Cellular toxicity of 2,4,5-trichlorophenoxyacetic acid: formation of 2,4,5-trichlorophenoxyacetylcholine.

One of the toxic symptoms of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) is reduction in metabolic rate and subsequent growth retardation. Acetylcholine (ACh) serves as an essential growth factor to facilitate amino acid transport and to promote fetal growth. Hydatidiform mole lacks the capacity for synthesis of ACh, and inhibition of ACh synthesis depresses placental amino acid transport. Therefore, we studied the formation of 2,4,5-acetylcoenzyme A (2,4,5-T-CoA) by acetylcoenzyme A synthase (ACoAS) and the formation of 2,4,5-T-ACh by human placental choline acteyltransferase (ChA) from 2,4,5-T-CoA and choline. In these studies, the widely used analog of 2,4,5-T as an herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), was also included. These studies have the following results (M +/- S.D.; N,6):1) The enzymatic rates of formation of acetyl-CoA, 2,4,5-T-CoA, and 2,4-D-CoA by ACoAS were 32 +/- 4, 23 +/- 3 and 26 +/- 8 nmol/mg protein/5 min., respectively; 2) There were no significant differences in the maximal amounts (nmol/mg protein) of acetyl-CoA (128 +/- 4), 2,4,5-T-CoA (125 +/- 8) and 2,4-D-CoA (96 +/- 6) formed during the reaction period of 50 min.; 3) 14C-2,4-ACh was formed from 14C-2,4-D-CoA and choline by placental-ChA; 4) Low concentrations (EC50 1-2 microM) of synthetic 2,4,5-T-ACh and 2,4-D-ACh decreased the contraction heights of the rat phrenic nerve-hemidiaphragm when the nerve or the muscle was electrically stimulated, and 5) Similar results were obtained with 2,4,6-T-ACh, an analog of 2,4,5-T-ACh. These observations indicate that chlorophenoxyherbicides form false cholinergic messengers in the nerve, muscle and placenta. These false cholinergic messengers can be formed at both muscarinic and nicotinic synaptic sites and also in non-neuronal cells, where ACh plays an important regulatory role as a local hormone, and act as blocking agents. These results will partially explain myotonia, ventricular fibrillation and fetal growth retardation induced by these herbicides.

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.

2-(alpha-Naphthoyl)ethyltrimethylammonium iodide and its beta-isomer: new selective, stable and fluorescent inhibitors of choline acetyltransferase.

The activities of 2-(alpha-naphthoyl)ethyltrimethylammonium (alpha-NETA) and its beta-isomer (beta-NETA) were studied at various sites of the cholinergic system using isolated enzyme and organ systems. They were selective inhibitors (I50: alpha-NETA, 9 microM; beta-NETA, 76 microM) of choline acetyltransferase (ChA). The inhibition of ChA by both alpha- and beta-NETA was noncompetitive with acetylcoenzyme A or choline as the variable substrate. In these experiments, the inhibitor and both substrates were added simultaneously to the reaction medium, and short reaction times of 10 min were used to determine initial linear velocities. Under these experimental conditions in the presence of substrates, the degree of inhibition of ChA by alpha-NETA was independent of enzyme concentration indicating the reversibility of the inhibition. If ChA was incubated with alpha-NETA for 10 min in the absence of substrates, the degree of inhibition was higher and was not reversible by dialysis of the inhibited ChA. These observations indicate that alpha-NETA is a pseudo-reversible or slowly reversible inhibitor. Neither alpha- nor beta-NETA exhibited significant effects at muscarinic receptors, ganglionic nicotinic receptors, skeletal muscular nicotinic receptors, cholinesterases or carnitine acetyltransferase at concentrations which inhibited ChA. At concentrations higher than their I50 values to inhibit ChA, both antagonized the effects of acetylcholine (ED50: alpha-NETA, 70-80 microM; beta-NETA, 100 microM), histamine and KCl-induced contractions in the guinea pig longitudinal ileal muscle. At high concentrations, alpha-NETA activated acetylcholinesterase (EC50, 360 microM) and inhibited cholinesterase (EC50, 1100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Peptides from human placenta: methionine enkephalin and substance P.

The occurrence of methionine enkephalin (379 pg/g tissue), beta-endorphin (448 pg/g tissue) and Substance P (2.4 pg/g tissue) in human placental villus were demonstrated by sensitive and specific radioimmunoassays. Conditions for the bioassay of placental extracts for enkephalin-like activities using the rat vas deferens were described. Substance P did not interfere in this bioassay. Comparison of the enkephalin-like activities determined by bioassay and the content of beta-endorphin and methionine enkephalin determined by radioimmunoassays indicated that placental villus extracts contain other unidentified potent opioid-like peptides or substances. It is suggested that methionine enkephalin and/or beta-endorphin and Substance P regulate release of acetylcholine or hormones from placental villus. Alternatively, these peptides may regulate sensory transmission (pain impulses) locally from the distended uterus during pregnancy or from the vaginal tract during childbirth.