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The effects of secretin and cholecystokinin on exocrine pancreas secretion in the guinea pig were investigated. The putative potentiating effect of these two hormones was studied in various settings to elucidate the effect of cholinergic stimuli in such interaction. In anesthetized guinea pig, intravenous infusion of cholecystokinin (0.75 pmol.kg-1.min-1) or secretin (0.5 pmol.kg-1.min-1) resulted in a marked and rapid increase of pancreatic juice flow and protein output. When cholecystokinin was combined with secretin, there was a significant increase in pancreatic, compared with cholecystokinin alone. This increase in pancreatic juice secretion and protein output was significantly suppressed by the prior administration of 100 micrograms/kg atropine. Similar results were obtained when trypsinogen release from pancreatic segments was measured in response to cholecystokinin (32 nM-32 pM) and (or) secretin (1 microM-32 nM). When we assayed the hormonal interaction on amylase release from dispersed pancreatic acini, we found that secretin (32 nM) failed to influence the secretory response to cholecystokinin (1 pM-10 nM). Thus we conclude that a combination of cholecystokinin and secretin resulted in a marked potentiation of the secretory responses in the exocrine guinea pig pancreas by a mechanism that involves cholinergic interactions present at the tissue level but not at the dispersed secretory cell level.

An investigation was made of the effects of histamine on the K + concentration in the effluent in superfused guinea pig and mouse pancreatic segments. The effect of acetylcholine (ACh) was examined for comparison. Histamine evoked a dose-dependent and transient increase in the K + concentration in the effluent (K + release) but is less potent compared to the cholinergic agonist, ACh. At the same doses histamine and ACh evoke a much larger K + release from mouse superfused pancreatic segments followed in the poststimulus period by a reuptake of K + . However, this reuptake of K + was not observed in guinea pig superfused pancreatic segments. On the other hand, the cholinergic antagonist, atropine, completely abolished the K + release in response to ACh and histamine from mouse and guinea pig pancreatic segments. Our results show the involvement of histamine in the control of K + release in pancreatic tissue, with significant differences in the observed responses between species.

This study investigates the effect of magnesium (Mg2+) on the secretory responses and the mobilization of calcium (Ca2+) and Mg2+ evoked by cholecystokinin-octapeptide (CCK-8) in the exocrine rat pancreas. In the isolated intact perfused pancreas CCK-8 (10(-10) M) produced marked increases in juice flow and total protein output in zero and normal (1.1 mM) extracellular Mg2+ [Mg2+]o compared to a much reduced secretory response in elevated (5 mM and 10 mM) [Mg2+]o. Similar effects of perturbation of [Mg2+]o on amylase secretion and 45 Ca2+ uptake (influx) were obtained in isolated pancreatic segments. In pancreatic acinar cells loaded with the fluorescent bioprobe fura-2 acetomethylester (AM), CCK-8 evoked marked increases in cytosolic free Ca2+ concentration [Ca2+]i in zero and normal [Mg2+]o compared to a much reduced response in elevated [Mg2+]o. Pretreatment of acinar cells with either dibutyryl cyclic AMP (DB2 cAMP) or forskolin had no effect on the CCK-8 induced changes in [Ca2+]i. In magfura-2-loaded acinar cells CCK-8 (10(-8) M) stimulated an initial transient rise in intracellular free Mg2+ concentration [Mg2+]i followed by a more prolonged and sustained decrease. This response was abolished when sodium (Na+) was replaced with N-methyl-D-glucamine (NMDG). Incubation of acinar cells with 10 mM Mg2+ resulted in an elevation in [Mg2+]i. Upon stimulation with CCK-8, [Mg2+]i decreased only slightly compared with the response obtained in normal [Mg2+]o. CCK-8 caused a net efflux of Mg2+ in pancreatic segments; this effect was abolished when extracellular sodium [Na+]o was replaced with either NMDG or choline. The results indicate that Mg2+ can regulate CCK-8-evoked secretory responses in the exocrine pancreas possibly via Ca2+ mobilization. Moreover, the movement of Mg2+ in pancreatic acinar cells is dependent upon extracellular Na+.

We have investigated the effects of histamine on motility of the gallbladder and characterized the receptor types involved. Histamine and the histamine H1-receptor agonist, 2-thiazolylethylamine (2-TEA) contracted the isolated guinea-pig gallbladder strip in a dose dependent manner. The contractile response to histamine was shifted to the right by the H1-receptor antagonist, mepyramine. In pre-contracted gallbladder strips, the H2-receptor agonist dimaprit reduced the tension generated in a dose dependent fashion. The histamine H2-receptor antagonist, ranitidine shifted the histamine concentration effect curve to the left and attenuated the dose dependent relaxations elicited at high concentrations. The histamine H3-receptor agonist, (R)-alpha-methylhistamine (RMHA) elicited dose dependent contraction of the tissue which was significantly inhibited in the presence of mepyramine. The effects of electrical field stimulation (EFS) on the strips were not significantly altered by the presence of RMHA (10(-10) - 10(-7) M) indicating little pre-synaptic H3 activity in this tissue. Histamine immunoreactivity (IR) was detected in gallbladder whole mount preparations of the mucosa and the muscularis/serosa. The histamine IR appeared cell bound in cells of varying morphological characteristics but no IR was detected in nerve fibres or cell bodies (ganglia). Alcian blue staining was consistent with the distribution of histamine IR cells as mast cells. The results indicate that histamine is distributed in the guinea-pig gallbladder and it can regulate contractile activity via activation of H1 and H2 but not H3 receptors.

This study investigates the interaction between histamine and the adenylate cyclase systems involved in the secretion of amylase from the guinea-pig pancreatic lobules. Histamine increased amylase release, reaching a maximum response at 10 −5 M. Similarly, vasoactive intestinal peptide (VIP) evoked significant increase in amylase release, though not in a dose-dependent fashion. When the pancreatic lobules were incubated with histamine in combination with VIP, forskolin or 3-isobutyl-1-methylxanthine (IBMX), amylase secretion was increased as compared to histamine alone. The stimulatory effect of VIP was also increased by the presence of forskolin or IBMX. These findings suggest that in guinea-pig pancreatic lobules, VIP, forskolin and IBMX, agents involved in the cyclic adenosine monophosphate (cAMP) pathway, potentiate histamine stimulated amylase release.

The Homer family of proteins consists of three adaptor proteins, Homer1, Homer2 and Homer3, each with various isoforms. Homer1 family presents an EVH1 domain, a coiled coil domain and two leucine zipper domains. Homer proteins regulate a number of Ca2+-handling proteins, including transient receptor potential channels and other Ca2+-permeable channels, ionotropic and metabotropic glutamate receptors, shank scaffolding proteins or endoplasmic reticulum Ca2+ release channels. This review article focuses on the association of Homer 1 proteins with Ca2+-handling proteins and their role on intracellular Ca2+-homeostasis.

The TRP family of cation-permeable channels owes its name to a Drosophila TRP mutant with impaired vision due to transient rather than sustained receptor potential. Mammalian TRP channels can be grouped into 6 subfamilies, including TRPC, TRPM, TRPV, TRPA, TRPP and TRPML and a number of TRP family members have been identified in the vasculature. TRP channels play an important functional role in the vasculature as mediators of cation influx across the plasma membrane, thus contributing to a large number of processes such as vascular smooth muscle contraction and vascular pressure or the responses to oxidative stress, mechanical stimuli, heat and hypoxia-induced vascular remodelling. TRP channelopaties are involved in the pathogenesis of different disorders including hypertension and cardiomyopathy. A number of identified natural compounds and synthetic agents have been reported to modulate TRP function, and are the base for therapeutical strategies.

The role of cAMP in spermatic functions was classically thought to be mediated exclusively through the activation of Protein Kinase A (PKA). However, it has recently been shown that cAMP also exerts its effects through a PKA-independent pathway activating a family of proteins known as Epac proteins. Therefore, many of the spermatic functions thought to be regulated by cAMP through the activation of PKA are again under study. We aimed to identify and to investigate the role of Epac proteins in spermatozoa using a specific permeable analog (8-Br-2'-O-Me-cAMP). Also, we aimed to study its relationship with E-cadherin, an adhesion protein involved in fertility. Our results demonstrate the presence and sub-cellular distribution of Epac 1 and Epac 2 in mammalian spermatozoa. Capacitation and the acrosome reaction induced a change in the localization of Epac proteins in sperm. Moreover, incubation with 8-Br-2'-O-Me-cAMP prompted an increase in Rap1 activation, in the scrambling of plasma membrane phospholipids (necessary for the capacitation process), the acrosome reaction, motility, and calcium mobilization, when spermatozoa were incubated in acrosome reaction conditions. Finally, the activation of Epac proteins induced a change in the distribution of E-cadherin. Therefore, the increase in the acrosome reaction, together with the increase in calcium (which is known to be essential for fertilization) and the Epac nteraction with E-cadherin, might indicate that Epac proteins have an important role in gamete recognition and fertilization.

This study investigates the effects of the gut peptide, secretin, on nerve-mediated and acetylcholine-evoked amylase secretion and calcium (Ca2+) and magnesium (Mg2+) mobilization in the in vitro rat pancreas. Both electrical field stimulation (EFS; 50 V, 20 Hz, 1 ms) and acetylcholine (ACh; 10(-6) M) caused marked increases in amylase output in isolated pancreatic segments. These responses were inhibited by atropine (10(-5) M). Secretin (10(-10) and 10(-8) M) evoked only a small increase in amylase secretion, which was unaffected by atropine. Combining either EFS or ACh with secretin resulted in an attenuation in amylase output compared with the response obtained with either EFS or ACh alone. In a Mg(2+)-free medium, secretin failed to inhibit the amylase output evoked by EFS. When forskolin (10(-5) M) was combined with EFS there was a marked potentiation of amylase output. In fura-2-loaded acinar cells, ACh (10(-6) M) elevated cytosolic free Ca2+ concentration ([Ca2+]i). Secretin alone had no detectable effect on [Ca2+]i compared with basal values, but it attenuated the ACh-induced elevation of [Ca2+]i. Both EFS and ACh elicited 45Ca2+ influx, whereas secretin had no significant effect. In the presence of secretin the EFS- and ACh-induced 45Ca2+ influx was reduced compared with responses obtained with either EFS or ACh alone. EFS caused a net efflux of Mg2+, whereas secretin alone evoked a net uptake of Mg2+ in pancreatic segments. Combining secretin with EFS resulted in a net uptake of Mg2+. In mag-fura-2-loaded acini ACh (10(-5) M) stimulated an initial transient rise in cytosolic free Mg2+ concentration ([Mg2+]i) followed by a sustained decrease in [Mg2+]i. Stimulation of acinar cells with secretin (10(-8) M) resulted in an elevation in [Mg2+]i compared with the resting level. When ACh (10(-5) M) was combined with secretin there was an initial increase in [Mg2+]i followed by a marked decrease to the pre-secretin-stimulated value. The results indicate that secretin may control nerve-mediated and ACh-evoked secretory responses in the rat pancreas, possibly by an interaction between cellular Ca2+ and Mg2+.

The discovery of molecular players in capacitative calcium (Ca(2+)) entry, also referred to as store-operated Ca(2+) entry (SOCE), supposed a great advance in the knowledge of cellular mechanisms of Ca(2+) entry, which are essential for a broad range of cellular functions. The identification of STIM1 and STIM2 proteins as the sensors of Ca(2+) stored in the endoplasmic reticulum unraveled the mechanism by which depletion of intracellular Ca(2+) stores is communicated to store-operated Ca(2+) channels located in the plasma membrane, triggering the activation of SOCE and intracellular Ca(2+)-dependent signaling cascades. Initial studies suggested a dominant function of STIM1 in SOCE and SOCE-dependent cellular functions compared to STIM2, especially those that participate in immune responses. Consequently, most of the subsequent studies focused on STIM1. However, during the last years, STIM2 has been demonstrated to play a more relevant and complex function than initially reported, being even important to sustain normal life in mice. These studies have led to reconsider the role of STIM2 in SOCE and its relevance in cellular physiology. This review is intended to summarize and provide an overview of the current data available about this exciting isoform, STIM2, and its actual position together with STIM1 in the mechanism of SOCE.

Background All identified mammalian TRPC channels show a C-terminal calmodulin (CaM)- and inositol 1,4,5-trisphosphate receptors (IP 3 Rs)-binding (CIRB) site involved in the regulation of TRPC channel function. Objectives To assess the basis of CaM/IP 3 Rs binding to the CIRB site of TRPC6 and its role in platelet physiology. Methods Protein association was detected by co-immunoprecipitation and Western blotting, Ca 2+ mobilization was measured by fluorimetric techniques and platelet function was analyzed by aggregometry. Results Co-immunoprecipitation of TRPC6 with CaM or the IP 3 Rs at different cytosolic free Ca 2+ concentrations ([Ca 2+ ] c ) indicates that the association between these proteins is finely regulated by cytosolic Ca 2+ via association of CaM and displacement of the IP 3 Rs at high [Ca 2+ ] c . Thrombin-stimulated association of TRPC6 with CaM or the IP 3 Rs was sensitive to 2-APB and partially inhibited by dimethyl BAPTA loading, thus suggesting that the association between these proteins occurs through both Ca 2+ -dependent and -independent mechanisms. Incorporation of an anti-TRPC6 C-terminal antibody, whose epitope overlaps the CIRB region, impaired the dynamics of the association of TRPC6 with CaM and the IP 3 Rs, which lead to both inhibition and enhancement of thrombin- and thapsigargin-evoked Ca 2+ entry in the presence of low or high, respectively, extracellular Ca 2+ concentrations, as well as altered thrombin-evoked platelet aggregation. Conclusions Our results indicate that the CIRB site of TRPC6 plays an important functional role in platelets both modulating Ca 2+ entry and aggregation through its interaction with CaM and IP 3 Rs.

Platelets from patients with type 2 diabetes show abnormalities in intracellular Ca(2+) homeostasis that are involved in platelet hyperaggregability and the development of thrombotic complications. Different Ca(2+) transport mechanisms have been reported to be altered in platelets from patients with type 2 diabetes, including the sarcoendoplasmic and plasma membrane Ca(2+)-ATPases, plasma membrane Ca(2+) channels, or the Na(+)/Ca(2+) exchanger. Here, we have investigated whether passive Ca(2+) leak from the stores is altered in platelets from patients with type 2 diabetes. Resting cytosolic Ca(2+) concentration ([Ca(2+)](i)) was found to be greater in platelets from patients with type 2 diabetes than in healthy controls. In a Ca(2+)-free medium, platelet stimulation with thrombin or ADP evokes a rapid and transient increase in [Ca(2+)](i) that was found to be greater in patients with diabetes than in healthy controls. Sequential or combined inhibition of Ca(2+) extrusion and Ca(2+) sequestration into the stores reduced the difference between the responses to agonists in patients with diabetes and healthy controls, although agonist-induced Ca(2+) efflux from the stores was still significantly greater in patients with diabetes. Ca(2+) leak from the dense tubular system or the acidic stores, induced by a low concentration of thapsigargin or 2,5-di-(t-butyl)-1,4-hydroquinone (TBHQ), respectively, was clearly greater in patients with diabetes than in controls, and was not significantly modified by treatment with 2-APB. These findings indicate that passive Ca(2+) leakage rate from the intracellular stores in platelets is significantly enhanced in patients with type 2 diabetes mellitus and this might explain the increased resting [Ca(2+)](i).

Store-operated Ca 2+ entry (SOCE) is a major pathway for Ca 2+ influx in non-excitable cells. Recent studies favour a conformational coupling mechanism between the endoplasmic reticulum (ER) Ca 2+ sensor STIM1 and Ca 2+ permeable channels in the plasma membrane to explain SOCE. Previous studies have reported a role for the cytoskeleton modulating the activation of SOCE; therefore, here we have investigated whether the interaction between STIM1 and the Ca 2+ permeable channels is modulated by the actin or microtubular network. In HEK-293 cells, treatment with the microtubular disrupter colchicine enhanced both the activation of SOCE and the association between STIM1 and Orai1 or TRPC1 induced by thapsigargin (TG). Conversely, stabilization of the microtubules by paclitaxel attenuated TG-evoked activation of SOCE and the interaction between STIM1 and the Ca 2+ channels Orai1 and TRPC1, altogether suggesting that the microtubules act as a negative regulator of SOCE. Stabilization of the cortical actin filament layer results in inhibition of TG-evoked both association between STIM1, Orai1 and TRPC1 and SOCE. Interestingly, disruption of the actin filament network by cytochalasin D did not significantly modify TG-evoked association between STIM1 and Orai1 or TRPC1 but enhanced TG-stimulated SOCE. Finally, inhibition of calmodulin by calmidazolium enhances TG-evoked SOCE and disruption of the actin cytoskeleton results in inhibition of TG-evoked association of calmodulin with Orai1 and TRPC1. Thus, we demonstrate that the cytoskeleton plays an essential role in the regulation of SOCE through the modulation of the interaction between their main molecular components.

10 páginas, 7 figuras.-- et al.-- El pdf del artículo es la versión post-print., Agonist-sensitive intracellular Ca2+ stores may be heterogeneous and exhibit distinct functional features. We have studied the properties of intracellular Ca2+ stores using targeted aequorins for selective measurements in different subcellular compartments. Both, HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and HeLa cells accumulated Ca2+ into the ER (endoplasmic reticulum) to near millimolar concentrations and the IP3-generating agonists, carbachol and ATP, mobilized this Ca2+ pool. We find in HEK-293T, but not in HeLa cells, a distinct agonist-releasable Ca2+ pool insensitive to the SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) inhibitor TBH [2,5-di-(t-butyl)-benzohydroquinone]. TG (thapsigargin) and CPA (cyclopiazonic acid) completely emptied this pool, whereas lysosomal disruption or manoeuvres collapsing endomembrane pH gradients did not. Our results indicate that SERCA3d is important for filling the TBH-resistant store as: (i) SERCA3d is more abundant in HEK-293T than in HeLa cells; (ii) the SERCA 3 ATPase activity of HEK-293T cells is not fully blocked by TBH; and (iii) the expression of SERCA3d in HeLa cells generated a TBH-resistant agonist-mobilizable compartment in the ER. Therefore the distribution of SERCA isoforms may originate the heterogeneity of the ER Ca2+ stores and this may be the basis for store specialization in diverse functions. This adds to recent evidence indicating that SERCA3 isoforms may subserve important physiological and pathophysiological mechanisms., This work was supported by The Spanish Ministerio de Ciencia e Innovación [grant numbers MICINNFEDER BFU2007-60157, BFU2007-60104, BFU2010-17379, BFU2010- 21043-C02-01 and RD06/0010/0000]; and the Junta de Castilla y León [grant number GR175]. F. J. A. was supported by a fellowship from the Ministerio de Ciencia e Innovación.

The phagocytic activity of pronephric granulocytes of spring tench at 22° C was evaluated. The head kidney was chosen because it is known to be the principal hematopoietic organ in teleosts. The granulocytes were isolated by Percoll density gradient centrifugation. Head kidney granulocytes were found capable of adhering both to nylon fibre and to a smooth plastic surface, to have a mobility capacity which is both spontaneous and oriented to a chemoattractant, and be able to attach and ingest Candida albicans (with and without serum) as well as latex beads (inert particles). Moreover, they show candidicidal activity and a capacity to increase NBT (nitroblue tetrazolium) reduction.

In many tissues the cellular responses mediated through different intracellular messenger systems are mutually interactive. In the exocrine pancreas the secretagogues acting via adenosine cyclic monophosphate (cAMP) and those acting via calcium-phosphoinositides can potentiate one another. On the other hand, protein kinase C (PK-C) modulates receptor-induced responses in exocrine pancreatic cells and other cell types. Recording total protein output, monitored on-line at 280 nm, from superfused rat pancreatic segments, we demonstrate that secretin (a cAMP-acting hormone) reduces the efficacy of the calcium-mediated secretagogue cholecystokinin-octapeptide (CCK-8). Likewise, the PK-C activator 12,O,tetradecanoyl phorbol 13 acetate (TPA) reduces both the efficacy of secretin and the potency of cholecystokinin. Thus, the hypothesis of potentiation between different stimulus-secretion coupling mechanisms must be revised, and receptor-activated responses in the exocrine pancreas must be considered a complex model with multiple inhibitory and stimulatory interactions.

Agonist-evoked Ca2+ entry has been reported to be enhanced in platelets from type 2 diabetic patients, which results in altered platelet responsiveness and cardiovascular complications. The present study is aimed to investigate whether store-operated divalent cation entry, a major Ca2+ entry pathway, is altered in platelets from diabetic patients. Store-operated divalent cation entry was estimated by determination of Mn2+ entry. Association between STIM1, Orai1, hTRPC1 and hTRPC6 was detected by co-immunoprecipitation and Western blotting. In the presence of specific purinergic and serotoninergic receptor antagonists Mn2+ entry, induced by thapsigargin (TG), was reduced in platelets from diabetic donors as compared to healthy controls. Treatment with TG or the agonist thrombin enhanced co-immunoprecipitation of STIM1 with Orai1, hTRPC1 and hTRPC6 in platelets from healthy donors, a response that was significantly reduced in platelets from diabetic patients. Our results indicate that store-operated divalent cation entry is reduced in platelets from type 2 diabetic subjects, which is likely mediated by impairment of the association of STIM1 with the channel subunits Orai1, hTRPC1 and hTRPC6 and might be involved in the pathogenesis of the altered platelet responsiveness observed in diabetic patients.

In this study we have used rat hippocampal astrocytes in culture to investigate the effect of ethanol on kainate-induced glutamate secretion. Our results show that kainate (10 μM to 500 μM) stimulated glutamate release from astrocytes. Preincubation of astrocytes in the presence of ethanol induced a concentration-dependent (1mM-50mM) inhibition of glutamate release caused by stimulation of cells with 100 μM kainate. Inhibition of alcohol-dehydrogenase, by preincubation of astrocytes in the presence of 4-methylpyrazole (1mM), abolished ethanol-induced inhibition of glutamate release in response to kainate. On the other hand, preincubation of astrocytes in the presence of the antioxidant cinnamtannin B-1 (10 μM) also blocked ethanol inhibitory action on glutamate release in response to kainate. Ethanol (50mM) reduced Ca(2+) mobilization in response to kainate, whereas cinnamtannin B-1 reversed the inhibitory action of ethanol on Ca(2+) mobilization by kainate. Our results are consistent with an inhibitory action of ethanol on glutamate secretion from hippocampal astrocytes. The inhibitory effects of ethanol are probably due to its oxidative metabolization, involves reactive oxygen species production, and a lower Ca(2+) mobilization by kainate. Taking into account the pivotal role that astrocytes play within the central nervous system, especially in relation to neurons, the negative effects of ethanol on the release of glutamate might affect neuron-glia communication in the hippocampus, which might lead to functional defects in the brain.

Different studies have reported that proteins involved in Ca(2+) entry are localized in discrete plasma membrane domains known as lipid rafts, which have been suggested to support store-operated Ca(2+) entry by facilitating STIM1 clustering in endoplasmic reticulum-plasma membrane junctions as well as the interaction of STIM1 with TRPC1. Here we report that treatment of HEK293 cells with thapsigargin (TG) results in the activation of Ca(2+) entry with two components, an early, La(3+)-sensitive, component and a late component that shows both La(3+)-sensitive and -insensitive constituents. Preincubation with methyl-beta-cyclodextrin (MbetaCD) prevented TG-induced activation of Ca(2+) entry but, in contrast, enhanced this process after its activation. Addition of MbetaCD after store depletion did not modify the La(3+)-sensitive store-operated divalent cation entry but increased La(3+)-insensitive non-capacitative Ca(2+) entry. Cell stimulation with TG results in a transient increase in Orai1 co-immunoprecipitation with STIM1, TRPC1 and TRPC6. TG-induced association of these proteins was significantly attenuated by preincubation for 30 min with MbetaCD, without altering surface expression of Orai1 or TRPCs. In contrast, the association of Orai1 with STIM1 or TRPC1 was unaffected when MbetaCD was added after store depletion with TG. Addition of MbetaCD to TG-treated cells promoted dissociation between Orai1 and TRPC6, as well as non-capacitative Ca(2+) entry. TRPC6 expression silencing indicates that MbetaCD-enhanced non-capacitative Ca(2+) entry was mediated by TRPC6. In conclusion, lipid raft domains are necessary for the activation but not the maintenance of SOCE probably due to the support of the formation of Ca(2+) signalling complexes involving Orai1, TRPCs and STIM1.

Homocysteine, a sulphur-containing amino acid derived from methionine, has been presented as an independent risk factor for cardiovascular disorders, including atherosclerosis and thrombogenesis. The mechanisms underlying homocysteine-induced effects have been intensively investigated over the last two decades. Homocysteine can induce oxidative stress promoting oxidant injury to vascular and blood cells. Hyperhomocysteinemia often results in intracellular Ca2+ mobilization, endoplasmic reticulum (ER) stress, with the subsequent development of apoptotic events, chronic inflammation leading to endothelial dysfunction and remodeling of the extracellular matrix. Homocysteine has also been reported to induce modulation of gene expression through alteration of the methylation status. The effects of elevated concentrations of circulating homocysteine on the vascular wall, platelet function and coagulation factors promote the development of a pro-coagulant state. The pathophysiological significance of homocysteine in the development of vascular disorders through the induction of endothelial dysfunction and abnormal platelet activity and blood coagulation is discussed in this review.

This study investigates the interaction between physiological doses of the synthetic gut hormones, cholecystokinin-octapeptide (CCK8) and secretin on pancreatic juice secretion in the anaesthetized rat and on amylase secretion and Ca2+ and Mg2+ mobilization in isolated pancreatic segments and acinar cells. CCK8 (150 pmol kg-1 h-1) and secretin (100 pmol kg-1 h-1) evoked marked time course increases in pancreatic juice flow, total protein output and amylase secretion in the anaesthetized rat when administered separately compared to saline controls. Simultaneous intravenous infusion of CCK8 and secretin did not yield either an additive response or a potentiation but instead it caused a decrease in secretory responses. Administration of either polymyxin B (10(-8) mol kg-1 h-1) or staurosporine (10(-8) mol kg-1 h-1), two protein kinase C inhibitors, simultaneously with both CCK8 and secretin caused a further decrease in all secretory parameters. Superfusing pancreatic segments with either CCK8 (10(-11) M) or secretin (10(-11) M) elevated amylase output compared to the smaller response with a combination of CCK8 and secretin. Combining staurosporine (10(-6) M) with CCK8 and secretin resulted in a further decrease in amylase output. CCK8 (10(-11) M) evoked a large increase in radiolabelled Ca2+ influx into pancreatic segments and elevated cytosolic free Ca2+ concentration ([Ca2+]i) in acinar cells loaded with the fluorescent dye, Fura-2. Secretin (10(-11) M) alone had no significant effect on Ca2+ mobilization but it markedly attenuated the increases in radiolabelled Ca2+ influx and [Ca2+]i elicited by CCK8. In superfused pancreatic segments CCK8 (10(-11) M) evoked a net efflux of Mg2+ whereas secretin (10(-11) M) induced a net uptake of Mg2+. Combining secretin with CCK8 also resulted in a net uptake of Mg2+. The results indicate that both Ca2+ and Mg2+ mobilization may be associated with the interaction between CCK8 and secretin in the rat pancreas.

1. In the present time-course study, we have examined the interactions between the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and the synthetic gut hormones, cholecystokinin-octapeptide (CCK-8) and secretin on pancreatic juice secretion in anaesthetized rat. 2. Administration of either TPA (10(-8) mol kg-1 hr-1), secretin (100 pmol kg-1 hr-1) or CCK-8 (150 pmol kg-1 hr-1) in the anaesthetized rat resulted in marked time-course increases in pancreatic juice flow, amylase secretion and total protein output compared to saline controls. The effect of secretin on juice flow was more pronounced and sustained compared to the smaller responses obtained with either CCK-8 or TPA. Similarly, CCK-8 evoked increases in protein output and amylase secretion compared to the responses obtained with either secretin or TPA. 3. Simultaneous infusion of TPA with either CCK-8 or secretin resulted in a marked reduction in pancreatic juice flow, total protein output and amylase secretion compared to the responses obtained with either CCK-8 or secretin alone. 4. Administration of polymyxin B (10(-8) mol kg-1 hr-1), a protein kinase C inhibitor with either TPA and CCK-8 or TPA and secretin caused a partial reduction of the inhibitory effect of TPA on CCK-8 and secretin-evoked secretory responses. 5. The present study further implicates the involvement of protein kinase C in the modulation of CCK-8 and secretin-induced pancreatic juice secretion in the anaesthetized rat.

There is a body of evidence suggesting that Ca(2+) handling proteins assemble into signaling complexes required for a fine regulation of Ca(2+) signals, events that regulate a variety of critical cellular processes. Canonical transient receptor potential (TRPC) and Orai proteins have both been proposed to form Ca(2+)-permeable channels mediating Ca(2+) entry upon agonist stimulation. A number of studies have demonstrated that inositol 1,4,5-trisphosphate receptors (IP(3)Rs) interact with plasma membrane TRPC channels; however, at present there is no evidence supporting the interaction between Orai proteins and IP(3)Rs. Here we report that treatment with thapsigargin or cellular agonists results in association of Orai1 with types I and II IP(3)Rs. In addition, we have found that TRPC3, RACK1 (receptor for activated protein kinase C-1), and STIM1 (stromal interaction molecule 1) interact with Orai1 upon stimulation with agonists. TRPC3 expression silencing prevented both the interaction of Orai1 with TRPC3 and, more interestingly, the association of Orai1 with the type I IP(3)R, but not with the type II IP(3)R, thus suggesting that TRPC3 selectively mediates interaction between Orai1 and type I IP(3)R. In addition, TRPC3 expression silencing attenuated ATP- and CCh-stimulated interaction between RACK1 and the type I IP(3)R, as well as Ca(2+) release and entry. In conclusion, our results indicate that agonist stimulation results in the formation of an Orai1-STIM1-TRPC3-RACK1-type I IP(3)R complex, where TRPC3 plays a central role. This Ca(2+) signaling complex might be important for both agonist-induced Ca(2+) release and entry.

Polyphenols, such as rosmarinic acid, are widely distributed natural products with relevant antioxidant activity. Oxidative stress plays an important role in the pathogenesis of a number of disorders. Here, we report on the synthesis and biological effects of the polyphenolic esters hydroxytyrosyl gallate (1), hydroxytyrosyl protocatechuate (2) and hydroxytyrosyl caffeate (3), structurally related to rosmarinic acid. The three compounds showed a greater free radical scavenging activity than their precursors and also than rosmarinic acid. Esters 1 and 3 significantly reduced thrombin-evoked platelet aggregation, which is likely mediated to the attenuation of thrombin-stimulated Ca(2+) release and entry. The three compounds reduced the ability of platelets to accumulate Ca(2+) in the intracellular stores, probably by enhancing the Ca(2+) leakage rate and reduced store-operated Ca(2+) entry in these cells. These observations suggest that the structurally-simplified analogs to rosmarinic acid, compounds 1 and 3, might be the base of therapeutic strategies to prevent thrombotic complications associated to platelet hyperaggregability due to oxidative stress.

In the central nervous system (CNS), both neurones and astrocytes play crucial roles. On a cellular level, brain activity involves continuous interactions within complex cellular circuits established between neural cells and glia. Although it was initially considered that neurones were the major cell type in cerebral function, nowadays astrocytes are considered to contribute to cerebral function too. Astrocytes support normal neuronal activity, including synaptic function, by regulating the extracellular environment with respect to ions and neurotransmitters. There is a plethora of noxious agents which can lead to the development of alterations in organs and functional systems, and that will end in a chronic prognosis. Among the potentially harmful external agents we can find ethanol consumption, whose consequences have been recognized as a major public health concern. Deregulation of cell cycle has devastating effects on the integrity of cells, and has been closely associated with the development of pathologies which can lead to dysfunction and cell death. An alteration of normal neuronal-glial physiology could represent the basis of neurodegenerative processes. In this review we will pay attention on to the recent findings in astrocyte function and their role toward neurons under ethanol consumption.

Platelet stimulation with thrombin induces an elevation in cytoplasmic free Ca(2+) concentration ([Ca(2+)]c) due to Ca(2+) release from intracellular stores and entry from the extracellular medium. Two different intracellular Ca(2+) stores have been described in human platelets: the dense tubular system and the lysosomal-like acidic stores. In the present study, we investigated the contribution of the acidic stores in thrombin-induced platelet aggregation. We have found that platelet aggregation induced by thrombin is reduced in a Ca(2+)-free medium. Discharge of the acidic Ca(2+) stores by treatment with the sarcoendoplasmic Ca(2+)-ATPase (SERCA)3 selective inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone reduced thrombin-evoked platelet aggregation. In the presence of 2,5-di-(tert-butyl)-1,4-hydroquinone, platelet aggregation induced by the protease-activated receptor (PAR)-1 and PAR-4 agonist peptides, SFLLRN and AYPGKF, respectively, was significantly reduced. In cells with depleted acidic stores, activation of GPIb-IX-V by thrombin resulted in reduced or no platelet aggregation in a medium containing 1 mmol/l Caor in a Ca(2+)-free medium, respectively. This finding suggests that Ca(2+) accumulation in the acidic Ca(2+) compartments is required for platelet aggregation induced by activation of the G-coupled PAR-1 and PAR-4 thrombin receptors and, by the occupation of the leucine-rich glycoprotein GPIb-IX-V and provide evidence supporting a functional role of the lysosomal-like acidic Ca(2+) stores in human platelets.

Summary. Background: Apoptosis or programmed cell death involves a number of biochemical events, including the activation of caspases, which lead to specific cell morphology changes and ultimately cell death. Traditionally, two apoptotic pathways have been described: the cell-surface death receptor-dependent extrinsic pathway and the mitochondria-dependent intrinsic pathway. Alternatively, apoptosis has been reported to be induced by endoplasmic reticulum (ER) stress, which is mainly induced by a reduction in intraluminal free Ca2+ concentration ([Ca2+]ER). Objectives: The present study aimed to investigate the development of apoptotic events after ER stress induced by N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), an ER Ca2+ chelator, in human platelets. Methods: Changes in cytosolic free Ca2+ concentration, caspase activity and phosphatidylserine externalization were determined by fluorimetric techniques. Results: Our results indicate that TPEN reduces the amount of free Ca2+ releasable by the Ca2+-mobilizing agonist thrombin. TPEN induced activation of caspase-3, -8 and -9 and subsequent phosphatidylserine externalization. The ability of TPEN to induce phosphatidylserine externalization was smaller than that of thrombin. In addition, TPEN was able to induce phosphorylation of the eukaryotic initiation factor 2α (eIF2α). TPEN-mediated caspase-3 activation requires functional caspase-8, but is independent of H2O2 generation. Activation of caspase-3 and -8 by TPEN was prevented by salubrinal, an agent that prevents ER stress-induced apoptosis. Conclusion: These findings provide experimental evidence for the existence of ER stress-mediated apoptosis in human platelets, a process that might limit platelet life span upon prolonged stimulation with agonists.

Apoptosis, an essential physiological process that is required for the normal development and maintenance of tissue homeostasis, is mediated by active intrinsic mechanisms, although extrinsic factors can also contribute. Aerobic metabolism induces the production of reactive oxygen species (ROS), which are able to induce oxidative stress that promotes cellular apoptosis. The mechanisms of ROS-induced modifications in ion transport pathways involves oxidation of sulphydryl groups located in the ion transport proteins, peroxidation of membrane phospholipids, inhibition of membrane-bound regulatory enzymes and modification of the oxidative phosphorylation and ATP levels. Alterations in the ion transport mechanisms lead to changes in a second messenger system, primary Ca2+ homeostasis. Ca2+ disregulation induces mitochondrial depolarization, which further augments the abnormal electrical activity and disturbs signal transduction, causing cell dysfunction and apoptosis. Control of ROS levels in cells is important, because cellular dysfunction triggered by ROS is a major factor contributing to the development of many diseases. Available evidences show that ROS can induce increases in cytosolic free Ca2+ concentration ([Ca2+]c) by release of the divalent cation from internal stores and impairment of Ca2+ clearance systems. In fact, [Ca2+]c increase is a constant feature of pathological states associated with oxidative stress and apoptosis.

In the central nervous system (CNS), both neurones and astrocytes play crucial roles. On a cellular level, brain activity involves continuous interactions within complex cellular circuits established between neural cells and glia. Although it was initially considered that neurones were the major cell type in cerebral function, nowadays astrocytes are considered to contribute to cerebral function too. Astrocytes support normal neuronal activity, including synaptic function, by regulating the extracellular environment with respect to ions and neurotransmitters. There is a plethora of noxious agents which can lead to the development of alterations in organs and functional systems, and that will end in a chronic prognosis. Among the potentially harmful external agents we can find ethanol consumption, whose consequences have been recognized as a major public health concern. Deregulation of cell cycle has devastating effects on the integrity of cells, and has been closely associated with the development of pathologies which can lead to dysfunction and cell death. An alteration of normal neuronal–glial physiology could represent the basis of neurodegenerative processes. In this review we will pay attention on to the recent findings in astrocyte function and their role toward neurons under ethanol consumption.

List of Contributors Preface G.M. Salido and J.A. Rosado 1. Oxidative stress, intracellular calcium signals and apoptotic processes G.M. Salido 2. Apoptosis M.L. Campo 3. Apoptosis in exocrine acinar cells J.A. Pariente 4. Apoptosis in the liver J. Gonzalez-Gallego and M.J. Tunon 5. Apoptosis in nervous cells A. Gonzalez Mateos 6. Apoptotic events in blood cells J.A. Rosado 7. Apoptotic events in endothelial and smooth muscle cells J. Garcia-Estan and N.M. Atucha 8. Apoptotic events in male germ cells and in mature mammalian spermatozoa J.A. Tapia and F.J. Pena Glossary of Terms Index

Our major aims in this study were to determine the possible existence of ultradian rhythms related to migrating motor complex (MMC), as well as to characterize the existence of circadian rhythms in gallbladder bile concentration in fasting and feeding dogs. Conscious dogs with chronic indwelling gallbladder catheter to allow the periodic collection of bile by means of an automatized device, were used to determine if the composition of gallbladder bile shows rhythmic changes. During feeding, gallbladder bile concentration shows circadian rhythms peaking immediately before meal time in all biliary compounds. On the other hand, during interdigestive state an ultradian rhythm of periodic dilution (90 min period) of gallbladder bile during the bile concentrating process is observed. These findings suggest that periodic food intake plays a role in the circadian rhythms of gallbladder bile composition and reveal that the interdigestive ultradian rhythms shows a frequency similar to those MMC.

An investigation was made on the effects of histamine and related receptor agonists and antagonists on amylase secretion and Ca2+ mobilization in isolated guinea pig pancreatic segments and

Oleuropein and (+)-cycloolivil are natural polyphenolic compounds with a significant radical scavenging activity present in olive tree. We have investigated the antiaggregant effects of oleuropein and (+)-cycloolivil isolated from an ethyl acetate extract of olive tree wood. Oleuropein and (+)-cycloolivil reduced the ability of thrombin to stimulate platelet aggregation. Both compounds reduced thrombin-evoked Ca 2+ release and entry to a similar extent to hydroxytyrosol. This effect was greater in platelets from patients with type 2 diabetes mellitus than in controls. Thrombin-, thapsigargin- and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ)-evoked protein tyrosine phosphorylation, which is involved in Ca 2+ signalling and platelet aggregation, is inhibited by oleuropein and (+)-cycloolivil. oleuropein and (+)-cycloolivil are natural oxygen radical scavengers that reduce thrombin-induced protein tyrosine phosphorylation, Ca 2+ signalling and platelet aggregation. These observations suggest that oleuropein and (+)-cycloolivil may prevent thrombotic complications associated to platelet hyperaggregability and be the base for the development of antiaggregant therapeutic strategies.

Store-operated Ca2+ entry (SOCE) is a mechanism regulated by the filling state of the intracellular Ca2+ stores that requires the participation of the Ca2+ sensor STIM1, which communicates the Ca2+ content of the stores to the plasma membrane Ca2+-permeable channels. We have recently reported that Orai1 mediates the communication between STIM1 and the Ca2+ channel hTRPC1. This event is important to confer hTRPC1 store depletion sensitivity, thus supporting the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of SOCE. Here we have explored the relevance of lipid rafts in the formation of the STIM1-Orai1-hTRPC1 complex and the activation of SOCE. Disturbance of lipid raft domains, using methyl-beta-cyclodextrin, reduces the interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 upon depletion of the intracellular Ca2+ stores and attenuates thapsigargin-evoked Ca2+ entry. These findings suggest that TRPC1, Orai1 and STIM1 form a heteromultimer associated with lipid raft domains and regulated by the intracellular Ca2+ stores.

Alcohol consumption has long been associated with cell damage, and it is thought that it is involved in approximately 40% of cases of acute pancreatitis. In the present study, we have investigated the early effects of acute ethanol exposure on cholecystokinin octapeptide (CCK-8)-evoked calcium (Ca2+) signals in mouse pancreatic acinar cells. Cells were loaded with fura-2 and the changes in fluorescence were monitorized using a spectrofluorimeter. Our results show that stimulation of cells with 1 nM CCK-8 led to a transient increase in [Ca2+]c, which consisted of an initial increase followed by a decrease of [Ca2+]c toward a value close to the prestimulation level. In the presence of 50mM ethanol, CCK-8 lead to a greater Ca2+ mobilization compared to that obtained with CCK-8 alone. The peak of CCK-8-evoked Ca2+ response, the "steady-state level" reached 5 min after stimulation, the rate of decay of [Ca2+]c toward basal values and the total Ca2+ mobilization were significantly affected by ethanol pretreatment. Thapsigargin (Tps) induced an increase in [Ca2+]c due to its release from intracellular stores. After stimulation of cells with CCK-8 or Tps in the presence of 50mM ethanol, a greater [Ca2+]c peak response, a slower rate of decay of [Ca2+]c, and higher values of [Ca2+]c were observed. The effects of ethanol might result from a delayed or reduced Ca2+ extrusion from the cytosol toward the extracellular space by plasma membrane Ca2+ adenosine triphosphatase (ATPase), or into the cytosolic stores by the sarcoendoplasmic reticulum Ca2+-ATPase. Participation of mitochondria in Ca2+ handling is also demonstrated. The actions of ethanol on CCK-8 stimulation of cells create a situation potentially leading to Ca2+ overload, which is a common pathological precursor that mediates pancreatitis.

Ca(2+) mobilization regulates a wide variety of cellular functions. Platelets possess agonist-releasable Ca(2+) stores in acidic organelles where sarcoendoplasmic reticulum Ca(2+)-ATPase-3 (SERCA) pump is involved in store refilling. Stromal interaction molecule 1 (STIM1), which has been presented as a central regulator of platelet function, is a Ca(2+) sensor of the intracellular Ca(2+) stores. Here we present that STIM1 is required for acidic store refilling. Electrotransjection of cells with anti-STIM1 (Y(231)-K(243)) antibody, directed towards a cytoplasmic sequence of STIM1, significantly reduced acidic store refilling, which was tested by remobilizing Ca(2+) from the acidic stores using 2,5-di-(t-butyl)-1,4-hydroquinone (TBHQ) after a brief refilling period that followed thrombin stimulation. Platelet treatment with thrombin or thapsigargin in combination with ionomycin, to induce extensive Ca(2+) store depletion, resulted in a transient increase in the interaction between STIM1 and SERCA3, reaching a maximum 30 s after stimulation. The coupling between STIM1 and SERCA3 was abolished by electrotransjection with anti-STIM1 antibody. The interaction between STIM1 and SERCA3 induced by thrombin or by treatment with thapsigargin plus ionomycin is reduced in platelets from type 2 diabetic patients, as well as Ca(2+) reuptake into the acidic Ca(2+) stores. These findings provide evidence for a role of STIM1 in acidic store refilling in platelets probably acting as a Ca(2+) sensor and regulating the activity of SERCA3. This action is impaired in platelets from type 2 diabetics, which might lead to the enhanced cytosolic Ca(2+) concentration observed and, therefore, in platelet hyperactivity.

An investigation was made of the effects of H1 and H2 receptor agonists and antagonists on rabbit pancreatic exocrine secretion stimulated by secretin and cholecystokinin (CCK). The H1 agonist 2-thiazolylethylamine elicited dose-dependent increases in the rate of secretion. Increases in pancreatic juice flow and enzyme output were also noted after H2 antagonist cimetidine. In contrast, the H1 antagonist chlorpheniramine and H2 agonist dimaprit caused reductions in flow and enzyme output. The results suggest that H1 receptors have stimulative effects and H2 receptors have inhibitory effects on exocrine rabbit pancreas.

The effects of histamine alone and histamine plus cimetidine on basal pancreatic exocrine secretion were determined in anaesthetized rabbits with an acute pancreatic cannula. Intravenous histamine administration (0.2-0.8 n mol/kg/min) increased pancreatic enzyme secretion. A much greater stimulative effect on pancreatic secretion was observed when histamine was administered against a constant background of H2 antagonist (cimetidine 4 mumol/kg/min). The results indicate that in the rabbit pancreas the stimulatory effect of histamine is mediated by H1 receptors.

1. 1. The effects of the anticholinergic compound pirenzepine on pancreatic secretion stimulated by intravenous infusions of increasing doses of CCK (0.025-0.4 Ivy Dog Unit/kg/min) were investigated in fasting anaesthetized dogs. 2. 2. Pirenzepine (0.75 mg/kg i.v.) slightly reduced the increases in the pancreatic juice secretion evoked by CCK. 3. 3. Similar results were obtained with others anticholinergic compounds as atropine (3 mg/kg i.v.) and hexametonium (5 mg/kg i.v.).

Effects of cholecystokinin (CCK) on bile flow through the sphincter of Oddi (SO) were studied in anaesthetized dogs. Intravenous injection of CCK (0.25, 0.5, 1 and 2 IDU/Kg) elicited a dose-dependent reduction in flow through the SO in the first minutes after CCK administration. Pirenzepine and atropine decreased significantly (P less than 0.05) by 29% and a 40% respectively the inhibitory effect induced by 1 IDU/Kg of CCK, whereas hexamethonium elicited an increase in the inhibitory effect induced by 0.5 IDU/Kg of CCK (P less than 0.05). Intravenous infusion of cummulative doses of CCK had different effects according to the dose infused. Lower doses (0.025 and 0.05 IDU/Kg/min) increased transphincteric flow, however, high doses (0.1, 0.2 and 0.4 IDU/Kg/min) were inhibitory. These finding indicated that CCK had two effects on the SO : firstly, a contractile effect, probably mediated through a direct myogenic action and neuronal release of ACh, and secondly a relaxant effect, probably mediated by stimulation of inhibitory postganglionic neurons.

Type 2 diabetes mellitus induces a characteristic platelet hyperactivity that might be due to several factors including oxidative stress and abnormal intracellular Ca(2+) homeostasis. Hyperhomocysteinaemia is considered a risk factor in the development of thrombosis although its effect on platelet function and the mechanisms involved are still poorly understood. Here we show that homocysteine induce a concentration-dependent increase in endogenous production of reactive oxygen species (ROS), which was significantly greater in platelets from diabetic patients than in controls. Platelet treatment with homocysteine resulted in Ca2+ release from the dense tubular system and the acidic stores. Ca2+ mobilization-induced by homocysteine consisted in two components, an initial slow increase in intracellular free Ca (+) concentration ([Ca2+]i) and a rapid and marked increase in [Ca2+]i, th second leading to the activation of platelet aggregation. As well as ROS generation, Ca2+ mobilization and platelet aggregation were significantly greater in platelets from diabetic donors than in controls, which indicate that platelets from diabetic donors are more sensitive to homocysteine. These findings, together with the hyperhomocysteinaemia reported in diabetic patients, strongly suggest that homocysteine might be considered a risk factor in the development of cardiovascular complications associated to type 2 diabetes mellitus.

Cinnamtannin B-1 is a naturally occurring trimeric A-type proanthocyanidin, present in a limited number of plants, which exhibits a large number of cellular actions mostly derived from its antioxidant properties. Cinnamtannin B-1 modulates several biological processes such as changes in cytosolic free Ca2+ concentration, endogenous reactive oxygen species generation, protein tyrosine phosphorylation and platelet aggregation. Proanthocyanidins, such as cinnamtannin B-1, have been reported to exert antitumoral activity mediated by a selective proapoptotic action in a number of tumoral cell lines associated with antiapoptotic activity in normal cells. The opposite effects of proanthocyanidins in normal and tumoral cells suggest that these compounds might be the base for therapeutic strategies directed selectively against tumoral cells. In addition, cinnamtannin B-1 shows antithrombotic actions through inhibition, in platelets, of endogenous ROS generation, Ca2+ mobilization and, subsequently, aggregation. This has been reported to be especially relevant in platelets from diabetic patients, where cinnamtannin B-1 reverses both platelet hypersensitivity and hyperactivity. Considering the large number of cellular effects of cinnamtannin B-1 the development of therapeutic strategies for thrombotic disorders or certain types of cancer deserves further studies. This review summarizes the current knowledge on the actions and relevance of the signalling pathways modulated by cinnamtannin B-1.

Endogenously expressed human canonical transient receptor potential 1 (hTRPC1) and human canonical transient receptor potential 6 (hTRPC6) have been shown to play a role in store-operated Ca2+ entry (SOCE) in human platelets, where two mechanisms for SOCE, regulated by the dense tubular system (DTS) or the acidic granules, have been identified. In cells preincubated for 1 min with 100 µM flufenamic acid we show that hTRPC6 is involved in SOCE activated by both mechanisms, as demonstrated by selective depletion of the DTS or the acidic stores, using thapsigargin (TG) (10 nM) or 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ) (20 µM), respectively, although it is more relevant after acidic store depletion. Co-immunoprecipitation experiments indicated that depletion of both stores separately results in time-dependent interaction between hTRPC1 and hTRPC6, and also between both hTRPCs and the type II IP3 receptor (IP3RII). The latter was greater after treatment with TG. TBHQ-induced coupling between hTRPC1 and 6 was transient and decreased after 30s of treatment, while that induced by TG increased for at least 3 min. TBHQ induced association between SERCA3, located in the acidic stores, hTRPC1, hTRPC6 and Orai1. TBHQ also evoked coupling between SERCA3 and IP3RII, presumably located in the DTS, thus suggesting interplay between both Ca2+ stores. Similarly, TG induces the interaction of SERCA2b with hTRPC1 and 6 and the IP3RII. The interactions between hTRPC1, hTRPC6, IP3RII and SERCA3 were impaired by disruption of the microtubules, supporting a role for microtubules in Ca2+ homeostasis. In conclusion, the present data demonstrate for the first time that hTRPC1, hTRPC6, IP3RII and SERCA3 are parts of a macromolecular protein complex activated by depletion of the intracellular Ca2+ stores in human platelets.

We have investigated the effect of the seleno-organic compound and radical scavenger ebselen on rat hippocampal astrocytes in culture. Throughout our study we carried out determinations of [Ca2+]c in fura-2-loaded cells by single cell imaging, glutamate secretion employing an enzymatic-based assay and GFAP expression, which was monitorized by immunocytochemistry and confocal microscopy. Our results show that ebselen (1–20 μM) dose dependently increases [Ca2+]c, stimulates glutamate release and increases GFAP content, a hallmark of astrocyte reactivity. Ebselen did not alter significantly cell viability as assayed by determination of LDH release into the extracellular medium. Ebselen-evoked glutamate release and increase in GFAP content were Ca2+-dependent, because incubation of astrocytes in the absence of extracellular Ca2+ (medium containing 0.5 mM EGTA) and in the presence of the intracellular Ca2+ chelator BAPTA (10 μM) significantly reduced ebselen-evoked changes in these parameters. The effects of ebselen we have observed may underline various signalling pathways which are important for cell proliferation, differentiation and function. However, aberrations in astroglial physiology could significantly compromise brain function, due to their role as modulators of neuron activity. Therefore, we consider that careful attention should be paid when employing ebselen as a prophylactic agent against brain damage.

Cinnamtannin B-1, a natural A-type proanthocyanidin recently identified as a radical scavenger component of Laurus nobilis L., exerts antiaggregant and antiapoptotic effects in human platelets. Here, we have investigated the intracellular mechanisms involved in the antiaggregant effects of cinnamtannin B-1. Cinnamtannin B-1 showed a greater free radical scavenging activity than vitamin C, vitamin E, or Trolox, among other antioxidants and reduced thrombin-evoked tubulin reorganization and platelet aggregation. Thrombin-evoked activation of Btk and pp60(src) was also inhibited by cinnamtannin B-1. In conclusion, we show that cinnamtannin B-1 is a powerful oxygen radical scavenger that reduces thrombin-evoked microtubular remodeling and activation of the tyrosine kinases Btk and pp60(src), which leads to inhibition of platelet aggregation. These observations suggest that cinnamtannin B-1 may prevent thrombotic complications associated to platelet hyperaggregability and hyperactivity, although further studies are necessary to establish appropriate therapeutic strategies.

Platelet hyperaggregability plays a pivotal role in the pathogenesis of cardiovascular diseases. Thrombin evokes aggregation through Ca 2+ mobilization, tyrosine phosphorylation and generation of reactive oxygen species (ROS). We have investigated the antiaggregant properties of Arbutus unedo extracts in human platelets. Changes in cytosolic Ca 2+ concentration and intracellular oxidants production were registered by espectrofluorimetry using fura-2 and dichlorodihydrofluorescein, respectively, platelet aggregation was assessed by aggregometry and protein tyrosine phosphorylation was detected by Western blotting. Platelet treatment with increasing concentrations (0.015–1.5 mg/mL) of crude aqueous, ethyl acetate or diethyl ether extracts reduced platelet aggregation evoked by thrombin (0.5 U/mL) and show a potent ROS scavenger activity, preventing thrombin-evoked endogenous generation of ROS. Treatment with Arbutus unedo extracts did not alter thrombin-evoked Ca 2+ release from the intracellular stores but reduced store-operated Ca 2+ entry induced by thrombin or by selective depletion of the two Ca 2+ stores in platelets, the dense tubular system and the acidic stores. In addition, platelet treatment with extracts reduced both basal and thrombin-stimulated protein tyrosine phosphorylation. We conclude that Arbutus unedo extracts show antiaggregant actions due to attenuation of Ca 2+ mobilization, ROS production and protein tyrosine phosphorylation and might be used for the treatment and/or prevention of cardiovascular diseases.

We have employed rat hippocampal astrocytes in culture to investigate the effect of ethanol on reactive oxygen species (ROS) production as well as its effect on [Ca2+]c and GFAP expression. Cells were loaded with the fluorescent probes fura-2 and H2DCFDA for the determination of changes in [Ca2+]c and ROS production respectively, employing spectrofluorimetry. GFAP content was determined by immunocytochemistry and confocal scanning microscopy. Our results show ROS production in response to 50 mM ethanol, that was reduced in Ca2+-free medium (containing 0.5 mM EGTA) and in the presence of the intracellular Ca2+ chelator BAPTA (10 microM). The effect of ethanol on ROS production was significantly reduced in the presence of the alcohol dehydrogenase inhibitor 4-methylpyrazole (1 mM), and the antioxidants resveratrol (100 microM) or catalase (300 U/ml). Preincubation of astrocytes in the presence of 10 microM antimycin plus 10 microM oligomycin to inhibit mitochondria completely blocked ethanol-evoked ROS production. In addition, ethanol led to a sustained increase in [Ca2+]c that reached a constant level over the prestimulation values. Finally, incubation of astrocytes in the presence of ethanol increased the content of GFAP that was significantly reduced in the absence of extracellular Ca2+ and by resveratrol and catalase pretreatment. The data obtained in the present study suggest that astrocytes are able to metabolize ethanol, which induces two effects on intracellular homeostasis: an immediate response (Ca2+ release and ROS generation) and later changes involving GFAP expression. Both effects may underline various signaling pathways which are important for cell proliferation, differentiation and function.