Permissive Modulation of Sphingosine-1-Phosphate-Enhanced Intracellular Calcium on BKCa Channel of Chromaffin Cells.

Sphingosine-1-phosphate (S1P), is a signaling sphingolipid which acts as a bioactive lipid mediator. We assessed whether S1P had multiplex effects in regulating the large-conductance Ca2+-activated K+ channel (BKCa) in catecholamine-secreting chromaffin cells. Using multiple patch-clamp modes, Ca2+ imaging, and computational modeling, we evaluated the effects of S1P on the Ca2+-activated K+ currents (IK(Ca)) in bovine adrenal chromaffin cells and in a pheochromocytoma cell line (PC12). In outside-out patches, the open probability of BKCa channel was reduced with a mean-closed time increment, but without a conductance change in response to a low-concentration S1P (1 µM). The intracellular Ca2+ concentration (Cai) was elevated in response to a high-dose (10 µM) but not low-dose of S1P. The single-channel activity of BKCa was also enhanced by S1P (10 µM) in the cell-attached recording of chromaffin cells. In the whole-cell voltage-clamp, a low-dose S1P (1 µM) suppressed IK(Ca), whereas a high-dose S1P (10 µM) produced a biphasic response in the amplitude of IK(Ca), i.e., an initial decrease followed by a sustained increase. The S1P-induced IK(Ca) enhancement was abolished by BAPTA. Current-clamp studies showed that S1P (1 µM) increased the action potential (AP) firing. Simulation data revealed that the decreased BKCa conductance leads to increased AP firings in a modeling chromaffin cell. Over a similar dosage range, S1P (1 µM) inhibited IK(Ca) and the permissive role of S1P on the BKCa activity was also effectively observed in the PC12 cell system. The S1P-mediated IK(Ca) stimulation may result from the elevated Cai, whereas the inhibition of BKCa activity by S1P appears to be direct. By the differentiated tailoring BKCa channel function, S1P can modulate stimulus-secretion coupling in chromaffin cells.

Vesicular Transmitter Content in Chromaffin Cells Can Be Regulated via Extracellular ATP.

The energy carrying molecule adenosine triphosphate (ATP) has been implicated for its role in modulation of chemical signaling for some time. Despite this, the precise effects and mechanisms of action of ATP on secretory cells are not well-known. Here, bovine chromaffin cells have been used as a model system to study the effects of extracellular ATP in combination with the catecholamine transmitter norepinephrine (NE). Both transmitter storage and exocytotic release were quantified using complementary amperometric techniques. Although incubation with NE alone did not cause any changes to either transmitter storage or release, coincubation with NE and ATP resulted in a significant increase that was concentration dependent. To probe the potential mechanisms of action, a slowly hydrolyzable version of ATP, ATP-γ-S, was used either alone or together with NE. The result implicates two different behaviors of ATP acting on both the purinergic autoreceptors and as a source of the energy needed to load chromaffin cell vesicles.

Chromaffin cells as a model to evaluate mechanisms of cell death and neuroprotective compounds.

In this review, we show how chromaffin cells have contributed to evaluate neuroprotective compounds with diverse mechanisms of action. Chromaffin cells are considered paraneurons, as they share many common features with neurons: (i) they synthesize, store, and release neurotransmitters upon stimulation and (ii) they express voltage-dependent calcium, sodium, and potassium channels, in addition to a wide variety of receptors. All these characteristics, together with the fact that primary cultures from bovine adrenal glands or chromaffin cells from the tumor pheochromocytoma cell line PC12 are easy to culture, make them an ideal model to study neurotoxic mechanisms and neuroprotective drugs. In the first part of this review, we will analyze the different cytotoxicity models related to calcium dyshomeostasis and neurodegenerative disorders like Alzheimer's or Parkinson's. Along the second part of the review, we describe how different classes of drugs have been evaluated in chromaffin cells to determine their neuroprotective profile in different neurodegenerative-related models.

Bud extracts from Salix caprea L. inhibit voltage gated calcium channels and catecholamines secretion in mouse chromaffin cells.

BACKGROUND: Salix caprea L. is an ornamental plant with prominent antioxidant activity. In the last decades Salix caprea bud extracts (SCBEs) have been used for the treatment of oxidative stress related disorders. PURPOSE: A large part of cellular functions depends on the amount of intracellular Ca2+ concentration which in turn is mainly determined by Ca2+ ions movements across plasma membrane as well as by Ca2+ released from the stores. For better evaluating the mechanism of action of SCBEs, we focused on the effect of SCBEs on voltage gated Ca2+ channels (VGCCs) functioning and related catecholamines secretion in mouse chromaffin cells (MCCs). These latter are neuroendocrine cells that share a wide variety of functions with neurons. They are particularly interesting for studying the relationship between VGCCs activation and catecholamines secretion both in control and under stressful conditions. STUDY DESIGN AND METHODS: We focused on the effect of SCBEs on VGCCs being these latter considered one of the main pathway of Ca2+ influx across plasma membrane. Ca2+ currents and capacitance changes were measured in patch clamp experiments performed in voltage clamp configuration. RESULTS: We show that SCBEs inhibited VGCCs in a dose dependent manner. On average, the saturating concentration of SCBEs (SCBEsmax) is able to block 36% of the maximum Ca2+ current amplitude (ICa) without selectivity for L (ICa, L) or non-L type (ICa, non-L) Ca2+ channels. Furthermore, ICa inhibition is not followed by alteration of VGCCs gating kinetics, but is responsible for a marked decrease of Ca2+ dependent catecholamines secretion. CONCLUSION: We conclude that the ability of SCBEs to inhibit VGCCs function, known to be potentiated during oxidative stress, could contribute to the already known antioxidant properties of Salix caprea L. We finally suggest that the inhibitory effect of SCBEs on catecholamines secretion may contribute to treat stress dependent cellular dysfunctions.

Benzothiazepine CGP37157 Analogues Exert Cytoprotection in Various in Vitro Models of Neurodegeneration.

Mitochondria regulate cellular Ca(2+) oscillations, taking up Ca(2+) through its uniporter and releasing it through the mitochondrial sodium/calcium exchanger. The role of mitochondria in the regulation of Ca(2+) cycle has received much attention recently, as it is a central stage in neuronal survival and death processes. Over the last decades, the 4,1-benzothiazepine CGP37157 has been the only available blocker of the mitochondrial sodium/calcium exchanger, although it targets several other calcium transporters. We report the synthesis of 4,1-benzothiazepine derivatives with the goal of enhancing mitochondrial sodium/calcium exchanger blockade and selectivity, and the evaluation of their cytoprotective effect. The compound 4c presented an interesting neuroprotective profile in addition to an important blockade of the mitochondrial sodium/calcium exchanger. The use of this benzothiazepine could help to understand the physiological functions of the mitochondrial sodium/calcium exchanger. In addition, we hypothesize that a moderate blockade of the mitochondrial sodium/calcium exchanger would provide enhanced neuroprotection in neurons.

Dietary unsaturated fatty acids differently affect catecholamine handling by adrenal chromaffin cells.

Catecholamines (CA) play an important role in cardiovascular (CDV) disease risk. Namely, noradrenaline (NA) levels positively correlate whereas adrenaline (AD) levels negatively correlate with obesity and/or CDV disease. Western diets, which are tipically rich in Ω-6 fatty acids (FAs) and deficient in Ω-3 FAs, may contribute to the development of obesity, type 2 diabetes and/or coronary artery disease. Taking this into consideration and the fact that our group has already described that saturated FAs affect catecholamine handling by adrenal chromaffin cells, this work aimed to investigate the effect of unsaturated FAs upon catecholamine handling in the same model. Our results showed that chronic exposure to unsaturated FAs differently modulated CA cellular content and release, regardless of both FA series and number of carbon atoms. Namely, the Ω-6 arachidonic and linoleic acids, based on their effect on CA release and cellular content, seemed to impair NA and AD vesicular transport, whereas γ-linolenic acid selectively impaired AD synthesis and release. Within the Ω-9 FAs, oleic acid was devoid of effect, and elaidic acid behaved similarly to γ-linolenic acid. Eicosapentaenoic and docosahexaenoic acids (Ω-3 series) impaired the synthesis and release of both NA and AD. These results deserve attention and future development, namely, in what concerns the mechanisms involved and correlative effects in vivo.

Protonophore properties of hyperforin are essential for its pharmacological activity.

Hyperforin is a pharmacologically active component of the medicinal plant Hypericum perforatum (St. John's wort), recommended as a treatment for a range of ailments including mild to moderate depression. Part of its action has been attributed to TRPC6 channel activation. We found that hyperforin induces TRPC6-independent H(+) currents in HEK-293 cells, cortical microglia, chromaffin cells and lipid bilayers. The latter demonstrates that hyperforin itself acts as a protonophore. The protonophore activity of hyperforin causes cytosolic acidification, which strongly depends on the holding potential, and which fuels the plasma membrane sodium-proton exchanger. Thereby the free intracellular sodium concentration increases and the neurotransmitter uptake by Na(+) cotransport is inhibited. Additionally, hyperforin depletes and reduces loading of large dense core vesicles in chromaffin cells, which requires a pH gradient in order to accumulate monoamines. In summary the pharmacological actions of the "herbal Prozac" hyperforin are essentially determined by its protonophore properties shown here.

Enhancement of catecholamine release from PC12 cells by the traditional Japanese medicine, rikkunshito.

BACKGROUND: Rikkunshito is a traditional Japanese herbal medicine that is used to treat appetite loss associated with cancer and other disorders. The formulation contains various constituents that influence cell signaling, and rikkunshito may accordingly affect human homeostasis through multiple regulatory pathways, including those governed by the endocrine system. We investigated the actions of rikkunshito on catecholamine release from PC12 cells, an adrenal chromaffin cell line. METHODS: The actions of rikkunshito on PC12 cells were evaluated by measuring intracellular cAMP levels, tyrosine hydroxylase (TH) and vasoactive intestinal peptide (VIP) mRNA expression levels, and catecholamine levels in the culture medium. The transcriptional activation of VIP gene by rikkunshito was assessed by using a VIP promoter-driven reporter gene assay. RESULTS: Rikkunshito dose-dependently enhanced forskolin-induced elevations in cAMP in PC12 cells, and also increased the gene expression of TH and VIP. The transcriptional activation of VIP gene by rikkunshito was confirmed. Norepinephrine and dopamine secretion into the culture medium of PC12 cells were also dose-dependently augmented by rikkunshito and/or forskolin, but experiments with a protein kinase C (PKC) activator and a phosphodiesterase inhibitor revealed that the effects of rikkunshito were not simply due to the modulation of PKC or phosphodiesterase activity. CONCLUSIONS: These findings suggest that rikkunshito enhances the release of catecholamines by a novel mechanism involving cAMP.

Garlic (Allium sativum) down-regulates the expression of angiotensin II AT(1) receptor in adrenal and renal tissues of streptozotocin-induced diabetic rats.

The up-regulation of angiotensin II AT1 receptors has been implicated as a major mediator in the development of hypertension and progressive nephropathy in experimental diabetes. In spite of the documented potential of garlic treatments in ameliorating diabetic complications, the possible involvement of the angiotensin II AT1 receptor, as a central target for the anti-diabetic potential of garlic, has not been explored. Three groups of rats were studied after 8 weeks following diabetes induction: normal, streptozotocin-induced diabetic (control diabetic), and garlic-treated diabetic rats. A polyclonal antibody of proven specificity to the AT1 receptor, as verified by western blotting, indicated in immunohistochemical assays that AT1 receptor labeling was significantly increased in adrenal and renal tissues of control diabetic rats compared to the normal group. The increased AT1 receptor labeling involved all cortical zones and medullary chromaffin cells of the adrenal gland. Except for glomerulii, increased AT1 receptor labeling was also evident in proximal convoluted tubules in the renal cortex, and all tubular segments and interstitial cells outlining the vasa recta bundles in the inner stripe of the outer renal medulla. Compared with control diabetic rats, the labeling of the AT1 receptor in the garlic-treated diabetic group was significantly reduced throughout adrenal and renal regions to levels comparable to those observed in normal rats. The capacity of garlic to modulate diabetes-induced AT1 receptor up-regulation may be implicated in reversing the detrimental consequences of excessive Ang II signaling, manifested by the development of hypertension and nephropathy.

Ca(V)1.3-driven SK channel activation regulates pacemaking and spike frequency adaptation in mouse chromaffin cells.

Mouse chromaffin cells (MCCs) fire spontaneous action potentials (APs) at rest. Ca(v)1.3 L-type calcium channels sustain the pacemaker current, and their loss results in depolarized resting potentials (V(rest)), spike broadening, and remarkable switches into depolarization block after BayK 8644 application. A functional coupling between Ca(v)1.3 and BK channels has been reported but cannot fully account for the aforementioned observations. Here, using Ca(v)1.3(-/-) mice, we investigated the role of Ca(v)1.3 on SK channel activation and how this functional coupling affects the firing patterns induced by sustained current injections. MCCs express SK1-3 channels whose tonic currents are responsible for the slow irregular firing observed at rest. Percentage of frequency increase induced by apamin was found inversely correlated to basal firing frequency. Upon stimulation, MCCs build-up Ca(v)1.3-dependent SK currents during the interspike intervals that lead to a notable degree of spike frequency adaptation (SFA). The major contribution of Ca(v)1.3 to the subthreshold Ca(2+) charge during an AP-train rather than a specific molecular coupling to SK channels accounts for the reduced SFA of Ca(v)1.3(-/-) MCCs. Low adaptation ratios due to reduced SK activation associated with Ca(v)1.3 deficiency prevent the efficient recovery of Na(V) channels from inactivation. This promotes a rapid decline of AP amplitudes and facilitates early onset of depolarization block following prolonged stimulation. Thus, besides serving as pacemaker, Ca(v)1.3 slows down MCC firing by activating SK channels that maintain Na(V) channel availability high enough to preserve stable AP waveforms, even upon high-frequency stimulation of chromaffin cells during stress responses.

Dual effects of nobiletin, a citrus polymethoxy flavone, on catecholamine secretion in cultured bovine adrenal medullary cells.

Nobiletin, a compound of polymethoxy flavones found in citrus fruits, possesses a wide range of pharmacological activities. Here we report the effects of nobiletin on catecholamine secretion in cultured bovine adrenal medullary cells. Nobiletin (1.0-100 microM) concentration-dependently stimulated catecholamine secretion and (45)Ca(2+) influx. Its stimulatory effect of nobiletin on catecholamine secretion was abolished by deprivation of extracellular Ca(2+) and partially inhibited by specific inhibitors of voltage-dependent Ca(2+) channels and Na(+)/Ca(2+) exchangers. On the other hand, nobiletin suppressed catecholamine secretion and (22)Na(+) and (45)Ca(2+) influx induced by acetylcholine, an agonist of nicotinic acetylcholine receptors, in a concentration-dependent manner. It also inhibited catecholamine secretion, (22)Na(+) influx and/or (45)Ca(2+) influx induced by veratridine, an activator of voltage-dependent Na(+) channels, and 56 mM K(+), an activator of voltage-dependent Ca(2+) channels. In Xenopus oocytes expressing alpha3beta4 neuronal acetylcholine receptors, nobiletin directly inhibited the current evoked by acetylcholine in a concentration-dependent manner similar to that observed in catecholamine secretion. The present findings suggest that nobiletin, by itself, stimulates catecholamine secretion via activation of voltage-dependent Ca(2+) channels or Na(+)/Ca(2+) exchangers, whereas it inhibits catecholamine secretion induced by acetylcholine through the suppression of Na(+) influx and Ca(2+) influx in cultured bovine adrenal medullary cells.

Retinol activates tyrosine hydroxylase acutely by increasing the phosphorylation of serine40 and then serine31 in bovine adrenal chromaffin cells.

Tyrosine hydroxylase is the rate-limiting enzyme in the biosynthesis of the catecholamines. It has been reported that retinol (vitamin A) modulates tyrosine hydroxylase activity by increasing its expression through the activation of the nuclear retinoid receptors. In this study, we observed that retinol also leads to an acute activation of tyrosine hydroxylase in bovine adrenal chromaffin cells and this was shown to occur via two distinct non-genomic mechanisms. In the first mechanism, retinol induced an influx in extracellular calcium, activation of protein kinase C and serine40 phosphorylation, leading to tyrosine hydroxylase activation within 15 min. This effect then declined over time. The retinol-induced rise in intracellular calcium then led to a second slower mechanism; this involved an increase in reactive oxygen species, activation of extracellular signal-regulated kinase 1/2 and serine31 phosphorylation and the maintenance of tyrosine hydroxylase activation for up to 2 h. No effects were observed with retinoic acid. These results show that retinol activates tyrosine hydroxylase via two sequential non-genomic mechanisms, which have not previously been characterized. These mechanisms are likely to operate in vivo to facilitate the stress response, especially when vitamin supplements are taken or when retinol is used as a therapeutic agent.

Synthesis and biological evaluation of new 4H-pyrano[2,3-b]quinoline derivatives that block acetylcholinesterase and cell calcium signals, and cause neuroprotection against calcium overload and free radicals.

The synthesis and biological evaluation of ethyl 5-amino-4-(3-pyridyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-b]quinoline-3-carboxylates (9-11) is described. We have found that these compounds inhibit AChE with a mild potency, mitigates the [Ca(2+)](c) triggered by high K(+), and cause neuroprotection against Ca(2+) overloading and free radical-induced neuronal death.

Constitutive activity of glycogen synthase kinase-3beta: positive regulation of steady-state levels of insulin receptor substrates-1 and -2 in adrenal chromaffin cells.

In cultured bovine adrenal chromaffin cells, 12-h treatment with 1-20 mM LiCl, an inhibitor of glycogen synthase kinase-3 (GSK-3), increased Ser(9) phosphorylation of GSK-3beta by approximately 44%, while decreasing insulin receptor substrate-1 (IRS-1) and IRS-2 protein levels by approximately 38 and approximately 62% in a concentration-dependent manner. Treatment with SB216763 (0.1-30 microM for 12 h), a selective inhibitor of GSK-3, lowered IRS-1 and IRS-2 levels by approximately 38 and approximately 48%, while increasing beta-catenin protein level by approximately 47%, due to the prevention of GSK-3-induced degradation of beta-catenin by SB216763. Insulin (100 nM for 24 h) increased Ser(9) phosphorylation of GSK-3beta by approximately 104%, while decreasing IRS-1 and IRS-2 levels by approximately 41 and approximately 72%; the insulin-induced Ser(9) phosphorylation of GSK-3beta, as well as down-regulations of IRS-1 and IRS-2 levels were restored to the control levels of nontreated cells at 24 h after the washout of the insulin (100 nM for 12 h)-treated cells. Either clasto-lactacystin beta-lactone or lactacystin (an inhibitor of proteasome) prevented LiCl- or SB216763-induced decreases of IRS-1 and IRS-2 levels by approximately 100 and approximately 69%, respectively. In contrast, calpastatin (an inhibitor of calpain) and leupeptin (an inhibitor of lysosome) failed to prevent the decreases of IRS-1 and IRS-2 levels caused by LiCl or SB216763. LiCl or SB216763 lowered IRS-2 mRNA level, with no effect on IRS-1 mRNA level. These results suggest that constitutive activity of GSK-3beta in quiescent cells positively maintains steady-state levels of IRS-1 and IRS-2 via regulating proteasomal degradation and/or synthesis of IRS-1 and IRS-2 proteins.

Inhibition by selenium compounds of catecholamine secretion due to inhibition of Ca2+ influx in cultured bovine adrenal chromaffin cells.

Selenium is an essential trace metal element, whereas large doses of selenium exert adverse effects to the human body. We examined the effects of selenium compounds, sodium selenite (Na2SeO3) and sodium selenate (Na2SeO4), on catecholamine secretion from cultured bovine adrenal chromaffin cells. Treatment of chromaffin cells with sodium selenite for 72, 48, and 24 h caused decreases in protein and catecholamine contents, in association with cell damage, at concentrations over 30, 300, and 300 microM, respectively. The cells treated with subtoxic conditions (<100 microM, 48 h) of sodium selenite were used for further experiments. Sodium selenite treatment for 48 h inhibited carbachol (CCh)-induced catecholamine secretion in a concentration-dependent and non-competitive manner, while it did not affect high K+- and veratridine-induced catecholamine secretion. Sodium selenite (100 microM) did not affect CCh- and veratridine-induced 22Na+ influx, while the compound inhibited 45Ca2+ influx induced only by CCh, but not high K+ and veratridine. Sodium selenate even at higher concentrations (1000 microM) did not affect any stimulus-induced catecholamine secretion and 45Ca2+ influx. Thus, sodium selenite may specifically exert adverse effects, such as inhibition of physiological stimulus-induced catecholamine secretion from adrenal chromaffin cells due to inhibition of Ca2+ influx.

New twist on neuronal insulin receptor signaling in health, disease, and therapeutics.

Long after the pioneering studies documenting the existence of insulin (year 1967) and insulin receptor (year 1978) in brain, the last decade has witnessed extraordinary progress in the understanding of brain region-specific multiple roles of insulin receptor signalings in health and disease. In the hypothalamus, insulin regulates food intake, body weight, peripheral fat deposition, hepatic gluconeogenesis, reproductive endocrine axis, and compensatory secretion of counter-regulatory hormones to hypoglycemia. In the hippocampus, insulin promotes learning and memory, independent of the glucoregulatory effect of insulin. Defective insulin receptor signalings are associated with the dementia in normal aging and patients with age-related neurodegenerative diseases (e.g., Alzheimer's disease); the cognitive impairment can be reversed with systemic administration of insulin in the euglycemic condition. Intranasal administration of insulin enhances memory and mood and decreases body weight in healthy humans, without causing hypoglycemia. In the hypothalamus, insulin-induced activation of the phosphoinositide 3-kinase pathway followed by opening of ATP-sensitive K+ channel has been shown to be related to multiple effects of insulin. However, the precise molecular mechanisms of insulin's pleiotropic effects still remain obscure. More importantly, much remains unknown about the quality control mechanisms ensuring correct conformational maturation of the insulin receptor, and the cellular mechanisms regulating density of cell surface functional insulin receptors.

Phytoestrogen cimicifugoside-mediated inhibition of catecholamine secretion by blocking nicotinic acetylcholine receptor in bovine adrenal chromaffin cells.

We investigated the effect of the phytoestrogen cimicifugoside, one of the pharmacologically active ingredients of the medicinal plant Cimicifuga racemosa (black cohosh) that has been used to treat many kinds of neuronal and menopausal symptoms, such as arthritis, menopausal depression, and nerve pain. Cimicifugoside inhibited calcium increase induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), a nicotinic acetylcholine receptor (nAChR) agonist in bovine adrenal chromaffin cells with a half-maximal inhibitory concentration (IC(50)) of 18 +/- 2 microM. In contrast, cimicifugoside did not affect the calcium increases evoked by high K(+), veratridine, and bradykinin. The DMPP-induced sodium increase was also inhibited by cimicifugoside with an IC(50) of 2 +/- 0.3 microM, suggesting that the activity of nAChRs is inhibited by cimicifugoside. Cimicifugoside did not affect the KCl-induced secretion but markedly inhibited the DMPP-induced catecholamine secretion that was monitored by carbon-fiber amperometry in real time and high-performance liquid chromatography through electrochemical detection. The results suggest that cimicifugoside selectively inhibits nAChR-mediated response in bovine chromaffin cells.

In vitro inhibition of adrenal catecholamine secretion by steroidal metabolites of ginseng saponins.

We reported previously that the protopanaxatriol saponins in Panax ginseng greatly reduce the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh). However, protopanaxadiol saponins showed only slight inhibitory effects. Recent studies have demonstrated that oligosaccharides connected to the hydroxyl groups of the aglycone in ginseng saponins (ginsenosides) are in turn hydrolyzed in the digestive tract and absorbed into the circulation following oral administration of ginseng. Therefore, the present study was performed to investigate the effects of the major ginsenoside metabolites (M1, M2, M3, M4, M5, M11, and M12) on catecholamine secretion. All of these metabolites were shown to be potent inhibitors of ACh-evoked secretion, and M4 was the most effective. M4 blocked not only the ACh-induced Na(+) influx into the chromaffin cells but also the ACh-induced inward current into Xenopus oocytes expressing human alpha 3 beta 4 neuronal nicotinic ACh receptors. M4 reduced the secretion induced by high K(+), an activator of voltage-sensitive Ca(2+) channels, to a much lesser extent than that evoked by ACh. M1, M2, M3, M5, and M12 are protopanaxadiol saponin-derived metabolites. Therefore, these results imply that the protopanaxadiol saponins are prodrugs, and they show more potent inhibitory activity following metabolism in the digestive tract. The results further suggest that the metabolites act on nicotinic ACh receptors, blocking Na(+) influx through the receptors, and consequently reduce the catecholamine secretion from bovine adrenal chromaffin cells. The inhibitory effect of ginsenoside metabolites is probably one of the mechanisms of action responsible for the pharmacological effects of ginseng.

Martentoxin, a novel K+-channel-blocking peptide: purification, cDNA and genomic cloning, and electrophysiological and pharmacological characterization.

Martentoxin, a novel K+-channel-specific peptide has been purified and characterized from the venom of the East-Asian scorpion (Buthus martensi Karsch). The whole cDNA precursor sequence suggested that martentoxin was composed of 37 residues with a unique sequence compared with other scorpion neurotoxins. The genomic DNA of martentoxin showed an additional intron situated unexpectedly in the 5' UTR region, besides one located close to the C-terminal of the signal peptide. The patch-clamp recording found that martentoxin at the applied dose of 100 nm could strongly block large-conductance Ca2+-activated K+ (BKCa) currents in adrenal medulla chromaffin cells, and BKCa currents blocked by martentoxin could be fully recovered within 30 seconds after washing, which is at least 10 times faster than recovery after charybdotoxin. Meanwhile, a biosensor binding assay showed a fast association rate and a slow dissociation rate of martentoxin binding on rat brain synaptosomes. The binding of martentoxin on rat brain synaptosomes could be inhibited regularly by charybdotoxin, and gradually by toosendanin in a concentration-dependent manner, but not by either apamin or P03 from Buthus martensi. The results thus indicate that martentoxin is a new member in the family of K+-channel-blocking ligands.

A novel conotoxin from Conus betulinus, kappa-BtX, unique in cysteine pattern and in function as a specific BK channel modulator.

A novel conotoxin, kappa-conotoxin (kappa-BtX), has been purified and characterized from the venom of a worm-hunting cone snail, Conus betulinus. The toxin, with four disulfide bonds, shares no sequence homology with any other conotoxins. Based on a partial amino acid sequence, its cDNA was cloned and sequenced. The deduced sequence consists of a 26-residue putative signal peptide, a 31-residue mature toxin, and a 13-residue extra peptide at the C terminus. The extra peptide is cleaved off by proteinase post-processing. All three Glu residues are gamma-carboxylated, one of the two Pro residues is hydroxylated at position 27, and its C-terminal residue is Pro-amidated. The monoisotopic mass of the toxin is 3569.0 Da. Electrophysiological experiments show that: 1) among voltage-gated channels, kappa-BtX is a specific modulator of K(+) channels; 2) among the K channels, kappa-BtX specifically up-modulates the Ca(2+)- and voltage-sensitive BK channels (252 +/- 47%); 3) its EC(50) is 0.7 nm with a single binding site (Hill = 0.88); 4) the time constant of wash-out is 8.3 s; and 5) kappa-BtX has no effect on single channel conductance, but increases the open probability of BK channels. It is concluded that kappa-BtX is a novel specific biotoxin against BK channels.