A new chelation method for determining ATPase activity in skeletal muscle.

Traditional methods for visualizing ATPase in sections use heavy metals that generate visible metal sulfide products. These methods use unpleasant and toxic reagents. We report a safer method using a novel ferric ion chelating agent to produce highly specific, low background, and permanent staining of muscle fiber enzymes.

A technique for assessing the effects of pH and photodegradation on the stability of the iron chelate of ethyl N-methyl-4-hydroxy-5-oxo-3-pyrroline-3-carboxylate.

Ethyl N-methyl-4-hydroxy-5-oxo-3-pyrroline-3-carboxylate forms a deep red chelate with iron salts. The color intensity is directly related to the iron concentration. The photostability of the red color was determined at pH 1.2 and 5 by spectrophotometric assay at 484 nm at intervals during irradiation by tungsten light at 1020 microW/cm2. After 528 hr of continuous irradiation in deionized water, 90.9% of the iron chelate had decomposed. The reaction followed zero order kinetics. Maximal stability was observed at pH 5 at both 10(-1) and 10(-2) molar concentrations of the iron chelate; no detectable decomposition occurred after 192 hr of continuous irradiation. The iron chelate in biological tissues is stable for 18 months. The staining technique is superior to other histological methods for estimating low concentrations of iron in tissue.

Investigation of the 5-hydroxytryptamine receptor mechanism mediating the short-circuit current response in rat colon.

1. 5-Hydroxytryptamine (5-HT) stimulated an increase in short-circuit current (SCC) in rat isolated colonic mucosa with an EC50 value of approximately 4 microM. The purpose of the present study was to investigate the 5-HT receptor mechanism(s) involved in this response. 2. The relatively selective 5-HT receptor agonists 5-carboxamidotryptamine (5-CT) and alpha-methyl-5-HT stimulated SCC and were 6 to 8 times less potent than 5-HT. 2-Methyl-5-HT was inactive both as an agonist and an antagonist. 3. The following compounds produced no significant inhibition of the SCC response to 5-HT: ketanserin (1 microM), methysergide (1 microM), methiothepin (0.3 microM), GR38032F (0.3 microM), tetrodotoxin (0.3 microM) and sulpiride (1 microM). 4. Both metoclopramide (3 and 10 microM) and cisapride (0.1 and 1 microM) inhibited the SCC responses to 5-HT in a concentration-related manner, and the higher doses similarly inhibited the responses to 5-CT. With both agonists the inhibitory effects of metoclopramide and cisapride were insurmountable. However, these inhibitory actions appeared to be selective since neither metoclopramide nor cisapride affected the basal SCC or the SCC response to prostaglandin E2. 5. The SCC responses to 5-HT and 5-methoxytryptamine were selectively inhibited by ICS205-930 at 3 microM, and respective pKB values of 6.0 and 6.6 were calculated. 6. It is concluded that 5-HT stimulates an SCC response in rat colon via a receptor mechanism that cannot be clearly identified as 5-HT1-like, 5-HT2 or 5-HT3. This receptor is selectively antagonized by ICS 205-930 and by the benzamides, metoclopramide and cisapride. The 5-HT receptor in rat colon therefore exhibits some of the properties associated with the so-called 5-HT4 receptor.