PKA delivery to the distal lung air spaces increases alveolar liquid clearance after isoproterenol-induced alveolar epithelial PKA desensitization

Abstract

Isoproterenol (Iso) infusion for 48 h in rats decreases the ability of beta-adrenoceptor (beta-AR) agonists to increase alveolar liquid clearance (ALC). An impairment in protein kinase A (PKA) function appears to be critical in producing the desensitized ALC response. To test this hypothesis, we used a novel protein delivery reagent (Chariot, Active Motif) to deliver either the PKA catalytic subunit or the PKA holoenzyme to the distal lung epithelium of Iso-infused rats (400 microg.kg(-1).h(-1), 48 h). After this infusion, ALC was measured by mass balance over 2 h. ALC in Iso-infused rats was 27.9% (SD 5.8) of instilled volume absorbed. Delivery of the catalytic PKA subunit to Iso-infused rats increased ALC to 47.7% (SD 8.9) (P < 0.05). ALC in Iso-infused rats delivered the inactive PKA holoenzyme [29.6% (SD 2.5)] was not increased above baseline values. Subsequent holoenzyme activation by intravenous infusion of the stable cAMP analog Sp-8-Bromo-cAMPS increased ALC to 41.7% (SD 8.8) (P < 0.05). Immunohistochemical localization of Chariot-delivered PKA revealed staining in the alveolar and distal airway epithelium. These data indicate that protein delivery reagents can be used to rapidly deliver biologically active proteins to the distal lung epithelium and that PKA desensitization may be an important rate-limiting event in the development of Iso-induced desensitization of the alveolar epithelial beta-AR signaling pathway.

Mechanotransduction Drives Post Ischemic Revascularization Through KATP Channel Closure and Production of Reactive Oxygen Species

“…We investigated whether the restoration of VEGF expression in the null mice would rescue the revascularization to WT levels. For this, we delivered VEGF protein to K IR 6.2 null mice in vivo, post-hind limb ischemia (by Chariot delivery method, see Materials and Methods section) (4,41). Introduction of VEGF in K IR 6.2 -/ -mice restored revascularization to WT levels (Fig.…”

“…Chariot has been reported to deliver biologically active proteins, peptides, and antibodies directly into cells in vitro and tissues in vivo (4,39,41). Our protocol involved incubation of the Chariot agent with protein (1 lg VEGF/6 ll chariot stock) for 30 min at room temperature followed by injecting it into the ischemic region (between the sutures) about 30 min post-hind limb ischemia and once each day (1 lg/day) thereafter for the next 5 days.…”

Protein vaccination with HPV16 E7/Pep‐1 nanoparticles elicits a protective T‐helper cell‐mediated immune response

“…A major key for successful protein transfer into cells is the development of protein delivery systems with high efficiency and low cytotoxicity (19). Pep-1 as a cell penetrating peptide has been effectively used to transfer different cargoes (peptide nucleic acids or PNAs, peptides, and proteins) into the cells (20)(21)(22)(23)(24)(25)(26)Gros et al,personal communication). Several advantages of the noncovalent complexes of Pep-1/cargo contain: (a) rapid delivery of proteins into cells with high efficiency, (b) stability in physiological buffers, and (c) lack of toxicity and sensitivity to serum.…”

“…It involves transient membrane disorganization along with folding of Pep-1 into a helical structure within the phospholipids membrane (27,28). Many types of proteins, antibodies, and peptides were successfully delivered into different cell lines using Pep-1 strategy and were represented to retain their full biological activity (20)(21)(22)(23)(24)(25)(26)Gros et al,personal communication). In this study, the potency of Pep-1 cell penetrating peptide was assessed for HPV16 E7 protein delivery in vitro.…”

Alveolar Epithelial Ion and Fluid Transport

“…Although one study indicated that ENaC can be phosphorylated by PKA (66), the functional relevance of phosphorylation is not yet known and PKA does not activate an amiloride-sensitive current in Xenopus oocytes injected with ENaC mRNAs (67). However, delivery of PKA catalytic subunits to the lung by protein transfection increased the AFC rate in rats (68). ENaC subunits, like other ion channels and transporters, are associated with cytoskeletal proteins such as actin, ankyrin, fodrin, or spectrin, which can serve as signaling molecules.…”