Genetic analysis of peptide amidation and opioid receptor function in mice

This thesis is a compilation of three individual projects that overlap two broad fields of research: protein processing and the endogenous opioid system.; We have generated a null mutant of the peptide amidating enzyme, peptidyl-glycine alpha-amidating monooxygenase (PAM), via traditional gene targeting methods in the mouse to determine if there are any required functions of PAM during embryogenesis. Homozygous mutant mice die at embryonic day (e) 14.5 with severe generalized edema. Histological analysis of mutant PAM embryos indicates that there are cardiovascular deficits, including left ventricular hypertrophy, that is consistent with the severe edema. No amidation activity was detected in homozygous mutant embryos demonstrating that PAM is the sole peptide amidation mechanism in the fetal mouse. Heterozygous mutant mice are viable and fertile. They have one half the wildtype levels of amidation activity and PAM protein. Adult male heterozygotes are hyperglycemic in a glucose tolerance test suggesting that there are metabolic abnormalities in these mice.; We have generated combinatorial opioid receptor knockout (KO) mice by interbreeding single gene KOs of the mu, kappa, and delta opioid receptors. Double and triple opioid receptor KO mice are viable and fertile and have no gross developmental abnormalities. These mice were used to determine the contributions of each individual opioid receptor in the analgesia induced by nalaxone benzoylhydrazone (NalBzoH) and nociceptin (OFQ) NalzoH analgesia is fully retained in triple KO mice. However, the analgesia of intrathecally administered OFQ is attenuated only in combinatorial mutants lacking the kappa opioid receptor (KOR-1).; We have previously generated KOR-1 KO mice, allowing for the genetic analysis of KOR-1 in the behavioral, neurochemical and molecular effects of cocaine. Preliminary studies have indicated that repeated injections of 10 or 20 mg/kg of cocaine may fail to illicit an increase in locomotor behavior to the extent that it does in wildtype mice. The induction of the immediate early genes c-fos, jun B, and fos B 30 minutes after an injection of 20 mg/kg of cocaine was attenuated in the brain of KOR-1 mutants as compared to wildtype mice. KOR-1 KO mice, therefore, exhibit accelerated behavioral and molecular sensitization to cocaine.