Modulating the Activity of the Human Organic Cation Transporter 2 Emerges as a Potential Strategy to Mitigate Unwanted Toxicities Associated with Cisplatin Chemotherapy.

Cisplatin (CDDP) stands out as an effective chemotherapeutic agent; however, its application is linked to the development of significant adverse effects, notably nephro- and ototoxicity. The human organic cation transporter 2 (hOCT2), found in abundance in the basolateral membrane domain of renal proximal tubules and the Corti organ, plays a crucial role in the initiation of nephro- and ototoxicity associated with CDDP by facilitating its uptake in kidney and ear cells. Given its limited presence in cancer cells, hOCT2 emerges as a potential druggable target for mitigating unwanted toxicities associated with CDDP. Potential strategies for mitigating CDDP toxicities include competing with the uptake of CDDP by hOCT2 or inhibiting hOCT2 activity through rapid regulation mediated by specific signaling pathways. This study investigated the interaction between the already approved cationic drugs disopyramide, imipramine, and orphenadrine with hOCT2 that is stably expressed in human embryonic kidney cells. Regarding disopyramide, its influence on CDDP cellular transport by hOCT2 was further characterized through inductively coupled plasma isotope dilution mass spectrometry. Additionally, its potential protective effects against cellular toxicity induced by CDDP were assessed using a cytotoxicity test. Given that hOCT2 is typically expressed in the basolateral membrane of polarized cells, with specific regulatory mechanisms, this work studied the regulation of hOCT2 that is stably expressed in Madin-Darby Canine Kidney (MDCK) cells. These cells were cultured in a matrix to induce the formation of cysts, exposing hOCT2 in the basolateral plasma membrane domain, which was freely accessible to experimental solutions. The study specifically tested the regulation of ASP+ uptake by hOCT2 in MDCK cysts through the inhibition of casein kinase II (CKII), calmodulin, or p56lck tyrosine kinase. Furthermore, the impact of this manipulation on the cellular toxicity induced by CDDP was examined using a cytotoxicity test. All three drugs-disopyramide, imipramine, and orphenadrine-demonstrated inhibition of ASP+ uptake, with IC50 values in the micromolar (µM) range. Notably, disopyramide produced a significant reduction in the CDDP cellular toxicity and platinum cellular accumulation when co-incubated with CDDP. The activity of hOCT2 in MDCK cysts experienced a significant down-regulation under inhibition of CKII, calmodulin, or p56lck tyrosine kinase. Interestingly, only the inhibition of p56lck tyrosine kinase demonstrated the capability to protect the cells against CDDP toxicity. In conclusion, certain interventions targeting hOCT2 have demonstrated the ability to reduce CDDP cytotoxicity, at least in vitro. Further investigations in in vivo systems are warranted to ascertain their potential applicability as co-treatments for mitigating undesired toxicities associated with CDDP in patients.

Activation of Hippo Pathway Damages Slit Diaphragm by Deprivation of Ajuba Proteins.

SIGNIFICANCE STATEMENT: Nuclear exclusion of the cotranscription factor YAP, which is a consequence of activation of the Hippo signaling pathway, leads to FSGS and podocyte apoptosis. Ajuba proteins play an important role in the glomerular filtration barrier by keeping the Hippo pathway inactive. In nephrocytes from Drosophila melanogaster , a well-established model system for podocyte research, Ajuba proteins ensure slit diaphragm (SD) formation and function. Hippo pathway activation leads to mislocalization of Ajuba proteins, decreased SD formation, rearrangement of the actin cytoskeleton, and increased SD permeability. Targeting the kinases of the Hippo pathway with specific inhibitors in the glomerulus could, therefore, be a promising strategy for therapy of FSGS. BACKGROUND: The highly conserved Hippo pathway, which regulates organ growth and cell proliferation by inhibiting transcriptional cofactors YAP/TAZ, plays a special role in podocytes, where activation of the pathway leads to apoptosis. The Ajuba family proteins (Ajuba, LIM domain-containing protein 1 (LIMD1) and Wilms tumor protein 1-interacting protein [WTIP]) can bind and inactivate large tumor suppressor kinases 1 and 2, (LATS1/2) two of the Hippo pathway key kinases. WTIP, furthermore, connects the slit diaphragm (SD), the specialized cell-cell junction between podocytes, with the actin cytoskeleton. METHODS: We used garland cell nephrocytes of Drosophila melanogaster to monitor the role of Ajuba proteins in Hippo pathway regulation and structural integrity of the SD. Microscopy and functional assays analyzed the interplay between Ajuba proteins and LATS2 regarding expression, localization, interaction, and effects on the functionality of the SD. RESULTS: In nephrocytes, the Ajuba homolog Djub recruited Warts (LATS2 homolog) to the SD. Knockdown of Djub activated the Hippo pathway. Reciprocally, Hippo activation reduced the Djub level. Both Djub knockdown and Hippo activation led to morphological changes in the SD, rearrangement of the cortical actin cytoskeleton, and increased SD permeability. Knockdown of Warts or overexpression of constitutively active Yki prevented these effects. In podocytes, Hippo pathway activation or knockdown of YAP also decreased the level of Ajuba proteins. CONCLUSIONS: Ajuba proteins regulate the structure and function of the SD in nephrocytes, connecting the SD protein complex to the actin cytoskeleton and maintaining the Hippo pathway in an inactive state. Hippo pathway activation directly influencing Djub expression suggests a self-amplifying feedback mechanism.

A SEC61A1 variant is associated with autosomal dominant polycystic liver disease.

BACKGROUND AND AIMS: Autosomal dominant polycystic liver and kidney disease is a spectrum of hereditary diseases, which display disturbed function of primary cilia leading to cyst formation. In autosomal dominant polycystic kidney disease a genetic cause can be determined in almost all cases. However, in isolated polycystic liver disease (PLD) about half of all cases remain genetically unsolved, suggesting more, so far unidentified genes to be implicated in this disease. METHODS: Customized next-generation sequencing was used to identify the underlying pathogenesis in two related patients with PLD. A variant identified in SEC61A1 was further analysed in immortalized patients' urine sediment cells and in an epithelial cell model. RESULTS: In both patients, a heterozygous missense change (c.706C>T/p.Arg236Cys) was found in SEC61A1, which encodes for a subunit of the translocation machinery of protein biosynthesis at the endoplasmic reticulum (ER). While kidney disease is absent in the proposita, her mother displays an atypical polycystic kidney phenotype with severe renal failure. In immortalized urine sediment cells, mutant SEC61A1 is expressed at reduced levels, resulting in decreased levels of polycystin-2 (PC2). In an epithelial cell culture model, we found the proteasomal degradation of mutant SEC61A1 to be increased, whereas its localization to the ER is not affected. CONCLUSIONS: Our data expand the allelic and clinical spectrum for SEC61A1, adding PLD as a new and the major phenotypic trait in the family described. We further demonstrate that mutant SEC61A1 results in enhanced proteasomal degradation and impaired biosynthesis of PC2.

Vascular and Liver Homeostasis in Juvenile Mice Require Endothelial Cyclic AMP-Dependent Protein Kinase A.

During vascular development, endothelial cAMP-dependent protein kinase A (PKA) regulates angiogenesis by controlling the number of tip cells, and PKA inhibition leads to excessive angiogenesis. Whether this role of endothelial PKA is restricted to embryonic and neonatal development or is also required for vascular homeostasis later on is unknown. Here, we show that perinatal (postnatal days P1-P3) of later (P28-P32) inhibition of endothelial PKA using dominant-negative PKA expressed under the control of endothelial-specific Cdh5-CreERT2 recombinase (dnPKAiEC mice) leads to severe subcutaneous edema, hypoalbuminemia, hypoglycemia and premature death. These changes were accompanied by the local hypersprouting of blood vessels in fat pads and the secondary enlargement of subcutaneous lymphatic vessels. Most noticeably, endothelial PKA inhibition caused a dramatic disorganization of the liver vasculature. Hepatic changes correlated with decreased gluconeogenesis, while liver albumin production seems to be unaffected and hypoalbuminemia is rather a result of increased leakage into the interstitium. Interestingly, the expression of dnPKA only in lymphatics using Prox1-CreERT2 produced no phenotype. Likewise, the mosaic expression in only endothelial subpopulations using Vegfr3-CreERT2 was insufficient to induce edema or hypoglycemia. Increased expression of the tip cell marker ESM1 indicated that the inhibition of PKA induced an angiogenic response in the liver, although tissue derived pro- and anti-angiogenic factors were unchanged. These data indicate that endothelial PKA is a gatekeeper of endothelial cell activation not only in development but also in adult homeostasis, preventing the aberrant reactivation of the angiogenic program.

Properties of Transport Mediated by the Human Organic Cation Transporter 2 Studied in a Polarized Three-Dimensional Epithelial Cell Culture Model.

The renal secretory clearance for organic cations (neurotransmitters, metabolism products and drugs) is mediated by transporters specifically expressed in the basolateral and apical plasma membrane domains of proximal tubule cells. Here, human organic cation transporter 2 (hOCT2) is the main transporter for organic cations in the basolateral membrane domain. In this study, we stably expressed hOCT2 in Madin-Darby Canine Kidney (MDCK) cells and cultivated these cells in the presence of an extracellular matrix to obtain three-dimensional (3D) structures (cysts). The transport properties of hOCT2 expressed in MDCK cysts were compared with those measured using human embryonic kidney cells (HEK293) stably transfected with hOCT2 (hOCT2-HEK cells). In the MDCK cysts, hOCT2 was expressed in the basolateral membrane domain and showed a significant uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) with an affinity (Km) of 3.6 ± 1.2 µM, similar to what was measured in the hOCT2-HEK cells (Km = 3.1 ± 0.2 µM). ASP+ uptake was inhibited by tetraethylammonium (TEA+), tetrapentylammonium (TPA+), metformin and baricitinib both in the hOCT2-HEK cells and the hOCT2- MDCK cysts, even though the apparent affinities of TEA+ and baricitinib were dependent on the expression system. Then, hOCT2 was subjected to the same rapid regulation by inhibition of p56lck tyrosine kinase or calmodulin in the hOCT2-HEK cells and hOCT2- MDCK cysts. However, inhibition of casein kinase II regulated only activity of hOCT2 expressed in MDCK cysts and not in HEK cells. Taken together, these results suggest that the 3D cell culture model is a suitable tool for the functional analysis of hOCT2 transport properties, depending on cell polarization.

Lack of WWC2 Protein Leads to Aberrant Angiogenesis in Postnatal Mice.

The WWC protein family is an upstream regulator of the Hippo signalling pathway that is involved in many cellular processes. We examined the effect of an endothelium-specific WWC1 and/or WWC2 knock-out on ocular angiogenesis. Knock-outs were induced in C57BL/6 mice at the age of one day (P1) and evaluated at P6 (postnatal mice) or induced at the age of five weeks and evaluated at three months of age (adult mice). We analysed morphology of retinal vasculature in retinal flat mounts. In addition, in vivo imaging and functional testing by electroretinography were performed in adult mice. Adult WWC1/2 double knock-out mice differed neither functionally nor morphologically from the control group. In contrast, the retinas of the postnatal WWC knock-out mice showed a hyperproliferative phenotype with significantly enlarged areas of sprouting angiogenesis and a higher number of tip cells. The branching and end points in the peripheral plexus were significantly increased compared to the control group. The deletion of the WWC2 gene was decisive for these effects; while knocking out WWC1 showed no significant differences. The results hint strongly that WWC2 is an essential regulator of ocular angiogenesis in mice. As an activator of the Hippo signalling pathway, it prevents excessive proliferation during physiological angiogenesis. In adult animals, WWC proteins do not seem to be important for the maintenance of the mature vascular plexus.

The Hippo pathway component Wwc2 is a key regulator of embryonic development and angiogenesis in mice.

The WW-and-C2-domain-containing (WWC) protein family is involved in the regulation of cell differentiation, cell proliferation, and organ growth control. As upstream components of the Hippo signaling pathway, WWC proteins activate the Large tumor suppressor (LATS) kinase that in turn phosphorylates Yes-associated protein (YAP) and its paralog Transcriptional coactivator-with-PDZ-binding motif (TAZ) preventing their nuclear import and transcriptional activity. Inhibition of WWC expression leads to downregulation of the Hippo pathway, increased expression of YAP/TAZ target genes and enhanced organ growth. In mice, a ubiquitous Wwc1 knockout (KO) induces a mild neurological phenotype with no impact on embryogenesis or organ growth. In contrast, we could show here that ubiquitous deletion of Wwc2 in mice leads to early embryonic lethality. Wwc2 KO embryos display growth retardation, a disturbed placenta development, impaired vascularization, and finally embryonic death. A whole-transcriptome analysis of embryos lacking Wwc2 revealed a massive deregulation of gene expression with impact on cell fate determination, cell metabolism, and angiogenesis. Consequently, a perinatal, endothelial-specific Wwc2 KO in mice led to disturbed vessel formation and vascular hypersprouting in the retina. In summary, our data elucidate a novel role for Wwc2 as a key regulator in early embryonic development and sprouting angiogenesis in mice.

Non-canonical chemical feedback self-limits nitric oxide-cyclic GMP signaling in health and disease.

Nitric oxide (NO)-cyclic GMP (cGMP) signaling is a vasoprotective pathway therapeutically targeted, for example, in pulmonary hypertension. Its dysregulation in disease is incompletely understood. Here we show in pulmonary artery endothelial cells that feedback inhibition by NO of the NO receptor, the cGMP forming soluble guanylate cyclase (sGC), may contribute to this. Both endogenous NO from endothelial NO synthase and exogenous NO from NO donor compounds decreased sGC protein and activity. This effect was not mediated by cGMP as the NO-independent sGC stimulator, or direct activation of cGMP-dependent protein kinase did not mimic it. Thiol-sensitive mechanisms were also not involved as the thiol-reducing agent N-acetyl-L-cysteine did not prevent this feedback. Instead, both in-vitro and in-vivo and in health and acute respiratory lung disease, chronically elevated NO led to the inactivation and degradation of sGC while leaving the heme-free isoform, apo-sGC, intact or even increasing its levels. Thus, NO regulates sGC in a bimodal manner, acutely stimulating and chronically inhibiting, as part of self-limiting direct feedback that is cGMP independent. In high NO disease conditions, this is aggravated but can be functionally recovered in a mechanism-based manner by apo-sGC activators that re-establish cGMP formation.

cAMP-dependent protein kinase A (PKA) regulates angiogenesis by modulating tip cell behavior in a Notch-independent manner.

cAMP-dependent protein kinase A (PKA) is a ubiquitously expressed serine/threonine kinase that regulates a variety of cellular functions. Here, we demonstrate that endothelial PKA activity is essential for vascular development, specifically regulating the transition from sprouting to stabilization of nascent vessels. Inhibition of endothelial PKA by endothelial cell-specific expression of dominant-negative PKA in mice led to perturbed vascular development, hemorrhage and embryonic lethality at mid-gestation. During perinatal retinal angiogenesis, inhibition of PKA resulted in hypersprouting as a result of increased numbers of tip cells. In zebrafish, cell autonomous PKA inhibition also increased and sustained endothelial cell motility, driving cells to become tip cells. Although these effects of PKA inhibition were highly reminiscent of Notch inhibition effects, our data demonstrate that PKA and Notch independently regulate tip and stalk cell formation and behavior.

Parasympathetic innervation regulates tubulogenesis in the developing salivary gland.

A fundamental question in development is how cells assemble to form a tubular network during organ formation. In glandular organs, tubulogenesis is a multistep process requiring coordinated proliferation, polarization and reorganization of epithelial cells to form a lumen, and lumen expansion. Although it is clear that epithelial cells possess an intrinsic ability to organize into polarized structures, the mechanisms coordinating morphogenetic processes during tubulogenesis are poorly understood. Here, we demonstrate that parasympathetic nerves regulate tubulogenesis in the developing salivary gland. We show that vasoactive intestinal peptide (VIP) secreted by the innervating ganglia promotes ductal growth, leads to the formation of a contiguous lumen, and facilitates lumen expansion through a cyclic AMP/protein kinase A (cAMP/PKA)-dependent pathway. Furthermore, we provide evidence that lumen expansion is independent of apoptosis and involves the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated Cl(-) channel. Thus, parasympathetic innervation coordinates multiple steps in tubulogenesis during organogenesis.

Cyclic AMP regulates formation of mammary epithelial acini in vitro.

Epithelial cells form tubular and acinar structures notable for a hollow lumen. In three-dimensional culture utilizing MCF10A mammary epithelial cells, acini form due to integrin-dependent polarization and survival of cells contacting extracellular matrix (ECM), and the apoptosis of inner cells of acini lacking contact with the ECM. In this paper, we report that cyclic AMP (cAMP)-dependent protein kinase A (PKA) promotes acinus formation via two mechanisms. First, cAMP accelerates redistribution of α6-integrin to the periphery of the acinus and thus facilitates the polarization of outer acinar cells. Blocking of α6-integrin function by inhibitory antibody prevents cAMP-dependent polarization. Second, cAMP promotes the death of inner cells occupying the lumen. In the absence of cAMP, apoptosis is delayed, resulting in perturbed luminal clearance. cAMP-dependent apoptosis is accompanied by a posttranscriptional PKA-dependent increase in the proapoptotic protein Bcl-2 interacting mediator of cell death. These data demonstrate that cAMP regulates lumen formation in mammary epithelial cells in vitro, both through acceleration of polarization of outer cells and apoptosis of inner cells of the acinus.

Transcriptional profiling identifies TNS4 function in epithelial tubulogenesis.

Hepatocyte growth factor (HGF) plays central roles in tubulogenesis and metastasis [1-4]. HGF treatment of Madin-Darby canine kidney (MDCK) cells grown as cysts in three-dimensional culture induces tubulogenesis [5, 6], which like most tubulogenic processes proceeds through distinct intermediate phases. Identification of genes associated with these phases is central to understanding the molecular mechanisms of tubulogensis; however, because of inefficient, asynchronous tubule formation, isolating such genes has been unfeasible. Here we developed a synchronous, efficient tubulogenesis system and used time-course transcriptional profiling to identify genes temporally regulated in developmental intermediates. Knockdown (KD) of tensin 4 (TNS4), a particularly highly upregulated gene, leads to a decrease in formation of extensions and tubules, two sequential intermediates in tubulogenesis. Exogenous expression of TNS4 marks invasive cells in an epithelial sheet. A mutation in the SH2 domain of TNS4 prevents the transition from extension formation to invasive migration during tubule formation and leads to increased basal activation of STAT3. Exogenous expression of a constitutively active STAT3 mimics the defect by the mutation. Our study highlights the role of the TNS4-STAT3 axis in epithelial sheet invasion and tubulogenesis.

Reciprocal regulation of aquaporin-2 abundance and degradation by protein kinase A and p38-MAP kinase.

Arginine-vasopressin (AVP) modulates the water channel aquaporin-2 (AQP2) in the renal collecting duct to maintain homeostasis of body water. AVP binds to vasopressin V2 receptors (V2R), increasing cAMP, which promotes the redistribution of AQP2 from intracellular vesicles into the plasma membrane. cAMP also increases AQP2 transcription, but whether altered degradation also modulates AQP2 protein levels is not well understood. Here, elevation of cAMP increased AQP2 protein levels within 30 minutes in primary inner medullary collecting duct (IMCD) cells, in human embryonic kidney (HEK) 293 cells ectopically expressing AQP2, and in mouse kidneys. Accelerated transcription or translation did not explain this increase in AQP2 abundance. In IMCD cells, cAMP inhibited p38-mitogen-activated protein kinase (p38-MAPK) via activation of protein kinase A (PKA). Inhibition of p38-MAPK associated with decreased phosphorylation (serine 261) and polyubiquitination of AQP2, preventing proteasomal degradation. Our results demonstrate that AVP enhances AQP2 protein abundance by altering its proteasomal degradation through a PKA- and p38-MAPK-dependent pathway.

Regulation of aquaporin-2 trafficking.

Principal cells lining renal collecting ducts control the fine-tuning of body water homeostasis by regulating water reabsorption through the water channels aquaporin-2 (AQP2), aquaporin-3 (AQP3), and aquaporin-4 (AQP4). While the localization of AQP2 is subject to regulation by arginine-vasopressin (AVP), AQP3 and AQP4 are constitutively expressed in the basolateral plasma membrane. AVP adjusts the amount of AQP2 in the plasma membrane by triggering its redistribution from intracellular vesicles into the plasma membrane. This permits water entry into the cells and water exit through AQP3 and AQP4. The translocation of AQP2 is initiated by an increase in cAMP following V2R activation through AVP. The AVP-induced rise in cAMP activates protein kinase A (PKA), which in turn phosphorylates AQP2, and thereby triggers the redistribution of AQP2. Several proteins participating in the control of cAMP-dependent AQP2 trafficking have been identified; for example, A kinase anchoring proteins (AKAPs) tethering PKA to cellular compartments; phosphodiesterases (PDEs) regulating the local cAMP level; cytoskeletal components such as F-actin and microtubules; small GTPases of the Rho family controlling cytoskeletal dynamics; motor proteins transporting AQP2-bearing vesicles to and from the plasma membrane for exocytic insertion and endocytic retrieval; SNAREs inducing membrane fusions, hsc70, a chaperone, important for endocytic retrieval. In addition, cAMP-independent mechanisms of translocation mainly involving the F-actin cytoskeleton have been uncovered. Defects of AQP2 trafficking cause diseases such as nephrogenic diabetes insipidus (NDI), a disorder characterized by a massive loss of hypoosmotic urine.This review summarizes recent data elucidating molecular mechanisms underlying the trafficking of AQP2. In particular, we focus on proteins involved in the regulation of trafficking, and physiological and pathophysiological stimuli determining the cellular localization of AQP2. The identification of proteins and protein-protein interactions may lead to the development of drugs targeting AQP2 trafficking. Such drugs may be suitable for the treatment of diseases associated with dysregulation of body water homeostasis, including NDI or cardiovascular diseases (e.g., chronic heart failure) where the AVP level is elevated, inducing excessive water retention.

Heat shock protein 90 regulates stabilization rather than activation of soluble guanylate cyclase.

Endothelium-derived nitric oxide (NO) activates the heterodimeric heme protein soluble guanylate cyclase (sGC) to form cGMP. In different disease states, sGC levels and activity are diminished possibly involving the sGC binding chaperone, heat shock protein 90 (hsp90). Here we show that prolonged hsp90 inhibition in different cell types reduces protein levels of both sGC subunits by about half, an effect that was prevented by the proteasome inhibitor MG132. Conversely, acute hsp90 inhibition affected neither basal nor NO-stimulated sGC activity. Thus, hsp90 is a molecular stabilizer for sGC tonically preventing proteasomal degradation rather than having a role in short-term activity regulation.

Microtubules are needed for the perinuclear positioning of aquaporin-2 after its endocytic retrieval in renal principal cells.

Water reabsorption in the renal collecting duct is regulated by arginine vasopressin (AVP). AVP induces the insertion of the water channel aquaporin-2 (AQP2) into the plasma membrane of principal cells, thereby increasing the osmotic water permeability. The redistribution of AQP2 to the plasma membrane is a cAMP-dependent process and thus a paradigm for cAMP-controlled exocytic processes. Using primary cultured rat inner medullary collecting duct cells, we show that the redistribution of AQP2 to the plasma membrane is accompanied by the reorganization of microtubules and the redistribution of the small GTPase Rab11. In resting cells, AQP2 is colocalized with Rab11 perinuclearly. AVP induced the redistribution of AQP2 to the plasma membrane and of Rab11 to the cell periphery. The redistribution of both proteins was increased when microtubules were depolymerized by nocodazole. In addition, the depolymerization of microtubules prevented the perinuclear positioning of AQP2 and Rab11 in resting cells, which was restored if nocodazole was washed out and microtubules repolymerized. After internalization of AQP2, induced by removal of AVP, forskolin triggered the AQP2 redistribution to the plasma membrane even if microtubules were depolymerized and without the previous positioning of AQP2 in the perinuclear recycling compartment. Collectively, the data indicate that microtubule-dependent transport of AQP2 is predominantly responsible for trafficking and localization of AQP2 inside the cell after its internalization but not for the exocytic transport of the water channel. We also demonstrate that cAMP-signaling regulates the localization of Rab11-positive recycling endosomes in renal principal cells.

A Role of myosin Vb and Rab11-FIP2 in the aquaporin-2 shuttle.

Arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells. Its binding to Gs-coupled vasopressin V2 receptors increases cyclic AMP (cAMP) and subsequently elicits the redistribution of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane (AQP2 shuttle), thereby facilitating water reabsorption from primary urine. The AQP2 shuttle is a paradigm for cAMP-dependent exocytic processes. Using sections of rat kidney, the AQP2-expressing cell line CD8, and primary principal cells, we studied the role of the motor protein myosin Vb, its vesicular receptor Rab11, and the myosin Vb- and Rab11-binding protein Rab11-FIP2 in the AQP2 shuttle. Myosin Vb colocalized with AQP2 intracellularly in resting and at the plasma membrane in AVP-treated cells. Rab11 was found on AQP2-bearing vesicles. A dominant-negative myosin Vb tail construct and Rab11-FIP2 lacking the C2 domain (Rab11-FIP2-DeltaC2), which disrupt recycling, caused condensation of AQP2 in a Rab11-positive compartment and abolished the AQP2 shuttle. This effect was dependent on binding of myosin Vb tail and Rab11-FIP2-DeltaC2 to Rab11. In summary, we identified myosin Vb as a motor protein involved in AQP2 recycling and show that myosin Vb- and Rab11-FIP2-dependent recycling of AQP2 is an integral part of the AQP2 shuttle.

Targeting the heme-oxidized nitric oxide receptor for selective vasodilatation of diseased blood vessels.

ROS are a risk factor of several cardiovascular disorders and interfere with NO/soluble guanylyl cyclase/cyclic GMP (NO/sGC/cGMP) signaling through scavenging of NO and formation of the strong oxidant peroxynitrite. Increased oxidative stress affects the heme-containing NO receptor sGC by both decreasing its expression levels and impairing NO-induced activation, making vasodilator therapy with NO donors less effective. Here we show in vivo that oxidative stress and related vascular disease states, including human diabetes mellitus, led to an sGC that was indistinguishable from the in vitro oxidized/heme-free enzyme. This sGC variant represents what we believe to be a novel cGMP signaling entity that is unresponsive to NO and prone to degradation. Whereas high-affinity ligands for the unoccupied heme pocket of sGC such as zinc-protoporphyrin IX and the novel NO-independent sGC activator 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino) methyl [benzoic]acid (BAY 58-2667) stabilized the enzyme, only the latter activated the NO-insensitive sGC variant. Importantly, in isolated cells, in blood vessels, and in vivo, BAY 58-2667 was more effective and potentiated under pathophysiological and oxidative stress conditions. This therapeutic principle preferentially dilates diseased versus normal blood vessels and may have far-reaching implications for the currently investigated clinical use of BAY 58-2667 as a unique diagnostic tool and highly innovative vascular therapy.

Effects of chronic endothelin ET(A) receptor blockade on blood pressure and vascular formation of cyclic guanosine-3',5'-monophosphate in spontaneously hypertensive rats.

Endothelin (ET) mediates vasoconstriction in intact arterial blood vessels with functional endothelium via stimulation of ET(A) receptors, while ET(B) receptor stimulation leads to vasodilation via nitric oxide (NO) release and formation of cyclic guanosine-3',5'-monophosphate (cGMP). In spontaneously hypertensive rats (SHR) the cGMP-forming NO-receptor guanylyl cyclase (sGC) is downregulated. It is unclear whether ET contributes to the hypertensive phenotype of SHR, and whether this involves the disturbed cGMP signaling. The selective ETA receptor antagonist darusentan (CAS 171714-84-4), given orally via drinking water (10 mg kg(-1) d(-1)) for 12 weeks, significantly lowered systolic blood pressure of SHR as determined by radiotelemetry. Neither impaired endothelium-dependent relaxation to acetylcholine was restored nor sGC expression and activity affected when compared to control SHR. While these findings show a role for ETA receptors in blood pressure regulation in genetically elevated blood pressure, downregulation of sGC expression and cGMP-mediated vasorelaxant response in SHR were shown to be independent of ETA receptors. The findings suggest distinct mechanisms of gene expression affecting ET and cGMP mediated vasomotor functions.

Distribution of soluble guanylyl cyclase in the rat brain.

The diffusible messenger nitric oxide (NO) acts in the brain largely through activation of soluble guanylyl cyclase (sGC), a heterodimer comprising alpha and beta subunits. We used immunohistochemistry to study the distribution of both sGC subunits in the brain of adult rats. alpha and beta subunits gave similar widespread staining throughout the CNS, which was strongest in neostriatum, olfactory tubercle, and supraoptic nucleus. Double-labeling experiments showed striking cellular colocalization in most brain regions, suggesting that the two subunits may be organized into enzymatically active alpha/beta heteromers. Mismatches were observed in cerebellar cortex: Purkinje cells and Bergmann glia were positive for both subunits, whereas granule cells and interneurons in the molecular layer were strongly immunopositive for beta but only weakly stained for the alpha subunit. By using multiple labeling, we compared the localization of sGC with neuronal nitric oxide synthase (NOS-I, the NO-producing enzyme in neurons). In forebrain, the distribution of sGC and NOS-I was complementary, with only occasional colocalization. In contrast, cellular colocalization was common in midbrain and cerebellum. These data support a widespread role for the NO/sGC/cGMP pathway in the CNS and suggest that, in addition to its role as paracrine messenger, NO may also be an intracellular autocrine agent.