Differences in the signaling pathways of α1A- and α1B-adrenoceptors are related to different endosomal targeting

Aims: To compare the constitutive and agonist-dependent endosomal trafficking of α1A- and α1B-adrenoceptors (ARs) and to establish if the internalization pattern determines the signaling pathways of each subtype. Methods: Using CypHer5 technology and VSV-G epitope tagged α1A- and α1B-ARs stably and transiently expressed in HEK 293 cells, we analyzed by confocal microscopy the constitutive and agonist-induced internalization of each subtype, and the temporal relationship between agonist induced internalization and the increase in intracellular calcium (determined by FLUO-3 flouorescence), or the phosphorylation of ERK1/2 and p38 MAP kinases (determined by Western blot). Results and Conclusions: Constitutive as well as agonist-induced trafficking of α1A and α1B ARs maintain two different endosomal pools of receptors: one located close to the plasma membrane and the other deeper into the cytosol. Each subtype exhibited specific characteristics of internalization and distribution between these pools that determines their signaling pathways: α1A-ARs, when located in the plasma membrane, signal through calcium and ERK1/2 pathways but, when translocated to deeper endosomes, through a mechanism sensitive to β-arrestin and concanavalin A, continue signaling through ERK1/2 and also activate the p38 pathway. α1B-ARs signal through calcium and ERK1/2 only when located in the membrane and the signals disappear after endocytosis and by disruption of the membrane lipid rafts by methyl-β-cyclodextrin

Potential Relevance of α1-Adrenergic Receptor Autoantibodies in Refractory Hypertension

-AAB might have a mechanistic role and could represent a therapeutic target. in cardiomyocytes and induce mesentery artery segment contraction.-AAB in hypertensive patients, and the notion of immunity as a possible cause of hypertension

alpha(1)-adrenoceptor subtypes differentially couple to growth promotion and inhibition in Chinese hamster ovary cells

We have compared the coupling of human alpha(1A)-, alpha(1B)-, and alpha(1D)-adrenoceptors (expressed at approximately 2000 fmol/mg protein in Chinese hamster ovary cells) to cellular growth promotion (as assessed by [(3)H]thymidine incorporation) and related signaling mechanisms. Maximum elevation of intracellular Ca(2+) by the three subtypes occurred with the rank order alpha(1A) (1691 nM) > alpha(1D) (1215 nM) > alpha(1B) (360 nM). In contrast, activation of the ERK, JNK, and p38 forms of mitogen-activated protein kinases occurred with the rank order alpha(1D) > alpha(1A) > alpha(1B). alpha(1A)-Adrenoceptor stimulation inhibited basal and growth factor-stimulated [(3)H]thymidine incorporation by 74%, and this was mitigated by p38 inhibition. In contrast, alpha(1D)-adrenoceptor stimulation enhanced cellular growth by 136%, and this was blocked by two distinct inhibitors of ERK activation. We conclude that within a given cell type alpha(1)-adrenoceptor subtypes can have opposite effects on cellular growth, although their proximal signal transduction displays only quantitative difference

Differential regulation of 46 and 54 kDa jun N-terminal kinases and p38 mitogen-activated protein kinase by human alpha(1A)-adrenoceptors expressed in Rat-1 cells

We have investigated the alpha(1A)-adrenoceptor-mediated activation of 46 and 54 kDa isoforms of c-jun N-terminal kinase (JNK) and of p38 mitogen-activated protein kinase. The alpha(1)-adrenoceptor agonist phenylephrine activated all three kinases but with different time courses and maximal effects. Activation of all three kinases was insensitive to the phosphatidylinositol-3-kinase inhibitor wortmannin but was enhanced by the protein kinase C inhibitor bisindolylmaleimide I; a protein kinase C-activating phorbol ester inhibited JNK but not p38 activation. Activation of 54 kDa JNK, but not of the other two kinases, was inhibited by pertussis toxin and the phospholipase C inhibitor U 73,122. We conclude that alpha(1)-adrenoceptor stimulation activates 46 kDa JNK, 54 kDa JNK and p38 but uses at least partly different pathways to do s

Stimulation of alpha1A-adrenoceptors in Rat-1 cells inhibits extracellular signal-regulated kinase by activating p38 mitogen-activated protein kinase

In Rat-1 fibroblasts, endothelin-1 and a protein kinase C-stimulating phorbol ester stimulated extracellular signal-regulated kinase (ERK), whereas phenylephrine, acting at stably transfected human alpha1A-adrenoceptors, inhibited basal and endothelin-1- and phorbol ester-stimulated ERK. On the other hand, phenylephrine stimulated p38 mitogen-activated protein kinase (MAPK). Anisomycin caused p38 activation and ERK inhibition quantitatively similar to those produced by phenylephrine. SB 203,580, an inhibitor of p38, significantly attenuated phenylephrine- and anisomycin-induced ERK inhibition. The ERK inhibition by phenylephrine was not affected by the cytosolic phospholipase A2 inhibitor arachidonyltrifluoromethyl ketone or the cyclooxygenase inhibitor indomethacin but was significantly attenuated by a combination of the phosphatase inhibitors Na3VO4 and okadaic acid. Neither SB 203,580 nor the phosphatase inhibitors significantly affected ERK inhibition by the adenylyl cyclase activator forskolin. We conclude that there is a previously unrecognized interaction between ERK and p38 MAPK, in which activation of p38 causes inhibition of ERK; this may at least partly involve MAPK phosphatases that inactivate ER

Neuropeptide-Y stimulation of extracellular signal-regulated kinases in human erythroleukemia cells

We have used human erythroleukemia (HEL) cells to investigate distal signaling mechanisms of neuropeptide-Y (NPY) receptors. NPY did not activate phospholipase D, determined as a phosphatidylethanol formation, or protein kinase C (PKC) determined enzymatically as a translocation to the plasma membrane. However, NPY caused a rapid (already maximal after 30 s) and concentration-dependent (maximum at 10-100 nM) activation of extracellular signal-regulated kinase (ERK) as assessed by immunoblotting with epitope-specific, antiphosphotyrosine antibodies and in some cases enzymatically. ERK activation by 100 nM NPY was abolished by the Y(1) NPY receptor antagonist BIBP 3226 (1 microM), pertussis toxin treatment (100 ng ml(-1) overnight), the mitogen-activated protein kinase (MAPK) kinase inhibitor PD 98059 (100 microM), and the phosphatidylinositol-3-kinase inhibitor wortmannin (100 nM). Whereas the PKC inhibitor staurosporine (3 microM) inhibited ERK activation by NPY, the chemically distinct PKC inhibitors calphostin C (3 microM), Gö 6976 (3 microM), and bisindolylmaleimide I (3 microM) did not. NPY did not activate other MAPK such as jun N-terminal kinase or p38 MAPK. We conclude that NPY does not activate phospholipase D, PKC, jun N-terminal kinase, or p38 MAPK in HEL cells. However, NPY activates ERK by a pathway involving Y(1) receptors, pertussis toxin-sensitive G proteins, and phosphatidylinositol-3-kinase, whereas PKC may not be involved. Staurosporine may have PKC-independent effects on ERK activatio

Baggergut der Hafengruppe Bremen-Stadt: Modelluntersuchungen zur Schwermetallmobilitaet und Moeglichkeiten der Verwertung von Hafenschlick aus Bremischen Haefen Endbericht zum Forschungsvorhaben 060 des Landes Bremen

Available from TIB Hannover: RO 7630(107) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Intracellular signaling mechanisms mediating catecholamine release upon activation of NPY Y1 receptors in mouse chromaffin cells

The adrenal chromaffin cells synthesize and release catecholamine (mostly epinephrine and norepinephrine) and different peptides, such as the neuropeptide Y (NPY). NPY stimulates catecholamine release through NPY Y1 receptor in mouse chromaffin cells. The aim of our study was to determine the intracellular signaling events coupled to NPY Y1 receptor activation that lead to stimulation of catecholamine release from mouse chromaffin cells. The stimulatory effect of NPY mediated by NPY Y1 receptor activation was lost in the absence of extracellular Ca2+. On the other hand, inhibition of nitric oxide synthase and guanylyl cyclase also decreased the stimulatory effect of NPY. Moreover, catecholamine release stimulated by NPY or by the nitric oxide donor (NOC-18) was inhibited by mitogen-activated protein kinase (MAPK) and protein kinase C inhibitors. In summary, in mouse chromaffin cells, NPY evokes catecholamine release by the activation the NPY Y1 receptor, in a Ca2+-dependent manner, by activating mitogen-activated protein kinase and promoting nitric oxide production, which in turn regulates protein kinase C and guanylyl cyclase activation