Neutralization of Apis mellifera bee venom activities by suramin.

In this work we evaluated the ability of suramin, a polysulfonated naphthylurea derivative, to antagonize the cytotoxic and enzymatic effects of the crude venom of Apis mellifera. Suramin was efficient to decrease the lethality in a dose-dependent way. The hemoconcentration caused by lethal dose injection of bee venom was abolished by suramin (30 µg/g). The edematogenic activity of the venom (0.3 µg/g) was antagonized by suramin (10 µg/g) in all treatment protocols. The changes in the vascular permeability caused by A. mellifera (1 µg/g) venom were inhibited by suramin (30 µg/g) in the pre- and posttreatment as well as when the venom was preincubated with suramin. In addition, suramin also inhibited cultured endothelial cell lesion, as well as in vitro myotoxicity, evaluated in mouse extensor digitorum longus muscle, which was inhibited by suramin (10 and 25 µM), decreasing the rate of CK release, showing that suramin protected the sarcolemma against damage induced by components of bee venom (2.5 µg/mL). Moreover, suramin inhibited the in vivo myotoxicity induced by i.m. injection of A. mellifera venom in mice (0.5 µg/g). The analysis of the area under the plasma CK vs. time curve showed that preincubation, pre- and posttreatment with suramin (30 µg/g) inhibited bee venom myotoxic activity in mice by about 89%, 45% and 40%, respectively. Suramin markedly inhibited the PLA2 activity in a concentration-dependent way (1-30 µM). Being suramin a polyanion molecule, the effects observed may be due to the interaction of its charges with the polycation components present in A. mellifera bee venom.

Changes in the vascular beta-adrenoceptor-activated signalling pathway in 2Kidney-1Clip hypertensive rats.

1. beta-Adrenoceptor (beta-AR)-mediated vasodilation, which plays an important physiological role in the regulation of vascular tone, is decreased in two-kidney, one clip (2K-1C) renal hypertension. In this study, downstream pathways related to vascular beta-AR activation were evaluated in 2K-1C rats. 2. Relaxation responses to isoprenaline, forskolin and 8-Br-cAMP were diminished in aortas without endothelium from 2K-1C when compared to those in normotensive two kidney (2K). Basal adenosine-3',5'-monophosphate (cAMP), as well as isoprenaline-induced increase in cAMP levels, was not different between 2K and 2K-1C aortas. 3. Contractile responses to caffeine, after depletion and reloading of intracellular Ca(2+) stores, were greater in 2K-1C than in 2K. The presence of isoprenaline during the Ca(2+)-reloading period abolished the differences between groups by increasing caffeine contraction in 2K without changing this response in 2K-1C aortas. Inhibition of the sarcolemmal Ca(2+)ATPase with thapsigargin markedly attenuated isoprenaline vasodilation in both 2K and 2K-1C and abolished the differences between groups. 4. Blockade of ATP-sensitive K(+) channels (K(ATP)) channels with glibenclamide significantly decreased isoprenaline vasodilation in 2K-1C without affecting this response in 2K. Both vascular gene and protein expression of protein kinase A (PKA), as well as phosphoserine-containing proteins, were increased in 2K-1C vs 2K rats. 5. In conclusion, decreased isoprenaline vasodilation in 2K-1C hypertensive rats is related to impaired modulation of the sarcolemmal Ca(2+)ATPase activity. Moreover, K(ATP) channels may play a compensatory role on isoprenaline-induced relaxation in renal hypertension. Both Ca(2+)ATPase and K(ATP) channel functional alterations, associated with decreased beta-AR vasodilation, are paralleled by an upregulation of protein kinase A (PKA) and phosphoserine proteins expression.

Immunolocalisation of neuronal nitric oxide synthase at the neuromuscular junction of MDX mice: a confocal microscopy study.

The distribution of nitric oxide synthase at the neuromuscular junction (NMJ) of normal, denervated and mdx mice was studied using a specific antibody against the neuronal isoform of nitric oxide synthase (nNOS). Fluorescence confocal microscopy demonstrated that nNOS immunoreactivity was localised mainly in the sarcolemma and presynaptic region covering acetylcholine receptor branches. The expression of presynaptic nNOS was greatly reduced in dystrophin-deficient muscles. In normal denervated muscles, nNOS was still present in the presynaptic region and there were no qualitative changes in the expression of this protein. These results suggest that the presynaptic distribution of nNOS is associated with terminal Schwann cells. The relationship between nNOS and the presynaptic components of the neuromuscular junction may open new perspectives for improving our understanding of the pathogenesis of dystrophic muscles.

Characterization and localization of an ATP diphosphohydrolase activity (EC 3.6.1.5) in sarcolemmal membrane from rat heart.

In the present report we describe an ATP diphosphohydrolase (apyrase EC 3.6.1.5) in rat cardiac sarcolemma. It is Ca2+ dependent and is insensitive to ouabain, orthovanadate, N-ethylmaleimide (NEM), lanthanum, and oligomycin that are classical ATPase inhibitors. Sodium azide that is a mitochondrial inhibitor at low concentrations, did not affect the enzyme activity at 5.0 mM or below. In contrast, at high concentrations (> 10 mM) sodium azide inhibited the enzyme. Levamisole, a specific inhibitor of alkaline phosphatase and P1, P5-di(adenosine 5'-)pentaphosphate (Ap5A), a specific inhibitor of adenylate kinase did not inhibit the enzyme. Mercury chloride showed a parallel inhibition of the hydrolysis of both substrates of apyrase. Similar inhibition profiles are powerful evidence for a common catalytic site for the hydrolysis of both substrates. The enzyme has an optimum pH range of 7.5-8.0 and catalyzes the hydrolysis of triphospho- and diphosphonucleosides other than ATP or ADP. The apparent Km (Michaelis constant) and Vmax (maximal velocity) are 62.1 +/- 5.2 microM and 1255.7 +/- 178 micromol inorganic phosphate liberated/min/mg with ATP and 59.4 +/- 4.3 microM and 269.2 +/- 39 micromol inorganic phosphate liberated/min/mg with ADP. Enzyme markers indicated that this apyrase is associated with the plasma membrane. A deposition of lead phosphate granules on the outer surface of the sarcolemmal vesicles was observed by electron microscopy in the presence of either ATP or ADP as substrate. It is suggested that the ATP diphosphohydrolase could regulate the concentration of extracellular adenosine, and thus is important in the control of vascular tone and coronary flow.

ATP-diphosphophydrolase activity in rat heart tissue.

Extracellular nucleotides interact with specific receptors on the cell surface and are locally metabolized by ecto-nucleotidases. Biochemical characterization of the ATPase and ADPase activities detected in rat heart sarcolemma, under conditions where mitochondrial ATPase and adenylate kinase were blocked, supports our proposal that both activities correspond to a single enzyme, known as ATP-diphosphohydrolase or apyrase. The physiological function of this enzyme could be dephosphorylation of the nucleotides present in the interstitial heart compartment acting together with 5'-nucleotidase. Both hydrolytic activities have similarities in: sarcolemma localization, bivalent metal ion dependence, optimum pH, effect of several amino acid residue modifiers, competitive inhibition of nucleotide analogs, and broad nucleoside di-and triphosphate specificity. The ATPase activity could not be separated from the ADPase either through isoelectrofocusing or electrophoresis under acid conditions.

Distinct mechanisms of inhibition of purified cardiac sarcolemma Ca(2+)-ATPase by two calmodulin antagonists.

The effects of calmidazolium and compound 48/80 were studied in four different states of activation of the purified Ca(2+)-ATPase from cardiac sarcolemma: "basal" or unactivated, activated by calmodulin, activated by phosphatidylserine, and activated by controlled trypsinization. When assayed in the presence of phosphatidylcholine as the sole phospholipid (basal state), the purified enzyme was resistant to inhibition by calmidazolium (0.1 to 3 microM). In the same range, calmidazolium inhibited the enzyme activated by controlled proteolysis as well as the calmodulin-activated enzyme regardless of the calmodulin concentration. The phosphatidylserine-activated enzyme was inhibited at higher calmidazolium concentrations due to non-specific trapping of the inhibitor by the excess of phospholipid. Addition of calmidazolium did not modify the K0.5 for calcium activation of ATP hydrolysis by the enzyme. The inhibition by calmidazolium was counteracted by Pi. Compound 48/80 also had no effect on the enzyme when only phosphatidylcholine was present and, like calmidazolium, it inhibited the calmodulin-activated enzyme and the phosphatidylserine-activated enzyme. The apparent Ki for inhibition by compound 48/80 was dependent on the calmodulin concentration. However, the enzyme activated by controlled trypsinization was insensitive to compound 48/80. Binding of 48/80 to the enzyme in the presence of phosphatidylserine or calmodulin reversed the increased affinity for Ca2+ caused by these activators.

Regulation of the nucleotide dependence of the cardiac sarcolemma Ca(2+)-ATPase.

The nucleotide dependence of the Ca(2+)-ATPase purified from cardiac sarcolemma by calmodulin-affinity chromatography was investigated for preparations either in the basal state or activated by three procedures: (i) addition of calmodulin; (ii) addition of phosphatidylserine and (iii) controlled proteolysis. Upon activation, the maximal velocity of ATP hydrolysis increases by a factor of 4-5, while the curves of ATP dependence of ATP hydrolysis change from hyperbolic to biphasic, revealing the presence of two Kmapp for ATP. A tight coupling between Ca2+ and ATP binding sites was also observed. At high ATP concentration, the ATPase activity of the basal state shows a complex dependence on Ca2+ concentration, increasing sharply at millimolar Ca2+. Our results indicate that this increase in ATPase activity is paralleled by the appearance of a second, low affinity Kmapp for ATP. When only the high affinity site for ATP is occupied the ATPase activity of the basal state displays a simple, hyperbolic dependence on the Ca2+ concentration. In addition, increasing Ca2+ concentration appears to decrease the ATP binding at the low affinity site of the enzyme. The effect of ADP on ATP hydrolysis was also examined. The finding that ADP is a potent inhibitor of the purified Ca(2+)-ATPase from heart suggests that the stimulatory action of ADP observed in cardiac sarcolemmal vesicles is not an intrinsic property of the enzyme.

Morphological, immunological and biochemical characterization of purified transverse tubule membranes isolated from rabbit skeletal muscle.

A microsomal fraction consisting of membranes of transverse tubule origin has been purified by a modification of the calcium-loading procedure initially described by Rosemblatt et al. (J Biol Chem 256:8140-8, 1981). Enzymatic analysis of this fraction shows an enrichment of the vesicles in the Mg++ ATPase (basal) activity characteristic of the T-tubules and an absent or very low Ca++-dependent-ATPase activity. Stereological analysis of freeze fracture replica of the membranes in the purified fraction indicates that they have a very low density of particles in their P faces and lack the structural manifestation of the caveolae typical of the sarcolemma. Immunological analysis performed with monoclonal antibodies prepared against purified T-tubule and sarcoplasmic reticulum membranes define some T-tubule specific antigens and confirm the morphological and biochemical data regarding the origin and purity of the T-tubule preparation.