Victoria E. Janson

Distribution and Retention of Nicotine and Its Metabolite, Cotinine, in the Rat as a Function of Time

Nicotine is oxidized to its major metabolite, cotinine, which has a long biological half-life (19-24 h). The plasma concentration of cotinine has been used as an index of tobacco smoke exposure. Cotinine possibly increases the turnover rate of platelet-activating factor (PAF) because it is a potent activator of PAF hydrolase, and it may play a significant role in tobacco-induced arterial thrombosis. Therefore, we studied the distribution and retention of nicotine as it was metabolized to cotinine in the rat. Nicotine (1 mg/kg, 5 microCi/kg) was administered into the femoral vein of male Sprague-Dawley rats under nembutal anesthesia. At different times (5-60 min) after nicotine administration, nicotine and its metabolite, cotinine, were determined by HPLC in plasma, liver, kidney, heart and brain. Within 5-10 min after administration, nicotine concentrations reached peak values in plasma (2,160 pmol/ml) and the organs analyzed. The plasma level of nicotine decreased by 50% within 20 min (half-time) after its intravenous administration. The half-time of nicotine in the brain was about 50 min. The half-times of nicotine for the other organs were about 20-25 min. The major metabolite, cotinine, accumulated in plasma, and by about 30 min the concentrations of nicotine and cotinine in plasma were about equal (890-1,000 pmol/ml). While cotinine accumulated in plasma, nicotine was eliminated by the kidney. While the nicotine concentrations decreased with time in all organs, cotinine concentrations remained constant. These observations indicate that nicotine is renally eliminated or metabolized to cotinine while cotinine exhibits a long retention time and accumulates in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in Enzymes of the Cholinergic System and Acetylcholine Release in the Cerebra of Aging Male Fischer Rats

The functional decline of memory in the aging human brain has been partially attributed to defects in cholinergic transmission. Therefore, we have investigated various components of the cholinergic system in cerebra of Fischer 344 male rats, ages 3-33 months. Choline acetyltransferase (ChA), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) activities were determined in homogenates of the cerebra using specific radiometric assays. For measuring the release of acetylcholine (ACh), cerebral slices were incubated for one hour in Krebs buffer containing 3H-choline chloride to label ACh formed in situ, washed, and transferred to a microbath for superfusion. 3H-ACh released into the superfusate was determined. The levels of ChA in the cerebra of 9- to 27-month-old rats were lower (33%) than those in 3-month-old rats. Only 1% of these rats survive to the age of 33 months. In rats of this age, there was no decrease in ChA levels. AChE decreased while BChE increased with advancing age. The rate of spontaneous release of 3H-ACh decreased gradually by 63% from 3 to 33 months of age. The evoked release of ACh decreased by 50% in 33-month-old rats. Alterations in the levels of ChA, AChE (or BChE) and cholinergic receptors are not large enough to account for losses in cholinergic transmission in the cerebrum. The large decreases in the rates of spontaneous or evoked release of ACh in the aging cerebrum indicates that the functional defect in the cholinergic transmission of the aging cerebrum is possibly due to a defective release mechanism of this transmitter.

Significance of substance P- and enkephalin-peptide systems in the male genital tract.

The plexi of the male reproductive tract have components of both the autonomic and sensory nervous systems. Rat epididymis was found to be a rich source of substance P. Substance P levels in the epididymis were higher by about 2.8 and 19.3 times than those in the prostate and seminal vesicles, respectively. Seminal vesicles were found to be a rich source of enkephalins. They had about 2.9 and 2.6 times higher leucine enkephalin levels than epididymis and prostate, respectively. Human seminal plasma contained about 47 times higher levels of leucine enkephalin than substance P. Using the split ejaculate technique, it has been demonstrated that early fractions of the human ejaculate contain fluids from prostate (and possibly epididymis), whereas later fractions represent seminal vesicle secretions. A low exogenous concentration of substance P (400 nM) increased sperm motility, whereas leucine enkephalin (100 microM) depressed it. Substance P (1-10 micrograms/mL) and muscarinic agonists enhanced the adrenergic transmission of the rat vas deferens to electrical stimulation. Leucine enkephalin (1-10 micrograms/mL) depressed adrenergic transmission and antagonized the effects of substance P and muscarinic agonists. These studies suggest that substance P-like tachykinins may play a role in sperm maturation, in expulsion of fluid from the epididymis, and in initiation of motility, whereas leucine enkephalin-like peptides may contribute to the orgasmic experience and detumescence.

Depression of human sperm motility by inhibition of enzymatic methylation

Alteration of membrane fluidity during enzymatic methylation of membrane phosphatidyl-ethanolamine (PE) and neutralization of negative charges of membrane proteins due to methylation of carboxyl groups may contribute to sperm motility. Therefore, enzymatic phospholipid methylation and carboxymethylation, and the consequences of their inhibition on motility, were studied using human sperm. These studies gave the following results. Human sperm homoganates contained two phospholipid N-methyltransferases (PMT) which converted PE to phosphatidylcholine (PC) in the presence of S-adenosylmethionine (SAM). The first PMT converted PE to phosphatidyl-N-methylethanolamine (PME). In had a Km of 4.0 microM and a pH optimum of 8.0. The second PMT converted PME to phosphatidyl-N,N-dimethylethanolamine and PC. It had a Km of 71 microM and a pH optimum of 10.0. Spermatozoa also contained protein carboxymethylase (PCM) and methyl aceptor protein (MAP). The intracellular levels of S-adenosylhomocysteine (SAH), an inhibitor of SAM-mediated methylations, were increased by adding adenosine (100 microM), L-homocysteine thiolactone (L-HCT, 10 microM), and erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA, 10 microM), an inhibitor of adenosine deaminase, to human sperm ejaculates that had been diluted with sodium phosphate buffer at pH 7.4 and 25 degrees. The motility index of each sperm suspension was determined every hour for 4 hr. In the presence of the mixture of adenosine, L-HCT and EHNA, the motility index was depressed by 57%. Under similar conditions, phospholipid methylation was depressed by 48%. Similar experiments were also conducted in the presence of 3-deazaadenosine (Deaza, 80 microM), a selective inhibitor of SAH hydrolase. In the presence of adenosine and L-HCT, Deaza depressed the motility index by 60% and phospholipid methylation by 86%. The potencies of SAH in the inhibition of phospholipid methylation and protein carboxymethylation in sperm homogenates had the following order: PMT I greater than PCM greater than PMT II. These observations indicate that the PMT system and/or the PCM-MAP system play a significant role in the regulation of human sperm motility.

HPLC Analysis of Amino Acids with Ion Exchange Chromatography and O-Phthalaldehyde Post-Column Derivatization: Applications to the Assay of Endogenous Free Amino Acids and Their Transport in Human Placental Villus

Abstract A method using high performance liquid chromatography (HPLC) for the analysis of primary amino acids in human placenta is described. This method involves separation of primary amino acids by high performance ion-exchange chromatography followed by post column derivatization using O-phlthalaldehyde (OPA) and 2-mercaptoethanol and fluorescence (excitation 340 nm and emission 410 nm) detection of derivatives. Waters 840 HPLC Amino Acid System was used for this purpose. For analysis, villus tissue was extracted with acetonitrile, and the recovered amino acids were reconstituted in a sodium diluent (pH 2.2). The complete profile of the primary amino acids in the sample could be constructed in about 90 minutes. Up to 44 samples can be analyzed without special attention. Using this method, essential amino acids (threonine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine) and nonessential amino acids (aspartic acid, serine, glutamic acid, glycine, alanine, arginine) we...