Butanol Isomers Exert Distinct Effects on Voltage-Gated Calcium Channel Currents and Thus Catecholamine Secretion in Adrenal Chromaffin Cells

Butanol (C₄H₁₀OH) has been used both to dissect the molecular targets of alcohols/general anesthetics and to implicate phospholipase D (PLD) signaling in a variety of cellular functions including neurotransmitter and hormone exocytosis. Like other primary alcohols, 1-butanol is a substrate for PLD and thereby disrupts formation of the intracellular signaling lipid phosphatidic acid. Because secondary and tertiary butanols do not undergo this transphosphatidylation, they have been used as controls for 1-butanol to implicate PLD signaling. Recently, selective pharmacological inhibitors of PLD have been developed and, in some cases, fail to block cellular functions previously ascribed to PLD using primary alcohols. For example, exocytosis of insulin and degranulation of mast cells are blocked by primary alcohols, but not by the PLD inhibitor FIPI. In this study we show that 1-butanol reduces catecholamine secretion from adrenal chromaffin cells to a much greater extent than tert-butanol, and that the PLD inhibitor VU0155056 has no effect. Using fluorescent imaging we show the effect of these drugs on depolarization-evoked calcium entry parallel those on secretion. Patch-clamp electrophysiology confirmed the peak amplitude of voltage-gated calcium channel currents (I_Ca) is inhibited by 1-butanol, with little or no block by secondary or tert-butanol. Detailed comparison shows for the first time that the different butanol isomers exert distinct, and sometimes opposing, effects on the voltage-dependence and gating kinetics of I_Ca. We discuss these data with regard to PLD signaling in cellular physiology and the molecular targets of general anesthetics.     

Digestion of haemoglobin by schistosomes: 35 years on

Adult schistosomes obtain nutrients by digesting haemoglobin, which they obtain from ingested host red blood cells. Here John Dalton, Angela Smith, Karen Clough and Paul Brindley argue that a cathepsin L proteinase recently identified in their laboratory as the predominant cysteine proteinase activity of Schistosoma mansoni may play the leading role in haemoglobin digestion. They discuss the importance of elucidating the roles of both cathepsin B and cathepsin L in the digestion of haemoglobin, since both should be considered important targets to which novel schistosomicidal agents could be directed.     

Regulation of toxocariasis in mice selectively reared for high and low immune responses to Nematospiroides dubius

Test mice have been selectively reared for high (H) or low (L) immune responses to Nematospiroides dubius. After secondary infection with N. dubius, the L mice voided ten times as many eggs in their faeces as the H mice, and at necropsy, 71% versus 20% of the inoculum of N. dubius were recovered as adult worms from the L and H mice respectively. Furthermore, N. dubius were more fecund in the L than in H mice. High or low immune responsiveness was not restricted to N. dubius infection in these mice but was also observed during Toxocara canis infection. The migration of T. canis larvae from gut via the liver to skeletal muscle and CNS was inhibited in H versus L mice. Many more larvae were recovered from the livers of H compared with L mice which was indicative of greater immunity in the H mice. The protective immune response in H compared with L mice to both N. dubius and T. canis included pronounced eosinophilia and elevated antiparasite antibody titres.     

Effects of cyclic AMP, glucocorticoids and insulin on the activities of phosphatidate phosphohydrolase, tyrosine aminotransferase and glycerol kinase in isolated rat hepatocytes in relation to the control of triacylglycerol synthesis and gluconeogenesis.

Rat hepatocytes were incubated in monolayer culture in modified Leibovitz L-15 medium containing either 10% (v/v) newborn-calf serum or 0.2% (w/v) fatty-acid-poor bovine serum albumin. The addition of 100 nM-dexamethasone increased the activities of both phosphatidate phosphohydrolase and tyrosine aminotransferase by about 3.5-fold after 8h, and these activities continued to rise until at least 24h. Incubating the hepatocytes in the albumin-containing medium with 10 microM- or 100 microM-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate increased the activities of the phosphohydrolase and aminotransferase by 2.6- and 3.4-fold respectively after 8h. These increases were blocked by actinomycin D. The increases in the activities that were produced by the cyclic AMP analogue and dexamethasone were independent and approximately additive. Insulin when added alone did not alter the phosphohydrolase activity, but it increased the aminotransferase activity by 34%. The dexamethasone-induced increase in the phosphohydrolase activity was completely blocked by 7-144 microM-insulin, whereas that of the aminotransferase was only partly suppressed. Insulin had no significant Effects on the increases in the activities of phosphatidate phosphohydrolase and tyrosine aminotransferase that were produced by the cyclic AMP analogue, but this may be because the analogue is fairly resistant to degradation by the phosphodiesterase. The activity of glycerol kinase was not significantly changed by incubating the hepatocytes with insulin, dexamethasone and the cyclic AMP analogue alone or in combinations. It is proposed that high concentrations of cyclic AMP and glucocorticoids increase the total activity of phosphatidate phosphohydrolase in the liver and provide it with an increased capacity for synthesizing triacylglycerols and very-low-density lipoproteins, which is expressed when the availability of fatty acids is high. There appears to be a co-ordinated hormonal control of triacyglycerol synthesis and gluconeogenesis in diabetes and in metabolic stress to enable the liver to supply other organs with energy.     

Drugs affecting the synthesis of glycerides and phospholipids in rat liver. The effects of clofibrate, halofenate, fenfluramine, amphetamine, cinchocaine, chlorpromazine, demethylimipramine, mepyramine and some of their derivatives.

The effects on glycerolipid synthesis of a series of compounds including many drugs were investigated in cell-free preparations and slices of rat liver. p-Chlorobenzoate, p-chlorophenoxyisobutyrate, halofenate, D-amphetamine, adrenaline, procaine and N-[2-(4-chloro-3-sulphamoylbenzoyloxy)ethyl]norfenfluramine had little inhibitory effect on any of the systems investigated. Two amphiphilic anions, clofenapate and 2-(p-chlorophenyl)-2-(m-trifluoromethylphenoxy)acetate, both inhibited glycerol phosphate acyltransferase and diacylglycerol acyltransferase at approx. 1.6 and 0.7 mm respectively. Clofenapate (1 mm) also inhibited the incorporation of glycerol into lipids by rat liver slices without altering the relative proportions of the different lipids synthesized. The amphilic amines, mepyramine, fenfluramine, norfenfluramine, hydroxyethylnorfenfluramine, N-(2-benzoyloxyethyl)norfenfluramine, cinchocaine, chlorpromazine and demethylimipramine inhibited phosphatidate phosphohydrolase by 50% at concentrations between 0.2 and 0.9 mm. The last four compounds inhibited glycerol phosphate acyltransferase by 50% at concentrations between 1 and 2.6 mm. None of the amines examined appeared to be an effective inhibitor of diacylglycerol acyltransferase. Norfenfluramine, hydroxyethylnorfenfluramine and N-(2-benzoyloxyethyl)norfenfluramine produced less inhibition of glycerol incorporation into total lipids than was observed with equimolar clofenapate. The major effect of these amines in liver slices was to inhibit triacylglycerol and phosphatidylcholine synthesis and to produce a marked accumulation of phosphatidate. The results are discussed in terms of the control of glycerolipid synthesis. They partly explain the observed effects of the various drugs on lipid metabolism. The possible use of these compounds as biochemical tools with which to investigate the reactions of glycerolipid synthesis is considered.