Rab8 Interacts with Distinct Motifs in α2B- and β2-Adrenergic Receptors and Differentially Modulates Their Transport

The molecular mechanism underlying the post-Golgi transport of G protein-coupled receptors (GPCRs) remains poorly understood. Here we determine the role of Rab8 GTPase, which modulates vesicular protein transport between the trans-Golgi network (TGN) and the plasma membrane, in the cell surface targeting of α_2B- and β₂-adrenergic receptors (AR). Transient expression of GDP- and GTP-bound Rab8 mutants and short hairpin RNA-mediated knockdown of Rab8 more potently inhibited the cell surface expression of α_2B-AR than β₂-AR. The GDP-bound Rab8(T22N) mutant attenuated ERK1/2 activation by α_2B-AR, but not β₂-AR, and arrested α_2B-AR in the TGN compartment. Co-immunoprecipitation revealed that both α_2B-AR and β₂-AR physically interacted with Rab8 and glutathione S-transferase fusion protein pulldown assays demonstrated that Rab8 interacted with the C termini of both receptors. Interestingly, mutation of the highly conserved membrane-proximal C terminus dileucine motif selectively blocked β₂-AR interaction with Rab8, whereas mutation of residues Val⁴³¹-Phe⁴³²-Asn⁴³³-Gln⁴³⁴, Pro⁴⁴⁷-Trp⁴⁴⁸, Gln⁴⁵⁰-Thr⁴⁵¹, and Trp⁴⁵³ in the C terminus impaired α_2B-AR interaction with Rab8. Furthermore, transport inhibition by Rab8(T22N) of a chimeric β₂-AR carrying the α_2B-AR C terminus was similar to α_2B-AR. These data provide strong evidence indicating that Rab8 GTPase interacts with distinct motifs in the C termini of α_2B-AR and β₂-AR and differentially modulates their traffic from the TGN to the cell surface.     

Impaired Ca2+ Signaling in β-Cells Lacking Leptin Receptors by Cre-loxP Recombination

Obesity is a major risk factor for diabetes and is typically associated with hyperleptinemia and a state of leptin resistance. The impact of chronically elevated leptin levels on the function of insulin-secreting β-cells has not been elucidated. We previously generated mice lacking leptin signaling in β-cells by using the Cre-loxP strategy and showed that these animals develop increased body weight and adiposity, hyperinsulinemia, impaired glucose-stimulated insulin secretion and insulin resistance. Here, we performed several in vitro studies and observed that β-cells lacking leptin signaling in this model are capable of properly metabolizing glucose, but show impaired intracellular Ca²⁺ oscillations and lack of synchrony within the islets in response to glucose, display reduced response to tolbutamide and exhibit morphological abnormalities including increased autophagy. Defects in intracellular Ca²⁺ signaling were observed even in neonatal islets, ruling out the possible contribution of obesity to the β-cell irregularities observed in adults. In parallel, we also detected a disrupted intracellular Ca²⁺ pattern in response to glucose and tolbutamide in control islets from adult transgenic mice expressing Cre recombinase under the rat insulin promoter, despite these animals being glucose tolerant and secreting normal levels of insulin in response to glucose. This unexpected observation impeded us from discerning the consequences of impaired leptin signaling as opposed to long-term Cre expression in the function of insulin-secreting cells. These findings highlight the need to generate improved Cre-driver mouse models or new tools to induce Cre recombination in β-cells.     

Orexin-A Promotes Cell Migration in Cultured Rat Astrocytes via Ca2+-Dependent PKCα and ERK1/2 Signals

Orexin-A is an important neuropeptide involved in the regulation of feeding, arousal, energy consuming, and reward seeking in the body. The effects of orexin-A have widely studied in neurons but not in astrocytes. Here, we report that OX1R and OX2R are expressed in cultured rat astrocytes. Orexin-A stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), and then induced the migration of astrocytes via its receptor OX1R but not OX2R. Orexin-A-induced ERK1/2 phosphorylation and astrocytes migration are Ca²⁺-dependent, since they could be inhibited by either chelating the extracellular Ca²⁺ or blocking the pathway of store-operated calcium entry (SOCE). Furthermore, both non-selective protein kinase C (PKC) inhibitor and PKCα selective inhibitor, but not PKCδ inhibitor, prevented the increase in ERK1/2 phosphorylation and the migration of astrocytes, indicating that the Ca²⁺-dependent PKCα acts as the downstream of the OX1R activation and mediates the orexin-A-induced increase in ERK1/2 phosphorylation and cell migration. In conclusion, these results suggest that orexin-A can stimulate ERK1/2 phosphorylation and then facilitate the migration of astrocytes via PLC-PKCα signal pathway, providing new knowledge about the functions of the OX1R in astrocytes.     

Endogenous and Exogenous Regulation of Human α- and β-Adrenergic Receptors

Abstract

Regulation of human β2-adrenergic receptors in lymphocytes (determined by (±)-125 iodocyanopindolol (ICYP) binding) and α₂-adrenergic receptors in platelets (determined by ³H-yohimbine binding) was studied. While α₂-adrenergic receptor number did not change with age, a significant negative correlation between the number of α₂-adrenergic receptors and age was found; plasma catecholamines, on the contrary, were elevated in the elderly.In healthy women during normal menstrual cycle the number of α₂-adrenergic receptors decreased with increasing plasma estradiol levels.Incubation of lymphocyte membranes with isoprenaline (100 μM) and of platelet membranes with clonidine (1-100 μM) led to a reduction of the number of β₂- and α₂-receptors, respectively, without changes in the K_D-values. Treatment of hypertensive patients with clonidine (3x150 μg/die) for 7 days reduced the number of α₂-adrenergic receptors in platelets. In platelet membranes from such treated patients inhibition of ³H-yohimbine binding by clonidine and adrenaline was not affected by 10-⁴MGTP. It is concluded, that human α- and β-adrenergic receptors undergo regulatory mechanisms similar to those recently described for adrenergic receptors in a variety of animal models.     

Reduction of Tumorigenicity by α3 Integrin in a Rhabdomyosarcoma Cell Line

The expression levels of integrin adhesion receptors have often been correlated with neoplastic transformation and invasiveness. To investigate more definitively the role of the integrin VLA-3 (α³β₁) in tumor cell behavior, we transfected α³ subunit cDNA into human rhabdomyosarcoma (RD) cells. Transi ectants expressing high levels of α³β₁, on their cell surface displayed an altered morphology and decreased anchorage-dependent growth in vitro. Cells expressing α³ also displayed marked reduction in anchorage-independent growth in soft agar and in their ability to form tumors when injected subcutaneously into athymic nude mice. Thus, VLA-3 can repress the transformed phenotype of rhabdomyosarcoma tumor cells. Similar changes in morphology and growth characteristics were observed in cells expressing a chimeric molecule X3C4 in which the α³ cytoplasmic domain had been exchanged with that of the α⁴ integrin subunit. Therefore, α³ inhibitory effects in RD cells appear not to require specific signalling through the α³ cytoplasmic domain.     

Glucose Evokes Rapid Ca2+ and Cyclic AMP Signals by Activating the Cell-Surface Glucose-Sensing Receptor in Pancreatic β-Cells

Glucose is a primary stimulator of insulin secretion in pancreatic β-cells. High concentration of glucose has been thought to exert its action solely through its metabolism. In this regard, we have recently reported that glucose also activates a cell-surface glucose-sensing receptor and facilitates its own metabolism. In the present study, we investigated whether glucose activates the glucose-sensing receptor and elicits receptor-mediated rapid actions. In MIN6 cells and isolated mouse β-cells, glucose induced triphasic changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c); glucose evoked an immediate elevation of [Ca²⁺]_c, which was followed by a decrease in [Ca²⁺]_c, and after a certain lag period it induced large oscillatory elevations of [Ca²⁺]_c. Initial rapid peak and subsequent reduction of [Ca²⁺]_c were independent of glucose metabolism and reproduced by a nonmetabolizable glucose analogue. These signals were also blocked by an inhibitor of T1R3, a subunit of the glucose-sensing receptor, and by deletion of the T1R3 gene. Besides Ca²⁺, glucose also induced an immediate and sustained elevation of intracellular cAMP ([cAMP]_c). The elevation of [cAMP]_c was blocked by transduction of the dominant-negative G_s, and deletion of the T1R3 gene. These results indicate that glucose induces rapid changes in [Ca²⁺]_c and [cAMP]_c by activating the cell-surface glucose-sensing receptor. Hence, glucose generates rapid intracellular signals by activating the cell-surface receptor.     

The effects of α- and β-adrenergic agents,Ca2+ and insulin on 2-deoxyglucose uptake and phosphorylation in perfused rat heart

Insulin (0.1 μM) and 1 μM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent V_max without altering the K_m. Isoproterenol (10 μM), 50 μM methoxamine and 10 mM CaCl₂ also increased uptake. Lowering of the perfusate Ca²⁺ concentration from 1.27 to 0.1 mM Ca²⁺, addition of the Ca²⁺ channel blocker nifedipine (1 μM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 μM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 μM insulin was only partly inhibited by Ca²⁺ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca²⁺, respectively. Maximal concentrations of insulin (0.1 μM) and epinephrine (1 μM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 μM but maximal concentrations of epinephrine (e.g., 1 μM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca²⁺ in signal reversal was also studied. Removal of 1 μM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca²⁺ concentration was lowered to 0.1 mM or nifedipine (1 μM) was added. Similarly, removal of 0.1 μM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca²⁺ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca²⁺ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca²⁺-dependent increase in 2-deoxyglucose transport from either α or β receptors. Insulin has both a Ca²⁺-dependent and a Ca²⁺-independent component. Reversal studies suggest an additional role for Ca²⁺ in maintaining the activated transport state when activated by either epinephrine or insulin.     

Central Opioidergic and Adrenergic systems Mediates Food Intake via α1, α2 and β2 Receptors in Neonatal Layer-Type Chicken

International Journal of Peptide Research and Therapeutics - The aim of the current study was to determine possible interaction of central Opioidergic and Adrenergic systems on food intake...     

?-Adrenergic Stimulation Increases RyR2 Activity via Intracellular Ca2+ and Mg2+ Regulation

Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca²⁺ and Mg²⁺ and the role of these changes in SR Ca²⁺ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N₂ to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca²⁺] <1 µM, ß-adrenergic stimulation increased luminal Ca²⁺ activation of single RyR channels, decreased luminal Mg²⁺ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg²⁺. At cytoplasmic [Ca²⁺] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg²⁺ and Ca²⁺ inhibition of RyRs. The K_a and maximum levels of cytoplasmic Ca²⁺ activation site were not affected by ß-adrenergic stimulation.Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca²⁺ binding to the luminal Ca²⁺ site and decreasing its affinity for luminal Mg²⁺ and 2) decreasing affinity of the low-affinity Ca²⁺/Mg²⁺ cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter.     

Signals and Responses: Choreographing the Complex Interaction between Legumes and α- and β-Rhizobia

The nitrogen-fixing symbiosis between bacteria in the family Rhizobiaceae and members of the legume family (Fabaceae) has been well studied, particularly from the perspective of the early signaling and recognition events. Recent studies of non-nodulating legume mutants have resulted in the identification of a number of genes that are responsive to signal molecules from the bacteria. However, a second group of nodule-forming bacteria, completely unrelated to the Rhizobiaceae, which are α-Proteobacteria, has been discovered. These bacteria belong to the β-Proteobacteria and have been designated β-rhizobia to distinguish them from the better-known α-rhizobia. Here, we review what is known in this economically important symbiosis about the interaction between legumes and α-rhizobia, and we incorporate information, where known, about the β-rhizobia.Key Words: biological nitrogen fixation, recognition, specificity, α-rhizobia, β-rhizobia     

Independent and Coordinate Regulation of β1- and β2-Adrenergic Receptors in Rat C6 Glioma Cells

Abstract

Rat C6 glioma cells have both β₁- and β₂-adrenergic receptors in ～ 7:3 ratio. When the cells were exposed to the β-adrenergic agonist isoproterenol, there was a rapid sequestration of up to 50% of the surface receptor population over a 30-min period as measured by the loss of binding of the hydrophilic ligand [³H] CGP-12177 to intact cells. Using the β₂-selective antagonist CGP 20712A to quantify the proportion of the two subtypes, it was found that although both β₁ and β₂ receptors were sequestered, the latter were sequestered initially twice as fast as the former. More prolonged agonist exposure led to a down-regulation of ～ 90% of the total receptor population by 6 h as measured by the loss of binding of the more hydrophobic ligand [¹²⁵I] iodocyanopindolol to cell lysates. The two subtypes, however, underwent down-regulation with similar kinetics. Treatment of the cells with agents that raise cyclic AMP levels such as cholera toxin and forskolin resulted in a slower, but still coordinated down-regulation of both subtypes. Thus, there appears to be both independent and coordinate regulation of endogenous β₁-and β₂-adrenergic receptors in the same cell line.     

Intracellular transport and processing of a tobacco vacuolar β-1,3-glucanase

The class I β-1,3-glucanases are basic, vacuolar enzymes implicated in the defense of plants against pathogen infection. The tobacco (Nicotiana tabacum L.) enzyme is synthesized as a preproprotein with an N-terminal signal peptide for targeting to the lumen of the endoplasmic reticulum and an N-glycosylated C-terminal extension which is lost during protein maturation. The transport and processing of β-1,3-glucanase in cellsuspension cultures of the tobacco cultivar Havana 425 was investigated by pulse-chase labelling and cell fractionation. We verified that mature β-1,3-glucanase is localized in the vacuole of the suspension-cultured cells. Comparison of the time course of processing in homogenates, the soluble fraction, and membrane fractions indicates that proglucanase is transported from the endoplasmic reticulum via the Golgi compartment to the vacuole. Processing to the mature form occurs in the vacuole. Treatment of cells with tunicamycin, which inhibits N-glycosylation, and digestion of the ³⁵S-labelled processing intermediates with endoglycosidase H indicate that β-1,3-glucanase has a single N-glycan attached to the C-terminal extension. Glycosylation is not required for proteolytic processing or correct targeting to the vacuole.     

Control by Ca2+ of mitochondrial structure and function in pancreatic β-cells

Mitochondria play a central role in glucose metabolism and the stimulation of insulin secretion from pancreatic β-cells. In this review, we discuss firstly the regulation and roles of mitochondrial Ca²⁺ transport in glucose-regulated insulin secretion, and the molecular machinery involved. Next, we discuss the evidence that mitochondrial dysfunction in β-cells is associated with type 2 diabetes, from a genetic, functional and structural point of view, and then the possibility that these changes may in part be mediated by dysregulation of cytosolic Ca²⁺. Finally, we review the importance of preserved mitochondrial structure and dynamics for mitochondrial gene expression and their possible relevance to the pathogenesis of type 2 diabetes.     

Insights into Distinct Modulation of α7 and α7β2 Nicotinic Acetylcholine Receptors by the Volatile Anesthetic Isoflurane

Nicotinic acetylcholine receptors (nAChRs) are targets of general anesthetics, but functional sensitivity to anesthetic inhibition varies dramatically among different subtypes of nAChRs. Potential causes underlying different functional responses to anesthetics remain elusive. Here we show that in contrast to the α7 nAChR, the α7β2 nAChR is highly susceptible to inhibition by the volatile anesthetic isoflurane in electrophysiology measurements. Isoflurane-binding sites in β2 and α7 were found at the extracellular and intracellular end of their respective transmembrane domains using NMR. Functional relevance of the identified β2 site was validated via point mutations and subsequent functional measurements. Consistent with their functional responses to isoflurane, β2 but not α7 showed pronounced dynamics changes, particularly for the channel gate residue Leu-249(9′). These results suggest that anesthetic binding alone is not sufficient to generate functional impact; only those sites that can modulate channel dynamics upon anesthetic binding will produce functional effects.     

A Low-Dose β1-Blocker in Combination with Milrinone Improves Intracellular Ca2+ Handling in Failing Cardiomyocytes by Inhibition of Milrinone-Induced Diastolic Ca2+ Leakage from the Sarcoplasmic Reticulum

Objectives

The purpose of this study was to investigate whether adding a low-dose β1-blocker to milrinone improves cardiac function in failing cardiomyocytes and the underlying cardioprotective mechanism.

Background

The molecular mechanism underlying how the combination of low-dose β1-blocker and milrinone affects intracellular Ca²⁺ handling in heart failure remains unclear.

Methods

We investigated the effect of milrinone plus landiolol on intracellular Ca²⁺ transient (CaT), cell shortening (CS), the frequency of diastolic Ca²⁺ sparks (CaSF), and sarcoplasmic reticulum Ca²⁺ concentration ({Ca²⁺}_SR) in normal and failing canine cardiomyocytes and used immunoblotting to determine the phosphorylation level of ryanodine receptor (RyR2) and phospholamban (PLB).

Results

In failing cardiomyocytes, CaSF significantly increased, and peak CaT and CS markedly decreased compared with normal myocytes. Administration of milrinone alone slightly increased peak CaT and CS, while CaSF greatly increased with a slight increase in {Ca²⁺}_SR. Co-administration of β1-blocker landiolol to failing cardiomyocytes at a dose that does not inhibit cardiomyocyte function significantly decreased CaSF with a further increase in {Ca²⁺}_SR, and peak CaT and CS improved compared with milrinone alone. Landiolol suppressed the hyperphosphorylation of RyR2 (Ser2808) in failing cardiomyocytes but had no effect on levels of phosphorylated PLB (Ser16 and Thr17). Low-dose landiolol significantly inhibited the alternans of CaT and CS under a fixed pacing rate (0.5 Hz) in failing cardiomyocytes.

Conclusion

A low-dose β1-blocker in combination with milrinone improved cardiac function in failing cardiomyocytes, apparently by inhibiting the phosphorylation of RyR2, not PLB, and subsequent diastolic Ca²⁺ leak.     

Characteristics of an Adenylate Cyclase Coupled β-Adrenergic Receptor in a Smooth Muscle Tumor Cell Line

Abstract

We have shown that binding of ³H-dihydroalprenolol ([³H] DHA) to DDT₁ MF-2 cells and cell membranes was of high affinity, saturable, stereoselective and reversible. The [³H]DHA dissociation constants were 0.63 ± 0.15 nM (n=6) and 0.83 ± 0.04 nM (n=5) for intact cells and cell membranes, respectively, with a binding site concentration for cells of 27,300 ± 5,200 sites/ cell (n=6) and for membranes 468 ± 24 fmoles/mg protein (n=5). The order of agonist competition for the [³H]-DHA binding site of DDT₁ cell membranes was isoproterenol (K_i = 0.20 ± 0.07 μM) > epinephrine (K_i = 0.4 ± 0.2 μM) > norepinephrine (K_i = 66.5 ± 5.15 μM) consistent with a β₂-selective receptor interaction. Zinterol, a β₂-selective antagonist, (K_i = 0.05 ± 0.01 μM) was 18x more effective than metoprolol, a β₁-selective antagonist (K_i = 0.9 ± 0.1 μM), in competing for the DHA binding site. A nonlinear iterative curve fitting analysis of zinterol and metoprolol binding isotherms indicated that (p>0.05) DDT₁ cells possess a pure population of β₂-adrenergic receptors. Finally, we have shown that DDT₁ MF-2 cell β₂-adrenergic receptor is functionally coupled to adenylate cyclase via a G/F protein complex as demonstrated in part by a guanine nucleotide requirement for isoproterenol stimulation of adenylate cyclase activity. In addition, guanine nucleotide mediated a reduction in the affinities of isoproterenol and epinephrine for the [³H]DHA binding site.     

Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca2+ Release via ROS-Stimulated Ryanodine Receptors

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells requires an increase in intracellular free Ca²⁺ concentration ([Ca²⁺]). Glucose uptake into β-cells promotes Ca²⁺ influx and reactive oxygen species (ROS) generation. In other cell types, Ca²⁺ and ROS jointly induce Ca²⁺ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca²⁺ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H₂O₂ in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H₂O₂-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca²⁺] produced by incubation of dissociated β-cells with H₂O₂. Addition of stimulatory glucose or H₂O₂ (in basal glucose) to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca²⁺ release, induced by the concomitant increases in [Ca²⁺] and ROS produced by stimulatory glucose, is an essential step in GSIS.