85 research outputs found
Role of protein kinase C in functional selectivity for desensitization at the µ-opioid receptor: from pharmacological curiosity to therapeutic potential
Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72412/1/j.1476-5381.2009.00198.x.pd
A Small Molecule Inhibitor of ITK and RLK Impairs Th1 Differentiation and Prevents Colitis Disease Progression
In T cells, the Tec kinases IL-2-inducible T cell kinase (ITK) and resting lymphocyte kinase (RLK) are activated by TCR stimulation and are required for optimal downstream signaling. Studies of CD4(+) T cells from Itk(-/-) and Itk(-/-)Rlk(-/-) mice have indicated differential roles of ITK and RLK in Th1, Th2, and Th17 differentiation and cytokine production. However, these findings are confounded by the complex T cell developmental defects in these mice. In this study, we examine the consequences of ITK and RLK inhibition using a highly selective and potent small molecule covalent inhibitor PRN694. In vitro Th polarization experiments indicate that PRN694 is a potent inhibitor of Th1 and Th17 differentiation and cytokine production. Using a T cell adoptive transfer model of colitis, we find that in vivo administration of PRN694 markedly reduces disease progression, T cell infiltration into the intestinal lamina propria, and IFN-gamma production by colitogenic CD4(+) T cells. Consistent with these findings, Th1 and Th17 cells differentiated in the presence of PRN694 show reduced P-selectin binding and impaired migration to CXCL11 and CCL20, respectively. Taken together, these data indicate that ITK plus RLK inhibition may have therapeutic potential in Th1-mediated inflammatory diseases
Role of protein kinase C and μ-opioid receptor (MOPr) desensitization in tolerance to morphine in rat locus coeruleus neurons
In morphine tolerance a key question that remains to be answered is whether μ-opioid receptor (MOPr) desensitization contributes to morphine tolerance, and if so by what cellular mechanisms. Here we demonstrate that MOPr desensitization can be observed in single rat brainstem locus coeruleus (LC) neurons following either prolonged (> 4 h) exposure to morphine in vitro or following treatment of animals with morphine in vivo for 3 days. Analysis of receptor function by an operational model indicated that with either treatment morphine could induce a profound degree (70–80%) of loss of receptor function. Ongoing PKC activity in the MOPr-expressing neurons themselves, primarily by PKCα, was required to maintain morphine-induced MOPr desensitization, because exposure to PKC inhibitors for only the last 30–50 min of exposure to morphine reduced the MOPr desensitization that was induced both in vitro and in vivo. The presence of morphine was also required for maintenance of desensitization, as washout of morphine for > 2 h reversed MOPr desensitization. MOPr desensitization was homologous, as there was no change in α2-adrenoceptor or ORL1 receptor function. These results demonstrate that prolonged morphine treatment induces extensive homologous desensitization of MOPrs in mature neurons, that this desensitization has a significant PKC-dependent component and that this desensitization underlies the maintenance of morphine tolerance
Co-expression of mu and delta opioid receptors as receptor-G protein fusions enhances both mu and delta signalling via distinct mechanisms
Abstract Mu and delta opioid receptors were co-expressed as fusion proteins between a receptor and a pertussis insensitive mutant Galphai/o protein in HEK 293 cells. Signalling efficiency was then monitored following inactivation of endogenous Galphai/o proteins by pertussis toxin. Co-expression resulted in increased delta opioid signalling which was insensitive to the mu specific antagonist CTAP. Under these conditions, mu opioid signalling was also increased and insensitive to the delta specific antagonist Tic-deltorphin. In this latter case, however, no G protein activation was observed in the presence of the delta specific inverse agonist N,N(CH3)2-Dmt-Tic-NH2. When a mu opioid receptor fused to a non-functional Galpha subunit was co-expressed with the delta opioid receptor-Galpha protein fusion, delta opioid signalling was not affected whereas mu opioid signalling was restored. Altogether our results suggest that increased delta opioid signalling is due to enhanced delta opioid receptor coupling to its tethered Galpha subunit. On the other hand, our data indicate that increased mu opioid signalling requires an active conformation of the delta opioid receptor and also results in activation of the Galpha subunit fused the delta opioid recepto
Morphine induces terminal μ-opioid receptor desensitization by sustained phosphorylation of serine-375
Morphine is a poor inducer of μ-opioid receptor (MOR) internalization, but a potent inducer of cellular tolerance. Here we show that, in contrast to full agonists such as [D-Ala(2)-MePhe(4)-Gly-ol]enkephalin (DAMGO), morphine stimulated a selective phosphorylation of the carboxy-terminal residue 375 (Ser(375)). Ser(375) phosphorylation was sufficient and required for morphine-induced desensitization of MOR. In the presence of full agonists, morphine revealed partial agonistic properties and potently inhibited MOR phosphorylation and internalization. Upon removal of the drug, DAMGO-desensitized receptors were rapidly dephosphorylated. In contrast, morphine-desensitized receptors remained at the plasma membrane in a Ser(375)-phosphorylated state for prolonged periods. Thus, morphine promotes terminal MOR desensitization by inducing a persistent modification of Ser(375)
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