Activation of multiple signaling pathways in cells expressing recombinant opioid receptors
thesisposted on 15.12.2014, 10:29 by Charlotte Harrison
Although opioids have been used for centuries in the management of pain, the opioid receptor family has only recently been cloned, thus allowing detailed studies on opioid receptor mediated signal transduction pathways to be performed. In addition, two novel opioid peptides, endomorphin-1 and -2, were identified in 1997, and it is believed that these represent new endogenous u-opioid receptor ligands. This thesis represents a study into opioid receptor mediated increases in Ca2+ j and provides an investigation into the cellular signaling pathways of endomorphin-1 and -2. Activation of the recombinant 6-opioid receptor expressed in CHO cells (CH05) by D- Pen2'5 -enkephalin produced a concentration dependent, pertussis toxin and thapsigargin sensitive increase in Ca2+ i in whole cell suspensions. Truncation of this receptor by the 37 C-terminal amino acids produced a rightward shift in the concentration response curve for Ca j release. In single adherent CHOu, CHOk (CHO cells expressing recombinant u- or K-opioid receptors respectively) or CH05 cells, application of fentanyl, spiradoline or D-Pen2' -enkephalin respectively produced an increase in Ca i in some cells. The putative u.-opioid receptor endogenous ligands endomorphin- 1 and endomorphin-2 bound with high affinity and selectivity to u-opioid receptors from CHOu, and SH-SY5Y cells. Endomorphin-1 and -2 concentration-dependently inhibited forskolin stimulated cAMP formation in CHOp. and SH-SY5Y cells and endomorphin-1 and -2 produced an increase in Ca2+ j in CHOji cells. Prolonged endomorphin-1 pretreatment desensitized the -opioid receptor in CHO cells, characterized by a reduction in maximal endomorphin-1 mediated cAMP inhibition, an up-regulation of cAMP formation and was due to receptor - G protein uncoupling. Endomorphin-1 pre-treatment produced a rapid loss of cell surface receptors from CHOp. cells, which was possibly accompanied by receptor degradation. Collectively these data add to our understanding of opioid receptor-mediated signal transduction pathways.