Lipopeptide-based micellar and liposomal carriers: Influence of surface charge and particle size on cellular uptake into blood brain barrier cells.

Lipopeptide-based micelles and liposomes were found to differ in cell recognition and uptake mode into blood brain barrier (BBB) endothelial cells. Here we analyse the role of size and surface charge of micelles and liposomes composed of different lipopeptide sequences with respect to uptake into human brain capillary (HBMEC) and aortic (HAoEC) endothelial cells. Comparable to the dipalmitoylated apolipoprotein E-derived P2A2, lipopeptides of cationic poly-arginine (P2Rn), poly-lysine (P2Kn) and an anionic glutamic-acid sequence (P2En) self assemble into micelles (12-14nm in diameter) with high surface charge density, and bind to small (SUVs, about 24nm in diameter) and large (LUV, about 100nm in diameter) liposomes at variable lipid to peptide ratios. The interaction pattern of the resulting particles with endothelial cells is highly variable as revealed by confocal laser scanning microscopic (CLSM) and fluorescence assisted cell sorting (FACS) studies. Micelles and SUVs with high P2A2 density are efficiently and selectively internalized into HBMEC. P2Kn micelles strongly accumulate in both the cytosol and at the cell membrane, while the interaction of liposomes tagged with a low amount of P2A2 and P2Kn with the cells was reduced. Anionic micelles seem to dissociate in the presence of cells and P2En molecules incorporate into the cellular membrane whereas the negatively charged liposomes hardly interact with cells. Surprisingly, all poly-R-based particles show high selectivity for HBMEC compared to HAoEC, independent of particle size and peptide surface density. The P2Rn-mediated internalization is highly efficient and partially clathrin-dependent. The oligo-R lipopeptide is considered to be most promising to selectively transport different drug carriers into the blood brain barrier.

Structural disorder of monomeric α-synuclein persists in mammalian cells.

Intracellular aggregation of the human amyloid protein α-synuclein is causally linked to Parkinson's disease. While the isolated protein is intrinsically disordered, its native structure in mammalian cells is not known. Here we use nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy to derive atomic-resolution insights into the structure and dynamics of α-synuclein in different mammalian cell types. We show that the disordered nature of monomeric α-synuclein is stably preserved in non-neuronal and neuronal cells. Under physiological cell conditions, α-synuclein is amino-terminally acetylated and adopts conformations that are more compact than when in buffer, with residues of the aggregation-prone non-amyloid-ß component (NAC) region shielded from exposure to the cytoplasm, which presumably counteracts spontaneous aggregation. These results establish that different types of crowded intracellular environments do not inherently promote α-synuclein oligomerization and, more generally, that intrinsic structural disorder is sustainable in mammalian cells.

Is transmission electron microscopy (TEM) a promising approach for qualitative and quantitative investigations of polymyxin B and miconazole interactions with cellular and subcellular structures of Staphylococcus pseudintermedius, Escherichia coli, Pseudomonas aeruginosa and Malassezia pachydermatis?

Antimicrobial therapy using a combination of polymyxin B and miconazole is effective against the main bacterial pathogens associated with otitis externa in dogs, and a synergistic effect of both drugs has been shown previously. The objective of the present investigation was to visualize ultrastructural changes after exposure of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus pseudintermedius and Malassezia pachydermatis to polymyxin B and miconazole by transmission electron microscopic (TEM). For this, cultures of E. coli, P. aeruginosa, S. pseudintermedius and M. pachydermatis were exposed to polymyxin B and miconazole, alone or in combination for 24 h. Ultrastructural changes were observed most frequently in the cell envelope of the four microorganisms. Exposure to polymyxin B seemed to cause more damage than miconazole within the range of concentrations applied. Treatment resulted in changes of the cell size: in E. coli, cell size increased significantly after treatment with either compound alone; in P. aeruginosa, cell size decreased significantly after treatment with polymyxin B and with miconazole; exposure of S. pseudintermedius to miconazole caused a decrease in cell size; in M. pachydermatis, cell size increased significantly after treatment with polymyxin B.; in E.coli, S. pseudintermedius and M. pachydermatis, cell size changed highly significant, in P. aeruginosa significantly after exposure to the combination of both compounds. In conclusion, by using a different approach than previous investigations, this study confirmed a clear combinatory effect of polymyxin B and miconazole against the tested microorganisms involved in canine otitis externa. It is the first time that visualization technologies were applied to compare the effect of single drugs to their combinatory effects on cellular and subcellular entities of selected bacterial and yeast species.

Redox Regulation of Cell Contacts by Tricellulin and Occludin: Redox-Sensitive Cysteine Sites in Tricellulin Regulate Both Tri- and Bicellular Junctions in Tissue Barriers as Shown in Hypoxia and Ischemia.

UNLABELLED: Tight junctions (TJs) seal paracellular clefts in epithelia/endothelia and form tissue barriers for proper organ function. TJ-associated marvel proteins (TAMPs; tricellulin, occludin, marvelD3) are thought to be relevant to regulation. Under normal conditions, tricellulin tightens tricellular junctions against macromolecules. Traces of tricellulin occur in bicellular junctions. AIMS: As pathological disturbances have not been analyzed, the structure and function of human tricellulin, including potentially redox-sensitive Cys sites, were investigated under reducing/oxidizing conditions at 3- and 2-cell contacts. RESULTS: Ischemia, hypoxia, and reductants redistributed tricellulin from 3- to 2-cell contacts. The extracellular loop 2 (ECL2; conserved Cys321, Cys335) trans-oligomerized between three opposing cells. Substitutions of these residues caused bicellular localization. Cys362 in transmembrane domain 4 contributed to bicellular heterophilic cis-interactions along the cell membrane with claudin-1 and marvelD3, while Cys395 in the cytosolic C-terminal tail promoted homophilic tricellullar cis-interactions. The Cys sites included in homo-/heterophilic bi-/tricellular cis-/trans-interactions contributed to cell barrier tightness for small/large molecules. INNOVATION: Tricellulin forms TJs via trans- and cis-association in 3-cell contacts, as demonstrated electron and quantified fluorescence microscopically; it tightens 3- and 2-cell contacts. Tricellulin's ECL2 specifically seals 3-cell contacts redox dependently; a structural model is proposed. CONCLUSIONS: TAMP ECL2 and claudins' ECL1 share functionally and structurally similar features involved in homo-/heterophilic tightening of cell-cell contacts. Tricellulin is a specific redox sensor and sealing element at 3-cell contacts and may compensate as a redox mediator for occludin loss at 2-cell contacts in vivo and in vitro. Molecular interaction mechanisms were proposed that contribute to tricellulin's function. In conclusion, tricellulin is a junctional redox regulator for ischemia-related alterations.

Anchoring dipalmitoyl phosphoethanolamine to nanoparticles boosts cellular uptake and fluorine-19 magnetic resonance signal.

Magnetic resonance (MR) methods to detect and quantify fluorine ((19)F) nuclei provide the opportunity to study the fate of cellular transplants in vivo. Cells are typically labeled with (19)F nanoparticles, introduced into living organisms and tracked by (19)F MR methods. Background-free imaging and quantification of cell numbers are amongst the strengths of (19)F MR-based cell tracking but challenges pertaining to signal sensitivity and cell detection exist. In this study we aimed to overcome these limitations by manipulating the aminophospholipid composition of (19)F nanoparticles in order to promote their uptake by dendritic cells (DCs). As critical components of biological membranes, phosphatidylethanolamines (PE) were studied. Both microscopy and MR spectroscopy methods revealed a striking (at least one order of magnitude) increase in cytoplasmic uptake of (19)F nanoparticles in DCs following enrichment with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). The impact of enriching (19)F nanoparticles with PE on DC migration was also investigated. By manipulating the nanoparticle composition and as a result the cellular uptake we provide here one way of boosting (19)F signal per cell in order to overcome some of the limitations related to (19)F MR signal sensitivity. The boost in signal is ultimately necessary to detect and track cells in vivo.

Clostridium difficile toxin B inhibits the secretory response of human mast cell line-1 (HMC-1) cells stimulated with high free-Ca²âº and GTPγS.

Clostridium difficile toxins A and B (TcdA and TcdB) belong to the class of large clostridial cytotoxins and inactivate by glucosylation some low molecular mass GTPases of the Rho-family (predominantly Rho, Rac and Cdc42), known as regulators of the actin cytoskeleton. TcdA and B also represent the main virulence factors of the anaerobic gram-positive bacterium that is the causal agent of pseudomembranous colitis. In our study, TcdB was chosen instead of TcdA for the well-known higher cytotoxic potency. Inactivation of Rho-family GTPases by this toxin in our experimental conditions induced morphological changes and reduction of electron-dense mast cell-specific granules in human mast cell line-1 (HMC-1) cells, but not cell death or permeabilisation of plasma-membranes. Previously reported patch-clamp dialysis experiments revealed that high intracellular free-Ca(2+) and GTPγS concentrations are capable of inducing exocytosis as indicated by significant membrane capacitance (Cm) increases in HMC-1 cells. In this study, we investigated the direct effects of TcdB upon HMC-1 cell "stimulated" Cm increase, as well as on "constitutive" secretion of hexosaminidase and interleukin-16 (IL-16). Compared to untreated control cells, HMC-1 cells incubated with TcdB for 3-24h exhibited a significant reduction of the mean absolute and relative Cm increase in response to free-Ca(2+) and GTPγS suggesting an inhibition of secretory processes by TcdB. In conclusion, the HMC-1 cell line represents a suitable model for the study of direct effects of C. difficile toxins on human mast cell secretory activity.

Interferon-γ safeguards blood-brain barrier during experimental autoimmune encephalomyelitis.

The function of blood-brain barrier is often disrupted during the progression of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the molecular mechanism of blood-brain barrier modulation during neuroinflammation remains unclear. Herein, we show that the expression of interferon-γ (IFNγ) receptor on endothelial cells (ECs) protected mice from the brain inflammation during EAE. IFNγ stabilized the integrity of the cerebral endothelium and prevented the infiltration of leukocytes into the brain. Further analysis revealed that IFNγ increased the expression of tight junction proteins zonula occludens protein 1 and occludin, as well as membranous distribution of claudin-5, in brain ECs. Silencing claudin-5 abolished the IFNγ-mediated improvement of EC integrity. Taken together, our results show that IFNγ, a pleiotropic proinflammatory cytokine, stabilizes blood-brain barrier integrity and, therefore, prevents brain inflammation during EAE.

Clathrin/AP-2 mediate synaptic vesicle reformation from endosome-like vacuoles but are not essential for membrane retrieval at central synapses.

Neurotransmission depends on presynaptic membrane retrieval and local reformation of synaptic vesicles (SVs) at nerve terminals. The mechanisms involved in these processes are highly controversial with evidence being presented for SV membranes being retrieved exclusively via clathrin-mediated endocytosis (CME) from the plasma membrane or via ultrafast endocytosis independent of clathrin. Here we show that clathrin and its major adaptor protein 2 (AP-2) in addition to the plasma membrane operate at internal endosome-like vacuoles to regenerate SVs but are not essential for membrane retrieval. Depletion of clathrin or conditional knockout of AP-2 result in defects in SV reformation and an accumulation of endosome-like vacuoles generated by clathrin-independent endocytosis (CIE) via dynamin 1/3 and endophilin. These results together with theoretical modeling provide a conceptual framework for how synapses capitalize on clathrin-independent membrane retrieval and clathrin/AP-2-mediated SV reformation from endosome-like vacuoles to maintain excitability over a broad range of stimulation frequencies.

Human mast cell line-1 (HMC-1) cells exhibit a membrane capacitance increase when dialysed with high free-Ca(2+) and GTPγS containing intracellular solution.

An increase in cytosolic free calcium concentration [Ca(2+)]i initiates the exocytotic activity in various types of secretory cells. The guanosine 5'-O-[3-thio]triphosphate (GTPγS), a non-hydrolysable analogue of GTP (guanosine 5'-triphosphate), is an effective secretagogue for different cell types of different species, like mast cells, neutrophils or eosinophils. Consequently, the internal administration of GTPγS causes degranulation of mouse and rat mast cells. Regarding rat mast cells, it is proved that Ca(2+) can cooperate with GTP or GTPγS in accelerating and increasing amplitude of the secretory response. All the previous studies with respect to capacitance recordings and mast cells were performed using mouse or rat mast cells, usually derived from peritoneum or the rat basophilic leukaemia cell line RBL. In this study, we applied the capacitance measurement technique to the human mast cell line-1 (HMC-1) cells, an immature cell line established from a patient with mast cell leukaemia. Patch-clamp dialysis experiments revealed that high intracellular free Ca(2+) and GTPγS concentrations are both required for considerable capacitance increases in HMC-1 cells. During degranulation of HMC-1 cells, the total membrane capacitance (Cm) increase appeared continuously and, in some cases, as a discrete capacitance change, developing in a stepwise manner. Then, we tested the effect of latrunculin B upon HMC-1 cell capacitance increase as well as of some classic mast cell stimulators like PMA, A23187 and IL-1ß in hexosaminidase release. Finally, we could conclude that the HMC-1 cell line represents a suitable model for the study of human mast cell degranulation.

Light-controlled toxicity of engineered amyloid ß-peptides.

Aggregation of amyloid ß (Aß(1-42)), causing toxicity, is a critical step in Alzheimer's disease (AD). AD studies are difficult to compare because Aß(1-42) aggregation is poorly controllable under physiological conditions. To control aggregation and toxicity, we engineered light-switchable Aß(1-42) analogues that enable controllable conversion of nontoxic fibrils into toxic oligomers simply by illumination.

Overexpression of claudin-5 but not claudin-3 induces formation of trans-interaction-dependent multilamellar bodies.

Tight junctions (TJs) regulate paracellular barriers and claudins (Cld) form the backbone of TJ strands. To elucidate the molecular mechanism of claudin polymer formation, TJs were reconstituted by claudin transfection of TJ-free HEK293 cells. Therewith, typical TJ stands can be found at cell-cell contacts. In addition, overexpression of Cld5-YFP induces formation of huge intracellular multilamellar bodies. In contrast, Cld3 does not induce similar structures. Inhibition of trans-interaction of Cld5 by Y148A substitution diminished formation of multilamellar bodies. These results demonstrate claudin subtype-specific oligomerization. Cld3 and Cld5 localize to the plasma membrane differentially. Phosphorylation at T207 of Cld5 was suggested to participate in regulation of Cld5 internalization. However, prevention of potential phosphorylation by T207A substitution did not increase Cld5 amount in the plasma membrane of transfected cells. Taken together, if carefully evaluated, transfection of claudin constructs in nonpolar cells is a powerful strategy to improve understanding of subcellular targeting and assembly of TJ proteins.

Treatment of experimental brain metastasis with MTO-liposomes: impact of fluidity and LRP-targeting on the therapeutic result.

PURPOSE: To test targeted liposomes in an effort to improve drug transport across cellular barriers into the brain. METHODS: Therefore we prepared Mitoxantrone (MTO) entrapping, rigid and fluid liposomes, equipped with a 19-mer angiopeptide as ligand for LDL lipoprotein receptor related protein (LRP) targeting. RESULTS: Fluid, ligand bearing liposomes showed in vitro the highest cellular uptake and transcytosis and were significantly better than the corresponding ligand-free liposomes and rigid, ligand-bearing vesicles. Treatment of mice, transplanted with human breast cancer cells subcutaneously and into the brain, with fluid membrane liposomes resulted in a significant reduction in the tumor volume by more than 80% and in a clear reduction in drug toxicity. The improvement was mainly depended on liposome fluidity while the targeting contributed only to a minor degree. Pharmacokinetic parameters were also improved for liposomal MTO formulations in comparison to the free drug. So the area under the curve was increased and t(1/2) was extended for liposomes. CONCLUSION: Our data show that it is possible to significantly improve the therapy of brain metastases if MTO-encapsulating, fluid membrane liposomes are used instead of free MTO. This effect could be further enhanced by fluid, ligand bearing liposomes.

Small molecule AKAP-protein kinase A (PKA) interaction disruptors that activate PKA interfere with compartmentalized cAMP signaling in cardiac myocytes.

A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating ß-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.

Interaction of angiotensin-converting enzyme (ACE) with membrane-bound carboxypeptidase M (CPM) - a new function of ACE.

Angiotensin-converting enzyme (ACE) demonstrates, besides its typical dipeptidyl-carboxypeptidase activity, several unusual functions. Here, we demonstrate with molecular, biochemical, and cellular techniques that the somatic wild-type murine ACE (mACE), stably transfected in Chinese Hamster Ovary (CHO) or Madin-Darby Canine Kidney (MDCK) cells, interacts with endogenous membranal co-localized carboxypeptidase M (CPM). CPM belongs to the group of glycosylphosphatidylinositol (GPI)-anchored proteins. Here we report that ACE, completely independent of its known dipeptidase activities, has GPI-targeted properties. Our results indicate that the spatial proximity between mACE and the endogenous CPM enables an ACE-evoked release of CPM. These results are discussed with respect to the recently proposed GPI-ase activity and function of sperm-bound ACE.

AKAP complex regulates Ca2+ re-uptake into heart sarcoplasmic reticulum.

The beta-adrenergic receptor/cyclic AMP/protein kinase A (PKA) signalling pathway regulates heart rate and contractility. Here, we identified a supramolecular complex consisting of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2), its negative regulator phospholamban (PLN), the A-kinase anchoring protein AKAP18delta and PKA. We show that AKAP18delta acts as a scaffold that coordinates PKA phosphorylation of PLN and the adrenergic effect on Ca(2+) re-uptake. Inhibition of the compartmentalization of this cAMP signalling complex by specific molecular disruptors interferes with the phosphorylation of PLN. This prevents the subsequent release of PLN from SERCA2, thereby affecting the Ca(2+) re-uptake into the sarcoplasmic reticulum induced by adrenergic stimuli.

Compartmentalization of cAMP-dependent signaling by phosphodiesterase-4D is involved in the regulation of vasopressin-mediated water reabsorption in renal principal cells.

The cAMP/protein kinase A (PKA)-dependent insertion of water channel aquaporin-2 (AQP2)-bearing vesicles into the plasma membrane in renal collecting duct principal cells (AQP2 shuttle) constitutes the molecular basis of arginine vasopressin (AVP)-regulated water reabsorption. cAMP/PKA signaling systems are compartmentalized by A kinase anchoring proteins (AKAP) that tether PKA to subcellular sites and by phosphodiesterases (PDE) that terminate PKA signaling through hydrolysis of localized cAMP. In primary cultured principal cells, AVP causes focal activation of PKA. PKA and cAMP-specific phosphodiesterase-4D (PDE4D) are located on AQP2-bearing vesicles. The selective PDE4 inhibitor rolipram increases AKAP-tethered PKA activity on AQP2-bearing vesicles and enhances the AQP2 shuttle and thereby the osmotic water permeability. AKAP18delta, which is located on AQP2-bearing vesicles, directly interacts with PDE4D and PKA. In response to AVP, PDE4D and AQP2 translocate to the plasma membrane. Here PDE4D is activated through PKA phosphorylation and reduces the osmotic water permeability. Taken together, a novel, compartmentalized, and physiologically relevant cAMP-dependent signal transduction module on AQP2-bearing vesicles, comprising anchored PDE4D, AKAP18delta, and PKA, has been identified.

High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides.

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.

Studies on the cellular uptake of substance P and lysine-rich, KLA-derived model peptides.

In the last decade many peptides have been shown to be internalized into various cell types by different, poorly characterized mechanisms. This review focuses on uptake studies with substance P (SP) aimed at unravelling the mechanism of peptide-induced mast cell degranulation, and on the characterization of the cellular uptake of designed KLA-derived model peptides. Studies on structure-activity relationships and receptor autoradiography failed to detect specific peptide receptors for the undecapeptide SP on mast cells. In view of these findings, a direct interaction of cationic peptides with heterotrimeric G proteins without the participation of a receptor has been proposed. Such a process would require insertion into and translocation of peptides across the plasma membrane. In order to clarify whether a transport of cationic peptides into rat peritoneal mast cells is possible, transport studies were performed by confocal laser scanning microscopy (CLSM) using fluorescence-labeled Arg(3),Orn(7)-SP and its D-amino acid analog, all-D-Arg(3),Orn(7)-SP, as well as by electron microscopic autoradiography using (3)H-labelled SP and (125)I-labelled all-D-SP. The results obtained by CLSM directly showed translocation of SP peptides into pertussis toxin-treated cells. Kinetic experiments indicated that the translocation process was rapid, occurring within a few seconds. Mast cell degranulation induced by analog of magainin 2 amide, neuropeptide Y and the model peptide acetyl-KLALKLALKALKAALKLA-amide was also found to be very fast, pointing to an extensive translocation of the peptides. In order to learn more about structural requirements for the cellular uptake of peptides, the translocation behavior of a set of systematically modified KLA-based model peptides has been studied in detail. By two different protocols for determining the amount of internalized peptide, evidence was found that the structure of the peptides only marginally affects their uptake, whereas the efflux of cationic, amphipathic peptides is strikingly diminished, thus allowing their enrichment within the cells. Although the mechanism of cellular uptake, consisting of energy-dependent and -independent contributions, is not well understood, KLA-derived peptides have been shown to deliver various cargos (PNAs, peptides) into cells. The results obtained with SP- and KLA-derived peptides are discussed in the context of the current literature.

Identification of a novel A-kinase anchoring protein 18 isoform and evidence for its role in the vasopressin-induced aquaporin-2 shuttle in renal principal cells.

Arginine vasopressin (AVP) increases the water permeability of renal collecting duct principal cells by inducing the fusion of vesicles containing the water channel aquaporin-2 (AQP2) with the plasma membrane (AQP2 shuttle). This event is initiated by activation of vasopressin V2 receptors, followed by an elevation of cAMP and the activation of protein kinase A (PKA). The tethering of PKA to subcellular compartments by protein kinase A anchoring proteins (AKAPs) is a prerequisite for the AQP2 shuttle. During the search for AKAP(s) involved in the shuttle, a new splice variant of AKAP18, AKAP18delta, was identified. AKAP18delta functions as an AKAP in vitro and in vivo. In the kidney, it is mainly expressed in principal cells of the inner medullary collecting duct, closely resembling the distribution of AQP2. It is present in both the soluble and particulate fractions derived from renal inner medullary tissue. Within the particulate fraction, AKAP18delta was identified on the same intracellular vesicles as AQP2 and PKA. AVP not only recruited AQP2, but also AKAP18delta to the plasma membrane. The elevation of cAMP caused the dissociation of AKAP18delta and PKA. The data suggest that AKAP18delta is involved in the AQP2 shuttle.