Effects of a new cholecystokinin antagonist (GE 410) on the smooth muscle of the guinea pig ileum

Summary Suc-Tyr-(SE)-Met-Gly-Trp-Met-Asp--phenethylamide (GE 410) competitively antagonized the contractions of smooth muscle strips from guinea pig ileum (pA₂=7.6, n=0.95) induced by cholecystokinin-octapeptide (CCK8). GE 410 inhibited the electrically-induced cholinergically mediated contractile responses and the [³H]ACh release in the ileum, as well as the CCK-stimulated electrical contractile responses and the [³H]ACh release in the cholinergic nerve terminals. The results suggest the existence of CCK-receptors not only in the smooth muscles but also on the neurons.     

Inhibition by tetanus and botulinum A toxin of the release of [3H]noradrenaline and [3H]GABA from rat brain homogenate

Rat brain homogenate was preloaded with [3H]noradrenaline or [3H]GABA and stimulated with high K+. Tetanus toxin and botulinum A neurotoxin partially prevent the evoked [3H]noradrenaline release in the same range of toxin concentrations starting below 10(-10) M. In contrast, release of gamma-amino butyric acid (GABA) is much more sensitive to tetanus than to botulinum A toxin.     

Cyclic nucleotide-dependent relaxation pathways in vascular smooth muscle

Vascular smooth muscle tone is controlled by a balance between the cellular signaling pathways that mediate the generation of force (vasoconstriction) and release of force (vasodilation). The initiation of force is associated with increases in intracellular calcium concentrations, activation of myosin light-chain kinase, increases in the phosphorylation of the regulatory myosin light chains, and actin-myosin crossbridge cycling. There are, however, several signaling pathways modulating Ca²⁺ mobilization and Ca²⁺ sensitivity of the contractile machinery that secondarily regulate the contractile response of vascular smooth muscle to receptor agonists. Among these regulatory mechanisms involved in the physiological regulation of vascular tone are the cyclic nucleotides (cAMP and cGMP), which are considered the main messengers that mediate vasodilation under physiological conditions. At least four distinct mechanisms are currently thought to be involved in the vasodilator effect of cyclic nucleotides and their dependent protein kinases: (1) the decrease in cytosolic calcium concentration ([Ca²⁺]c), (2) the hyperpolarization of the smooth muscle cell membrane potential, (3) the reduction in the sensitivity of the contractile machinery by decreasing the [Ca²⁺]c sensitivity of myosin light-chain phosphorylation, and (4) the reduction in the sensitivity of the contractile machinery by uncoupling contraction from myosin light-chain phosphorylation. This review focuses on each of these mechanisms involved in cyclic nucleotide-dependent relaxation of vascular smooth muscle under physiological conditions.     

Serotonin-stimulated protein phosphorylation in aortic smooth muscle cells

The effects of serotonin on the formation of inositol phosphates and protein phosphorylation were examined in cultured smooth muscle cells. Serotonin stimulated the formation of [3H]inositol monophosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. This effect was prevented by 5-HT2 specific antagonist, 6-methyl-1-(1-methylethyl)ergoline-8-carboxylic acid, 2-hydroxy-1-methylpropyl ester [Z]-2-butenedioate (LY53857). Serotonin stimulated the phosphorylation of many polypeptides, among which a 20 kDa polypeptide was the most prominent. The phosphorylation was also inhibited by LY53857. LY53857 alone produced no effects on protein phosphorylation. The 20 kDa polypeptides were also phosphorylated by the addition of 12-O-tetradecanoylphorbol-13-acetate. These results suggest that serotonin stimulates protein phosphorylation through 5-HT2 receptors and possibly activates protein kinase C in intact vascular smooth muscle cells.     

Inhibition by tetanus and botulinum A toxin of the release of [3H]noradrenaline and [3H]GABA from rat brain homogenate

Summary Rat brain homogenate was preloaded with [³H]noradrenaline or [³H]GABA and stimulated with high K⁺. Tetanus toxin and botulinum A neurotoxin partially prevent the evoked [³H]noradrenaline release in the same range of toxin concentrations starting below 10^–10M. In contrast, release of -amino butyric acid (GABA) is much more sensitive to tetanus than to botulinum A toxin.     

On the synthesis of serine and homoserine samples asymmetrically labelled with tritium and deuterium in the hydroxymethylene group.

(1R) [1-3H, 2H1] 3-Phenylpropanol, the key intermediate in the synthesis of (4R) [4-3H, 2H1] D,L-homoserine and of the (4S)-isomer, is obtained from (1S) [1-2H1] 3-phenylpropanol and (1RS) [1-3H] ethanol upon incubation with yeast alcohol dehydrogenase and NAD+; under similar conditions 2-phenylethanol undergoes very small exchange with [1-2H2] ethanol.     

The endocannabinoid system in rat gliosomes and its role in the modulation of glutamate release

The endocannabinoid system and endocannabinoid receptor-driven modulation of glutamate release were studied in rat brain cortex astroglial gliosomes. These preparations contained the endocannabinoids N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol, as well their major biosynthetic (N-acyl-phosphatidylethanolamines-hydrolyzing-phospholipase D and diacylglycerol-lipase) and catabolic (fatty acid amide-hydrolase and monoacylglycerol-lipase) enzymes. Gliosomes expressed type-1 (CB1R), type-2 (CB2R) cannabinoid, and type-1 vanilloid (TRPV1) receptors, as ascertained by Western blotting and confocal microscopy. Methanandamide, a stable analogue of anandamide acting as CB1R, CB2R, and TRPV1 agonist, stimulated or inhibited the depolarization-evoked gliosomal [³H]d-aspartate release, at lower and higher concentrations, respectively. Experiments with ACEA (arachidonyl-2′-chloroethylamide), JWH133 ((6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]-pyran) and capsaicin, selective agonists at CB1R, CB2R and TRPV1, respectively, demonstrated that potentiation of [³H]d-aspartate release was due to CB1R while inhibition to CB2R and TRPV1 engagement. These findings were confirmed by using selective receptor antagonists. Furthermore, CB1R activation caused increase of intracellular IP3 and Ca²⁺ concentration, suggesting an involvement of phospholipase C.     

Evidence against prostaglandin E having a physiological role in acetylcholine liberation from Auerbach's plexus of guinea-pig ileum.

The effect of INDO, a PGE synthesis inhibitor, on ACh output from Auerbach's plexus of guinea-pig ileum was investigated. INDO (15-45 mum/ml) failed to alter significantly either spontaneous ACh output or ACh output induced by field stimulation. It is concluded that PGE plays no physiological role in ACh liberation from this tissue.     

Serotonin-stimulated protein phosphorylation in aortic smooth muscle cells

Summary The effects of serotonin on the formation of inositol phosphates and protein phosphorylation were examined in cultured smooth muscle cells. Serotonin stimulated the formation of [³H]inositol monophosphate, [³H]inositol bisphosphate and [³H]inositol trisphosphate. This effect was prevented by 5-HT₂ specific antagonist, 6-methyl-1-(1-methylethyl)ergoline-8-carboxylic acid, 2-hydroxy-1-methylpropyl ester [Z]-2-butenedioate (LY53857). Serotonin stimulated the phosphorylation of many polypeptides, among which a 20 kDa polypeptide was the most prominent. The phosphorylation was also inhibited by LY53857. LY53857 alone produced no effects on protein phosphorylation. The 20 kDa polypeptides were also phosphorylated by the addition of 12-O-tetradecanoylphorbol-13-acetate. These results suggest that serotonin stimulates protein phosphorylation through 5-HT₂ receptors and possibly activates protein kinase C in intact vascular smooth muscle cells.Part of the data contained in this paper was presented at the 74th local meeting of the Japanese Society of Pharmacology at Kanagawa.     

3[H]-Sulpiride labels mesolimbic non-dopaminergic sites that bind antidepressant drugs

3[H]-(-)-Sulpiride and 3[H]-spiperone binding was compared in rat amygdala, nucleus accumbens and striatum, using (+/-)-sulpiride to define specific binding. 3[H]-(-)-Sulpiride bound to twice as many sites in amygdala and nucleus accumbens as 3[H]-spiperone. 3[H]-(-)-Sulpiride binding was directed to these additional sites by using 1 microM spiperone to mask dopaminergic binding. The binding of 3[H]-(-)-sulpiride to these sites was high affinity, reversible, Na+-dependent, but not stereospecific. Metoclopramide, tiapride and antidepressant medications, but not other neuroleptics, ADTN, or serotonin displaced 3[H]-(-)-sulpiride binding to these sites. These data suggest that 3[H]-(-)-sulpiride labels mesolimbic sites other than dopamine receptors which may mediate antidepressant effects.     

Inhibition of cholesterol synthesis ex vivo and in vivo by fluvastatin,a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase

The inhibitory effect of fluvastatin sodium (fluvastatin), a new type of 3-hydroxy-3-methylglutaryl (HMG) coenzyme A inhibitor, on de novo cholesterol synthesis was investigated and compared with that of pravastatin. Fluvastatin at a concentration of 12.5 mg/kg inhibited sterol synthesis ex vivo from [¹⁴C]acetate in rat liver and ileum by 97–99% with respect to the control, while the inhibition in kidney was 55%. The inhibition by fluvastatin in the liver and ileum persisted for approximately 9 h after administration. Significant differences between fluvastatin also had an inhibitory effect on cholesterol synthesis in vivo in various tissues of rats given [¹⁴C]acetate intraperitoneally. Sterol synthesis in the liver, ileum and kidney was inhibited by over 95% 3 h after administration of 6.25 mg/kg of fluvastatin. Significant differences between fluvastatin and pravastatin were found in the liver and ileum. Fluvastatin was more potent than pravastatin in inhibiting both ex vivo and in vivo sterol synthesis in the ileum (but not in kidney) and liver.     

Comparative study of the 3 beta-hydroxy-delta 5-steroid dehydrogenase activity of the mitochondrial and microsomal fractions of human and cat placentas

Delta 5, 3 beta hydroxysteroid dehydrogenase activity was studied on mitochondrial and microsomal fractions of human and cat placentas. This activity was assessed by measuring the rate of [3H] progesterone formation from [3H] pregnenolone and of [3H] delta 4-androstenedione from [3H] dehydroepiandrosterone (DHA), by a double isotope dilution method. Specific activities measured under initial velocity conditions were similar in both human and cat placentas. However, the kinetics of the reaction with DHA as substrate exhibit a zoological specificity.     

Effects of bunaphtine on 45Ca movements in rat aortic smooth muscle

The effect of bunaphtine (BNA, 5 X 10(-5)M) on La3+ -resistant 45Ca content and 45Ca efflux was studied on rat aortic smooth muscle. BNA decreased both control and norepinephrine-stimulated La3+-resistant 45Ca content and increased the 45Ca efflux. These effects could explain the inhibition of the contractile responses induced by BNA.     

Changes with hypertension and maturation in the response of stomach fundus to acetylcholine

The purpose of these experiments was to compare the contractile response to ACh of stomach fundal strips from hypertensive (SHR) and normotensive (WKY) rats during the development of hypertension. The results indicate that the reactivity to ACh is the same in fundal strips from young SHR and WKY rats; however, with maturation strips from WKY rats undergo a reduction in responsiveness which does not occur in the SHR. Therefore, strips from older SHR rats are more reactive to ACh than are those from age matched WKY rats.     

Lack of participation of cytoplasmic choline in the synthesis of acetylcholine in the striatal synaptosomes of the rat]

Synaptosomes prepared from Rat striatum were loaded with (14C) choline at 0 degrees C in the course of a 60 min. preincubation. After the removal, by washing, of the extracellular molecules, synaptosomes were incubated 5 min. at 37 degrees C in the presence of (3H) choline. The measurement of the amounts of (3H) ACh and of (14C) ACh formed allowed us to conclude that the acetylation of the molecules of choline was directly coupled to their passage through the synaptosomal membrane.     

Effect of enkephalins in the presence of the antibiotic bacitracin in the longitudinal muscle strip preparation from guinea-pig ileum

Summary The antibiotic bacitracin (5×10^–5–4×10^–4 M) increases the inhibition of the contractile response caused by both enkephalin release and direct application of Met-enkephalin 5×10^–7 M in the longitudinal muscle strip preparation from guinea-pig ileum. This effect is attributed to an inhibition of enkephalin degrading peptidases by bacitracin.     

Failure to identify 'thrombocytolysin' (a spasmogenic factor released from platelets immunoreaction) with anaphylatoxin

Horse platelets release an unidentified smooth muscle contracting substance after lysis by antiserum and complement. Since the active factor (thrombocytolysin) does not produce tachyphylactic response of the guinea-pig ileum it seems that it is not related to anaphylatoxins.     

Failure to identify ‘thrombocytolysin’ (a spasmogenic factor released from platelets by immunoreaction) with anaphylatoxin

Summary Horse platelets release an unidentified smooth muscle contracting substance after lysis by antiserum and complement. Since the active factor (thrombocytolysin) does not produce tachyphylactic response of the guinea-pig ileum it seems that it is not related to anaphylatoxins.     

Studies on the serotonin transporter in platelets

[3H]-Imipramine and [3H]-paroxetine label with high affinity a recognition site which is associated with the serotonergic transporter in blood platelets. The pharmacological profile of [3H]-imipramine and [3H]-paroxetine binding is highly correlated with the potency of drugs to inhibit the uptake of serotonin. Dissociation kinetic experiments suggest that the substrate recognition site for serotonin may be different from the modulatory site which is labeled with [3H]-imipramine or [3H]-paroxetine. The existence of an endocoid acting on the imipramine receptor to modulate the serotonin transporter has been proposed by several laboratories. In clinical studies most laboratories have reported a decrease in Bmax of [3H]-imipramine binding in platelets from depressed untreated patients when compared with matched healthy volunteers. The Bmax of [3H]-imipramine binding in platelets appears to be a state-dependent biological marker in depression.