Biomedical subjects
Publications and source records attributed to A V Wallace.
3-Phenyl-3.4-dihydro-1-isoquinolinamine is a weak inhibitor of iNOS and nNOS. Structural variation of 5a results in inhibitors with a range of potency and selectivity for the NOS enzymes, including a potent and very selective iNOS inhibitor 5j.
5-Substituted 7-amino-4,5-tetrahydrothieno[2,3-c]pyridines and 6-substituted 4-amino-6,7-dihydrothieno[3,2-c]pyridines were shown to be exceptionally potent inhibitors of inducible and neuronal nitric oxide synthase. Selectivity and potency could be modulated by variation of the 5- or 6-substituent. Compound 3e showed potent in vivo inhibition of iNOS.
Reperfusion of the ischaemic myocardium leads to intracellular calcium overload followed by mitochondrial dysfunction, resulting in insufficient energy supply and ultimately myocardial necrosis. Ruthenium red (RR), a potent mitochondrial calcium uptake inhibitor, prevents this disruption to mitochondrial metabolism and improves post reperfusion recovery. This therefore suggested that mitochondrial calcium influx is an attractive target for the treatment of reperfusion injury. However, RR is unsuitable for therapeutic use, so we undertook a search for novel compounds which inhibit mitochondrial calcium uptake. The most potent compounds discovered were simple tris(ethylenediamine) transition metal complexes and dinuclear Co complexes. The structure-activity relationship (SAR) of these small molecules has helped to define the structural requirements for inhibition of calcium transport by outlining the size and charge dependency of the interactive site on the mitochondrial calcium uniporter.
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Absence of physiological concentrations of extracellular Ca2+ in the Krebs-Henseleit incubation buffer did not affect the ability of 10 nM glucagon (< 5%) to increase hepatocyte intracellular cyclic AMP concentrations, but severely ablated (by approximately 70%) the ability of 10 nM insulin to decrease these elevated concentrations. Cyclic AMP metabolism is determined by production by adenylate cyclase and degradation by cyclic AMP phosphodiesterase (PDE). In the absence of added extracellular Ca2+ (2.5 mM), insulin's ability to activate PDE activity was selectively compromised, showing a failure of insulin to activate two of the three insulin-stimulated activities, namely the 'dense-vesicle' and peripheral plasma-membrane (PPM) PDEs. In the absence of added Ca2+, insulin's ability to inhibit adenylate cyclase activity in intact hepatocytes was decreased dramatically. Vasopressin and adrenaline (+ propranolol) failed to elicit the activation of either the 'dense-vesicle' or the PPM-PDEs. The presence of physiological concentrations of extracellular Ca2+ in the incubation medium is shown to be important for the appropriate generation of insulin's actions on cyclic AMP metabolism.
The angiotensin II receptor antagonist, DUP 753 (Losartan), was compared with diazepam for antianxiety properties in the rat using the elevated plus-maze. Oral diazepam (5 mg kg-1) resulted in a significantly greater number of entries of rats into the open arms of the maze, an increase in time spent in the open arms and a decreased time spent in the closed arms. Oral doses of DUP 753 likewise resulted in significantly greater numbers of entries into the open arms (active at 0.0001, 0.001, 0.01 and 0.1 mg kg-1), increased time spent on the open arms (active at 0.0001-0.01 mg kg-1) and a decreased time spent in the closed arms (active at 0.0001, 0.01 and 0.1 mg kg-1). Larger (1.0 mg kg-1) or smaller (0.00001 mg kg-1) doses of DUP 753 were not active.
Radiation inactivation of the two high affinity cyclic AMP phosphodiesterases (PDE) found in liver plasma membranes afforded an estimation of their molecular target sizes in situ. The activity of the peripheral plasma membrane PDE decayed as a single exponential with a target size corresponding to a monomer of circa 54 kDa. The integral, cyclic GMP-stimulated PDE decayed as a dimer of circa 125 kDa. Preincubation of plasma membranes with insulin (10nM), prior to irradiation, caused the target size of only the peripheral plasma membrane PDE to increase. We suggest that insulin addition causes the peripheral plasma membrane PDE to alter its coupling to an integral plasma membrane protein with a target size of circa 90 kDa.
The ability of rat mammary-gland Golgi membranes to produce monosaccharide-specific pores in phospholipid vesicles was investigated. The apparent ability of Triton X-100 extracts of Golgi membranes to form such pores was re-evaluated, since we have now found that an apparent pore is produced by the detergent alone. We therefore incorporated intact Golgi membranes (1 mg of protein) into egg-yolk phospholipid vesicles by direct sonication in the absence of any detergent. These vesicles retained about 0.6% of the total sucrose, but demonstrated selective permeability towards glucose compared with sucrose, with 19.8% of the glucose being lost during gel filtration on Sepharose 4B. This phenomenon seemed to be enhanced by the presence of acidic phospholipids and lysophosphatidylcholine, but was inhibited by inclusion of cholesterol in the vesicles. The best mixture of phospholipids comprised 6.5 mg of egg-yolk phospholipid, 1 mg of phosphatidylserine and 0.05 mg of lysophosphatidylcholine, where 32.9% of the glucose was lost. By using this optimum phospholipid mixture the pores were shown to be permeable to both glucose and mannitol, whereas sucrose and lactose were retained by the vesicles. Chaps (3- [(3-cholamidopropyl)dimethylammonio] propane-1-sulphonate)-solubilized membranes produced similar permeability in vesicles produced by dialysis of a solution of the phospholipids mixed with the membrane extract. This technique resulted in a greater loss of glucose, 33% loss requiring about 0.25 mg of protein. The pore-forming ability of both intact Golgi membranes and Chaps extracts was sensitive to boiling and proteolysis, indicating that a membrane protein was likely to be involved in pore formation.
Glucagon (10nM) prevented insulin (10nM) from activating the plasma-membrane cyclic AMP phosphodiesterase. This effect of glucagon was abolished by either PIA [N6-(phenylisopropyl)adenosine] (100nM) or adenosine (10 microM). Neither PIA nor adenosine exerted any effect on the plasma-membrane cyclic AMP phosphodiesterase activity either alone or in combination with glucagon. Furthermore, PIA and adenosine did not potentiate the action of insulin in activating this enzyme. 2-Deoxy-adenosine (10 microM) was ineffective in mimicking the action of adenosine. The effect of PIA in preventing the blockade by glucagon of insulin's action was inhibited by low concentrations of theophylline. Half-maximal effects of PIA were elicited at around 6nM-PIA. It is suggested that adenosine is exerting its effects on this system through an R-type receptor. This receptor does not appear to be directly coupled to adenylate cyclase, however, as PIA did not affect either the activity of adenylate cyclase or intracellular cyclic AMP concentrations. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase, in the presence of both glucagon and PIA, was augmented by increasing intracellular cyclic AMP concentrations with either dibutyryl cyclic AMP or the cyclic AMP phosphodiesterase inhibitor Ro-20-1724. PIA also inhibited the ability of glucagon to uncouple (desensitize) adenylate cyclase activity in intact hepatocytes. This occurred at a half-maximal concentration of around 3 microM-PIA. However, if insulin (10 nM) was also present in the incubation medium, PIA exerted its action at a much lower concentration, with a half-maximal effect occurring at around 4 nM.
Treatment of hepatocytes with either NH4Cl (10mM) or fructose (10mM) blocks insulin's activation of the 'dense-vesicle' cyclic AMP phosphodiesterase. The ability of insulin (10 nM) to decrease intracellular cyclic AMP concentrations raised by glucagon (10 nM) was unaffected by pre-treatment with either NH4Cl (10 mM) or fructose (10 mM). It is concluded that the 'dense-vesicle' enzyme does not play a significant role in this action of insulin and that as yet unidentified cyclic AMP phosphodiesterase(s) must be activated by insulin. Treatment of hepatocytes with either NH4Cl or fructose appeared to increase, reversibly, cyclic AMP phosphodiesterase activity. When N6-(phenylisopropyl)adenosine was used to prevent glucagon from blocking insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase activity, insulin's ability to decrease intracellular cyclic AMP concentrations in glucagon-treated hepatocytes was increased markedly. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase activity can exert a potent effect in decreasing intracellular cyclic AMP concentrations elevated by glucagon.
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Insulin elicits the activation of two distinct membrane-bound cyclic AMP phosphodiesterases when incubated at 37 degrees C for 5 min with intact hepatocytes: the 'dense-vesicle' enzyme and the peripheral-plasma-membrane enzyme. In hepatocytes the lysosomotropic agents chloroquine, methylamine and NH4Cl, as well as intracellular ATP depletion elicited by fructose or incubation with insulin at 22 degrees C, blocks selectively the activation of the 'dense-vesicle' enzyme. Incubation of hepatocytes with bacitracin, leupeptin and a variety of proteinase inhibitors failed to affect insulin's activation of these two cyclic AMP phosphodiesterases by distinct routes. It is suggested that activation of the 'dense-vesicle' enzyme occurs through a pathway triggered by the endocytosis, processing and recycling of the insulin receptor. This might involve the delivery, with subsequent activation, of a latent phosphodiesterase into this fraction.
Glucagon (10 nM) caused a transient elevation of intracellular cyclic AMP concentrations, which reached a peak in around 5 min, and slowly returned to basal values in around 30 min. When 1 mM-3-isobutyl-1-methylxanthine (IBMX) was present, this process yielded a Ka of 1 nM for glucagon. The addition of insulin (10 nM) after 5 min exposure to glucagon (10 nM) caused intracellular cyclic AMP concentrations to fall dramatically, attaining basal values within 10 min. The regulation of this process was dose-dependent, exhibiting a Ka of 0.4 nM for insulin. If insulin and glucagon were added together to hepatocytes, then insulin decreased the magnitude of the cyclic AMP response to glucagon. IBMX (1 mM) prevented insulin antagonizing the action of glucagon in both of these instances. A gentle homogenization procedure followed by a rapid subcellular fractionation of hepatocytes on a Percoll gradient was developed. This was used to resolve subcellular membrane fractions and to identify cyclic AMP phosphodiesterase activity in both membrane and cytosol fractions. Glucagon and insulin only affected the activity of two distinct membrane-bound species, a plasma-membrane enzyme and a 'dense vesicle' enzyme. Glucagon (10 nM), insulin (10 nM), IBMX (1 mM), dibutyryl cyclic AMP (10 microM) and cholera toxin (1 microgram/ml) all elicited the activation of the 'dense vesicle' enzyme. The plasma-membrane enzyme was not activated by glucagon, IBMX or dibutyryl cyclic AMP, although insulin and cholera toxin both led to its activation. The degree of activation of the plasma-membrane enzyme produced by insulin was increased in the presence of IBMX or dibutyryl cyclic AMP. Glucagon pretreatment (5 min) of hepatocytes blocked the ability of insulin to activate the plasma-membrane enzyme. The activity state of these phosphodiesterases is discussed in relation to the observed changes in intracellular cyclic AMP concentrations. It is suggested that insulin exerts its action on the plasma-membrane phosphodiesterase through a mechanism involving a guanine nucleotide-regulatory protein.
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