Michael J. Rindler

cAMP sensor Epac as a determinant of ATP-sensitive potassium channel activity in human pancreatic β 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 β cells. Because Epac2 is reported to interact in vitro with the isolated nucleotide‐binding fold‐1 (NBF‐1) of the β‐cell sulphonylurea receptor‐1 (SUR1), we hypothesized that cAMP might act via Epac1 and/or Epac2 to inhibit β‐cell ATP‐sensitive K+ channels (KATP channels; a hetero‐octomer of SUR1 and Kir6.2). If so, Epac‐mediated inhibition of KATP channels might explain prior reports that cAMP‐elevating agents promote β‐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 KATP channels in human β cells and rat INS‐1 insulin‐secreting cells. Inhibition of KATP 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 KATP 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 KATP channel activity in pancreatic β cells.

A cAMP and Ca2+ coincidence detector in support of Ca2+-induced Ca2+ release in mouse pancreatic β 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 β 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 β 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 β 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 β cell glucose metabolism to stimulate insulin secretion.

Carboxypeptidase E, a Peripheral Membrane Protein Implicated in the Targeting of Hormones to Secretory Granules, Co-aggregates with Granule Content Proteins at Acidic pH

Carboxypeptidase E (CPE) is a prohormone-processing enzyme and peripheral membrane protein of endocrine/neuroendocrine secretory granules. CPE has been shown to bind to an amino-terminal peptide of pro-opiomelanocortin (N-POMC) at pH 5.5 and hypothesized to be critically involved in the targeting of hormones such as POMC to the regulated secretory pathway [Cool, D. R., Normant, E., Shen, F., Chen, H. C., Pannell, L., Zhang, Y., and Loh, Y. P. (1997) Cell 88, 73–83]. To further explore the possibility that CPE serves to mediate the association of content proteins with the membrane during granule biogenesis, the binding of CPE to granule content proteins was investigated using anin vitro aggregation assay in which the selective precipitation of granule content proteins is induced by titration of the pH to <6.0. CPE was observed to co-aggregate efficiently with pituitary and chromaffin granule content proteins at concentrations well below those that promote its self-aggregation. In addition, CPE co-precipitated at pH 5.8 with purified prolactin and with insulin, which homophillically self-aggregate yet are structurally distinct from N-POMC. N-POMC when added to the assays did not inhibit the aggregation of CPE with prolactin or insulin, indicating that these interactions do not involve a binding site for N-POMC. The data show that CPE interacts at acidic pH with a variety of different content proteins, resembling in this regard other granule membrane proteins. The results support the idea that co-aggregation of abundant membrane proteins with content proteins is an important general mechanism for the sorting and retention of secretory granule proteins during granule maturation.

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 Bt2cAMP-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 μm) of Bt2cAMP-AM as compared with 8-pCPT-2′-O-Me-cAMP-AM, and because the action of Bt2cAMP-AM but not 8-pCPT-2′-O-Me-cAMP-AM was nearly abrogated by treatment with 3 μm 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 μm 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 μm 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.

Epac2‐dependent mobilization of intracellular Ca2+ by glucagon‐like peptide‐1 receptor agonist exendin‐4 is disrupted in β‐cells of phospholipase C‐ɛ knockout mice

Calcium can be mobilized in pancreatic β‐cells via a mechanism of Ca2+‐induced Ca2+ release (CICR), and cAMP‐elevating agents such as exendin‐4 facilitate CICR in β‐cells by activating both protein kinase A and Epac2. Here we provide the first report that a novel phosphoinositide‐specific phospholipase C‐ɛ (PLC‐ɛ) is expressed in the islets of Langerhans, and that the knockout (KO) of PLC‐ɛ gene expression in mice disrupts the action of exendin‐4 to facilitate CICR in the β‐cells of these mice. Thus, in the present study, in which wild‐type (WT) C57BL/6 mouse β‐cells were loaded with the photolabile Ca2+ chelator NP‐EGTA, the UV flash photolysis‐catalysed uncaging of Ca2+ generated CICR in only 9% of the β‐cells tested, whereas CICR was generated in 82% of the β‐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 β‐cells of PLC‐ɛ 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‐ɛ KO β‐cells with recombinant PLC‐ɛ rescued the action of 8‐pCPT‐2′‐O‐Me‐cAMP‐AM to facilitate CICR, whereas a K2150E PLC‐ɛ with a mutated Ras association (RA) domain, or a H1640L PLC‐ɛ that is catalytically dead, were both ineffective. Since 8‐pCPT‐2′‐O‐Me‐cAMP‐AM failed to facilitate CICR in WT β‐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‐ɛ exists in β‐cells, and that the activities of Epac2 and PLC‐ɛ are key determinants of CICR in this cell type.

Synthesis and secretion of apolipoprotein E by human placenta and choriocarcinoma cell lines

Abstract The synthesis and secretion of apolipoproteins (apos) by cells from a human choriocarcinoma cell line, JAR, were examined by [35S]-methionine labeling followed by immunoprecipitation and SDS/PAGE. Apo E, but not aposA-I, A -IV, or B, was synthesized and secreted. Apo E was also synthesized by fragments of chorionic villi from human placenta and by another choriocarcinoma line, BeWo. Pulse-chase experiments with JAR cells revealed that apo E was initially synthesized as a 33 kDa protein followed by a 34 KDa protein, probably the result of glycosylation. The latter was secreted into the medium where it was detected coincident with a 21 22 kDa doublet, possibly proteolyticfragments ofapo E. Approximately 50 per cent of the apo E in the medium was complexed with lipid as indicated by ultracentrifugation at a density of 1.21 g/ml. The amount of apo E produced by JAR was not affected by preincubation with dibutyryl cAMP and theophylline, or by the cholesterol content of the cells. Following perfusion ofan isolated lobule ofhuman placenta with [14C]-amino acids, [14C]-apo E was detected by immunoprecipitation of the maternal and fetal perfusates with 88 per cent in the maternal perfusate. These studies suggest that apo E, which promotes receptor-mediated lipoprotein uptake, is secreted by the trophoblast to facilitate uptake of maternal lipoproteins.

Two Mechanisms for IgG Uptake in Cultured Human Trophoblast: Evidence for a Novel High Affinity Fc Receptor

ABSTRACT: The mechanism of IgG transport by the placental trophoblast was examined by studying IgG uptake by purified trophoblast maintained in culture. This model retains the ability to bind and endocytose human IgG from human serum. Comparison of the relative IgG uptake by the trophoblast among the four subclasses of both human and mouse IgG indicates that the trophoblast IgG receptor has different affinities from those described for the three known human Fcγ receptors, FcRγI, FcRγII, and FcRγIII. These results suggest the presence of a novel trophoblast Fcγ receptor. Although FcγRIII has been reported to be present on trophoblasts, immunocytochemical studies failed to detect binding to the cell surface of antibody-specific for FcγRIII, 3G8 MAb. In addition, blocking studies with MAb 3G8 did not interfere with IgG uptake. Scatchard analysis of human IgG uptake revealed a biphasic curve consistent with two distinct mechanisms for the transport of IgG by the trophoblast. The first is a higher affinity system (Ka = 1.7 × 107 M-1 1.7 × 104 binding sites/cell) which exhibits IgG subclass and species specificity, and the second is a low affinity system (Ka = 6.9 × 103 M-1, 7.5 × 107 binding sites/cell).

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.

Biogenesis of storage granules and vesicles

Abstract The production of storage granules and vesicles that undergo regulated exocytosis occurs via biosynthetic and endocytotic pathways, respectively. Prominent in the formation of secretory granules in the Golgi apparatus is selective protein aggregation, which may account for the biogenesis of a multiple granules in a single cell. New cDNA transfection and immunolocalization studies have helped to further refine our understanding of the relationship between the formation of regulated secretory vesicles and the earlyh stages of the endocytotic pathway. Recent insights into the targeting of membrane proteins to these organelles have implicated both specific sortingb signals and protein-protein aggregation.

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

BackgroundCardiac 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.ResultsThe 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.ConclusionOur 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.