Cytotoxicity of spin trapping compounds.

Spin trapping compounds are used frequently to detect free radicals released by cells. Their cytotoxicity has to be considered in order to prevent perturbations of normal cell growth and viability. Eleven spin traps (eight nitrones and three nitroso traps) have been tested for their effects on bovine aortic endothelial cells (toxicity range, 50% survival rate). The lowest cytotoxicity was found for 5,5-dimethylpyrroline-1-oxide and 2,2,4-trimethyl-2H-imidazole-1-oxide whereas nitrosobenzene and 2-methyl-2-nitrosopropane exerted the strongest cytotoxic effects. In addition, three nitronyl nitroxides were tested. Their cytotoxicity was found to be dependent on substitution, and the toxic concentration of a lipophilic derivative was found to be more than two orders lower as compared to a hydrophilic derivative. The results of this study indicate that most spin traps can be used in cell cultures at customary (i.e. millimolar) concentrations; caution is recommended when nitroso spin traps are applied to cells.

Gonadotropin-releasing hormone (GnRH) analogs: relationship between their structure, proteolytic inactivation and pharmacokinetics in rats.

There are two types of superactive agonists of gonadotropin-releasing hormone (GnRHa-I: (D-amino acid)6-GnRH and GnRHa-II: (D-amino acid)6-(desGly)10-GnRH- ethylamide) the high hormonal activity of which is understood to be due to their higher receptor affinity and their higher proteolytic stability as compared with the native GnRH sequence. Using the soluble fractions of various rat tissues in studies on the inactivation of GnRH peptides, we confirmed the higher proteolytic resistance of GnRHa-II, but not of D-Phe6-GnRH (GnRHa-I) and of another analog, D-Trp3-D-Phe6-GnRH, as compared with GnRH. The exact behaviour of the peptides during degradation was found to be dependent on the peptide concentrations used, showing the importance of using conditions as near to the physiological ones a possible. Towards the membrane fractions, however, the order of degradability was found to be GnRH much greater than D-Phe6-GnRH much greater than D-Trp3-D-Phe6-GnRH. The pharmacokinetic consequences of the different proteolytic degradabilities of the GnRH peptides, observed in rats, were a moderate increase in the biological half-life of D-Phe6-GnRH by 2.5-fold, as compared with GnRH, and a small increase in half-life of D-Trp3-D-Phe6-GnRH by 1.4-fold when compared with D-Phe6-GnRH. Whereas no intact GnRH was recovered in rat urine, small amounts of D-Phe6-GnRH (about 1% of dose) and high amounts of D-Trp3-D-Phe6-GnRH (25.5%) were excreted into urine. Combining the biochemical and pharmacokinetic data, it is concluded that proteolytic stability of GnRH analogs in pharmacological terms means stability towards membrane enzymes (pharmacologically-related stability) and that designing analogs with further increased proteolytic stability will be of only limited consequences with respect to their biological half-lives, the glomerular filtration rate of the kidney becoming the determining factor in the peptide clearance.

Gonadotropin-releasing hormone (GnRH) pharmacokinetics: peptide hormone pharmacokinetics needs clarification.

The plasma level curves of the peptide hormone gonadotropin-releasing hormone (GnRH) after its intravenous, intramuscular, and intraperitoneal administration into rats were fitted according to a two- (i.v.) and one-compartment model (i.m., i.p.), respectively. From the pharmacokinetic parameters it is concluded that urinary excretion and proteolytic degradation by kidney and liver are not sufficient to fully account for the clearance of the hormone and that, therefore, proteolytic degradation by tissues may play a role for the elimination of GnRH. This may be generally true with other short peptide hormones. The GnRH pharmacokinetics is shown as an example to underline that there presently exist problems of interpreting pharmacokinetic data of peptide hormones and that there is a need for a close interplay between biochemical and pharmacokinetic studies on peptide hormones for their pharmacokinetic behaviour to be understood.

Influence of continuous levels of fentanyl in rats on the mu-opioid receptor in the central nervous system.

The highly potent and efficacious mu-opioid agonist fentanyl was SC infused into rats with submaximal analgesic doses (0-1.14 mumol/kg/day) continuously for 8 days, checked by the constant daily urinary recovery of intact drug (0.43 +/- 0.031% of the daily dose). Tail-flick latencies measured at 24 (day 1) and 48 h (day 2) after starting the infusion were increased in a dose-dependent fashion compared with those before the infusion (day 0). However, at day 8, the latencies were increased only weakly, not significantly, revealing tolerance to the antinociceptive activity of fentanyl. Fentanyl at all doses showed no significant effect on the capacity (Bmax) and affinity (Kd) of the mu-opioid receptor binding of DAMGO to whole brain (Bmax 126.2 +/- 3.00 fmol/mg protein, Kd 1.00 +/- 0.04 nM) and spinal cord (Bmax 48.24 +/- 2.71 fmol/mg protein, Kd 1.93 +/- 0.13 nM) membranes gained from the rats after killing them at day 8. Gpp(NH)p increased the Kd for brain and spinal cord sites by 3.09 and 2.65, respectively, independent of the fentanyl dose. The infusion with fentanyl did not after the basal and forskolin-stimulated adenylate cyclase activity in the whole brain membranes, nor did it change the inhibition of the forskolin-stimulated activity by DAMGO. It is concluded that, in rats, constant long-term body levels of highly potent mu-agonists result in a tolerant state that, however, does not produce overall changes in the parameters of their specific receptor sites in the CNS, i.e., receptor capacity and affinity, and in the events closely related to them, i.e., their regulation by GTP and of adenylate cyclase. This does not exclude such possible changes to be restricted to specific regions in the CNS.

Novel caged compounds of hydrolysis-resistant 8-Br-cAMP and 8-Br-cGMP: photolabile NPE esters.

The application of the 1-(2-nitrophenyl)ethyl (NPE) moiety as a photolabile ligand for the release of hydrolysis-resistant 8-Br-cAMP and 8-Br-cGMP was examined. NPE-caged 8-Br-cAMP and 8-Br-cGMP liberate 8-Br-cAMP and 8-Br-cGMP during irradiation with ultraviolet light. The synthesis procedure resulted in diastereoisomeric mixtures, which were chromatographically separated into the axial and equatorial isomers of NPE-caged 8-Br-cAMP and 8-Br-cGMP. The hydrolytic stability, solubility and photochemical properties of these derivatives were compared to the previously reported 4.5-dimethoxy-2-nitrobenzyl (DMNB) compounds. We found that the axial isomers of NPE-caged 8-Br-cAMP and 8-Br-cGMP had a considerably better solvolytic stability than the respective equatorial isomers as well as the DMNB-caged derivatives. Their usefulness for physiological studies was examined in a mammalian cell line expressing the cyclic nucleotide-gated (CNG) ion channel of bovine olfactory sensory neurons.

Does a relationship exist between the quality of stress and the motivation to ingest alcohol?

Previous experimental findings on the relationship between emotional stress and motivation to ingest alcohol are contradictory. To obtain information about this relationship we tested the effects on alcohol consumption in rats subjected to two types of chronic unavoidable stressors, intermittent immobilization and social isolation, which differ in their influence on the functional state of the endogenous opioid system. To characterize the nature and magnitude of the stress induced by these stressors, we measured their effects on functional parameters which have a close relationship to the regulatory influence of endogenous opioid peptides (endogenous opioid dependence, pain sensitivity, blood pressure). Our investigations have shown that chronic intermittent immobilization, which induced development of endogenous opioid dependence, presumably due to activation of endogenous opioid systems, did not produce increased alcohol consumption. On the contrary, chronic social isolation, which did not induce development of endogenous opioid dependence, was followed by a significant increase in alcohol consumption. It is concluded that not all types of stress produce increased alcohol consumption, but that the effect on the endogenous opioid system may be a decisive factor in determining whether a stressor produces increased alcohol consumption.

[Is the prediction of LD50 using cell cultures generally valid?]. / Besitzt die Vorhersage der LD50 mit Hilfe der Zellkultur Allgemeingültigkeit?

In this article we use the linear regression model to compare the values of IC50 and LD50 p.o. (mice and rats) taken from the literature and estimated in our laboratory. By this analysis it was found that for different substance classes a similar relationship exists between IC50 and LD50, even if different cell types or cellular activities were used for estimating the IC50 on the one hand and different modes of substance application in animal toxicity tests on the other hand. The minimum and maximum values of the measured LD50 (LD50G) registered in animal experiments deviated from the theoretical LD50 values (LD50T) on the (log transformed) regression line in approximately 77 per cent of the cases by the factor FG less than or equal to log 5 only. The linear correlation between IC50 and LD50 is to be expected only for such substances which do not act specifically via higher levels of integration, cell type specific reactions or metabolites, respectively. These and previously reported results indicate a certain general validity not only for the positive linear correlation between IC50 and LD50 but also for the predicting the LD50 in a relatively narrow dosage range on the basis of in vitro estimated IC50 values. With these results new possibilities were opened for reducing animal experiments to estimate LD50.

Oxygen radicals attenuate the contractility of skinned muscle fibres from the pig myocardium.

This study deals with the influence of oxygen radicals on the contraction of skinned muscle fibres from pig myocardium. The radicals were generated by xanthine-xanthine oxidase (X/XO) or by Fe2+/H2O2 (Fenton system). Addition of the X/XO to the incubation medium (KCl/imidazole) induced a depression of the contractility which was dependent from the incubation time and the X/XO concentration. The maximum contraction in the presence of high concentrations of free calcium ions (pCa 4.32) decreased to 52.0 +/- 15.5% (p < 0.01). The EC50 of calcium ions inducing fibre contraction increased from 2.82 +/- 0.66 mumol/l to 5.47 +/- 2.06 mumol/l (p < 0.05). The Hill plot of contraction versus concentration of calcium ions was shifted to the right and the maximum of contractility was attenuated. Replacement of X/XO by the Fenton system was without significant effects on the fibre contractility. Addition of 5.10(-4) mol/l APP 210-533 (3-amino-6-methyl-5-phenyl-1,2- dihydropyrid-2-on), a known "calcium sensitizer", increased the fibre contractility in radical impaired fibres, too. This may indicate that the radicals did not impair the troponine complex. Oxygen radicals were detected by electron spin resonance spectroscopy spin trapping using 5,5-dimethylpyrroline-1-oxide. Superoxide radicals were found in the presence of X/XO whereas addition of Fe2/H2O2 to the incubation medium resulted in the formation of hydroxyl radical adducts. The appearance of additional adducts observed in both system is discussed. The experiments indicate that free radicals can interact with components of the skinned fibre (probably with contractile proteins of the myocardial muscle cells) resulting in an impairment of the contractility.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+ sensitization of myocardial force and actomyosin ATPase by (D-Ala2, Met5) enkephalinamide.

The presence of proenkephalin mRNA and proenkephalin peptides in cardiac muscle cells suggests the local production of enkephalins in the myocardium. Yet, the effects of these peptides on the function of the contractile proteins are unknown. The effects of (D-Ala2, Met5) enkephalinamide (DALA) on the activity of the actin stimulated Ca, Mg-myosin ATPase in myofibrils and on the contractility and the activity of the related actomyosin ATPase of chemically skinned muscle fibres from pig myocardium were studied. In this article, it is shown that the myofibrillar actomyosin ATPase as well as the contractility and the actomyosin ATPase in skinned fibres are sensitized to Ca2+ ions by DALA. 10(-11) -10(-6) mol/l DALA decrease the effective concentration of Ca2+ stimulating the myofibrillar ATPase activity by 50% (EC50) from 4.0.10(-5) to 1.5.10(-5) mol/l (p < 0.05). The magnesium dependent myosin ATPase activity at low Ca2+ concentration (10(-9) mol/l) is increased. The EC50 values of Ca2+ for both force development and the related actomyosin ATPase activity of skinned fibres are decreased by DALA (10(-11) -10(-5) mol/l) from 2.5.10(-6) to 2.0.10(-6) mol/l (contractions; p < 0.01) and from 2.0.10(-6) to 1.3.10(-6) mol/l (ATPase activity; p < 0.01). The tension cost (ATPase/tension) of the fibres is unchanged by DALA. In conclusion, the results demonstrate a Ca2+ sensitization of the contractile proteins by low concentrations of DALA, indicating a direct regulatory involvement of enkephalins in the regulation of myocardial contractility. These results correspond with the positive inotropic effects of enkephalins in isolated heart muscle cells.

Angiotensin-converting enzyme and other peptidolytic enzymes in human semen and relations to its spermatologic parameters.

Angiotensin-converting enzyme (ACE) and other enzymes of the renin-angiotensin system (RAS) occur in human semen in high activities. In contrast to bull ejaculates, not all zinc-dependent metallopeptidases are found to be in close correlation to the microscopically determined semen parameters; such a relationship was established only partly for the ACE. On the other hand, the RAS-dependent spermatozoa-bound enzymes, inclusive ACE, uniformly show negative correlations to the spermatologic parameters of human semen. These results, for the first time, point to different functions of the sperm-cell-bound (testicular) and of the seminal plasma (pulmonary) ACE activities.

Nociceptin (orphanin FQ): high-affinity and high-capacity binding site coupled to low-potency stimulation of guanylyl-5'-O-(gamma-thio)-triphosphate binding in rat brain membranes.

G protein activation by the agonist-occupied nociceptin- (orphanin FQ-) receptor in rat cerebral cortex was studied by characterizing the nociceptin-stimulated binding of the radiolabeled guanylyl triphosphate (GTP) analog 35S-guanylyl-5'-O-(gamma-thio)-triphosphate (GTPgammaS). Using 3H-Tyr14- and 125I-Tyr14-nociceptin in saturation and displacement receptor binding studies, a single high-affinity (Kd 21.6-116.7 pM) and high-capacity binding site for nociceptin (orphanin FQ) in membranes and sections of rat cerebral cortex was identified. Stable GTP analogs and NaCl lowered the affinity only moderately by 2- to 3-fold, but under these conditions nociceptin stimulated the binding of 35S-GTPgammaS to G proteins in the membranes with a potency about 100-fold lower (EC50 9.11 nM). It was estimated that this stimulation was due to a 29-fold increase in the affinity from Kd 45. 8 to 1.57 nM of only about 6.5% of the basal binding sites for GTPgammaS, and that at least 10 G protein binding sites could be stimulated by one receptor site. The link of this nociceptin-stimulated binding of GTP to the nociceptin receptor was further evidenced by the specificity of stimulation, as seen with nociceptin, nociceptin(1-13), D-Ala7-nociceptin and nociceptin(1-9), which paralleled that of their receptor affinities. Furthermore, the distribution in rat brain regions of the binding of 35S-GTPgammaS stimulated by nociceptin differed from that stimulated by the mu opioid agonist [D-Ala2, N-Me-Phe4, Gly5-ol)]-enkephalin. Especially, no stimulation by nociceptin was observed in caudate putamen, where also the absence of ORL1 receptors had been reported. The putative coupling of the high-affinity nociceptin receptor to the low-potency stimulation of GTPgammaS binding in rat cerebral cortex might be explained by the switch of a low part of occupied nociceptin binding sites to a very low-affinity state being stabilized at high peptide concentrations and catalytically stimulating the GTP binding.

[Effect of growth factor preparations on the proliferation of in vitro cultivated aortic endothelial cells]. / Wirkung von Wachstumsfaktor-Präparationen auf die Proliferation in vitro kultivierter Aorten-Endothelzellen.

A functionally and structurally well-defined calf aortic endothelial cell line has been used to characterize tissue preparations with mitogenic activity by means of cell proliferation kinetics. The following preparations were tested: An extract from corpora lutea (pig), produced by stepwise ammonium sulfate precipitation (CL-S3), a fraction obtained from CL-S3 extract by ion exchange chromatography (CM-S4), and a chorioallantoic membrane preparation (CAM preparation). Under optimal cultivation conditions 100 micrograms CL-S3, 5 micrograms CM-S4, or 100 micrograms CAM preparation per ml medium increased the cell number and growth rate of the endothelial cells in approximately like manner after an incubation period of 48 hours. Compared with CL-S3 these results demonstrate a 20 fold enrichment of the mitogenic activity in the CM-S4 fraction. Under suboptimal cultivation conditions (i.e. a low cell number at the seeding and a reduced nutritive quality) 100 micrograms CL-S3 per ml medium stimulated the cell proliferation up to 12 times at the end of a 6-days-cultivation-period. These results are discussed in connection with the use of these preparations in the angiogenesis research and with respect to the application of selected preparations in cell culture media in order to reduce the serum concentrations.

Plasma and urine salsolinol in humans: effect of acute ethanol intake on the enantiomeric composition of salsolinol.

The tetrahydroisoquinoline (TIQ) salsolinol (SAL), a condensation product of dopamine and pyruvate or acetaldehyde, is one of the neuropharmacologically active alkaloids in mammals. Previous HPLC studies have shown that the R-enantiomer of SAL is largely predominant, or is the only enantiomer in the urine of healthy subjects, whereas the S-enantiomer was found predominant in the urine of alcoholics. An enzymatic pathway for SAL formation that is influenced by chronic alcohol intake was proposed. However, our analyses showed that the SAL detectable in human urine and plasma is racemic, at least in healthy subjects. No change of the enantiomeric distribution was observed after an acute alcohol ingestion (1 g alcohol/kg body weight). Using a new method for the resolution of the SAL enantiomers and gas chromatography mass spectrometry analysis, the SAL enantiomers were quantified in the urine and plasma of 24 subjects before and after the intake of alcohol. Special dietary conditions were observed to avoid interferences by the SAL of the foodstuff. Although the distribution of SAL enantiomers was not changed after alcohol intake, the total urinary SAL output and the plasma concentration of SAL were significantly influenced in different ways. Only five subjects showed a significant increase both in plasma SAL concentration and in the total urinary SAL output, whereas 19 subjects showed decreased or unchanged SAL levels after alcohol administration. Data also show that only the subjects with low baseline levels (mean of 0.148 ng SAL/ml plasma) tend to increase SAL levels after ethanol ingestion, which may imply some genetic basis for the response.