Volume 16, Issue 2, Pages 302-307 (February 2008) Modulation of Adrenal Catecholamine Secretion by In Vivo Gene Transfer and Manipulation of G Protein–coupled Receptor Kinase-2 Activity  Anastasios Lymperopoulos, Giuseppe Rengo, Carmela Zincarelli, Stephen Soltys, Walter J Koch  Molecular Therapy  Volume 16, Issue 2, Pages 302-307 (February 2008) DOI: 10.1038/sj.mt.6300371 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 The retrograde suprarenal rat vein–mediated gene delivery technique.See text for details. Note the difference between the venous circulations in the two adrenal glands in the rat. LSG, left suprarenal gland; RSG, right suprarenal gland; 1, inferior vena cava; 2, right renal vein; 3, left renal vein; 4, right suprarenal vein; 5, left suprarenal vein. Red arrows indicate direction of blood flow and green arrows indicate direction of adenoviral injection (retrograde delivery). The black-dotted circles indicate the sites of the vein ligations preceding the virus injection. Molecular Therapy 2008 16, 302-307DOI: (10.1038/sj.mt.6300371) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Evaluation of adrenal gene delivery efficiency using (a) β -galactosidase (β -Gal) staining and (b) green fluorescent protein (GFP) fluorescence. (a)Representative confocal images (at ×10 or ×40 magnification) of β -Gal staining of rat adrenal sections infected in vivo with AdlacZ either by the direct intra-adrenal (direct injection) method or by the suprarenal vein-mediated (vein injection) method (see also “Materials and Methods”). Adrenal medulla section-specific images are also shown (medulla). (b)Representative confocal images of GFP fluorescence [fluorescein isothiocyanate (FITC)], and its overlay with transmission images, of rat adrenal sections infected in vivo with AdGFP, either by the direct intra-adrenal (direct injection) method or by the suprarenal vein-mediated (vein injection) method (see “Materials and Methods”). Images of adrenal sections injected in vivo with saline are also shown as negative controls. Molecular Therapy 2008 16, 302-307DOI: (10.1038/sj.mt.6300371) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Adrenal-specific β ARKct transgene expression in vivo. (a) Immunoblotting for β ARKct with a specific anti-GRK2 antibody (see “Materials and Methods”) in protein extracts from excised rat adrenal glands injected in vivo with saline (lane 1), with Adβ ARKct through the suprarenal vein (lane 2), or with Adβ ARKct directly injected into the gland (lane 3), 7 days after in vivo infection. Shown is a representative blot of three separate gene delivery experiments carried out using each of the two methods. The blot for actin is also shown as a loading control. (b) Immunoblotting for β ARKct in protein extracts from hearts, kidneys, lungs, and livers of rats in which adrenal-specific in vivo infection with Adβ ARKct was conducted. A representative blot is shown indicating the absence of any appreciable β ARKct peptide expression in these tissues. Equal protein (50 μg of protein) was loaded in each lane, as also indicated by the blot for actin as loading control. GRK2, G protein–coupled receptor kinase-2. Molecular Therapy 2008 16, 302-307DOI: (10.1038/sj.mt.6300371) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Manipulation of adrenal G protein–coupled receptor kinase-2 (GRK2) levels modulates plasma catecholamine levels in vivo. (a)Plasma catecholamine levels in rats 7 days after adrenal-specific in vivo gene delivery of AdGFP, AdGRK2, or Adβ ARKct (see “Materials and methods”).*P < 0.05, compared to AdGFP; **P < 0.05, compared to AdGRK2; n = 5 rats/group. (b)Immunoblotting for GRK2 and β ARKct in protein extracts from the excised adrenal glands of these rats at 7 days after in vivo gene delivery. Representative blots are shown, including blots for glyceraldehyde-3-phosphate dehydrogenase as loading control. Molecular Therapy 2008 16, 302-307DOI: (10.1038/sj.mt.6300371) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 In vivo modulation of adrenal G protein–coupled receptor kinase-2 (GRK2) activity regulates chromaffin cell α 2AR function. In vitro epinephrine and norepinephrine secretion from chromaffin cells isolated from the adrenals of rats infected in vivo with AdGFP, AdGRK2, or Adβ ARKct in response to 20 μmol/l nicotine treatment, following pretreatment with vehicle (Nicotine) or with 10 μmol/l UK14304 (UK+Nicotine) (see “Materials and Methods”). UK14304 pretreatment alone had no effect (data not shown). *P < 0.05, compared to AdGFP-UK+Nicotine, **P < 0.05, compared to Adβ ARKct-UK+Nicotine, n = 9. Molecular Therapy 2008 16, 302-307DOI: (10.1038/sj.mt.6300371) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions