Haematological and biochemical reference intervals for farmed channel catfish

The reference intervals for biochemical variables and red blood cell indices of healthy intensively bred channel catfish Ictalurus punctatus were determined. The blood variables were determined using standardized clinical methods. The reference intervals (25th and 75th percentiles) were established using a non-parametric method. Reference intervals for plasma glucose, serum total protein, sodium, potassium, calcium, magnesium, chloride concentration, primary and secondary red blood cell indices were established. The haematological and biochemical reference intervals established may allow important clinical decisions about channel catfish. (c) 2007 the Authors Journal compilation (C) 2007 the Fisheries Society of the British Isles.

Down-regulation of the cardiac sarcoplasmic reticulum ryanodine channel in severely food-restricted rats

We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2), phospholamban (PLB), and ryanodine channel (RYR2) mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats) or 50% diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50% food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 +/- 0.48 vs food-restricted group = 4.84 +/- 0.33, P < 0.01). The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 +/- 0.44 vs food-restricted group = 7.96 +/- 0.45, and control = 1.52 +/- 0.06 vs food-restricted group = 1.53 +/- 0.10, respectively). Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.

N-methyl-D-aspartate channel and consciousness: From signal coincidence detection to quantum computing

Research on Blindsight, Neglect/Extinction and Phantom limb syndromes, as well as electrical measurements of mammalian brain activity, have suggested the dependence of vivid perception on both incoming sensory information at primary sensory cortex and reentrant information from associative cortex. Coherence between incoming and reentrant signals seems to be a necessary condition for (conscious) perception. General reticular activating system and local electrical synchronization are some of the tools used by the brain to establish coarse coherence at the sensory cortex, upon which biochemical processes are coordinated. Besides electrical synchrony and chemical modulation at the synapse, a central mechanism supporting such a coherence is the N-methyl-D-aspartate channel, working as a 'coincidence detector' for an incoming signal causing the depolarization necessary to remove Mg 2+, and reentrant information releasing the glutamate that finally prompts Ca 2+ entry. We propose that a signal transduction pathway activated by Ca 2+ entry into cortical neurons is in charge of triggering a quantum computational process that accelerates inter-neuronal communication, thus solving systemic conflict and supporting the unity of consciousness. © 2001 Elsevier Science Ltd.

Evidence for the role of calcineurin in morphogenesis and calcium homeostasis during mycelium-to-yeast dimorphism of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a dimorphic fungus that causes paracoccidioidomycosis, the most prevalent human deep mycosis in Latin America. The dimorphic transition from mycelium to yeast (M-Y) is triggered by a temperature shift from 25°C to 37°C and is critical for pathogenicity. Intracellular Ca 2+ levels increased in hyphae immediately after temperature-induced dimorphism. The chelation of Ca 2+ with extracellular (EGTA) or intracellular (BAPTA) calcium chelators inhibited temperature-induced dimorphism, whereas the addition of extracellular Ca 2+ accelerated dimorphism. The calcineurin inhibitor cyclosporine A (CsA), but not tacrolimus (FK506), effectively decreased cell growth, halted the M-Y transition that is associated with virulence, and caused aberrant growth morphologies for all forms of P. brasiliensis. The difference between CsA and FK506 was ascribed by the higher levels of cyclophilins contrasted to FKBPs, the intracellular drug targets required for calcineurin suppression. Chronic exposure to CsA abolished intracellular Ca 2+ homeostasis and decreased mRNA transcription of the CCH1 gene for the plasma membrane Ca 2+ channel in yeast-form cells. CsA had no detectable effect on multidrug resistance efflux pumps, while the effect of FK506 on rhodamine excretion was not correlated with the transition to yeast form. In this study, we present evidence that Ca 2+/calmodulin-dependent phosphatase calcineurin controls hyphal and yeast morphology, M-Y dimorphism, growth, and Ca 2+ homeostasis in P. brasiliensis and that CsA is an effective chemical block for thermodimorphism in this organism. The effects of calcineurin inhibitors on P. brasiliensis reinforce the therapeutic potential of these drugs in a combinatory approach with antifungal drugs to treat endemic paracoccidioidomycosis. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Long-term obesity promotes alterations in diastolic function induced by reduction of phospholamban phosphorylation at serine-16 without affecting calcium handling

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

STIM 1/Orai 1 contributes to sex differences in vascular responses to calcium in spontaneously hypertensive rats

Sex differences in Ca2+-dependent signalling and homoeostasis in the vasculature of hypertensive rats are well characterized. However, sex-related differences in SOCE (store-operated Ca2+ entry) have been minimally investigated. We hypothesized that vascular protection in females, compared with males, reflects decreased Ca2+ mobilization due to diminished activation of Orai 1/STIM 1 (stromal interaction molecule I). In addition, we investigated whether ovariectomy in females affects the activation of the Orai 1/STIM 1 pathway. Endothelium-denuded aortic rings from male and female SHRSP (stroke-prone spontaneously hypertensive rats) and WKY (Wistar Kyoto) rats and from OVX (ovariectomized) or sham female SHRSP and WKY rats were used to functionally evaluate Ca2+ influx-induced contractions. Compared with females, aorta from male SHRSP displayed: (i) increased contraction during the Ca2+-loading period; (ii) similar transient contraction during Ca2+ release from the intracellular stores; (iii) increased activation of STIM 1 and Orai1, as shown by the blockade of STIM 1 and Orai1 with neutralizing antibodies, which reversed the sex differences in contraction during the Ca2+-loading period; and (iv) increased expression of STIM I and Orai I. Additionally, we found that aortas from OVX-SHRSP showed increased contraction during the Ca2+-loading period and increased Orai1 expression, but no changes in the SR (sarcoplasmic reticulum)-buffering capacity or STIM I expression. These findings suggest that augmented activation of STIM 1/Orai 1 in aortas from male SHRSP represents a mechanism that contributes to sex-related impaired control of intracellular Ca2+ levels. Furthermore, female sex hormones may negatively modulate the STIM/Orai 1 pathway, contributing to vascular protection observed in female rats.

Impaired beta-adrenoceptor-induced relaxation in small mesenteric arteries from DOCA-salt hypertensive rats is due to reduced K-Ca channel activity

beta-Adrenoceptor (beta-AR)-mediated relaxation plays an important role in the regulation of vascular tone. beta-AR-mediated vascular relaxation is reduced in various disease states and aging. We hypothesized that beta-AR-mediated vasodilatation is impaired in DOCA-salt hypertension due to alterations in the cAMP pathway. beta-AR-mediated relaxation was determined in small mesenteric arteries from DOCA-salt hypertensive and control uninephrectomized (Uni) rats. To exclude nitric oxide (NO) and cyclooxygenase (COX) pathways, relaxation responses were determined in the presence of L-NNA and indomethacin, NO synthase inhibitor and COX inhibitors, respectively. Isoprenaline (ISO)-induced relaxation was reduced in arteries from DOCA-salt compared to Uni rats. Protein kinase A (PKA) inhibitors (H89 or Rp-cAMPS) or adenylyl cyclase inhibitor (SQ22536) did not abolish the difference in ISO-induced relaxation between the groups. Forskolin (adenylyl cyclase activator)-induced relaxation was similar between the groups. The inhibition of IKCa/SKCa channels (TRAM-34 plus UCL1684) or BKCa channels (iberiotoxin) reduced ISO-induced relaxation only in Uni rats and abolished the relaxation differences between the groups. The expression of SKCa channel was decreased in DOCA-salt arteries. The expression of BKCa channel a subunit was increased whereas the expression of BKCa channel p subunit was decreased in DOCA-salt arteries. The expression of receptor for activated C kinase 1 (RACK1), which is a binding protein for BKG, channel and negatively modulates its activity, was increased in DOCA-salt arteries. These results suggest that the impairment of beta-AR-mediated relaxation in DOCA-salt mesenteric arteries may be attributable to altered IKCa/SKCa and/or BKCa channels activities rather than cAMP/PKA pathway. Impaired beta-AR-stimulated BKCa channel activity may be due to the imbalance between its subunit expressions and RACK1 upregulation. (C) 2012 Elsevier Ltd. All rights reserved.

Chronic Stress Improves the Myocardial Function without Altering L-type Ca+2 Channel Activity in Rats

Background: Chronic stress is associated with cardiac remodeling; however the mechanisms have yet to be clarified. Objective: The purpose of this study was test the hypothesis that chronic stress promotes cardiac dysfunction associated to L-type calcium Ca2+ channel activity depression. Methods: Thirty-day-old male Wistar rats (70 - 100 g) were distributed into two groups: control (C) and chronic stress (St). The stress was consistently maintained at immobilization during 15 weeks, 5 times per week, 1h per day. The cardiac function was evaluated by left ventricular performance through echocardiography and by ventricular isolated papillary muscle. The myocardial papillary muscle activity was assessed at baseline conditions and with inotropic maneuvers such as: post-rest contraction and increases in extracellular Ca2+ concentration, in presence or absence of specific blockers L-type calcium channels. Results: The stress was characterized for adrenal glands hypertrophy, increase of systemic corticosterone level and arterial hypertension. The chronic stress provided left ventricular hypertrophy. The left ventricular and baseline myocardial function did not change with chronic stress. However, it improved the response of the papillary muscle in relation to positive inotropic stimulation. This function improvement was not associated with the L-type Ca2+ channel. Conclusion: Chronic stress produced cardiac hypertrophy; however, in the study of papillary muscle, the positive inotropic maneuvers potentiated cardiac function in stressed rats, without involvement of L-type Ca2+ channel. Thus, the responsible mechanisms remain unclear with respect to Ca2+ influx alterations. (Arq Bras Cardiol 2012;99(4):907-914)

Heavy metal cations permeate the TRPV6 epithelial cation channel

TRPV6 belongs to the vanilloid family of the transient receptor potential channel (TRP) superfamily. This calcium-selective channel is highly expressed in the duodenum and the placenta, being responsible for calcium absorption in the body and fetus. Previous observations have suggested that TRPV6 is not only permeable to calcium but also to other divalent cations in epithelial tissues. In this study, we tested whether TRPV6 is indeed also permeable to cations such as zinc and cadmium. We found that the basal intracellular calcium concentration was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells, and that this difference almost disappeared in nominally calcium-free solution. Live cell imaging experiments with Fura-2 and NewPort Green DCF showed that overexpression of human TRPV6 increased the permeability for Ca(2+), Ba(2+), Sr(2+), Mn(2+), Zn(2+), Cd(2+), and interestingly also for La(3+) and Gd(3+). These results were confirmed using the patch clamp technique. (45)Ca uptake experiments showed that cadmium, lanthanum and gadolinium were also highly efficient inhibitors of TRPV6-mediated calcium influx at higher micromolar concentrations. Our results suggest that TRPV6 is not only involved in calcium transport but also in the transport of other divalent cations, including heavy metal ions, which may have toxicological implications.

Calcium-modulated chloride pathways contribute to chloride flux in murine cystic fibrosis-affected macrophages

Cystic fibrosis (CF), a common lethal inherited disorder defined by ion transport abnormalities, chronic infection, and robust inflammation, is the result of mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a cAMP-activated chloride (Cl-) channel. Macrophages are reported to have impaired activity in CF. Previous studies suggest that Cl- transport is important for macrophage function; therefore, impaired Cl- secretion may underlie CF macrophage dysfunction. To determine whether alterations in Cl- transport exist in CF macrophages, Cl- efflux was measured using N-[ethoxycarbonylmethyl]- 6-methoxy-quinolinium bromide (MQAE), a fluorescent indicator dye. The contribution of CFTR was assessed by calculating Cl- flux in the presence and absence of cftr(inh)-172. The contribution of calcium (Ca(2+))-modulated Cl- pathways was assessed by examining Cl- flux with varied extracellular Ca(2+) concentrations or after treatment with carbachol or thapsigargin, agents that increase intracellular Ca(2+) levels. Our data demonstrate that CFTR contributed to Cl- efflux only in WT macrophages, while Ca(2+)-mediated pathways contributed to Cl- transport in CF and WT macrophages. Furthermore, CF macrophages demonstrated augmented Cl- efflux with increases in extracellular Ca(2+). Taken together, this suggests that Ca(2+)-mediated Cl- pathways are enhanced in CF macrophages compared with WT macrophages.

Regulation of Cx45 hemichannels mediated by extracellular and intracellular calcium

Connexin45 (Cx45) hemichannels (HCs) open in the absence of Ca(2+) and close in its presence. To elucidate the underlying mechanisms, we examined the role of extra- and intracellular Ca(2+) on the electrical properties of HCs. Experiments were performed on HeLa cells expressing Cx45 using electrical (voltage clamp) and optical (Ca(2+) imaging) methods. HCs exhibit a time- and voltage-dependent current (I(hc)), activating with depolarization and inactivating with hyperpolarization. Elevation of [Ca(2+)](o) from 20 nM to 2 μM reversibly decreases I(hc), decelerates its rate of activation, and accelerates its deactivation. Our data suggest that [Ca(2+)](o) modifies the channel properties by adhering to anionic sites in the channel lumen and/or its outer vestibule. In this way, it blocks the channel pore and reversibly lowers I(hc) and modifies its kinetics. Rapid lowering of [Ca(2+)](o) from 2 mM to 20 nM, achieved early during a depolarizing pulse, led to an outward I(hc) that developed with virtually no delay and grew exponentially in time paralleled by unaffected [Ca(2+)](i). A step increase of [Ca(2+)](i) evoked by photorelease of Ca(2+) early during a depolarizing pulse led to a transient decrease of I(hc) superimposed on a growing outward I(hc); a step decrease of [Ca(2+)](i) elicited by photoactivation of a Ca(2+) scavenger provoked a transient increase in I(hc). Hence, it is tempting to assume that Ca(2+) exerts a direct effect on Cx45 hemichannels.

Continuous Formation and Separation of Calcium-Alginate Capsules Containing Viable Mammalian Cells in Microfluidic Devices

Microfluidic devices can be used for many applications, including the formation of well-controlled emulsions. In this study, the capability to continuously create monodisperse droplets in a microfluidic device was used to form calcium-alginate capsules.Calcium-alginate capsules have many potential uses, such as immunoisolation of cells and microencapsulation of active drug ingredients or bitter agents in food or beverage products. The gelation of calcium-alginate capsules is achieved by crosslinking sodiumalginate with calcium ions. Calcium ions dissociated from calcium carbonate due to diffusion of acetic acid from a sunflower oil phase into an aqueous droplet containing sodium-alginate and calcium carbonate. After gelation, the capsules were separated from the continuous oil phase into an aqueous solution for use in biological applications. Typically, capsules are separated bycentrifugation, which can damage both the capsules and the encapsulated material. A passive method achieves separation without exposing the encapsulated material or the capsules to large mechanical forces, thereby preventing damage. To achieve passiveseparation, the use of a microfluidic device with opposing channel wa hydrophobicity was used to stabilize co-laminar flow of im of hydrophobicity is accomplished by defining one length of the channel with a hydrogel. The chosen hydrogel was poly (ethylene glycol) diacrylate, which adheres to the glass surface through the use of self-assembled monolayer of 3-(trichlorosilyl)-propyl methacrylate. Due to the difference in surface energy within the channel, the aqueous stream is stabilized near a hydrogel and the oil stream is stabilized near the thiolene based optical adhesive defining the opposing length of the channel. Passive separation with co-laminar flow has shown success in continuously separating calcium-alginatecapsules from an oil phase into an aqueous phase. In addition to successful formation and separation of calcium alginate capsules,encapsulation of Latex micro-beads and viable mammalian cells has been achieved. The viability of encapsulated mammalian cells was determined using a live/dead stain. The co-laminar flow device has also been demonstrated as a means of separating liquid-liquidemulsions.

CSTX-1, a toxin from the venom of the hunting spider Cupiennius salei, is a selective blocker of L-type calcium channels in mammalian neurons

The inhibitor cystine-knot motif identified in the structure of CSTX-1 from Cupiennius salei venom suggests that this toxin may act as a blocker of ion channels. Whole-cell patch-clamp experiments performed on cockroach neurons revealed that CSTX-1 produced a slow voltage-independent block of both mid/low- (M-LVA) and high-voltage-activated (HVA) insect Ca(v) channels. Since C. salei venom affects both insect as well as rodent species, we investigated whether Ca(v) channel currents of rat neurons are also inhibited by CSTX-1. CSTX-1 blocked rat neuronal L-type, but no other types of HVA Ca(v) channels, and failed to modulate LVA Ca(v) channel currents. Using neuroendocrine GH3 and GH4 cells, CSTX-1 produced a rapid voltage-independent block of L-type Ca(v) channel currents. The concentration-response curve was biphasic in GH4 neurons and the subnanomolar IC(50) values were at least 1000-fold lower than in GH3 cells. L-type Ca(v) channel currents of skeletal muscle myoballs and other voltage-gated ion currents of rat neurons, such as I(Na(v)) or I(K(v)) were not affected by CSTX-1. The high potency and selectivity of CSTX-1 for a subset of L-type channels in mammalian neurons may enable the toxin to be used as a molecular tool for the investigation of this family of Ca(v) channels.

Modeling Calcium Concentration and Biochemical Reactions

Almost all regions of the brain receive one or more neuromodulatory inputs, and disrupting these inputs produces deficits in neuronal function. Neuromodulators act through intracellular second messenger pathways to influence the electrical properties of neurons, integration of synaptic inputs, spatio-temporal firing dynamics of neuronal networks, and, ultimately, systems behavior. Second messengers pathways consist of series of bimolecular reactions, enzymatic reactions, and diffusion. Calcium is the second messenger molecule with the most effectors, and thus is highly regulated by buffers, pumps and intracellular stores. Computational modeling provides an innovative, yet practical method to evaluate the spatial extent, time course and interaction among second messenger pathways, and the interaction of second messengers with neuron electrical properties. These processes occur both in compartments where the number of molecules are large enough to describe reactions deterministically (e.g. cell body), and in compartments where the number of molecules is small enough that reactions occur stochastically (e.g. spines). – In this tutorial, I explain how to develop models of second messenger pathways and calcium dynamics. The first part of the tutorial explains the equations used to model bimolecular reactions, enzyme reactions, calcium release channels, calcium pumps and diffusion. The second part explains some of the GENESIS, Kinetikit and Chemesis objects that implement the appropriate equations. In depth explanation of calcium and second messenger models is provided by reviewing code, both in XPP, Chemesis and Kinetikit, that implements simple models of calcium dynamics and second messenger cascades.

Characterization of calcium signaling mechanisms in osteoblasts

Bone remodeling is controlled by the osteoclast, which resorbs bone, and the osteoblast, which synthesizes and secretes proteins that are eventually mineralized into bone. Ca$\sp{2+}$ homeostasis and signaling contribute to the function of nearly all cell types, and understanding both in the osteoblast is of importance given its secretory properties and interaction with osteoclasts. This study was undertaken to identify and investigate the physiology of the Ca$\sp{2+}$ signaling mechanisms present in osteoblasts. The Ca$\sp{2+}$ pumps, stores and channels present in osteoblasts were studied. RT-PCR cloning revealed that osteoblast-like cells express PMCA1b, an alternatively spliced transcript of the plasma membrane Ca$\sp{2+}$-ATPase. The PMCA1b isoform contains a consensus phosphorylation site for cAMP-dependent protein kinase A and a modified calmodulin binding domain. The regulation of osteoblast function by agents that act via cAMP-mediated pathways may involve alterations in the activity of the plasma membrane Ca$\sp{2+}$-ATPase.^ Calcium release from intracellular stores is a signaling mechanism used universally by cells responding to hormones and growth factors, and the compartmentalization and regulated release of calcium is cell-type specific. Fura-2 was employed to monitor intracellular Ca$\sp{2+}$. Thapsigargin and 2,5,-di-(tert-butyl)-1,4-benzohydroquinone (tBuHQ), two inhibitors of endoplasmic reticulum Ca$\sp{2+}$-ATPase activity, both emptied a single intracellular calcium pool which was released in response to either ATP or thrombin, identifying it as the inositol 1,4,5-trisphosphate-sensitive calcium store. The Ca$\sp{2+}$ storage system present in osteoblasts is typical of a non-excitable cell type, despite these cells sharing characteristics of excitable cells such as voltage-sensitive Ca$\sp{2+}$ channels (VSCCs).^ VSCCs are important cell surface regulators of membrane permeability to Ca$\sp{2+}$. In non-excitable cells VSCCs act as cellular transducers of stimulus-secretion coupling, activators of intracellular proteins, and in control of cell growth and differentiation. Functional VSCCs have been shown to exist in osteoblasts, however, no molecular cloning has been reported. To obtain information concerning the molecular identity of the osteoblastic VSCC, we used an RT-PCR regional amplification approach. Sequencing of the products indicated that osteoblasts express at least two isoforms of the L-type VSCC, $\alpha 1\sb{\rm C-a}$ and the $\alpha 1\sb{\rm C-d}$, which share regions of identity to the $\alpha \sb{\rm 1C}$ isoform first identified in cardiac myocytes. The ability of $1,25(\rm OH)\sb2D\sb3$ and structural analogs to modulate expression of Ca$\sp{2+}$ channel mRNA was then investigated. Cells were cultured for 48 hr in the presence of $1,25(\rm OH)\sb2D\sb3$ or vitamin D analogs, and the levels of mRNA encoding VSCC $\alpha \sb{\rm 1C}$ were quantitated using a competitive RT-PCR assay. It was found that $1,25(\rm OH)\sb2D\sb3$ and analog BT reduced steady state levels of $\alpha \sb{\rm 1C}$ mRNA. Conversely, analog AT did not alter steady state levels of Ca$\sp{2+}$ channel mRNA. Since it has been shown previously that analog BT, but not AT, binds and activates the nuclear vitamin D receptor, these findings suggest that the down regulation of channel mRNA involves the nuclear receptor for $1,25(\rm OH)\sb2D\sb3$. ^