Effects of LF-14, THA and physostigmine in rat hippocampus and cerebral cortex

The effects of physostigmine, tetrahydroaminoacridine (THA) and LF-14 [3,3-dimethyl-1(4- amino-3-pyridyl)urea], a 3,4-diaminopyridine derivative, were compared on inhibition of acetyl- cholinesterase (AChE) activity, and release of [³H]acetylcholine (ACh) from rat brain cortical and hippocampal slices. All three compounds caused a concentration dependent inhibition of AChE, with an order of potency physostigmine > THA > LF-14. The electrically stimulated release of ACh from hippocampal and cortical slices was decreased by 10⁻⁵M physostigmine, although the effect was significant only in cortex. THA (5 × 10⁵M) caused a slight, but not significant, decrease in ACh release from both tissues. In contrast, LF-14 (5 × 10⁻⁵ M) caused an approx. 3-fold enhancement of stimulated release. When AChE was inhibited by prior addition of physostigmine, THA caused only a slight enhancement of ACh release, whereas LF-14 greatly increased release. ACh release was also reduced by stimulation of presynaptic muscarinic receptors with oxotremorine. In this case, THA had no effect on ACh release, while LF-14 was able to reverse the inhibition. This study suggests that LF-14 acts to promote ACh release through blocking K⁺ channels, and has a less potent AChE inhibitory effect. It is possible that a compound like LF-14 could be useful in treating diseases of cholinergic dysfunction such as Alzheimer's disease, by both promoting the release of ACh and inhibiting its breakdown.     

Does cholinesterase participate in the intercellular interactions in pollen-pistil system?

Hydrolysis of acetylthiocholine and butyrylthiocholine has been observed in aqueous extracts from petunia pollen and pistils. The reproductive organs of self-compatible clone showed a higher rate of choline ester hydrolysis than those of self-incompatible clone. The highest rate of acetylthiocholine hydrolysis blocked by the cholinesterase inhibitors (physostigmine and neostigmine) was characteristic for the pollen of self-compatible clone. The incomplete (25 - 40 %) inhibition of hydrolysis in pistil extracts of self-compatible clone suggests the presence of unspecific esterases. The eight-fold lower hydrolysis was observed in the pistils of self-incompatible clone as compared to the pistils of compatible clone; neostigmine completely blocked this low hydrolytic activity. The treatment of flower buds with physostigmine and neostigmine (10-5 - 10-3 M) decreased the seed production by 10 - 20 % in compatible clone. When the surfaces of pistil stigmae were treated with physostigmine and neostigmine (10-5 - 10-3 M) before pollination, the seed formation was inhibited by 95 % after both self- and cross-pollination. This revised version was published online in July 2006 with corrections to the Cover Date.     

Acetylcholine causes rooting in leaf explants of in vitro raised tomato (Lycopersicon esculentum Miller) seedlings

The animal neurotransmitter acetylcholine (ACh) induces rooting and promotes secondary root formation in leaf explants of tomato (Lycopersicon esculentum Miller var. Pusa Ruby), cultured in vitro on Murashige and Skoog's medium. The roots originate from the midrib of leaf explants and resemble taproot. ACh at 10(-5) M was found to be the optimum over a wide range of effective concentrations between 10(-7) and 10(-3) M. The breakdown products, choline and acetate were ineffective even at 10(-3) M concentration. ACh appears to have a natural role in tomato rhizogenesis because exogenous application of neostigmine, an inhibitor of ACh hydrolysis, could mimic the effect of ACh. Neostigmine, if applied in combination with ACh, potentiated the ACh effect.     

Compensatory mechanisms enhance hippocampal acetylcholine release in transgenic mice expressing human acetylcholinesterase

Central cholinergic neurotransmission was studied in learning-impaired transgenic mice expressing human acetylcholinesterase (hAChE-Tg). Total catalytic activity of AChE was approximately twofold higher in synaptosomes from hippocampus, striatum and cortex of hAChE-Tg mice as compared with controls (FVB/N mice). Extracellular acetylcholine (ACh) levels in the hippocampus, monitored by microdialysis in the absence or presence of 10(-8)-10(-3) M neostigmine in the perfusion fluid, were indistinguishable in freely moving control and hAChE-Tg mice. Muscarinic receptor functions were unchanged as indicated by similar effects of scopolamine on ACh release and of carbachol on inositol phosphate formation. However, when the mice were anaesthetized with halothane (0.8 vol. %), hippocampal ACh reached significantly lower levels in AChE-Tg mice as compared with controls. Also, the high-affinity choline uptake (HACU) in hippocampal synaptosomes from awake hAChE-Tg mice was accelerated but was reduced by halothane anaesthesia. Moreover, hAChE-Tg mice displayed increased motor activity in novel but not in familiar environment and presented reduced anxiety in the elevated plus-maze test. Systemic application of a low dose of physostigmine (100 microgram/kg i.p.) normalized all of the enhanced parameters in hAChE-Tg mice: spontaneous motor activity, hippocampal ACh efflux and hippocampal HACU, attributing these parameters to the hypocholinergic state due to excessive AChE activity. We conclude that, in hAChE-Tg mice, hippocampal ACh release is up-regulated in response to external stimuli thereby facilitating cholinergic neurotransmission. Such compensatory phenomena most likely play important roles in counteracting functional deficits in mammals with central cholinergic dysfunctions.     

Regulation of self-incompatibility by acetylcholine and cAMP in Lilium longiflorum

Elongation of pollen tubes in pistils of Lilium longiflorum cv. Hinomoto after self-incompatible pollination was here found to be promoted by acetylcholine (ACh) and other choline derivatives, such as acetylthiocholine, l-alpha-phosphatidylcholine and chlorocholinechloride [CCC; (2-chloroethyl) trimethyl ammonium chloride]. Moreover, the elongation was promoted by neostigmine, a potent inhibitor of acetylcholinesterase (AChE; acetylcholine-decomposing enzyme) (EC 3.1.1.7.) and activities of this and choline acetyltransferase (ChAT; acetylcholine-forming enzyme) (EC 2.3.1.6.) in pistils were associated with self-incompatibility. The activity of ChAT was lower after self-incompatible as compared with cross-compatible pollination. Application of cAMP promoted ChAT activities in both cases, whereas activity of AChE in pistils after self-pollination was higher than that after cross-compatible pollination and was suppressed by cAMP in both cases. Furthermore, AChE activity was inhibited by treatment with neostigmine or heating. Our results indicate that the self-incompatibility with self-pollination is due to decrease of ACh and cAMP, causing reduction of ChAT and AC (adenylate cyclase) and concise elevation of AChE and PDE (cAMP phosphodiesterase), and therefore suppressed growth of pollen tubes.     

Depression of potassium-evoked striatal acetylcholine release by delta-receptor activation: inhibition by cholinoactive agents

To examine the role of delta-opioid receptors in the modulation of striatal acetylcholine (ACh) release, the action of D-Pen2,L-Pen5-enkephalin, a selective delta-opioid receptor agonist, was tested on [3H]ACh release from slices of the rat caudate-putamen. Slices, incubated with [3H]choline, were superfused with a physiological buffer and stimulated twice by exposure to a high potassium (K+) concentration. In the absence of a cholinesterase inhibitor, 1 microM D-Pen2,L-Pen5-enkephalin produced a 46 and 35% decrease in the release of [3H]ACh evoked by 15 and 25 mM K+, respectively. The depressant action of the enkephalin analogue was concentration dependent, with a maximal effect on K+-evoked [3H]ACh release occurring at 1.0 microM, and was completely blocked in the presence of the delta-opioid receptor selective antagonist, ICI 174864 (1 microM). In the presence of the cholinesterase inhibitors physostigmine (10 microM) and neostigmine (10 microM), or the muscarinic receptor agonist oxotremorine (10 microM), D-Pen2,L-Pen5-enkephalin did not depress the K+-evoked release of [3H]ACh. Atropine (1 microM) blocked the inhibitory effect of physostigmine on the depressant action of D-Pen2,L-Pen5-enkephalin. The results of this study indicate that delta-opioid receptor activation is associated with an inhibition of striatal ACh release, but this opioid-cholinergic interaction is not apparent under conditions of presynaptic muscarinic receptor activation.     

Microdialysis measures of functional increases in ACh release in the hippocampus with and without inclusion of acetylcholinesterase inhibitors in the perfusate

Because brain extracellular acetylcholine (ACh) levels are near detection limits in microdialysis samples, an acetylcholinesterase (AChE) inhibitor such as neostigmine is often added to microdialysis perfusates to increase ACh levels in the dialysate, a practice that raises concerns that the inhibitor might alter the results. Two experiments compared functional differences in ACh release with and without neostigmine. In the first experiment, 30-60% increases in extracellular ACh concentrations in the hippocampus were evident during food-rewarded T-maze training with 20-500 nm neostigmine in the perfusate but no increases were seen without neostigmine. In the second experiment, 78% increases in ACh release in the hippocampus were seen after injections of the GABA(A) receptor antagonist, bicuculline, into medial septum only if neostigmine (50 nm) was included in the perfusate. These findings suggest that, in the hippocampus, endogenous brain AChEs are very efficient at removing extracellular ACh, obscuring differences in ACh release in these experiments. Therefore, inclusion of AChE inhibitors in the microdialysis perfusate may be necessary under some conditions for observations of functional changes in release of ACh in the hippocampus.     

Assay of acetylcholinesterase activity by potentiometric monitoring of acetylcholine

An acetylcholine-selective electrode based on a plasticized polymeric membrane has been developed. The electrode exhibited good selectivity for acetylcholine (ACh) over choline and some common ions, low drift, and a fast response to ACh. The response was linear over an ACh concentration range of 1×10(-6) to 1×10(-3) M with a slope of 59.1±0.1 and a detection limit of 1.5×10(-7)±1.2×10(-8) M. The electrode was used to monitor enzymatic ACh hydrolysis catalyzed by acetylcholinesterase (AChE) at different substrate and enzyme concentrations. A kinetic data analysis permitted the determination of the Michaelis-Menten constant of the enzymatic hydrolysis and AChE activity in the range of 2×10(-5) to 3.8×10(-1)U ml(-1).     

The effects of pretreatments with carbamates,atropine and mecamylamine on survival and on Soman-induced alterations in rat and rabbit brain acetylcholine

A three component pretreatment regimen composed of a carbamate, atropine and mecamylamine offered complete protection against a multiple lethal doses of Soman in rats. In animals, given chemical pretreatment containing physostigmine in the drug regimen, Soman-induced cerebral acetylcholine (ACh) levels were initially elevated but were back down to normal by 30 min post Soman, but in rats given neostigmine in the pretreatment regimen, ACh concentrations were found to be the highest at 30 min after Soman exposure. The data suggest that peripheral acetylcholinesterase (AChE) and nicotinic and muscarinic ACh receptors are critical sites in organophosphorus (OP) anticholinesterase exposure in rats and should be protected to maximize efficacy against OP intoxication. The data also suggest that carbamates which penetrate the blood-brain barrier may be superior to quaternary carbamates in antagonizing OP exposure in that they could be expected to dampen and rapidly abolish OP-induced rises in total brain ACh which in turn should restore normal neural activity in the brain.     

Cardiac responses of Pacific oyster Crassostrea gigas to agents modulating cholinergic function

To examine the functional effects of cholinergic modulation compounds in oyster hearts and to explore their possible use in monitoring intoxication with acetylcholine-esterase (AChE) inhibitors such as organophosphates, tests were performed with in situ oyster heart preparations. The endogenous cholinergic agonist acetylcholine (ACh), AChE-resistant synthetic agonist carbachol, and the reversible carbamate type of AChE inhibitor physostigmine, all potently depressed spontaneous cardiac contractility. The depression was reversed by extensive washout, or prevented by muscarinic cholinergic antagonist atropine. The irreversible organophosphate type AChE inhibitor parathion or its active metabolite paraoxon at concentrations up to 100 microM failed to depress cardiac contractility. While other reversible AChE inhibitors such neostigmine and pyridostigmine also depressed the contractility, organophosphate AChE inhibitors malathion, diazinon, or phenthoate did not. Despite the differential effect in depressing cardiac function between the reversible and irreversible inhibitors, both of these inhibitors effectively inhibited cardiac AChE activity. The results suggest that the activation of muscarinic cholinergic receptors is coupled to inhibitory cardiac modulation, and organophosphate AChE inhibitors may inhibit only an AChE isozyme located at sites that are not important for control of cardiac activity in oysters.     

Dysfunctional Presynaptic M2 Receptors in the Presence of Chronically High Acetylcholine Levels: Data from the PRiMA Knockout Mouse

The muscarinic M₂ receptor (M2R) acts as a negative feedback regulator in central cholinergic systems. Activation of the M₂ receptor limits acetylcholine (ACh) release, especially when ACh levels are increased because acetylcholinesterase (AChE) activity is acutely inhibited. Chronically high ACh levels in the extracellular space, however, were reported to down-regulate M2R to various degrees. In the present study, we used the PRiMA knockout mouse which develops severely reduced AChE activity postnatally to investigate ACh release, and we used microdialysis to investigate whether the function of M2R to reduce ACh release in vivo was impaired in adult PRiMA knockout mice. We first show that striatal and hippocampal ACh levels, while strongly increased, still respond to AChE inhibitors. Infusion or injection of oxotremorine, a muscarinic M₂ agonist, reduced ACh levels in wild-type mice but did not significantly affect ACh levels in PRiMA knockout mice or in wild-type mice in which ACh levels were artificially increased by infusion of neostigmine. Scopolamine, a muscarinic antagonist, increased ACh levels in wild-type mice receiving neostigmine, but not in wild-type mice or in PRiMA knockout mice. These results demonstrate that M2R are dysfunctional and do not affect ACh levels in PRiMA knockout mice, likely because of down-regulation and/or loss of receptor-effector coupling. Remarkably, this loss of function does not affect cognitive functions in PRiMA knockout mice. Our results are discussed in the context of AChE inhibitor therapy as used in dementia.     

Effect of local acetylcholinesterase inhibition on sweat rate in humans.

ACh is the neurotransmitter responsible for increasing sweat rate (SR) in humans. Because ACh is rapidly hydrolyzed by acetylcholinesterase (AChE), it is possible that AChE contributes to the modulation of SR. Thus the primary purpose of this project was to identify whether AChE around human sweat glands is capable of modulating SR during local application of various concentrations of ACh in vivo, as well as during a heat stress. In seven subjects, two microdialysis probes were placed in the intradermal space of the forearm. One probe was perfused with the AChE inhibitor neostigmine (10 microM); the adjacent membrane was perfused with the vehicle (Ringer solution). SR over both membranes was monitored via capacitance hygrometry during microdialysis administration of various concentrations of ACh (1 x 10(-7)-2 M) and during whole body heating. SR was significantly greater at the neostigmine-treated site than at the control site during administration of lower concentrations of ACh (1 x 10(-7)-1 x 10(-3) M, P < 0.05), but not during administration of higher concentrations of ACh (1 x 10(-2)-2 M, P > 0.05). Moreover, the core temperature threshold for the onset of sweating at the neostigmine-treated site was significantly reduced relative to that at the control site. However, no differences in SR were observed between sites after 35 min of whole body heating. These results suggest that AChE is capable of modulating SR when ACh concentrations are low to moderate (i.e., when sudomotor activity is low) but is less effective in governing SR after SR has increased substantially.     

Effects of Choline Administration on In Vivo Release and Biosynthesis of Acetylcholine in the Rat Striatum as Studied by In Vivo Brain Microdialysis

The purpose of the present study is to clarify the effects of the administration of choline on the in vivo release and biosynthesis of acetylcholine (ACh) in the brain. For this purpose, the changes in the extracellular concentration of choline and ACh in the rat striatum following intracerebroventricular administration of choline were determined using brain microdialysis. We also determined changes in the tissue content of choline and ACh. When the striatum was dialyzed with Ringer solution containing 10 microM physostigmine, ACh levels in dialysates rapidly and dose dependently increased following administration of various doses of choline and reached a maximum within 20 min. In contrast, choline levels in dialysates increased after a lag period of 20 min following the administration. When the striatum was dialyzed with physostigmine-free Ringer solution, ACh could not be detected in dialysates both before and even after choline administration. After addition of hemicholinium-3 to the perfusion fluid, the choline-induced increase in ACh levels in dialysates was abolished. Following administration of choline, the tissue content of choline and ACh increased within 20 min. These results suggest that administered choline is rapidly taken up into the intracellular compartment of the cholinergic neurons, where it enhances both the release and the biosynthesis of ACh.     

Choline availability and acetylcholine synthesis in the hippocampus of acetylcholinesterase-deficient mice

Mice deficient for acetylcholinesterase (AChE) have strongly increased extracellular levels of acetylcholine (ACh) in the dorsal hippocampus [Hartmann, J., Kiewert, C., Duysen, E.G., Lockridge, O., Greig, N.H., Klein, J., 2007. Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity. J. Neurochem. 100, 1421-1429]. Using microdialysis, we found that increased ACh levels are accompanied by decreased levels of extracellular choline which were 1.60 microM in AChE-deficient mice and 4.36 microM in wild-type mice. Addition of choline (10 microM) to the perfusion fluid, while ineffective in wild-type animals, more than doubled extracellular ACh levels in AChE-deficient mice. High-affinity choline uptake (HACU), as measured ex vivo in corticohippocampal synaptosomes, was more than doubled in AChE-deficient mice. Inhibition of HACU by hemicholinium-3 (HC-3) in vivo reduced extracellular levels of ACh by 60% in wild-type mice but by more than 90% in AChE-deficient mice. Decreased ACh levels caused by infusion of HC-3 or tetrodotoxin (TTX) were accompanied by increased levels of free choline. Infusion of scopolamine (1 microM) caused a fivefold increase of ACh levels in wild-type animals but only a 50% increase in AChE-deficient mice. In conclusion, absence of AChE causes dynamic changes in the ratio of choline to ACh. High levels of extracellular ACh are accompanied by reduced levels of extracellular choline, and ACh release becomes strongly dependent on choline availability. Similar changes may take place in patients chronically exposed to AChE inhibitors.     

Acetylcholinesterase and the ADH-dependent transport of water in the amphibian bladder]

It was found that acetylcholine (ACh) at the concentration of 10(-3) M inhibited ADH-stimulated water transport through the wall of amphibian urinary bladder. This effect was suggested to be caused by an interaction of ACh with acetylcholinesterase (AChE) rather than by a stimulation of the M- or N-cholinoreceptor. The inhibitory action of ACh was completely suppressed in the presence of various AChE inhibitors (physostigmine, proserine, armine, Gd-42, acridine-iodmethylate), while an inhibitor of butyrylcholinesterase (BuChE), AD-4, failed to affect it. In accord with this observation the activity of AChE (but not of BuChE) was demonstrated in the urinary bladder epithelium. Since, in addition to the hydrosmotic effects of pituitrine, 8-arginine-vasopressin or oxytocin, ACh blocked also effects of forskolin or cyclic AMP, one may conclude that it acts at some post-cyclic AMP production stage. AChE-dependent inhibition of the ADH-stimulated water transport decreased significantly when the serosal pH was raising from 7.2 to 8.0, but was augmented by serosal acidification (pH 6.8), whereas such pH alterations did not affect the activity of the epithelium AChE. The effect of ACh under consideration was suppressed by adding amiloride (10(-4) M) to the serosal solution. Similarly, the ACh effect was blocked by an inhibitor of Ca-dependent K+ channels, 4-aminopyrdine, which in addition prevented the inhibition of the ADH-stimulated water transport by the serosal acidification. It was noteworthy that some other K+ channel blockers (Ba2+, Cs+, tetraethylammonium, apamine, quinine) did not affect either the water transport or the antipituitrine effect of ACh. In conclusion, we suggest that the inhibitory action of ACh on the ADH-stimulated water transport in the urinary bladder is mediated through the intracellular acidification resulting from ACh interaction with AChE. It is unlikely that the acidification is merely a consequence of the ACh hydrolysis, rather the ACh-AChE interaction induces directly an increase in the proton conductivity of the basolateral membrane of the urinary bladder epithelium.     

Multienzyme microbiosensor based on electropolymerized o-phenylenediamine for simultaneous in vitro determination of acetylcholine and choline

The electrochemical biosensors based on poly(o-phenylenediamine) (PoPD) and acetylcholinesterase (AChE) and choline oxidase (ChO) enzymes were fabricated on carbon fibre (CF) substrate. The electropolymerized PoPD was used to reduce the interfering substances. The electrode assembly was completed by depositing functionalized carbon nano tubes (FCNTs) and Nafion (Naf). Amperometric detection of acetylcholine (ACh) and choline (Ch) were realized at an applied potential of +750 mV vs Ag/AgCl (saturated KCl). At pH 7.4, the final assembly, Naf-FCNTs/AChE-ChO((10:1))/PoPD/CF(Elip), was observed to have high sensitivity towards Ch (6.3±0.3 μA mM(-1)) and ACh (5.8±0.3 μA mM(-1)), linear range for Ch (K(M)=0.52±0.03 mM) and ACh (K(M)=0.59±0.07 mM), and for Ch the highest ascorbic acid blocking capacity (97.2±2 1mM AA). It had a response time of <5s and with 0.045 μM limit of detection. Studies on different ratio (ACh/Ch) revealed that 10:1, gave best overall response.     

Optical detection of choline and acetylcholine based on H₂O₂-sensitive quantum dots

In this paper, we have constructed a simple, rapid and sensitive biosensor for detection of choline and acetylcholine (ACh) based on the hydrogen peroxide (H(2)O(2))-sensitive quantum dots (QDs). The detection limit for choline was 0.1 μM and the linear range was 0.1-0.9 μM and 5-150 μM, respectively. The detection limit for ACh was found to be 10 μM and the linear range was 10-5000 μM. The wide linear ranges were shown to be suitable for routine analyses of choline and ACh. Possible mechanism of the fluorescence of QDs quenched by H(2)O(2) was an electron transfer (ET) process. The experimental conditions of biosensors were optimized, and anti-interference ability was also presented. We also detected the choline in milk samples and the linear range was 5-150 μM. The detection linear range of ACh in serum was 10-140 μM. Most importantly, the recovery of choline in milk and ACh in serum samples were both close to 99%. The excellent performance of this biosensor showed that the method can be used in practice detection of choline and ACh.     

Endogenous acetylcholine release and labelled acetylcholine formation from [3H]choline in the myenteric plexus of the guinea-pig ileum.

The spontaneous release of acetylcholine (ACh) from the guinea-pig myenteric plexus - longitudinal muscle preparation superfused at a constant rate in the presence of physostigmine was 10 nmol-g-1-h-1. This release was decreased to one-third by tetradotoxin or by MnCl2 and increased 2.5 times by 0.1 Hz and 20 times by 16 Hz stimulation. The formation of [3H]ACh from [3H]choline increased from 3 to 33 nmol-g(-1)-h(-1) when the concentration of [3H]choline was increased from 1 muM to 50 muM. The rate of [3H]ACh formation was not affected by tetrodotoxin, MnCl2, or physostigmine in the absence of stimulation. It was increased by 50% by 0.1 Hz and by 100% by 16 Hz stimulation during the first 9 min of exposure to [3H]choline but not subsequently. The myenteric plexus - longitudinal muscle preparation contains 200 nmol/g choline. Results suggest that the apparent small [3H]ACh formation from low concentrations of [3H]choline is due to the dilution of [3H]choline by endogenous choline. The major part of [3H]ACh formation appears to be due to the intracellular turnover of ACh while the evoked release of [3H]ACh appears to originate from a small pool.     

The effect of heptyl-physostigmine,a new cholinesterase inhibitor,on the central cholinergic system of the rat

Heptyl-physostigmine (Heptyl-Phy; MF-201) is a new carbamate derivative of physostigmine (Phy) with greater lipophilicity and longer inhibitory action on cholinesterase (ChE) activity than the parent compound. Following single dose administration of 5 mg/kg heptyl-Phy i.m., maximal whole brain acetylcholinesterase (AChE) inhibition (82%) if reached at 60 min. Inhibition of plasma BuChE butyrylcholinesterase (BuChE) remains close to the steady state level (60%) between 120 and 360 min. At 360 min, whole brain AChE activity is still 67% inhibited compared to controls. Inhibition of AChE activity displays brain regional differences which are more significant at 360 min. At this time point, AChe activity in cerebellum is only 40% inhibited while frontal cortex and medial septum are still 80% inhibited. Increases in acetycholine (ACh) levels also show regional differences, however, there is no direct relationship between AChE inhibition and ACh increase. The electrically evoked [³H]ACh release in cortical slices was inhibited only by the highest concentration of heptyl-Phy tested (10^–4M). At this concentration ChE activity was 97% inhibited in vitro. In conclusion, our results demonstrate that heptyl-Phy compares favorably to other reversible cholinesterase inhibitors (ChEI), particularly to Phy as far as producing a more long-lasting inhibition of AChE and a more prolonged increase of ACh in brain with less severe side effects. Therefore, it represents an interesting candidate for cholinomimetic therapy of Alzheimer disease (AD).Dept. of Pharmacology, Shanghai Institute of Materia Medica Chinese Academy of Sciences Shanghai 20031 China.Special issue dedicated to Dr. Paola S. Timiras     

Distinct cell-specific expression patterns of early and late gibberellin biosynthetic genes during Arabidopsis seed germination

Gibberellins (GAs) are biosynthesized through a complex pathway that involves several classes of enzymes. To predict sites of individual GA biosynthetic steps, we studied cell type-specific expression of genes encoding early and late GA biosynthetic enzymes in germinating Arabidopsis seeds. We showed that expression of two genes, AtGA3ox1 and AtGA3ox2, encoding GA 3-oxidase, which catalyzes the terminal biosynthetic step, was mainly localized in the cortex and endodermis of embryo axes in germinating seeds. Because another GA biosynthetic gene, AtKO1, coding for ent-kaurene oxidase, exhibited a similar cell-specific expression pattern, we predicted that the synthesis of bioactive GAs from ent-kaurene oxidation occurs in the same cell types during seed germination. We also showed that the cortical cells expand during germination, suggesting a spatial correlation between GA production and response. However, promoter activity of the AtCPS1 gene, responsible for the first committed step in GA biosynthesis, was detected exclusively in the embryo provasculature in germinating seeds. When the AtCPS1 cDNA was expressed only in the cortex and endodermis of non-germinating ga1-3 seeds (deficient in AtCPS1) using the AtGA3ox2 promoter, germination was not as resistant to a GA biosynthesis inhibitor as expression in the provasculature. These results suggest that the biosynthesis of GAs during seed germination takes place in two separate locations with the early step occurring in the provasculature and the later steps in the cortex and endodermis. This implies that intercellular transport of an intermediate of the GA biosynthetic pathway is required to produce bioactive GAs.