Mitochondrial lipid droplet formation as a detoxification mechanism to sequester and degrade excessive urothelial membranes.

The apical surface of the terminally differentiated mammalian urothelial umbrella cell is mechanically stable and highly impermeable, in part due to its coverage by urothelial plaques consisting of 2D crystals of uroplakin particles. The mechanism for regulating the uroplakin/plaque level is unclear. We found that genetic ablation of the highly tissue-specific sorting nexin Snx31, which localizes to plaques lining the multivesicular bodies (MVBs) in urothelial umbrella cells, abolishes MVBs suggesting that Snx31 plays a role in stabilizing the MVB-associated plaques by allowing them to achieve a greater curvature. Strikingly, Snx31 ablation also induces a massive accumulation of uroplakin-containing mitochondria-derived lipid droplets (LDs), which mediate uroplakin degradation via autophagy/lipophagy, leading to the loss of apical and fusiform vesicle plaques. These results suggest that MVBs play an active role in suppressing the excessive/wasteful endocytic degradation of uroplakins. Failure of this suppression mechanism triggers the formation of mitochondrial LDs so that excessive uroplakin membranes can be sequestered and degraded. Because mitochondrial LD formation, which occurs at a low level in normal urothelium, can also be induced by disturbance in uroplakin polymerization due to individual uroplakin knockout and by arsenite, a bladder carcinogen, this pathway may represent an inducible, versatile urothelial detoxification mechanism.

The trafficking protein, EHD2, positively regulates cardiac sarcolemmal KATP channel surface expression: role in cardioprotection.

ATP-sensitive K+ (KATP) channels uniquely link cellular energy metabolism to membrane excitability and are expressed in diverse cell types that range from the endocrine pancreas to neurons and smooth, skeletal, and cardiac muscle. A decrease in the surface expression of KATP channels has been linked to various disorders, including dysregulated insulin secretion, abnormal blood pressure, and impaired resistance to cardiac injury. In contrast, up-regulation of KATP channel surface expression may be protective, for example, by mediating the beneficial effect of ischemic preconditioning. Molecular mechanisms that regulate KATP channel trafficking are poorly understood. Here, we used cellular assays with immunofluorescence, surface biotinylation, and patch clamping to demonstrate that Eps15 homology domain-containing protein 2 (EHD2) is a novel positive regulator of KATP channel trafficking to increase surface KATP channel density. EHD2 had no effect on cardiac Na+ channels (Nav1.5). The effect is specific to EHD2 as other members of the EHD family-EHD1, EHD3, and EHD4-had no effect on KATP channel surface expression. EHD2 did not directly affect KATP channel properties as unitary conductance and ATP sensitivity were unchanged. Instead, we observed that the mechanism by which EHD2 increases surface expression is by stabilizing KATP channel-containing caveolar structures, which results in a reduced rate of endocytosis. EHD2 also regulated KATP channel trafficking in isolated cardiomyocytes, which validated the physiologic relevance of these observations. Pathophysiologically, EHD2 may be cardioprotective as a dominant-negative EHD2 mutant sensitized cardiomyocytes to ischemic damage. Our findings highlight EHD2 as a potential pharmacologic target in the treatment of diseases with KATP channel trafficking defects.-Yang, H. Q., Jana, K., Rindler, M. J., Coetzee, W. A. The trafficking protein, EHD2, positively regulates cardiac sarcolemmal KATP channel surface expression: role in cardioprotection.

Plasticity of sarcolemmal KATP channel surface expression: relevance during ischemia and ischemic preconditioning.

Myocardial ischemia remains the primary cause of morbidity and mortality in the United States. Ischemic preconditioning (IPC) is a powerful form of endogenous protection against myocardial infarction. We studied alterations in KATP channels surface density as a potential mechanism of the protection of IPC. Using cardiac-specific knockout of Kir6.2 subunits, we demonstrated an essential role for sarcolemmal KATP channels in the infarct-limiting effect of IPC in the mouse heart. With biochemical membrane fractionation, we demonstrated that sarcolemmal KATP channel subunits are distributed both to the sarcolemma and intracellular endosomal compartments. Global ischemia causes a loss of sarcolemmal KATP channel subunit distribution and internalization to endosomal compartments. Ischemia-induced internalization of KATP channels was prevented by CaMKII inhibition. KATP channel subcellular redistribution was also observed with immunohistochemistry. Ischemic preconditioning before the index ischemia reduced not only the infarct size but also prevented KATP channel internalization. Furthermore, not only did adenosine mimic IPC by preventing infarct size, but it also prevented ischemia-induced KATP channel internalization via a PKC-mediated pathway. We show that preventing endocytosis with dynasore reduced both KATP channel internalization and strongly mitigated infarct development. Our data demonstrate that plasticity of KATP channel surface expression must be considered as a potentially important mechanism of the protective effects of IPC and adenosine.

Sequential and compartmentalized action of Rabs, SNAREs, and MAL in the apical delivery of fusiform vesicles in urothelial umbrella cells.

Uroplakins (UPs) are major differentiation products of urothelial umbrella cells and play important roles in forming the permeability barrier and in the expansion/stabilization of the apical membrane. Further, UPIa serves as a uropathogenic Escherichia coli receptor. Although it is understood that UPs are delivered to the apical membrane via fusiform vesicles (FVs), the mechanisms that regulate this exocytic pathway remain poorly understood. Immunomicroscopy of normal and mutant mouse urothelia show that the UP-delivering FVs contained Rab8/11 and Rab27b/Slac2-a, which mediate apical transport along actin filaments. Subsequently a Rab27b/Slp2-a complex mediated FV-membrane anchorage before SNARE-mediated and MAL-facilitated apical fusion. We also show that keratin 20 (K20), which forms a chicken-wire network ∼200 nm below the apical membrane and has hole sizes allowing FV passage, defines a subapical compartment containing FVs primed and strategically located for fusion. Finally, we show that Rab8/11 and Rab27b function in the same pathway, Rab27b knockout leads to uroplakin and Slp2-a destabilization, and Rab27b works upstream from MAL. These data support a unifying model in which UP cargoes are targeted for apical insertion via sequential interactions with Rabs and their effectors, SNAREs and MAL, and in which K20 plays a key role in regulating vesicular trafficking.

Measuring and evaluating the role of ATP-sensitive K+ channels in cardiac muscle.

Since ion channels move electrical charge during their activity, they have traditionally been studied using electrophysiological approaches. This was sometimes combined with mathematical models, for example with the description of the ionic mechanisms underlying the initiation and propagation of action potentials in the squid giant axon by Hodgkin and Huxley. The methods for studying ion channels also have strong roots in protein chemistry (limited proteolysis, the use of antibodies, etc.). The advent of the molecular cloning and the identification of genes coding for specific ion channel subunits in the late 1980s introduced a multitude of new techniques with which to study ion channels and the field has been rapidly expanding ever since (e.g. antibody development against specific peptide sequences, mutagenesis, the use of gene targeting in animal models, determination of their protein structures) and new methods are still in development. This review focuses on techniques commonly employed to examine ion channel function in an electrophysiological laboratory. The focus is on the K(ATP) channel, but many of the techniques described are also used to study other ion channels.

Endosomal KATP channels as a reservoir after myocardial ischemia: a role for SUR2 subunits.

ATP-sensitive K(+) (K(ATP)) channels, composed of inward rectifier K(+) (Kir)6.x and sulfonylurea receptor (SUR)x subunits, are expressed on cellular plasma membranes. We demonstrate an essential role for SUR2 subunits in trafficking K(ATP) channels to an intracellular vesicular compartment. Transfection of Kir6.x/SUR2 subunits into a variety of cell lines (including h9c2 cardiac cells and human coronary artery smooth muscle cells) resulted in trafficking to endosomal/lysosomal compartments, as assessed by immunofluorescence microscopy. By contrast, SUR1/Kir6.x channels efficiently localized to the plasmalemma. The channel turnover rate was similar with SUR1 or SUR2, suggesting that the expression of Kir6/SUR2 proteins in lysosomes is not associated with increased degradation. Surface labeling of hemagglutinin-tagged channels demonstrated that SUR2-containing channels dynamically cycle between endosomal and plasmalemmal compartments. In addition, Kir6.2 and SUR2 subunits were found in both endosomal and sarcolemmal membrane fractions isolated from rat hearts. The balance of these K(ATP) channel subunits shifted to the sarcolemmal membrane fraction after the induction of ischemia. The K(ATP) channel current density was also increased in rat ventricular myocytes isolated from hearts rendered ischemic before cell isolation without corresponding changes in subunit mRNA expression. We conclude that an intracellular pool of SUR2-containing K(ATP) channels exists that is derived by endocytosis from the plasma membrane. In cardiac myocytes, this pool can potentially play a cardioprotective role by serving as a reservoir for modulating surface K(ATP) channel density under stress conditions, such as myocardial ischemia.

Epac2-dependent mobilization of intracellular Ca²+ by glucagon-like peptide-1 receptor agonist exendin-4 is disrupted in ß-cells of phospholipase C-ε knockout mice.

Calcium can be mobilized in pancreatic ß-cells via a mechanism of Ca(2+)-induced Ca(2+) release (CICR), and cAMP-elevating agents such as exendin-4 facilitate CICR in ß-cells by activating both protein kinase A and Epac2. Here we provide the first report that a novel phosphoinositide-specific phospholipase C- (PLC-) is expressed in the islets of Langerhans, and that the knockout (KO) of PLC- gene expression in mice disrupts the action of exendin-4 to facilitate CICR in the ß-cells of these mice. Thus, in the present study, in which wild-type (WT) C57BL/6 mouse ß-cells were loaded with the photolabile Ca(2+) chelator NP-EGTA, the UV flash photolysis-catalysed uncaging of Ca(2+) generated CICR in only 9% of the ß-cells tested, whereas CICR was generated in 82% of the ß-cells pretreated with exendin-4. This action of exendin-4 to facilitate CICR was reproduced by cAMP analogues that activate protein kinase A (6-Bnz-cAMP-AM) or Epac2 (8-pCPT-2'-O-Me-cAMP-AM) selectively. However, in ß-cells of PLC- KO mice, and also Epac2 KO mice, these test substances exhibited differential efficacies in the CICR assay such that exendin-4 was partly effective, 6-Bnz-cAMP-AM was fully effective, and 8-pCPT-2'-O-Me-cAMP-AM was without significant effect. Importantly, transduction of PLC- KO ß-cells with recombinant PLC- rescued the action of 8-pCPT-2'-O-Me-cAMP-AM to facilitate CICR, whereas a K2150E PLC- with a mutated Ras association (RA) domain, or a H1640L PLC- that is catalytically dead, were both ineffective. Since 8-pCPT-2'-O-Me-cAMP-AM failed to facilitate CICR in WT ß-cells transduced with a GTPase activating protein (RapGAP) that downregulates Rap activity, the available evidence indicates that a signal transduction 'module' comprised of Epac2, Rap and PLC- exists in ß-cells, and that the activities of Epac2 and PLC- are key determinants of CICR in this cell type.

Enhanced Rap1 activation and insulin secretagogue properties of an acetoxymethyl ester of an Epac-selective cyclic AMP analog in rat INS-1 cells: studies with 8-pCPT-2'-O-Me-cAMP-AM.

To ascertain the identities of cyclic nucleotide-binding proteins that mediate the insulin secretagogue action of cAMP, the possible contributions of the exchange protein directly activated by cAMP (Epac) and protein kinase A (PKA) were evaluated in a pancreatic beta cell line (rat INS-1 cells). Assays of Rap1 activation, CREB phosphorylation, and PKA-dependent gene expression were performed in combination with live cell imaging and high throughput screening of a fluorescence resonance energy transfer-based cAMP sensor (Epac1-camps) to validate the selectivity with which acetoxymethyl esters (AM-esters) of cAMP analogs preferentially activate Epac or PKA. Selective activation of Epac or PKA was achieved following exposure of INS-1 cells to 8-pCPT-2'-O-Me-cAMP-AM or Bt(2)cAMP-AM, respectively. Both cAMP analogs exerted dose-dependent and glucose metabolism-dependent actions to stimulate insulin secretion, and when each was co-administered with the other, a supra-additive effect was observed. Because 2.4-fold more insulin was secreted in response to a saturating concentration (10 microm) of Bt(2)cAMP-AM as compared with 8-pCPT-2'-O-Me-cAMP-AM, and because the action of Bt(2)cAMP-AM but not 8-pCPT-2'-O-Me-cAMP-AM was nearly abrogated by treatment with 3 microm of the PKA inhibitor H-89, it is concluded that for INS-1 cells, it is PKA that acts as the dominant cAMP-binding protein in support of insulin secretion. Unexpectedly, 10-100 microm of the non-AM-ester of 8-pCPT-2'-O-Me-cAMP failed to stimulate insulin secretion and was a weak activator of Rap1 in INS-1 cells. Moreover, 10 microm of the AM-ester of 8-pCPT-2'-O-Me-cAMP stimulated insulin secretion from mouse islets, whereas the non-AM-ester did not. Thus, the membrane permeability of 8-pCPT-2'-O-Me-cAMP in insulin-secreting cells is so low as to limit its biological activity. It is concluded that prior reports documenting the failure of 8-pCPT-2'-O-Me-cAMP to act in beta cells, or other cell types, need to be re-evaluated through the use of the AM-ester of this cAMP analog.

Short-term pacing in the mouse alters cardiac expression of connexin43.

BACKGROUND: Cardiac insults such as ischemia, infarction, hypertrophy and dilatation are often accompanied by altered abundance and/or localization of the connexin43 gap junction protein, which may predispose towards arrhythmic complications. Models of chronic dyssynchronous cardiac activation have also been shown to result in redistribution of connexin43 in cardiomyocytes. We hypothesized that alterations in connexin43 expression and localization in the mouse heart might be induced by ventricular pacing over a short period of time. RESULTS: The subdiaphragmatic approach was used to pace a series of wild type mice for six hours before the hearts were removed for analysis. Mice were paced at 10-15% above their average anesthetized sinus rate and monitored to ensure 1:1 capture. Short-term pacing resulted in a significant reduction in connexin43 mRNA abundance, a partial redistribution of connexin43 from the sarcolemma to a non-sarcolemmal fraction, and accumulation of ubiquitinated connexin43 without a significant change in overall connexin43 protein levels. These early pacing-induced changes in connexin43 expression were not accompanied by decreased cardiac function, prolonged refractoriness or increased inducibility into sustained arrhythmias. CONCLUSION: Our data suggest that short-term pacing is associated with incipient changes in the expression of the connexin43 gap junction, possibly including decreased production and a slowed rate of degradation. This murine model may facilitate the study of early molecular changes induced by pacing and may ultimately assist in the development of strategies to prevent gap junction remodeling and the associated arrhythmic complications of cardiac disease.

Calsyntenins are secretory granule proteins in anterior pituitary gland and pancreatic islet alpha cells.

Calsyntenins are members of the cadherin superfamily of cell adhesion molecules. They are present in postsynaptic membranes of excitatory neurons and in vesicles in transit to neuronal growth cones. In the current study, calsyntenin-1 (CST-1) and calsyntenin-3 (CST-3) were identified by mass spectrometric analysis (LC-MS/MS) of integral membrane proteins from highly enriched secretory granule preparations from bovine anterior pituitary gland. Immunofluorescence microscopy on thin frozen sections of rat pituitary revealed that CST-1 was present only in gonadotropes where it colocalized with follicle-stimulating hormone in secretory granules. In contrast, CST-3 was present not only in gonadotrope secretory granules but also in those of somatotropes and thyrotropes. Neither protein was detected in mammatropes. In addition, CST-1 was also localized to the glucagon-containing secretory granules of alpha cells in the pancreatic islets of Langerhans. Results indicate that calsyntenins function outside the nervous system and potentially are modulators of endocrine function.

Proteomic analysis of pancreatic zymogen granules: identification of new granule proteins.

The composition of zymogen granules from rat pancreas was determined by LC-MS/MS. Enriched intragranular content, peripheral membrane, and integral membrane protein fractions were analyzed after one-dimensional SDS-PAGE and tryptic digestion of gel slices. A total of 371 proteins was identified with high confidence, including 84 previously identified granule proteins. The 287 remaining proteins included 37 GTP-binding proteins and effectors, 8 tetraspan membrane proteins, and 22 channels and transporters. Seven proteins, pantophysin, cyclic nucleotide phosphodiesterase, carboxypeptidase D, ecto-nucleotide phosphodiesterase 3, aminopeptidase N, ral, and the potassium channel TWIK-2, were confirmed by immunofluorescence microscopy or by immunoblotting to be new zymogen granule membrane proteins.

cAMP sensor Epac as a determinant of ATP-sensitive potassium channel activity in human pancreatic beta cells and rat INS-1 cells.

The Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs, also known as Epac1 and Epac2) mediate stimulatory actions of the second messenger cAMP on insulin secretion from pancreatic beta cells. Because Epac2 is reported to interact in vitro with the isolated nucleotide-binding fold-1 (NBF-1) of the beta-cell sulphonylurea receptor-1 (SUR1), we hypothesized that cAMP might act via Epac1 and/or Epac2 to inhibit beta-cell ATP-sensitive K+ channels (K(ATP) channels; a hetero-octomer of SUR1 and Kir6.2). If so, Epac-mediated inhibition of K(ATP) channels might explain prior reports that cAMP-elevating agents promote beta-cell depolarization, Ca2+ influx and insulin secretion. Here we report that Epac-selective cAMP analogues (2'-O-Me-cAMP; 8-pCPT-2'-O-Me-cAMP; 8-pMeOPT-2'-O-Me-cAMP), but not a cGMP analogue (2'-O-Me-cGMP), inhibit the function of K(ATP) channels in human beta cells and rat INS-1 insulin-secreting cells. Inhibition of K(ATP) channels is also observed when cAMP, itself, is administered intracellularly, whereas no such effect is observed upon administration N6-Bnz-cAMP, a cAMP analogue that activates protein kinase A (PKA) but not Epac. The inhibitory actions of Epac-selective cAMP analogues at K(ATP) channels are mimicked by a cAMP agonist (8-Bromoadenosine-3', 5'-cyclic monophosphorothioate, Sp-isomer, Sp-8-Br-cAMPS), but not a cAMP antagonist (8-Bromoadenosine-3', 5'-cyclic monophosphorothioate, Rp-isomer, Rp-8-Br-cAMPS), and are abrogated following transfection of INS-1 cells with a dominant-negative Epac1 that fails to bind cAMP. Because both Epac1 and Epac2 coimmunoprecipitate with full-length SUR1 in HEK cell lysates, such findings delineate a novel mechanism of second messenger signal transduction in which cAMP acts via Epac to modulate ion channel function, an effect measurable as the inhibition of K(ATP) channel activity in pancreatic beta cells.

Isolation of zymogen granules from rat pancreas.

This unit describes methods for preparing zymogen granules from rat pancreas. Zymogen granules are storage organelles in pancreatic acinar cells containing digestive enzymes that are released into the pancreatic duct. The protocols in this unit take advantage of the large size (up to 1 microm diameter) and high density (>1.20 g/cm(3) on sucrose gradients) of the granules as compared to other cellular organelles. They use a combination of differential sedimentation and density gradient separation to accomplish the purification. Similar procedures can be used to isolate zymogen granules from mouse pancreas and canine pancreas. A protocol for preparing zymogen granules from dog pancreas is also included.

A cAMP and Ca2+ coincidence detector in support of Ca2+-induced Ca2+ release in mouse pancreatic beta cells.

The blood glucose-lowering hormone glucagon-like peptide-1 (GLP-1) stimulates cAMP production, promotes Ca2+ influx, and mobilizes an intracellular source of Ca2+ in pancreatic beta cells. Here we provide evidence that these actions of GLP-1 are functionally related: they reflect a process of Ca2+-induced Ca2+ release (CICR) that requires activation of protein kinase A (PKA) and the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs). In rat insulin-secreting INS-1 cells or mouse beta cells loaded with caged Ca2+ (NP-EGTA), a GLP-1 receptor agonist (exendin-4) is demonstrated to sensitize intracellular Ca2+ release channels to stimulatory effects of cytosolic Ca2+, thereby allowing CICR to be generated by the uncaging of Ca2+ (UV flash photolysis). This sensitizing action of exendin-4 is diminished by an inhibitor of PKA (H-89) or by overexpression of dominant negative Epac. It is reproduced by cell-permeant cAMP analogues that activate PKA (6-Bnz-cAMP) or Epac (8-pCPT-2'-O-Me-cAMP) selectively. Depletion of Ca2+ stores with thapsigargin abolishes CICR, while inhibitors of Ca2+ release channels (ryanodine and heparin) attenuate CICR in an additive manner. Because the uncaging of Ca2+ fails to stimulate CICR in the absence of cAMP-elevating agents, it is concluded that there exists in beta cells a process of second messenger coincidence detection, whereby intracellular Ca2+ release channels (ryanodine receptors, inositol 1,4,5-trisphosphate (IP3) receptors) monitor a simultaneous increase of cAMP and Ca2+ concentrations. We propose that second messenger coincidence detection of this type may explain how GLP-1 interacts with beta cell glucose metabolism to stimulate insulin secretion.