Fusion of bolaamphiphile micelles: a method to prepare stable supported biomimetic membranes.

Supported biomimetic membranes (SBMs) on solid substrates have been commonly prepared from vesicle-forming double-tail lipids, such as zwitterionic phospholipids, using the method of vesicle fusion. Here we report on the preparation of SBMs on silica surfaces via a similar process of "micelle fusion" from a cationic single-tail bolaamphiphile GLH-20 that forms spherical and elongated thread-like micelles in solution. We demonstrate that, in contrast to zwitterionic phospholipids, GLH-20 self-assembles into a stable contiguous SBM at both low and high ionic strengths. The cationic charge of GLH-20 promotes the formation of a stable SBM through enhanced double-layer interactions with the negatively charged silica surface. It is also shown that spinach aquaporin PM-28 was successfully incorporated within bolaamphiphile SBM in a manner similar to SBMs prepared by vesicle/proteoliposome fusion; thereby the inherent curvature of the micelle surface does not inhibit protein reconstitution. The results suggest that SBMs based on charged bolaamphiphiles might be an attractive platform for applications such as water purification and biosensors, where the stability and low defect rate of SBMs in diverse conditions are crucial for achieving desired performance.

Synthesis of novel cationic bolaamphiphiles from vernonia oil and their aggregated structures.

The present study describes the synthesis of a novel class of vesicle-forming bolaamphiphiles with choline ester head groups. These bolaamphiphiles were derived from vernonia oil, whose main constituent is vernolic acid, a fatty acid with a unique combination of epoxy, carboxy and unsaturated double bonds. A series of bolaamphiphiles containing amido or ester groups within the hydrophobic domain were synthesized from N,N'-alkylenebis (vernolamides) and alpha,omega-alkylene divernolate ester in a two-stage synthesis comprising opening of the epoxy ring with chloroacetic acid, followed by quaternization with N,N-dimethylaminoethyl acetate to form choline ester head groups. The products were characterized by FT-IR, (1)H and (13)C NMR, and ESI-MS. Vesicles prepared from these bolaamphiphiles have the potential to serve as a targeted drug delivery systems with selective decapsulation in the presence of the enzyme acetylcholine esterase, resulting in site-specific release of the drug.

Osmotic strength differentiates between two types of calcium transport pathways regulating catecholamine secretion from cultured bovine chromaffin cells.

Calcium transport and catecholamine secretion was measured in cultured bovine chromaffin cells. Calcium ions which entered the cells following stimulation with either nicotine or 50 mM KCl (high potassium) triggered catecholamine release, but then inactivated the secretory process. The nicotine and the high potassium-induced calcium transport mechanisms were mechanistically distinct, but functionally dependent on each other. The specific evidence is that whereas the high potassium-induced Ca2+ influx was found to be inhibited by hyperosmotic medium, the nicotine-stimulated calcium influx was unaffected under these conditions. High potassium and nicotine-stimulated catecholamine release were also differently affected by hyperosmotic medium. While potassium-stimulated catecholamine release was profoundly inhibited by hyperosmolarity, nicotine-stimulated release was only moderately inhibited. Sequential treatments of cells with nicotine and high potassium, under isotonic physiological conditions, indicate that there is a functional, biochemical communication between the otherwise mechanistically distinct calcium channels. Calcium ions which were found to inactivate these channels may be the basis for such communication.

A common origin of voltage noise and generator potentials in statocyst hair cells.

Voltage noise, generator potentials, and hair movements in the Hermissenda statocyst were analyzed. Motile hairs on the cyst's luminal surface moved as rods through +/- 10 degrees Hz when free and at 7 Hz when loaded with the weight of the statoconia (at 120 degrees C). For hair cells oriented opposite to a centrifugal force vector, rotation caused depolarization and increase of voltage noise variance. The depolarizing generator potential and the increase in voltage noise variance were similarly reduced by perfusion with zero external sodium or chloral hydrate. Cooling, perfusion with zero external sodium or chloral hydrate reduced the movement frequencies of the hairs but increased their range of motion. The same treatments reduced voltage noise variance and increased input resistance of the hair cell membrane. The results indicate that voltage noise and hair cell generator potential have a common origin: exertion of force on statocyst hairs by the weight of statoconia. The collision of statoconia with the motile hairs, not the hairs' bending, produces most of the voltage noise.

Real-time measurements of acetylcholine-induced release of ATP from bovine medullary chromaffin cells.

The luminescent oxidation of luciferin has been used to monitor acetylcholine-induced ATP release from cultured bovine chromaffin cells. Acetylcholine (1-100 microM) evoked ATP release of up to 30% of the total cellular ATP. This secretion required external free calcium and could also be elicited by K+-induced membrane depolarization. The size of the cytosolic ATP compartment was estimated as 5% of the ATP in the cell by solubilising the cell membrane using digitonin (20 microM) or by application to the cells of brief pulses (2 microseconds) of high electric field (2000 V/cm). Blockers of the voltage-gated Ca2+ channel effectively blocked K+-induced ATP release, while the acetylcholine antagonists d-tubocurarine and beta-bungarotoxin inhibited the acetylcholine-induced release of ATP. These data support the concept that ATP is released together with the catecholamines by exocytosis of chromaffin granule contents.

Release of clotting factors from photosensitized endothelial cells: a possible trigger for blood vessel occlusion by photodynamic therapy.

Photodynamic treatment of solid tumors results in the occlusion of blood vessels in the treated tissue. We hypothesize that this process is triggered by the release of one or more clotting factors from the photodamaged endothelial cells. Experimental evidence is presented that immediately after photodynamic treatment, cultured endothelial cells start releasing clotting factors into the medium in a dose range of minimal cytotoxicity.

A novel immunoassay with direct relevance to protection against organophosphate poisoning.

Antiparaoxon immune sera were employed in a new immunoassay based on competition between acetylcholinesterase and antibodies for the binding of paraoxon. Unlike radioimmunoassay, the new assay described herein can be extended to predict the feasibility of antibodies to confer in vivo protection of acetylcholinesterase against organophosphate poisoning. The toxicity of paraoxon was reduced in mice which were preinjected with the immune sera.

Contrasting monoamine oxidase activity and tyramine induced catecholamine release in PC12 and chromaffin cells.

PC12 (phaeochromocytoma derived) cells possess the catecholamine synthesizing enzymes as well as the ability to store and release the catecholamines in response to K+. However, their monoamine oxidase activity and catecholamine release in response to tyramine has not been examined previously. PC12 cells have monoamine oxidase activity which oxidizes type A (noradrenaline and serotonin) and type A-B (dopamine, tyramine and kynuramine) substrates, and is selectively inhibited by clorgyline (IC50 approximately 10(-6) M). In contrast, PC12 cell monoamine oxidase hardly oxidizes phenylethylamine a type B substrate, and is relatively insensitive to inhibition by the selective monoamine oxidase type B inhibitor, 1-deprenyl (IC50 approximately 10(-6) M). By the above criteria it is apparent that the monoamine oxidase in PC12 is solely type A. The kinetics of the oxidase are similar to those of monoamine oxidase type A reported in other tissues including the adrenergic neuron, having apparent Km values of 400, 280, 170 and 227 microM for noradrenaline, dopamine, serotonin and tyramine. The apparent Km value for phenylethylamine is 235 microM. On the other hand, isolated chromaffin cells have the B form of monoamine oxidase with high affinity (Km approximately 25 microM) for phenylethylamine and low affinities for noradrenaline (Km approximately 1100 microM) and adrenaline (Km approximately 1700 microM). This enzyme form is selectively inactivated by the monoamine oxidase type B inhibitor, 1-deprenyl. In similar fashion to peripheral adrenergic neurons, PC12 cells share the capacity to express a tyramine releasable pool of catecholamines, a property entirely lacking in mature cultured chromaffin cells, even though the latter cells are capable of taking up tyramine by a cocaine sensitive process.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and localization of muscarinic receptors in P19-derived neurons.

The muscarinic acetylcholine receptors are important in a variety of physiological processes such as induction of secretion from various glands and regulation of pacemaker activity, muscle tone, and neurotransmission. To date, the muscarinic receptor family includes five members (designated m1-m5), of which m1-m4 are abundant in brain and in peripheral tissues, and m5 is found exclusively in brain, and even there at very low levels. The expression of m1-m5 receptor subtypes was studied in neurons derived from the murine embryonal carcinoma cell line P19. These cells serve as a model system for differentiation and maturation of neurons resembling CNS neurons. Our results show that P19 neurons express mainly the m2, m3, and m5 subtypes. Low levels of m1 receptors are also detected and m4 subtype is practically absent. Furthermore, muscarinic receptors in P19 neurons are functional in activating second messenger signaling pathways. The localization of m2 receptors is predominantly presynaptic, whereas the m5 subtype is mainly postsynaptic. Consequently, P19 cells provide a model system for the study of pre- and postsynaptic muscarinic acetylcholine-receptor subtypes in a proper neuronal context. This is particularly valid for the rare m5 receptors.

Inhibition of choline efflux results in enhanced acetylcholine synthesis and release in the guinea-pig corticocerebral synaptosomes.

Synthesis and release of [3H]acetylcholine ([3H]ACh) were measured in synaptosomes from the guinea pig cerebral cortex after preloading with [3H]choline ([3H]Ch). We demonstrate here that inhibition of choline (Ch) efflux results in an increase in acetylcholine (ACh) synthesis and release. Our findings are as follows: (1) inhibition of [3H]Ch efflux by hemicholinium-3 (HC-3) (100 microM), increased the levels of both the released (116% of control) and the residing (115% of control) [3H]ACh. (2) The muscarinic agonist, McN-A-343 (100 microM), which was previously shown to inhibit Ch efflux, also increased the released (121% of control) and the residing (109% of control) [3H]ACh. (3) Omission of Na+ ions (which are required for Ch transport) from the incubation medium had similar effects to those observed with McN-A-343 and HC-3. These results suggest inverse relationships between Ch efflux on one hand, and ACh synthesis and release on the other hand. (4) Depolarization with 50 mM K+, or with the K+ channel blocker, 4-aminopyridine (100 microM), also increased the total level of [3H]ACh (113 and 107% of nondepolarized synaptosomes, respectively). However, whereas conditions that inhibit Ch transport such as HC-3, McN-A-343 and "no sodium" increased both the residing and the released [3H]ACh depolarization with high K+ or 4-aminopyridine reduced the residing (79 and 87% of control, respectively) and increased only the released [3H]ACh (182 and 148% of control, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between secretagogue-induced Ca2+ influx, intracellular Ca2+ levels and secretion of catecholamines in cultured adrenal chromaffin cells.

Catecholamine secretion induced by various secretagogues in cultured bovine chromaffin cells has been correlated with Ca2+ influx and intracellular Ca2+ concentrations. Nicotine and high K+ caused prompt secretion of catecholamines from cells. Coincidently, both secretagogues evoked 45[Ca2+] influx with a parallel increase in free intracellular Ca2+ concentration, as determined by Quin 2 fluorescence. However, the rate of return of Ca2+ level to baseline after nicotine stimulation was more rapid than after K+ stimulation. In comparison, stimulation with veratridine produced a slow and prolonged Ca2+ influx accompanied by lower levels of intracellular Ca2+ than those observed after nicotine or K+ stimulation. Yet, during 15 min of stimulation, veratridine induced a substantial catecholamine release, which was larger than that obtained after nicotine or K+ stimulations. The Ca2+ ionophore A23187 (1 microM) induced a pronounced increase in intracellular Ca2+ levels, but did not evoke any significant catecholamine release. Finally, addition of the Ca2+ channel blocker verapamil following stimulation, at a time when intracellular Ca2+ concentration was at its peak level, did not affect the rate of decline in intracellular free Ca2+ concentration but promptly blocked Ca2+ uptake and catecholamine secretion. These findings suggest that the rate of Ca2+ influx, rather than the absolute level of intracellular Ca2+ concentration, determines the rate and extent of catecholamine release.

Selective signaling via unique M1 muscarinic agonists.

Rigid analogs of acetylcholine (ACh) were designed for selective actions at muscarinic receptor (mAChR) subtypes and distinct second messenger systems. AF102B, AF150, and AF151 are such rigid analogs of ACh. AF102B, AF150 and AF151 are centrally active M1 agonists. AF102B has a unique agonistic profile showing, inter alia: only part of the M1 electrophysiology of ACh and unusual binding parameters to mAChRs. AF150 and AF151 are more efficacious agonists than AF102B for M1 AChRS in rat cortex and in CHO cells stably transfected with the m1 AChR subtype. Notably, the selectivity of the new m1 agonists is reflected also by activation of select second messenger systems via distinct G-proteins. These compounds reflect a new pharmacological concept, tentatively defined as ligand-selective signaling. Thus, agonist/m1AChR complexes may activate different combinations of signaling pathways, depending on the ligand used. Rigid agonists may activate a limited repertoire of signaling systems. In various animal models for Alzheimer's disease (AD) the agonists AF102B, AF150 and AF151, exhibited positive effects on mnemomic processes and a wide safety margin. Such agonists, and especially AF102B, can be considered as a rational treatment strategy for AD.

Dehydroepiandrosterone augments M1-muscarinic receptor-stimulated amyloid precursor protein secretion in desensitized PC12M1 cells.

Epidemiologic studies suggest that the age-related decline in dehydroepiandrosterone (DHEA) levels may be associated with Alzheimer's disease (AD). Cholinergic markers also decline with age, and are associated with AD pathology. Activation of m1AChR-transfected PC12 cells (PC12M1) with cholinergic agonists results in secretion of Alzheimer's beta-amyloid precursor protein (APP) which in turn reduces beta-amyloid production. This study examined whether DHEA affects APP processing in m1AChR-transfected PC12 cells. DHEA treatment did not significantly alter basal or m1AChR-stimulated APP secretion. However, DHEA (0.1 microM) significantly diminished the desensitization of APP secretion in cells exposed to carbachol for 24 h. The effect of DHEA on APP processing is probably not related to up-regulation of m1AChR or increased m1AChR-activated phosphoinositide hydrolysis since these parameters did not change following DHEA treatment. These findings imply a possible involvement of DHEA in APP processing. Thus, the age-associated decline in DHEA levels may contribute to decreased APP secretion and a consecutive increase in beta-amyloid deposition, which in turn may play a role in the development of AD.

M1 agonists for the treatment of Alzheimer's disease. Novel properties and clinical update.

The AF series compounds, AF102B and congeners of AF150(S), are functionally selective agonists for m1 muscarinic receptors (m1AChRs). This is shown in stable transfected CHO and PC12 cells (PC12M1) with m1m5AChRs and m1AChRs, respectively. AF102B and AF150(S) are partial agonists, but AF150, AF151, and AF151 (S) are full agonists in stimulating phosphoinositides hydrolysis or arachidonic acid release in these cells. Yet, all these compounds behave as antagonists when compared with carbachol in elevating cAMP levels. In PC12M1 cells, unlike carbachol, the AF series compounds induce only minimal to moderate neurite outgrowth. Yet, these agonists synergize strongly with NGF, which by itself mediates only a mild response. Stimulation of m1AChRs by AF102B, AF150(S) and AF151(S) in PC12M1 cells enhances secretion of beta/A4 amyloid precursor protein derivatives (APPs). The enhanced APPs secretion induced by AF102B is potentiated by NGF. AF102B also stimulates APPs secretion from rat cortical slices. Stimulation of m1AChR in PC12M1 cells with carbachol or AF102B decreases tau phosphorylation as indicated by specific tau-1 mAb and alkaline phosphatase treatment. Due to the above mentioned properties m1 agonists may be of unique value in delaying the progression of Alzheimer's disease (AD). The AF series compounds show a wide safety margin and improve memory and learning deficits in animal models for AD. There is a dearth of clinical reports on m1 agonists. These include studies on AF102B and xanomeline, another m1 selective agonist. We tested AF102B in escalating doses of 20, 40, 60 mg, tid, po, (each dose for 2 weeks) for a total of 10 weeks. This was a single-blind placebo-controlled, parallel-group study in patients with probable AD. AF102B was significantly effective at 40 and 60 mg, tid in the ADAS, ADAS-cognitive and ADAS-word recognition scales.

NGF-dependent neurotrophic-like effects of AF102B, an M1 muscarinic agonist, in PC12M1 cells.

The non-selective muscarinic agonist oxotremorine induces atropine-sensitive neurite outgrowth in PC12 cells stably transfected with m1 muscarinic receptors. In contrast, AF102B, an M1-selective muscarinic agonist, mediated minimal neurite outgrowth in these cells. In the presence of nerve growth factor (NGF) however, it induced atropine-sensitive neurite outgrowth in almost half the cell population. AF102B mediated phosphoinositide hydrolysis, but unlike carbachol, it did not stimulate cyclic AMP accumulation in these cells. These signals were not affected by NGF, indicating that they were not directly responsible for the cholinergic neurotrophic-like response. Our observations suggest that AF102B may improve neuronal responsiveness to neurotrophic factors, and thus may provide another beneficial aspect for treating Alzheimer's disease.

The role of neurochemical modulation in learning.

Tsukahara creatively exploited the advantages of a "simple system" approach in a vertebrate context to gain cellular insights into the learning process. The molluscs Aplysia and Hermissenda have provided useful invertebrate examples of this approach. For classical conditioning of Hermissenda a temporal sequence of cellular transformations has been found to correspond to and to substantially account for a learning-specific behavioral transformation. For at least days after the conditioning a biophysical record persists: two voltage-dependent K+ currents, IA and ICa2+-K+, remain reduced in amplitude and at least IA shows an increased rate of inactivation. More recently, a similar biophysical record of associative memory has been identified in the mammalian brain (Disterhoft et al., 1986). Other experiments suggest that a synergistic interaction of C-kinase activation with Ca2+/CaM-kinase activation enhances and prolongs Ca2+-mediated K+ current reduction. The effects of alpha-receptor agonists to enhance depolarization of type B cells (a site of visual-vestibular convergence) and in turn acquisition of classical conditioning are in contrast to the effects of serotonin which can hyperpolarize and thereby reduce depolarization during the acquisition process. For both LTP and LTD, application of a neurotransmitter itself is not sufficient to produce long-lasting neural modification. In this respect, both the LTP and LTD models are more similar to the biochemical sequence implicated in Hermissenda conditioning than to the mechanism initiated by serotonin-like substances proposed for Aplysia sensitization.

Novel m1 muscarinic agonists in treatment and delaying the progression of Alzheimer's disease: an unifying hypothesis.

M1 selective agonists from the AF series (e.g. AF102B, AF150(S)), via m1 muscarinic receptors, activate distinct signal transductions, enhance amyloid precursors proteins secretion from transfected cells and primary cell cultures, show neurotrophic effects and are beneficial in a variety of animal models for Alzheimer's disease. Such m1 agonists may be effective in the treatment and therapy of Alzheimer's disease.

Differential long-term effect of AF64A on [3H]ACh synthesis and release in rat hippocampal synaptosomes.

The activities of various presynaptic cholinergic parameters were determined in hippocampal synaptosomes of rats 29 weeks after intracerebroventricular injection of ethylcholine aziridinium (AF64A) (3 nmol/2 microliters/side) or vehicle (saline). Synaptosomes were preloaded with [3H]choline ([3H]Ch), treated with diisopropyl fluorophosphate to inhibit cholinesterase activity and then were assayed for their content of [3H]Ch and [3H]acetylcholine ([3H]ACh) and for their ability to synthesize and release [3H]ACh. In synaptosomes from AF64A-treated rats compared with synaptosomes from vehicle-treated rats we observed that: (i) specific uptake of [3H]Ch was reduced to 60% of control; (ii) residing [3H]ACh levels were 43% of control while residing [3H]Ch levels were 72% of control; (iii) basal and K(+)-induced [3H]ACh release were 77% and 73% of control, respectively; (iv) high K(+)-induced synthesis of [3H]ACh was only 9% of control; (v) but, choline acetyltransferase activity remained relatively high, being 80% of control. These results suggest that AF64A-induced cholinergic hypofunction is expressed by both loss of some cholinergic neurons and impairment in the functioning of the spared neurons.

Cholinotoxicity of the ethylcholine aziridinium ion in primary cultures from rat central nervous system.

The cytotoxic effects of ethylcholine aziridinium ion (AF64A) were studied in primary cultures prepared from either whole brain, septum, or midbrain of fetal rats. AF64A, at concentrations up to 22.5 microM, significantly reduced the number of acetylcholinesterase-stained cells without affecting the number of dopaminergic neurons or their ability to take up and release [3H]dopamine. Many of the survived acetylcholinesterase-stained cells appeared with intact somata but damaged processes, indicating a retrograde degeneration starting at the nerve terminal. Higher concentrations of AF64A (greater than 22.5 microM), caused general toxicity which was expressed by degeneration of various neuronal and glial cells. Choline (500 microM), significantly protected the cells from AF64A induced cytotoxicity. The results are consistent with a previously described kinetic model, that predicted a dual action of AF64A: selective cholinotoxicity at low concentrations and non-selective cytotoxicity at higher concentrations.

Muscarinic control of amyloid precursor protein secretion in rat cerebral cortex and cerebellum.

It was previously shown by us and by others that activation of muscarinic acetylcholine receptors evoke amyloid precursor protein (APP) secretion in various cell lines. Here we examined if such muscarinic control of APP secretion occurs also in normal brain tissues. We found that the secretion of APP from rat cerebrocortical slices (rich in M1 receptors) was significantly increased by K+ depolarization, the non-selective agonist, carbachol (CCh), and the M1-selective agonist, AF102B. CCh also increased APP secretion from cerebellar slices (rich in M2 receptors) while AF102B had no significant effect in this brain region. Despite of its stimulatory effect on APP release in the cerebellum, CCh had no effect on phosphoinositide (PI) metabolism in this brain region. In the cerebral cortex PI metabolism was significantly increased by CCh but only partially increased by AF102B. These results suggest that APP secretion in the brain is mediated via muscarinic receptors. In the cerebral cortex APP secretion seems to be regulated via M1 receptors. Our results also suggest that PI metabolism is not a pronounced step in mediating APP processing.