Synergistic Interaction of Y1-Neuropeptide Y and α1b-Adrenergic Receptors in the Regulation of Phospholipase C, Protein Kinase C, and Arachidonic Acid Production

Abstract

Neuropeptide Y (NPY) and norepinephrine, found colocalized in sympathetic neurons innervating blood vessels, exert synergistic responses on vasoconstriction. To examine the signaling mechanisms involved, free of complications associated with mixed receptor populations, we have established a stable Chinese hamster ovary cell line expressing both Y1-NPY and alpha 1b-adrenergic receptors. Occupation of either receptor species, with 100 nM peptide YY (PYY) or 10 microM phenylephrine (PE), respectively, resulted in a rapid increase in the cytoplasmic free calcium concentration ([Ca2+]i) as assessed with Fura-2/AM. The rise due to PYY, but not that due to PE, was abolished by pretreatment with pertussis toxin. Both responses were largely maintained in the absence of extracellular Ca2+, but abolished by prior depletion of intracellular Ca2+ pools with either thapsigargin or 2,5-di-(t-butyl)-1,4-benzohydroquinone. Using cells prelabeled with myo-[3H]inositol, PE promoted a rapid (5 s) rise in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) as analyzed by anion-exchange high pressure liquid chromatography, whereas the response to PYY (first significant at > 15 s post-stimulation) was too slow to play a causative role in Ca2+ mobilization. Combination of PE and PYY resulted in increases in [Ca2+]i which were at best additive, whereas they promoted a clearly synergistic rise in Ins(1,4,5)P3 at both 15 and 60 s. Co-stimulation also resulted in a synergistic activation of both protein kinase C (PKC) and [3H]arachidonic acid release. In either instance PYY alone was without effect. The potentiation of arachidonic acid release was abolished by depletion of cellular PKC following chronic treatment with phorbol esters. It is suggested that the ability of PYY to mobilize Ca2+ in an Ins(1,4,5)P3-independent fashion minimizes the functional importance of the capacity to potentiate PE-stimulated Ins(1,4,5)P3 generation. Instead the major consequences of the synergistic activation of phospholipase C are mediated via PKC, the other route of the signaling pathway.

Protein Kinase C-θ Phosphorylation of Moesin in the Actin-binding Sequence

“…PKC-is the main novel isozyme expressed in skeletal muscle, testes, platelets, and their megakaryoblastic precursor cells, T-lymphocytes, and neoplastic hemopoietic cells (7,48,49). We have found moesin phosphorylation activity in HL-60 cells, spleen cells, and AML cells, which is consistent with the known distribution of PKC- (29) and in contrast to the tissue distribution of the (50) and the / isoforms, which are not found in spleen (51,52). We have previously described modulation of this activity under various conditions affecting cellular proliferation and differentiation (4,5).…”

Antipeptide antibodies directed against the C‐terminus of protein kinase Cζ (PKCζ) react with a Ca2+‐ and TPA‐sensitive PKC in HT‐29 human intestinal epithelial cells

“…For this reason this peptide was chosen to raise PKC [-specific antibodies by Gibco and several authors, and used by many groups to determine the presence of this isoform in human [7-91 and other species [lo-171. Very recently, it has been reported that this antipeptide antibody also recognizes a newly described PKC, aPKCz [4]. This isoform is clearly different from the 77 kDa cPKC since it has a lower molecular weight and does not respond to TPA or Ca2' [4].…”

“…Very recently, it has been reported that this antipeptide antibody also recognizes a newly described PKC, aPKCz [4]. This isoform is clearly different from the 77 kDa cPKC since it has a lower molecular weight and does not respond to TPA or Ca2' [4].…”

“…The novel PKC subfamily (nPKC), composed of isoforms 6, E, q and 19, lacks the Ca2'-binding domain and the activity of its members is independent of Ca2' [l-3]. Recently, two other PKC isoforms, c and z, have been gathered into a new subfamily, atypical PKCs (aPKC), in accordance with their insensitivity to diacylglycerol, the natural activator of the other PKCs [1,4]. Antibodies raised against peptides specific for the different isoforms of PKC have been used as a powerful tool to examine the presence of the members of the PKC family in different cells and tissues.…”

NO2‐induced expression of specific protein kinase C isoforms and generation of phosphatidylcholine‐derived diacylglycerol in cultured pulmonary artery endothelial cells

“…For example, the conventional PKC isoforms (c&r) are activated by calcium, PS, and DAG or PDBu. The novel PKC isoforms (6,~,rl,t3) are also activated by PS and DAG or PDBu but lack the calcium binding domain, whereas atypical PKC isoforms (&t,y,u) are activated by PS but not by DAG, PDBu, or calcium [9,20,21]. Very little is known about the distribution and expression of PKC isoforms in vascular endothelium.…”