Fenofibrate decreases asymmetric dimethylarginine level in cultured endothelial cells by inhibiting NF-κB activity

Previous investigations have demonstrated that endogenous inhibitors of nitric oxide synthase (NOS), such as asymmetric dimethylarginine (ADMA), contribute importantly to endothelial dysfunction, and that fenofibrate has a protective effect on the endothelium in rats treated with low-density lipoprotein (LDL) by reducing ADMA levels. In the present study, we explored further the possible mechanism underlying inhibition of ADMA generation by fenofibrate in cultured human umbilical vein endothelial cells (HUVECs). Endothelial injury was induced in cultured HUVECs by incubation with oxidative LDL (ox-LDL) and the levels of ADMA, lactate dehydrogenase (LDH), NO and tumour necrosis factor- (TNF-) in the conditioned medium were measured. Cell viability and the activity of dimethylarginine dimethylaminohydrolase (DDAH) and nuclear factor-B (NF-B) in the cultured HUVECs were also determined. Incubation of HUVECs with ox-LDL (100 g/ml) for 24 h markedly elevated ADMA, LDH and TNF- in the conditioned medium and significantly increased the activity of NF-B, concomitantly with a significant decrease in the activity of DDAH and the content of NO. Pretreatment with fenofibrate (3, 10 or 30 M) significantly inhibited the increases in ADMA, LDH and TNF-, attenuated the decreased levels of NO and the decreased activity of DDAH and prevented the activation of NF-B. Similar effects were observed in the presence of pyrrolidine dithiocarbamate (PDTC, 10 M), an antagonist of NF-B. The beneficial effects of fenofibrate on cultured endothelial cells were abolished by MK-886, a specific peroxisome proliferator-activated receptor- (PPAR) antagonist. The present results suggest that fenofibrate inhibits ox-LDL-induced endothelial cell damage by decreasing ADMA and increasing DDAH activity, and the protective effects of fenofibrate on endothelial cells may be related to reduction of NF-B activity by activation of the PPAR receptor.     

Comparative behavioral characterization of the neuroactive steroids 3α-OH,5α-pregnan-20-one and 3α-OH,5β-pregnan-20-one in rodents

Pregnan steroids have been shown to possess anesthetic, hypnotic, anticonvulsant and anxiolytic properties. In this study, two endogenous neuroactive steroid isomers, 3-hydroxy-5-pregnan-20-one (3,5-P) and 3-hydroxy-5-pregnan-20-one 3,5-P), were studied for differences in their pharmacological properties using behavioral assays. 3,5-P and 3,5-P were similar in their potencies and efficacies in blocking pentylenetetrazol-induced seizures in mice (ED₅₀: 3,5-P=2.8 mg/kg and 3,5-P=3.0 mg/kg). Similarly, both neuroactive steroids produced roto-rod deficits within the same range of potency (TD₅₀:3,5-P=18.8 mg/kg and 3,5-P=21.2 mg/kg). However, in animal models of anxiety, subtle differences were observed between the two isomers. In both the light/dark transition test and elevated plus-maze, 3,5-P was more efficacious than 3,5-P, though both compounds had similar potencies. In the Geller-Seifter test, 3,5-P was more potent and efficacious than 3,5-P. Neither compound had significant effects on unpunished responding within the dose range tested. Both compounds produced similar biphasic curves in the locomotor test. All together, the data indicate that 3,5-P and 3,5-P have similar anticonvulsant activity, but the 5-isomer possesses more potent and efficacious anxiolytic properties than the 5-isomer.     

Subclassification of presynaptic α2-adrenoceptors: α2D-autoreceptors and α2D-adrenoceptors modulating release of acetylcholine in guinea-pig ileum

The study was designed to classify in terms of _2A, _2B, _2C and _2D the presynaptic ₂-autoreceptors, as well as the ₂-receptors modulating the release of acetylcholine, in the myenteric plexus-longitudinal muscle (MPLM) preparation of the guinea-pig ileum. A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the MPLM preparation were preincubated with ³H-noradrenaline or ³H-choline and then superfused and stimulated electrically.The stimulation periods used (3H-noradrenaline: 3 trains of 20 pulses, 50 Hz, train interval 60 s; ³H-choline: single trains of 30 pulses, 0.2 Hz) did not lead to ₂-autoinhibition or inhibition of ³H-acetylcholine release by endogenous noradrenaline. The ₂-selective agonist 5-bromo6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) reduced the evoked overflow of tritium in both ³H-noradrenaline and ³H-choline experiments. Most (³H-noradrenaline) or all (³H-choline) of the 10 antagonists shifted the concentration-inhibition curves of UK 14,304 to the right. pK_d values of the antagonists were calculated from the shifts. pK_d values from ³H-noradrenaline experiments correlated with pK_d values from ³H-choline experiments (r = 0.981).It is concluded that ₂-autoreceptors and ₂-heteroreceptors modulating the release of acetylcholine in the MPLM preparation are of the same subtype. Comparison with antagonist affinities for prototypic native ₂ binding sites, binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that both are _2D. The results are consonant with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree, across mammalian or at least across rodent and lagomorph species. The same may hold true for ₂-adrenoceptors on non-noradrenergic neurones.     

Protein Aggregates Seem to Play a Key Role Among the Parameters Influencing the Antigenicity of Interferon Alpha (IFN-α) in Normal and Transgenic Mice

Purpose. During long-term treatment of various malignant or viral diseases with IFN- up to 20% of patients develop anti-IFN- antibodies for as yet unknown reasons. Methods. To address this issue, a mouse model using Balb/C mice was established and the relevance of several potentially anti-IFN- antibodies inducing factors was studied. Results. The model revealed that both a higher frequency of injections and a higher dosage of IFN- were more immunogenic and that the route of administration affected the antibody response to IFN-. The intrinsic immunostimulatory activity of IFN- itself also enhanced the immune response. IFN- protein aggregates (IFN--IFN- and human serum albumin (HSA)-IFN- aggregates), which were recently identified in all marketed IFN- products, were significantly more immunogenic than IFN- monomers. These aggregates broke the tolerance against human IFN- monomers in human IFN- transgenic mice. Conclusions. Based on these animal studies it is proposed that the immune response to IFN- in humans is most probably elicited by a combination of several factors among which IFN- protein aggregates seem to play a key role.     

Comparison of cloned and pharmacologically defined rat tissue α1-adrenoceptor subtypes

Multiple ₁-adrenoceptor subtypes have been defined by pharmacological and receptor cloning techniques, but the precise alignment of cloned and pharmacologically-defined subtypes is still unclear. We have compared the affinities of 8 subtype-selective compounds at three cloned ₁-adrenoceptor subtypes (rat _1B, bovine _1C rat _1A/D) with those previously determined by the same methods in rat spleen, cerebral cortex, and kidney (Naunyn-Schmiedeberg's Arch. Pharmacol. 348: 385–395, 1993). Among all compounds tested to date at cloned ₁-adrenoceptor subtypes (+)-tamsulosin appears to be the most selective with a rank order of potency _1C > _{1A/D 1B}. Affinities for the _1A-selective 5-methyl-urapidil, methoxamine, oxymetazoline, phentolamine and (–)- and (+)-tamsulosin and for noradrenaline and SDZ NVI-085 at the splenic _1B-adrenoceptors and at their low affinity sites in cerebral cortex and kidney correlated best with those at the cloned _1B-adrenoceptor. Affinities of these drugs at their high affinity sites in cerebral cortex (pharmacologically-defined _1A-adrenoceptor) were matched best by those at the cloned _1C-adrenoceptor. Rat kidney appears to contain two chloroethylclonidine-resistant ₁-adrenoceptor subtypes one of which is similar to the cloned at _1C- and one to the cloned _1A/D-adrenoceptor. We conclude that the cloned _1B-adrenoceptor is the genetic correlate of the pharmacologically-defined _1B-adrenoceptor. An ₁-adrenoceptor subtype corresponding to the cloned _1A/D-adrenoceptor appears to exist in rat kidney. Among cloned ₁-adrenoceptor subtypes, the bovine _1C-adrenoceptor bears the closest resemblance to the pharmacologically-defined _1A-adrenoceptor in rat cortex and to one of the chloroethylclonidine-insensitive subtypes in rat kidney.     

Involvement of interleukin-4 in down-regulation of endothelin-1 during gastric ulcer healing: Effect of ebrotidine

Aims: Healing gastric mucosal injury implies an orderly involvement of the signaling molecules that exert their influence on the processes leading to the restoration of the mucosal integrity. In this study, we investigated the effect of antiulcer agent, ebrotidine, on the course of events during gastric ulcer healing by analyzing the expression of interleukin-4 (IL-4), endothelin-1 (ET-1), tumor necrosis factor- (TNF-), and the mucosal activity of caspase-3, and constitutive (cNOS) and inducible (NOS-2) nitric oxide synthase. Methods: Rats with experimentally induced chronic gastric ulcers were administered twice daily for 14 days with either ebrotidine at 100 mg/kg or vehicle, and at different stages of treatment their stomachs were used for macroscopic and biochemical assessments. Results: The ulcer onset was characterized by a massive epithelial apoptosis associated with a significant increase in caspase-3, NOS-2, TNF- and ET-1, and a decline in the mucosal expression of cNOS and IL-4. The healing was reflected in a rapid recovery in IL-4, decrease in apoptosis, caspase-3, TNF-, ET-1 and NOS-2, and a slow recovery in cNOS, and the process was accelerated in the ebrotidine-treated group. In the absence of ebrotidine the expression of IL-4 returned to the level of normal mucosa by the seventh day of healing, and that of ET-1 and TNF- by the fourteenth day. An accelerated ulcer healing (7 days) with ebrotidine treatment was associated with IL-4 recovery by the fourth day, ET-1 by the seventh day, and TNF- and cNOS by the 10th day. However, in both groups of animals the apoptotic DNA fragmentation rate, and the expression of caspase-3 and NOS-2 remained significantly elevated even after the ulcer healed. Conclusions: Our findings suggest that a drop in the mucosal expression of IL-4 at the ulcer onset causes dysregulation of ET-1 production, induction of TNF- and triggers the apoptotic events that affect the efficiency of the repair process. Ebrotidine, by eliciting rapid recovery in IL-4, is capable of suppression the mucosal apoptotic events and accelerate the ulcer healing.     

Plasma concentration of α-methyldopa and sulphate conjugate after oral administration of methyldopa and intravenous administration of methyldopa and methyldopa hydrochloride ethyl ester

Summary The plasma concentrations of free -methyldopa and methyldopa sulphate conjugate were measured in 7 hypertensive patients with normal renal function following -methyldopa (1 g) orally. Five of these patients subsequently received -methyldopa ethyl ester (250 mg) (methyldopate) intravenously and two further patients received 250 mg of -methyldopa intravenously. After oral administration a large amount of total plasma -methyldopa was present as sulphate conjugate. There were wide interindividual differences in the ratio of free: conjugated -methyldopa in plasma (ratio at 4 hours ranged from 3.73 – 0.83) suggesting that individual differences in the extent of sulphate conjugation may occur. There was no close correlation between the degree of conjugation and the fall in arterial pressure. At all time intervals examined, plasma concentrations were higher following intravenous -methyldopa than -methyldopate. The plasma concentration of -methyldopa (free and esterified) 60 minutes after i.v. -methyldopate was 1.7±0.3 µg/ml wile at the same time after the same dose of methyldopa by the same route the mean concentration was 5.9 µg/ml. Although small amounts of sulphate conjugate were detected after i.v. -methyldopate, insignificant quantities of conjugate were found after i.v. -methyldopa. The average fall in mean arterial pressure was 27 mm Hg following i.v. -methyldopa but only 2.7 mm Hg following -methyldopate. These results suggest that sulphate conjugation of -methyldopa occurs in the gastrointestinal tract during absorption. Hydrolysis of -methyldopa ethyl ester does not appear to be instantaneous and pharmacokinetic differences between the ester and free -methyldopa have been demonstrated.     

Possible subdivision of postsynaptic α-adrenoceptors mediating pressor responses in the pithed rat

Summary Additional evidence has been obtained indicating a possible subclassification of postsynaptic -adrenoceptors into ₁ and ₂-subtypes. The pressor responses to the -adrenoceptor agonists L-phenylephrine and guanfacine were quantified after i.v. administration to pithed rats. The -sympatholytic drug yohimbine (1 mg/kg) displaced both dose-response curves to the right, but the effect was greatest for guanfacine. After prazosin (0.1 mg/kg) a 53-fold shift to the right was noticed for the dose-response characteristic of L-phenylephrine. Prazosin antagonized the effect of only the higher doses of guanfacine. The findings indicate that L-phenylephrine and prazosin preferentially interact with ₁-adrenoceptors as agonist and antagonist, respectively. Yohimbine proved less selective than prazosin, but preferentially blocks postjunctional ₂-adrenoceptors in the vascular wall. The results obtained with guanfacine may be interpreted to indicate that this drug acts on ₂-adrenoceptors at lower doses and additionally stimulates ₁-adrenoceptors at higher ones. Preliminary findings with corynanthine and rauwolscine support this interpretation.     

α-Adrenoceptor activity of arylalkylimidazoles is improved byα-methylation and impaired byα-hydroxylation

Summary To investigate the effects of hydroxyl and methyl substitution of the alkyl bridge bond on the-adrenoceptor activity of arylalkylimidazole derivatives, the cardiovascular effects of the molecules were studied in anaesthetized and pithed rats. The compounds studied were 4(5)-substituted imidazole derivatives with a methano, ethano or etheno bridge between the imidazole and the 2-, 2,3- or 2,6-methyl substituted phenyl rings. The hypotensive and bradycardic activities of the molecules in the anaesthetized rat were always reduced by-hydroxylation and usually augmented by-methylation of the bridge between the imidazole and phenyl rings. Hydroxylation was associated with a consistent, marked decrease in vasopressor and sympatho-inhibitory activity in the cardiovascular system of the pithed rat, but a methyl moiety as a bulky substituent in the-position of the alkyl bridge did not decrease but even caused an increase in-adrenoceptor activity in this test system. The detrimental effect of-hydroxylation of the compounds at ₁- and ₂-adrenoceptors supports the notion that the interaction of the imidazoles at-adrenoceptor is different from that of the classical, noradrenaline-like phenethylamines. The results also suggest that the alkyl bridge between the phenyl and imidazole rings of the imidazoles may contribute directly to the binding process.     

Effect of α-hexachlorocyclohexane on metabolism and excretion of pentetrazol (Cardiazol®) in the rat

Summary -Hexachlorocyclohexan (-HCH) has been shown to be a potent anticonvulsant when tested with pentetrazol. In the experiments reported here the question was examined whether or not the anticonvulsant properties of -HCH are based on an acceleration of pentetrazol breakdown in the rat.In this study the rat has been shown to metabolize pentetrazol extensively. The enzymatic activity is located in the microsomal fraction of the liver and requires NADPH and oxygen. It is inhibited by SKF-525 A and carbon monoxide. This is taken as evidence that P-450 containing mixed function oxidases are involved in the breakdown of pentetrazol.-HCH pretreatment leads to an acceleration of pentetrazol break-down by microsome preparations of about 140%. In vivo -HCH pretreated rats eliminate pentetrazol from brain and serum with a half life of 1.3 h, while this is 3.8 h in untreated rats.The earliest point in time at which -HCH pretreatment causes diminished brain levels of pentetrazol has been found 60 min after pentetrazol injection. Prior to this brain levels of pentetrazol were identically in untreated and -HCH-treated rats.As pentetrazol elicits convulsions within the first 30 min following oral or subcutaneous administration of a convulsive dose and -HCH is clearly active as an anticonvulsant under these conditions, the accelerated breakdown of pentetrazol cannot be the cause of the anticonvulsive action of -HCH.Supported by Deutsche Forschungsgemeinschaft.H. W. V. was the holder of a grant from Deutsche Forschungsgemeinschaft.     

Cloricromene in endotoxemia: role of NF-κB

In this study we investigated, for the first time in vivo, the effect of cloricromene, a cumarine derivative, on NF-B activation in endotoxin-treated rats. Endotoxemia was induced in male rats by the intravenous injection of Salmonella typhosa lipopolysaccharide (LPS; 2 mg/kg/i.v.). In vivo treatment with cloricromene (2 mg/kg/i.v.) 30 min before lipopolysaccharide administration reversed the LPS-induced loss in tone of the aortic rings, improved their reactivity to phenylephrine, decreased both nitric oxide (NO) and TNF- serum levels by inhibiting LPS-induced inducible NO synthase and TNF- mRNA expression, and interestingly inhibited LPS-induced NF-B activation. Our data suggest that cloricromene protects rats from LPS by blocking LPS-induced NF-B activation, leading to inhibition of NO and TNF- overproduction and thereby reversing the LPS-induced vascular hyporeactivity.     

Prodrugs of Peptides. 13. Stabilization of Peptide Amides Against α-Chymotrypsin by the Prodrug Approach

Various derivatives of the C-terminal amide group in N-protected amino acid and peptide amides were synthesized to assess their suitability as prodrug forms with the aim of protecting the amide or peptide bond against cleavage by -chymotrypsin. Whereas N-acetylation, N-hydroxymethylation, and N-phthalidylation did not afford any protection but, in fact, accelerated the terminal amide bond cleavage, condensation with glyoxylic acid to produce peptidyl--hydroxyglycine derivatives and, to a minor extent, N-aminomethylation were found to improve the stability of the parent amides. Besides protecting the terminal, derivatized amide moiety toward cleavage by -chymotrypsin, -hydroxyglycine derivatization resulted in a significant protection, by a factor ranging from 5 to 75, of the internal peptide bond in various N-protected dipeptide amides. These derivatives are readily bioreversible, the conversion to the parent peptide or amino acid amide taking place either by spontaneous hydrolysis at physiological pH, as demonstrated for the N-Mannich bases, or by catalysis by plasma, as for peptidyl--hy-droxyglycine derivatives.     

Postjunctional α-adrenoceptors

Summary The postsynaptic -adrenoceptors in rat aorta and in pithed rat were investigated according to their sensitivity to nine -adrenergic agonists and to the selective antagonists yohimbine (₂) and prazosin (₁) and the non-selective one, phentolamine. In addition, in radioligand binding studies, the affinity and selectivity of the drugs were determined on rat cerebral cortex using [³H] yohimbine and [³H] prazosin.On rat aorta, prazosin is 1,000 times more potent than yohimbine against each -adrenoceptor agonist, whether ₁- or ₂-selective. Rat aorta probably contains only ₁-adrenoceptors.Pressor effects in pithed rats are mediated by post-junctional ₁- and ₂-adrenoceptors. The dose-response curve for -methylnorepinephrine in the presence of prazosin, using Hofstee's plots, revealed ₁- and ₂-adrenoceptors, respective proportions being 80.5 and 19.5%     

Vasoconstriction mediated by postsynaptic α2-adrenoceptor stimulation

Summary The postsynaptic -adrenoceptors involved in vasoconstriction brought about by B-HT 933 (2-amino-6-ethyl-4,5,7,8-tetrahydro-6H-oxazolo-[5,4-d]-azepin) administered i.v. to pithed, normotensive rats were characterized. The rate of onset of the hypertensive response to i.v. B-HT 933 is slower than that induced by (–)-phenylephrine, an agonist of ₁-adrenoceptor. The antagonism of the -adrenoceptor blocking drugs rauwolscine, yohimbine and corynanthine was quantified towards B-HT 933-induced increases in diastolic pressure. Rauwolscine (pA₂=7.06) and yohimbine (pA₂=6.83) were effective antagonists, whereas corynanthine proved much less potent (pA₂=5.03). On the basis of the reported selectivity of yohimbine and its two diastereoisomers rauwolscine and corynanthine for ₁- and ₂-adrenoceptor, it is concluded that the postsynaptic -adrenoceptors triggered by B-HT 933 are of the ₂-type. B-HT 933 identifies a subclass of postsynaptic ₂-adrenoceptor in vascular smooth muscle distinct from postsynaptic ₁-adrenoceptor. Both types of -adrenoceptors are likely to be involved in the mediation of vasoconstriction.Preliminary data were presented at the 21th Spring Meeting of the German Pharmacological Soceity, Mainz, March 18–21, 1980 (Timmermans, 1980)     

Chronic effects of miaserin on noradrenaline metabolism in the rat brain: Evidence for a pre-synaptic α-adrenolytic action in vivo

Chronic administration of the pre-synaptic -adrenoreceptor agonist clonidine decreases the concentration of the extra-neuronal metabolite of noradrenaline normetanephrine in the amygdaloid cortex and increases it in the mid-brain. Conversely, blockade of these pre-synaptic receptors by yohimbine increases the normetanephrine concentration in the amygdaloid cortex and decreases it in the mid-brain. Mianserin had a qualitatively similar action to that of yohimbine. When given clinically to rats in combination with clonidine, mianserin antagonizes both the depression of behaviour of the rats in the open field apparatus and also the effects of the -agonist in reducing the concentration of normetanephrine in the amygdaloid cortex. It thus appears that the chronic effects of mianserin are due to an increase in noradrenaline release as a consequence of the inhibition of pre-synaptic -adrenoreceptors.     

Cardiovascular effects of alpha-adrenergic drugs: Differences between clonidine and guanabenz

Summary Guanabenz induced a pressor effect in pithed rats through postsynaptic ₂-adrenoceptors whereas clonidine activated both vascular ₁ and ₂-adrenoceptors. Previous treatment with prazosin, and ₁-antagonist, or depletion of the noradrenergic stores by reserpine produced supersensitivity to the pressor response to clondine only, probably through postsynaptic ₁-adrenoceptors.The hypotension and bradycardia developed in normotensive rats after intravenous guanabenz administration were abolished by prazosin, whereas the central effects of clonidine were antagonized by both prazosin and yohimbine.Selective destruction of central noradrenergic neurons by [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine] (DSP 4) or reserpine plus blockade of catecholamine synthesis by -methyl-p-tyrosine abolished the hypotension and bradycardia produced by guanabenz but merely reduced the bradycardia from clonidine.The present results suggest that, in rats, guanabenz is a selective stimulant of central -autoadrenoceptors antagonized by prazosin whereas at a vascular level guanabenz preferentially activates -adrenoceptors antagonized by yohimbine. The differences observed between the mechanisms by which guanabenz and clonidine produce their central cardiovascular responses might be attributed to their acting on different nuclei.     

Effect of K+ channel blockers on the α2-adrenoceptor-coupled regulation of electrically evoked noradrenaline release in hippocampus

Summary The question whether presynaptic ₂-adrenoceptors regulating noradrenaline release in hippocampus directly couple to tetraethylammonium chloride (TEA) or -dendrotoxin (-DTX)-sensitive K⁺ channels was investigated. Hippocampal slices, prelabelled with [³H] noradrenaline, were superfused in the presence of (+)-oxaprotiline and electrically stimulated with 4 pulses delivered at 100 Hz, in order to avoid autoinhibition due to released noradrenaline.TEA enhanced the evoked [³H]noradrenaline release in rabbit hippocampus in a concentration-dependent manner, yielding an approximately 4-fold increase at 30 mmol/l, whereas the spontaneous outflow of tritium was only slightly affected at this concentration. The ₂-adrenoceptor agonist clonidine, at 10–100 nmol/l inhibited the evoked [³H]noradrenaline release between 77% and 96%. The inhibitory effect of the ₂-agonist was distinctly diminished in the presence of 30 mmol/l TEA but was restored in low Ca²⁺/high Mg²⁺ buffer. Therefore, the diminution of the ₂-agonist effect by TEA observed in experiments with normal Ca²⁺ can be explained by an increase of the Ca²⁺ availability for the release process due to the prolongation of action potentials. In rabbit hippocampus -DTX (10–200 nmol/l) did neither affect the evoked release of [³H]noradrenaline nor its ₂-agonist-induced modulation. However, in rat hippocampus -DTX significantly increased the evoked transmitter release and diminished the effect of clonidine.Taken together, the present data for the rabbit hippocampus exclude the possibility that activation of presynaptic ₂-adrenoceptors inhibits depolarization-evoked [³H]noradrenaline release by inducing an outward K⁺ current through TEA- or -DTX-sensitive K⁺ channels. However, there are species differences between the rabbit and the rat so that in the rat the ₂-adrenoceptors could actually be coupled to K⁺ channels — provided that the release-enhancing properties of -DTX do not account for the ₂-antagonism observed.Correspondence to C. Allgaier at the above address     

α1A and α1B-adrenoceptors enhance inositol phosphate generation in rat renal cortex

Summary We have studied the role of _1A and _1B-adrenoceptors in noradrenaline- and methoxamine-stimulated inositol phosphate accumulation in rat renal cortical slices. [³H]Prazosin binding studies with and without inactivation of _1B-adrenoceptors by chloroethylclonidine treatment suggested that noradrenaline lacks relevant selectivity for ₁-adrenoceptor subtypes. Both agonists stimulated [³H]inositol phosphate accumulation with similar maximal effects. The _1A-selective antagonists 5-methyl-urapidil and (+)-niguldipine inhibited inositol phosphate formation by both agonists with shallow biphasic curves but the high affinity component was only 15%–31% and 38%–41%, respectively. The irreversible _1B-selective antagonist chloroethylclonidine inhibited inositol phosphate generation by both agonists by 54%–57%. In contrast to our previous data in rat cerebral cortical slices; we conclude that in rat renal cortex both _1A- and _1B-adrenoceptors are involved in noradrenaline-and methoxamine-stimulated inositol phosphate generation.     

Tumor necrosis factor α decreases inositol phosphate formation and phosphatidylinositol-bisphosphate (PIP2) synthesis in rat cardiomyocytes

Treatment of neonatal rat cardiomyocytes for 72 h in the presence of tumor necrosis factor (TNF) (10 U/ml) lead to a decrease in basal and ₁-adrenoceptor-induced formation of the calcium-mobilizing second messenger inositol trisphosphate (IP₃) and its metabolites, IP₂ and IP₁, by 35 and 26%, respectively. The synthesis of phosphatidylinositol bisphosphate (PIP₂), the substrate of PI-specific phospholipase C, was decreased by 45% following the TNF (10 U/ml) exposure. Time courses of TNF (10 U/ml)-induced alterations in rat cardiomyocytes showed a parallel decline of basal inositol phosphate formation and PIP₂ synthesis suggesting that the decrease in inositol phosphate formation was due to the reduction in PIP₂ synthesis. As the TNF-induced decrease of PIP₂ synthesis was associated with a decreased synthesis of the phospholipid phosphatidylinositol (PI), the precursor of PIP₂, by 33%, the decreased availability of PIP₂ is apparently, at least in part, the result of the decreased synthesis of PI. As an apparent functional consequence of the decrease in IP₃ formation following the TNF exposure, the ₁-adrenoceptor-mediated induction of arrhythmias by 100 mol/l noradrenaline + 10 mol/l timolol was abolished in TNF-pretreated rat cardiomyocytes.To investigate one of the possible mechanisms of the TNF-induced decrease of PIP₂ formation, the effect of TNF pretreatment on glycerol-3-phosphate dehydrogenase (GDH), a key enzyme of lipogenesis, was studied: Exposure of the rat cardiomyocytes for 72 h to TNF induced a concentration-dependent decrease in GDH activity by maximally 55%.The result presented are consistent with the hypothesis that the decreased basal and ₁-adrenoceptor-induced formation of the second messenger IP₃ observed in chronic endotoxinemia and sepsis may be mediated by a TNF-induced decrease in the synthesis of PIP₂, the substrate of PI-specific phospholipase C. This mechanism occurs following long-term exposure to low TNF/ha concentrations and is apparently distinct from the short-term cardiac effects induced by high concentrations of TNF. Correspondence to: C. Reithmann at the above address     

Involvement of tumor necrosis factor-α in sulfur mustard-induced skin lesion; effect of topical iodine

Sulfur mustard (SM), also termed mustard gas, is a potent vesicant that elicits an inflammatory response upon exposure of the skin. Evaluation of mouse ear 3 h after SM exposure revealed acute inflammatory-cell aggregates in the vascular beds accompanied by strongly TNF--positive neutrophils. Eight hours after SM exposure, this phenomenon became intensified and associated with infiltration into the adjacent dermis. In ear skin topically treated with iodine, however, no inflammatory cells were observed 3 h after SM exposure; 8 h postexposure, blood vessels contained very few TNF--positive inflammatory cells. Since TNF- induction was shown to be associated with reactive oxygen species production, we studied the effect of iodine on activated peritoneal mouse neutrophils. Iodine elicited a concentration-dependent reduction in the oxidative burst of activated neutrophils. Iodine also scavenged hydroxyl radicals generated by glucose oxidase in a concentration-dependent manner. The involvement of TNF- in SM-induced skin toxicity was confirmed by reduction of 49 and 30% in ear edema following administration of 1 and 2 g anti-TNF- antibodies, respectively. These findings were corroborated by quantitative analysis of the histological findings showing 46% reduction in acute inflammation and no signs of subacute inflammation in the treated group, in contrast to the control group treated with SM only. Other epidermal (microblister formation, ulceration, and necrosis) and dermal (neutrophilia, hemorrhage, and necrosis) parameters also showed marked reductions in the antibodies-treated group in comparison to controls. The combination of iodine and antiTNF- antibodies might constitute a new approach for treatment of SM-exposed individuals.U. Wormser is affiliated with David R. Bloom Center for Pharmacy at The Hebrew University.