Vascular responses after alpha adrenergic receptor blockade: II. Responses of venous and arterial segments to adrenergic stimulation in the forelimb of dog.

The effects of nerve stimulation and of intraarterial injections of norepinephrine on arterial and venous resistances were studied in the perfused forelimb of dog before and after administration of the alpha adrenergic receptor blocker phenoxybenzamine.Pressures were recorded from the perfused brachial artery and a small metacarpal vein in the forepaw. Blood flow to the whole forelimb was maintained constant. Changes in perfusion pressure in the brachial artery reflected primarily changes in arterial resistance and changes in small vein pressure reflected changes in resistance of venous segments downstream from the point of pressure measurement.Alpha receptor blockade reduced vasoconstrictor responses to both nerve stimulation and norepinephrine. Responses to angiotensin, used in these experiments as an internal control, were not blocked consistently in a dose-related manner indicating that the effects of phenoxybenzamine were specific to adrenergic stimuli.Increases in venous pressure in response to norepinephrine and to nerve stimulation were blocked almost completely whereas increases in arterial pressure were reduced only in part by the blocker. The more effective reduction of pressor responses in the small vein was not caused by a passive reduction in blood flow through the paw nor was it caused by a reduction in the concentration of norepinephrine in the venous effluent reaching the venous segments.This differential effect of alpha receptor blockade on increases in venous and arterial resistances may account for the beneficial effect of phenoxybenzamine in shock.

Vascular responses after alpha adrenergic receptor blockade: I. Responses of capacitance and resistance vessels to norepinephrine in man.

Experiments were done to test the hypothesis that alpha receptor blockers antagonize more effectively venous than arterial responses to norepinephrine in man.Systemic arterial blood pressure, venous pressure in the forearm, blood flow through the forearm, and the volume of the forearm at a venous pressure of 30 mm Hg were measured using pressure transducers and a mercury strain-gauge plethysmograph. Infusions of norepinephrine into the brachial artery reduced forearm blood flow and venous distensibility without changing arterial pressure. After intraarterial infusion of phentolamine the decrease in venous distensibility during administration of norepinephrine was blocked almost completely whereas the decrease in blood flow through the forearm was not altered.The results indicate that alpha adrenergic receptor blockade can antagonize constriction of capacitance vessels more effectively than constriction of resistance vessels.

Comparison of effects of deoxycorticosterone and dexamethasone on cardiovascular responses to norepinephrine.

Cardiovascular responses to graded iv infusions of norepinephrine were observed in 24 dogs that had been treated for 1 week with either placebo, dexamethasone, or deoxycorticosterone. Eight dogs served as control and received daily iv injections of placebo; eight dogs received the mineralocorticoid, deoxycorticosterone; and eight received the glucocorticoid, dexamethasone. The three groups did not differ with respect to base-line hemodynamic variables either before administration of norepinephrine or after autonomic reflexes had been inhibited by ganglionic blockade. Comparisons of the three groups' hemodynamic responses to norepinephrine were made both before and after ganglionic blockade with the parallel line bioassay as a statistical test. Dogs given deoxycorticosterone had much greater increases in mean arterial pressure and peripheral resistance with norepinephrine than did dogs given dexamethasone or placebo. Dogs given dexamethasone had slightly greater increases in mean arterial pressure than did dogs given placebo; changes in peripheral resistance were similar in the two groups. The augmented response of mean arterial pressure was apparent only after ganglionic blockade in the dexamethasone group. The vascular effects of norepinephrine, therefore, were markedly augmented by treatment with doxycorticosterone and only slightly augmented by treatment with dexamethasone. The effect of norepinephrine on mean right atrial pressure was augmented in both groups treated with steroid before hexamethonium but only in the group treated with dexamethasone after hexamethonium. The results indicate that deoxycorticosterone and dexamethasone have different qualitative and quantitative effects on circulatory responses to norepinephrine.

Responses of saphenous and mesenteric veins to administration of dopamine.

Others have observed that dopamine (3,4-dihydroxyphenylethylamine) constricts resistance vessels in skin, but dilates these vessels in the mesentery. We studied the effects of dopamine on cutaneous and mesenteric veins of dogs to see if this agent also produced qualitatively different effects on the tone of capacitance vessels (veins) in these vascular beds. The lateral saphenous or the left colic vein was perfused at constant flow with blood from a femoral artery. Pressures at the tip of the perfusion cannula and at the tip of a catheter 15 cm downstream were recorded continuously. Increases in the pressure gradient between these two points indicated venoconstriction; decreases indicated venodilatation. Dopamine and norepinephrine injected into the perfusion tubing caused constriction of both veins. The constriction was antagonized by blockade of alpha receptors. A dilator action of dopamine was not seen, even after alpha receptor blockade or in the presence of increased venous tone produced by serotonin, norepinephrine, or nerve stimulation. Reserpine and cocaine did not alter responses to dopamine in the saphenous vein; this suggests that the venoconstrictor action of dopamine results mainly from a direct effect on alpha receptors and that uptake into sympathetic nerve endings may not be important in regulating the amount of dopamine available to receptors in the saphenous vein.

Identification of an essential acidic residue in Cdc25 protein phosphatase and a general three-dimensional model for a core region in protein phosphatases.

The reaction mechanism of protein tyrosine phosphatases (PTPases) and dual-specificity protein phosphatases is thought to involve a catalytic aspartic acid residue. This residue was recently identified by site-directed mutagenesis in Yersinia PTPase, VHR protein phosphatase, and bovine low molecular weight protein phosphatase. Herein we identify aspartic acid 383 as a potential candidate for the catalytic acid in human Cdc25A protein phosphatase, using sequence alignment, structural information, and site-directed mutagenesis. The D383N mutant enzyme exhibits a 150-fold reduction in kcat, with Kw only slightly changed. Analysis of sequence homologies between several members of the Cdc25 family and deletion mutagenesis substantiate the concept of a two-domain structure for Cdc25, with a regulatory N-terminal and a catalytic C-terminal domain. Based on the alignment of catalytic residues and secondary structure elements, we present a three-dimensional model for the core region of Cdc25. By comparing this three-dimensional model to the crystal structures of PTP1b, Yersinia PTPase, and bovine low molecular weight PTPase, which share only very limited amino acid sequence similarities, we identify a general architecture of the protein phosphatase core region, encompassing the active site loop motif HCXXXXXR and the catalytic aspartic acid residue.

Cdc25 as a potential target of anticancer agents.

Ever since its discovery in yeast more than a decade ago [1], Cdc25 has continued to surprise and intrigue researchers. This dual-specificity protein tyrosine phosphatase (dsPTPase) and other members of the protein tyrosine phosphatase family (PTPases) have only recently joined the protease and kinase enzyme families in drug discovery efforts. The role of phosphatases in tumourigenesis was reviewed recently by Parsons [2]. He is arguing that the phosphatase family of enzymes is involved in a variety of cancers and thus poses both a challenge and an opportunity for new therapeutics. The general biology and biochemistry of Cdc25 were recently reviewed [3]. Here I shall first summarize the recent literature on the role of Cdc25 in disease, as well as on new insights into the regulation of this family of proteins. In the second part, I will review current knowledge of the Cdc25 protein structure and the chemical structures and activities of published Cdc25 inhibitors.

Mechanism of bacterial bioluminescence: 4a,5-dihydroflavin analogs as models for luciferase hydroperoxide intermediates and the effect of substituents at the 8-position of flavin on luciferase kinetics.

Bioluminescence catalyzed by bacterial luciferases was measured using FMN, iso-FMN (6-methyl-8-nor-FMN), and FMN analogs carrying the following substituents at position 8: -H, -Cl, -F, SMe, SOMe, -SO2Me, or -OMe. The first-order rate constants for the decay of light emission correlate with the one-electron oxidation potentials of the 4a,5-dihydro forms of the FMN analogs. To determine the values of these potentials, isoalloxazine (flavin) derivatives having the 4a,5-propano-4a,5-dihydro structure and -H, -CH3, -Cl, -OCH3, and -NH2 as substituents at position 8 have been synthesized as models for the 4a-peroxy-4a,5-dihydroflavin intermediates occurring during catalysis by the flavin-dependent monooxygenase luciferase. The tetrahydropyrrole ring between positions 4a and 5 of these isoalloxazine derivatives stabilizes the 4a,5-dihydroflavin by impeding formation of the thermodynamically more stable 1,5-dihydro form. One-electron oxidation potentials (Eobs) were measured by cyclic voltammetry and used to determine the empirical coefficients in the Swain equation. On the basis of this, the one-electron oxidation potentials of 4a,5-propano-4a,5-dihydro analogs with other substituents in position 8 were calculated (Ecalc). The bioluminescence reaction rate is fastest with FMN analogs of lowest oxidation potential; i.e., the slope of the correlation is negative. This indicates that in the rate-limiting step the 4a,5-dihydroflavin moiety donates negative charge. The results are compatible with an intramolecular, chemically initiated electron exchange luminescence mechanism for the bacterial luciferase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism-based inhibition of thymidylate synthase by 5-(trifluoromethyl)-2'-deoxyuridine 5'-monophosphate.

Thymidylate synthase (TS) from Lactobacillus casei is inhibited by 5-(trifluoromethyl)-2'-deoxyuridine 5'-monophosphate (CF3dUMP). CF3dUMP binds to the active site of TS in the absence of 5,10-methylenetetrahydrofolate, and attack of the catalytic nucleophile cysteine 198 at C6 of the pyrimidine leads to activation of the trifluoromethyl group and release of fluoride ion. Subsequently, the activated heterocycle reacts with a nucleophile of the enzyme to form a moderately stable covalent complex. Proteolytic digestion of TS treated with [2'-3H]CF3dUMP, followed by sequencing of the labeled peptides, revealed that tyrosine 146 and cysteine 198 are covalently bound to the inhibitor in the enzyme-inhibitor complex. The presence of dithiothreitol (DTT) or beta-mercaptoethanol resulted in the breakdown of the covalent complex, and products from the breakdown of the complex were isolated and characterized. The three-dimensional structure of the enzyme-inhibitor complex was determined by X-ray crystallography, clearly demonstrating covalent attachment of the nucleotide to tyrosine 146. A chemical reaction mechanism for the inhibition of TS by CF3dUMP is presented that is consistent with the kinetic, biochemical, and structural results.

A time-dependent bacterial bioluminescence emission spectrum in an in vitro single turnover system: energy transfer alone cannot account for the yellow emission of Vibrio fischeri Y-1.

Yellow fluorescent protein (YFP), which has a bound FMN, was isolated from the marine bacterium Vibrio fischeri strain Y-1b. Its presence in a luciferase [alkanal monooxygenase (FMN-linked); alkanal, reduced-FMN:oxygen oxidoreductase (1-hydroxylating, luminescing), EC 1.14.14.3] reaction mixture causes a striking color change, and an increase in bioluminescence intensity, as well as a faster rate of intensity decay, so that the quantum yield is not changed. The emission spectrum shows two distinct color bands, one at 490 nm attributed to the unaltered emission of the luciferase system, the other peaking in the yellow around 540 nm due to YFP emission. The kinetics of the two color bands differ, so the spectrum changes with time. The yellow emission reaches its initial maximum intensity later than the blue, and then both blue and yellow emissions decay exponentially with nearly the same pseudo-first-order rate constants, linearly dependent on [YFP] (from 0.01 sec-1 with no YFP to a maximum of approximately 0.1 sec-1 at 4 degrees C) but exhibiting a saturation behavior. The data can be interpreted by assuming the interaction of YFP with the peroxyhemiacetal intermediate in the luciferase reaction to form an unstable new complex whose breakdown gives the yellow emitter in its excited state. This simple model fits well the data at [YFP] less than 15 microM. The results indicate that a single primary excited state cannot be responsible for the blue and the yellow emissions.

Kinetic analysis of the catalytic domain of human cdc25B.

The Cdc25 cell cycle regulator is a member of the dual-specificity class of protein-tyrosine phosphatases that hydrolyze phosphotyrosine- and phosphothreonine-containing substrates. To study the mechanism of Cdc25B, we have overexpressed and purified the catalytic domain of human Cdc25B (Xu, X., and Burke, S. P. (1996) J. Biol. Chem. 271, 5118-5124). In the present work, we have analyzed the kinetic properties of the Cdc25B catalytic domain using the artificial substrate 3-O-methylfluorescein phosphate (OMFP). Steady-state kinetic analysis indicated that the kcat/Km for OMFP hydrolysis is almost 3 orders of magnitude greater than that for p-nitrophenyl phosphate hydrolysis. Like other dual-specificity phosphatases, Cdc25 exhibits a two-step catalytic mechanism, characterized by formation and breakdown of a phosphoenzyme intermediate. Pre-steady-state kinetic analysis of OMFP hydrolysis indicated that formation of the phosphoenzyme intermediate is approximately 20 times faster than subsequent phosphoenzyme breakdown. The resulting burst pattern of product formation allowed us to derive rate constants for enzyme phosphorylation (26 s-1) and dephosphorylation (1.5 s-1) as well as the dissociation constant for OMFP (0.3 mM). Calculations suggest that OMFP binds with higher affinity and reacts faster with Cdc25B than does p-nitrophenyl phosphate. OMFP is a highly efficient substrate for the dual-specificity protein-tyrosine phosphatases VHR and rVH6, but not for two protein-tyrosine phosphatases, PTP1 and YOP. The ability to observe distinct phases of the reaction mechanism during OMFP hydrolysis will facilitate future analysis of critical catalytic residues in Cdc25 and other dual-specificity phosphatases.