Stimulation Of Influx Research Articles

Stimulation of calcium influx and CK1α by NF-κB antagonist [6]-Gingerol reprograms red blood cell longevity.

Chemotherapy-induced anemia (CIA) is a major obstacle in cancer management. Although the mechanisms governing CIA are poorly understood, recent efforts have identified suicidal erythrocyte (red blood cell, RBC) death as a possible cause of CIA. [6]-Gingerol (GNG), a polyphenol extracted from Zingiber officinale plant, exhibits a wide array of biological activities including antimicrobial, antioxidant, anti-inflammatory, immunomodulatory, and anticancer activities, in vitro and in vivo. However, the potential toxicity of GNG to human RBCs remains unexplored. RBCs from heparinized blood were isolated by centrifugation and exposed to antitumor concentrations (10-100µM) of GNG for 24hr at 37°C. Hemolysis was calculated from hemoglobin leakage in the supernatant (λmax =405nm), while cytofluorometric analysis of eryptosis employed Annexin-V-FITC to detect phosphatidylserine (PS) exposure, forward scatter (FSC) to estimate cell volume, Fluo4/AM to measure calcium activity, and H2 DCFDA to assess oxidative stress. Moreover, zVAD(OMe)-FMK, SB203580, necrostatin-2, staurosporin, and D4476 were used to identify signaling pathways responsive to GNG. GNG induced significant hemolysis at 100µM, independently of extracellular calcium, and increased Annexin-V-FITC fluorescence that was thoroughly abrogated without extracellular calcium. GNG also enhanced Fluo4 fluorescence and reduced FSC, but had no significant effect on DCF fluorescence. Importantly, the presence of D4476 significantly attenuated GNG-induced hemolysis. In conclusion, GNG stimulates premature RBC death characterized by loss of membrane asymmetry, elevated cytosolic calcium, cell shrinkage, and casein kinase 1α activation. Blocking the activity of calcium channels or CK1α may, therefore, ameliorate the toxic effects of GNG on RBCs. PRACTICAL APPLICATIONS: This report presents a safety assessment of GNG as a chemotherapeutic agent and highlights the novel toxicity of GNG to human RBCs. Our findings provide novel insights that may lead to more efficient utilization of GNG in chemotherapy. Specifically, our data revealed the involvement of calcium channels and casein kinase 1α in mediating GNG-induced premature RBC death, and, therefore, inverse agonists or inhibitors of either pathway may be used as pharmaceutical adjuvants to attenuate the toxic effects of GNG.

Benzodiazepine exposure induces transcriptional down-regulation of GABAA receptor α1 subunit gene via L-type voltage-gated calcium channel activation in rat cerebrocortical neurons

GABAA receptors are targets of different pharmacologically relevant drugs, such as barbiturates, benzodiazepines, and anesthetics. In particular, benzodiazepines are prescribed for the treatment of anxiety, sleep disorders, and seizure disorders. Benzodiazepines potentiate GABA responses by binding to GABAA receptors, which are mainly composed of α (1–3, 5), β2, and γ2 subunits. Prolonged activation of GABAA receptors by endogenous and exogenous modulators induces adaptive changes that lead to tolerance. For example, chronic administration of benzodiazepines produces tolerance to most of their pharmacological actions, limiting their usefulness. The mechanism of benzodiazepine tolerance is still unknown. To investigate the molecular basis of tolerance, we studied the effect of sustained exposure of rat cerebral cortical neurons to diazepam on the GABAA receptor. Flunitrazepam binding experiments showed that diazepam treatment induced uncoupling between GABA and benzodiazepine sites, which was blocked by co-incubation with flumazenil, picrotoxin, or nifedipine. Diazepam also produced selective transcriptional down-regulation of GABAA receptor α1 subunit gene through a mechanism dependent on the activation of L-type voltage-gated calcium channels. These findings suggest benzodiazepine-induced stimulation of calcium influx through L-type voltage-gated calcium channels triggers the activation of a signaling pathway that leads to uncoupling and an alteration of receptor subunit expression. Insights into the mechanism of benzodiazepine tolerance will contribute to the design of new drugs that can maintain their efficacies after long-term treatments.

Transcription of specific auxin efflux and influx carriers drives auxin homeostasis in tobacco cells.

Auxin concentration gradients are informative for the transduction of many developmental cues, triggering downstream gene expression and other responses. The generation of auxin gradients depends significantly on cell-to-cell auxin transport, which is supported by the activities of auxin efflux and influx carriers. However, at the level of individual plant cell, the co-ordination of auxin efflux and influx largely remains uncharacterized. We addressed this issue by analyzing the contribution of canonical PIN-FORMED (PIN) proteins to the carrier-mediated auxin efflux in Nicotiana tabacum L., cv. Bright Yellow (BY-2) tobacco cells. We show here that a majority of canonical NtPINs are transcribed in cultured cells and in planta. Cloning of NtPIN genes and their inducible overexpression in tobacco cells uncovered high auxin efflux activity of NtPIN11, accompanied by auxin starvation symptoms. Auxin transport parameters after NtPIN11 overexpression were further assessed using radiolabelled auxin accumulation and mathematical modelling. Unexpectedly, these experiments showed notable stimulation of auxin influx, which was accompanied by enhanced transcript levels of genes for a specific auxin influx carrier and by decreased transcript levels of other genes for auxin efflux carriers. A similar transcriptional response was observed upon removal of auxin from the culture medium, which resulted in decreased auxin efflux. Overall, our results revealed an auxin transport-based homeostatic mechanism for the maintenance of endogenous auxin levels. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at http://osf.io/ka97b/.

Acute stimulation of glucose influx upon mitoenergetic dysfunction requires LKB1, AMPK, Sirt2 and mTOR-RAPTOR.

Mitochondria play a central role in cellular energy production, and their dysfunction can trigger a compensatory increase in glycolytic flux to sustain cellular ATP levels. Here, we studied the mechanism of this homeostatic phenomenon in C2C12 myoblasts. Acute (30 min) mitoenergetic dysfunction induced by the mitochondrial inhibitors piericidin A and antimycin A stimulated Glut1-mediated glucose uptake without altering Glut1 (also known as SLC2A1) mRNA or plasma membrane levels. The serine/threonine liver kinase B1 (LKB1; also known as STK11) and AMP-activated protein kinase (AMPK) played a central role in this stimulation. In contrast, ataxia-telangiectasia mutated (ATM; a potential AMPK kinase) and hydroethidium (HEt)-oxidizing reactive oxygen species (ROS; increased in piericidin-A- and antimycin-A-treated cells) appeared not to be involved in the stimulation of glucose uptake. Treatment with mitochondrial inhibitors increased NAD+ and NADH levels (associated with a lower NAD+:NADH ratio) but did not affect the level of Glut1 acetylation. Stimulation of glucose uptake was greatly reduced by chemical inhibition of Sirt2 or mTOR-RAPTOR. We propose that mitochondrial dysfunction triggers LKB1-mediated AMPK activation, which stimulates Sirt2 phosphorylation, leading to activation of mTOR-RAPTOR and Glut1-mediated glucose uptake.

Multi target therapy instead of mono receptor approach: Motility modulation beyond MCP

Motility-modulating drugs are important in functional GI diseases, formely with a mono receptor approach as a gold standard. Important examples were cisaprid, which stimulates 5-HT4-receptors, and metoclopramide (MCP), a dopamine D2 antagonist and additionally an 5-HT3-antagonist and 5-HT4-agonist, enhancing gastrointestinal motility. This changed by the withdrawal of cisapride due to cardiac, and recently the referral for metoclopramide (MCP) due to neurological side effects, with omission of functional dyspepsia from its therapeutic indications. It is therefore important to identify other options with proven clinical efficacy but more favorable safety. Herbal medicinal products like the multi drug combination STW 5 have shown to be a safe and effective treatment option in these indications, which raises the question for the – obviously different – underlying mechanisms of action. For identifying effects on GI motility, a systematic database search was conducted in accordance to the PRISMA statement. Studies on STW 5 in small intestinal preparations [1] showed spasmolysis in spastic, tonicising effects in the relaxed state. This was confirmed in inflamed intestinal tissue in vitro and in vivo [2]. In the stomach, a region specific action was described in vitro, based on an inhibition of Ca influx via SOC channels in the gastric fundus and on a stimulation of Ca influx via L type Ca channels in the antrum and was confirmed in a clinical pharmacological study in vivo [3]. A tonicising effect on the lower esophageal sphincter is based on the same mechanisms of action[1]. Components of STW 5 have been shown to act synergistically [4]. While MCP or cisaprid, despite acting via only few specific targets, have been shown to have serious side effects, STW 5 has, in contrast, multiple targets, which potentially contribute to its clinical effects, and an excellent safety profile [1], documented by its use for more than 50 years and in more than 25 mio patients.

KNOXing on the Door of Selective Insulin Resistance

Insulin controls systemic nutrient homeostasis by promoting anabolic processes in various tissues, including the stimulation of glucose influx (into muscle and adipose), protein and glycogen synthesis (in muscle and liver), lipid synthesis and storage (in liver and adipose), and the inhibition of fatty acid oxidation, glycogenolysis, gluconeogenesis, and apoptosis and autophagy (especially in a damaged heart).1–3 Resistance to the action of insulin is associated with chronic physiological and inflammatory stress that develops during obesity and inactivity. In too many instances it progresses to type 2 diabetes mellitus when compensatory hyperinsulinemia fails to maintain glucose homeostasis. A combination of compensatory hyperinsulinemia and inconsistent levels of insulin resistance across body tissues can cause life-threatening sequela, especially nonalcoholic fatty liver disease and atherosclerosis.3,4 Thus, dissection of the insulin signaling pathways and the molecular mechanisms of tissue-specific insulin resistance might reveal novel strategies to arrest or reverse the progression of metabolic disease. See accompanying article on page 1236 Cell-based studies initiated decades ago and extended most recently with mouse genetics reveal a common insulin signaling cascade that begins by activation of the insulin receptor tyrosine kinase (IR) and propagates through the insulin receptor substrates (IRS1 and IRS2) to the phosphatidyl inositol 3 kinase→ v-akt murine thymoma viral oncogene homolog (AKT) cascade 1. AKT plays a particularly broad role as it phosphorylates many protein substrates—including the direct phosphorylation and inactivation of FoxO (forkhead box protein O1 family of transcription factors, FoxO1 and FoxO3) and the indirect phosphorylation of CRTC2 (cAMP response element binding protein–regulated transcription coactivator 2) that inactivates cAMP response element binding protein. Inactivation of these factors suppresses the expression of many hepatic genes, including phosphoenolpyruvate carboxykinase and glucose-6-phosphatase that promote gluconeogenesis (Figure).5,6 Genetic disruption of hepatic insulin signaling by the ablation of the …

Adipokine ganglioside GM2 activator protein stimulates insulin secretion

Recently, we identified ganglioside GM2 activator protein (GM2AP) as a novel adipokine, and revealed that treatment of cultured cells with GM2AP impairs insulin signal transduction. The aim of this study was to examine the impact of GM2AP on glucose metabolism in vivo. Injection of recombinant GM2AP in mice significantly lowered blood glucose levels in glucose tolerance tests. Administration of GM2AP to mice for 10days increased serum insulin levels, whereas the contents of glucose, leptin and FFA were significantly decreased. Stimulation of calcium influx and insulin secretion by GM2AP was observed in hamster insulinoma HIT-T15 cells. Blockage of GM2AP function by specific antibodies inhibited GM2AP-induced insulin secretion. These results provide novel insights into the physiological functions of GM2AP in obesity.

Mechanisms of Pyrethroid Insecticide-Induced Stimulation of Calcium Influx in Neocortical Neurons

Pyrethroid insecticides bind to voltage-gated sodium channels (VGSCs) and modify their gating kinetics, thereby disrupting neuronal function. Pyrethroids have also been reported to alter the function of other channel types, including activation of voltage-gated calcium channels. Therefore, the present study compared the ability of 11 structurally diverse pyrethroids to evoke Ca(2+) influx in primary cultures of mouse neocortical neurons. Nine pyrethroids (tefluthrin, deltamethrin, λ-cyhalothrin, β-cyfluthrin, esfenvalerate, S-bioallethrin, fenpropathrin, cypermethrin, and bifenthrin) produced concentration-dependent elevations in intracellular calcium concentration ([Ca(2+)](i)) in neocortical neurons. Permethrin and resmethrin were without effect on [Ca(2+)](i). These pyrethroids displayed a range of efficacies on Ca(2+) influx; however, the EC(50) values for active pyrethroids all were within one order of magnitude. Tetrodotoxin blocked increases in [Ca(2+)](i) caused by all nine active pyrethroids, indicating that the effects depended on VGSC activation. The pathways for deltamethrin- and tefluthrin-induced Ca(2+) influx include N-methyl-D-aspartic acid receptors, L-type Ca(2+) channels, and reverse mode of operation of the Na(+)/Ca(2+) exchanger inasmuch as antagonists of these sites blocked deltamethrin-induced Ca(2+) influx. These data demonstrate that pyrethroids stimulate Ca(2+) entry into neurons subsequent to their actions on VGSCs.

Regulated ADAM10-dependent Ectodomain Shedding of γ-Protocadherin C3 Modulates Cell-Cell Adhesion

Gamma-protocadherins (Pcdh gamma) are type I transmembrane proteins, which are most notably expressed in the nervous system. They are enriched at synapses and involved in synapse formation, specification, and maintenance. In this study, we show that Pcdh gamma C3 and Pcdh gamma B4 are specifically cleaved within their ectodomains by the disintegrin and metalloprotease ADAM10. Analysis of ADAM10-deficient fibroblasts and embryos, inhibitor studies, as well as RNA interference-mediated down-regulation demonstrated that ADAM10 is not only responsible for the constitutive but also for the regulated shedding of these proteins in fibroblasts and in neuronal cells. In contrast to N-cadherin shedding, which was activated by N-methyl-D-aspartic acid receptor activation in neuronal cells, Pcdh gamma shedding was induced by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrate stimulation, suggesting differential regulation mechanisms of cadherin-mediated functions at synapses. Cell aggregation assays in the presence or absence of metalloprotease inhibitors strongly suggest that the ectodomain shedding events modulate the cell adhesion role of Pcdh gamma. The identification of ADAM10 as the protease responsible for constitutive and regulated Pcdh gamma shedding may therefore provide new insight into the regulation of Pcdh gamma functions.

Phosphorylation of dystrophin Dp71d by Ca2+/calmodulin‐dependent protein kinase II modulates the Dp71d nuclear localization in PC12 cells

We have shown that the splicing isoform of Dp71 (Dp71d) localizes to the nucleus of PC12 cells, an established cell line derived from a rat pheochromocytoma; however, the mechanisms governing its nuclear localization are unknown. As protein phosphorylation modulates the nuclear import of proteins, and as Dp71d presents several potential sites for phosphorylation, we analyzed whether Dp71d is phosphorylated in PC12 cells and the role of phosphorylation on its nuclear localization. We demonstrated that Dp71d is phosphorylated under basal conditions at serine and threonine residues by endogenous protein kinases. Dp71d phosphorylation was activated by 2-O-tetradecanoyl phorbol 13-acetate (TPA), but this effect was blocked by EGTA. Supporting the role of intracellular calcium on Dp71d phosphorylation, we observed that the stimulation of calcium influx by cell depolarization increased Dp71d phosphorylation, and that the calcium-calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7) blocked such induction. The blocking action of bisindolylmaleimide I (Bis I), a specific inhibitor for Ca2+/diacylglicerol-dependent protein kinase (PKC), on Dp71d phosphorylation suggested the participation of PKC in this event. In addition, transfection experiments with Ca2+/calmodulin-dependent protein kinase II (CaMKII) expression vectors as well as the use of KN-62, a CaMKII-specific inhibitor, demonstrated that CaMKII is also involved in Dp71d phosphorylation. Stimulation of Dp71d phosphorylation by cell depolarization and/or the overexpression of CaMKII favored the Dp71d nuclear accumulation. Overall, our results indicate that CAMKII-mediated Dp71d phosphorylation modulates its nuclear localization.

Imidazole-induced elevations of intracellular calcium in HL-60 cells: effect of inhibition of phospholipase C by the steroidal maleimide U73122

A variety of agents, including imidazoles, such as miconazole, elicit elevations of calcium in human leukemic HL-60 cells through a stimulation of influx of calcium and/or a release of intracellular calcium. Release of intracellular calcium by such agents may involve stimulation of IP3 formation. The steroidal maleimide U73122, a potent phospholipase C (PLC) inhibitor, at 3–10 µM blocked elevations of calcium elicited in HL-60 cells by ATP. At a 10&!hyphen; µM concentration, U73122 either blocked or altered elevations of calcium elicited by imidazoles, such as miconazole, SKF 96365, and clotrimazole that are often used to block capacitative calcium entry, and by calmidazolium, a quaternary imidazole that can trigger massive influx of calcium in HL-60 cells. Elevations of calcium elicited by thapsigargin were not affected by 3 µM U73122, but were reduced by higher concentrations. Remarkably, U73122 at 10 µM did not block, but instead slowed and potentiated the elevation of calcium elicited by higher concentrations of imidazoles, an effect that may involve inhibition of Ca2+ -ATPases of the endoplasmic reticulum. The steroidal succinimide U73343 had minimal effects on calcium elevations in HL-60 cells. The results indicate that U73122 requires cautious use in assessing the role of PLC in controlling calcium levels in HL-60 cells, in particular when used with imidazoles, such as SKF 96365, which are used to block capacitative calcium entry. The effects of U73122 on cell calcium are reviewed. In toto, the results suggest that many lipophilic agents may affect intracellular calcium by activating PLC through unknown mechanisms. Drug Dev. Res. 67:519–534, 2006. Published 2006 Wiley-Liss, Inc.

Effects of prolactin on ionic membrane conductances in the human malignant astrocytoma cell line U87-MG.

Prolactin (PRL) is involved in numerous biological processes in peripheral tissues and the brain. Although numerous studies have been conducted to elucidate the signal transduction pathways associated with the PRL receptor, very few have examined the role of ion conductances in PRL actions. We used the patch-clamp technique in "whole cell" configuration and microspectrofluorimetry to investigate the effects of PRL on membrane ion conductances in the U87-MG human malignant astrocytoma cell line, which naturally expresses the PRL receptor. We found that a physiological concentration (4 nM) of PRL exerted a biphasic action on membrane conductances. First, PRL activated a Ca(2+)-dependent K(+) current that was sensitive to CTX and TEA. This current depended on PRL-induced Ca(2+) mobilization, through a JAK2-dependent pathway from a thapsigargin- and 2-APB-sensitive Ca(2+) pool. Second, PRL also activated an inwardly directed current, mainly due to the stimulation of calcium influx via nickel- and 2-APB-sensitive calcium channels. Both phases resulted in membrane hyperpolarizations, mainly through the activation of Ca(2+)-dependent K(+) channels. As shown by combined experiments (electrophysiology and microspectrofluorimetry), the PRL-induced Ca(2+) influx increased with cell membrane hyperpolarization and conversely decreased with cell membrane depolarization. Thus PRL-induced membrane hyperpolarizations facilitated Ca(2+) influx through voltage-independent Ca(2+) channels. Finally, PRL also activated a DIDS-sensitive Cl(-) current, which may participate in the PRL-induced hyperpolarization. These PRL-induced conductance activations are probably related to the PRL proliferative effect we have already described in U87-MG cells.

Nicotine exposure and bronchial epithelial cell nicotinic acetylcholine receptor expression in the pathogenesis of lung cancer.

Forty-eight million Americans smoke because of addiction to nicotine, and prevention of smoking initiation and new methods for aiding in smoking cessation are some of the most important opportunities for disease prevention available (1). This smoking addiction results from nicotine acting on neuronal nicotinic acetylcholine receptors (nAchRs) in the brain in key regions controlling behavior. There is detailed structural and functional information on neuronal nAchRs that are the prototype of ligand-gated ion channels mediating transmission of endogenous acetylcholine (Ach) and exogenous nicotine signals in the central and peripheral nervous system (2). In addition to the well-formulated role of nAchRs in the etiology of addiction, emerging evidence suggests that nicotine contributes directly to lung carcinogenesis through stimulation of nAchRs in non-neuronal cells. Our group showed that many lung cancers expressed nAchRs and that low concentrations of nicotine blocked the induction of apoptosis in these cells (3). Conti-Fine’s group demonstrated that functional nAchRs are expressed in human and rodent bronchial epithelial cells (4, 5). While not in the bronchial epithelium, Grando and colleagues showed that human epidermal keratinocytes synthesize, secrete, and degrade Ach which is used to mediate stimulation of calcium influx and cell differentiation through α7 nAchRs on these same cells (6, 7). nAchRs bind not only nicotine, but also nicotine-derived nitrosamines such as the tobacco specific carcinogens N′-Nitrosonornicotine (NNN) and 4(methylnitrosamino)-1-(3–pyridyl)-1-butanone (NNK), which are potent lung carcinogens. There is evidence from Schuller’s group that lung cancer and fetal pulmonary neuroendocrine cells express nAchRs that bind these ligands with higher affinity than nicotine stimulating the growth of these cells (8–10).

Phospholipases D1 and D2 regulate different phases of exocytosis in mast cells.

The rat mast cell line RBL-2H3 contains both phospholipase D (PLD)1 and PLD2. Previous studies with this cell line indicated that expressed PLD1 and PLD2 are both strongly activated by stimulants of secretion. We now show by use of PLDs tagged with enhanced green fluorescent protein that PLD1, which is largely associated with secretory granules, redistributes to the plasma membrane in stimulated cells by processes reminiscent of exocytosis and fusion of granules with the plasma membrane. These processes and secretion of granules are suppressed by expression of a catalytically inactive mutant of PLD1 or by the presence of 50 mM 1-butanol but not tert-butanol, an indication that these events are dependent on the catalytic activity of PLD1. Of note, cholera toxin induces translocation of PLD1-labeled granules to the plasma membrane but not fusion of granules with plasma membrane or secretion. Subsequent stimulation of calcium influx with Ag or thapsigargin leads to rapid redistribution of PLD1 to the plasma membrane and accelerated secretion. Also of note, PLD1 is recycled from plasma membrane back to granules within 4 h of stimulation. PLD2, in contrast, is largely confined to the plasma membrane, but it too participates in the secretory process, because expression of catalytically inactive PLD2 also blocks secretion. These data indicate a two-step process: translocation of granules to the cell periphery, regulated by granule-associated PLD1, and a calcium-dependent fusion of granules with the plasma membrane, regulated by plasma membrane-associated PLD2 and possibly PLD1.

Two-way arginine transport in human endothelial cells: TNF-α stimulation is restricted to system y+

Human umbilical vein endothelial cells transport arginine through two Na(+)-independent systems. System y(+)L is insensitive to N-ethylmaleimide (NEM), inhibited by L-leucine in the presence of Na(+), and referable to the expression of SLC7A6/y(+)LAT2, SLC7A7/y(+)LAT1, and SLC3A2/4F2hc. System y(+) is referable to the expression of SLC7A1/CAT1 and SLC7A2/CAT2B. Tumor necrosis factor-alpha (TNF-alpha) and bacterial lipopolysaccharide induce a transient stimulation of arginine influx and efflux through system y(+). Increased expression of SLC7A2/CAT2B is detectable from 3 h of treatment, while SLC7A1 expression is inhibited at later times of incubation. System y(+)L activity and expression remain unaltered. Nitric oxide synthase type 2 mRNA is not detected in the absence or presence of TNF-alpha, while the latter condition lowers nitric oxide synthase type 3 expression at the mRNA and the protein level. Nitrite accumulation is comparable in cytokine-treated and control cells up to 48 h of treatment. It is concluded that modulation of endothelial arginine transport by TNF-alpha or lipopolysaccharide occurs exclusively through changes in CAT2B and CAT1 expression and is dissociated from stimulation of nitric oxide production.

Zinc Phytoextraction in Thlaspi caerulescens

ABSTRACT The existence of metal hyperaccumulator species demonstrates that plants have the genetic potential to remove toxic metals from contaminated soil. Possibly, one of the best-known hyperaccumulators is Thlaspi caerulescens. This species has been shown to accumulate very high Zn concentrations without manifesting any sign of toxicity. Thus, T. caerulescens represents an excellent experimental system for studying metal hyperaccumulation in plants as it relates to phytoremediation. In this article, we review the results of an investigation into the physiology, biochemistry, and molecular regulation of Zn transport and accumulation in T. caerulescens compared with a nonaccumulator relative T. arvense. Physiological studies focused on the use of 65Zn radiotracer flux techniques to characterize zinc transport and compartmentation in the root, and translocation to the shoot. Transport studies indicated that a number of Zn transport sites were stimulated in T. caerulescens, contributing to the hyperaccumulation trait. Thus, Zn influx into root and leaf cells, and Zn loading into the xylem was greater in T. caerulescens compared with the nonaccumulator T. arvense. The 4.5-fold stimulation of Zn influx into the roots of T. caerulescens was hypothesized to be due to an overexpression of Zn transporters in this species. Additionally, compartmental analysis (radiotracer wash out or efflux techniques) was used to show that Zn was sequestered in the root vacuole of T. arvense inhibiting Zn translocation to the shoot in this nonaccumulator species. Molecular studies focused on the cloning and characterization of Zn transport genes in T. caerulescens. Functional complementation of a yeast Zn transport-defective mutant with a T. caerulescens cDNA library constructed in a yeast expression vector resulted in the cloning of a Zn transport cDNA, ZNT1. Expression of ZNT1 in yeast allowed for a physiological characterization of this transporter. ZNT1 was shown to encode a high-affinity Zn transporter that can also mediate low-affinity Cd transport. Biochemical analyses indicated that enhanced Zn transport in T. caerulescens results from a constitutively high expression of ZNT1 in roots and shoots. These results suggest that overexpression of ZNT1 may be linked to an alteration of the Zn tolerance mechanism in this species.

Inhibition of nitric oxide synthesis augments pulmonary oedema in isolated perfused rabbit lung

The role of nitric oxide (NO) in precipitating pulmonary oedema in acute lung injury remains unclear. We have investigated the mechanism of involvement of NO in the maintenance of liquid balance in the isolated rabbit lung. Thirty pairs of lungs were perfused with colloid for up to 6 h, during which pulmonary vascular resistance (PVR) and capillary pressure (PCP) were measured frequently, and time to gain 5 g in weight (t5) was recorded. Four protocols with different perfusate additives were studied: (i) none (control, n = 11); (ii) 10 mmol NG-nitro-L-arginine methyl ester (L-NAME) (n = 6); (iii) 10 mmol L-NAME with 100 mumol lodoxamide, an inhibitor of mast cell degranulation (n = 7); (iv) 10 mmol L-NAME with 10 mumol 8-bromo-3',5'-cyclic guanosine monophosphate (8Br-cGMP), an analogue of cGMP that may reduce vascular permeability by relaxing contractile elements in endothelial cells (n = 6). Neither PVR nor PCP differed between protocols. L-NAME markedly reduced t5 from 248 (27) min (mean (SEM)) in protocol (i) to 144 (5) min in protocol (ii) (P < 0.05). Both lodoxamide (t5 = 178 (7) min) and 8Br-cGMP (t5 = 204 (10) min) substantially corrected the effect of L-NAME (P < 0.005). Results suggest that maintenance of a low permeability by NO may involve mast cell stabilization and endothelial cell relaxation.

Voltage-dependent, pertussis toxin insensitive inhibition of calcium currents by histamine in bovine adrenal chromaffin cells.

Histamine is a known secretagogue in adrenal chromaffin cells. Activation of G-protein linked H(1) receptors stimulates phospholipase C, which generates inositol trisphosphate leading to release of intracellular calcium stores and stimulation of calcium influx through store operated and other channels. This calcium leads to the release of catecholamines. In chromaffin cells, the main physiological trigger for catecholamine release is calcium influx through voltage-gated calcium channels (I(Ca)). Therefore, these channels are important targets for the regulation of secretion. In particular N- and P/Q-type I(Ca) are subject to inhibition by transmitter/hormone receptor activation of heterotrimeric G-proteins. However, the direct effect of histamine on I(Ca) in chromaffin cells is unknown. This paper reports that histamine inhibited I(Ca) in cultured bovine adrenal chromaffin cells and this response was blocked by the H(1) antagonist mepyramine. With high levels of calcium buffering in the patch pipette solution (10 mM EGTA), histamine slowed the activation kinetics and inhibited the amplitude of I(Ca). A conditioning prepulse to +100 mV reversed the kinetic slowing and partially relieved the inhibition. These features are characteristic of a membrane delimited, voltage-dependent pathway which is thought to involve direct binding of G-protein betagamma subunits to the Ca channels. However, unlike virtually every other example of this type of inhibition, the response to histamine was not blocked by pretreating the cells with pertussis toxin (PTX). The voltage-dependent, PTX insensitive inhibition produced by histamine was modest compared with the PTX sensitive inhibition produced by ATP (28% vs. 53%). When histamine and ATP were applied concomitantly there was no additivity of the inhibition beyond that produced by ATP alone (even though the agonists appear to activate distinct G-proteins) suggesting that the inhibition produced by ATP is maximal. When experiments were carried out under conditions of low levels of calcium buffering in the patch pipette solution (0.1 mM EGTA), histamine inhibited I(Ca) in some cells using an entirely voltage insensitive pathway. We demonstrate that activation of PTX insensitive G-proteins (most likely Gq) by H(1) receptors inhibits I(Ca). This may represent a mechanism by which histamine exerts inhibitory (in addition to previously identified stimulatory) effects on catecholamine release.

Molecular physiology of zinc transport in the Zn hyperaccumulator Thlaspi caerulescens

In this manuscript, recent research from this laboratory into physiological and molecular aspects of heavy metal (Zn) transport in the hyperaccumulating plant species, Thlaspi caerulescens is reviewed. This research is aimed at elucidating the processes that underlie the accumulation of extraordinarily high levels of Zn in the T. caerulescens shoot (up to 3% Zn dry wt.) without any associated toxicity symptom. Physiological studies focused on the use of radiotracer flux techniques (65Zn2+) to characterize zinc transport and compartmentation in the root, and translocation and accumulation in the shoot of T. caerulescens in comparison with a related non-accumulator, T. arvense. These studies indicated that Zn transport was stimulated at a number of sites in T. caerulescens, contributing to the hyperaccumulation trait. The transport processes that were stimulated included Zn influx into both root and leaf cells, and Zn loading into the xylem. The 4- to 5-fold stimulation of Zn influx into the root was hypothesized to be due to an increased abundance of Zn transporters in T. caerulescens root cells. Additionally, compartmental analysis (radiotracer wash out or efflux techniques) was used to show that Zn was sequestered in the vacuoles of T. arvense root cells which retarded Zn translocation to the shoot in this non-accumulator species. Molecular studies have focused on the cloning and characterization of Zn transport genes in T. caerulescens. Complementation of a yeast Zn transport-defective mutant with a T. caerulescens cDNA library resulted in the recovery of a cDNA, ZNT1, that encodes a Zn transporter. Sequence analysis of ZNT1 indicated it is a member of a recently discovered micronutrient transport gene family which includes the Arabidopsis Fe transporter, IRT1, and the ZIP Zn transporters. Expression of ZNT1 in yeast allowed for a physiological characterization of this transporter. It was shown to encode a high affinity Zn transporter which can also mediate low affinity Cd transport. Northern analysis of ZNT1 and its homologue in the two Thlaspi species indicated that enhanced Zn transport in T. caerulescens results from a constitutively high expression of the ZNT1 gene in roots and shoots. In T. arvense, ZNT1 is expressed at far lower levels and this expression is stimulated by imposition of Zn deficiency.

Dialdehyde sesquiterpenes and other terpenoids as vanilloids

Selected naturally occurring unsaturated dialdehyde sesquiterpenes and related bioactive terpenoids were assayed for vanilloid-like activity. Out of the 25 compounds tested, eight inhibited completely the specific binding of [ 3 H ]resiniferatoxin by rat spinal cord membranes: binding affinities ranged from 0.6 μM for cinnamodial to 19.0 μM for hebelomic acid F. These values were comparable to the binding affinity of capsaicin (2.7 μM). With the exception of four ligands, compounds that inhibited resiniferatoxin binding to rat spinal cord membranes were also pungent on the human tongue where they showed cross-tachyphylaxis with capsaicin. As expected from their reactive nature, these compounds possess additional sites of action, as reflected in the complex behavior of the stimulation of calcium influx by cinnamodial and cinnamosmolide at high concentrations. This observation might explain the unexpectedly weak membrane depolarization by cinnamodial compared to capsaicin. We conclude that a range of sesquiterpene dialdehydes and related terpenoids, both pungent and non-pungent, may function as vanilloids. These compounds may represent a new chemical lead for the development of vanilloid drugs, structurally unrelated to either capsaicin or resiniferatoxin.