Hippocampal acetylcholine release during memory testing in rats: augmentation by glucose.

Several lines of evidence indicate that a modest increase in circulating glucose levels enhances memory. One mechanism underlying glucose effects on memory may be an increase in acetylcholine (ACh) release. The present experiment determined whether enhancement of spontaneous alternation performance by systemic glucose treatment is related to an increase in hippocampal ACh output. Samples of extracellular ACh were assessed at 12-min intervals using in vivo microdialysis with HPLC-EC. Twenty-four minutes after an intraperitoneal injection of saline or glucose (100, 250, or 1000 mg/kg), rats were tested in a four-arm cross maze for spontaneous alternation behavior combined with microdialysis collection. Glucose at 250 mg/kg, but not 100 or 1000 mg/kg, produced an increase in spontaneous alternation scores (69.5%) and ACh output (121.5% versus baseline) compared to alternation scores (44.7%) and ACh output (58.9% versus baseline) of saline controls. The glucose-induced increase in alternation scores and ACh output was not secondary to changes in locomotor activity. Saline and glucose (100-1000 mg/kg) treatment had no effect on hippocampal ACh output when rats remained in the holding chamber. These findings suggest that glucose may enhance memory by directly or indirectly increasing the release of ACh. The results also indicate that hippocampal ACh release is increased in rats performing a spatial task. Moreover, because glucose enhanced ACh output only during behavioral testing, circulating glucose may modulate ACh release only under conditions in which cholinergic cells are activated.

Synaptophysin, a major synaptic vesicle protein, is not essential for neurotransmitter release.

Synaptophysin (syp I) is a synaptic vesicle membrane protein that constitutes approximately 7% of the total vesicle protein. Multiple lines of evidence implicate syp I in a number of nerve terminal functions. To test these, we have disrupted the murine Syp I gene. Mutant mice lacking syp I were viable and fertile. No changes in the structure and protein composition of the mutant brains were observed except for a decrease in synaptobrevin/VAMP II. Synaptic transmission was normal with no detectable changes in synaptic plasticity or the probability of release. Our data demonstrate that one of the major synaptic vesicle membrane proteins is not essential for synaptic transmission, suggesting that its function is either redundant or that it has a more subtle function not apparent in the assays used.

Ethinylestradiol does not enhance the expression of nitric oxide synthase in bovine endothelial cells but increases the release of bioactive nitric oxide by inhibiting superoxide anion production.

Estradiol is known to exert a protective effect against the development of atherosclerosis, but the mechanism by which this protection is mediated is unclear. Since animal studies strongly suggest that production of endothelium-derived relaxing factor is enhanced by estradiol, we have examined the effect of estrogens on nitric oxide (NO) synthase (NOS) activity, protein, and mRNA in cultured bovine aortic endothelial cells. In reporter cells rich in guanylate cyclase, it has been observed that long-term treatment (> or = 24 hr) with ethinylestradiol (EE2) dose-dependently increased guanylate cyclase-activating factor activity in the conditioned medium of endothelial cells. However, conversion of L-[14C]arginine to L-[14C]citrulline by endothelial cell homogenate or quantification of nitrite and nitrate released by intact cells in the conditioned medium did not reveal any change in NOS activity induced by EE2 treatment. Similarly, Western and Northern blot analyses did not reveal any change in the endothelial NOS protein and mRNA content in response to EE2. However, EE2 dose- and time-dependently decreased superoxide anion production in the conditioned medium of endothelial cells with an EC50 value (0.1 nM) close to that which increased guanylate cyclase-activating factor activity (0.5 nM). Both of these effects were completely prevented by the antiestrogens tamoxifen and RU54876. Thus, endothelium exposure to estrogens appears to induce a receptor-mediated antioxidant effect that enhances the biological activity of endothelium-derived NO. These effects could account at least in part for the vascular protective properties of these hormones.

FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals.

Previous work has shown that the fluorescent styryl dye FM1-43 stains nerve terminals in an activity-dependent fashion. This dye appears to label the membranes of recycled synaptic vesicles by being trapped during endocytosis. Stained terminals can subsequently be destained by repeating nerve stimulation in the absence of dye; the destaining evidently reflects escape of dye into the bathing medium from membranes of exocytosing synaptic vesicles. In the present study we tested two key aspects of this interpretation of FM1-43 behavior, namely: (i) that the dye is localized in synaptic vesicles, and (ii) that it is actually released into the bathing medium during destaining. To accomplish this, we first photolyzed the internalized dye in the presence of diaminobenzidine. This created an electron-dense reaction product that could be visualized in the electron microscope. Reaction product was confined to synaptic vesicles, as predicted. Second, using spectrofluorometry, we quantified the release of dye liberated into the medium from tubocurarine-treated nerve-muscle preparations. Nerve stimulation increased the amount of FM1-43 released, and we estimate that normally a stained synaptic vesicle contains a few hundred molecules of the dye. The key to the successful detection of released FM1-43 was to add the micelle-forming detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), which increased FM1-43 quantum yield by more than two orders of magnitude.

Nucleotide binding-promoted conformational changes release a nonnative polypeptide from the Escherichia coli chaperonin GroEL.

The Escherichia coli chaperonins GroEL and GroES facilitate the refolding of polypeptide chains in an ATP hydrolysis-dependent reaction. The elementary steps in the binding and release of polypeptide substrates to GroEL were investigated in surface plasmon resonance studies to measure the rates of binding and dissociation of a normative variant of subtilisin. The rate constants determined for GroEL association with and dissociation from this variant yielded a micromolar dissociation constant, in agreement with independent calorimetric estimates. The rate of GroEL dissociation from the nonnative chain was increased significantly in the presence of 5'-adenylylimidodiphosphate (AMP-PNP), ADP, and ATP, yielding maximal values between 0.04 and 0.22 s(-1). The sigmoidal dependence of the dissociation rate on the concentration of AMP-PNP and ADP indicated that polypeptide dissociation is limited by a concerted conformational change that occurs after nucleotide binding. The dependence of the rate of release on ATP exhibited two sigmoidal transitions attributable to nucleotide binding to the distal and proximal toroid of a GroEL-polypeptide chain complex. The addition of GroES resulted in a marked increase in the rate of nonnative polypeptide release from GroEL, indicating that the cochaperonin binds more rapidly than the dissociation of polypeptides. These data demonstrate the importance of nucleotide binding-promoted concerted conformational changes for the release of chains from GroEL, which correlate with the sigmoidal hydrolysis of ATP by the chaperonin. The implications of these findings are discussed in terms of a working hypothesis for a single cycle of chaperonin action.

Molecular cloning and characterization of SCaMPER, a sphingolipid Ca2+ release-mediating protein from endoplasmic reticulum.

Release of Ca2+ stored in endoplasmic reticulum is a ubiquitous mechanism involved in cellular signal transduction, proliferation, and apoptosis. Recently, sphingolipid metabolites have been recognized as mediators of intracellular Ca2+ release, through their action at a previously undescribed intracellular Ca2+ channel. Here we describe the molecular cloning and characterization of a protein that causes the expression of sphingosyl-phosphocholine-mediated Ca2+ release when its complementary RNA is injected into Xenopus oocytes. SCaMPER (for sphingolipid Ca2+ release-mediating protein of endoplasmic reticulum) is an 181 amino acid protein with two putative membrane-spanning domains. SCaMPER is incorporated into microsomes upon expression in SO cells or after translation in vitro. It mediates Ca2+ release at 4 degrees C as well as 22 degrees C, consistent with having ion channel function. The EC50 for Ca2+ release from Xenopus oocytes is 40 microM, similar to sphingosyl-phosphocholine-mediated Ca2+ release from permeabilized mammalian cells. Because Ca2+ release is not blocked by ryanodine or La3+, the activity described here is distinct from the Ca2+ release activity of the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor. The properties of SCaMPER are identical to those of the sphingolipid-gated Ca2+ channel that we have previously described. These findings suggest that SCaMPER is a sphingolipid-gated Ca2+-permeable channel and support its role as a mediator of this pathway for intracellular Ca2+ signal transduction.

Ascorbic acid is essential for the release of insulin from scorbutic guinea pig pancreatic islets.

Pancreatic islets from young normal and scorbutic male guinea pigs were examined for their ability to release insulin when stimulated with elevated D-glucose. Islets from normal guinea pigs released insulin in a D-glucose-dependent manner showing a rapid initial secretion phase and three secondary secretion waves during a 120-min period. Islets from scorbutic guinea pigs failed to release insulin during the immediate period, and only delayed and decreased responses were observed over the 40-60 min after D-glucose elevation. Insulin release from scorbutic islets was greatly elevated if 5 mM L-ascorbic acid 2-phosphate was supplemented in the perifusion medium during the last 60 min of perifusion. When 5 mM L-ascorbic acid 2-phosphate was added to the perifusion medium concurrently with elevation of medium D-glucose, islets from scorbutic guinea pigs released insulin as rapidly as control guinea pig islets and to a somewhat greater extent. L-Ascorbic acid 2-phosphate without elevated D-glucose had no effect on insulin release by islets from normal or scorbutic guinea pigs. The pancreas from scorbutic guinea pigs contained 2.4 times more insulin than that from control guinea pigs, suggesting that the decreased insulin release from the scorbutic islets was not due to decreased insulin synthesis but due to abnormal insulin secretion.

Nitric oxide inhibits the release of norepinephrine and dopamine from the medial basal hypothalamus of the rat.

Previous research indicates that norepinephrine and dopamine stimulate release of luteinizing hormone (LH)-releasing hormone (LHRH), which then reaches the adenohypophysis via the hypophyseal portal vessels to release LH. Norepinephrine exerts its effect via alpha 1-adrenergic receptors, which stimulate the release of nitric oxide (NO) from nitricoxidergic (NOergic) neurons in the medial basal hypothalamus (MBH). The NO activates guanylate cyclase and cyclooxygenase, thereby inducing release of LHRH into the hypophyseal portal vessels. We tested the hypothesis that these two catecholamines modulate NO release by local feedback. MBH explants were incubated in the presence of sodium nitroprusside (NP), a releaser of NO, and the effect on release of catecholamines was determined. NP inhibited release of norepinephrine. Basal release was increased by incubation of the tissue with the NO scavenger hemoglobin (20 micrograms/ml). Hemoglobin also blocked the inhibitory effect of NP. In the presence of high-potassium (40 mM) medium to depolarize cell membranes, norepinephrine release was increased by a factor of 3, and this was significantly inhibited by NP. Hemoglobin again produced a further increase in norepinephrine release and also blocked the action of NP. When constitutive NO synthase was inhibited by the competitive inhibitor NG-monomethyl-L-arginine (NMMA) at 300 microM, basal release of norepinephrine was increased, as was potassium-evoked release, and this was associated in the latter instance with a decrease in tissue concentration, presumably because synthesis did not keep up with the increased release in the presence of NMMA. The results were very similar with dopamine, except that reduction of potassium-evoked dopamine release by NP was not significant. However, the increase following incubation with hemoglobin was significant, and hemoglobin, when incubated with NP, caused a significant elevation in dopamine release above that with NP alone. In this case, NP increased tissue concentration of dopamine along with inhibiting release, suggesting that synthesis continued, thereby raising the tissue concentration in the face of diminished release. When the tissue was incubated with NP plus hemoglobin, which caused an increase in release above that obtained with NP alone, the tissue concentration decreased significantly compared with that in the absence of hemoglobin, indicating that, with increased release, release exceeded synthesis, causing a fall in tissue concentration. When NO synthase was blocked by NMMA, the release of dopamine, under either basal or potassium-evoked conditions, was increased. Again, in the latter instance the tissue concentration declined significantly, presumably because synthesis did not match release. Therefore, the results were very similar with both catecholamines and indicate that NO acts to suppress release of both amines. Since both catecholamines activate the release of LHRH, the inhibition of their release by NO serves as an ultra-short-loop negative feedback by which NO inhibits the release of the catecholamines, thereby reducing the activation of the NOergic neurons and decreasing the release of LHRH. This may be an important means for terminating the pulses of release of LHRH, which generate the pulsatile release of LH that stimulates gonadal function in both male and female mammals.

Evidence for synaptotagmin as an inhibitory clamp on synaptic vesicle release in Aplysia neurons.

While previous studies have demonstrated that synaptotagmin plays an essential role in evoked neurotransmitter release, it has been difficult to determine whether it acts to facilitate or inhibit release. To address this question, we used acute genetic manipulations to alter the expression of synaptotagmin in Aplysia neurons. Transient overexpression of synaptotagmin in acutely dissected cholinergic neurons and in cultured glutaminergic neurons decreased the amplitude of the excitatory postsynaptic potential (EPSP) by 32% and 26%, respectively. In contrast, treatment of cultured presynaptic neurons with synaptotagmin antisense oligonucleotides increased the amplitude of the EPSP by 50-75%. These results are consistent with a role of synaptotagmin as an inhibitor of release.