George D. Webb

Contractile and Vasorelaxant Effects of Hydrogen Sulfide and Its Biosynthesis in the Human Internal Mammary Artery

This study aimed to test these hypotheses: cystathionine γ-lyase (CSE) is expressed in a human artery, it generates hydrogen sulfide (H2S), and H2S relaxes a human artery. H2S is produced endogenously in rat arteries from cysteine by CSE. Endogenously produced H2S dilates rat resistance arteries. Although CSE is expressed in rat arteries, its presence in human blood vessels has not been described. In this study, we showed that both CSE mRNA, determined by reverse transcription-polymerase chain reaction, and CSE protein, determined by Western blotting, apparently occur in the human internal mammary artery (internal thoracic artery). Artery homogenates converted cysteine to H2S, and the H2S production was inhibited by dl-propargylglycine, an inhibitor of CSE. We also showed that H2S relaxes phenylephrine-precontracted human internal mammary artery at higher concentrations but produces contraction at low concentrations. The latter contractions are stronger in acetylcholine-prerelaxed arteries, suggesting inhibition of nitric oxide action. The relaxation is partially blocked by glibenclamide, an inhibitor of KATP channels. The present results indicate that CSE protein is expressed in human arteries, that human arteries synthesize H2S, and that higher concentrations of H2S relax human arteries, in part by opening KATP channels. Low concentrations of H2S contract the human internal mammary artery, possibly by reacting with nitric oxide to form an inactive nitrosothiol. The possibility that CSE, and the H2S it generates, together play a physiological role in regulating the diameter of arteries in humans, as has been demonstrated in rats, should be considered.

Apparent dissociation constants for several inhibitors of acetylcholinesterase in the intact electroplax of the electric eel.

Abstract Apparent dissociation constants were determined for d -tubocurarine, benzoquinonium, ambenonium, WIN 3286, and WIN 7789 as inhibitors of acetylcholinesterase (AChE) in the intact electroplax of Electrophorus electricus . These five compounds showed nearly the same order of potency for inhibiting AChE in intact electroplax cells as had been determined earlier using AChE purified from the electric organ of the electric eel. However, the constants were 140–510 times higher for cellular AChE than for purified AChE. The order of effectiveness of these five compounds as inhibitors of cellular AChE was completely different from the order previously determined for inhibition of the acetylcholine receptor of the intact electroplax of the electric eel. The ratio of the apparent dissociation constants determined for cellular AChE compared to the constants for the acetylcholine receptor varied from 0.16 to 3500. It is concluded that the active site of AChE is different from that of the acetylcholine receptor.

Studies of the blood chemistry of Electrophorus electricus and a new physiological saline solution based thereon

Abstract Based upon analyses of the composition of electric eel blood serum we suggest a new physiological saline solution as follows: 188 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , 2 mM CaCl 2 , 0.15 mM NaH 2 PO 4 , 1.45 mM Na 2 HPO 4 and 5 mM glucose; pH 7.4. The major difference between this saline and that used in most of the previous investigations using eel electroplaques is that the total Na + concentration is increased from between 162.7 and 171.7 mequiv/l to 191 mequiv./l. This increase does not appear to affect the electrophysiological properties of the electroplaque.

Interactions of calcium with purified and intact cell acetylcholinesterase of Electrophorus electricus

The effects of lanthanum on the activity of purified preparations of acetylcholinesterase (AChE) from the electric organ ofE. electricus and on the activity of AChE in intact electro-plaques from the same species were studied. 0.1mm LaCl3 produced an initial inhibition of purified AChE which was followed by a delayed activation of the enzyme. Upon pretreatment of purified enzyme with LaCl3, initial activity was markedly increased. LaCl3 exerted a marked, concentration-dependent inhibition of intact cell AChE.

Effect of insecticides on the short-circuit current and resistance of isolated frog skin

Abstract The effects of aldrin, carbaryl, α- and γ-chlordane, dichlorodiphenyldichloroethane (DDD), dichlorodiphenyltrichloroethane (DDT), dieldrin, endrin, heptachlor, lindane, methoxychlor, and nonachlor on the short-circuit current and resistance of the isolated intact frog skin were studied. The short-circuit current is approximately equivalent to the rate of active transport of sodium, while the resistance indicates the summed ionic permeability of the skin. At a concentration of 2 × 10 −4 M , only carbaryl, DDD, dieldrin, and lindane produced significant ( P t test) changes in the short-circuit current. Nonachlor produced a decrease ( P =0.12) in the short-circuit current and also increased the resistance ( P =0.07). DDD, dieldrin, and carbaryl caused significant increases in short-circuit current while at the same time the resistance was significantly decreased. Lindane increased both the short-circuit current and the resistance. It was concluded that the frog skin probably contains effective permeability barriers that prevent externally applied insecticides from reaching the site of active sodium transport. It appeared likely that most of the insecticides produced the observed effects on the frog skin by altering the sodium permeability of the outer barrier.

Influence of physiological factors on the age-related increase in blood pressure in healthy men

The independent and collective influences of several physiological factors on the age-related increase in blood pressure in healthy men were examined. Twenty-seven younger and 25 older, mostly normotensive, healthy men were studied. Blood pressure, body fat, body fat distribution, maximal oxygen consumption (VO2max), plasma norepinephrine, dietary Na, and erythrocyte Na-K pump activity were measured. Older men showed 57% higher percent body fat, 40% higher plasma norepinephrine concentration, 14% greater mean arterial blood pressure (MAP), and 5% higher plasma K concentration than younger men (all p < 0.01). Older men showed a 38% (p < 0.01) lower VO2max, 19% (p < 0.05) lower energy intake, 18% (p < 0.05) lower Na-K pump rate constant, and a 17% (p < 0.05) lower Na-K pump rate. Group means for MAP were adjusted for combinations of plasma norepinephrine, waist:thigh ratio, VO2max, and the Na-K pump rate constant, to determine if any one variable or combination could account for the age related increase in MAP. Statistical adjustment for plasma norepinephrine, waist:thigh ratio, and Na-K pump rate constant eliminated the significant difference between MAPs for the two groups. Thus, alterations in sympathetic nervous system activity, body fat distribution, and the membrane Na-K pump activity independently contribute to the age-related increase in MAP in healthy men.

The effects of calcium and lanthanum on the interaction of decamethonium with soluble acetylcholinesterase from Electrophorus electricus

THERE is considerable evidence supporting a regulatory role for non-catalytic anionic sites on the AChE (EC 3.1.1.7) molecule. Early evidence for the presence of negative sites distinct from the active centre was presented by BERCMANN et al. (1950) and later by WILSON (1960). CHANCEUX et al. (1968) measured 25G300 ACh molecules bound per molecule of AChE in the presencc of active site inhibitors such as eserine, supporting the notion of a large number of non-catalytic substrate-binding sites. Topographically-distinct non-catalytic sites were also postulated by KUHNEN (1972) in studies on the inhibition of AChE by his-pyridinium compounds. Further characterization of a peripheral anionic site has been obtained with 10-carbon bis-quaternary ammonium ligands where the quaternary nitrogens are separated by 14w. CHANGEUX (1966) demonstrated that decamethonium (C, O)r a his-quaternary ammonium ligand which produces a depolarizing block of cholinergic receptors, inhibits a solubilized Torpedo AChE preparation. ROUFOGALIS & QUIST (1972) obtained similar results with erythrocyte AChE and suggested that C , , is bound in a bridge-like manner to both the active site and to peripheral anionic sites. More recently. TAYLOR & LAPPI (1975) have shown that C,,, displaces the fluorescent probe, propidium diiodide, from a peripheral anionic centre on purified Torpedo AChE. These findings support previous work indicating the existence of two discrete anionic sites on AChE (BELLEAU et al. (1970); KITZ et al. (1970; WOMBACH~R & WOLF

DDT and sodium transport in the eel electroplaque

Abstract DDT inhibits the ATPase activity of the intact eel electroplaque. At a concentration of 10 −5 M , DDT inhibited 46% of the total ATPase activity, and 10 −4 M DDT inhibited 62% of the total ATPase activity and 62% of the ouabain-sensitive ATPase activity. The latter concentration of DDT reduced the rate of Na efflux from intact electroplaques and slowed the rate of recovery of the membrane potential following a large depolarization produced by carbamylcholine application. Repetitive direct stimulation of the innervated membrane at 10 Hz during the application of 10 −4 M DDT produced a significant irreversible depolarization. Ouabain, 10 −4 M , produced similar effects. The possible role of the inhibition of active NaK transport in producing the symptoms of DDT poisoning is discussed.