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WOS: 000258628400032, PubMed ID: 18538317, The pathophysiology of pre-eclampsia is still unknown thus effective primary prevention is not possible at the stage. The present study was conducted to research the smooth muscle responses in the pre-eclampsia model with suramin treated rats and the effect of phosphodiesterase-5 (PDE5) inhibitor on these responses. Rats of three groups; control, suramin and suramin+sildenafil were given intraperitoneal injections of saline, suramin or sildenafil citrate. Suramin injections caused increased blood pressure, protein in urine and caused fetal growth retardation.The use of sildenafil citrate straightened significantly both blood pressure and average fetus weight, but did not reach to control values. At the end of pregnancy, thoracic aorta rings were exposed to contractile and relaxant agents. KCl contraction responses, sodium nitroprusside and papaverine relaxation responses were similar in three groups. Contraction responses of phenylephrine, increased significantly in suramin group. Relaxation responses of acethylcholine and bradykinin decreased in suramin group. The use of sildenafil citrate partially straightened both relaxation and contraction responses, but did not reach to control values. In all groups in the presence of L-nitromonomethylarginine (L-NAME), 1H-(1, 2, 4) oxadiazole (4, 3-a) guinoxalin-1-one (ODQ) and indomethacin decreased the relaxation responses of acetylcholine and bradykinin. The cyclic guanosine monophosphate (cGMP) content of thoracic aorta tissue was determined by radioimmunoassay technique. The content of cGMP in suramin group decreased and use of sildenafil citrate increased the cGMP content but did not reach to control values. We conclude that in pre-eclampsia, the increase of contraction responses, the decrease of relaxation responses and the decrease of cGMP content can depend on insufficiency about synthesis or release of relaxant factors which was released from the vessel endothelium. The results in this study show that in pre-eclampsia: PDE5 inhibitors enhance endothelial function and may be used for protection. Further studies are needed to clear the efficiency and safety of PDE5 inhibitors. (c) 2008 Elsevier B.V. All rights reserved., Cumhuriyet University Scientific Research [T-282], This study was supported by Cumhuriyet University Scientific Research Project T-282 (CUBAP, Sivas, Turkey).

The metabolic syndrome is a cluster of more or less related metabolic and cardiovascular derangements including visceral obesity, insulin resistance, blood and tissue dislipidemia, high blood pressure and it is often associated with neuroendocrine and immunological dysregulations. The aetiology of this syndrome is clinically highly relevant because it predisposes to life-threatening complications, such as Diabetes Mellitus, kidney failure, cardiovascular disease, and certain types of cancer. Contributing factors include a sedentary life-style combined with increased dietary fat intake and psychosocial stress. From a biological viewpoint, however, the metabolic syndrome can be considered as a maladaptive consequence of an initially successful adaptation to high environmental demands. As opposed to pre-historic times - when environmental demands were usually energy-costly (e.g., fight/flight/hunt) and nutritional resource often inadequate - energy-utilizing actions serve no longer an optimal solution to deal with environmental demands of current human society. This paper describes the interactions between psychosocial stress and nutrition and how these may affect emotional and metabolic components of the metabolic syndrome. A deeper understanding of these interactions is necessary to come to effective treatment and prevention of the metabolic syndrome in the future. (C) 2008 Elsevier B.V. All rights reserved.

Selective serotonin uptake inhibitors (SSRIs) exert their effects by inhibiting serotonin (5-HT) re-uptake. Although blockade occurs almost immediately, the neurochemical effects on 5-HT, as measured by in vivo microdialysis, have been a matter of considerable debate. In particular, literature reports yield conflicting neurochemical results in the rat frontal cortex. Thus, while some groups consistently find increases in extracellular 5-HT levels following acute SSRI administration, others reproducibly report an absence of these acute serotonergic effects. In an attempt to unravel this apparent discrepancy, we combined published literature with in-house microdialysis experiments. When we plotted the lateral stereotaxic coordinate of the dialysis probe against published reports on the acute effects of fluoxetine a clear correlation was revealed. Whereas pronounced increases in SSRI-induced 5-HT were observed when the dialysis probe was placed 0 to 1 mm from the midline, effects diminished when the lateral probe placement was greater than 3 mm from the midline. In-house microdialysis studies corroborated these reports. Overall, these results illustrate - for the first time - that the midline stereotaxic coordinate is critical for interpreting the acute serotonergic effects of SSRIs within the frontal cortex. Moreover, the common observation that the clinical efficacy of SSRIs is not evident following acute administration complements preclinical microdialysis results in the lateral frontal cortex. The significance of this observation, along with potential explanations for the disparate neurochernical findings in the medial versus lateral cortices, will be discussed. (C) 2007 Elsevier B.V. All rights reserved.

Using conflict drinking and forced swimming tests in rats, we examined the anxiolytic- and the antidepressant-like activity, respectively, of (2 R )-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]-pyrrolidine (SB 269970), a selective 5-HT 7 receptor antagonist, after its intrahippocampal administration. SB 269970 at doses of 0.3, 1 and 3 μg showed an anticonflict effect which was weaker than that of diazepam (40 μg), whereas SB 269970 at doses of 3 and 10 μg had marked anti-immobility action comparable to that of imipramine (0.1 μg). Importantly, the anxiolytic- and antidepressant-like activity of SB 269970 seemed to be specific, since that agent − when given by the same route in doses effective in either model – affected neither the shock threshold, nor the non-punished water consumption, nor the exploratory activity of rats. The obtained results indicate that the hippocampus is one of the neuroanatomical structures involved in the potential anxiolytic and, in particular, antidepressant activity of SB 269970.

International audience; This study shows for the first time, that dexfenfluramine, a 5-HT 2 receptor agonist, attenuates the development of chronic hypoxia-induced pulmonary hypertension. Chronic exposure to hypoxia, 4 weeks, induced hypoxic pulmonary hypertension in adult rat as haemodynamic and cardiac measurements showed significant modifications in right ventricle parameters (free wall right ventricle thickness; pulmonary acceleration time and velocity time integral) in chronic hypoxic control when compared to normoxic control animals. We observed that free wall right ventricle thickness and pulmonary velocity time integral were significantly less in chronic hypoxic rats treated with dexfenfluramine when compared to chronic hypoxic control rats. Similarly, rats exposed to chronic hypoxia exhibited an increase in both right ventricle pressure and weight by comparison to normoxic control animals but those variations were significantly diminished in dexfenfluramine-treated rats, indicating the moderating influence exerted by dexfenfluramine on chronic hypoxia-induced pulmonary hypertension and cardiac alterations. Thus, we report here the ability of dexfenfluramine to limit chronic hypoxia-induced pulmonary hypertension, emphasizing the importance of the time after the dexfenfluramine treatment discontinuation to assess the influence of this 5-HT receptor agonist on the development of chronic hypoxia-induced pulmonary hypertension.

WOS: 000235343900010, PubMed ID: 16388799, This study was designed to compare the effects of beta-adrenoccptoy agonists formoterol and BRL 37344 on spontaneous contractions and the levels of cAMP and cGMP of myometrial strips isolated from timed-pregnant rats. Myometrial strips were obtained from term-pregnant Wistar albino rats (n = 12), mounted in organ baths and tested for changes in isometric tension in response to formoterol and BRL 37344. We evaluated the effect of increasing concentrations of formoterol and BRL 37344 on oxytocin-induced myometrial contractions and on contractions of myometrial smooth muscle pretreated with metoprolol, ICI 118.551 and SR 59230A (beta(1), beta(2), beta(3)-adrenoceptor antagonist, respectively, 10-6 M). Effects of formoterol and BRL 37344 on cAMP and cGMP levels in isolated myornetrial strips (n=6) were evaluated by radio immunoassay kits. Formoterol (10(-12) - 10(-8) M) and BRL 37344 (10(-11)-10(-5) M) concentration-dependently decreased the amplitude of oxytocin-induced contractions. E-max value (100%) of formoterol was increased significantly more than E-max value (70.6%) of BRL 37344 (P < 0.05), with no change in pD(2) value (9.54 +/- 0.12 and 9.12 +/- 0.12, respectively). The inhibition of the amplitude of oxytocin-induced contractions by formoterol was antagonized with ICI 118.551 (10(-6) M), but they were not changed by nictoprolol (10(-6) M) or SR 59230A (10(-6) M). The inhibition of the amplitude of oxytocin-induced contractions by BRL 37344 were antagonized with SR 59230A (10(-6) M), but they were not changed by metoprolol (10(-6) M) or ICT 118.551 (10(-6) M). Formoterol and BRL 37344 increased cAMP levels. BRL 37344 increased cGMP levels in BRL 37344 group more than control group, but this increase is less significant than cAMP levels (P > 0.05). Fon-noterol and BRL 37344 decreased amplitude of myometrial contractions with similar potency, but efficacy of forinoterol was better than BRL 37344. (c) 2005 Elsevier B.V. All rights reserved.

We used two experimental models to prove that resveratrol (trans-3,4',5-trihydroxystilbene) reduces cardiac ischemic-reperfusion injury by means of a nitric oxide- and adenosine-dependent mechanism. (1). ACUTE EX VIVO: resveratrol (10 microM, 10 min) infusion in Langendorff-perfused normoxic rat hearts significantly increased adenosine release and coronary flow compared with baseline. After 30-min low-flow ischemia, vasodilation, still present at reperfusion, was completely abolished by resveratrol plus adenosine antagonist 8-(p-sulfophenyl)theophylline (SPT, 50 microM) administration. (2). CHRONIC IN VIVO: rats received tap water containing 25 mg/l resveratrol for 15 days or normal water. Twenty-four hours after, their hearts were Langendorff-perfused and submitted to 60-min low-flow ischemia and reperfusion. The resveratrol-treated hearts showed better functional recovery at reperfusion and significant vasodilation, but no variation in high-energy phosphates (31P Nuclear Magnetic Resonance). N(G)-nitro-L-arginine methyl ester (L-NAME, 30 microM), a nonselective nitric oxide synthase inhibitor, or SPT (50 microM) administered for 10 min prior to the low-flow ischemia cancelled the effects. This suggests that long-term moderate resveratrol consumption could play an important role in late cardioprotective effects.

Rats were chronically treated with the selective serotonin re-uptake inhibitor citalopram [1-(3-dimethylaminopropyl)-1-(4-fluorophenyl)-5-phtalancarbonitril], by means of osmotic minipumps. Using an infusion concentration of 50 mg/ml citalopram, steady-state plasma concentrations of approximately 0.3 mu M citalopram were maintained for 15 days. Citalopram plasma levels dropped below pharmacologically active concentrations 48 h after removal of the minipumps. Although chronic treatment with citalopram did induce an attenuated response by extracellular levels of 5-hydroxytryptamine (5-HT) after systemic administration of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), no effect of chronic citalopram treatment was observed when 5-HT1B receptor function was evaluated with a local infusion of 5-HT1B/D receptor agonist, sumatriptan (3-[2-dimethylamino]ethyl-N-methyl-1 H-indole-5methane sulphonamide). Controversially, no augmentation of the increase of 5-HT levels was observed upon systemic administration of citalopram It is concluded that, although chronic treatment with citalopram does induce desensitisation of 5-HT1A receptors, the absence of augmented effects of citalopram on 5-HT levels indicates that other mechanisms compensate for the loss of autoreceptor control. (C) 2000 Elsevier Science B.V. All rights reserved.

This report describes the structure, function, and tissue distribution pattern of rat OCTN1 (novel organic cation transporter 1). The rat OCTN1 cDNA was isolated from a rat placental cDNA library. The cDNA is 2258 bp long and codes for a protein of 553 amino acids. Its amino acid sequence bears high homology to human OCTN1 (85% identity) and rat OCTN2 (74% identity). When expressed heterologously in mammalian cells, rat OCTN1 mediates Na+-independent and pH-dependent transport of the prototypical organic cation tetraethylammonium. The transporter interacts with a variety of structurally diverse organic cations such as desipramine, dimethylamiloride, cimetidine, procainamide, and verapamil. Carnitine, a zwitterion, interacts with rat OCTN1 with a very low affinity. However, the transport of carnitine via rat OCTN1 is not evident in the presence or absence of Na+. We conclude that rat OCTN1 is a multispecific organic cation transporter. OCTN1-specific mRNA transcripts are present in a wide variety of tissues in the rat, principally in the liver, intestine, kidney, brain, heart and placenta. In situ hybridization shows the distribution pattern of the transcripts in the brain (cerebellum, hippocampus and cortex), kidney (cortex and medulla with relatively more abundance in the cortical-medullary junction), heart (myocardium and valves) and placenta (labyrinthine zone).

Constitutive nitric oxide synthase (cNOS) may play an important protective role in the intestine, since our previous study has shown that the degree of bowel injury induced by platelet-activating factor (PAF), a potent inflammatory mediator, is inversely related to the cNOS content of the intestine. This study aims to examine the composition of the cNOS system in rat small intestine, and its regulation by PAF. We found that an approximately 120 kDa NOS I (neuronal NOS) is the predominant NOS in rat intestine, as evidenced by the following: (a) immunoblotting with specific antibodies detected a NOS I of approximately 120 kDa, but little NOS III; (b) the Ca 2+ -dependent, constitutive NOS (cNOS) activity of the rat intestine was removed by immunoprecipitation with the anti-NOS I, but not anti-NOS II or anti-NOS III antibodies; (c) RT–PCR revealed constitutive expression of NOS I in the intestinal tissue, but only a minute amount of NOS III. Immunofluorescent staining with anti-NOS I located NOS in the Auerbach plexus and nerve fibers in the muscle layer. We also found that this 120 kDa NOS I is rapidly (within 1 h) down-regulated in response to PAF administration. The protein level, enzyme activity as well as mRNA of nNOS were all decreased in the intestine.

The inhibitory action of the natural selective tachykinin NK3 receptor agonist, PG-KII, (pGlu-Pro-Asn-Pro-Asp-Glu-Phe-Val-Gly-Leu-Met-NH2), on colonic propulsion was studied in rats after central administration. Intracerebroventricular injection of PG-KII (0.1, 1, 10 and 100 ng/rat) produced a dose-related inhibition of colonic propulsion, measured as the increase in the mean expulsion time of a 5-mm glass bead placed in the distal colon. At the same doses as PG-KII, the selective tachykinin NK3 receptor agonist, senktide, (succ-[Asp6-MePhe8] substance P-(6-11)), induced a similar dose-related inhibition. Conversely, substance P (0.1, 1 and 10 microg/rat), a tachykinin NK1-preferring receptor agonist, had weaker antipropulsive effects, neurokinin A (0.1, 1 and 10 microg/rat), a tachykinin NK2-preferring receptor agonist, at the highest dose used only slightly inhibited colonic propulsion and neurokinin B (0.1, 1 and 10 microg/rat), a tachykinin NK3-preferring receptor agonist, left propulsion unchanged. Pretreatment with the selective tachykinin NK3 receptor antagonist, 3-indolycarbonyl-Hyp-Phg-N(me)-Bzl, referred as to R820 (6.2 microg/rat), prevented PG-KII-induced colonic antipropulsion, whereas the tachykinin NK1 receptor antagonist, (S)-1-(2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)pi peridin-3-yl] ethyl)-4-phenyl-1-azoniabicyclo[2.2.2] octane chloride, referred to as SR 140,333 (1 microg/rat), and the tachykinin NK2 receptor antagonist, ([Tyr5,D-Trp6,8,9, Arg10] neurokinin A-(4-10)), referred to as Men 10,376 (5 microg/rat), left it unchanged. These findings show that of the tachykinins tested, PG-KII and senktide are the most potent central inhibitors of colonic propulsion in the rat, suggesting that the central tachykinin NK3 receptor system plays an inhibitory role in modulating colonic transit. As well as confirming the selectivity of PG-KII for tachykinin NK3 receptors, we show that PG-KII provides useful information about the physiological role of central tachykinin NK3 receptors and that glass bead expulsion test is a reliable non-invasive in vivo method for evaluating the tachykinin NK3 receptor selectivity of new synthetic or natural tachykinins.

We investigated the interaction of rat PEPT2, a high-affinity peptide transporter, with neutral, anionic, and cationic dipeptides using electrophysiological approaches as well as tracer uptake methods. d-Phe-L-Gln (neutral), d-Phe-L-Glu (anionic), and d-Phe-L-Lys (cationic) were used as representative, non-hydrolyzable, dipeptides. All three dipeptides induced H+-dependent inward currents in Xenopus laevis oocytes heterologously expressing rat PEPT2. The H+:peptide stoichiometry was 1:1 in each case. A simultaneous measurement of radiolabeled dipeptide influx and charge transfer in the same oocyte indicated a transfer of one net positive charge into the oocyte per transfer of one peptide molecule irrespective of the charged nature of the peptide. We conclude that the zwitterionic peptides are preferentially recognized by PEPT2 as transportable substrates and that the proton/peptide stoichiometry is 1 for the transport process.

In addition to Ca2+ and K+ fluxes, angiotensin II (Ang II) has been shown to influence sperm motility. The present study investigated the involvement of angiotensin II type 1 receptor (AT1) in mediating the modulatory effect of Ang II on a sperm Ca2+-activated K+ channel expressed in Xenopus oocytes injected with RNAs of spermatogenic cells. Ang II at a concentration of 1 μM was found to potentiate the ionomycin-induced current, previously demonstrated to be mediated by a ‘Maxi’ Ca2+-activated K+ channel. However, at higher concentration, 20 μM, Ang II was found to suppress the ionomycin-induced current. Both potentiating and inhibitory effects of Ang II were blocked by losartan, a specific antagonist of AT1 receptors. Immunohistochemical studies further confirmed the presence of AT1 receptors in spermatogenic cells while expression of AT1 receptor mRNA was demonstrated by RT–PCR. These results suggest that Ang II may influence sperm motility as well as other sperm function by acting on AT1 receptors, and exerting potentiating and inhibitory effects on the Ca2+-activated K+ channels.

The present study investigated the feasibility of using Xenopus oocytes to express sperm ion channel by injection of RNAs extracted from the rat testis. The RNA-injected oocytes expressed an outwardly rectifying current which was dependent on K+ concentration and inhibitable by K+ channel blockers, charybdotoxin (CTX) and tetraethylammonium (TEA). The Ca2+ ionophore, ionomycin, could also stimulate current activation with similar current characteristics in the RNA-injected oocytes, suggesting the expression of a Ca2+-activated K+ channel. Immunolocalization indicated predominant Ca2+-activated K+ channel immunoreactivity associated with spermatogenic cells. Reverse transcriptase-polymerase chain reaction studies confirmed the expression of the Ca2+-activated K+ channel mRNA in isolated spermatogenic cells. Our results suggest that ion channels and/or receptors of spermatogenic cells could be investigated using the Xenopus oocyte as an expression system. The present study also suggests that sperm may possess a Ca2+-activated K+ channel which has been implicated in the process of sperm activation and gamete interaction.

Na+/H+ exchange (NHE) activity varies with ontogenic state in rat intestinal basolateral membrane vesicles (BLMV). The current investigation sought to determine if these observations are due to differential expression of BLM NHE isoforms, NHE-1 and NHE-4. In rat kidney, BLMV sodium uptake levels were similar in 2, 3 and 6 week rats (13.28+/-0.68, 14.03+/-0.84, and 11.71+/-0.66 nmol Na+/mg protein/30 s, respectively), and lower in adults (5.53+/-0.24) (n=4; p0.001 between 2 week rats and adults, and between 3 week rats and adults; p0.01 between 6 week rats and adults). In rat jejunum, BLMV uptake was highest in adults (13.07+/-0.86 nmol Na+/mg protein/30 s), and decreased in 6, 3, and 2 week rats (4.48+/-0.75, 2.94+/-0.68, and 1.59+/-0.58, respectively) (n=4; p0.001 between all groups and adults). Control immunoblot experiments with NHE-3 antiserum showed that BLMV preps were not contaminated with significant amounts of this brush-border membrane specific protein. Northern blots with isoform-specific probes showed highest renal NHE-1 hybridization intensities in 2 and 3 week rats (11.00+/-0.25 and 12.07+/-0.16 phosphorimage units, respectively), and lower intensities in 6 week and adult animals (4.30+/-0.95, and 4.40+/-1.40, respectively) (n=4; p0.01 between 2 week animals and 6 week and adult animals, and between 3 week animals and 6 week and adult animals). NHE-1 probes in the intestine showed no hybridization intensity differences between groups: 2 week-7.09+/-1.10, 3 week-5.39+/-0.56, 6 week-8. 24+/-1.57, and adult-8.99+/-2.20 (n=3). NHE-4 specific probes in the kidney showed hybridization intensity levels of 9.22+/-0.35 in 2 week animals, 12.12+/-1.26 in 3 week animals, 5.63+/-0.81 in 6 week animals, and 3.52+/-0.57 in adults (n=4; p0.05 between 2 week and adults; p0.01 between 3 week and 6 week animals, and between 3 week and adults). No NHE-4 message was detected in rat jejunum by Northern blot analysis or by reverse transcriptase-PCR. These results suggest that ontogenic NHE activity at the jejunal BLM is not related to differential expression of NHE-1, while NHE activity at the renal BLM may in part be related to differential ontogenic expression of NHE-1 and NHE-4.

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and a vascular permeability factor. In this study we found that the addition of angiotensin II (AII) to rat heart endothelial cells induced VEGF mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of AII and decreased after 4 h. The induction was superinduced by cycloheximide and blocked by actinomycin D. Losartan, an AT1 receptor antagonist, abolished the induction of VEGF mRNA by AII, whereas PD 123319, an AT2 receptor antagonist, had no effect on VEGF mRNA induction. H7, a protein kinase C inhibitor, blocked the induction. RT-PCR experiments showed two mRNA species (VEGF 120 and VEGF 164) in these cells and both species were stimulated by AII. Transient transfection experiment showed that VEGF promoter activity was increased 2.2-fold upon AII stimulation. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-kappa B. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 8 h after AII stimulation. Our results demonstrate for the first time that the upregulation of VEGF by AII may play a significant role in AII-induced hyperpermeability.

Using brain microdialysis, it was demonstrated that the release of 5-hydroxytryptamine (5-HT) in the central nucleus of the amygdala is under inhibitory control of somatodendritic and postsynaptic 5-HT1A receptors. Systemic administration of flesinoxan, a selective 5-HT1A receptor agonist, significantly reduced the extracellular levels of 5-HT in the central nucleus of the amygdala. This effect could be completely antagonized by the 5-HT1A receptor antagonist N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)-N-(2-pyridyl)cyclohexane carboxamine trihydrochloride (WAY 100635). Local administration of these compounds by reversed microdialysis into the raphe nuclei revealed that extracellular 5-HT levels in the central nucleus of the amygdala can be regulated through 5-HT1A receptors in the caudal linear raphe nucleus, but not in the dorsal and median raphe nuclei. Interestingly, administration of flesinoxan into the central nucleus of the amygdala also decreased dialysate 5-HT levels both locally and in the caudal linear raphe nucleus. The former effect could be blocked by pretreatment with WAY 100635 when applied into the central nucleus of the amygdala, but not when applied into the caudal linear raphe nucleus. These data provide circumstantial evidence for the existence of a 5-HT1A receptor mediated feedback loop from the central nucleus of the amygdala to the caudal linear raphe nucleus. (C) 1997 Elsevier Science B.V.

The aim of this study was to investigate the [3H]arachidonic acid metabolism of rat peritoneal macrophages, induced by allergen (ovalbumin) and the impact of interleukin-4 on this process. We established that ovalbumin induces an increase of [3H]arachidonic acid mobilisation from membrane lipids and of [3H]arachidonic acid catabolism, principally by the 5-lipoxygenase pathway, when the macrophages are sensitized and when serum is present. The allergen effect is not modified by the presence of interleukin-4 in the culture medium of macrophages 15 h before the allergen challenge. We also showed that, whereas the basal [3H]arachidonic acid metabolism of macrophages from control and actively sensitized rats is not different, interleukin-4 increases the [3H]arachidonic acid mobilisation and catabolism by cyclooxygenase and 5-lipoxygenase pathways in macrophages from control rats although it does not in macrophages from actively sensitized rats. In macrophages from control rats, the interleukin-4 effect is diminished by the addition of IgEs to their culture medium. In summary, interleukin-4 has an enhancer effect on the macrophage arachidonic acid catabolism that depends on the sensitization condition of the cell but that has no consequences on the further increased arachidonic acid metabolism induced by the allergen.

Treatment of Parkinson patients with l -DOPA (3,4-dihydroxy- l -phenylalanine) leads to endproduct inhibition of tyrosine hydroxylase, the key enzyme in dopamine biosynthesis and the enzyme needing tetrahydrobiopterin and iron as cofactors. To overcome this problem an alternative treatment was investigated which attempted to stimulate endogenous dopamine biosynthesis. Incubation of rat PC 12 cells with NADH (β-nicotinamide adeninedinucleotide) leads to increased dopamine production. We investigated the possibility that this increase of dopamine biosynthesis was due to stimulation of quinonoid dihydropteridine reductase, the enzyme which recycles the inactive dihydrobiopterin to the active tetrahydrobiopterin. The experiments showed that whereas NADH is able to increase dopamine production in PC 12 cells (rat phaeochromocytoma cells, clone PC 12) up to three-fold, no influence is exerted by NADH on pteridine metabolism; neither are tetrahydrobiopterin concentrations nor the de novo-biosynthesis of pteridines from guanosine triphosphate altered by NADH. Further no influence of NADH on protein de novo synthesis of quinonoid dihydropteridine reductase was observed. However, NADH was able to directly increase the catalytic activity of this enzyme. Our results suggest that the stimulation of dopamine biosynthesis by NADH is due to more rapid regeneration of quinonoid dihydrobiopterin to tetrahydrobiopterin.

Reticuloendothelial phagocytic and serum opsonic activity was evaluated at terminal stages of tumour growth in rats transplanted subcutaneously with chondrosarcoma in an attempt to evaluate the role of opsonic protein(s) in governing liposome recognition and clearance by the macrophage system. The liver of the tumour-bearing animals manifested a decline in the uptake of multilamellar vesicles composed of egg phosphatidylcholine: cholesterol: dicetyl phosphate (mole ratio 7:2:1) from the blood when compared to healthy animals. In contrast, an increase in splenic clearance of liposomes was encountered in tumour-bearing rats. Studies with isolated liver non-parenchymal cells suggested that liposome recognition in both health and at terminal stages of cancer growth is influenced by a serum opsonin, which can be precipitated by 35–50% ammonium sulphate, as well as the concentration of calcium levels in serum. Serum of healthy animals equally enhanced liposome recognition by the hepatic macrophages of both normal and tumour-bearing rats. In contrast, both cell populations manifested poor liposome recognition in the presence of serum pooled from tumour-bearing animals and the results were comparable to the corresponding liposome-cell interaction in the absence of serum. The opsonic activity of serum derived from tumour-bearing rats could be demonstrated either by prior dialysis of serum against de-ionized water or by addition of EGTA. Liver phagocytes of healthy animals recognized more liposome in the presence of dialysed or EGTA-chelated tumour-serum than that of liver cells derived from tumour transplanted rats. A significant increase in serum calcium concentration was found in all tumour-bearing rats. When the concentration of calcium in the serum of normal animals was increased to the level that is encountered in tumour-bearing rats, a sharp drop in liposome recognition by liver phagocytes was observed. This drop in opsonic activity was not related to changes in the ionic strength of serum. The ammonium sulphate precipitated opsonin was also calcium-sensitive and its opsonic activity was abolished in the presence of calcium. Studies with isolated splenic phagocytes suggested that an increase in the opsonic activity of serum, but not the elevated calcium level, was responsible for hyperphagocytosis of liposomes by the splenic phagocytes of tumour-transplanted animals. The opsonic molecule which enhanced liposome recognition by liver non-parenchymal cells failed to enhance liposome clearance by the splenic phagocytes. These findings suggest that the alteration in macrophage clearance of liposomes during the terminal growth of cancer may be mediated in part by changes in the opsonic capacity of serum.

To explore the spatial and temporal localization of PKC isoforms during ischemia, we quantified PKC isoforms in the subcellular fractions in perfused rat heart by immunoblotting using specific antibodies against PKC isoforms. PKCs-alpha and epsilon translocated from the 100000 x g supernatant (S, cytosolic) fraction to the 1000 x g pellet (PI, nucleus-myofibril) and the 1000-100000 x g pellet (P2, membrane) fractions during 5-40 min of ischemia. PKC-delta redistributed from the P2 to the S fraction. A 50-kDa fragment of PKC-alpha appeared during ischemia possibly through calpain action. Immunohistochemical observations showed the different localizations of PKC-alpha, delta, and epsilon in the myocytes. The PKC assay displayed high basal levels of Ca(2+)-independent PKC, the activation of Ca(2+)-dependent PKC in the P1 and P2 fractions, and the activation of Ca(2+)-independent PKC in the P1 fraction after 20 min of ischemia. These observations show that ischemia induces different patterns of translocation of the three PKC isoforms, suggesting differences in their roles.

Treatment of rat parotid tissues with 1 microM isoproterenol (IPR) for 10 min caused a 60% decrease in pertussis toxin (IAP)-catalyzed ADP-ribosylation of Gi alpha and resulted in supersensitivity of amylase secretion from the tissues. However, conversely, IPR treatment for 30 min caused a 40% increase in IAP-catalyzed ADP-ribosylation of Gi alpha, coupled with desensitization of amylase secretion. No changes in Gs function were observed in IPR-induced phenomena. Pretreatment with okadaic acid induced enhancement of the supersensitivity of amylase secretion and disappearance of the desensitization. These phenomena were accompanied with decreases in IAP-catalyzed ADP-ribosylation of Gi alpha. IPR treatment for 30 min caused a 50% decrease in phosphorylation of Gi2 alpha immunoprecipitated with anti-G protein antiserum (AS/7) from [32P]Pi-labeled cells, but such treatment for 10 min caused a 40% increase in phosphorylation in the cells pretreated with okadaic acid. Phosphorylation and dephosphorylation of immunoprecipitates with AS/7 by protein kinase A (PKA) and alkaline phosphatase caused decreases and increases in IAP-catalyzed ADP-ribosylation, respectively, indicating the presence of PKA-mediated phosphorylation sites on Gi2 alpha. Thus, the control of the phosphorylation of Gi2 alpha is of importance and relevance in the regulation of biological processes and cellular responses.

Hyperplasia of the pancreatic tissue during late lactation (third week) and lasting for at least the first two weeks after weaning has been observed by several authors. Since the tetradecapeptide somatostatin (SS) inhibits pancreatic growth and its plasma levels are elevated during these periods, the aim of the present study was to determine the possible implication of the somatostatinergic system in the pancreatic changes cited above. Thus, the present study investigated 125I-Tyr(11)-somatostatin (125I-Tyr(11)-SS) binding and the effects of SS on guanylate cyclase activity as well as pancreatic somatostatin-like immunoreactivity (SSLI) levels in pancreatic acinar membranes from control, lactating and weaning rats. SS receptors were identified using 125I-Tyr(11)-SS and isolated pancreatic acinar membranes in vitro. There was an increase in the number of SS receptors after the third week of lactation (244 +/- 6 vs. 155 +/- 12 fmol/mg protein, P0.01) and the first two weeks after weaning (327 +/- 8 vs. 164 +/-10 fmol/mg protein, P0.001). No change in the affinity of the receptor site was detected at either study time. In addition, SS-stimulated guanylate cyclase activity was markedly increased at the third week of lactation (119%) and at the second week after weaning (158%) when compared with the control group. In contrast, basal guanylate cyclase activity was not modified at either study period. Thus, SS-stimulated guanylate cyclase activity is increased in pancreatic acinar membranes at late lactation and at the second week after beginning weaning probably due to an increase in the number of SS receptors. Significant decreases in SSLI content were observed at the third week of lactation (69%) and the second week after weaning (37%) when compared with the respective controls. The present results suggest that pancreatic acinar cell growth observed at the third week of lactation and the second week after weaning is associated with up-regulation of SS receptors which would represent a mechanism promoted by the cell that would negatively regulate the mitogenic activity of the increased number of pancreatic growth factors observed during both periods.

Effects of fusogenic and DNA-binding amphiphilic compounds on the receptor-mediated gene transfer using asialofetuin-labeled liposomes (AF-liposomes) were examined with HepG2 cells and rat hepatocytes in primary culture. AF-liposomes were sufficiently taken up by both types of cells through the asialoglycoprotein receptor-mediated endocytosis. In HepG2 cells, bacterial beta-galactosidase (beta-Gal) gene expression was observed by transfection using AF-liposomes encapsulating plasmid pCMV beta DNA (AF-liposome-pCMV beta). By addition of dioleoylphosphatidylethanolamine (DOPE) to the liposomal lipid composition (AF-liposome(DOPE)-pCMV beta), the transfection efficiency was clearly increased. The effects of DOPE were more conspicuous in the presence of chloroquine in the medium throughout the transfection. When pCMV beta complexed with gramicidin S (pCMV beta (GrS)) was encapsulated (AF-liposome(DOPE)-pCMV beta (GrS) and was transfected to HepG2 cells, an significantly high beta-Gal activity in the cells was observed as compared with that in the cells transfected with AF-liposome(DOPE)-pCMV beta. No effects of GrS were found in the transfection using AF-non-labeled control liposomes. In primary culture of rat hepatocytes, no beta-Gal gene expression was observed even though AF-liposome(DOPE)-pCMV beta was introduced into the cells prepared from adult rats. However, following the transfection with AF-liposome(DOPE)-pCMV beta, the beta-Gal activity was expressed in the cells from immature rats cultured in the medium supplemented with epidermal growth factor and insulin, and the transfection efficiency was 2-fold higher than that transfected with pCMV beta encapsulated in AF-non-labeled control liposomes. By the complex formation of pCMV beta with GrS, the transfection efficiency of AF-liposome(DOPE)-pCMV beta (GrS) increased according to the increase of GrS in the complex. It was shown that AF-liposome(DOPE)-pCMV beta (GrS) did efficiently introduce and express beta-Gal gene in both HepG2 cells and primary hepatocytes in the receptor mediated manner.

WOS: A1996TQ75900006, PubMed ID: 8822266, Altered responses to several agonists have been reported in various smooth muscles from experimentally-diabetic animals suggesting a defective contractile process of smooth muscle Recently, decreased smooth muscle calmodulin levels have been reported in streptozotocin-diabetic rats. However, the effectiveness of insulin on the decreased calmoduiin levels in diabetic rats has not been questioned. Therefore, the present study was designed to examine the effect of insulin on smooth muscle calmodulin levels from streptozotocin-diabetic rats. Calmodulin levels of the smooth muscle were measured by a radioimmunoassay technique. Streptozotocin diabetes caused a significant decrease in tissue calmodulin levels of smooth muscles. Insulin therapy for 20 days did not correct the changes in calmodulin levels of rat smooth muscles, although it normalised blood glucose in streptozotocin-diabetic rats. These findings suggest that the altered smooth muscle calmodulin may contribute the defective contractile responses in diabetes and these changes may be resistant to insulin therapy.

The non-steroidal anti-inflammatory drug diclofenac is reported to cause, in rare cases, fulminant heaptic necrosis associated with chronic use of the drug. In order to investigate the possibility that covalent protein adducts of reactive metabolites of diclofenac might be responsible for the hepatotoxicity produced by this drug, we developed an antibody against a diclofenac-keyhole limpet hemocyanin adduct. The anti-diclofenac antibody did recognize diclofenac-protein adducts on Western blots of homogenates of cultured human hepatocytes exposed to diclofenac. The major detected adduct was a 60 kDa protein, which was present in both human and rat hepatocytes. These results suggest that binding of diclofenac to human hepatocytes is, like in rats, selective and that a 60 kDa protein appears to be the major target for alkylation. Immunoblots f homogenates of liver sinusoidal lining cells (LSLC) from rats treated with diclofenac also exhibited adducts with a 60 kDa protein. This fact suggest a role for LSLC in processing of chemically altered proteins in the liver.

PCR was used to amplify the coding region of CHO MCT1 cDNA. This was then used to screen a rat skeletal muscle cDNA library which lead to the isolation of a full length cDNA encoding MCT1 from rat. The cDNA derived amino acid sequence shows 94% and 86% identity to CHO and human MCT1, respectively.

We studied how the release of histamine and prostaglandin D2 (PGD2) were connected in stimulated rat peritoneal mast cells, and to what extent these processes were controlled by the cytosolic Ca2+ concentration, [Ca2+]i, and protein tyrosine kinases. In the presence of 1 mM CaCl2, the G-protein activating compound 48/80 (10 micrograms/ml) evoked a transient rise in [Ca2+]i and a relatively high secretion of histamine, but only a low release of PGD2. In contrast, 5 microM thapsigargin (an inhibitor of endomembrane Ca(2+)-ATPases) and 5 microM ionomycin evoked high and prolonged rises in [Ca2+]i, and stimulated the cells to release relatively small amounts of histamine and high amounts of PGD2. Stimulation of the cells with CaCl2 and 10 microM ATP4- gave only minor quantities of histamine and PGD2, despite of the micromolar level of [Ca2+]i reached. When CaCl2 was replaced by EGTA, rises in [Ca2+]i as well as release of histamine and PGD2 were reduced with each agonist, but the preference of agonists to release more histamine or PGD2 remained unchanged. In mast cells with depleted Ca2+ stores, the addition of CaCl2 stimulated the store-regulated Ca2+ entry resulting in a prolonged rise in [Ca2+]i. However, simultaneous addition of compound 48/80 and CaCl2 was required for release of histamine and PGD2. In cells with full stores, PGD2 release evoked by compound 48/80 was greatly reduced by genistein and methyl-2,5-dihydroxycinnamate, two structurally unrelated inhibitors of protein tyrosine kinases, whereas histamine secretion was not influenced by these inhibitors. Similarly, with thapsigargin or ionomycin as agonist, PGD2 release was more sensitive to the tyrosine kinase inhibitors than histamine secretion. We conclude that in activated rat peritoneal mast cells: (i) the influx of extracellular Ca2+ potentiates agonist-evoked rises in [Ca2+]i as well as histamine secretion and PGD2 release; (ii) the amplitude of the [Ca2+]i rise does not determine the preferential effect of agonists to release more histamine or more PGD2; (iii) the relatively high PGD2 release evoked by thapsigargin and ionomycin is probably due to their potency to evoke a prolonged rise in [Ca2+]i and to activate protein tyrosine kinases.

En: Biochimica et biophysica acta, ISSN 0006-3002 1993. Vol. 1167, pp 70-78 This study was addressed to determine whether the tissue-specific LPL activity response to fasting differs between nonpregnant and pregnant rats over the course of pregnancy. Fed and 24-h fasted rats were studied at days 12, 15 or 20 of gestation and were compared to virgin controls. In fed rats at days 15 and 20 of gestation LPL activity decreased in lumbar adipose tissue and the heart and liver, and increased in mammary gland tissue. Fasting decreased LPL activity in lumbar adipose tissue in 12 day pregnant and virgin rats and in mammary gland tissue in pregnant rats at 15 and 20 days of gestation and in virgin rats, whereas it increased LPL activity in heart tissue in rats at day 15 and 20 and in liver at day 20 of gestation. Plasma triacylglycerols were higher in 20 day pregnant rats than in the other groups when fed and this difference was even more noticeable in the fasting condition where the plasma ,B-hydroxybutyrate level also reached the highest value in the 20 day pregnant rats. Since tissue LPL activity controls the hydrolysis and uptake of circulating macylgylcerols, the present results indicate that in fed rats after the 15th day of gestation circulating triacylglycerols are preferentially [taken up by the mammary gland instead of being taken up by adipose tissue and heart. However, after fasting, circulating triacylglycerols ~e driven to the heart and liver in the late pregnant rat, and become a major source for fatty acid oxidation, an effect that seems to be ~pecially evident in the liver of the 20 day pregnant rat where there is an intense increase in LPL activity and the triacylglycerols become preferential substrates for ketone body production.

The effects of chronic intragastric administration of the anorectic agent d-fenfluramine on energy metabolism and nutrient concentrations were investigated at rest and during swimming. Rats were provided with permanent cannulas for blood sampling and intragastric administration of d-fenfluramine or saline. Energy expenditure and nutrient and hormone concentrations were determined. Under baseline conditions, d-fenfluramine increased carbohydrate utilization (14.2 vs. 7.0 mg/kg·min) and decreased fat oxidation (2.8 vs. 5.5 mg/kg·min). Plasma free fatty acid concentration was decreased (0.29 vs. 0.55 mmol/l) and lactate and insulin concentrations were increased after d-fenfluramine treatment (0.64 vs. 0.37 mmol/l and 61 vs. 33 mU/l, respectively). The shift in nutrient utilization also occurred during swimming. The exercise-induced increase in blood glucose was reduced after d-fenfluramine (+0.8 vs. +2.0 mmol/l). During swimming, free fatty acid, lactate and insulin concentrations were similar in the two groups. It is hypothesized that chronic d-fenfluramine treatment increases in the oxidation of carbohydrates and decreases the oxidation of fat as a result of a decrease in the transport of fatty acids over the mitochondrial membrane.

The present microdialysis study investigated whether nociceptin/orphanin FQ exerts a tonic inhibition of the release of noradrenaline in the basolateral nucleus of the amygdala in awake rats. The non-peptide competitive nociceptin/orphanin FQ (N/OFQ) peptide receptor antagonist J-113397 (20 mg/kg i.p.) induced an increase in the release of noradrenaline to about 150-200%. The increase was strongly suppressed by local infusion of an endogenous N/OFQ peptide receptor agonist, nociceptin/orphanin FQ (1 muM) via retrograde microdialysis, into the basolateral nucleus of the amygdala. Local infusion of nociceptin/orphanin FQ 1 muM) itself reduced noradrenaline release in the basolateral nucleus of the amygdala to about 70% of basal levels. These results indicate that a large part of basal release of noradrenaline in the basolateral nucleus of the amygdala is under tonic inhibitory control by endogenous nociceptin/orphanin FQ through the N/OFQ peptide receptors localized within the basolateral nucleus of the amygdala. (C) 2003 Elsevier B.V. All rights reserved.

It has been previously described that gamma-hydroxybutyrate (GHB) provides significant protection against transient global cerebral ischemia in the rat (four vessel occlusion model), when given 30 min before or 10 min after artery occlusion. Here, we show that in the same rat model, significant protection can also be obtained when treatment is started 2 h after the ischemic episode. In saline-treated animals, 30 min of global ischemia followed by reperfusion caused a massive loss of neurons in the hippocampal CA1 subfield (examined 63 days after the ischemic episode), and an impairment of sensory-motor performance (tested on the 51st and 63rd days after ischemia) and of spatial learning and memory (evaluated starting 46 days after the ischemic episode). Treatment with GHB--300 mg/kg intraperitoneally (i.p.) 2 h after the ischemia-reperfusion episode, followed by 100 mg/kg i.p. twice daily for the following 10 days--afforded a highly significant protection, against both histological damage and sensory-motor and learning-memory impairments. These data further suggest the possible therapeutic effectiveness of GHB in brain ischemia, and indicate that the underlying mechanism of action involves non-immediate steps of the ischemia-induced cascade of events.

Nociceptin (also called orphanin FQ), a 17-amino-acid peptide, is the natural ligand of the nociceptin opioid peptide (NOP) receptor. This peptide shows similarities, in its structure, to opioid peptides, mainly to dynorphin A. However, unlike opioid peptides, it does not produce a conditioned place preference or aversion but inhibits rewarding effect of drugs of abuse. The present study was designed to examine the ability of nociceptin to block the acquisition of amphetamine-induced place preference, and the development of amphetamine-induced sensitization to stereotypy in rats. Our experiments indicated that repeated administration of nociceptin at increasing doses during conditioning significantly attenuated the reinforcing effect of amphetamine in conditioned place preference paradigm. Nociceptin did not change the acute effect of amphetamine-induced stereotypy but prevented the development of sensitization to stereotypy measured on the challenge day. Our results suggest the involvement of nociceptin in long-lasting neuronal adaptation after repeated amphetamine treatment.

Activation of peripheral cannabinoid CB1 receptors contributes to hemorrhagic hypotension, and endocannabinoids produced by macrophages and platelets may be mediators of this effect. A number of studies have provided evidence that functional links exist in the mechanisms of action of cannabinoids and opioid peptides; and opioids too play an important role in the pathophysiology of hemorrhagic hypotension and shock. On the other hand, melanocortin peptides, which are the main endogenous functional antagonists of opioid peptides, have an antishock effect in animals and humans. Thus, we investigated whether an interaction exists between endocannabinoids and the endogenous opioid/antiopioid system also in a condition of hemorrhagic shock and, particularly, whether the blockade of cannabinoid CB1 receptors potentiates the antishock effect of melanocortins. Urethane-anesthetized rats were stepwise bled until mean arterial pressure decreased to, and stabilized at, 21–23 mm Hg. In this model of hemorrhagic shock, which caused the death of all control rats within 30 min after vehicle (tween 80, 5% in saline) injection, the intravenous (i.v.) bolus injection of the cannabinoid CB1 receptor antagonist N-piperidino-5-[4-chlorophenyl]-1-[2,4 dichlorophenyl]-4-methyl-3-pyrazolecarboxamide (SR141716A) increased mean arterial pressure, pulse pressure, respiratory rate and survival rate in a dose-related manner (0.1–3 mg/kg), an almost complete recovery of mean arterial pressure, pulse pressure and respiratory rate, and 100% survival at the end of the observation period (2 h), occurring with the dose of 3 mg/kg. The melanocortin ACTH-(1–24) (adrenocorticotropin) also produced in a dose-related manner (0.02–0.16 mg/kg i.v.) a restoration of cardiovascular and respiratory functions, and increased survival rate, an almost complete recovery and 100% survival at the end of the observation period (2 h) occurring with the dose of 0.16 mg/kg. When a subactive dose of SR141716A (0.2 mg/kg; 30% survival) was associated with a subactive dose of ACTH-(1–24) (0.02 mg/kg; 12% survival), a complete reversal of the shock condition was obtained with 100% survival at the end of the 2-h observation period. The present results show that the concurrent inhibition of both endogenous opioid and cannabinoid systems produces a reversal of hemorrhagic shock more effective than that produced by the inhibition of either of them. These data suggest that functional interactions between endocannabinoids and opioid/antiopioid are at work also in the pathophysiology of hemorrhagic shock.

WOS: 000176080900017, PubMed ID: 12020692, The aim of this study was to determine the effects of nimesulide and pentoxifylline on the contractile effects of KCl, carbachol and substance P in the longitudinal muscle of rat ileum during peritonitis. Peritonitis was induced in rat ileum by cecal ligation and puncture. Thirty rats were operated on to induce peritonitis, 10 of which received nimesulide (5 mg/kg, subcutaneously) and 10 of which received pentoxifylline (25 mg/kg, subcutaneously) before the operation; 10 other rats underwent a sham operation and acted as controls. Twenty-four hours after the operation, ileum segments were transferred to isolated organ baths and responses to KCl, carbachol and substance P were recorded. E-max values of KCl, carbachol and substance P were markedly lower (P < 0.05), with no change in the pD(2) values, in the peritonitis group than in the controls. Peritonitis-induced changes in the KCl, carbachol and substance P responses of ileum were significantly restored by nimesulide (P

The effect of GAB-D-Trp(2-Me)-D-Trp(2-Me)-LysNH(2) (EP 80661), GAB-D-Trp(2-Me)-D-Trp(2-Me)-D-Trp(2-Me)-LysNH(2) (EP 60761), GAB-D-Trp(2-Me)-LysNH(2) (EP 91071) and GAB-D-Trp(2-Me)-D-beta Nal-Phe-LysNH(2) (EP 50885), four hexarelin peptide analogues that induce penile erection when injected into the paraventricular nucleus of the hypothalamus of male rats, on the concentration of NO(2)(-) and NO(3)(-) in the paraventricular dialysate was studied in male rats. EP peptides (1 microg) induced penile erection and increased the concentration of NO(2)(-) and NO(3)(-) in the paraventricular dialysate. In contrast, hexarelin (1 microg) was ineffective on either penile erection or paraventricular NO(2)(-) and NO(3)(-). EP peptide-induced penile erection was prevented by the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methylester given into the paraventricular nucleus (20 microg), which also reduced the concomitant increase of NO(2)(-) and NO(3)(-) concentration in the paraventricular dialysate. In contrast, the oxytocin receptor antagonist [d(CH(2))(5)Tyr(Me)(2)-Orn(8)]vasotocin (1 microg) given into the paraventricular nucleus, was ineffective on penile erection and on the NO(2)(-) and NO(3)(-) increase induced by EP peptides, despite its ability to prevent the sexual response induced by the above peptides when given into the lateral ventricles. The present results show that EP peptides induce penile erection by activating nitric oxide synthase in the paraventricular nucleus of the hypothalamus, possibly in the cell bodies of oxytocinergic neurons that control penile erection.

The effects of the oxidant tert-butylhydroperoxide (t-buOOH) on carbachol-stimulated pancreatic secretion in the vascularly perfused rat pancreas have been studied in parallel with [Ca2+]i signalling and amylase output in perifused rat pancreatic acinar cells. Perfusion of the pancreas with t-buOOH (0.1–1 mM) caused a rapid and irreversible inhibition of carbachol-stimulated (3×10−7 M) amylase and fluid secretion. Pre-perfusion of the pancreas with vitamin C and dithiothreitol or a cocktail of GSH and GSH-precursor amino acids provided only marginal protection against the deleterious effects of t-buOOH, even though GSH levels were elevated significantly. In perifused pancreatic acini, repetitive [Ca2+]i spikes evoked by carbachol (3×10−7 M) were sustained for 40 min. t-buOOH (1 mM) acutely increased the amplitude and duration of Ca2+ spikes, then attenuated Ca2+ spiking and subsequently caused a marked and sustained rise in [Ca2+]i. t-buOOH-induced alterations in carbachol-stimulated [Ca2+]i signalling and amylase release in perifused pancreatic acini were prevented by vitamin C. Although vitamin C restored impaired Ca2+ signalling and maintained amylase output in pancreatic acini, it seems likely that oxidative stress inhibits fluid secretion irreversibly in the intact pancreas, resulting in a loss of amylase output. Thus, perturbations in [Ca2+]i signalling may not fully explain the secretory block caused by oxidative stress in acute pancreatitis.

An up to 14-fold increase in total hexokinase activity induced by low-frequency stimulation in rat fast-twitch muscle was followed by a rapid decay in enzyme activity after cessation of stimulation. In vivo labeling revealed that these alterations were related to rapid changes in [35S]methionine incorporation into hexokinase II. A recovery period of 15 h after cessation of stimulation was sufficient to normalize the approximately 30-fold elevated [35S]methionine incorporation.

Ageing leads to an impairment of muscle performance that may result from alteration of sarcolemma excitability. Therefore, we compare sodium channels of native fast-twitch skeletal muscle fibres of 21–26-month-old aged rats and 4–6-month-old young-adult rats, using the patch-clamp method. Extrajunctional sarcolemma of aged-rat fibres presented a higher sodium current density than that of young-rat fibres, which resulted from the presence of a higher number of available channels per membrane area. Open probability and availability voltage-dependence of sodium channels were similar in aged- and young-rat fibres, but permeation property was altered during ageing: aged-rat muscles showed a bimodal distribution of fibres with two values of sodium-channel conductance measured between −40 and 0 mV; a young phenotype with a conductance close to 18 pS overlapping that found in young-rat fibres and an aged phenotype with a lower approximately half conductance. Current–voltage curves extended to −60 and +20 mV showed that the aged-phenotype conductance level resulted from an outward rectification occurring in these aged-rat fibres. Furthermore, in these aged-rat fibres belonging to the aged phenotype, ensemble average sodium currents showed slower activation and inactivation kinetics. Sodium currents of the two phenotypes were blocked by 100 nM tetrodotoxin, therefore excluding possible denervation effect. These age-related modifications in sodium current may contribute to the alteration of muscle excitability and function observed during the ageing process.

The mechanism of the inhibitory effect of imipramine, a lipophilic organic cation on the Na + -dependent transport of l -glutamic acid across intestinal brush-border membrane was investigated. The uptake of l -glutamic acid by intestinal brush-border membrane vesicles was dependent on the concentration of Na + . Fitting of the uptake data in the presence of various concentrations of Na + using Hill equation yielded a Hill coefficient of 2.18. This result suggest that the carrier system of l -glutamic acid has at least two sites for Na + -binding. By the analysis of double reciprocal plot and Dixon-type plot, it was found that imipramine inhibits the transport of l -glutamic acid by interacting competitively with the binding sites of Na + , but not inhibit l -glutamic acid binding site. Moreover, the effect of imipramine on the transport of l -alanine and d -glucose which are co-transported with only one Na + molecule was also suggestive of interaction with the Na + -binding sites on the carrier. These results indicate that the mechanism of the inhibitory effect of imipramine on the Na + -dependent carrier systems is common for all systems regardless of the stoichiometry or substrates.

The dependence of macroscopic gap junctional conductance (g(j)) upon transjunctional voltage (Vj) was examined in 39 paired osteoblast-like (OB) cells from primary cultures using the double whole cell patch clamp technique. OB cells were derived from calvarial explants of new-born rats. Instantaneous current-voltage (Ij-Vj) relationships of OB cell pairs (n = 6) were linear in the entire voltage range (-150Vj150 mV) examined. The steady-state Ij-Vj relationship was non-linear for Vor = +/-60 mV. The curve for the normalised steady-state junctional conductance-voltage relationship (Gss/G0-Vj) was bell-shaped, and was fitted with a two-state Boltzmann equation with a minimum conductance (Gmin) of 0.2-0.3, and a half deactivation voltage (Vo) of +/-83 mV. In two recordings unitary gap junction channel activity was observable. The linear I-V relationships revealed a single channel conductance of approximately 100 pS. Application of parathyroid hormone (10(-8) M) had no effect on the voltage dependence nor the magnitude of macroscopic currents (n = 7).

We have cloned and characterized a new diacylglycerol kinase (DGK) isozyme which is expressed in the retina and the brain of rat. The cDNA contains an open reading frame of 567 amino acid residues with a predicted protein of 64 kDa and shows very high homology to human DGK epsilon. The new DGK isozyme contains two distinctive zinc-finger structures and a putative catalytic domain. This DGK expressed predominantly in the inner and outer nuclear layers of retina. This expression pattern is different from those of the previously cloned DGKs including the human DGK epsilon, suggesting that this DGK isozyme has potential importance in visual functions as was the case in Drosophila retinal cells.

Guanidinopropionic acid (GPA), an analogue of creatine (Cr), is known to inhibit Cr uptake by cells. The metabolic effects of chronic Cr depletion on brain, heart and soleus muscle of rats were studied. In GPA hearts and soleus muscle, total specific creatine kinase (CK) activity was decreased by approx. 40% compared to controls, whereas in brain this same activity was elevated by a factor of two. Immunoblot analysis of soleus mitochondria from GPA rats showed an approximate 4-fold increase in Mi-CK protein and a concomitant 3-fold increase in adenine nucleotide translocator (ANT) protein, when compared to control. In GPA-fed rats, the specific activities of adenylate kinase (ADK) and succinate dehydrogenase were significantly higher in brain and soleus (2-fold), but heart remained the same. However, hexokinase (HK) decreased by approx. 50% both in heart and soleus, indicating that muscle and brain follow different strategies to compensate the energy deficit caused by creatine depletion. Skinned muscle fibres from Cr-depleted soleus attained approx. only 70% maximum state 3 respiration with 0.1 M ADP in the presence of 10 mM Cr compared to 100% in control fibres. This defect in Cr stimulated respiration was also seen in isolated heart mitochondria, but was normal in those from brain. The observed deficit of Cr-stimulated respiration, the significant accumulation of Mib-CK and ANT, concomitant with the formation of Mib-CK rich intra-mitochondrial inclusions shown by electron microscopy, indicate that Mib-CK function and coupling to oxidative phosphorylation (OXPHOS), is impaired in these abnormal mitochondria. In addition, our results show tissue-specific metabolic compensations to Cr depletion.

Previous studies have demonstrated that reactive oxygen species are involved in ischemic injury. The present work was undertaken to determine in vivo the role of xanthine oxidase in the oxygen free radical production during rat liver ischemia and to examine the activity of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) during the same period. Our results indicate a 4-fold increase in xanthine oxidase activity between 2 and 3 hours of normothermic ischemia, in parallel with a decrease in cell viability. Moderate hypothermia delays both events. Under the same conditions, the activity of oxygen radical scavenging enzymes remains unchanged. Moreover, we have compared in vitro the susceptibility of isolated liver cells to an oxidative stress induced by O2·−, H2O2 and OH. Our results reveal that endothelial cells are much more susceptible to reactive oxygen species than hepatocytes, probably because they lack H2O2-detoxifying enzymes. These findings suggest that xanthine oxidase might play a major role in the ischemic injury mainly at the level of the sinusoidal space where most endothelial cells are located.

In intracellular recordings with conventional microelectrodes on rat IMCD cells, we find that hypotonic stress depolarizes membrane voltage and decreases cell input resistance. Ion substitution experiments reveal that these effects are largely due to the activation of a prominent Cl−-conductance. After block of this conductance with dideoxyforskolin a smaller concomitant increase in K+ conductance becomes detectable.

The redox gradient along the sinusoid in the rat liver was studied using a redox scanner, a device based on tissue fluorescence scanning spectroscopy measuring the fluorescence signals of oxidized flavoprotein (FP) and reduced pyridine nucleotide (PN). The FP/(FP + PN) ratio reflects the mitochondrial redox state in the liver tissue. The distribution of mitochondrial redox state on the scanned area is expressed as two-dimensional gray-scale images with a 20 μm resolution. Using this instrument, we have scanned a 2.5 × 2.5 mm area of the frozen rat liver sample to investigate the redox gradient within acini and the effects of glucagon on the changes in the redox distribution. The redox images obtained in the perfused livers showed mosaic patterns implicating a regular heterogeneity of redox state in an acinus. The analysis of gradient curve, furthermore, clarified that the redox level in an acinus decreased sigmoidally from the periportal to the pericentral region. Glucagon, which has been reported to reduce the intracellular redox state, decreased the redox potential in whole acini, especially, in the periportal region, when compared with the perfusion without glucagon. These results strongly indicate an intraacinus heterogeneity of glucagon function, with glucagon selectively operating in the upstream of the sinusoid.

The pathophysiological mechanism of Campylobacter jejuni (enterotoxigenic) induced secretory diarrhoea remains least understood. To investigate the mechanism(s) involved, the unidirectional fluxes of Na+ and Cl− were measured across the C. jejuni live culture infected and control (non infected) rat ileum (unstriped), in vitro by Ussing technique under short circuit conditions, in the presence or absence of: Ca2+ ionophore A23187 (5 μM), 1-verapamil (100 μM), calmodulin (CaM) antagonist W-7 (100 μM), dantrolene (25 μM), protein kinase C (PKC) activator PMA (100 ng/ml) and H-7 (60 μM), selective inhibitor of PKC. There was net absorption of Na+ and enhanced Cl− secretion in infected animals while in control animals there was net absorption of Na+ and marginal secretion Cl−. Ca2+ ionophore A23187 mimicked the effects of C. jejuni infection whereas 1-verapamil had significant antisecretory effect on Na+ and Cl− secretion in infected animals. In vitro measurement of unidirectional 45Ca fluxes in Ussing chamber experiments revealed net absorption of Ca2+ in infected rat ileum as compared to net secretion of Ca2+ in control rat ileum. These observations clearly indicate that there is increased stimulation of Ca2+ uptake from extracellular milieu to the enterocytes during C. jejuni-induced diarrhoea. The intracellular calcium levels (Ca2+]i (as measured by fluorescent probe Fura-2AM) were found to be raised significantly (P < 0.001) in enterocytes isolated from C. jejuni infected ileum as compared to the enterocytes from control ileum. The observed increase in [Ca2+]i in enterocytes isolated from C. jejuni live culture supernatant treated rat ileum further shows the involvement of enterotoxin in diarrhoeal process. Dantrolene decreased significantly C. jejuni-induced net Na+ and Cl− secretion but it could not reverse it to absorption suggesting the partial involvement of Ca2+ mobilised from intracellular stores in mediating secretion. W-7 failed to inhibit the C. jejuni-induced net Na+ and Cl− secretion. In addition the CaM activity estimated in intestinal microvillar core remained same in both the control and C. jejuni infected animals. This indicates that C. jejuni-induced diarrhoea is not mediated through the activation of Ca2+-CaM complex pathway of the Ca2+ messenger system. The PKC activator PMA, induced net secretion of Na+ and Cl− in the control animals but it could not enhance further the C. jejuni-induced Na+ and Cl− secretion, suggesting that there is overlapping effect of PMA and C. jejuni live culture infection. H-7 significantly inhibited (P < 0.001) net Na+ and Cl− secretion in the infected animals but had no effect on basal rat ileal Na+ and Cl− transport in control animals. This again suggest the activation of PKC signal in C. jejuni-induced ileal Na+ and Cl− secretion. Although intracellular cAMP levels were found to be significantly raised in the enterocytes isolated from C. jejuni live culture infected and C. jejuni culture supernatant treated rat ileum, the mechanism of interaction between cAMP and Ca2+ messenger system in infection remains undefined. Taken together, the present results therefore, indicate that enterotoxigenic C. jejuni live culture-induced Na+ and Cl− secretion is a calcium-dependent process where both increased Ca2+ entry from extracellular milieu and Ca2+ mobilization from intracellular stores result in increased enterocyte (Ca2+]i. The activation of PKC further appears to be the important intracellular mediator pathway involved in Ca2+-dependent stimulation of C. jejuni-induced diarrhoea.

Hypercholesterolemia is associated with a higher risk for developing atherosclerotic coronary heart disease. During the past few years, evidence has been increasing that modification of lipoproteins, particularly low-density lipoprotein (LDL) oxidation, might be involved in the pathogenesis of atherosclerosis. To compare these factors metal-dependent and -independent photodynamic methods were used for the screening of several indexes of LDL oxidation. Lipid oxidation has been continuously monitored by the increase of conjugated dienes and verified by iodometric and thiobarbituric reaction assay. A close association between LDL cholesterol concentration (and/or serum cholesterol concentration) and LDL maximum diene formation was found using both methods and different sources of sera. With copper-induced oxidation, highly significant correlation coefficient r = 0.86, and with photo-sensitive oxidation r = 0.84 were noted. The data standardized to protein unit showed a reduced but still significant correlation. The extent of LDL oxidation was also closely related to preformed dienes, i.e., to the data obtained before the start of oxidation (r = 0.91). The rate of LDL oxidation was positively linked to LDL cholesterol using both oxidation methods but with photo-sensitive oxidation the rate was much higher. The lag time was inversely related to LDL cholesterol (standardized data) with Cu2+ induced oxidation but it was absent in the photosensitive oxidation. In animals known to be resistant to spontaneous atherosclerosis (rats, guinea pigs) a prolonged lag time, markedly reduced diene formation and lower LDL cholesterol in LDL in parallel was demonstrated. The fact that, using various methods (epidemiology, arteriography, autopsy), the cholesterol level in men was found positively linked to atherosclerosis development on the one hand, and positively associated to oxidation of human LDL on the other, strongly supports the concept on the important role of LDL oxidative modification in this pathological process.

Subcutaneous injection of a dilute formaldehyde solution (5 or 10%) into a hind paw induced, in the majority of rats, the appearance of ‘mirror pain’: licking the contralateral untreated hind paw 10–60 min after injection. Contralateral licking activity was much less frequent than the ipsilaterally directed one, but the overall intensities of the two responses were positively correlated. Qualitatively, the two behaviours were similar. Functional activity levels of the lumbar spinal cord, as revealed by the 2-deoxyglucose (2-DG) technique, were increased bilaterally over the first hour after unilateral hind limb formalin injection in unanesthetized, freely moving rats. The enhancement of the [14C]2-DG uptake could be detected both in dorsal and ventral horns, as well as in the gray matter surrounding the central canal, and the anterolateral and dorsolateral funiculi. These metabolic changes may reflect an enhancement of the functional activity of both interneuronal pools and units projecting to supraspinal centers, giving rise to a referred contralateral pain.

Rat aortic smooth muscle rings without endothelial cells were subjected to α 1 -adrenoceptor stimulation. We measured the contractile state of the smooth muscle cells and the formation of inositol phosphates (InsPs) on receptor stimulation. Using different extracellular calcium-containing solutions (2.5 mM, 0.1 mM and Ca 2+ -free) enabled us to discriminate three contractile phases after noradrenaline (10 −5 M) stimulation: an initial fast contraction (15 s) and a fast and slow component of the sustained contraction, which was established 10 min after stimulation. Under normal calcium conditions in the presence of 10 mM LiCl the formation of Ins(1,4,5)P 3 was increased predominantly after stimulation, while the formation of Ins(1,3,4)P 3 , Ins(1,3,4,6)P 4 , Ins(1,3,4,5)P 4 , Ins(3,4,5,6)P 4 and InsP 5 /InsP 6 was also stimulated. The cAMP-inducing agent forskolin (0.5 μ M) induced a relaxation of the basal tone and increased the level of the InsP 4 isomers. The noradrenaline-induced contractile responses as well as the formation of InsP fractions mentioned were inhibited by forskolin. Further an increase in the formation of phosphatidylinositol bisphosphate was observed. It is concluded that in rat aorta InsPs and in particular Ins(1,4,5)P 3 is involved in the different contractile phases caused by α 1 -adrenoceptor stimulation. The relaxation induced by forskolin under these circumstances could be explained by an interaction of forskolin, most likely via the formation of cAMP, with InsPs formation at the level of phospholipase C activation.