Oligo-[alpha]-deoxynucleotides covalently linked to an intercalating agent. Double helices with parallel strands are formed with complementary oligo-[beta]-deoxynucleotides.

An oligo-[alpha]-deoxynucleotide of sequence (5')d(TCTAAACTC) (3') was synthesized using the alpha-anomers of deoxynucleosides and its 5'-phosphate was covalently linked to a 9-amino acridine derivative via a pentamethylene linker. Two oligo-[beta]-deoxynucleotides containing the complementary sequence in either the 5'----3' or the 3'----5' orientation were synthesized using natural [beta]-deoxynucleosides. Complex formation was investigated by absorption and fluorescence spectroscopies. No change in spectroscopic properties was detected with the anti-parallel [beta] sequence. Absorption changes were induced in the visible absorption band of the acridine derivative at 2 degrees C when the acridine-substituted oligo-[alpha]-deoxynucleotide was mixed in equimolecular amounts with the complementary [beta]-sequence in the parallel orientation. Hypochromism was observed in the UV range. The fluorescence of the acridine derivative was quenched by the guanine base present in the second position of the complementary sequence. Cooperative dissociation curves were observed and identical values of melting temperatures were obtained by absorption and fluorescence. An increase in salt concentration stabilized the complex with a delta Tm of 8 degrees C when NaCl concentration increased from 0.1 to 1 M. These results demonstrate that an oligo-[alpha]-deoxynucleotide covalently linked to an intercalating agent is able to form a double helix with an oligo-[beta]-deoxynucleotide. The two strands of this [alpha]-[beta] double helix adopt a parallel 5'----3' orientation. The acridine ring is able to intercalate between the first two base pairs on the 5'-side of the duplex structure.     

Rate of degradation of [alpha]- and [beta]-oligodeoxynucleotides in Xenopus oocytes. Implications for anti-messenger strategies.

End-labelled oligodeoxynucleotides were injected into Xenopus laevis oocytes and their degradation products were analysed by high-performance ion-exchange liquid chromatography after various times of incubation. The oligonucleotides were synthesised with either the natural [beta] anomers or the synthetic [alpha] anomers of deoxynucleotide units. Oligo-[beta] deoxynucleotides are short-lived inside oocytes (half-life approximately equal to 10 min). Covalent attachment of an intercalating agent to the 3'-phosphate and of a methylthiophosphate group at the 5'-end protects oligodeoxynucleotides against 3'- and 5'-exonucleases, respectively. The half-life of such substituted oligodeoxynucleotides is increased to 40 minutes. Oligo-[alpha]-deoxynucleotides are quite resistant to both endo and exonucleases inside Xenopus oocytes. After 8 hours only 40% of a 16-mer oligo-[alpha]-deoxynucleotide were hydrolysed. The rapid degradation of oligo-[beta]-deoxynucleotides suggests that efficient inhibition of translation in Xenopus oocytes involves an RNase H-induced hydrolysis of mRNAs hybridized to oligo-[beta]-deoxynucleotides.     

Sequence-specific recognition, photocrosslinking and cleavage of the DNA double helix by an oligo-[alpha]-thymidylate covalently linked to an azidoproflavine derivative.

A 3-azidoproflavine derivative was covalently linked to the 5'-end of an octathymidylate synthesized with the [alpha]-anomers of the nucleoside. Two target nucleic acids were used for this substituted oligo-[alpha]-thymidylate: a 27-mer single-stranded DNA fragment containing an octadeoxyadenylate sequence and a 27-mer duplex containing eight contiguous A.T base pairs with all adenines on the same strand. Upon visible light irradiation the octa-[alpha]-thymidylate was photocrosslinked to the single-stranded 27-mer. Chain breaks were induced at the crosslinked sites upon piperidine treatment. From the location of the cleavage sites on the 27-mer sequence it was concluded that a triple helix was formed by the azidoproflavine-substituted oligo-[alpha]-thymidylate with its complementary oligodeoxyadenylate sequence. When the 27-mer duplex was used as a substrate cleavage sites were observed on both strands after piperidine treatment of the irradiated sample. They were located at well defined positions which indicated that the octathymidylate was bound to the (dA)8.(dT)8 sequence in parallel orientation with respect to the (dA)8-containing strand. Specific binding of the [alpha]-octathymidylate involved local triple strand formation with the duplex (dA)8.(dT)8 sequence. This result shows that it is possible to synthesize sequence-specific molecules which specifically bind oligopurine-oligopyrimidine sequences in double-stranded DNA via recognition of the major groove hydrogen bonding sites of the purines.     

Enzymatic amplification of translation inhibition of rabbit beta-globin mRNA mediated by anti-messenger oligodeoxynucleotides covalently linked to intercalating agents.

The effects of anti-messenger oligodeoxynucleotides, covalently linked to an intercalating agent, on translation of rabbit beta-globin mRNA, were investigated both in wheat germ extract and in microinjected Xenopus oocytes. A specific inhibition of beta-globin synthesis was observed in both expression systems with a modified 11-mer covalently linked to an acridine derivative. In injected oocytes a more efficient block was observed with this modified oligonucleotide than with its unsubstituted homolog. This was ascribed to stacking interactions of the intercalating agent with base pairs which provide an additional stabilization of the [mRNA/DNA] hybrid. We demonstrated that in wheat germ extract, the modified and unmodified oligonucleotides behaved similarly due to the presence of a high RNaseH activity. RNaseH was also present, although to a lesser extent, in the oocyte cytoplasm. This anti-messenger DNA-induced degradation of target mRNA resulted in amplified efficiency of hybrid-arrested translation. This additional mechanism might provide anti-sense DNAs with an advantage over anti-sense RNAs.     

Synthesis and binding properties of oligo-2'-deoxyribonucleotides conjugated with triple-helix-specific intercalators: benzo[e] and benzo[g] pyridoindoles

DNA binding compounds, such as benzo[e] (BePI) and benzo[g] pyridoindole (BgPI) derivatives, exhibit preferential stabilization of triple helices. We report here the synthesis of a series of pyrimidine triple-helix-forming oligo-2'-deoxyribonucleotides conjugated with these molecules. BePI was coupled to the 5-position of 2'-deoxyuridine via two linkers of different sizes attached to its 11-position and placed at either the 5'-end, inside the sequence, or at both the 5'-end and the internal positions using periodate oxidation of a diol-containing oligonucleotide followed by reductive coupling with amino-linked BePI. The same BePI derivatives were also linked to the oligonucleotide chain via internucleotidic phosphorothiolate or phosphoramidate linkages. A mixture of diastereoisomers was prepared as well as separate pure Rp and Sp isomers. A BePI derivative, with two different linkers attached to its 3-position, and BgPI derivatives were also linked to the 5-position of a 2'-deoxyuridine located at either the 5'-end or inside the sequence, as well as to the beta- anomeric position of an additional 2'- deoxyribose placed inside the sequence. The binding properties of these oligonucleotide-benzopyridoindoles conjugates with their double-stranded DNA target was studied by absorption spectroscopy.     

Changes in the Accumulation of [alpha]- and [beta]-Tubulin Isotypes during Cotton Fiber Development

The expression of [alpha]- and [beta]-tubulin proteins in developing fibers and several other tissues of cotton (Gossypium hirsutum, cv Texas Marker 1) have been analyzed by immunoblots of one- and two-dimensional gels utilizing anti-tubulin antibodies as probes. As a percentage of total protein, fibers had greater amounts of tubulin than did hypocotyls, roots, leaves, or cotyledons. Both [alpha]- and [beta]-tubulin, having apparent molecular masses of approximately 50 kD and isoelectric points between pH 5 and pH 6, were resolved on a single two-dimensional gel. Under the conditions used, [alpha]-tubulin was less acidic in the isoelectric focusing dimension and migrated slightly faster in the sodium dodecyl sulfate dimension than did [beta]-tubulin. Nine [alpha]-tubulin isotypes that formed two distinct groups were identified on immunoblots of two-dimensional gels. The three most abundant [alpha]-tubulin isotypes were common to all tissues examined. Seven distinct [beta]-tubulin isotypes were also identified. Although their level of accumulation differed, four of the [beta]-tubulin isotypes were common to all tissues. Preferential accumulation of isotypes was more apparent in fibers than in the other tissues examined. Two [alpha]-tubulin isotypes and two [beta]-tubulin isotypes showed preferential accumulation in 10- and 20-d postanthesis fibers, respectively.     

An acridine-linked oligodeoxynucleotide targeted to the common 5' end of trypanosome mRNAs kills cultured parasites

Anti-messenger oligodeoxynucleotides covalently linked to an intercalating agent were tested for their ability to inhibit translation of Trypanosoma brucei mRNAs in a cell-free system. The sequence of these oligodeoxynucleotides was complementary to part of the 35-nucleotide (nt) sequence which is present at the 5' end of all trypanosome mRNAs (the so-called mini-exon sequence). In a rabbit reticulocyte lysate, a nonadeoxynucleotide linked to an acridine derivative, specifically inhibited protein synthesis from T. brucei mRNAs much more efficiently than unmodified oligodeoxynucleotides of similar length. These oligodeoxynucleotides were tested on cultured trypanosomes. The acridine-linked nonadeoxynucleotide had a lethal effect on the parasites. No effect was observed with the homologous unmodified 9-mer nor with those 9-mers linked to the acridine derivative which were not complementary to the mini-exon sequence. These effects are probably a result of hybrid formation between the anti-messenger and mini-exon sequence. Trypanocidal activity of the acridine-modified nonadeoxynucleotide is most likely due to (i) increased affinity for its target, (ii) improved resistance to 3' exonucleases, and (iii) promoted membrane penetration of living parasites.     

Poly(rA) binding of alpha-anomeric and beta-anomeric tetrathymidylates covalently linked to an intercalating oxazolopyridocarbazolium. Determination of the binding parameters

We have investigated by means of absorbance measurements at 310 nm the binding of alpha-anomeric or beta-anomeric tetrathymidylates covalently substituted at their 3' end by an intercalating agent (oxazolopyridocarbazolium), to poly(rA). Taking into account the strong autoaggregation of the free ligands, we have derived the binding parameters corresponding to the [alpha] and the [beta] ligands. The affinity of the alpha-anomer for poly(rA) is higher than the affinity of the beta-anomer in accordance with the Tm studies conducted on such a system.     

Triplex formation with alpha anomers of purine-rich and pyrimidine-rich oligodeoxynucleotides.

Nuclease-resistant alpha anomers of pyrimidine-rich CT- and purine-rich GA- and GT-containing oligonucleotides were investigated for their triplex-forming potential and compared with their corresponding nuclease-sensitive beta anomers. Both 23mer CT-alpha and 23mer CT-beta had quite similar triplex binding affinities. Synthetic 23mer GT-alpha oligonucleotides were capable of triplex formation with binding affinities slightly lower than corresponding 23mer GT-beta oligonucleotides. The orientation of third strand GT-alpha binding was parallel to the purine strand of the duplex DNA target, whereas the orientation of third strand GT-beta binding was found to be antiparallel. Triplex formation with both GT oligonucleotides showed the typical dependence on magnesium and temperature. In contrast, 23mer GA-alpha oligonucleotides did not support triplex formation in either orientation under a variety of experimental conditions, whereas the corresponding 23mer GA-beta oligonucleotides demonstrated strong triplex formation in the antiparallel orientation. GA-alpha oligonucleotides covalently conjugated to acridine were similarly unable to demonstrate triplex formation. GA-alpha oligonucleotides, in contrast to GT-alpha oligonucleotides, were capable of self-association, detectable by gel retardation and UV spectroscopy, but competing self-association could not fully account for the lack of triplex formation. Thus for in vivo triplex gene regulation strategies using GT oligonucleotides the non-natural alpha anomer may be a feasible alternative to the natural beta anomer, allowing for a comparable degree of triplex formation without rapid cellular degradation. However, alpha anomeric inversion does not appear to be a feasible alternative in applications involving GA oligonucleotides.     

Mapping of nucleotide-depleted mitochondrial F1-ATPase with 2-azido-[alpha-32P]adenosine diphosphate. Evidence for two nucleotide binding sites in the beta subunit

Photolabeling of nucleotide binding sites in nucleotide-depleted mitochondrial F1 has been explored with 2-azido [alpha-32P]adenosine diphosphate (2-N3[alpha-32P] ADP). Control experiments carried out in the absence of photoirradiation in a Mg2+-supplemented medium indicated the presence of one high affinity binding site and five lower affinity binding sites per F1. Similar titration curves were obtained with [3H]ADP and the photoprobe 3'-arylazido-[3H]butyryl ADP [( 3H]NAP4-ADP). Photolabeling of nucleotide-depleted F1 with 2-N3[alpha-32P]ADP resulted in ATPase inactivation, half inactivation corresponding to 0.6-0.7 mol of photoprobe covalently bound per mol F1. Only the beta subunit was photolabeled, even under conditions of high loading with 2-N3[alpha-32P]ADP. The identification of the sequences labeled with the photoprobe was achieved by chemical cleavage with cyanogen bromide and enzymatic cleavage by trypsin. Under conditions of low loading with 2-N3[alpha-32P]ADP, resulting in photolabeling of only one vacant site in F1, covalently bound radioactivity was located in a peptide fragment of the beta subunit spanning Pro-320-Met-358 identical to the fragment photolabeled in native F1 (Garin, J., Boulay, F., Issartel, J.-P., Lunardi, J., and Vignais, P. V. (1986) Biochemistry 25, 4431-4437). With a heavier load of photoprobe, leading to nearly 4 mol of photoprobe covalently bound per mol F1, an additional region of the beta subunit was specifically labeled, corresponding to a sequence extending from Gly-72 to Arg-83. The isolated beta subunit also displayed two binding sites for 2-N3-[alpha-32P]ADP. When F1 was first photolabeled with a low concentration of NAP4-ADP, leading to the covalent binding of 1.5 mol of NAP4-ADP/mol F1, with the bound NAP4-ADP distributed equally between the alpha and beta subunits, a subsequent photoirradiation in the presence of 2-N3[alpha-32P]ADP resulted in covalent binding of the 2-N3[alpha-32P]ADP to both alpha and beta subunits. It is concluded that each beta subunit in mitochondrial F1 contains two nucleotide binding regions, one of which belongs to the beta subunit per se, and the other to a subsite shared with a subsite located on a juxtaposed alpha subunit. Depending on the experimental conditions, the subsite located on the alpha subunit is either accessible or masked. Unmasking of the subsite in the three alpha subunits of mitochondrial F1 appears to proceed by a concerted mechanism.     

Monoclonal Antibodies to the [alpha]- and [beta]-Subunits of the Plant Mitochondrial F1-ATPase

We have generated nine monoclonal antibodies against subunits of the maize (Zea mays L.) mitochondrial F1-ATPase. These monoclonal antibodies were generated by immunizing mice against maize mitochondrial fractions and randomly collecting useful hybridomas. To prove that these monoclonal antibodies were directed against ATPase subunits, we tested their cross-reactivity with purified F1-ATPase from pea cotyledon mitochondria. One of the antibodies ([alpha]-ATPaseD) cross-reacted with the pea F1-ATPase [alpha]-subunit and two ([beta]-ATPaseD and [beta]-ATPaseE) cross-reacted with the pea F1-ATPase [beta]-subunit. This established that, of the nine antibodies, four react with the maize [alpha]-ATPase subunit and the other five react with the maize [beta]-ATPase subunit. Most of the monoclonal antibodies cross-react with the F1-ATPase from a wide range of plant species. Each of the four monoclonal antibodies raised against the [alpha]-subunit recognizes a different epitope. Of the five [beta]-subunit antibodies, at least three different epitopes are recognized. Direct incubation of the monoclonal antibodies with the F1-ATPase failed to inhibit the ATPase activity. The monoclonal antibodies [alpha]-ATPaseD and [beta]-ATPaseD were bound to epoxide-glass QuantAffinity beads and incubated with a purified preparation of pea F1-ATPase. The ATPase activity was not inhibited when the antibodies bound the ATPase. The antibodies were used to help map the pea F1-ATPase subunits on a two-dimensional map of whole pea cotyledon mitochondrial protein. In addition, the antibodies have revealed antigenic similarities between various isoforms observed for the [alpha]- and [beta]-subunits of the purified F1-ATPase. The specificity of these monoclonal antibodies, along with their cross-species recognition and their ability to bind the F1-ATPase without inhibiting enzymic function, makes these antibodies useful and invaluable tools for the further purification and characterization of plant mitochondrial F1-ATPases.     

High-yield synthesis of a [3H]ethylenediamine ditetrodotoxin derivative

[3H]Tetrodotoxin [( 3H]TTX) and a [3H]ethylenediamine derivative of TTX are the most widely used ligands for the study of the Na channel. The former ligand presents a low specific radioactivity (1 Ci/mmol) while the latter is highly labeled (30 Ci/mmol). However, its two-step synthesis, i.e., mild oxidation followed by coupling of [3H]ethylenediamine, has been described with a low overall yield of 1.7%. In this work, more favorable experimental conditions are defined for the limiting reaction, i.e., the oxidation step, using [14C]testosterone as a model molecule. Applied to the oxidation of tetrodotoxin, this procedure produces yield values of 30-50%, as determined by high-performance liquid chromatography. Moreover, two oxidized TTX molecules appear to be covalently linked to [3H]ethylenediamine, yielding a new labeled tetrodotoxin derivative with a specific radioactivity of 45 Ci/mmol and a dissociation constant of 0.6 nM for electroplax membranes.     

Synthesis and biological evaluation of pyrido[3',2':4,5]furo[3,2-d]pyrimidine derivatives as novel PI3 kinase p110alpha inhibitors

4-Morpholin-4-ylpyrido[3',2':4,5]thieno[3,2-d]pyrimidine 2a was discovered in our chemical library as a novel p110alpha inhibitor with an IC(50) of 1.4 microM. By structural modification of 2a, the 2-aryl-4-morpholinopyrido[3',2':4,5]furo[3,2-d]pyrimidine derivative 10e was discovered as a p110alpha inhibitor with approximately 400-fold greater potency than 2a. Evaluation of isoform selectivity showed that 10e is a potent inhibitor of p110beta. Furthermore, 10e showed anti-proliferative activity in various cell lines, including multi-drug resistant MCF7/ADR-res cells, and was effective against HeLa human cervical tumor xenografts in nude mice.     

Strand orientation of [alpha]-oligodeoxynucleotides in triple helix structures: dependence on nucleotide sequence.

The aims of the present theoretical study of the conformations of [alpha]-oligodeoxynucleotides forming triple helices with DNA duplexes are to understand the structural and energetic factors involved in [alpha]-triple helix formation by means of energy minimization, and to explain the experimentally observed dependence of strand orientation on the nucleotide sequence. It is found that the energetically preferred orientation of the [alpha]-oligonucleotide with respect to the homopurine strand depends on the sequence of the homopurine.homopyrimidine tracts. This is a consequence of the structural heteromorphism of base triplets in the intrinsically more stable reverse Hoogsteen hydrogen bonding configuration. Practical rules are proposed for determining the orientation of the nuclease-resistant [alpha]-oligodeoxynucleotide strand which will form the most stable triple helix.     

Rebeccamycin analogues from indolo[2,3-c]carbazole

Glycosylated indolocarbazoles related to the antibiotic rebeccamycin represent an important series of antitumor drugs. In the course of structure-activity relationship studies, we report the synthesis of two new derivatives containing an indolo[2,3-c]carbazole chromophore instead of the conventional indolo[2,3-a]carbazole unit found in the natural metabolites. The N-methylated compound 8 containing one glucose residue behaves as a typical DNA intercalating agent, as judged from circular and electric linear dichroism measurements with purified DNA. In contrast, the bis-glycosylated derivative 7 containing a glucose residue on each indole nitrogen has lost its capacity to form stable complexes with DNA. DNA relaxation experiments reveal that the two drugs 7 and 8 have weak effects on human DNA topoisomerase I. The modified conformation of the indolocarbazole chromophore is detrimental to the stabilization of topoisomerase I-DNA complexes. The lack of potent topoisomerase I inhibition leads to decreased cytotoxicity but, however, we observed that the DNA-intercalating mono-glycosyl derivative 8 is about 5 times more cytoxic than the bis-glycosyl analogue 7. The study suggests that the naturally-occurring indolo[2,3-a]carbazole skeleton should be preserved to maintain the topoisomerase I inhibitory and cytotoxic activities.     

The acridine ring selectively intercalated into a DNA helix at various types of abasic sites: double strand formation and photophysical properties.

The interactions between the intercalating agent and the three types of abasic sites: abasic frameshift, apurinic and apyrimidinic, were investigated. 9-amino-6-chloro-2-methoxyacridine (ACMA), whose spectroscopic properties are strongly perturbed by the environment, was selected as the intercalating agent. The optically pure threoninol derived from the reduction of L-threonine was used as an artificial abasic site mimicking the ring-opened natural ribose. In order to secure the selective intercalation to the adjacent abasic site, ACMA and the abasic site were connected through a tri- pentamethylene linker. These modified oligonucleotides covalently linked to an ACMA molecule at the internucleotide site having the same base-sequence were synthesized using the acridine-phosphoramidites. Although all the modified oligonucleotides lack a nucleobase at the intervening position, these double strands showed high thermal stability. The pentamethylene linker and the apyrimidinic systems were especially stabilized. At the same time, sharpness of the absorption spectra and a new fluorescent band of the acridine, due to the fixation of the environment around ACMA, were observed. Therefore, it is concluded that the acridine binds preferentially to the apyrimidinic site rather than the frameshift abasic site and that the surroundings of the acridine are strictly fixed at the microenvironmental level.     

Isolation and characterization of a new hemoglobin derivative cross-linked between the alpha chains (lysine 99 alpha 1----lysine 99 alpha 2)

Bis(3,5-dibromosalicyl) fumarate and a number of related bifunctional reagents react preferentially with oxyhemoglobin to cross-link the beta chains within the 2,3-diphosphoglycerate-binding site. In this report we describe a new derivative cross-linked between the alpha chains which is formed specifically in the reaction with deoxyhemoglobin. X-ray crystallographic studies show that the cross-link lies between Lys-99 alpha 1 and Lys-99 alpha 2, spanning the central cavity of the tetramer. Lys-99 alpha 1 and Lys-99 alpha 2 are located within a cluster of charged residues very near the middle of the hemoglobin molecule. In oxyhemoglobin, this site is completely inaccessible to the cross-linking agent. Competition experiments with inositol hexaphosphate indicate that the compound enters the central cavity in deoxyhemoglobin through the cleft between the alpha chains. Despite the presence of the cross-link between the alpha chains, the modified hemoglobin remains highly cooperative. The Hill coefficient for HbXL99 alpha is 2.6. The oxygen affinity of the cross-linked derivative is decreased by approximately 2-fold; at pH 7.0 in the presence of 0.1 M NaCl the P50 is 13.9 mm Hg compared to 6.6 mm Hg for HbA. This difference appears to be due to relatively small changes in both KR, the association constant for binding of oxygen to the R state, and the allosteric constant L. Surprisingly, the isoelectric point of oxyHbXL99 alpha is almost identical to that of oxyHbA, whereas in the deoxy form the isoelectric point of the cross-linked derivative is decreased relative to native hemoglobin as expected due to the loss of the two positive charges of the modified amino groups. In agreement with these findings, the alkaline Bohr effect of HbXL99 alpha is decreased by more than 50%. Earlier studies argue strongly against the possibility that Lys-99 alpha is directly responsible for this large fraction of the Bohr effect in HbA. Analysis of the structure suggests that in the cross-linked derivative Glu-101 beta, which is in close proximity to Lys-99 alpha in oxyhemoglobin, becomes an acid Bohr group.     

8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid stimulates GABA release from interneurons projecting to CA1 pyramidal neurons in the rat hippocampus via pre-synaptic alpha7 acetylcholine receptors

Nicotinic acetylcholine (ACh) receptors, such as alpha7, alpha3beta4 and alpha4beta2 receptors in the hippocampus, are suggested to modulate neurotransmitter release. 8-[2-(2-Pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) (100 nM), a linoleic acid derivative, potentiated responses of alpha7, alpha3beta4 and alpha4beta2 ACh receptors expressed in Xenopus oocytes that are blocked by 3-(1-[dimethylaminopropyl] indol-3-yl)-4-[indol-3-yl] maleimide (GF109203X), a selective inhibitor of protein kinase C (PKC), except for alpha3beta4 ACh receptors. DCP-LA enhanced the nicotine-triggered release of GABA from rat hippocampal slices in the presence of tetrodotoxin in a bell-shaped dose-dependent manner at concentrations ranging from 10 nM to 10 microM, although DCP-LA by itself had no effect on GABA release. The DCP-LA action was inhibited by GF109203X or alpha-bungarotoxin, an inhibitor of alpha7 ACh receptors, but not by mecamylamine or dihydro-beta-erithroidine, an inhibitor of alpha3beta4 and alpha4beta2 ACh receptors. A similar effect on GABA release was obtained with 12-O-tetradecanoylphorbol 13-acetate, a PKC activator. DCP-LA (100 nM) also enhanced GABA release triggered by choline, an agonist of alpha7 ACh receptors, but not 3-[2(s)-azetidinylmethoxy] pyridine, an agonist of alpha4beta2 ACh receptors. In addition, DCP-LA (100 nM) increased the rate of nicotine-triggered GABA(A) receptor-mediated miniature inhibitory post-synaptic currents, monitored from CA1 pyramidal neurons of rat hippocampal slices, and the effect was also inhibited by GF109203X or alpha-bungarotoxin but not by mecamylamine. Thus, the results of the present study indicate that DCP-LA stimulates GABA release by enhancing activity of pre-synaptic alpha7 ACh receptors present on the GABAergic terminals of interneurons that transmit to CA1 pyramidal neurons via a PKC pathway.