The effects of reverse micelles on the reaction mechanism of alpha-chymotrypsin

Reverse micelles were employed to test the accuracy of the widely accepted mechanism for alpha-chymotrypsin in a highly structured aqueous system similar to intracellular conditions. Results yielded from spectrophotometrical assays of the alpha-chymotrypsin catalyzed hydrolysis of both p-nitrophenyl acetate (p-NPA) and p-nitrophenyl trimethylacetate (p-NPTA) were kinetically analyzed to determine constants typical of the proposed mechanistic model. This was accomplished through the establishment of a control, i.e. the well studied buffer system, for comparison between the reverse micellular environment and a bulk aqueous solution. Control group results yielded kinetic constants in favor of the proposed mechanism (Km = 1.55 x 10(-5) +/- 1.40 x 10(-6) M for p-NPA and a Km = 4.97 x 10(-6) +/- 2.29 x 10(-7) M, Km(app) = 4.92 x 10(-6) +/- 2.33 x 10(-8) M, k2 = 4.34 x 10(-3) +/- 1.31 x 10(-3), k(cat) = 1.96 x 10(-3) +/- 2.47 x 10(-4), and Ks = 1.60 x 10(-5) +/- 4.61 x 10(-6) M for p-NPTA). In contrast, similar reactions of the enzyme in a reverse micellular system produced kinetic constants atypical to that representative of the textbook mechanism. (Km = 1.59 x 10(-4) +/- 2.70 x 10(-5) M, Ks = -8.67 x 10(-5) +/- 4.46 x 10(-5) M and Km(app) = -4.80 x 10(-5) +/- 7.05 x 10(-5) M for p-NPA and Km = 1.95 x 10(-4) +/- 9.28 x 10(-5) M, Km(app) = -1.79 x 10(-4) +/- 2.36 x 10(-5) M, and Ks = -3.95 x 10(-4) +/- 1.18 x 10(-4) M for p-NPTA). In addition to negative kinetic constants, alpha-chymotrypsin seemed to display characteristics indicative of super-activity and a hysteretic response. Overall, the widely accepted mechanism for alpha-chymotrypsin appeared to fail within the confines of reverse micelles, due to the direct influence of the system's highly structured form.     

MR angiography of aneurysms in Beh?et disease: a report of four cases

Two new pregnane-type steroidal alkaloids, saligcinnamide [(20S,2'E)-20-(N,N-dimethylamino)-3beta-(3'-phenyl-2'-propenyl-N-meth ylamido)pregnane](1) and N(a)-methyl epipachysamine-D [(20S)-20-(N,N-dimethylamino)-3beta-(N-methylbenzamido)pregnane](2 ), along with a known base, epipachysamine D [(20S)-20-(N,N-dimethylamino)-3beta-(benzamido)pregnane] (3), were isolated from the EtOH extracts of the roots and stems of Sarcococca saligna. The new bases exhibited antibacterial activity against several human pathogenic bacteria. Two derivatives of 1, dihydrosaligcinnarnide [(20S)-20-(N,N-dimethylamino)-3beta-(3'-phenylpropionoyl-N-meth ylamido)pregnane](4) and dihydrosaligcinnamine [(20S)-20-(N,N-dimethylamino)-3beta-N-(3'-phenylpropyl-N-methylamino)pre gnane](5), and a derivative of 2, N(a)-methyl epipachysamine [(20S)-20-(N,N-dimethylamino)-3beta-(N-benzyl,N-methylamino)pregnane](6) were prepared and their antibacterial activity determined.     

Comparison of high specific activity (-) and (+)-6-[18F]fluoronorepinephrine and 6-[18F]fluorodopamine in baboons: heart uptake, metabolism and the effect of desipramine

(-)-Norepinephrine is the principal neurotransmitter of the mammalian sympathetic nervous system and a major CNS neurotransmitter. The simple ring fluorinated derivatives of (-)- and (+)-norepinephrine [(-)- and (+)6-fluoronorepinephrine] and dopamine (6-fluorodopamine) have been labeled with 18F in high specific activity (2-5 Ci/mumol) and evaluated as tracers for (-)-norepinephrine. Comparative PET studies of (-) and (+)-6-[18F]fluoronorepinephrine [(-)-6-[18F]FNE and (+)-6-[18F]FNE] and 6-[18F]fluorodopamine (6-[18F]FDA) in the same baboon showed strikingly different kinetics in the heart. Analysis of plasma showed more rapid metabolism of 6-[18F]FDA with only 1%-2% of 18F remaining as parent tracer at 10 min after injection of 6-[18F]FDA, in contrast to 28% and 17% remaining after injection of (-) and (+)-6-[18F]FNE. No changes in vital signs were observed at any time during the study. Pretreatment with desipramine (0.5 mg/kg), a tricyclic antidepressant drug which interacts with a binding site associated with norepinephrine reuptake, markedly decreased cardiac uptake of 6-[18F]FDA and (-)-6-[18F]FNE. However, a greater blocking effect was observed for (-)-6-[18F]FNE. These studies show that (-) and (+)-6-[18F]FNE are similar to (-)- and (+)-norepinephrine in their patterns of metabolism and clearance in the heart and that (-)-6-[18F]FNE is a promising tracer for endogenous (-)-norepinephrine.     

Ranitidine effervescent and famotidine wafer in the relief of episodic symptoms of gastro-oesophageal reflux disease

Radioligands that specifically target dopamine uptake sites can provide a means of determining dopamine fiber loss at intrastriatal mesencephalic grafts in Parkinsonian patients, using Positron Emission Tomography (PET). The BTCP derivative, 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-4-(2-hydroxyethyl)-piperazine, shows in vitro high affinity and selectivity for the dopamine transporter. To evaluate the potential of such a compound as a potential dopaminergic PET tracer the positron-emitting analogues, 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-4-(2-[18F]fluoroethyl)-piperazine and 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-4-[11C]methylpiperazine, were synthesized. Radiofluorination was carried out by the reaction of 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-4-(2-chloroethyl)-piperazine with cyclotron-produced n.c.a. 18F-(half life 109.9 min) obtained by the (p,n) reaction on 18O-enriched water. Labelling with carbon-11 (half life 20.4 min) was achieved by 11C methylation of 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-piperazine with [11C]methyl iodide. After intravenous administration to rats these two compounds enter the brain, but despite their high in vitro affinity they display a high non specific binding in vivo which greatly limits their use as PET radioligands.     

Enzymatic synthesis of site-specifically (alpha 1-3)-fucosylated polylactosamines containing either a sialyl Lewis (x), a VIM-2, or a sialylated and internally difucosylated sequence

By using two different reaction pathways, we generated enzymatically three sialylated and site-specifically alpha 1-3-fucosylated polylactosamines. Two of these are isomeric hexasaccharides Neu5Ac(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)] GlcNAc and Neu5Ac(alpha 2-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4) GlcNAc, containing epitopes that correspond to VIM-2 and sialyl Lewis (x), respectively. The third one, nonasaccharide Neu5Ac(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)] GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc, is a sialylated and internally difucosylated derivative of a trimeric N-acetyllactosamine. All three oligosaccharides have one fucose-free N-acetyllactosaminyl unit and can be used as acceptors for recombinant alpha 1-3-fucosyltransferases in determining the biosynthesis pathways leading to polyfucosylated selectin ligands.     

Biosynthesis of scarab beetle pheromones

Chemical communication in scarab beetles (Coleoptera: Scarabaeidae) is achieved with a wide variety of pheromones, but one typical structure is the gamma-lactone having a long unsaturated hydrocarbon chain. Several species utilize (R, Z)-5-(-)-(oct-1-enyl)-oxacyclopentan-2-one (buibuilactone), (R, Z)-5-(-)-(dec-1-enyl)-oxacyclopentan-2-one and (S, Z)-5-(+)-(dec-1-enyl)-oxacyclopentan-2-one [(R)-japonilure and (S)-japonilure]. Using deuterated precursors, we have demonstrated that these compounds are biosynthesized from fatty acids. (9, 10-d4)-Palmitic acid, (9,10-d4)-stearic acid, (9,10-d2)-palmitoleic acid, (9,10-d2)-oleic acid, (9,10-d2)-8-hydroxypalmitoleic acid and (9,10-d2)-8-hydroxyoleic acid were readily incorporated by female Anomala cuprea into the pheromone molecules, while (Z)-(5, 6-d2)-5-dodecenoic acid and (Z)-(5,6-d2)-5-tetradecenoic acid were not. Therefore, the reaction pathway starts from saturated fatty acids, involves their desaturation, followed by 8-hydroxylation, chain shortening and cyclization. The products obtained from racemic (9,10-d2)-8-hydroxypalmitoleic acid and (9,10-d2)-8-hydroxyoleic acid were also racemic, implying that the steps following hydroxylation were not stereospecific. Perdeuterated palmitic acid was applied to disclose the mechanism of the unique hydroxylation reaction. Retention of all deuterium atoms implied that this reaction was a direct process mediated by a specific fatty acid hydroxylase, and preceding desaturation or epoxidation was not involved.     

Synthesis and cellular uptake of 2'-substituted analogues of (E)-5-(2-[125I]iodovinyl)-2'-deoxyuridine in tumor cells transduced with the herpes simplex type-1 thymidine kinase gene. Evaluation as probes for monitoring gene therapy

A useful synthetic methodology was developed to synthesize and radiolabel a series of (E)-5-(2-[125I]iodovinyl)uracil nucleoside substrates for herpes simplex virus type-1 thymidine kinase (HSV-1 TK). (E)-5-(2-[125I]Iodovinyl)-2'-deoxyuridine ([125I]IVDU, 10), (E)-5-(2-[125I]iodovinyl)-2'-fluoro-2'-deoxyuridine ([125I]IVFRU, 11), (E)-5-(2-[125I]iodovinyl)-2'-fluoro-2'-deoxyarabinouridine ([125I]IVFAU, 12), and (E)-5-(2-[125I]iodovinyl)arabinouridine ([125I]IVAU, 13) were synthesized in 63-83% radiochemical yield by reaction of the unprotected (E)-5-(2-(trimethylsilyl)vinyl) precursors (6-9) with [125I]ICl. Cellular uptake of these labeled compounds (10-13) was evaluated in vitro. All compounds showed minimal uptake in the KBALB cell line. However, increased uptake was observed for all compounds in KBALB-STK cells which are transduced with a replication incompetent Moloney murine leukemia virus vector encoding the HSV-1 TK gene. The results indicate that uptake of these compounds in KBALB-STK cells is variable and highly dependent on the nature of the sugar 2'-substituent. When a fluoro (12) or a hydroxy (13) substituent is present in the arabinofuranosyl (up) configuration at the 2'-position, there is diminished cellular uptake in KBALB-STK cells relative to hydrogen (10) or fluorine (11) in the ribofuranosyl (down) configuration at the 2'-position. Our results indicate that radiolabeled IVFRU (11) is most promising for further in vivo studies.     

Serum leptin and IGF-I levels in cystic fibrosis

(3S,4R)-4-(4-Fluorophenyl)-3-[[3,4-(methylenedioxy)phenoxy]methyl] piperidine [(3S,9R)-3, paroxetine] is a selective serotonin reuptake inhibitor (SSRI) used as an antidepressant in humans. In previous studies, we reported that certain (1R)-3 beta-(substituted phenyl)nortropane-2 beta-carboxylic acid methyl esters (2a) exhibited high affinity and reasonable selectivity for the serotonin transporter (5-HTT). The major structural differences between 2a and (3S,4R)-3 are that 2a possesses a different absolute stereochemistry and has an ethylene bridge not present in 3. In addition, 2a possesses a carbomethoxy substituent adjacent to the aryl ring, whereas (3S,4R)-3 contains a [3,4-(methylenedioxy)phenoxy]methyl group. In this study, we present the synthesis and biological evaluations of six of the possible eight isomers of 3-(4-fluorophenyl)-2-[[3,4-(methylenedioxy)phenoxy]methyl]nortropane+ ++ (4). The data for inhibition of [3H]paroxetine binding show that (1R)-2 beta, 3 alpha-4c, which has the same stereochemistry as paroxetine, has the highest affinity at the 5-HTT. Strikingly, the most potent compounds for inhibition of [3H]WIN-35,428 binding were not the (1R)-2 beta, 3 beta-isomers but rather (1R)-2 beta, 3 alpha-4c and (1S)-2 beta, 3 alpha-4f. Conformational analyses show that these isomers exist in a flattened boat conformation with pseudoequatorial substituents. Thus, the binding data show that this conformation is recognized by the DAT-associated binding site and also suggest that this conformation of paroxetine is recognized by the 5-HTT-associated binding site.     

Effects of type-selective phosphodiesterase inhibitors on glucose-induced insulin secretion and islet phosphodiesterase activity

1. We examined various type-selective phosphodiesterase (PDE) inhibitors on glucose-induced insulin secretion from rat isolated islets, on islet PDE activity and on islet cyclic AMP accumulation in order to assess the relationship between type-selective PDE inhibition and modification of insulin release. 2. The non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 10(-5)-10(-3) M), as well as the type III selective PDE inhibitors SK&F 94836 (10(-5)-10(-3) M), Org 9935 (10(-7)-10(-4) M), SK&F 94120 (10(-5)-10(-4) M) and ICI 118233 (10(-6)-10(-4) M) each caused concentration-dependent augmentation (up to 40% increase) of insulin release in the presence of a stimulatory glucose concentration (10 mM), but not in the presence of 3 mM glucose. 3. Neither the type IV PDE inhibitor rolipram (10(-4) M) nor the type I and type V PDE inhibitor, zaprinast (10(-4)-10(-3) M) modified glucose-induced insulin release when incubated with islets, although a higher concentration of rolipram (10(-3) M) inhibited secretion by 55%. However, when islets were preincubated with these drugs followed by incubation in their continued presence, zaprinast (10(-6)-10(-4) M) produced a concentration-dependent inhibition (up to 45% at 10(-4) M). Under these conditions, rolipram inhibited insulin secretion at a lower concentration (10(-4) M) than when simply incubated with islets. 4. A combination of SK&F 94836 (10(-5) M) and forskolin (5 x 10(-8) M) significantly augmented glucose-induced insulin secretion (30% increase), although neither drug alone, in these concentrations, produced any significant effect. 5. Islet cyclic AMP levels, which were not modified by forskolin (10-6 M), SK&F 94836 (10-4 M) or Org 9935 (10-5 M) were significantly elevated (approximately 3.7 fold increase) by forskolin inc ombination with either SK&F 94836 or Org 9935.6 Homogenates of rat islets showed a low Km (1.7 microM) and high Km (13 microM) cyclic AMP PDE in the supernatant fractions (from 48,000 g centrifugation), whereas the particulate fraction showed only a low Km (1.4 microM) cyclic AMP PDE activity.7. The PDE activity of both supernatant and pellet fractions were consistently inhibited by SK&F94836 or Org 9935, the concentrations required to reduce particulate PDE activity by 50% being 5.5 and 0.05 microM respectively.8 Rolipram (10-5 10-4 M) did not consistently inhibit PDE activity in homogenates of rat islets and zaprinast (10-4 M) consistently inhibited activity by 30% in the supernatant fraction, but not consistently in the pellet.9 These data are consistent with the presence of a type III PDE in rat islets of Langerhans.     

Endothelin receptors mediating vasoconstriction in rat pressurized small arteries

The effects of endothelin-1 (ET-1), a nonselective ETA and ETB receptor agonist, and sarafotoxin S6c, a selective ETB agonist, were investigated in the presence and absence of BQ123 and BQ788, ETA- and ETB-selective antagonists, respectively, in rat mesenteric small arteries, using a perfusion pressurized arteriograph in which segments of vessels were cannulated and exposed to constant pressure and flow. ET-1 (10(-13)-10(-7) M) induced vasoconstriction in both intact and endothelium-denuded arteries in a concentration-dependent manner. BQ123 (10(-7) and 10(-6) M) inhibited the effect of ET-1, displacing the concentration-response curve to the right in a concentration-dependent manner. The effect of ET-1 was not significantly affected by BQ788 (10(-7) and 10(-6) M), a selective antagonist of ETB receptors. Sarafotoxin S6c (10(-11)-10(-7) M) also induced a slight concentration-dependent vasoconstriction. The effect of sarafotoxin S6c (10(-8) M) was inhibited by the ETB-selective antagonist BQ788 (10(-7) M), but was not significantly changed by BQ123 (10(-7) M). Vasoconstriction induced by sarafotoxin S6c (10(-8) M) in a single bolus concentration was significantly greater than the contraction induced by the same concentration as part of a cumulative concentration-response curve, indicating desensitization or downregulation of ETB receptors during the latter. Repeated application of single concentrations of sarafotoxin S6c (10(-8) M) caused progressively smaller contraction of arteries. These results show the existence of both ETA and ETB vasoconstrictor receptors located on smooth muscle of small arteries. They also show that ETB receptors induce a smaller constrictor effect, and rapidly undergo desensitization after sustained or repeated activation.     

Synthesis and structure-activity relationships of a new series of benzimidazoles as H1-antihistaminic agents

New 2-(4-(4-azolylbutyl)piperazinyl)-, 2-(4-(4-azolylbutyl) piperazinylmethyl)-, 2-(4-(-azolylbutyl)homopiperazinyl)- and 2-(4-(4-azolylbutyl)homopiperazinylmethyl)benzimidazoles were synthesized, characterized and tested for in vitro and in vivo H1-antihistaminic activity. Structure-activity relationships implied that the best antihistaminic activity required the simultaneous presence of a homopiperazinylbenzimidazole system (or a methylene linker between the benzimidazole and the piperazine rings) and an unsubstituted pyrazole ring. 1-(2-Ethoxyethyl)-2-?4-[4-(pyrazol-1-yl)butyl] homopiperazin-1-yl?benzimidazole (17), as its dimaleate salt, has been chosen for further development.     

Inhibition of cAMP mediated relaxation in rat coronary vessels by block of Ca++ activated K+ channels

The hypothesis for this study is that block of calcium activated potassium (KCa) channels inhibits cAMP induced relaxation in pressurized rat coronary resistance arteries. Pressure-diameter experiments with septal arteries (200-270 microns internal diameter at 60 mmHg and maximum dilation) showed significant basal tone over a range of pressure from 40-120 mmHg. The level of tone was increased with the thromboxane A2 analogue 9,11-dideoxy-11 alpha, 9 alpha-epoxy-methanoprostaglandin F2 alpha (U46619) in all experiments. Receptor activation of the cAMP pathway was done with adenosine (ADO) and isoproterenol (ISO). Tetraethylammonium ion (TEA+), 1mM, significantly inhibited relaxation to ADO (10(-6)-10(-3)M) with a maximal inhibition of 75 +/- 7% (as a % of maximum diameter change with the vasodilator alone) at 10(-3)M ADO. TEA+ inhibited ISO (10(-6)M) relaxation by 63 +/- 9%. Direct activation of the cAMP pathway was done with forskolin and 8-bromo-cAMP. TEA+ significantly inhibited forskolin (10(-6)-10(-4)M) induced relaxation with a maximal inhibition of 81.3 +/- 1.2% at 10(-4)M forskolin. TEA+ and iberiotoxin (10(-7)M) significantly inhibited 8- bromo-cAMP (10(-3)M) induced relaxation by 72 +/- 5% and 56 +/- 3% respectively. The effect of TEA+ on relaxation induced by nitroprusside (a cGMP dependent vasodilator) was not significant. The results show that rat coronary resistance arteries possess significant myogenic tone and modulation of Kca channels plays a major role in cAMP mediated relaxation.     

Beneficial effects of S-adenosyl-L-methionine on blood-brain barrier breakdown and neuronal survival after transient cerebral ischemia in gerbils

In the present study, the effect of bradykinin on basal and precontracted mouse-isolated trachea was investigated. In basal conditions mouse-isolated tracheal rings do not respond to bradykinin. However, when the tracheal rings were precontracted with carbachol (10(-7) M) a relaxation with bradykinin (3 x 10(-9)-3 x 10(-7)) was found. The maximal response amounted 69.7+/-4.1% (n=15) with a pD2 value of 7.2+/-0.21. The selective bradykinin B2 receptor antagonist HOE 140 (10(-10)-10(-8) M) antagonized the bradykinin-induced relaxation, while the bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin (10(-6) M) had no influence. The selective bradykinin B1 receptor agonist des-Arg9-bradykinin (10(-6) M) caused a small relaxation (8.4+/-2.5%, n=6), which could be antagonized completely by the selective bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin (10(-6) M) while addition of the selective bradykinin B2 receptor antagonist HOE 140 (10(-8) M) was without effect. In the presence of indomethacin (10(-6) M) the relaxation of bradykinin was completely abolished. Pretreatment of the tracheal rings with capsaicin, or the presence of the selective NK1 receptor antagonist RP 67851 (10(-6) M) or the presence of the nitric oxide synthase inhibitor L-NAME (3 x 10(-4) M) had no effect on the bradykinin-induced relaxation. In conclusion, these results demonstrate that the mouse-isolated tracheal is a preparation in which bradykinin exerts a relaxant response via stimulation of bradykinin B2 receptors. This response is probably mediated by prostaglandins.     

Anticipating parental death in families with young children

Voltage-activated currents from adult honey bee antennal motor neurons were characterized with in vitro studies in parallel with recordings taken from cells in situ. Two methods were used to ensure unequivocal identification of cells as antennal motor neurons: 1) selective backfilling of the neurons with fluorescent markers before dissociation for cell culture or before recording from cells in intact brains, semiintact brains, or in brain slices or 2) staining with a fluorescent marker via the patch pipette during recordings and identifying antennal motor neurons in situ on the basis of their characteristic morphology. Four voltage-activated currents were isolated in these antennal motor neurons with pharmacological, voltage, and ion substitution protocols. The neurons expressed at least two distinct K+ currents, a transient current (IA) that was blocked by 4-aminopyridine (4-5 x 10(-3) M), and a sustained current (IK(V)) that was partially blocked by tetraethylammonium (2-3 x 10(-2) M) and quinidine (5 x 10(-5) M). IA activated above -40 to -30 mV and the half-maximal voltages for steady-state activation and inactivation were -8.8 and -43.2 mV, respectively. IK(V) activated above -50 to -40 mV and the midpoint of the steady-state activation curve was +11.2 mV. IK(V) did not show steady-state inactivation. Additionally, two inward currents were isolated: a tetrodotoxin (10(-7) M)-sensitive, transient Na+ current (INa) that activated above -35 mV, with a maximum around -5 mV and a half-maximal voltage for inactivation of -72.6 mV, and a CdCl2 (5 x 10(-5) M)-sensitive Ca2+ current that activated above -45 to -40 mV, with a maximum around -15 mV. This study represents the first step in our effort to analyze the cellular and ionic mechanisms underlying the intrinsic properties and plasticity of antennal motor neurons.     

Prevention of hypoxemia-induced renal dysfunction by perindoprilat in the rabbit

The role of angiotensin II, a potent postglomerular vasoconstrictor, in the hypoxemia-induced renal changes is still controversial. The ability of perindoprilat, an angiotensin converting-enzyme inhibitor, to prevent the acute renal effects of hypoxemia was assessed in 22 anesthetized-ventilated rabbits. In 8 untreated rabbits, hypoxemia induced a significant drop in mean blood pressure (MBP) (-12 +/- 2%), glomerular filtration rate (GFR) (-16 +/- 3%) and renal blood flow (RBF) (-12 +/- 3%) with a concomittant increase in renal vascular resistance (RVR) (+18 +/- 5%) and urine flow rate (+33 +/- 14%), and without any changes in filtration fraction (FF) (-4 +/- 2%). This suggests the occurrence of glomerular vasoconstriction during the hypoxemic stress. In 7 normoxemic rabbits, intravenous perindoprilat (20 microg/kg) induced an increase in urine flow rate (+17 +/- 4%) and RBF (+17 +/- 4%), and a decrease in MBP (-6 +/- 1%), RVR (-14 +/- 3%) and FF (-11 +/- 2%) without a significant change in GFR. The drop in FF and the increase in RBF suggests preferential postglomerular vasodilatation. In 7 rabbits, perindoprilat prevented the occurence of the hypoxemia-induced changes in RBF and RVR without improving MBP. FF decreased significantly (-18 +/- 2%), while the drop in GFR (-7 +/- 2%) was partially blunted and the increase in urine flow rate (+25 +/- 9%) was confirmed. These results could be explained by the inhibition of the angiotensin-mediated efferent vasoconstriction and by the inhibition of bradykinin degradation by perindoprilat. These data confirm the ability of converting-enzyme inhibitors to prevent the renal hypoperfusion induced by acute hypoxemia.     

Endothelin-1-induced in vitro cerebral venoconstriction is mediated by endothelin ETA receptors

The in vitro effects of endothelin-1 on cerebral veins were studied using cylindrical segments, 5 mm long, from dog pial veins. Isometric responses to endothelin-1 (10(-12)-10(-7) M) and to the endothelin ET(B) receptor agonist, IRL 1620 (Suc-[Glu9,Ala11,15]endothelin-1-(8-21), 10(-12) -10(-7) M), were recorded in veins under control conditions and pretreated with the endothelin ET(A) receptor antagonist, BQ-123 (cyclo-(D-Asp-Pro-D-Val-Leu-D-Trp), 10(-8) -10(-5) M), and the endothelin ETB receptor antagonist, BQ-788 (N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl]-1-(me thoxycarbonyl)-D-tryptophyl]-D-norleucine monosodium, 10(-6) and 10(-5) M). The response to endothelin-1 was also recorded in veins pretreated with the nitric oxide synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), or the cyclooxygenase inhibitor, meclofenamate (10(-5) M), and in veins without endothelium or placed in medium without Ca2+ but with EDTA (0.1 mM). In control veins, endothelin-1 produced a concentration-dependent contraction (EC50 = 2.0 x 10(-10) M; maximal contraction = 113 +/- 6 mg) and IRL 1620 induced no effects or a small contraction only with high concentrations (10(-8) - 10(-6) M) (EC50 = 1.5 x 10 (-8) M; maximal contraction = 9 +/- 3 mg). BQ-123 shifted the response to endothelin-1 to the right in a parallel, concentration-dependent way, whereas BQ-788, L-NAME or meclofenamate did not modify the response to endothelin-1. Compared with the control, veins in a medium without Ca2+ had similar EC50 values, but a lower maximal contraction induced by endothelin-1 (57 +/- 10 mg, P < 0.05), and veins without endothelium exhibited similar EC50 values. Thus, endothelin-1 produces marked cerebral venoconstriction that could be mainly mediated by activation of endothelin ETA receptors, may be dependent on extracellular Ca2+, and may be independent of endothelium, nitric oxide and prostanoids.     

Inhibitory effects of nitric oxide donors on nitric oxide synthesis in rat gastric myenteric plexus

We investigated whether nitric oxide (NO) exerts an inhibition on its own synthesis in the gastric myenteric plexus in rats. Nonadrenergic, noncholinergic relaxations in response to transmural electrical stimulation (TS) were markedly antagonized by NG-nitro-L-arginine methyl ester, (10(-4) M) and abolished by tetrodotoxin (10(-6) M). Pretreatment with various NO donors (3-morpholino-sydnonymide [SIN-1 (3 x 10(-7) to 3 x 10(-6) M)], S-nitroso-N-acetylpenicillamine (10(-6) to 10(-5) M), sodium nitroprusside (10(-8) to 3 x 10(-8) M) and 8-bromoquanosine 3', 5'-cyclic monophosphate [8-bromo-cGMP (10(-6) to 3 x 10(-6) M)]) significantly inhibited TS-evoked nonadrenergic, noncholinergic relaxations in a dose-dependent manner. In contrast, vasoactive intestinal polypeptide (10(-8) M)-induced relaxations were not affected by SIN-1 or 8-bromo-cGMP. TS evoked a significant increase in 3H-citrulline formation, which was completely abolished by calcium-free medium, NG-nitro-L-arginine methyl ester, (10(-4) M) and tetrodotoxin (10(-6) M). 3H-citrulline formation evoked by TS was significantly inhibited by SIN-1 (10(-7) to 10(-5) M) and 8-bromo-cGMP (10(-7) to 10(-5) M) in a dose-dependent manner. The inhibitory effect of SIN-1 was partially prevented by 1H-[1,2, 4]oxadiazolo[3,4-a]quinoxalin-1-one (10(-5) M), a guanylate cyclase inhibitor. We conclude that NO synthesis in the gastric myenteric plexus is negatively regulated by NO and cGMP. This suggests an autoregulatory feedback mechanism of NO synthesis in the gastric myenteric plexus.     

Heteroaryl analogues of AMPA. 2. Synthesis, absolute stereochemistry, photochemistry, and structure-activity relationships

We have previously shown that (S)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid [(S)-APPA, 2] is a weak agonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors, specifically activated by (S)-AMPA (1), whereas (S)-2-amino-3-[3-hydroxy-5-(2-pyridyl)-4-isoxazolyl]propionic acid [(S)-2-Py-AMPA, 5] and (RS)-2-amino-3-[3-hydroxy-5-(2-thiazolyl)-4-isoxazolyl]propionic acid (4) are potent AMPA agonists. On the other hand, (R)-APPA (3) and (R)-2-Py-AMPA (6) have been shown to be weak AMPA antagonists. We now report the synthesis of 2-Py-AMPA (7a) and the isomeric compounds 3-Py-AMPA (7b) and 4-Py-AMPA (7c) as well as the 7a analogues, (RS)-2-amino-3-[3-hydroxy-5-(6-methyl-2-pyridyl)-4-isoxazolyl]p ropion ic acid (7d) and (RS)-2-amino-3-[3-hydroxy-5-(2-quinolinyl)-4-isoxazolyl]propionic acid (7e). Furthermore, (RS)-2-amino-3-[3-hydroxy-5-(2-furyl)-4-isoxazolyl]propionic acid (2-Fu-AMPA, 7f) and its 5-bromo-2-furyl derivative (7g) were synthesized, and (S)-2-Fu-AMPA (8) and (R)-2-Fu-AMPA (9) were prepared by semipreparative chiral HPLC resolution of 7f. HPLC analyses and circular dichroism spectroscopy indicated the absolute stereochemistry of 8 and 9 to be S and R, respectively. This was confirmed by an X-ray crystallographic analysis of 9.HCl. In receptor binding (IC50 values) and rat cortical wedge electrophysiological (EC50 values) studies, 7c (IC50 = 5.5 +/- 0.6 microM; EC50 = 96 +/- 5 microM) was shown to be markedly weaker than 7a (IC50 = 0.57 +/- 0.16 microM; EC50 = 7.4 +/- 0.2 microM) as an AMPA agonist, whereas 7b,d,e were inactive. The very potent AMPA agonist effect of 7f (IC50 = 0.15 +/- 0.03 microM; EC50 = 1.7 +/- 0. 2 microM) was shown to reside exclusively in 8 (IC50 = 0.11 +/- 0.01 microM; EC50 = 0.71 +/- 0.11 microM), whereas 9 did not interact significantly with AMPA receptors, either as an agonist or as an antagonist. 8 was shown to be photochemically active and is a potential photoaffinity label for the recognition site of the AMPA receptors. Compound 7g turned out to be a very weak AMPA receptor agonist (IC50 = 12 +/- 0.7 microM; EC50 = 160 +/- 15 microM). None of these new compounds showed detectable effects at N-methyl-d-aspartic acid (NMDA) or kainic acid receptors in vitro. The present studies have emphasized that the presence of a heteroatom in the 2-position of the heteroaryl 5-substituent greatly facilitates AMPA receptor agonist activity.     

Synthesis, antiviral activity and enzymatic phosphorylation of 9-phosphonopentenyl derivatives of guanine

(E)-9-(5-Phosphonopent-4-enyl)guanine and (E)-9-[3-(hydroxymethyl)-5- phosphonopent-4-enyl]guanine which bear a vinyl phosphonate moiety as a mimic of the phosphate group were synthesized. Their activities against human immunodeficiency virus type-1 (HIV-1), herpes simplex virus type-1 (HSV-1) and human cytomegalovirus (HCMV) were evaluated in vitro in parallel with those of 9-(5-phosphonopentyl)guanine and 9-(5,5-difluoro-5- phosphonopentyl)guanine. Both vinyl phosphonates exhibited anti-HIV-1 and anti-HCMV activities, whereas the methyl- and difluoromethyl phosphonate analogues were inactive. The selectivity index, calculated as the ratio of the toxicity for the host cells (50% reduction in cell viability or in [methyl-3H]thymidine incorporation) to the 50% inhibitory concentration for HIV-1 replication, was the highest for (E)-9-[3-(hydroxymethyl)-5-phosphonopent-4-enyl]guanine. The acyclonucleotide analogues were also studied as substrates of guanylate kinase, an enzyme believed to play a critical role in the conversion of acyclic phosphate and phosphonate derivatives of guanine to their antivirally active diphosphate derivatives. (E)-9-(5-Phosphonopent-4- enyl)guanine and (E)-9-[3-(hydroxymethyl)-5-phosphonopent-4-enyl]guanine were good substrates of guanylate kinase, being phosphorylated with efficiencies of 14 and 36% of that determined for GMP, respectively. These results contrast with the poor efficiency found for 9-(5-phosphonopentyl)guanine (0.3%) and the lack of phosphorylation of 9-(5,5-difluoro-5-phosphonopentyl)guanine by guanylate kinase (Navé et al. (1992) Arch. Biochem. Biophys. 295, 253-257). The role of the vinyl phosphonate group in the expression of the anti-HIV-1 activity of the phosphonopentenyl derivatives of guanine is discussed.     

Sodium balance of hyper- and hypo-natriophilic rats under basal conditions and during sodium deprivation

Sodium and water balance was determined in two strains of Wistar rats selectively bred for high (hypernatriophilic, HR) or low salt preference (hyponatriophilic, HO) under basal conditions and during sodium deprivation. Male rats from each stain were selected for an average ingestion of 1.5% NaCl solution of more than (HR) or less than (HO) 4 ml 100 g body weight (-1) day (-1), during a 10-day period. HR rats (N = 17) presented markedly higher sodium intake under basal conditions (2.983 +/- 0.316 mEq 100 g body weight (-1) day (-1)) than HO rats (N = 12; 0.406 +/- 0.076 mEq 100 g body weight (-1) day (-1); Mann-Whitney test, P < 0.01). Water (HR: 8.6 +/- 0.57; HO: 7.7 +/- 0.32 ml 100 g body weight (-1) day (-1)) and sodium balances (HR: 0.936 +/- 0.153; HO: 0.873 +/- 0.078 mEq 100 g body weight (-1) day (-1)) were similar in both strains, despite a higher sodium and total fluid (HR: 16.3 +/- 1.06; HO: 10.8 +/- 0.49 ml 100 g body weight (-1) day (-1); P < 0.01) ingestion in HR rats. During sodium deprivation HR rats (N = 13) exhibited a sodium balance similar to that of HO rats (N = 13) (HR: -0.159 +/- 0.011; HO: -0.129 +/- 0.019 mEq 100 g body weight (-1) day (-1)), and, in addition, an adequate suppression of natriuresis (HR: 0.049 +/- 0.011; HO: 0.026 +/- 0.004 mEq 100 g body weight (-1) day (-1)). These data show that HR rats present hypernatriophilia as a primary trait, since their sodium-conserving mechanisms are intact. Therefore, these rats provide an adequate model to study factors that determine innate sodium preference.