Effects of azelastine on contractility, action potentials and L-type Ca(2+) current in guinea pig cardiac preparations.

Azelastine is used for symptomatic relief of allergic rhinitis and asthma bronchiale. In vitro studies in smooth muscle cells from guinea pig trachea and ileum demonstrate that the drug blocks L-type Ca(2+) current (I(Ca, L)). However, for safety reasons, it is important to know whether azelastine also affects cardiac I(Ca, L) in therapeutically relevant concentrations. We have therefore studied the effects of azelastine on I(Ca, L) in guinea pig ventricular myocytes using standard whole-cell patch-clamp technique. Force of contraction and action potentials from isolated papillary muscles of the same species were also investigated at physiological temperature (36 degrees C). Azelastine (30 microM) significantly reduced force of contraction, shortened action potential duration, and depressed maximum upstroke velocity. I(Ca, L) was elicited by 200-ms-long clamp steps from -100 to 0 mV (one pulse every 3 s). Azelastine blocked I(Ca, L) reversibly and concentration-dependently with an IC(50) of 20.2+/-1.3 microM and a Hill coefficient of 1.1. At 10 microM, azelastine shifted steady-state inactivation by 5 mV (n=7) to more negative potentials. The time course of I(Ca, L) inactivation could be described by a double exponential function. Azelastine (10 microM) significantly shortened the slow inactivation time constant (tau(s)) from 54.2+/-2.8 ms under control conditions to 38.7+/-2.9 ms (n=16) in the presence of drug. Azelastine also reduced low-voltage-activated Ca(2+) currents with a similar IC(50) value (24 microM, at -35 mV). Since the therapeutic plasma concentrations are in the order of 10-100 nM, we conclude that azelastine does indeed affect also cardiac I(Ca, L), but the concentrations required are at least two orders of magnitude larger than those obtained during drug therapy.

Inosine(15.1) hammerhead ribozymes for targeting the transthyretin-30 mutation.

The most common cause of hereditary amyloidosis (HA) is the val30met mutation in the transthyretin protein (TTR-met30). The mutation is caused by a mononucleic substitution from G to A (GUC to AUC) in the transthyretin gene resulting in the exchange for the amino acids valine to methionine in the corresponding protein sequence. The aim of our work was the development of a specific cleavage of TTR-30 mRNA using hammerhead ribozymes. We chemically modified nuclease stable hammerhead ribozymes to target the TTR-30 mRNA with high specificity. The exchange of adenosine(15.1) with inosine(15.1) in the catalytic core of the hammerhead ribozyme resulted in a change of the cleavable target sequence from N(16.2)U(16.1)H(17) to N(16. 2)C(16.1)H(17) without loss in ribozymal activity (Nucleic Acids Res. 26, 2279-2285, 1998). This modification allowed a specific cleavage of the TTR-30 mutation ("gCC Gug" to "gCC Aug"). In vitro experiments with TTR-30 mRNA demonstrated that the RNase stable inosine(15.1) hammerhead ribozyme cleaved the TTR-30 mRNA with 100% specificity and with a velocity of 0.23 min(-1), whereas no cleavage occured in the wildtype mRNA of TTR. In conclusion, the development of this NCH specific hammerhead ribozyme represents a promising tool for future in vivo therapeutic application for TTR-met30 induced hereditary amyloidosis.

Extending the cleavage rules for the hammerhead ribozyme: mutating adenosine15.1 to inosine15.1 changes the cleavage site specificity from N16.2U16.1H17 to N16.2C16.1H17.

In this paper, we show that an adenosine to inosine mutation at position 15.1 changes the substrate specificity of the hammerhead ribozyme from N16.2U16.1H17to N16.2C16.1H17(H represents A, C or U). This result extends the hammerhead cleavage triplet definition from N16.2U16.1H17to the more general N16.2Y16.1H17. Comparison of cleavage rates using I15.1ribozymes for NCH triplets and standard A15.1 ribozymes for NUH triplets under single turnover conditions shows similar or slightly enhanced levels of reactivity for the I15. 1-containing structures. The effect of I15.1 substitution was also tested in nuclease-resistant 2'- O -alkyl substituted derivatives (oligozymes), showing a similar level of activity for the NUH and NCH cleaving structures. The availability of NCH triplets that can be targeted without loss of efficiency increases the flexibility of ribozyme targeting strategies. This was demonstrated by an efficient cleavage of an HCV transcript at a previously inaccessible GCA site in codon 2.

Pharmacological differentiation between neuronal and recombinant glutamate receptor channels expressed in Xenopus oocytes.

To determine the molecular components of neuronal glutamate receptors, it is important to identify pharmacological tools that allow differentiation between different glutamate receptor types. Here, we utilized the naphthalene derivative Evans Blue (EB) and a collection of other subtype-specific compounds (polyamine toxins, concanavalin A, cyclothiazide) to compare the pharmacological profile of neuronal and recombinant glutamate receptors GluR1-GluR6 expressed in Xenopus oocytes. Submicromolar concentrations of EB selectively reduced the activity of homomeric glutamate receptors GluR1, GluR2(Q) and GluR4. Applied at concentrations above 100 microM, EB potentiated kainate responses of receptors GluR1, GluR3 and GluR4, while receptors GluR2(Q) and GluR6(Q) were completely blocked. Similar experiments were performed on identified neurones in brain slices and after injection of rat brain RNA in Xenopus oocytes. Neuronal kainate responses were (i) potentiated by 100 microM cyclothiazide, (ii) slightly blocked after preincubation in 10 microM concanavalin A, and (iii) not significantly affected by either low (< 1 microM) or high (> 100 microM) concentrations of EB. Their pharmacological properties were markedly different from those of recombinant glutamate receptor channels GluR1-GluR6 investigated in heterologous expression systems.

Pharmacokinetics of a synthetic, chemically modified hammerhead ribozyme against the rat cytochrome P-450 3A2 mRNA after single intravenous injections.

Modulation of gene expression via nucleic acid sequence-specific intervention represents a new paradigm for drug discovery and development. Ribozymes are small RNA structures capable of cleaving RNA target molecules in a catalytic fashion. A 2'-O-allyl-modified hammerhead ribozyme designed to cleave the messenger RNA of cytochrome P-450 3A2 was administered to rats via 0.25 mg intravenous injections to investigate the disposition of this compound. The chemically modified ribozyme binds to serum albumin and can be displaced by phosphorothioate oligonucleotides. A biphasic plasma clearance with a distribution half-life of 12 min and an elimination half-life of 6.5 h was observed. A volume of distribution of 2.1 l/kg indicates perfusion into tissues well beyond the vascular system. The chemically modified ribozyme can be detected intact in the plasma up to 48 h after injection. Metabolic degradation of the chemically modified ribozyme occurs at unmodified ribonucleotides, leaving the 2'-O-allyl-modified sites intact. Recovery of intact chemically modified ribozyme was 1.9% of the administered dose at 12 h along with significant metabolites. The renal clearance of the intact ribozyme is an average 34.3 ml/h. The tissue distribution of the chemically modified ribozyme at 48 h is primarily to kidney and liver but the only detected material is a single 27-mer metabolite that has been cut in the unmodified GAAA region. The brain concentration of the prominent 27-mer metabolite is greater than that observed in the lung or spleen. Examination of tissues reveals no morphological evidence of toxicity. These data strongly support the potential utility of synthetic, 2'-O-allyl-modified hammerhead ribozymes as therapeutic agents in vivo.

A single amino acid determines the subunit-specific spider toxin block of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor channels.

Joro spider toxin (JSTX) is one of the most potent antagonists of glutamatergic AMPA/KA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate) receptor channels in invertebrates and vertebrates. A differential blocking effect on certain types of glutamatergic synapses--e.g., parallel and climbing fiber synaptic inputs to rat cerebellar Purkinje neurons--has been shown by using a synthetic analog of the spider toxin. By investigating the molecular basis of the JSTX action on the recombinant AMPA/KA receptors GluR1-GluR4 and GluR6 expressed in Xenopus oocytes, we found that submicromolar concentrations of JSTX exert a subunit-specific block. Thus, receptor subunits forming a receptor channel with a linear current-voltage (I-V) relationship (GluR1/2, GluR2/3, and GluR6) were not affected, while receptor subunits with rectifying I-V relationships (GluR1, GluR3, GluR4, and GluR1/3) were reversibly blocked by JSTX. By using receptor-subunit mutants obtained by site-directed mutagenesis, we have identified a single amino acid position (glutamine in the proposed second transmembrane domain) that is critical for the JSTX block. Since this site has previously been shown to control the I-V relationship of the AMPA/KA receptor channel and to participate in the regulation of the channel's permeability for calcium ions, our findings suggest that JSTX binds close to the central pore region of the channel.

Identification of a subunit-specific antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor channels.

Excitatory synaptic transmission in the mammalian central nervous system is mediated predominantly by glutamate receptor (GluR) channels of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate (AMPA/KA) receptor type. A major improvement in our understanding of glutamatergic synaptic transmission has been achieved after the identification of quinoxalinediones (e.g., 6-cyano-7-nitroquinoxaline-2,3-dione) as specific antagonists of AMPA/KA receptors. In addition to their effects on neurons, quinoxalinediones were also shown to block glutamate-induced responses mediated by recombinant AMPA/KA receptor channels expressed in heterologous systems, irrespective of their particular subunit composition. Here we report the identification of an AMPA/KA receptor antagonist that selectively blocks a subset of AMPA/KA receptors. We found that Evans blue, a biphenyl derivative of naphthalene disulfonic acid, blocks at low concentrations (IC50 = 355 nM for the subunit combination GluR1,2) KA-mediated responses of the subunits GluR1, GluR1,2, GluR1,3, and GluR2,3 expressed in Xenopus oocytes but not responses of GluR3 or GluR6. The blocking action of Evans blue was partially reversible and did not compete with KA for the agonist binding site. These findings suggest not only that Evans blue is a potent tool for elucidating the functional role of specific AMPA/KA receptor subtypes for excitatory synaptic transmission but also that it may also represent a powerful starting point for clinically useful drugs that are able to reduce the excitatory drive in specific neuronal populations of the central nervous system.

Molybdenum-dependent degradation of quinoline by Pseudomonas putida Chin IK and other aerobic bacteria.

Eighteen different aerobic bacteria were isolated which utilized quinoline as sole source of carbon, nitrogen, and energy. Attempts were unsuccessful at isolating anaerobic quinoline-degrading bacteria. The optimal concentration of quinoline for growth was in the range of 2.5 to 5 mM. Some organisms excreted 2-hydroxyquinoline as the first intermediate. Hydroxylation of quinoline was catalyzed by a dehydrogenase which was induced in the presence of quinoline or 2-hydroxyquinoline. Quinoline dehydrogenase activity was dependent on the availability of molybdate in the growth medium. Growth on quinoline was inhibited by tungstate, an antagonist of molybdate. Partially purified quinoline dehydrogenase from Pseudomonas putida Chin IK indicated the presence of flavin, iron-sulfur centers, and molybdenum-binding pterin. Mr of quinoline dehydrogenase was about 300 kDa in all isolates investigated.

[Characterization and pharmacological effect on brain damage as a result of cold injury]. / Zur Charakterisierung und pharmakologischen Beeinflussung der durch Kryoläsion ausgelösten Hirnschäden.

Freezing lesion of brain damages the blood-brain-barrier and induces edema. Both injuries are partially separate processes with different progress and diverse response to pharmacological treatment. A very weak lesion fails in to induce edema despite of an enhanced permeation of Evans blue. The injured permeability responds to treatment with dexamethasone or piracetam whereas the edema does not.

Influence of nootropic drugs on the age-dependent potassium-coupling of transmitter release.

The potassium-induced dopamine release from rat striatum slices shows an age-dependent decline comparable to observations after hypoxia. Pretreatment of aged animals with antihypoxically active nootropic drugs for three weeks results in an improvement of the impaired transmitter release. Simultaneously the slope of the stimulus-release relation is increased and an age-related 50% decrease of the high affinity Ca(++)-ATPase activity (brain P2 fraction) is partially compensated. Like the antihypoxic effect, the effectiveness of nootropic drugs regarding age-dependent changes of neuronal functions probably will consist, above all, in vascular influences of the microcirculation, repair of phospholipids damaged by free radical triggered peroxidation and improvement of stimulus-release coupling.

[Peroxidative vulnerability of synaptosomal high affinity Ca++-ATPase and pharmacologic effects]. / Zur peroxidativen Schädigung der synaptosomalen hochaffinen Ca++-ATPase und ihrer pharmakologischen Beeinflussbarkeit.

The high affinity Ca++-ATPase participates essentially in the regulation of intrasynaptosomal calcium homeostasis. Related to posthypoxically restricted transmitter release, we examined the influence of newly-generated free radicals (ascorbic acid-ferric salt mixture) or sodium dodecyl sulfate in vitro and of a mild hypobaric hypoxia in vivo on the activity of synaptosomal high affinity Ca++-ATPase. Moreover we tested the effectiveness of piracetam, meclofenoxate hydrochloride, pyritinol and verapamil on the changed enzyme activity subsequent to a hypoxic exposure. The activity of synaptosomal high affinity Ca++-ATPase (1.04 +/- 0.03 mumol Pi/mg.h) is reduced by not more than 40% depending on the concentration of the ascorbic acid-ferric salt mixture used but is nearly totally inhibited by sodium dodecyl sulfate (0.2 mg/ml). Hypobaric hypoxia (18 h, 8.7 kPa) decreases the enzyme activity to 0.79 +/- 0.03 mumol Pi/mg.h. Piracetam, meclofenoxate hydrochloride and pyritinol are protectively effective on the decrease of enzyme activity induced by hypoxia. The results emphasize the importance of intact protein-phospholipid interactions for the enzyme activity and support relations between synaptosomal high affinity Ca++-ATPase and transmitter release.

[In vitro malondialdehyde formation in brain structure: a method to characterize antihypoxic properties]. / In vitro-Malondialdehyd-Bildung in Hirnstrukturen: Eine Methode zur Charakterisierung antihypoxischer Eigenschaften.

In vitro generated free radicals (ascorbic acid-ferric salt-mixture or hydrogen-peroxide) result in lipid peroxidation on brain cellular fractions and striatum slices an decrease of the stimulated dopamine release from rat striatum slices. Besides cysteamine, alpha-tocopherol, pyrogallol and chelating agents also tisochromide shows an antioxidative activity on lipid peroxidation induced by ascorbic acid-ferric salt-mixture. Nootropic drugs like piracetam, methylglucamine orotate, meclofenoxate hydrochloride and nicergoline are ineffective to mitochondrial malondialdehyde generation. On the other hand, piracetam exhibits a limited effect on oxidative damage of striatum slices. For that reason, the antihypoxic activity of these drugs is not accompanied by any antioxidative component and presumes a relatively high degree of tissular organization.

Characterization of the antihypoxic activity of tisochromid.

The antihypoxic activity of tisochromid is compared with the well-known restituting effect of piracetam on posthypoxic dopamine release inhibition. In addition to a distinct restituting effect which accelerates the normalization of dopamine release highly, tisochromid exhibits obviously an antioxidative potency which is responsible for a greater effectiveness of the drug when given prehypoxically. Therefore, tisochromid acts simultaneously as a protective and a restituting drug.

An enzyme-linked immunoassay for circulating immune complexes using solid phased goat Clq.

An ELISA procedure was developed for measuring circulating immune complexes (IC), using solid phased goat Clq. The use of purified Clq from this species significantly diminished the background uptake of the enzyme-labeled goat antibodies used in the assay, in comparison with Clq isolated from human, guinea pig or rabbit serum. The test specimen results are reported as micrograms equivalent (microgram eq)/ml of heat aggregated human immunoglobulin G (HAIgG), and are based on standard curves developed with this latter reagent for each assay. The 2 World Health Organization (WHO) reference preparations for immune complex determinations (HAIgG and a human tetanus antitoxin-toxoid immune complex) were assayed by this ELISA procedure, and the results obtained were in very good agreement with the WHO established values. All the reagents in the ELISA, including the lyophilized HAIgG standard and the solid phased Clq are stable for more than one year at 4 degrees C. The range of accurate quantitation in test serum is 2-500 micrograms/ml, when using a 1:100 specimen dilution. The total incubation is less than 2 h, with no preliminary preparation of test specimens. The average concentration of IC reactivity in 126 healthy adults was 6 micrograms eq/ml, and the normal upper limit was determined to be 12 micrograms eq/ml.

[Effect of reserpine on monoamine uptake by rat brain synaptosomes]. / Beeinflussung der Monaminaufnahme in Synaptosomen des Rattenhirns durch Reserpin.

With regard to the inhibition of monoamine-uptake the concentration response curve of reserpine shows a biphasice turn. The first phase produces a maximum dopamin-uptake inhibition of 55% (EC50:8 X 10(-9) mol X 1(-1)) which is of a competitive manner only in the presence of a very low reserpine concentration (1 X 10(-9) mol X 1(-1)). Higher concentrations of reserpine inhibit the dopamine-uptake non competitively. This specific irreversible vesicular reserpine effect used therapeutically forms a plateau within a concentration rank from 10(-7) mol X 1(-1) to 10(-5) mol X 1(-1). A second mode of action of reserpine affects obviously the carrier mechanism located at the synaptosomal membrane (EC50: 5 X 10(-5) mol X 1(-1)). This second phase differs both from the first vesicular reserpine effect and from the synaptosomal action of tricyclic antidepressant drugs by its kinetic behavior (Km, Vmax).

Effect of arecoline on synaptosomal K+-phosphatase and (Na+ + K+)-ATPase.

Synaptosomal fractions and synaptosomal membranes from rat brain tissue were prepared and characterized enzymatically. Arecoline increased both the activity of K+-phosphatase in incubated synaptosomal fractions and the (Na+ + K+)-ATPase activity of synaptosomal membranes by 40% and 78%, respectively. This activation of ion transport processes is believed to be associated with increased ACh synthesis produced by arecoline.