James L. Caffrey

Screening for opioids in dog heart

Dog hearts divided into right and left atria, right and left ventricles and intraventricular septum were homogenized in acid for extraction. Total opioids, and specific peptides (methionine-enkephalin, methionine-enkephalin-arg6-gly7-leu8) were determined by radioreceptor and radioimmunoassay, respectively. Catecholamines were quantitated amperometrically following HPLC. The effects of anesthetic agents (pentobarbital, alpha-chloralose), hemorrhage and ganglionic blockade (hexamethonium and atropine) were evaluated. Total opioids, enkephalins and epinephrine were distributed uniformly throughout the myocardium, while norepinephrine was preferentially concentrated in the atria. Immunoreactive methionine-enkephalin accounted for only 1 to 2% of the total cardiac opioids estimated by radioreceptor assay. Hemorrhage lowered methionine-enkephalin content throughout the myocardium with no significant effect on total opioids or catecholamines. Ganglionic blockade increased total opioid, methionine-enkephalin-arg6-gly7-leu8 and catecholamine content without altering methionine-enkephalin content. HPLC of left ventricular extracts demonstrated that 50% of met-enkephalin-immunoreactivity eluted at retention times equal to synthetic metenkephalin. In summary, there appears to be substantive opioid concentrations within canine myocardium which respond to physiological and pharmacological interventions. These cardiac opioid responses do not parallel changes observed for catecholamines under the same conditions.

Role of calcium and prostaglandins in the antidiuretic hormone response: Effect of ionophore A23187

The calcium ionophore A23187 (IP) inhibited the antidiuretic hormone (ADH)-stimulated hydro-osmotic response in toad urinary bladder but had no effect on the osmotic transfer of water in the absence of hormone. Extracellular calcium was necessary for this effect at lower but not at higher IP concentrations. The hydro-osmotic response to exogenous cyclic AMP was unaltered by IP, but the same response produced by inhibition of phosphodiesterase was reduced significantly. Cyclic AMP concentrations in isolated toad bladder epithelial cells were reduced by 50% with IP or exogenous prostaglandin E2 (PGE2). Indomethacin, a prostaglandin synthesis inhibitor, prevented the inhibitory actions of the IP on the ADH-mediated response. Collectively, these observations suggest a key role for cellular calcium in modulating the actions of antidiuretic hormone and are consistent with the hypothesis that the ionophore, through increasing intracellular calcium, stimulates the synthesis of prostaglandins which have a negative feedback on adenylcyclase. This effect would terminate the action of the hormone.

The assay of Met-enkephalin aminopeptidase with [125I]Met-enkephalin.

Presented here are procedural modifications which permit the utilization of 125I-labeled Met-enkephalin as substrate in the assay of rat brain enkephalin aminopeptidase. Th hydrolysis of enkephalin is monitored by the release of [125I]tyrosine separated on Porapak Q. The release of tyrosine is proportionate with both increasing time and tissue concentration. The estimated Km is near 10(-4) M and the enzyme activity can be inhibited more than 95% with puromycin. The majority of the enzyme activity remains in the 100,000 x g supernatant following differential centrifugation.