Melatonin protects against Fenoxaprop-ethyl exposure-induced meiotic defects in mouse oocytes.

Prolonged exposure of Fenoxaprop-ethyl (FE), a post-emergence herbicide, can cause serious damage to animals through food chain. Melatonin is synthesized by the pineal gland in mammals and believed to protect cells from oxidative stress damage. In this study, we aimed to investigate the effects of FE on mouse oocyte meiosis maturation and the protective roles of melatonin on FE-exposed oocytes by in vitro maturation model. FE exposure significantly caused defects of the first polar body extrusion, which could be protected by co-culture with melatonin. Furthermore, we examined the meiotic maturation details by performing the sperm binding, actin and tubulin immunofluorescence, ROS and apoptosis detection, and histone methylation assay. Our data showed that FE exposure to oocytes led to disrupted actin filament dynamics, mis-organized spindle, and reduced the sperm binding capacity. In addition, FE-exposure increased oxidative stress level and induced oocyte apoptosis. We also found that FE exposure resulted in histone methylation changes. Treatment with melatonin could significantly improve these phenotypes in oocytes exposed to FE. In conclusion, FE exposure can cause meiotic defects by disrupting the cytoskeletal integrality and inducing excessive ROS accumulation to initiate apoptosis in oocytes, while melatonin can reduce all these damages, suggesting that melatonin has protective effects on oocytes exposed to FE during meiotic maturation.

Distinct segment-specific functions of calyculin A-sensitive protein phosphatases in the regulation of cAMP-triggered events in ejaculated bull spermatozoa.

Livestock spermatozoa possess more tenacious suppressors of cAMP-triggered events-including capacitation-associated changes-than laboratory animal spermatozoa, leading to flagellar hyperactivation. In order to identify the suppressors, we examined effects of an inhibitor of serine/threonine protein phosphatases (calyculin A) on cAMP-triggered changes in the protein phosphorylation state, and subsequent occurrence of hyperactivation and acrosome reaction in ejaculated bull spermatozoa. Ejaculated spermatozoa were incubated in cAMP-supplemented medium, then assessed for motility, acrosome morphology, and phosphorylated protein localization. The addition of calyculin A greatly enhanced cAMP-triggered protein phosphorylation at serine/threonine and tyrosine residues in the connecting piece and induction of flagellar hyperactivation. Most hyperactivated spermatozoa exhibited extremely asymmetrical bends at the middle piece, which produced intensive twisting or figure-eight movements. In the sperm head, however, cAMP-triggered dephosphorylation of serine/threonine-phosphorylated proteins and subsequent acrosome reaction were abolished by the addition of calyculin A. Based on these results, we suggest that calyculin A-sensitive protein phosphatases in the connecting piece are suppressors of cAMP-triggered events leading to hyperactivation. By contrast, similar protein phosphatases in the sperm head accelerate cAMP-triggered events leading to the acrosome reaction. These findings are consistent with the indication that calyculin A-sensitive protein phosphatases have distinct functions in the regulation of cAMP-triggered events in different regions of ejaculated bull spermatozoa.

Transient inhibition of Calyculin A induced premature chromosome condensation by hyperthermia.

The analysis of chromosomal aberrations by premature chromosome condensation (PCC) induced by Calyculin A (Cal) is feasible in tumor biopsies from patients and has the potential to predict sensitivity to radiotherapy. As hyperthermia (HT) improves radiotherapy outcome in certain tumor sites, it was investigated whether PCC induction is still possible after temperatures reached in the clinic. Human cervical carcinoma (CaSki) and lung carcinoma (SW-1573) cells were incubated with Cal to induce PCC immediately after 1 h treatment at temperatures ranging from 41 degrees C to 43 degrees C and after recovery for up to 24 h after treatment with 43 degrees C. Levels of phosphorylated Cdc2 (at the Tyr15 residue), histone H3 (at the Ser10 residue) and Cyclin B1 were investigated by immunoblotting. The amount of cells positive for phosphorylated histone H3 was determined by flow cytometry. Temperatures > or =42.5 degrees C inhibited the induction of PCC by Cal, while recovery of PCC-induction was observed at >20 h after treatment in both cell lines. The phosphorylation status of Cdc2 as well as of histone H3 in cells treated with Cal directly after HT at 43 degrees C was similar to that of cells treated with Cal alone or treated with Cal 24 h after HT at 43 degrees C. HT alone did not affect the levels of phosphorylated Cdc2, while phosphorylation levels of histone H3 were increased as compared with control status of these two proteins. Phosphorylated and total Cyclin B1 levels were not influenced by any of the treatments. Flow cytometric analysis confirmed that HT at 43 degrees C did not interfere with phosphorylation of histone H3. Our data indicate that HT transiently inhibits PCC induction by Cal in a temperature-dependent manner. Therefore, an interval of at least 24 h after HT should be applied before taking tumor biopsies for karyogram analysis of patients treated with temperatures above 42.5 degrees C.

Selective inhibition of cyclooxygenase-2 inhibits colon cancer cell adhesion to extracellular matrix by decreased expression of beta1 integrin.

High level expression of cyclooxygenase (COX)-2 is reported in 80-90% of colorectal adenocarcinomas. In the recent years, selective inhibitors of COX-2 have been developed, and are shown to effectively protect against cancer development and progression. Colon cancer cells, as well as the epithelial cells in general, are dependent on appropriate interactions with the extracellular matrix (ECM) proteins to achieve a number of important functions, such as proliferation, differentiation, invasion and survival. These interactions are mediated via a family of cell-surface receptors called integrins, which interact with cytoskeletal proteins on the cytoplasmic side of the plasma membrane and thereby provide a link between the ECM and the cytoskeleton. In the present study, a high-COX-2 (high level COX-2 expression) colon cancer cell line, HT-29, and a low-COX-2 (low level COX-2 expression), DLD-1, were used to investigate the anticolon cancer effect of the selective COX-2 inhibitor, JTE-522. Moreover, to clarify its mechanisms of action, we focused especially on the ability to adhere to and to migrate on ECM. We could clearly demonstrate that, in addition to the decrease of the proliferative activity, JTE-522 caused a dose-dependent decrease in both the ability of colon cancer cells to adhere to and to migrate on ECM. These effects were, at least in part, dependent on the down-regulation of beta1-integrin expression, which was evident in HT-29, the high-COX-2 colon cancer cells, but not the low-COX-2, DLD-1. In addition, prostaglandin E2 almost completely reversed the effect of JTE-522, strongly suggesting the involvement of a COX-2-dependent pathway. In conclusion, for the first time, we could demonstrate the down-regulation of beta1 integrin caused by COX-2 inhibition, with consequent impairment of the ability of cancer cells to adhere to and to migrate on ECM, which are crucial steps for cancer metastases to develop.

Interactive effects of vinclozolin and testosterone propionate on pregnancy and sexual differentiation of the male and female SD rat.

In mammals, androgens are essential in directing mammalian sexual differentiation of the male phenotype. Administration of testosterone during this period alters female development in a male-like direction, whereas exposure to an androgen receptor antagonist like vinclozolin (V) demasculinizes and feminizes the male offspring. In the current study, we administered V (gavage at 200 mg/kg/day) and/or testosterone propionate (TP, sc, at 1 mg/rat/day), alone and in combination to Sprague-Dawley (SD) rats on days 14 through 19 of pregnancy, to determine if V would antagonize the effects of TP in the female and, conversely, if TP would antagonize the effects of V in the male offspring. These doses of TP and V were selected because they significantly alter sexual differentiation in the majority of female and male rat offspring, respectively, without producing severe toxicity in the dam or offspring. The study design is a 2 x 2 factorial (7 dams per group) including vehicle control, V, TP, and V + TP groups. As expected, individually, both V and TP reduced maternal weight gain and the V + TP group was affected in a cumulative fashion. Litter size on postnatal day (PND) 2 was reduced only by V + TP, whereas pup body weight was reduced in all three treated groups, the effect of V + TP again being cumulative. In female offspring, TP-induced alterations (i.e., increased anogenital distance [AGD] and fewer nipples, vaginal agenesis, hydrometrocolpos, induced prostate and bulbourethral glands, and levator ani muscle tissues) were all reversed by coadministration of V. In male offspring, V-induced alterations were only modestly antagonized by TP. At the dosage levels used herein, V + TP-treated male offspring had less well-developed nipples as infants and adults and a lower incidence of ectopic testis than did the V group. However, V-induced changes in reproductive organ weights, AGD, atrophic testes, vaginal pouch, and agenesis of the sex accessory tissues were not antagonized by concurrent TP treatment in male offspring. We observed that the combination of V and TP, two chemicals with opposing endocrine action, antagonized one another during sexual differentiation, especially in the female offspring and induced cumulative effects on maternal and neonatal toxicity. We suspect that antagonism of V by TP would be enhanced in the male if lower dose levels of V were used, but then the antagonism of TP by V in the female would likely be attenuated.

Effect of ethanol on reductions in norepinephrine electrochemical signal in the rostral ventrolateral medulla and hypotension elicited by I1-receptor activation in spontaneously hypertensive rats.

BACKGROUND: The mechanism of the antagonistic hemodynamic interaction between ethanol and centrally acting sympatholytics is not known. In this study, we tested the hypothesis that the imidazoline (I1)-receptor modulation of norepinephrine (NE) release within the rostral ventrolateral medulla (RVLM) plays a pivotal role in this clinically relevant hemodynamic interaction. METHOD In anesthetized spontaneously hypertensive rats, the effects of centrally acting sympatholytics on RVLM NE electrochemical signal were investigated by in vivo electrochemistry along with cardiovascular responses in the absence and presence of ethanol. In vivo microdialysis in conscious spontaneously hypertensive rats was used to confirm the electrochemical findings. RESULTS Clonidine (30 microg/kg, intravenously) or rilmenidine (400, 600, or 800 microg/kg) significantly reduced RVLM NE electrochemical signal (index of neuronal activity) and mean arterial pressure; rilmenidine effects were dose-related, and ethanol (1 g/kg) counteracted these responses. Ethanol (1 g/kg) pretreatment increased both RVLM NE electrochemical signal and blood pressure but did not influence the reductions in both variables elicited by subsequently administered clonidine. The alpha2-adrenergic antagonist 2-methoxyidazoxan (30 microg/kg) counteracted rilmenidine (800 microg/kg)-evoked responses. In vivo microdialysis in conscious spontaneously hypertensive rats confirmed the electrochemical findings since clonidine- (30 microg/kg, intravenously) evoked reductions in RVLM NE and the associated hypotension were counteracted by ethanol (1 g/kg). CONCLUSIONS (1) Ethanol counteracts centrally mediated hypotension, at least in part, by increasing RVLM NE; (2) the interaction involves the I1 receptor modulation of RVLM neuronal activity; (3) the alpha2-adrenergic receptor contributes to the electrochemical and cardiovascular effects of high doses of rilmenidine, and (4) the RVLM is a neuroanatomical target for systemically administered ethanol.

Possible involvement of optimally phosphorylated L-plastin in activation of superoxide-generating NADPH oxidase.

The involvement of protein phosphatases in the activation of superoxide (O2-)- generating enzyme in human neutrophils was examined using calyculin A, an inhibitor of protein phosphatase type 1 and 2A. Calyculin A inhibited the phorbol myristate acetate (PMA)- and opsonized zymosan (OZ)-activated O2- generation by human neutrophils. This inhibitory effect of calyculin A on PMA-activated O2- generation was reversed by the addition of KT5926, a specific inhibitor of myosin light chain kinase and Ca2+/calmodulin-dependent protein kinase II. These results suggest that the addition of calyculin A may cause hyperphosphorylation of some protein(s) that plays a crucial role in the PMA-dependent activation of O2- generating enzyme, and that this protein hyperphosphorylation may be evoked by a KT5926-sensitive kinase or its downstream kinase. Whereas two-dimensional analysis involving 32P revealed that calyculin A caused the hyperphosphorylation of many proteins, KT5926 mainly reduced the calyculin A-induced hyperphosphorylation of a 67 kDa protein in activated neutrophils, suggesting that the hyperphosphorylation of the 67 kDa protein might inhibit the PMA-dependent activation of NADPH oxidase. The 67 kDa cytosolic protein was moderately phosphorylated on the addition of PMA. On the other hand, in the absence of calyculin A, KT5926 inhibited both PMA-induced O2- generation and phosphorylation of the 67 kDa protein. Amino acid sequence analysis of peptides derived from the 67 kDa protein revealed that the 67 kDa protein was identical to L-plastin, an actin-bundling protein. We conclude that optimally phosphorylated L-plastin may play some crucial role in the activation of NADPH oxidase.

Three-dimensional common-feature hypotheses of inhibitors of calling behaviour and in vitro [14C]acetate incorporation by pheromone glands of Plodia interpunctella.

Some octopamine (OA) agonists were found to suppress the calling behaviour and pheromone biosynthesis in vitro of the Indian meal moth, Plodia interpunctella (Hübner), a stored-product pest. Compounds were screened using a calling behaviour bioassay of female P interpunctella. Three active derivatives, with activity at the nanomolar level, were identified. In order of decreasing pheromonostatic activity these were: 2-(2-ethyl-6-methylanilino)oxazolidine > 2-(2,6-diethylanilino)thiazolidine > 2-(2,6-diethylanilino)oxazolidine. These compounds showed also in vitro inhibitory activities in de novo pheromone biosynthesis. Three-dimensional pharmacophore hypotheses were built from a set of 19 compounds. Among the ten common-featured models generated by the program Catalyst/HipHop, a hypothesis including a ring aromatic group (RA), a positive ionizable group (PI) and two hydrophobic aliphatic (HpA1) features was considered to be essential for inhibitory activity in the calling behaviour and pheromone biosynthesis in vitro. Active compounds mapped well onto all the RA, PI and HpA1 features of the hypothesis. Less-active compounds were shown not to achieve the energetically favourable conformation which was found in the active molecules in order to fit the 3-D common-feature pharmacophore models. The present studies demonstrate that inhibition of calling behaviour and PBAN-stimulated incorporation of radioactivity is by OA-agonistic activity.

Involvement of imidazoline receptors in the baroreflex effects of rilmenidine in conscious rabbits.

OBJECTIVE: It has been suggested that imidazoline receptors rather than alpha2-adrenoceptors are involved in the sympathoinhibitory action of centrally acting antihypertensive drugs such as rilmenidine. In the present study, we examined the relative importance of alpha2-adrenoceptors and imidazoline receptors in modulating the renal sympathetic and heart rate (HR) baroreflex in response to central administration of rilmenidine in conscious normotensive rabbits. METHODS: In seven conscious rabbits, chronically instrumented with a fourth ventricular (4V) catheter, aortic and vena caval cuff occluders and a renal nerve electrode, we continuously recorded renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and HR and assessed baroreflex MAP-RSNA and MAP-HR relationships with balloon-induced ramp rises and falls in MAP. Rabbits were treated with 4V rilmenidine (22 microg/kg) followed by 4V idazoxan (30 microg/kg; a mixed alpha2-adrenoceptor and imidazoline receptor antagonist) or 4V 2-methoxy-idazoxan (1 microg/kg; an alpha2-adrenoceptor antagonist with little affinity for imidazoline receptors). RESULTS: Rilmenidine lowered blood pressure by 24% and reduced both upper and lower plateaus of the renal sympathetic baroreflex curve, such that the RSNA range (difference between plateaus) was reduced by 40% (-32 +/- 10 normalized units). Curves were shifted to the left with the fall in MAP. Idazoxan restored MAP, maximum RSNA and the RSNA baroreflex range. By contrast the alpha2-adrenoceptor antagonist 2-methoxy-idazoxan caused only a partial recovery of MAP and RSNA baroreflex upper plateau and range (-9 +/- 2 mmHg, 29 and 33% lower than control). Both antagonists partially restored the HR baroreflex. CONCLUSION: These findings suggest that in conscious rabbits, both imidazoline receptors and alpha2-adrenoceptors are involved in the central antihypertensive and baroreflex actions of rilmenidine, but that activation of imidazoline receptors is more important for its renal sympathoinhibitory action.

Preventive effect of rilmenidine on the occurrence of neurogenic ventricular arrhythmias in rabbits.

BACKGROUND: Centrally acting antihypertensive drugs bearing an imidazoline or a related chemical structure inhibit sympathetic nervous output to the heart and vascular beds, and enhance parasympathetic tone. Cardiac ischaemia and ventricular arrhythmias that can result from hypertension are likely to benefit from such effects. OBJECTIVE: To investigate the effects of rilmenidine, an oxazoline with antihypertensive properties, in a model of neurogenically induced ischaemic ventricular arrhythmias. METHODS AND RESULTS: Bicuculline, a alpha-aminobutyric acid (GABA(A)) receptor antagonist, was administered intracisternally in pentobarbitone anaesthetized rabbits; 10 microg/kg intracisternal bicuculline induced polymorphic ventricular ectopic beats and ventricular tachycardia, while blood pressure increased by about 50-60% and heart rate in sinus rhythm decreased by about 20%. Rilmenidine pretreatment (10 min), either administered intravenously (0.01, 0.1, 1 mg/kg) or intracisternally (3, 10, 30 microg/kg), dose-dependently prevented the occurrence of bicuculline-induced arrhythmias and, because of a lower baseline, the blood pressure values reached were less when compared with controls. Intracisternal idazoxan (15 microg/kg) had no significant antiarrhythmic effect but antagonized, in part, the haemodynamic and antiarrhythmic effects of rilmenidine (1 mg/kg intravenously; 30 microg/kg intracisternally). CONCLUSION: The antiarrhythmic effects observed with rilmenidine are mainly mediated by blunting the bicuculline-induced increase in the sympathetic nervous output to the heart and the vascular beds. These effects of rilmenidine are likely to originate from action on the central as well as on the peripheral nervous systems. Direct coronary or cardiac effects might also play a role, in particular at low non-hypotensive intravenous doses.

Pretreatment with quinpirole inhibits the central antihypertensive effects of rilmenidine and alpha-methyldopa in conscious rats.

Treatment of conscious spontaneously hypertensive rats (SHR) with the dopamine D2 receptor agonist quinpirole causes a short-lasting pressor response and apparent desensitisation to the effects of subsequent injections of quinpirole or central antihypertensives such as clonidine. In the present study, a number of aspects of this apparent desensitisation were investigated. Thirty minutes after intravenous injection of quinpirole into spontaneously hypertensive rats, treatment with the dopamine D2 receptors antagonist raclopride caused a significant fall in blood pressure. At this time point, circulating levels of vasopressin were not significantly different compared to controls. In Brattleboro rats, the pressor response to quinpirole was reduced in the first 15 min after injection, but not difference in blood pressure was observed at later time points. In SHR which had been treated with quinpirole, the central antihypertensive effects of rilmenidine or alpha-methyldopa were significantly inhibited. By contrast, the bradycardia induced by these drugs was similar in quinpirole-treated rats and controls. Quinpirole pretreatment caused an enhancement of the hypotension but a reduction of the reflex tachycardia after intravenous treatment with hydralazine. In SHR treated with methylatropine and quinpirole, the upper plateau of the sympathetic baroreceptor-heart rate reflex curve was reduced. These results show that treatment with quinpirole has marked effects on central sympathetic vasomotor mechanisms which are the target of antihypertensive drugs such as rilmenidine and alpha-methyldopa. At least some of these effects may occur at the level of the sympathetic baroreflex. Moreover, while the effects of quinpirole on sympathetic regulation are prolonged, the initial pressor response is counteracted by an as yet unidentified compensatory mechanism which can be unmasked when quinpirole is displaced from its receptor by dopamine D2 receptor antagonist treatment.

Antinociceptive effects of oral clonidine and S12813-4 in acute colon inflammation in rats.

Acute colonic inflammation was induced by perendoscopic injection of 50 microleters of dilute formalin (5%) in the depth of the colonic wall (c.w.) in rats. Compared to saline injection, the procedure was followed by nociceptive behaviors from which visceral nociception was quantified. The alpha 2-adrenoceptor agonist, clonidine 2-[2,6-dichlorophenylamine]-2-imidazole hydrochloride (75, 150 and 300 mg/kg), administered orally 15 min after c.w. injection of formalin significantly reduced the nociceptive responses at the high dose only. However, when administered 30 min prior to nociceptive stimulation, the compound exhibited an antinociceptive effect at the three doses. A novel analgesic, the compound "S12813-4' 3-[2-(4-phenylpiperazine-1-yl)-ethyl]-2-oxo-2,3-dihydro-oxazolo[b] pyridine, chlorydrate (10, 30 and 90 mg/kg), given orally displayed antinociceptive effects whatever the administration schedule, before or after c.w. injection of formalin. The antinociceptive effect of S12813-4 (30 mg/kg given orally) was prevented by subcutaneous (s.c.) injection of yohimbine or idazoxan (1 mg/kg). We conclude that visceral nociception elicited by formalin-induced colonic inflammation is attenuated by clonidine and S12813-4. The pharmacological profiles of the two compounds and the inhibition of the antinociceptive effect of S12813-4 by yohimbine and idazoxan suggest that noradrenergic mechanisms are involved in the transmission and/or modulation of the nociceptive influx arising from the inflamed colon.

The protein phosphatase inhibitor calyculin-A affects catecholamine secretion and granular distribution in cultured adrenomedullary chromaffin cells.

Calyculin-A, a potent inhibitor of types 1 and 2A protein phosphatases, increases basal catecholamine secretion in cultured chromaffin cells with a maximum effect observed at 100 nM. This effect was increased by forskolin and the calmodulin antagonist W7, but was modified neither by phorbol esters nor the protein kinase inhibitor, H7. The effect of the toxin, calyculin-A, on basal secretion was completely prevented by the protein kinase inhibitor K252a. In digitonin-permeabilized cells calyculin-A induced an increase in basal release, but, in contrast, it partially reduced calcium-induced secretion. Analysis of total proteins revealed that calyculin-A treatment of the cells increased the level of phosphorylation of different protein bands. Examination of the Triton X-100-insoluble fraction revealed a clear increase in the phosphorylation level of various proteins, including vimentin. Calyculin-A provoked a rapid morphological change in chromaffin cells in the same range of concentration (50-300 nM). Cells became rounder and were partially detached from the substratum forming clusters, this effect was also blocked by K252a. Transmission electron microscopy of calyculin-A-treated cells showed an increase in the proportion of chromaffin granules located closer to the membrane. These results suggest that calyculin-A induces changes both in the catecholamine secretory response and in the cytoskeletal elements of chromaffin cells by protein phosphorylation.

Comparison of the action of the stereoisomers of the psychostimulant 4-methylaminorex (4-MAX) on midbrain dopamine cells in the rat: an extracellular single unit study.

In this study, we examined and characterized the action of the stereoisomers of 2-amino-4-methyl-delta 2-5-phenyl-oxazoline (4-methylaminorex, 4-MAX) on spontaneously active dopamine (DA) neurons in the substantia nigra pars compacta (SNC or A9) and ventral tegmental area (VTA or A10) in anesthetized male rats. This was accomplished using the technique of extracellular single unit recording. The intravenous (i.v.) administration of the stereoisomers of 4-MAX (0.1-6.4 mg/kg) produced a dose-dependent suppression of the basal firing rate of A10 DA cells with the following rank order of potency: trans 4S,5S > cis 4R,5S approximately cis 4S,5R >> trans 4S,5S 4-MAX. The rank order of potency of the isomers of 4-MAX to suppress the firing of A9 DA cells was trans 4S,5S = cis 4R,5S = cis 4S,5R >> trans 4R,5R. The trans 4S,5S isomer was 5-fold more potent in suppressing DA cell firing in the A10 compared to the A9 area. The suppressant action of the isomers on A9 and A10 DA cells was reversed by the i.v. administration of haloperidol and the D2/D3 receptor antagonists (-)-sulpiride and (-)-eticlopride but not by the D1 receptor antagonists SCH 23390 and SCH 39166. In addition, the suppressant action of the trans 4S,5S isomer on A10 DA cells was not antagonized or reversed by the i.v. administration of the receptor antagonists granisetron (5-HT3), ritanserin (5-HT2A,C), idazoxan (alpha 2), phentolamine (peripheral alpha 1), (+/-)-pindolol (5-HT1A,B beta) or prazosin (alpha 1). The pretreatment of animals with either alpha-methyl-p-tyrosine (AMPT) or reserpine, but not p-chlorophenylalanine (PCPA), (+/-)-fluoxetine or tomoxetine, significantly attenuated the suppression of A10 DA cell firing produced by trans 4S,5S 4-MAX. Overall, our results suggest that the suppressant action of 4-MAX on midbrain DA cell firing may be mediated by the release of DA, which subsequently interacts with D2/D3 receptors.

Contrasting effects of calyculin A and okadaic acid on the respiratory burst of human neutrophils.

The involvement of serine/threonine protein-phosphatases in the production of superoxide (respiratory burst) by human neutrophils was investigated using calyculin A, a potent inhibitor of both protein phosphatases type 1 and 2A, and okadaic acid, which preferentially inhibits protein phosphatase type 2A. Treatment of neutrophils with calyculin A (25-75 nM) or okadaic acid (1-4 microM) had no stimulatory effect but potently enhanced total superoxide production induced by an optimal fMLP (N-formyl-methionyl-leucyl-phenylalanine) concentration (0.1 microM). The maximum increase plateaued with 50-75 nM calyculin A and 2-4 microM okadaic acid, reaching approximately 120 and 200% of control values, respectively. Unlike calyculin A, okadaic acid also primed the initial rate of superoxide production, suggesting that protein phosphatases may down-regulate both initiation and termination of respiratory burst. Optimal stimulation of the respiratory burst by PMA (160 nM) was inhibited by calyculin A and okadaic acid, with an IC50 of 60 nM and 2 microM, respectively, although both drugs caused protein hyperphosphorylation. The inhibition was partially prevented by a nonstimulatory concentration of A23187, indicating a role of calcium in the inhibitory effects of the drugs. Unlike the optimal respiratory burst, suboptimal respiratory burst induced by PMA (1-7 nM) was enhanced by calyculin A and okadaic acid. Unprimed and primed respiratory bursts were depressed by a selective antagonist of protein kinase C (GF 109203X), indicating positive regulation of these responses by protein kinase C. Thus, the use of calyculin A and okadaic acid distinguishes two regulatory processes of superoxide production.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of imidazoline-preferring receptors in regulation of sympathetic tone.

We examined the contribution of imidazoline-preferring receptors (IPR) and alpha 2-adrenoceptors at different levels of the central nervous system in the antihypertensive and sympathoinhibitory actions of rilmenidine in 2 conscious animal models, the spontaneously hypertensive rat (SHR) and the normotensive rabbit. In conscious SHRs, we compared the potency of rilmenidine and clonidine administered intravenously into the lateral cerebral ventricle, the cisterna magna, and into the subarachnoidal space of the thoracolumbar spinal cord. In SHRs, we found that rilmenidine was more potent and more effective by the intrathecal than the intracisternal route. By contrast, clonidine was most effective after administration into the cisterna magna. Intravenous administration of rilmenidine or clonidine induced dose-dependent and prolonged decreases in blood pressure and heart rate. Neither rilmenidine nor clonidine altered mean arterial pressure or heart rate when given into the lateral cerebral ventricle. These data suggest that in SHRs the spinal cord may be an important site for the antihypertensive action of rilmenidine. We therefore characterized the receptor type involved. We observed in conscious SHRs that intrathecal post-treatment with idazoxan, a mixed alpha 2-adrenoceptor and IPR antagonist, abolished the antihypertensive effect of rilmenidine, whereas 2-methoxyidazoxan, a selective alpha 2-adrenoceptor antagonist, caused only a partial reversal of the blood pressure effects of rilmenidine. These results suggest that rilmenidine acts mainly through IPR rather than alpha 2-adrenoceptors in the spinal cord. In view of these findings, we compared the hypotensive actions of rilmenidine and clonidine, administered into the lateral cerebral ventricle, the cisterna magna, and the subarachnoid space of the thoracolumbar spinal cord in conscious normotensive rabbits. Both drugs were less potent and effective when administered intrathecally than intracisternally. These experiments suggest that the hypotensive action of rilmenidine and clonidine in the rabbit is mediated through receptors mainly located in the brainstem. Further, we found that idazoxan reversed the hypotensive action of rilmenidine more readily than 2-methoxyidazoxan. Surprisingly, both idazoxan and 2-methoxyidazoxan completely reversed the depressor effects of clonidine. Therefore, in the rabbit, rilmenidine acts through IPR located in the brainstem and clonidine acts predominantly through alpha 2-adrenoceptors. In conclusion, our studies demonstrate that IPR are involved in the vasodepressor action of rilmenidine in both conscious SHRs and rabbits. However, although the main site of action of rilmenidine in SHRs may be located in the thoracolumbar spinal cord, in the rabbit it appears to be in the brainstem.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein phosphatase inhibitors induce the release of serotonin from rat basophilic leukemia cells (RBL-2H3).

Effects of okadaic acid (OA) and calyculin-A (CL-A), selective inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A), on the release of serotonin from the rat basophilic leukemia cell line (RBL-2H3) were investigated. Both OA and CL-A induced the long-lasting release of serotonin in an extracellular Ca(2+)-independent manner. CL-A did not increase intracellular Ca2+ concentration in the fura-2-loaded cells. CL-A was 100-fold more potent than OA in inducing the release, suggesting that PP1 is a dominant protein phosphatase in regulating RBL-2H3 cells. The CL-A-induced release of serotonin was completely inhibited by the nonselective protein kinase inhibitors, staurosporine and K-252a. CL-A induced phosphorylation of several cellular proteins in RBL-2H3 cells, which could be inhibited by staurosporine. These findings suggest that the release of serotonin is subject to tonic, Ca(2+)-independent, inhibition by PP1 in RBL-2H3 cells.

Sympathoinhibition by rilmenidine in conscious rabbits: involvement of alpha 2-adrenoceptors.

Cardiovascular and sympathetic nervous system effects of the mixed alpha 2-adrenoceptor and imidazoline receptor agonist rilmenidine were studied in conscious rabbits chronically instrumented for the recording of the firing rate of renal sympathetic fibers. Separate experiments were carried out on pithed rabbits with electrically stimulated (2 Hz) sympathetic outflow. Drugs were administered intravenously in a cumulative manner. In conscious rabbits, rilmenidine 0.1, 0.3 and 1.0 mg kg-1 dose-dependently lowered blood pressure, renal sympathetic nerve activity, heart rate and the plasma concentration of noradrenaline and adrenaline. The effect on blood pressure and plasma catecholamines was maximal after 0.3 mg kg-1 whereas heart rate and renal sympathetic nerve activity decreased further after rilmenidine 1.0 mg kg-1. Yohimbine 0.1 and 0.5 mg kg-1, when injected subsequently, attenuated and at the higher dose abolished all effects of rilmenidine. The effects of rilmenidine were also antagonized by the alpha 2-adrenoceptor antagonist 2-(2,3-dihydro-2-methoxy-1,4-benzodioxin-2-yl)-4,5-dihydro-1H-imid azole HCl (RX821002; 0.1 and 0.5 mg kg-1). Yohimbine 0.1 and 0.5 mg kg-1 did not attenuate or attenuated only slightly the decrease of heart rate and renal sympathetic nerve activity produced by infusion of vasopressin. In pithed rabbits with electrically-stimulated sympathetic outflow, yohimbine 0.1 submaximally and yohimbine 0.5 mg kg-1 maximally increased the plasma noradrenaline concentration. The experiments show by direct measurement of sympathetic nerve firing and plasma catecholamines that rilmenidine causes sympathoinhibition in conscious rabbits, presumably through central sites of action.(ABSTRACT TRUNCATED AT 250 WORDS)

[Selectivity of a new antihypertensive agent, rilmenidine, for imidazoline receptors in the human brain]. / Sélectivité d'un nouvel antihypertenseur, la rilmenidine, pour les récepteurs aux imidazolines du cerveau humain.

The involvement of the central nervous system in the global hypotensive effect of rilménidine (R) was already suspected after experiments on cats and dogs. Indeed, the injection of this drug in the vertebral artery provoked a decrease in the arterial blood pressure in these two species. Recently, we showed that intracisternal injections of cumulative doses (1 to 300 micrograms/kg) in anaesthetized rabbits significantly lowered the blood pressure. In that protocol the efficient dose 20% was 1.5 micrograms/kg when it was 70 micrograms/kg for intravenous injections. The hypotension was always associated with bradycardia. Thus we confirmed that the drop in the arterial blood pressure induced by R was, at least partially, due to a central inhibition of the vasomotor tone. When we pretreated anaesthetized rabbits, always by intracisternal injections, with identical doses of yohimbine or idazoxan (5 nmoles/kg), we observed that idazoxan prevented much more the hypotensive effects of R than yohimbine. This study demonstrated that R whose chemical structure is close to that of imidazolines was much better antagonized by a substance with an imidazoline-like structure than by a classical alpha 2-antagonist. These results were confirmed by binding studies realized with human brain membranes. Tritiated clonidine was bound to cortical membrane preparations, containing mainly alpha 2-adrenoceptors, as well as to medullary membrane preparations, containing mainly imidazoline receptors. We observed that R selectivity for the medullary imidazoline preferring receptors was 2.5 times higher than that of the reference substance, clonidine. So, it seems that the central hypotensive effect of R might be related to its interaction with imidazoline specific receptors.(ABSTRACT TRUNCATED AT 250 WORDS)