B), and complexes were collected and analyzed by SDS-PAGE and Western blotting with anti-CgB (A) and anti-VGF (B; asterisks indicate nonspecific bands). PC12 cell lysate (25 μg) was coanalyzed as a positive control and represented 5% of the input lysate immunoprecipitated. C) PC12 cell lysates were immunoprecipitated with anti-SgII, anti-CgA, or anti-SgIII, and analyzed by SDS-PAGE and Western blotting with anti-VGF; no coimmunoprecipitated VGF was identified (asterisks indicate nonspecific bands). D) Western blot analysis revealed VGF protein (arrows ∼85 and ∼90 kDa) in adrenal gland lysates from male and female wild-type mice and rat PC12 cells. E–J) VGF and CgB distributions in PC12 cells were analyzed by indirect immunofluorescence and confocal microscopy. Coexpression of VGF (red; E, H) and CgB (green; F, I) is shown in merge panels (yellow; G, J). K–Q) VGF (red; K, O) and CgB (green; L, P) were similarly coexpressed (yellow; M, N, Q) in sections of mouse adrenal gland (K–N) and, shown at lower magnification, rat adrenal gland (O–Q).

Figure 5.

Adrenal catecholamine content and plasma epinephrine levels are increased in VGF homozygous-knockout compared to wild-type mice. Norepinephrine (A, C, E) and epinephrine (B, D, F) levels were quantified in adrenal gland (A, B), plasma (C, D), and heart (E, F), from wild-type Vgf^+/+ (WT), heterozygous-knockout Vgf^+/− (HET), and homozygous-knockout Vgf^−/− (KO) mice, as described in Materials and Methods (means±sem; n=4–8 mice/group). *P < 0.05, **P < 0.005, ***P < 0.0001; ANOVA with Tukey's post hoc comparison.

Figure 6.

Germline VGF ablation increases BP that is rescued by germline knock-in of human VGF_1–615 into the mouse Vgf locus, consistent with reduced BP in VGF peptide TLQP-21-treated mice and rats. A, B) BP (A) and pulse (B) were recorded utilizing tail plethysmography in awake wild-type Vgf^+/+, heterozygous-knockout Vgf^+/−, and homozygous-knockout Vgf^−/− mice, as described in Materials and Methods. SBP, DBP, MAP, and pulse (bpm) are shown, and SBP, DBP, and MAP were significantly increased in Vgf^−/− (KO) compared to Vgf^+/+ (WT) and Vgf^+/− (HET) mice (means±sem; n=9–12 mice/group). *P < 0.05, ***P < 0.0001; 1-way ANOVA. C) Chronic 14-d treatment of obese, hypertensive mice with TLQP-21 (400 μg/d) decreased SBP and DBP, recorded by intracarotid catheterization under anesthesia, while a lower dose of TLQP-21 (40 μg/d) was ineffective. STD, standard diet; sal, saline. *P < 0.05, **P < 0.01 vs. HFD-sal group. D) Acute intravenous infusion of TLQP-21 (5 mg/kg) rapidly and transiently decreased MAP in freely moving Sprague-Dawley rats (F_(61,122)=4.8, P<0.0001; n=4) measured by telemetry, while infusion of a lower dose of TLQP-21 (1 mg/kg) was ineffective (n=2). Dashed line marks peptide infusion. **P < 0.01 vs. baseline; ANOVA with Tukey's post hoc comparison. E–H) Germline knock-in of human VGF_1–615 or an SNP-encoded, truncated human VGF_1–524 into the mouse Vgf locus rescued the hypertensive phenotype resulting from germline VGF ablation. E) Gene-targeting strategy used to generate hVGF and SNP lines. F) Western blotting with anti-VGF antisera was used to identify full-length human VGF_1–615 and truncated human VGF_1–524 in total brain lysates from homozygous hVGF and SNP mice, respectively. G, H) SBP, DBP, and MAP (G) and pulse (bpm; H) were measured by tail plethysmography in awake wild-type (WT), and homozygous hVGF_1–615 (HUM) and hVGF_1–524 (SNP) mice (means±sem; n=9–12 mice/group). Note that SBP in homozygous hVGF_1–615 is slightly but significantly lower than homozygous hVGF_1–524 mice that lack TLQP-21 (G). ***P < 0.0001; 1-way ANOVA.