Mechanism of mifepristone-induced spasmolytic effect on isolated rat uterus

Mifepristone, a synthetic 19-norsteroid, relaxed the KCl-induced tonic contraction in isolated rat uterus in a concentration-dependent way and CaCl2 (0.1 to 10 mM) counteracted it. This effect was similar to other steroids although the mechanisms involved are unclear. Before adding the contracturant, tissue was incubated with actinomycin D (10 microM), cycloheximide (300 microM), TPCK (3 and 10 microM), Rp-cAMPS (30 microM), DDA (100 microM) and H-7 (1 microM). None of these modified the relaxing effect of mifepristone. Incubation with drugs that interfere with cGMP such as a nucleotide analogue DDG (100 microM), a soluble guanylyl cyclase inhibitor ODQ (1 microM) and an inhibitor of protein kinase G 8pCPTcGMPS (1 microM) significantly modified the effect of mifepristone, increasing its IC50.     

Pharmacological investigations on the lipolytic and antilipolytic effects of growth hormone (GH) in chicken adipose tissue in vitro: evidence for involvement of calcium and polyamines

The involvement of RNA/protein synthesis, calcium, calmodulin, protein kinase C, and polyamines in the lipolytic and antilipolytic (inhibition of glucagon-stimulated lipolysis) responses to GH have been investigated employing chicken adipose tissue in vitro. The lipolytic, but not the antilipolytic, effect of GH was blocked by inhibitors of RNA/protein synthesis (actinomycin D, cycloheximide, and puromycin) and calcium uptake (verapamil) and low calcium concentrations (0.28 mM CaCl2). The antilipolytic, but not the lipolytic, effect of GH was blocked by alpha-difluoromethylornithine (DFMO), a polyamine synthesis inhibitor. DFMO-induced blockade of the antilipolytic GH response was reversed by the addition of spermidine. The lipolytic and antilipolytic effects of GH were not influenced by chlorpromazine (a calmodulin inhibitor) or phorbol 12-myristate 13-acetate (PMA) (an activator of protein kinase C).     

Vasodilatory action mechanisms of apigenin isolated from Apium graveolens in rat thoracic aorta.

The effect of apigenin, isolated from Apium graveolens, on the contraction of rat thoracic aorta was studied. Apigenin inhibited the contraction of aortic rings caused by cumulative concentrations of calcium (0.03-3 mM) in high potassium (60 mM) medium, with an IC50 of about 48 microM. After pretreatment it also inhibited norepinephrine (NE, 3 microM)-induced phasic and tonic contraction in a concentration (35-140 microM)-dependent manner with an IC50 of 63 microM. At the plateau of NE-induced tonic contraction, addition of apigenin caused relaxation. This relaxing effect of apigenin was not antagonized by indomethacin (20 microM) or methylene blue (50 microM), and still existed in endothelial denuded rat aorta or in the presence of nifedipine (2-100 microM). Neither cAMP nor cGMP levels were changed by apigenin. Both the formation of inositol monophosphate caused by NE and the phasic contraction induced by caffeine in the Ca(2+)-free solution were unaffected by apigenin. 45Ca2+ influx caused by either NE or K+ was inhibited by apigenin concentration-dependently. It is concluded that apigenin relaxes rat thoracic aorta mainly by suppressing the Ca2+ influx through both voltage- and receptor-operated calcium channels.     

Cellular mechanisms involved in iso-osmotic high K+ solutions-induced contraction of the estrogen-primed rat myometrium

The aim of the present study was to investigate the mechanisms involved in the contraction evoked by iso-osmotic high K+ solutions in the estrogen-primed rat uterus. In Ca2+-containing solution, iso-osmotic addition of KCl (30, 60 or 90 mM K+) induced a rapid, phasic contraction followed by a prolonged sustained plateau (tonic component) of smaller amplitude. The KCl (60 mM)-induced contraction was unaffected by tetrodotoxin (3 microM), omega-conotoxin MVIIC (1 microM), GF 109203X (1 microM) or calphostin C (3 microM) but was markedly reduced by tissue treatment with neomycin (1 mM), mepacrine (10 microM) or U-73122 (10 microM). Nifedipine (0.01-0.1 microM) was significantly more effective as an inhibitor of the tonic component than of the phasic component. After 60 min incubation in Ca2+-free solution containing 3 mM EGTA, iso-osmotic KCl did not cause any increase in tension but potentiated contractions evoked by oxytocin (1 microM), sodium orthovanadate (160 micrM) or okadaic acid (20 microM) in these experimental conditions. In freshly dispersed myometrial cells maintained in Ca2+-containing solution and loaded with indo 1, iso-osmotic KCl (60 mM) caused a biphasic increase in the intracellular Ca2+ concentration ([Ca2+]i). In cells superfused for 60 min in Ca2+-free solution containing EGTA (1 mM), KCl did not increase [Ca2+]i. In Ca2+-containing solution, KCl (60 mM) produced a 76.0 +/- 16.2% increase in total [3H]inositol phosphates above basal levels and increased the intracellular levels of free arachidonic acid. These results suggest that, in the estrogen-primed rat uterus, iso-osmotic high K+ solutions, in addition to their well known effect on Ca2+ influx, activate other cellular processes leading to an increase in the Ca2+ sensitivity of the contractile machinery by a mechanism independent of extracellular Ca2+.     

Effect of hydrophobic compounds on rRNA synthesis in intact cells and isolated nuclei

Papaverine, cycloheximide, 2,4-dinitrophenol (DNP) and actinomycin D at low concentration have been shown to suppress selectively rRNA synthesis in Ehrlich ascite carcinoma cells. rRNA synthesis in isolated nuclei is not sensible to wide range of concentration of papaverine (0,005-0,1 mM), cycloheximide (0,5-100 micrograms/ml) and DNP (5-500 microM). Actinomycin D at low concentration does not act on the rRNA synthesis in vitro either. To suppress rRNA synthesis in this system much higher concentration of this agent (10 micrograms/ml) producing inhibition of all classes of rRNA synthesis in intact cells is required. Selective sensitivity of rRNA synthesis in the cells to papaverine, cycloheximide, DNP and low concentration of actinomycin D does not connect with their direct action on the apparatus of rRNA synthesis.     

Progestin facilitation of lordosis in rodents involves adenylyl cyclase activity in the ventral tegmental area

Increasing cAMP, or activating dopamine type 1 (D(1)) or GABA(A)/benzodiazepine receptor complexes (GBRs), in the ventral tegmental area (VTA) enhances lordosis of rodents. Whether D(1)- and/or GBR-mediated increases in progestin-facilitated lordosis involve the cAMP-synthesizing enzyme, adenylyl cyclase, in the VTA, was investigated. In Experiment 1, ovariectomized estradiol (E(2); 10 microg at h 0)+progesterone (P; 250 microg at h 45)-primed hamsters first received bilateral infusions of the adenylyl cyclase inhibitor, 2',5'-dideoxyadenosine (DDA; 12 microM/side), or vehicle, and then were infused with the D(1) agonist, SKF38393 (100 ng/side), the GBR agonist, muscimol (100 ng/side), or vehicle, to the VTA. Lordosis was evaluated before and 30 min after each infusion. In Experiment 2, ovariectomized, E(2)-primed (10 microg at h 0) rats received VTA infusions of DDA (12 microM/side) or vehicle; SKF38393 (100 ng/side), muscimol (100 ng/side), or vehicle; and the neurosteroid, 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP; 100 or 200 ng/side), or beta-cyclodextrin vehicle. Lordosis was assessed before the series of infusions, immediately after drug infusions and 10 or 60 min after 3alpha,5alpha-THP infusions. Progestin- or progestin plus SKF38393-or muscimol-mediated increases in lordosis were blocked by DDA pretreatment. Thus, in the VTA, progestins' membrane action may involve adenylyl cyclase.     

D,L-alpha-difluoromethylornithine inhibits human cytomegalovirus replication.

D,L-alpha-Difluoromethylornithine (DFMO) is an inhibitor of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway. Exposure of human foreskin fibroblast cells to DFMO before their infection with human strains of cytomegalovirus (CMV) resulted in a reduction in the amount of infectious virus produced. A 3-day exposure to the drug was required to elicit maximal antiviral effect. Cells exposed to DFMO at the time of infection produced normal amounts of infectious virus. Preexposure to the drug for 1, 2, or 3 days before infection resulted in at least 10-, 100-, or 1,000-fold decreases, respectively, in the amount of infectious virus produced. This decrease paralleled the loss of intracellular spermidine and was partially spared by the addition of exogenous putrescine, spermidine, or spermine (10 microM). When added 3 days before infection, DFMO depressed production of herpes simplex virus and simian CMV, as well as wild-type and laboratory prototype strains of human CMV. Although some antiviral effect was observed at a drug concentration of 1 mM, 10 mM gave a stronger effect and was the amount routinely used. At 30 mM DFMO, growth of noninfected cells was slowed but not arrested. Studies to investigate the level at which DFMO interferes with CMV replication showed that DFMO-treated, infected cells (i) exhibit a typical CMV-specific cytopathic effect, (ii) synthesize both viral proteins and viral DNA, (iii) contain at least some capsid forms, and (iv) shed greatly reduced amounts of virus particles into the growth medium. Since CMV virions, like those of herpes simplex virus, contain the polyamines spermidine and spermine, and since DFMO essentially eliminates the pool of intracellular spermidine, the possibility is suggested that this drug may exert its antiviral effect by interfering with virus assembly, perhaps at the level of DNA packaging or capsid envelopment or both.     

Differential effects of protease inhibitors on 1,25-dihydroxyvitamin D3 receptors

We describe herein two different effects of protease inhibitors and substrates on receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) obtained from the intestinal mucosa of vitamin D-deficient chicks: inhibition of binding of 1,25(OH)2D3 to its receptor and stabilization of the receptor. Both L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), a chymotrypsin inhibitor, and N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), a trypsin inhibitor, block [3H]1,25(OH)2D3 binding to the receptor. Fifty per cent inhibition of binding occurs at 20 microM TPCK, and 100% inhibition at 100-200 microM; TLCK is about 25-fold less effective. At higher concentrations (10-100 mM), the chymotrypsin substrates N alpha-p-tosyl-L-arginine methyl ester and tryptophan methyl ester and the cathepsin B inhibitor leupeptin also inhibit [3H] 1,25(OH)2D3 binding to its receptor. Different inhibitors and substrates interact with the receptor differently: TPCK (20 microM) and N alpha-p-tosyl-L-arginine methyl ester (10 mM) are reversible, noncompetitive inhibitors, L-tryptophan methyl ester (20 mM) is a reversible competitive inhibitor, and phenylmethylsulfonyl fluoride (300 microM) shows no effect on [3H]1,25(OH)2D3 binding to its receptor. The most stable form of unoccupied 1,25(OH)2D3 receptors from chick intestinal mucosa was that obtained from a low salt chromatin preparation (t 1/2 = 6.0 h). The presence of KCl drastically decreased receptor stability (t 1/2 = 1.8 h); and the addition of 2.5 mM CaCl2 further reduced their stability. Phenylmethylsulfonyl fluoride and Trasylol inhibited the KCl-induced receptor instability, but did not prevent the additional instability in the presence of CaCl2. In summary, TPCK and TLCK exert direct effects on the 1,25(OH)2D3 receptor molecule, independent of their protease inhibitor function. These compounds may prove useful as covalent affinity labels for the receptor. On the other hand, phenylmethylsulfonyl fluoride and Trasylol stabilize 1,25(OH)2D3 receptors, probably via inhibition of KCl-activated nuclear protease(s). This receptor stabilization will be advantageous in receptor assays and/or purification procedures.     

EDRF-release and Ca+(+)-channel blockade by magnolol, an antiplatelet agent isolated from Chinese herb Magnolia officinalis, in rat thoracic aorta

Magnolol is an antiplatelet agent isolated from Chinese herb Magnolia officinalis. It inhibited norepinephrine (NE, 3 microM)-induced phasic and tonic contractions in rat thoracic aorta. At the plateau of the NE-induced tonic contraction, addition of magnolol caused two phases (fast and slow) of relaxation. These two relaxations were concentration-dependent (10-100 micrograms/ml), and were not inhibited by indomethacin (20 microM). The fast relaxation was completely antagonized by hemoglobin (10 microM) and methylene blue (50 microM), and disappeared in de-endothelialized aorta while the slow relaxation was not affected by the above treatments. Magnolol also inhibited high potassium (60 mM)-induced, calcium-dependent (0.03 to 3 mM) contraction of rat aorta in a concentration-dependent manner. 45Ca(+)+ influx induced by high potassium or NE was markedly inhibited by magnolol. Cyclic GMP, but not PGI2, was increased by magnolol in intact, but not in de-endothelialized aorta. It is concluded that magnolol relaxed vascular smooth muscle by releasing endothelium-derived relaxing factor (EDRF) and by inhibiting calcium influx through voltage-gated calcium channels.     

Glucose-stimulated insulin secretion is not dependent on activation of protein kinase A

The involvement of cyclic AMP-dependent protein kinase A (PKA) in the exocytotic release of insulin from rat pancreatic islets was investigated using the Rp isomer of adenosine 3',5'-cyclic phosphorothioate (Rp-cAMPS). Preincubation of electrically permeabilised islets with Rp-cAMPS (1 mM, 1 h, 4 degrees C) inhibited cAMP-induced phosphorylation of islet proteins of apparent molecular weights in the range 20-90 kDa, but did not affect basal (50 nM Ca2+) nor Ca2(+)-stimulated (10 microM) protein phosphorylation. Similarly, Rp-cAMPS (500 microM) inhibited both cAMP- (100 microM) and 8BrcAMP-induced (100 microM) insulin secretion from electrically permeabilised islets without affecting Ca2(+)-stimulated (10 microM) insulin release. In intact islets, Rp-cAMPS (500 microM) inhibited forskolin (1 microM, 10 microM) potentiation of insulin secretion, but did not significantly impair the insulin secretory response to a range of glucose concentrations (2-20 mM). These results suggest that cAMP-induced activation of PKA is not essential for either basal or glucose-stimulated insulin secretion from rat islets.     

Forskolin induction of S-100 protein in glioma and hybrid cells

The S-100 protein level in mouse neuroblastoma (N18TG-2 and NIE-115), rat glioma (C6, C6BU-1, and C6V-1), and hybrid (NG108-15, 140-3, 141-B, NBr10A, NBr20A, NCB20, and NX3IT) cells was determined with a sensitive enzyme immunoassay system that uses a rabbit antibody to bovine brain S-100 protein. S-100 protein was detected in glioma but not in neuroblastoma cells. All seven hybrid cells derived from neuroblastoma and glioma or other types of cells were found to possess a very little or undetectable S-100 protein. The induction of S-100 protein level in prestationary phase cultures of glioma C6BU-1 cells was examined by forskolin, which was a highly specific activator of adenylate cyclase of the cells and produced morphological differentiation. After incubation with 10 microM forskolin for 48 hr, the S-100 protein level increased 2-2.5-fold in C6BU-1 glioma cells whose mean control level was 60 +/- 26 ng/mg protein (+/- SD). The forskolin induction of S-100 protein in the cells was dose dependent, and the concentration of forskolin required for 50% activation of S-100 protein was about 0.6 microM. The increase by forskolin was initiated from 10-15 hr after incubation with it and was inhibited with cycloheximide and actinomycin D. In NG108-15 hybrid cells the induction of S-100 protein was also observed by forskolin as well as prostaglandin (PG) E1 plus theophylline which are known to activate adenylate cyclase of the cells. The results indicate that S-100 protein biosynthesis is genetically controlled in these clonal cells, and that S-100 protein can be regulated in a cAMP-dependent fashion in prestationary cultures.     

Androgen-sensitive spermine-binding protein of rat ventral prostate. Purification of the protein and characterization of the hormonal effect.

The rat ventral prostate contains a cytosol protein that can non-covalently bind spermine much more tightly than spermidine or other natural diamines. The protein has been purified to homogeneity, as judged by electrophoresis in urea- and sodium dodecyl sulphate-containing polyacrylamide gels. The protein, with or without spermine bound to it, sediments at 3 S in a sucrose gradient with or without 0.4 M-KCl. The molecular weight of the protein is about 30 000. Each molecule of the binding protein can bind one molecule of spermine. In the prostate of rats injected with cycloheximide, the protein appears to have a half-life of about 3.5 h. The spermine-binding activity of an acidic fraction obtained by DEAE-cellulose chromatography of the prostate cytosol proteins is reduced by about 40--60% within 20--40 h after castration. This effect is reversed very rapidly within 15--30 min by intraperitoneal injection of 5 alpha-dihydrotestosterone. The hormonal effect is androgen-specific and is not mimicked by dexamethasone or oestradiol-17 beta. The androgen effect was reduced significantly when rats were injected with cycloheximide or actinomycin D, suggesting that the acidic protein may be one of the earliest proteins induced by androgen in the rat ventral prostate.     

Acute relaxant effects of 17-beta-estradiol through non-genomic mechanisms in rabbit carotid artery

Estrogens could play a cardiovascular protective role not only by means of systemic effects but also by means of direct effects on vascular structure and function. We have studied the acute effects and mechanisms of action of 17-beta-estradiol on vascular tone of rabbit isolated carotid artery. 17-Beta-estradiol (10, 30, and 100 microM) elicited concentration-dependent relaxation of 50 mM KCl-induced active tone in male and female rabbit carotid artery. The stereoisomer 17-alpha-estradiol showed lesser relaxant effects in male rabbits. Endothelium removal did not modify relaxation induced by 17-beta-estradiol. The NO synthase inhibitor L-NAME (100 microM) only reduced significantly relaxation produced by 30 microM 17-beta-estradiol. Relaxation was not modified by the estrogen receptor antagonist ICI 182,780 (1 microM), the protein synthesis inhibitor cycloheximide (1 microM), and the selective K(+) channel blockers charybdotoxin (0.1 microM) and glibenclamide (1 microM). CaCl(2) (30 microM -10 mM) induced concentration-dependent contraction in rabbit carotid artery depolarized by 50 mM KCl in Ca(2+) free medium. Preincubation with 17-beta-estradiol (3, 10, 30, or 100 microM) or the L-type Ca(2+) channel blocker nicardipine (0.01, 0.1, 1, or 10 nM) produced concentration-dependent inhibition of CaCl(2)-induced contraction. In conclusion, 17-beta-estradiol induces endothelium-independent relaxation of rabbit carotid artery, which is not mediated by classic estrogen receptor and protein synthesis activation. The relaxant effect is due to inhibition of extracellular Ca(2+) influx to vascular smooth muscle, but activation of K(+) efflux is not involved. Relatively high pharmacological concentrations of estrogen causing relaxation preclude acute vasoactive effects of plasma levels in the carotid circulation.     

Polyamines and Adventitious Root Formation in the Juvenile and Mature Phase of English Ivy

The involvement of polyamines during adventitious root formationwas evaluated using a de-bladed petiole rooting assay for theeasy-to-root juvenile and difficult-to-root mature phase ofEnglish ivy (Hedera helix L.). Auxin (NAA 0.1 mM) stimulatedroot formation in juvenile phase cuttings, but failed to promoterooting in the mature phase. The addition of putrescine, spermineor spennidine (1.0 mM) with or without NAA (0.1 mM) did notaffect the rooting response in either the juvenile or maturephase cuttings. There was a significant increase in endogenouslevels of putrescine and spermidine in NAA-treated cuttings,but the only significant difference between the root formingjuvenile and the non-root forming mature phase cuttings wasan increase in putrescine levels. In NAA-treated juvenile cuttings,the polyamine biosynthesis inhibitor DFMA (1.0 mM) promotedroot formation from 9.2 to 14.5 roots per cutting, while DFMO(1.0 mM) reduced root formation from 9.1 to 1.4 roots per cutting.The promotion of rooting by DFMA was completely reversed byputrescine (1.0 mM), but putrescine, spermine or spermidine(1.0 mM) could not reverse the inhibitory effect of DFMO. NeitherDFMA nor DFMO promoted root formation in mature phase cuttings.DFMA was also added to NAA-treated juvenile petioles at variousstages during the root formation process. DFMA promoted rootingwhen applied during the early stages of root induction (0–3d), but became inhibitory to root formation when applied duringthe organization (6–9 d) or root elongation stages (9–12d). Key words: Hedera helix, organogenesis, root initiation, polyamines, DFMA, DFMO     

Plasmodium berghei: effect of protease inhibitors during gametogenesis and early zygote development

Plasmodium berghei: The effect of five protease inhibitors, TPCK, TLCK, PMSF, leupeptin, and 1,10-phenanthroline on in vitro gametogenesis and early zygote development of P. berghei was investigated. PMSF and leupeptin showed no effect. Cysteine/serine protease inhibitors TPCK/TLCK at concentrations of 75 and 100 microM were effective on inhibiting exflagellation center formation, and this effect was reversible with the addition of l-cysteine. Exflagellation center formation was most effectively blocked by 1,10-phenanthroline (1mM), and exflagellation center numbers were restored by the addition of Zn(2+). A reduction of ookinete production was observed when TPCK/TLCK (100 microM) was added at 2h after gametogenesis, but no effect was observed with 1,10-phenanthroline (1mM). Our results suggest that proteolysis is important in both gametocyte activation and sexual development of P. berghei.     

Excitatory Amino Acid-Induced Phosphoinositide Turnover in Guinea Pig Cerebral Cortical Slices: Selective Enhancement by Spermine of the Response to DL-1-Aminocyclopentane- trans-1, 3-Dicarboxylate

In the presence of 1 mM spermine, accumulations of 3H labelled inositol phosphates elicited by quisqualate (100 microM) and 1-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD, 300 microM) were significantly enhanced by 21 and 26%, respectively, without a significant alteration in the accumulation elicited by L-glutamate (10 mM) or DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionate (10 microM). Analysis of concentration-response data indicated that the presence of spermine led to an increase in the maximal response to t-ACPD without altering the EC50 value. The stimulatory effect of spermine on the accumulation of t-ACPD-elicited 3H-inositol phosphates was not reversed by ifenprodil or diethylenetriamine (putative polyamine site antagonists), by agents that activate or inhibit protein kinase C, or by calcium channel blockade, but was abolished in the presence of elevated extracellular calcium ion concentration. We conclude that spermine enhances the phosphoinositide turnover in guinea pig cerebral cortical slices elicited by the "metabotropic" excitatory amino acid receptor. The site through which the action of spermine is mediated remains to be defined, but it is apparently distinct from that suggested to modulate N-methyl-D-aspartate receptor activity.