Physiological Nanomolar Concentrations Research Articles

Trace amounts of pyroglutaminated Aβ-(3–42) enhance aggregation of Aβ-(1–42) on neuronal membranes at physiological concentrations: FCS analysis of cell surface

Minor species of amyloid β-peptide (Aβ), such as Aβ-(1–43) and pyroglutaminated Aβ-(3–42) (Aβ-(3pE–42)), have been suggested to be involved in the initiation of the Aβ aggregation process, which is closely associated with the etiology of Alzheimer's disease. They can play important roles in aggregation not only in the aqueous phase but also on neuroral membranes; however, the latter behaviors remain mostly unexplored. Here, initial aggregation processes of Aβ on living cells were monitored at physiological nanomolar concentrations by fluorescence correlation spectroscopy. Membrane-bound Aβ-(1–42) and Aβ-(1–40) formed oligomers composed of ~4 Aβ molecules during 48-h incubation, whereas the peptides remained monomeric in the culture medium, indicating that the membranes facilitated Aβ aggregation. The presence of 5 mol% Aβ-(3pE–42), but not Aβ-(1–43), significantly enhanced the aggregation of Aβ-(1–42) up to ~10-mers. On the other hand, neither trace amounts of Aβ-(1–42) nor Aβ-(3pE–42) enhanced the aggregation of Aβ-(1–40). The observed small Aβ oligomers are expected to act as pathogenic seeds for amyloid fibrils responsible for neurotoxicity. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.

Statin-conferred enhanced cellular resistance against bacterial pore-forming toxins in airway epithelial cells.

Statins are widely used to prevent cardiovascular disease. In addition to their inhibitory effects on cholesterol synthesis, statins have beneficial effects in patients with sepsis and pneumonia, although molecular mechanisms have mostly remained unclear. Using human airway epithelial cells as a proper in vitro model, we show that prior exposure to physiological nanomolar serum concentrations of simvastatin (ranging from 10-1,000 nM) confers significant cellular resistance to the cytotoxicity of pneumolysin, a pore-forming toxin and the main virulence factor of Streptococcus pneumoniae. This protection could be demonstrated with a different statin, pravastatin, or on a different toxin, α-hemolysin. Furthermore, through the use of gene silencing, pharmacological inhibitors, immunofluorescence microscopy, and biochemical and metabolic rescue approaches, we demonstrate that the mechanism of protection conferred by simvastatin at physiological nanomolar concentrations could be different from the canonical mevalonate pathways seen in most other mechanistic studies conducted with statins at micromolar levels. All of these data are integrated into a protein synthesis-dependent, calcium-dependent model showing the interconnected pathways used by statins in airway epithelial cells to elicit an increased resistance to pore-forming toxins. This research fills large gaps in our understanding of how statins may confer host cellular protection against bacterial infections in the context of airway epithelial cells without the confounding effect from the presence of immune cells. In addition, our discovery could be potentially developed into a host-centric strategy for the adjuvant treatment of pore-forming toxin associated bacterial infections.

Melatonin promotes Bax sequestration to mitochondria reducing cell susceptibility to apoptosis via the lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid

Extra-neurological functions of melatonin include control of the immune system and modulation of apoptosis. We previously showed that melatonin inhibits the intrinsic apoptotic pathway in leukocytes via stimulation of high affinity MT1/MT2 receptors, thereby promoting re-localization of the anti-apoptotic Bcl-2 protein to mitochondria. Here we show that Bcl-2 sequesters pro-apoptotic Bax into mitochondria in an inactive form after melatonin treatment, thus reducing cell propensity to apoptosis. Bax translocation and the anti-apoptotic effect of melatonin are strictly dependent on the presence of Bcl-2, and on the 5-lipoxygenase (5-LOX) metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), which we have previously shown to be produced as a consequence of melatonin binding to its low affinity target calmodulin. Therefore, the anti-apoptotic effect of melatonin requires the simultaneous, independent interaction with high (MT1/MT2) and low (calmodulin) affinity targets, eliciting two independent signal transduction pathways converging into Bax sequestration and inactivation. MT1/MT2 vs. lipoxygenase pathways are activated by 10−9 vs. 10−5M melatonin, respectively; the anti-apoptotic effect of melatonin is achieved at 10−5M, but drops to 10−9M upon addition of exogenous 5-HETE, revealing that lipoxygenase activation is the rate-limiting pathway. Therefore, in areas of inflammation with increased 5-HETE levels, physiological nanomolar concentrations of melatonin may suffice to maintain leukocyte viability.

Prostaglandin E2 regulation of amnion cell vascular endothelial growth factor expression: relationship with intramembranous absorption rate in fetal sheep.

We hypothesized that prostaglandin E2 (PGE2) stimulates amniotic fluid transport across the amnion by upregulating vascular endothelial growth factor (VEGF) expression in amnion cells and that amniotic PGE2 concentration correlates positively with intramembranous (IM) absorption rate in fetal sheep. The effects of PGE2 at a range of concentrations on VEGF 164 and caveolin-1 gene expressions were analyzed in cultured ovine amnion cells. IM absorption rate, amniotic fluid (AF) volume, and PGE2 concentration in AF were determined in late-gestation fetal sheep during control conditions, isovolumic fetal urine replacement (low IM absorption rate), or intra-amniotic fluid infusion (high IM absorption rate). In ovine amnion cells, PGE2 induced dose- and time-dependent increases in VEGF 164 mRNA levels and reduced caveolin-1 mRNA and protein levels. VEGF receptor blockade abolished the caveolin-1 response, while minimally affecting the VEGF response to PGE2. In sheep fetuses, urine replacement reduced amniotic PGE2 concentration by 58%, decreased IM absorption rate by half, and doubled AF volume (P < 0.01). Intra-amniotic fluid infusion increased IM absorption rate and AF volume (P < 0.01), while amniotic PGE2 concentration was unchanged. Neither IM absorption rate nor AF volume correlated with amniotic PGE2 concentration under each experimental condition. Although PGE2 at micromolar concentrations induced dose-dependent responses in VEGF and caveolin-1 gene expression in cultured amnion cells consistent with a role of PGE2 in activating VEGF to mediate AF transport across the amnion, amniotic PGE2 at physiological nanomolar concentrations does not appear to regulate IM absorption rate or AF volume.

TBid Undergoes Multiple Conformational Changes at the Membrane Required for Bax Activation

Bid is a Bcl-2 family protein that promotes apoptosis by activating Bax and eliciting mitochondrial outer membrane permeabilization (MOMP). Full-length Bid is cleaved in response to apoptotic stimuli into two fragments, p7 and tBid (p15), that are held together by strong hydrophobic interactions until the complex binds to membranes. The detailed mechanism(s) of fragment separation including tBid binding to membranes and release of the p7 fragment to the cytoplasm remain unclear. Using liposomes or isolated mitochondria with fluorescently labeled proteins at physiological concentrations as in vitro models, we report that the two components of the complex quickly separate upon interaction with a membrane. Once tBid binds to the membrane, it undergoes slow structural rearrangements that result in an equilibrium between two major tBid conformations on the membrane. The conformational change of tBid is a prerequisite for interaction with Bax and is, therefore, a novel step that can be modulated to promote or inhibit MOMP. Using automated high-throughput image analysis in cells, we show that down-regulation of Mtch2 causes a significant delay between tBid and Bax relocalization in cells. We propose that by promoting insertion of tBid via a conformational change at the mitochondrial outer membrane, Mtch2 accelerates tBid-mediated Bax activation and MOMP. Thus the interaction of Mtch2 and tBid is a potential target for therapeutic control of Bid initiated cell death.

1α,25(OH) 2 Vitamin D 3 actions on ion channels in osteoblasts

Membrane-initiated cellular responses to steroids include modulation of ion channel activities via signal transduction pathways. However, the molecular mechanisms involved in nongenomic actions remain only partially understood. Our research has focused on the rapid effects of 1α,25(OH) 2 Vitamin D 3 [1,25D] on L-type Ca 2+ [L-Ca] and DIDS-sensitive Cl − channels in osteoblasts. Physiological nanomolar concentrations of hormonally active 1,25D promote rapid (1–5 min) potentiation of outward Cl − currents in osteosarcoma ROS 17/2.8 cells and mouse primary osteoblasts. In addition, 1,25D increases inward barium currents through L-Ca channels at low depolarizing potentials within seconds in a fashion similar to the 1,4-dihydropyridine [DHP] agonist Bay K8644. We found that second messenger cAMP is involved in 1,25D potentiation of Cl − and Ca 2+ channels. Nongenomic 1,25D effects on ion channel activities in osteoblasts appear to involve different mechanisms that include a possible direct interaction with the L-Ca channel molecule, on one hand, and signaling through the cAMP pathway, on the other. Rapid 1,25D actions on Cl − and Ca 2+ currents seem to couple to secretory activities in osteoblasts, thus contributing to bone mass formation.

Saturated and cis/trans Unsaturated Acyl CoA Esters Differentially Regulate Wild-Type and Polymorphic β-Cell ATP-Sensitive K+ Channels

Metabolic regulation of pancreatic beta-cell ATP-sensitive K+ channel (K(ATP) channel) function plays a key role in the process of glucose-stimulated insulin secretion (GSIS). Modulation of K(ATP) channel activity by long-chain acyl CoAs represents an important endogenous regulatory mechanism. Elevated acyl CoA levels have been reported in obese and type 2 diabetic individuals and may contribute to reduced beta-cell excitability and impaired GSIS. Recent studies suggest that the composition of dietary fat may influence the effects of high-fat feeding on impaired GSIS. Therefore, we examined the effects of side-chain length and the degree of saturation of various acyl CoAs on K(ATP) channel activity. Macroscopic currents from either wild-type or polymorphic (Kir6.2[E23K/I337V]) recombinant beta-cell K(ATP) channels were measured in inside-out patches by exposing the inner surface of the membrane to acyl CoAs at physiological nanomolar concentrations. Acyl CoAs increased both wild-type and polymorphic K(ATP) channel activity with the following rank order of efficacy: C18:0, C18:1trans approximately C18:1cis, C20:4 = C16:0, C16:1, and C18:2. A significant correlation exists between activation and acyl CoA hydrophobicity, suggesting that both side-chain length and degree of saturation are critical determinants of K(ATP) channel activation. Our observations reveal a plausible mechanism behind the disparate effects of acyl CoA saturation on K(ATP) channel activation and suggest that dietary fat composition may determine the severity of impaired GSIS via differential activation of beta-cell K(ATP) channels.

Oxidative Metabolites Accelerate Alzheimer's Amyloidogenesis by a Two-Step Mechanism, Eliminating the Requirement for Nucleation

The process of amyloid formation by the amyloid beta peptide (Abeta), i.e., the misassembly of Abetapeptides into soluble quaternary structures and, ultimately, amyloid fibrils, appears to be at the center of Alzheimer's disease (AD) pathology. We have shown that abnormal oxidative metabolites, including cholesterol-derived aldehydes, modify Abeta and accelerate the early stages of amyloidogenesis (the formation of spherical aggregates). This process, which we have termed metabolite-initiated protein misfolding, could explain why hypercholesterolemia and inflammation are risk factors for sporadic AD. Herein, the mechanism by which cholesterol metabolites hasten Abeta 1-40 amyloidogenesis is explored, revealing a process that has at least two steps. In the first step, metabolites modify Abeta peptides by Schiff base formation. The Abeta-metabolite adducts form spherical aggregates by a downhill polymerization that does not require a nucleation step, dramatically accelerating Abeta aggregation. In agitated samples, a second step occurs in which fibrillar aggregates form, a step also accelerated by cholesterol metabolites. However, the metabolites do not affect the rate of fibril growth in seeded aggregation assays; their role appears to be in initiating amyloidogenesis by lowering the critical concentration for aggregation into the nanomolar range. Small molecules that block Schiff base formation inhibit the metabolite effect, demonstrating the importance of the covalent adduct. Metabolite-initiated amyloidogenesis offers an explanation for how Abeta aggregation could occur at physiological nanomolar concentrations.

Multiple molecular mechanisms of 1α,25(oh) 2-vitamin D 3 rapid modulation of three ion channel activities in osteoblasts

Rapid nongenomic responses to steroids include modulation of ion channel activities on the cell membrane of target cells, but little is known about the molecular mechanisms involved. In this paper we investigate the mechanisms underlying the combined action of the secosteroid hormone 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2D 3] on three different ion channel types in rat osteoblasts, which include a voltage-gated L-type Ca 2+ channel, a mechanosensitive Cl − channel, and a stretch-activated cation (SA-Cat) channel. We found that physiological nanomolar concentrations of 1α,25(OH) 2D 3 rapidly modify the overall electrical activity of the membrane in ROS 17/2.8 cells. 1α,25(OH) 2D 3 increases the osteoblast L-type Ca 2+ channel activity at low depolarizing voltages in a fashion similar to the 1,4-dihydropyridine (DHP) agonist Bay K8644. At highly depolarizing potentials 1α,25(OH) 2D 3 potentiates volume-sensitive Cl − currents through mechanisms that may involve a putative membrane receptor. We show for the first time that 1α,25(OH) 2D 3 also increases inward currents through SA-Cat channels at positive membrane voltages in a dose-dependent manner. Contrary to our expectations, the stereoisomer 1β,25(OH) 2D 3, which suppresses 1α,25(OH) 2D 3 activation of osteoblast Cl − currents, mimicked 1α,25(OH) 2D 3 agonist effects on Ca 2+ and SA-Cat channel activities. Cyclic AMP is involved in 1α,25(OH) 2D 3 effects on both Ca 2+ and SA-Cat channels, but not in Cl − channels. We conclude that 1α,25(OH) 2D 3 rapid effects on ion channel activities in ROS 17/2.8 cells occur through multiple mechanisms that, on the one hand, involve a possible direct interaction with the L-type Ca 2+ channel molecule and, on the other hand, molecular pathways that may include a putative membrane receptor.

Amyloid beta protein forms ion channels: implications for Alzheimer's disease pathophysiology.

Amyloid beta protein (AbetaP) is the major constituent of senile plaques associated with Alzheimer's disease (AD). However, its mechanistic role in AD pathogenesis is poorly understood. Globular and nonfibrillar AbetaPs are continuously released during normal metabolism. Using techniques of atomic force microscopy, laser confocal microscopy, electrical recording, and biochemical assays, we have examined the molecular conformations of reconstituted globular AbetaPs as well as their real-time and acute effects on neuritic degeneration. Atomic force microscopy (AFM) of AbetaP1-42 shows globular structures that do not form fibers in physiological-buffered solution for up to 8 h of continuous imaging. AFM of AbetaP1-42 reconstituted in a planar lipid bilayer reveals multimeric channel-like structures. Consistent with these AFM resolved channel-like structures, biochemical analysis demonstrates that predominantly monomeric AbetaPs in solution form stable tetramers and hexamers after incorporation into lipid membranes. Electrophysiological recordings demonstrate the presence of multiple single channel currents of different sizes. At the cellular level, AbetaP1-42 allows calcium uptake and induces neuritic abnormality in a dose- and time-dependent fashion. At physiological nanomolar concentrations, rapid neuritic degeneration was observed within minutes; at micromolar concentrations, neuronal death was observed within 3-4 h. These effects are prevented by zinc (an AbetaP channel blocker) and by the removal of extracellular calcium, but are not prevented by antagonists of putative AbetaP cell surface receptors. Thus, AbetaP channels may provide a direct pathway for calcium-dependent AbetaP toxicity in AD.

Somatostatin through its specific receptor inhibits spontaneous and TNF-alpha- and bacteria-induced IL-8 and IL-1 beta secretion from intestinal epithelial cells.

Intestinal epithelial cells secrete proinflammatory cytokines and chemokines that are crucial in mucosal defense. However, this secretion must be tightly regulated, because uncontrolled secretion of proinflammatory mediators may lead to chronic inflammation and mucosal damage. The aim of this study was to determine whether somatostatin, secreted within the intestinal mucosa, regulates secretion of cytokines from intestinal epithelial cells. The spontaneous as well as TNF-alpha- and Salmonella-induced secretion of IL-8 and IL-1beta derived from intestinal cell lines Caco-2 and HT-29 was measured after treatment with somatostatin or its synthetic analogue, octreotide. Somatostatin, at physiological nanomolar concentrations, markedly inhibited the spontaneous and TNF-alpha-induced secretion of IL-8 and IL-1beta. This inhibition was dose dependent, reaching >90% blockage at 3 nM. Furthermore, somatostatin completely abrogated the increased secretion of IL-8 and IL-1beta after invasion by Salmonella. Octreotide, which mainly stimulates somatostatin receptor subtypes 2 and 5, affected the secretion of IL-8 and IL-1beta similarly, and the somatostatin antagonist cyclo-somatostatin completely blocked the somatostatin- and octreotide-induced inhibitory effects. This inhibition was correlated to a reduction of the mRNA concentrations of IL-8 and IL-1beta. No effect was noted regarding cell viability. These results indicate that somatostatin, by directly interacting with its specific receptors that are expressed on intestinal epithelial cells, down-regulates proinflammatory mediator secretion by a mechanism involving the regulation of transcription. These findings suggest that somatostatin plays an active role in regulating the mucosal inflammatory response of intestinal epithelial cells after physiological and pathophysiological stimulations such as bacterial invasion.

The direct renal tubular effect of angiotensin II in chicken.

Taking advantage of the particular renal vascular arrangement in cocks, angiotensin II was injected (0.12-0.96 microgram/min) into the portal system of one kidney in order to increase the angiotensin II concentration in the physiological nanomolar range at the level of the renal tubules. Angiotensin II induced an increase in blood pressure of 5% and a bilateral rise in glomerular filtration rate and effective renal plasma flow of 28 and 22%, respectively. The urine volume increased five times on the infused side and three times on the control side. The Na+ excretion increased 14 times on the infused side and only seven times on the control side. During angiotensin infusion, the fractional water excretion was 4.9% on the infused side and 2.9% on the control side versus 1.1 and 1.2% during the control period. For the fractional Na+ excretion, the respective values were 2 and 1.2% versus 0.2 and 0.2% during the control period. The differences between the two kidneys demonstrate the direct tubular action of angiotensin II, inhibiting the tubular Na+ and water reabsorption at physiological nanomolar concentrations. Angiotensin seems thus to play an important intrarenal role.