The selective alpha7 nicotinic acetylcholine receptor agonist PNU-282987 [N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride] enhances GABAergic synaptic activity in brain slices and restores auditory gating deficits in anesthetized rats.

Schizophrenic patients are thought to have an impaired ability to process sensory information. This deficit leads to disrupted auditory gating measured electrophysiologically as a reduced suppression of the second of paired auditoryevoked responses (P50) and is proposed to be associated with decreased function and/or expression of the homomeric alpha7 nicotinic acetylcholine receptor (nAChR). Here, we provide evidence that N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987), a novel selective agonist of the alpha7 nAChR, evoked whole-cell currents from cultured rat hippocampal neurons that were sensitive to the selective alpha7 nAChR antagonist methyllycaconitine (MLA) and enhanced GABAergic synaptic activity when applied to hippocampal slices. Amphetamine-induced sensory gating deficit, determined by auditory-evoked potentials in hippocampal CA3 region, was restored by systemic administration of PNU-282987 in chloral hydrate-anesthetized rats. Auditory gating of rat reticular thalamic neurons was also disrupted by amphetamine; however, PNU-282987 normalized gating deficit only in a subset of tested neurons (6 of 11). Furthermore, PNU-282987 improved the inherent hippocampal gating deficit occurring in a subpopulation of anesthetized rats, and enhanced amphetamine-induced hippocampal oscillation. We propose that the alpha7 nAChR agonist PNU-282987, via modulating/enhancing hippocampal GABAergic neurotransmission, improves auditory gating and enhances hippocampal oscillatory activity. These results provide further support for the concept that drugs that selectively activate alpha7 nAChRs may offer a novel, potential pharmacotherapy in treatment of schizophrenia.

The DBA/2J strain and prepulse inhibition of startle: a model system to test antipsychotics?

RATIONALE: Prepulse inhibition (PPI) of the startle response in mice is increasingly used as a paradigm of sensory gating with potential predictive and construct validity towards schizophrenia. OBJECTIVES: Establishment of a mouse PPI paradigm in which typical and atypical antipsychotic drugs directly improve a low performance PPI. METHODS: Three strains of mice--C57Bl/6J, 129S6/SvEvTac and DBA/2J--were tested in a startle paradigm with three prepulse intensities, 2, 4 and 8 dB above background. RESULTS: Under these conditions, risperidone (0, 0.25, 0.5 and 1 mg/kg i.p.) and clozapine (0, 1, 3 and 9 mg/kg i.p.) improved PPI in all three strains, with order of effect in DBA/2J > 129S6SvEvTac > C57Bl/6J. The DBA/2J strain showed larger PPI-enhancing effects, without disturbing the basal startle response. Two alpha7 nicotinic receptor agonists, GTS-21 (1-10 mg/kg i.p.) and AR-R17779 (1-10 mg/kg i.p.) were inactive in the PPI procedure in DBA/2J mice. CONCLUSIONS: DBA/2J mice were very sensitive to the antipsychotic-like effects of atypical (clozapine) and typical (risperidone) antipsychotics, and this strain is proposed as a model to directly measure sensory gating properties of drugs. Alpha7 Nicotinergic receptor agonists were ineffective in this PPI paradigm.

Leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels.

Leptin, the protein encoded by the obese (ob) gene, is secreted from adipose tissue and is thought to act in the central nervous system to regulate food intake and body weight. It has been proposed that leptin acts in the hypothalamus, the main control centre for satiety and energy expenditure. Mutations in leptin or the receptor isoform (Ob-R[L]) present in hypothalamic neurons result in profound obesity and symptoms of non-insulin-dependent diabetes. Here we show that leptin hyperpolarizes glucose-receptive hypothalamic neurons of lean Sprague-Dawley and Zucker rats, but is ineffective on neurons of obese Zucker (fa/fa) rats. This hyperpolarization is due to the activation of a potassium current, and is not easily recovered on removal of leptin, but is reversed by applying the sulphonylurea, tolbutamide. Single-channel recordings demonstrate that leptin activates an ATP-sensitive potassium (K[ATP]) channel. Our data indicate that the K(ATP) channel may function as the molecular end-point of the pathway following leptin activation of the Ob-R(L) receptor in hypothalamic neurons.

Characterization of the steady-state and dynamic fluorescence properties of the potential-sensitive dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol (Dibac4(3)) in model systems and cells.

The steady-state and dynamic fluorescence properties of the membrane potential-sensitive bis-oxonol dye Dibac4(3) were characterized in vitro using model ligand systems and in vivo in A10 smooth muscle cells by fluorescence microscopy in conjunction with the ACAS imaging system. In the latter system the dye responds to potassium ion-induced jumps in membrane potential with changes in its fluorescence intensity, which follow pseudo-first-order kinetics. The relationship between the magnitude of the changes and the corresponding rate constants excludes the possibility that a simple, one-step equilibrium between extracellular and cytoplasmic dye would be sufficient to account for this phenomenon. The necessity of invoking an additional step suggested that the redistribution of the free dye between the cytoplasm and the exocellular medium is rapid and that the slow step associated with the fluorescence changes may be the interaction of the dye with proteins in the cytoplasm, along the lines proposed by Bräuner et al. (Biochim. Biophys. Acta 771 (1984), 208, 216). The interaction of the dye with BSA and with egg lecithin SUVs was studied as a model for the in vivo phenomenon. The dependence of fluorescence intensity changes on the concentrations of the reagents shows the formation of a reversible dye/albumin complex with a 2/1-stoichiometry, with Kd = 0.03 +/- 0.01 microM and a reversible adsorption to the SUVs with Kd 0.45 +/- 0.08 microM. The fluorescence lifetime of the dye in solution, < 100 ps, results in a high solution steady-state anisotropy. The latter decreases considerably upon binding to BSA, SUVs and A10 cells concomitant with a large increase in the lifetime. With such a short lifetime of the free dye, selective gating of the excitation source or the photodetector should eliminate the high background of the unbound dye and thereby enhance the sensitivity of the system.

ATP-sensitive K+ channel opener acts as a potent Cl- channel inhibitor in vascular smooth muscle cells.

We describe the activation of a K+ current and inhibition of a Cl- current by a cyanoguanidine activator of ATP-sensitive K+ channels (KATP) in the smooth muscle cell line A10. The efficacy of U83757, an analogue of pinacidil, as an activator of KATP was confirmed in single channel experiments on isolated ventricular myocytes. The effects of U83757 were examined in the clonal smooth muscle cell line A10 using voltage-sensitive dyes and digital fluorescent imaging techniques. Exposure of A10 cells to U83757 (10 nM to 1 microM) produced a rapid membrane hyperpolarization as monitored by the membrane potential-sensitive dye bis-oxonol ([diBAC4(3)], 5 microM). The U83757-induced hyperpolarization was antagonized by glyburide and tetrapropylammonium (TPrA) but not by tetraethlyl-ammonium (TEA) or charybdotoxin (ChTX). The molecular basis of the observed hyperpolarization was studied in whole-cell, voltage-clamp experiments. Exposure of voltage-clamped cells to U83757 (300 nM to 300 microM) produced a hyperpolarizing shift in the zero current potential; however, the hyperpolarizing shift in reversal potential was associated with either an increase or decrease in membrane conductance. In solutions where EK = -82 mV and ECl = 0 mV, the reversal potential of the U83757-sensitive current was approximately -70 mV in those experiments where an increase in membrane conductance was observed. In experiments in which a decrease in conductance was observed, the reversal potential of the U83757-sensitive current was approximately 0 mV, suggesting that U83757 might be acting as a Cl- channel blocker as well as a K+ channel opener. In experiments in which Cl- current activation was specifically brought about by cellular swelling and performed in solutions where Cl- was the major permeant ion, U83757 (300 nM to 300 microM) produced a dose-dependent current inhibition. Taken together these results (i) demonstrate the presence of a K(+)-selective current which is sensitive to KATP channel openers in A10 cells and (ii) indicate that the hyperpolarizing effects of K+ channel openers in vascular smooth muscle may be due to both the inhibition of Cl- currents as well as the activation of a K(+)-selective current.

Synthesis and biological activity of spirocyclic benzopyran imidazolone potassium channel openers.

A series of novel spirocyclic benzopyran imidazolones were synthesized as rigid analogues of cromakalim. These compounds cause a dose-dependent membrane hyperpolarization of A10 rat aorta cells. This hyperpolarization was blocked by pretreatment with glyburide, indicating that the spirocyclic benzopyran imidazolones were acting by increasing the open probability of ATP-sensitive potassium channels in A10 cells. Representative compounds also showed potent in vivo activity as hypotensive agents in normotensive rats. Many of the compounds described are much more potent than cromakalim both in vitro and in vivo, with one of the most potent compounds being 2,3-dihydro-2,2-dimethyl-6-nitro-2'-(propylamino)spiro[4H-1-benzopyran- 4,4'-[4H]imidazol]-5'(1'H)-one (5r). It is concluded that the N1' nitrogen of the imidazolone is an effective substitute for the carbonyl oxygen of cromakalim. The rigid spirocyclic ring fusion holds this nitrogen in an optimum orientation relative to the benzopyran ring.

Limitation of myocardial injury with the potassium channel opener cromakalim and the nonvasoactive analog U-89,232: vascular vs. cardiac actions in vitro and in vivo.

Cromakalim has been shown to have anti-ischemic properties, but it also produces profound hypotension upon systemic administration. We hypothesized that U-89,232, a cromakalim analog, would reduce infarct size in an ischemia-reperfusion injury model without hemodynamic alteration. Twenty-four anesthetized, open chest New Zealand White rabbits were instrumented for occlusion of a marginal branch of the left coronary artery. All animals were subjected to coronary artery occlusion (30 min) and reperfusion (2 hr). Study animals received either cromakalim (20 micrograms/kg, i.v.) or U-89,232 (20 micrograms/kg, i.v.), which was given as a pretreatment 30 min before occlusion. Control animals (n = 10) received vehicle (10% dimethyl sulfoxide). At termination of the experiment, the necrotic area and the area at risk were determined with tetrazolium and India ink staining, and infarct size was calculated using planimetry. Treatment with cromakalim produced profound hypotension (greater than 30% decrease in mean arterial pressure), whereas U-89,232 had no such hemodynamic effect. With comparable areas at risk, infarct size (as a percent of risk area) in the control animals was 46.8 +/- 3.4%. Treatment with cromakalim or U-89,232 reduced infarct size to 33.1 +/- 4.4 and 24.4 +/- 4.0%, respectively (P < .05, both compared to control). In vitro studies demonstrate that although both of these compounds shorten the duration of the cardiac action potential, only cromakalim is active in vascular smooth muscle. We conclude that U-89,232 exhibits myoprotection without hypotension, and that its mechanism of action is most likely due to ability to affect cardiac electrophysiology.

High-sulfur protein gene expression in a transgenic mouse.

We analyzed the effect of minoxidil on hair follicles isolated from transgenic mice. These transgenic animals synthesize the reporter enzyme CAT in their hair follicles only during the active phases of hair growth. The recombinant gene used to generate these mice contained the bacterial enzyme CAT under the control of the promoter from the gene of UHS protein. Studies using in situ hybridization showed that UHS proteins are expressed specifically in the matrix cells of the hair follicle during the terminal stages of hair differentiation. Hence the expression of the UHS proteins is a clear sign of active hair growth. With other in situ hybridization studies we demonstrated that CAT mRNA is expressed in differentiating matrix cells of the hair shaft in a location similar to that in which mRNA encodes UHS proteins. Thus we can use the levels of CAT activity as a measure of hair growth. We have confirmed that expression of the transgene is found in hair that is high in anagen and low in catagen follicles. The usefulness of our model was further demonstrated by showing that minoxidil, a drug that stimulates hair growth, increased the expression of CAT in cultured hair follicles. Thus we have demonstrated that expression of this reporter gene is sensitive, hair specific, and also useful for monitoring effects in cultured hair follicles. Hence these transgenic mice provide a model system for studying the biology of hair growth.

Hair-specific keratins: characterization and expression of a mouse type I keratin gene.

A genomic clone for a member of the mouse type I hair keratin protein family has been isolated and analyzed in order to study the regulation of this keratin during the hair growth cycle. The coding sequence is divided into seven exons. The gene structure is typical of keratins in particular and intermediate filaments in general in that the intron-exon borders are not located at the domain borders of the protein. Comparison with a sheep wool keratin gene shows that the splice sites in the two hair keratin genes are found at identical locations relative to the amino acid sequence of the proteins. Similarly, comparison of the promoter areas of these genes shows several areas of nucleotide sequence conservation, including the area around the TATA box and an SV40 core enhancer sequence. In addition, a high degree of sequence identity exists in the fourth intron. In situ hybridization shows that transcripts of this gene are first found in the relatively undifferentiated proximal cortex area in the keratogenous zone of mouse vibrissae.

Localization of TIMP in cycling mouse hair.

TIMP (tissue inhibitor of metalloproteinase) is a glycoprotein inhibitor of metalloproteinases that we hypothesize to be involved in the tissue remodeling that occurs during each hair growth cycle. We examined this hypothesis by studying the expression of TIMP at selected times during a single hair cycle using TIMP-lacZ transgenic mice to localize TIMP gene activity in the hair follicle. TIMP gene induction was visualized by staining mouse back skin for beta-galactosidase (beta-gal) activity. Paraffin sections were analyzed for the localization of TIMP expression. TIMP gene activation appears in hair follicles only during the mid-anagen (the growing stage of the hair cycle) primarily in Henle's layer of the inner root sheath. Some expression of TIMP is also seen in a few connective tissue cells, in the sebaceous gland and in cells at the proximity of the dermal papilla cells in catagen (regressing) and telogen (resting) follicles. These results are consistent with a role for TIMP in cyclic remodeling of connective tissue in hair follicles.

P388 leukaemia cells resistant to the anthracycline menogaril lack multidrug resistant phenotype.

Menogaril is an anthracycline presently in Phase II clinical trials. Menogaril-resistant mouse leukaemia P388 cells were developed in vitro by 4 months of exposure to step-wise increasing concentrations of menogaril after which resistant cells (P388/MEN) were cloned in 320 ng ml-1 menogaril. P388/MEN cells were 40-fold more resistant to menogaril in vitro compared to P388/O and were also resistant in vivo. Resistance to menogaril was stable for at least 2 months in the absence of the drug. The results indicate that P388/MEN, although resistant to an anthracycline, did not display the typical multidrug resistant phenotype. It was not cross-resistant to several structurally unrelated drugs such as actinomycin D, cisplatin, or vinblastine, but it was cross-resistant to the anthracycline, adriamycin. Uptake and efflux of menogaril was similar in sensitive and resistant cell lines. Also, resistance was not reversed by verapamil. No major karyotypic difference was noted between P388/O and P388/MEN. There was no significant amplification or overexpression of the mdr gene in P388/MEN compared to P388/O. In contrast to P388/MEN, P388 cells resistant to adriamycin displayed the typical multidrug resistant phenotype. Glutathione content of P388/MEN cells was similar to that of P388/O and depletion of glutathione did not potentiate menogaril cytotoxicity. Therefore, we conclude that glutathione is not likely to be involved in menogaril resistance to P388/MEN cells.

Hair-specific expression of chloramphenicol acetyltransferase in transgenic mice under the control of an ultra-high-sulfur keratin promoter.

We have generated a transgenic mouse line by microinjection of a chimeric DNA fragment (KER-CAT) containing a hair-specific, murine ultra-high-sulfur keratin promoter (KER) fused to the coding region of the bacterial chloramphenicol acetyltransferase (CAT) gene. A 671-base pair (bp) stretch of the 5' promoter region was used to direct the expression of the CAT gene in this construct. Of the tissues tested for CAT activity in these transgenic animals only skin with growing hair, isolated hair follicles, and microdissected vibrissae showed substantial levels of activity. These are the same tissues where the endogenous ultra-high-sulfur keratin gene is expressed as shown by in situ hybridization. Furthermore, analysis of the CAT activity during the developmental stages of the hair growth cycle shows that the chimeric gene is expressed during the anagen phase of the hair growth cycle; this is the expected time during development for its expression. From these results we conclude that 671 bp of the promoter sequence from the ultra-high-sulfur keratin gene is sufficient to direct the correct development-specific and tissue-specific expression of the reporter gene construct in transgenic mice. The appropriate expression of the KER-CAT construct in transgenic mice is an important step in understanding the regulation of this gene during hair organogenesis.

Exonuclease activity that degrades histone mRNA is stable when DNA or protein synthesis is inhibited.

The induction and repression of histone synthesis during the cell cycle are regulated, in part, by modulating histone mRNA stability. When DNA synthesis stops, histone mRNA seems to be destabilized, perhaps via an autoregulatory circuit triggered by cytoplasmic histones. We have used an in vitro mRNA decay system to determine whether differential histone mRNA turnover is linked to changes in the basal activity of cytoplasmic mRNA-degrading enzymes. The basal level of the polysome-associated exonuclease enzyme or enzymes that degrade histone mRNA was similar in untreated cells and in cells exposed to DNA or protein synthesis inhibitors. Histone mRNA decay was accelerated in reactions supplemented with histones and soluble cytoplasmic factor(s) (S130), but S130s from control and inhibitor-treated cells were indistinguishable in these assays. The data indicate that basal exonuclease activity is stable or constitutive. The putative factor(s) required for autoregulating histone mRNA decay also do not change appreciably when DNA or protein synthesis is inhibited. The implications of these results with regard to the autoregulation of histone mRNA turnover are discussed.

Platelet-derived growth factor does not induce c-fos in NIH 3T3 cells expressing the EJ-ras oncogene.

Platelet-derived growth factor (PDGF), the calcium ionophore A23187, and the tumor promoter phorbol myristate acetate stimulated c-fos mRNA levels in control NIH 3T3 cells. However, NIH 3T3 cells transformed by EJ-ras DNA transfection, which have diminished PDGF-stimulated phospholipase C activity, showed a 95% reduction in PDGF-stimulated c-fos mRNA levels. The responses to A23187 and phorbol myristate acetate were also attenuated, but not as severely as the PDGF-mediated induction. The reduction in PDGF-stimulated c-fos induction did not appear to be a general result of cellular transformation, since src-transformed NIH 3T3 cells displayed a strong PDGF-stimulated c-fos induction. Despite the reduction in PDGF-stimulated c-fos induction, EJ-ras-transformed cells still responded mitogenically to PDGF. These data suggest that the magnitude of c-fos induction cannot be directly correlated with PDGF-stimulated mitogenesis in EJ-ras-transformed NIH 3T3 cells.

Phorbol myristate acetate inhibits growth in S49 cells: isolation of resistant variants.

We have used S49 mouse lymphoma cells to study phorbol ester effects on growth. Treatment of wild-type (wt) cells with phorbol 12-myristate 13-acetate (PMA) results in growth arrest within 72 hr. We have selected variants that are resistant to PMA-induced growth arrest, based on a selection in the presence of 10 nM PMA. We have characterized one of these variants, termed 21.1, in detail. The 21.1 and wt cells contain similar levels of protein kinase C (PKC) as determined by [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding. Treatment of both wt and 21.1 cells with PMA results in translocation of PKC to the membrane, suggesting that the coupling between PKC and an immediate biological response is intact. PMA treatment leads to the phosphorylation of many similar proteins in wild-type and 21.1 cells. However, in the 21.1 cells there is a prominent substrate of approximately 70 kilodaltons (kD) which is no longer phosphorylated after PMA treatment. In wild-type cells ornithine decarboxylase (ODC) activity and mRNA levels are decreased within 1 hr of PMA treatment. Likewise, ODC levels are decreased in the 21.1 cells after exposure to PMA even though PMA only slightly modulates the growth of these cells. The 21.1 cells represent a unique line with a dominant phenotype in which ODC expression is uncoupled from the growth state of the cell. These cells may represent a good model system in which to examine the steps involved in phorbol ester growth regulation in S49 cells.

Separation and purification of S49 mouse lymphoma histones by reversed-phase high-performance liquid chromatography.

A rapid reversed-phase high-performance liquid chromatography procedure for the fractionation of histones from S49 mouse lymphoma cells is reported. The system utilizes a Vydac C4 macroporous column, heptafluorobutyric acid as solubilizing and ion-pairing agent, and an acetonitrile gradient. All five histone classes and several subclass species are separated, including two H1 species, H2B, two H2A species, H4, and two H3 species. Analytical to multimilligram semipreparative scale fractionations are demonstrated while maintaining resolution of all histone types.

Inhibition of protein synthesis stabilizes histone mRNA.

The inhibition of protein synthesis in exponentially growing S49 cells leads to a specific fivefold increase in histone mRNA in 30 min. The rate of transcription of histone mRNA, measured in intact or digitonin-permeabilized cells, is increased slightly, if at all, by cycloheximide inhibition of protein synthesis. Both approach-to-equilibrium labeling and pulse-chase experiments show that cycloheximide prolongs histone mRNA half-life from approximately 30 min to greater than 2 h. Histone mRNA made before the addition of cycloheximide becomes stable after the inhibition of protein synthesis, whereas removal of the inhibitor is followed by rapid degradation of histone mRNA. This suggests that the increased stability of histone mRNA during inhibition of protein synthesis results not from alteration of the structure of the mRNA, but from the loss of an activity in the cell which regulates histone mRNA turnover.