Increase In Cytosolic Free Calcium Research Articles

CPL207280, a Novel G Protein-Coupled Receptor 40/Free Fatty Acid Receptor 1-Specific Agonist, Shows a Favorable Safety Profile and Exerts Antidiabetic Effects in Type 2 Diabetic Animals.

G protein-coupled receptor (GPR) 40 is a free fatty acid receptor mainly expressed in pancreatic β-cells activated by medium- and long-chain fatty acids and regulating insulin secretion via an increase in cytosolic free calcium ([Ca2+]i). Activation of GPR40 in pancreatic β-cells may improve glycemic control in type 2 diabetes through enhancement of glucose-stimulated insulin secretion. However, the most clinically advanced GPR40 agonist-TAK-875 (fasiglifam)-was withdrawn from phase III because of its hepatotoxicity resulting from the inhibition of pivotal bile acid transporters. Here, we present a new, potent CPL207280 agonist and compare it with fasiglifam in numerous in vitro and in vivo studies. CPL207280 showed greater potency than fasiglifam in a Ca2+ influx assay with a human GPR40 protein (EC50 = 80 vs. 270 nM, respectively). At the 10 µM concentration, it showed 3.9 times greater enhancement of glucose-stimulated insulin secretion in mouse MIN6 pancreatic β-cells. In Wistar Han rats and C57BL6 mice challenged with glucose, CPL207280 stimulated 2.5 times greater insulin secretion without causing hypoglycemia at 10 mg/kg compared with fasiglifam. In three diabetic rat models, CPL207280 improved glucose tolerance and increased insulin area under the curve by 212%, 142%, and 347%, respectively. Evaluation of potential off-target activity (Safety47) and selectivity of CPL207280 (at 10 μM) did not show any significant off-target activity. We conclude that CPL207280 is a potent enhancer of glucose-stimulated insulin secretion in animal disease models with no risk of hypoglycemia at therapeutic doses. Therefore, we propose the CPL207280 compound as a compelling candidate for type 2 diabetes treatment. SIGNIFICANCE STATEMENT: GPR40 is a well-known and promising target for diabetes. This study is the first to show the safety and effects of CPL207280, a novel GPR40/free fatty acid receptor 1 agonist, on glucose homeostasis both in vitro and in vivo in different diabetic animal models. Therefore, we propose the CPL207280 compound as a novel, glucose-lowering agent, overcoming the unmet medical needs of patients with type 2 diabetes.

Live Animal Imaging of Calcium Signaling During Transendothelial Migration Demonstrates Tight Temporospatial Regulation Coordinated by Endothelial Cell TRPC6, IQGAP1, and CaMKIIδ

Proper regulation of inflammatory cell recruitment is critical to fighting infection and maintaining organism homeostasis. Misdirected inflammation can exacerbate pathology and underlies the pathology of virtually all diseases including arthritis, atherosclerosis, and multiple sclerosis. The transendothelial migration (TEM) of leukocytes out of the bloodstream and into tissue is the rate-limiting and putative committed step of the inflammatory process. As such, the regulation of TEM represents a key target for potential therapeutic intervention. Our lab has previously reported that the divalent cation channel TRPC6 was responsible for the increase in cytosolic free calcium (↑[Ca2+]i) in endothelial cells that is required for TEM. TRPC6 is activated downstream of PECAM engagement and is concentrated at the site of leukocyte-endothelial contact during TEM. However, the temporal and spatial regulation of ↑[Ca2+]i during TEM was not known. To visualize calcium signaling during TEM in live animals we developed an intravital microscopy system using the GCaMP3 reporter mouse. In this model, the fluorescence of a modified GFP construct increases with calcium concentration. Indeed, during leukocyte TEM we observed a highly localized increase in endothelial calcium signal surrounding the transmigrating leukocyte that corresponded temporally to the transmigration event. This is the first demonstration of endothelial cell calcium signaling during TEM in vivo. Our lab has also previously reported that the scaffolding protein IQ-motif containing GTPase Activating Protein 1 (IQGAP1) in endothelial cells is required for TEM and functions downstream of PECAM engagement. Here we show that the signals propagated by TRPC6 and IQGAP1 converge into one pathway that results in the activation of calmodulin kinase II δ (CaMKIIδ) that is required for TEM. More specifically, we show that the IQ domain of IQGAP1 binds calmodulin (CaM), a known calcium binding protein. This interaction is required for the downstream activation of CaMKIIδ, which binds to CaM in a calcium-dependent manner. Disruption of either the IQGAP1-CaM or the CaM-CaMKIIδ interaction substantially reduces TEM in vitro. To further validate these findings, we developed two separate genetically-altered mouse lines: one with an inducible endothelial-specific deletion of CaMKIIδ, the other with an inducible endothelial-specific expression an intracellular CaMKIIN inhibitory peptide. Both mice showed significantly reduced TEM in the croton oil dermatitis model of inflammation compared to uninduced controls, confirming that endothelial CaMKIIδ is required for efficient leukocyte TEM in vivo. Overall, these findings uncover a new role for CaMKIIδ and novel connections between it and TRPC6, IQGAP1, and CaM in coordinating the calcium signal that facilitates leukocyte TEM. These results also identify CAMKIIδ as a potential target for anti-inflammatory therapeutics.

Alzheimer's Risk Gene TREM2 Determines Functional Properties of New Type of Human iPSC-Derived Microglia.

Microglia are key in the homeostatic well-being of the brain and microglial dysfunction has been implicated in neurodegenerative disorders such as Alzheimer’s disease (AD). Due to the many limitations to study microglia in situ or isolated for large scale drug discovery applications, there is a high need to develop robust and scalable human cellular models of microglia with reliable translatability to the disease. Here, we describe the generation of microglia-like cells from human induced pluripotent stem cells (iPSC) with distinct phenotypes for mechanistic studies in AD. We started out from an established differentiation protocol to generate primitive macrophage precursors mimicking the yolk sac ontogeny of microglia. Subsequently, we tested 36 differentiation conditions for the cells in monoculture where we exposed them to various combinations of media, morphogens, and extracellular matrices. The optimized protocol generated robustly ramified cells expressing key microglial markers. Bulk mRNA sequencing expression profiles revealed that compared to cells obtained in co-culture with neurons, microglia-like cells derived from a monoculture condition upregulate mRNA levels for Triggering Receptor Expressed On Myeloid Cells 2 (TREM2), which is reminiscent to the previously described disease-associated microglia. TREM2 is a risk gene for AD and an important regulator of microglia. The regulatory function of TREM2 in these cells was confirmed by comparing wild type with isogenic TREM2 knock-out iPSC microglia. The TREM2-deficient cells presented with stronger increase in free cytosolic calcium upon stimulation with ATP and ADP, as well as stronger migration towards complement C5a, compared to TREM2 expressing cells. The functional differences were associated with gene expression modulation of key regulators of microglia. In conclusion, we have established and validated a work stream to generate functional human iPSC-derived microglia-like cells by applying a directed and neuronal co-culture independent differentiation towards functional phenotypes in the context of AD. These cells can now be applied to study AD-related disease settings and to perform compound screening and testing for drug discovery.

Phosphate Deprivation Can Impair Mechano-Stimulated Cytosolic Free Calcium Elevation in Arabidopsis Roots.

The root tip responds to mechanical stimulation with a transient increase in cytosolic free calcium as a possible second messenger. Although the root tip will grow through a heterogeneous soil nutrient supply, little is known of the consequence of nutrient deprivation for such signalling. Here, the effect of inorganic phosphate deprivation on the root’s mechano-stimulated cytosolic free calcium increase is investigated. Arabidopsis thaliana (cytosolically expressing aequorin as a bioluminescent free calcium reporter) is grown in zero or full phosphate conditions, then roots or root tips are mechanically stimulated. Plants also are grown vertically on a solid medium so their root skewing angle (deviation from vertical) can be determined as an output of mechanical stimulation. Phosphate starvation results in significantly impaired cytosolic free calcium elevation in both root tips and whole excised roots. Phosphate-starved roots sustain a significantly lower root skewing angle than phosphate-replete roots. These results suggest that phosphate starvation causes a dampening of the root mechano-signalling system that could have consequences for growth in hardened, compacted soils.

DORN1/P2K1 and purino-calcium signalling in plants: making waves with extracellular ATP

Background and AimsExtracellular ATP governs a range of plant functions, including cell viability, adaptation and cross-kingdom interactions. Key functions of extracellular ATP in leaves and roots may involve an increase in cytosolic free calcium as a second messenger (‘calcium signature’). The main aim here was to determine to what extent leaf and root calcium responses require the DORN1/P2K1 extracellular ATP receptor in Arabidopsis thaliana. The second aim was to test whether extracellular ATP can generate a calcium wave in the root.MethodsLeaf and root responses to extracellular ATP were reviewed for their possible links to calcium signalling and DORN1/P2K1. Leaves and roots of wild type and dorn1 plants were tested for cytosolic calcium increase in response to ATP, using aequorin. The spatial abundance of DORN1/P2K1 in the root was estimated using green fluorescent protein. Wild type roots expressing GCaMP3 were used to determine the spatial variation of cytosolic calcium increase in response to extracellular ATP.Key ResultsLeaf and root ATP-induced calcium signatures differed markedly. The leaf signature was only partially dependent on DORN1/P2K1, while the root signature was fully dependent. The distribution of DORN1/P2K1 in the root supports a key role in the generation of the apical calcium signature. Root apical and sub-apical calcium signatures may operate independently of each other but an apical calcium increase can drive a sub-apical increase, consistent with a calcium wave.ConclusionDORN1 could underpin several calcium-related responses but it may not be the only receptor for extracellular ATP in Arabidopsis. The root has the capacity for a calcium wave, triggered by extracellular ATP at the apex.

Danger signals activate a putative innate immune system during regeneration in a filamentous fungus.

The ability to respond to injury is a biological process shared by organisms of different kingdoms that can even result in complete regeneration of a part or structure that was lost. Due to their immobility, multicellular fungi are prey to various predators and are therefore constantly exposed to mechanical damage. Nevertheless, our current knowledge of how fungi respond to injury is scarce. Here we show that activation of injury responses and hyphal regeneration in the filamentous fungus Trichoderma atroviride relies on the detection of two danger or alarm signals. As an early response to injury, we detected a transient increase in cytosolic free calcium ([Ca2+]c) that was promoted by extracellular ATP, and which is likely regulated by a mechanism of calcium-induced calcium-release. In addition, we demonstrate that the mitogen activated protein kinase Tmk1 plays a key role in hyphal regeneration. Calcium- and Tmk1-mediated signaling cascades activated major transcriptional changes early following injury, including induction of a set of regeneration associated genes related to cell signaling, stress responses, transcription regulation, ribosome biogenesis/translation, replication and DNA repair. Interestingly, we uncovered the activation of a putative fungal innate immune response, including the involvement of HET domain genes, known to participate in programmed cell death. Our work shows that fungi and animals share danger-signals, signaling cascades, and the activation of the expression of genes related to immunity after injury, which are likely the result of convergent evolution.

Non-canonical pathway induced by Wnt3a regulates β-catenin via Pyk2 in differentiating human neural progenitor cells

Wnt/β-catenin and Wnt/Ca2+ pathways are involved in cellular processes during embryonic development and the interaction between them in the same cell decides the outcome of cellular functions. In this study, we showed that Wnt3a triggers the Wnt/Ca2+ signaling pathway, indicated by an increase of cytosolic free calcium ([Ca2+]i) and activation of calmodulin dependent kinase II (CaMKII) during the differentiation of human neuronal progenitor cells (hNPCs). Wnt3a via the increase of [Ca2+]i activates proline-rich tyrosine kinase 2 (Pyk2), which subsequently regulates phosphorylation of glycogen synthase kinase 3β (GSK3β) and β-catenin stabilization. Our findings suggest that Pyk2 plays an important role in the coordination of stabilization of β-catenin in the crosstalk between Wnt/β-catenin and Wnt/Ca2+ signaling pathways upon Wnt3a stimulation in differentiating hNPCs.

Dapper1 attenuates hepatic gluconeogenesis and lipogenesis by activating PI3K/Akt signaling

Studies have shown that hepatic insulin resistance, a disorder of glucose and lipid metabolism, plays a vital role in type 2 diabetes (T2D). To clarify the function of Dapper1 in glucose and lipid metabolism in the liver, we investigated the relationships between Dapper1 and adenosine triphosphate (ATP)- and Ca2+-mediated activation of PI3K/Akt. We observed a reduction in hepatic Dapper1 in db/db (mice that are homozygous for a spontaneous diabetes mutation) and HFD-induced diabetic mice with T2D. Hepatic overexpression of Dapper1 improved hyperglycemia, insulin resistance, and fatty liver. It also increased Akt (pAkt) signaling and repressed both gluconeogenesis and lipogenesis. Conversely, Ad-shDapper1-induced knockdown of hepatic Dapper1 promoted gluconeogenesis and lipogenesis. Furthermore, Dapper1 activated PI3K p110α/Akt in an insulin-independent manner by inducing ATP production and secretion in vitro. Blockade of P2 ATP receptors, the downstream phospholipase C (PLC), or the inositol triphosphate receptor (IP3R all reduced the Dapper1-induced increase in cytosolic free calcium and Dapper1-mediated PI3K/Akt activation, as did removal of calcium in the medium. In conclusion, Dapper1 attenuates hepatic gluconeogenesis and lipogenesis in T2D.

Mechanosensitive TRPM7 mediates shear stress and modulates osteogenic differentiation of mesenchymal stromal cells through Osterix pathway.

Microenvironments that modulate fate commitments of mesenchymal stromal cells (MSCs) are composed of chemical and physical cues, but the latter ones are much less investigated. Here we demonstrate that intermittent fluid shear stress (IFSS), a potent and physiologically relevant mechanical stimulus, regulates osteogenic differentiation of MSCs through Transient receptor potential melastatin 7 (TRPM7)-Osterix axis. Immunostaining showed the localization of TRPM7 near or at cell membrane upon IFSS, and calcium imaging analysis demonstrated the transient increase of cytosolic free calcium. Expressions of osteogenic marker genes including Osterix, but not Runx2, were upregulated after three-hour IFSS. Phosphorylation of p38 and Smad1/5 was promoted by IFSS as well. TRPM7 gene knockdown abolished the promotion of bone-related gene expressions and phosphorylation. We illustrate that TRPM7 is mechanosensitive to shear force of 1.2 Pa, which is much lower than 98 Pa pressure loading reported recently, and mediates distinct mechanotransduction pathways. Additionally, our results suggest the differential roles of TRPM7 in endochondral and intramembranous ossification. Together, this study elucidates the mechanotransduction in MSCs fate commitments and displays an efficient mechano-modulation for MSCs osteogenic differentiation. Such findings should be taken into consideration when designing relevant scaffolds and microfluidic devices for osteogenic induction in the future.

Ursolic acid induces autophagy in U87MG cells via ROS-dependent endoplasmic reticulum stress

Malignant gliomas are the most common primary brain tumors, and novel ways of treating gliomas are urgently needed. Ursolic acid (UA), a pentacyclic triterpenoid, has been reported to exhibit promising antitumor activity. Here, we evaluated the effects of UA on U87MG cells and explored the underlying molecular mechanisms. The results demonstrated that both G1-phase arrest and autophagy were induced by UA in U87MG cells. Evidence of UA-induced autophagy included the formation of acidic vesicular organelles, increase of autophagolysosomes and LC3-II accumulation. UA was also found to induce ER stress and an increase in intracellular calcium accompanied by ROS production. The increase in free cytosolic calcium induced by UA activated the CaMKK-AMPK-mTOR kinase signaling cascade, which ultimately triggered autophagy. Western blot analysis showed that UA promoted the phosphorylation of PERK and eIF2α; this was followed by the upregulation of the downstream protein CHOP, implying the involvement of the ER stress-mediated PERK/eIF2α/CHOP pathway in glioma cells. Meanwhile, UA activated IRE1α and subsequently increased the levels of phosphorylated JNK and Bcl-2, resulting in the dissociation of Beclin1 from Bcl-2. Furthermore, TUDCA and the silencing of either PERK or IRE1α partially blocked the UA-induced accumulation of LC3-II, suggesting that ER stress precedes the process of autophagy. Additionally, NAC attenuated the UA-induced elevation in cytosolic calcium, ER stress markers and autophagy-related proteins, indicating that UA triggered ER stress and autophagy via a ROS-dependent pathway. Collectively, our findings revealed a novel cellular mechanism triggered by UA and provide a molecular basis for developing UA into a drug candidate.

Hepatic Overexpression of ATP Synthase β Subunit Activates PI3K/Akt Pathway to Ameliorate Hyperglycemia of Diabetic Mice

ATP synthase β subunit (ATPSβ) had been previously shown to play an important role in controlling ATP synthesis in pancreatic β-cells. This study aimed to investigate the role of ATPSβ in regulation of hepatic ATP content and glucose metabolism in diabetic mice. ATPSβ expression and ATP content were both reduced in the livers of type 1 and type 2 diabetic mice. Hepatic overexpression of ATPSβ elevated cellular ATP content and ameliorated hyperglycemia of streptozocin-induced diabetic mice and db/db mice. ATPSβ overexpression increased phosphorylated Akt (pAkt) levels and reduced PEPCK and G6pase expression levels in the livers. Consistently, ATPSβ overexpression repressed hepatic glucose production in db/db mice. In cultured hepatocytes, ATPSβ overexpression increased intracellular and extracellular ATP content, elevated the cytosolic free calcium level, and activated Akt independent of insulin. The ATPSβ-induced increase in cytosolic free calcium and pAkt levels was attenuated by inhibition of P2 receptors. Notably, inhibition of calmodulin (CaM) completely abolished ATPSβ-induced Akt activation in liver cells. Inhibition of P2 receptors or CaM blocked ATPSβ-induced nuclear exclusion of forkhead box O1 in liver cells. In conclusion, a decrease in hepatic ATPSβ expression in the liver, leading to the attenuation of ATP-P2 receptor-CaM-Akt pathway, may play an important role in the progression of diabetes.

Shear Flow Regulates Osteogenic Differentiation of Mesenchymal Stem Cells through TRPM7-Mediated Osterix Pathway

Mesenchymal stem cells (MSCs) have the potential to differentiate into different cell types and are precursors of osteoblasts. Micro-environments composed of chemical and physical cues both can regulate MSCs commitments but the later ones are less investigated. Previous studies have indicated that shear flow, a potent and physiologically relevant mechanical force, affects MSCs differentiation. Here we demonstrated that shear flow regulated osteogenic differentiation and induced calcium signaling of MSCs. For the study, MSCs were cultured in homemade parallel-plate chambers with 0.08 mm thickness until desired confluency was reached. Gene expressions of transcription factor Osterix and homeoprotein Dlx5 were upregulated after MSCs exposed under 12 dyne/cm2 intermittent shear stress for three hours, but the expression of Runt-related transcription factor 2 (Runx2) was not changed significantly. On the contrary, continuous shear flow upregulated Runx2 without affecting Osterix gene expression. Moreover, ten minutes after the intermittent shear flow turned off, the upregulation of Osterix was abolished. Gene knockdown of Transient Receptor Potential Melastatin 7 (TRPM7) and 2-Aminoethoxydiphenyl borate blocking of TRPM7 attenuated the upregulation of Osterix as well. In addition, ratiometric analysis of real-time calcium imaging demonstrated that laminar fluid flow immediately caused transient cytosolic free calcium increase in MSCs. In summary, our study reveals two distinct signaling pathways that are regulated by different patterns of fluid flow. For the first time, we identify a novel TRPM7-Dlx5-Osterix axis which is activated by intermittent shear flow and related to calcium signaling. We are currently investigating the underlying mechanotransduction pathways and the responsive mechanosensors for intermittent shear flow. The underlying molecular mechanisms revealed by this study could provide insight for future drug developments.

The Third-Generation Beta-Blocker

In this issue of the Journal, Khan et al1 study the third-generation beta-blocker, nebivolol, in a rat model of aldosterone/salt treatment–induced hypertensive heart disease and show that myocyte necrosis is incited by oxidative stress secondary to an increase in cytosolic free calcium and mitochondrial calcium overload—a process that overwhelms the endogenous zinc (Zn)-based antioxidants. Using the first-generation beta-blocker atenolol as a control, treatment with nebivolol induced activation of endothelial nitric oxide synthase, nitric oxide (NO) generation, and a marked increase in intracellular Zn. Conversely, there was a concomitant decrease in intracellular and subsarcolemmal mitochondrial calcium levels. Likewise, there was attenuation of mitochondrial hydrogen peroxide production and lipid peroxidation, inhibition of microscopic myocardial scarring, and a reduction in collagen volume fractions. They postulate an intriguing pathophysiologic phenomenon whereby nebivolol, acting as a β3 agonist, increases NO formation leading to a rise in cytosolic free Zn. This inhibits intracellular and mitochondrial calcium overload, which ameliorates many of the detrimental effects of reactive oxygen species (ROS), lipid peroxidation, and the eventual myocardial scarring. Several important points can be gleaned from this study. First, this animal model of hypertensive cardiomyopathy serves as an effective model of sudden cardiac death as both myocardial fibrosis and adverse remodeling are key features of this disease process. This hypothesis, originally formulated by Weber and others, served as a fundamental basis for the RALES and EPHESUS trials. This concept was recently reviewed and confirmed by Esposito et al2 in a canine model of pacing-induced systolic heart failure, interstitial fibrosis, and myocardial electrical activation delays. They showed that mineralocorticoid receptor antagonism played a key role in preventing the activation of inflammatory pathways and in the development of ventricular arrhythmogenic foci. It is interesting to speculate that nebivolol may also induce similar cardioprotection. Nebivolol is approved for the treatment of hypertension in the United States and is marketed as Bystolic. In Europe, it is approved for the treatment of both hypertension and left ventricular failure. At low doses, it is a selective β1-receptor blocker, and as the dose is up-titrated, it loses its selectivity. It is unique in that unlike carvedilol (Coreg), it has a NO potentiating ability, a property that Khan et al1 have effectively shown in their study. Another plausible mechanism for the drug's efficacy is that it may act primarily within the mitochondria at the site of ROS production or as a ROS scavenger, inhibiting superoxide anion or hydrogen peroxide. Accordingly, the sequelae of pathology associated with excessive ROS production in hypertensive cardiomyopathy can effectively be targeted. Nebivolol is not the first beta-blocker to demonstrate antioxidant properties. Carvedilol, the first of the third-generation beta-blockers, has been shown in a number of studies to be both an antioxidant and free radical scavenger.3 Its antioxidant property resides in its carbazole moiety, a property partly shared by nebivolol. The apparent underlying protective mechanism of carvedilol is through inhibition of lipid peroxidation primarily through free radical scavenging. Khan et al1 showed that nebivolol could similarly protect against lipid peroxidation. This unique property has not received a great deal of attention from our clinical colleagues, except in the burgeoning field of Cardio-Oncology. The chemotherapeutic anthracyclines are known to be one of the most effective anticancer treatments available but produce an excessive amount of free radicals within the myocardium, leading to cardiotoxicity and heart failure. It is of no surprise then that carvedilol is currently considered the “beta-blocker of choice” for patients suffering from anthracycline-induced heart failure. Perhaps, it is time when nebivolol was investigated for its antioxidant properties in oxidative pathological conditions also? The use of the term “third-generation beta-blocker,” is a method we use to explain the hierarchy of beta-blockers in terms of development. Atenolol is a first-generation beta-blocker in terms of age, a drug known to have no antioxidant properties and a characteristic Khan et al1 use for control experiments. The second-generation beta-blockers include metoprolol, which although has cardioprotective effects, does not have any antioxidant effects. The current third-generation beta-blockers include carvedilol, a drug with antioxidant effects and free radical scavenging ability.4 Nebivolol can now be added to this list. It is rather interesting to recall that over the last several decades, both laboratory and clinical research with endogenous antioxidants such as superoxide dismutase, vitamin E, and catalase were all fraught with failure and frustration. Conceivably, the elusive and effective antioxidant has been right in front us for the past 20 years, and we were just a little slow in recognizing the potential expedient effects of these drugs.

ANP impairs the dose-dependent stimulatory effect of ANG II or AVP on H<sup>+</sup>-ATPase subcellular vesicle trafficking

The effect of angiotensin II (ANG II) or arginine vasopressin (AVP) alone or plus atrial natriuretic peptide (ANP) on H+-ATPase subcellular vesicle trafficking was investigated in MDCK cells following intracellular pH (pHi) acidification by exposure to20 mMNH4Cl for 2 min in a Na+-free solution containing Schering 28080, conditions under which H+-AT-Pase is the only cell mechanism for pHi recovery. Using the acridine orange fluorescent probe (5mM) and confocal microscopy, the vesicle movement was quantified by determining, for each experimental group, the mean slope of the line indicating the changes in apical/basolateral fluorescence density ratio over time during the first 5.30 min of the pHi recovery period. Under the control conditions, the mean slope was 0.079 ± 0.0033 min-1 (14) and it increased significantly with ANG II [10-12 and 10-7 M, respectively to 0.322 ± 0.038 min-1 (13) and 0.578 ± 0.061 min-1 (12)] or AVP [10-12 and 10-6 M, respectively to 0.301 ± 0.018 min-1 (12) and 0.687 ± 0.049 min-1 (11)]. However, in presence of ANP (10-6 M, decreases cytosolic free calcium), dimethyl-BAPTA/AM (5 × 10-5 M, chelates intracellular calcium) or colchicine (10-5 M, 2-h preincubation; inhibits microtubule-dependent vesicular trafficking) alone or plus ANG II or AVP the mean slopes were similar to the control values, indicating that such agents blocked the stimulatory effect of ANG II or AVP on vesicle trafficking. The results suggest that the pathway responsible for the increase in cytosolic free calcium and the microtu-bule-dependent vesicular trafficking are involved in this hormonal stimulating effect. Whether cytosolic free calcium reduction represents an important direct mechanism for ANP impairs the dose-dependent stimulatory effect of ANG II or AVP on H+-ATPase subcellular vesicle trafficking, or is a side effect of other signaling pathways which will require additional studies.

Role of the Immune System in Hypertension: Modulation by Dietary Antioxidants

Hypertension is a major health problem worldwide. Individuals with hypertension are at an increased risk for stroke, heart disease, and kidney failure. Although the etiology of essential hypertension has a genetic component, lifestyle factors such as diet play an important role. Insulin resistance is a common feature of hypertension in both humans and animal models affecting glucose and lipid metabolism producing excess aldehydes including methylglyoxal. These aldehydes react with proteins to form conjugates called advanced glycation end products (AGEs). This alters protein structure and function and can affect vascular and immune cells leading to their activation and secretion of inflammatory cytokines. AGEs also act via receptors for advanced glycation end products on these cells altering the function of antioxidant and metabolic enzymes, and ion channels. This results in an increase in cytosolic free calcium, decrease in nitric oxide, endothelial dysfunction, oxidative stress, peripheral vascular resistance, and infiltration of vascular and kidney tissue with inflammatory cells leading to hypertension. Supplementation with dietary antioxidants including vitamins C, E, or B(6), thiols such as cysteine and lipoic acid, have been shown to lower blood pressure and plasma inflammatory cytokines in animal models and humans with essential hypertension. A well-balanced diet rich in antioxidants that includes vegetables, fruits, low fat dairy products, low salt, and includes whole grains, poultry, fish and nuts, lowers blood pressure and vascular inflammation. These antioxidants may achieve their antihypertensive and anti-inflammatory/immunomodulatory effects by reducing AGEs and improving insulin resistance and associated alterations. Dietary supplementation with antioxidants may be a beneficial, inexpensive, front-line alterative treatment modality for hypertension.

Historical perspective on ADP-induced platelet activation

We have come a long way since 1960 when Hellem observed that a small molecule lost from red cells caused platelets to adhere to glass [1]. During the next year, Ollgaard [2] showed that this small molecule caused platelet aggregation, and it was identified as adenosine diphosphate (ADP) in Owren’s laboratory [3]. At that time, Marjory Zucker was grading ADP-induced aggregation visually as 1–4 after shaking citrated platelet-rich plasma by hand for 1 min [4]. She had been carrying out research on platelets since 1947 and was already a major figure in the field. The key development of the aggregometer in Gustav Born’s laboratory in 1962 provided a light transmission technique for assessing and recording the rate and extent of aggregation that is still widely used today (light transmission aggregometry [LTA]) [5]. In the next few years, Born and his colleagues used the aggregometer in detailed investigations of the changes in platelets during ADP-induced aggregation, and inhibitors of this process [6]. Much of the early work was well reviewed in the classic book The Physiology of Blood Platelets, subtitled Recent Biochemical, Morphologic and Clinical Research, that was written by Aaron Marcus and Marjorie Zucker in 1965 [7]. In 1970, another major review summarized the developments in the 1960s [6]. It had become obvious that ADP plays an important role in hemostasis and thrombosis. The much more recent findings that platelets possess two P2Y receptors (P2Y1 and P2Y12) for ADP, and a P2X1 receptor for ATP have made it possible to understand the reactions responsible for many of the early observations [8]. Our present knowledge about ADP-induced platelet activation is attributable to the work of thousands of investigators and this historical review can mention only some of them. In the late 1950s and early 1960s, several groups of investigators carried out in vitro experiments showing that thrombin or collagen caused platelet aggregation and that ADP was released during this process [9–13]. In vivo, ADP and ATP do not normally circulate in the plasma, but they are stored in the dense granules of the platelets. During the formation of hemostatic plugs or arterial thrombi, platelets are stimulated by collagen and thrombin to release the contents of these platelet storage granules. In vitro, at a normal platelet count of 250,000/μl, the concentrations of ATP and ADP in plasma immediately after release of granule contents induced by collagen or thrombin have been reported in the ranges of 4–7 μM for ATP and 3–4 μM for ADP [14, 15]. The released ADP adds to the response of platelets to the other aggregating agents. In addition to causing aggregation, the effects of ADP on platelets include shape change, refractoriness, potentiation of the effects of other aggregating agents, inhibition of platelet adenylyl cyclase, increase in cytosolic free calcium, and activation of specific receptors that stimulate intracellular signaling pathways that converge on the cytoplasmic domain of the integrin αIIbβ3 (glycoprotein (GP) IIb–IIIa), leading to its becoming able to bind extracellular fibrinogen and von Willebrand factor [16, 17].

Self-incompatibility in Papaver rhoeas activates nonspecific cation conductance permeable to Ca2+ and K+.

Cellular responses rely on signaling. In plant cells, cytosolic free calcium is a major second messenger, and ion channels play a key role in mediating physiological responses. Self-incompatibility (SI) is an important genetically controlled mechanism to prevent self-fertilization. It uses interaction of matching S-determinants from the pistil and pollen to allow "self" recognition, which triggers rejection of incompatible pollen. In Papaver rhoeas, the S-determinants are PrsS and PrpS. PrsS is a small novel cysteine-rich protein; PrpS is a small novel transmembrane protein. Interaction of PrsS with incompatible pollen stimulates S-specific increases in cytosolic free calcium and alterations in the actin cytoskeleton, resulting in programmed cell death in incompatible but not compatible pollen. Here, we have used whole-cell patch clamping of pollen protoplasts to show that PrsS stimulates SI-specific activation of pollen grain plasma membrane conductance in incompatible but not compatible pollen grain protoplasts. The SI-activated conductance does not require voltage activation, but it is voltage sensitive. It is permeable to divalent cations (Ba(2+) ≥ Ca(2+) > Mg(2+)) and the monovalent ions K(+) and NH(4)(+) and is enhanced at voltages negative to -100 mV. The Ca(2+) conductance is blocked by La(3+) but not by verapamil; the K(+) currents are tetraethylammonium chloride insensitive and do not require Ca(2+). We propose that the SI-stimulated conductance may represent a nonspecific cation channel or possibly two conductances, permeable to monovalent and divalent cations. Our data provide insights into signal-response coupling involving a biologically important response. PrsS provides a rare example of a protein triggering alterations in ion channel activity.

Increase of cytosolic calcium induced by trichosanthin suppresses cAMP/PKC levels through the inhibition of adenylyl cyclase activity in HeLa cells

Increase of cytosolic free calcium played a pivotal role in apoptotic cells induced by trichosanthin. However, little is known about the influence of cytosolic calcium increase on adenylyl cyclase activity and intracellular cAMP signaling pathway in HeLa cells. The present study showed that an influx of extracellular Ca2+ initiated by trichosanthin was required for the suppression of adenylyl cyclase activity and decrease of intracellular cAMP level. Furthermore, this inhibition was abolished by activation of PKC rather than PKA. Therefore, our results suggested that increase of cytosolic calcium induced by trichosanthin inhibits cAMP levels via suppression of adenylyl cyclase activity.

Astrocytic adrenoceptors and learning: α1-Adrenoceptors

Noradrenergic receptors are expressed on both on astrocytes and neurons and noradrenergic activation of astrocytic β2- and β3-adrenoceptors are necessary for memory consolidation. In this paper, we marshal evidence for astrocytic α1-adrenoceptor involvement in memory consolidation. We examine the role of α1-adrenoceptors in hippocampal and mesopallial (cortical) memory processing using a discriminative avoidance task in the day-old chick. The selective α1-adrenoceptor agonist, methoxamine, caused the consolidation of weakly-reinforced memory at the time of transition of short-term to intermediate memory and at the time of transition of intermediate to long-term memory. The selective antagonist prazosin prevented memory consolidation at these two times. Blockade of memory by injection of an α2-adrenoceptor agonist into the LoC could be overcome by mesopallial or hippocampal injection of α1-, β2- and β3-adrenoceptor agonists. The results of studies where we challenged the ability of methoxamine to promote consolidation by pre-administration of astrocytic metabolic inhibitors of glycogenolysis or oxidative metabolism, suggest that the α1-adrenoceptor effect is astrocytic. This conclusion is supported by the finding that co-administration of suboptimal doses of methoxamine and thrombin have an additive effect on promoting consolidation. Thrombin causes a calcium response in cultured chick astrocytes but not in neurons. Thrombin, like methoxamine, promotes consolidation at the transition points between short-term, intermediate memory and long-term memory stages. Thrombin enhancement of memory consolidation is blocked by an α1-adrenoceptor antagonist but not by antagonists of β2- or β3-adrenoceptors.In summary, noradrenaline activation of α1-adrenoceptors is necessary for consolidation from both short-term and intermediate memory in both the hippocampus and the mesopallium in the chick. Evidence is presented suggesting that the memory consolidating action of α1-adrenoceptor stimulation results from receptors located on astroctyes and involves an increase in free cytosolic calcium from internal stores.

Calcium Signaling Promotes Targeted Recycling of the Lateral Border Recycling Compartment (LBRC) During Diapedesis (140.1)

Abstract Leukocyte transendothelial migration (diapedesis, TEM) is a crucial step in both the innate and adaptive immune responses. We have shown that targeted recycling of membrane from a reticulum localized close to the endothelial cell (EC) lateral border (the lateral border recycling compartment, LBRC) surrounds the transmigrating leukocyte during TEM (Mamdouh, Z., et al 2003. Nature). Targeted recycling is required for TEM to proceed. However the mechanisms underlying recruitment of the LBRC are not entirely clear. An increase of cytosolic free calcium (Ca) in ECs and the resulting activation of Ca/Calmodulin-dependent myosin light chain kinase (MLCK) are crucial for diapedesis to occur. Here we address the role of Ca signaling in the process of targeted recycling. The cell permeant Ca chelator BAPTA-AM was loaded into ECs under conditions that blocked calcium flux in response to histamine, but did not impair cytoskeletal or junctional integrity. Under these conditions, blocking of cytosolic free Ca flux in ECs abolished targeted recycling of the LBRC, as well as leukocyte transendothelial migration to the same extent (> 80%). Furthermore, blockade of MLCK with ML7 (1uM) also blocked targeted recycling and TEM by > 80%. This study demonstrates that Ca signaling is upstream of LBRC targeted recycling, and MLCK activation (presumably by Ca/Calmodulin dependent mechanism) is required for targeted recruitment of the LBRC.