Expression of intracellular calcium signalling genes in cattle skin during tick infestation

It is widely acknowledged that changes in intracellular calcium ion (Ca2+) concentration provide dynamic signals that control a plethora of cellular processes, including triggering and mediating host defence mechanisms. In this study, quantitative real-time PCR was used to analyse gene expression of 14 Ca2+ signalling proteins in skin obtained from high tick-resistant (HR) and low tick-resistant (LR) cattle following artificial challenge with cattle tick (Rhipicephalus (Boophilus) microplus). Up-regulation of numerous genes was observed in both HR and LR skin following tick challenge, however substantially higher transcription activation was found in HR tissue. The elevated expression in HR skin of specific Ca2+ signalling genes such as AHNAK, CASQ, IL2, NFAT2CIP and PLCG1 may be related to host resistance. Our data suggest that Ca2+ and its associated proteins might play an important role in host response to ticks and that further investigation is warranted.

Effects of oral calcium sensitizers on experimental heart failure

Suun kautta annosteltava kalsiumherkistäjä parantaa sydämen vajaatoimintaan liittyvää pumppausvajetta kokeellisissa sydämen vajaatoimintamalleissa Huolimatta viime vuosikymmenien lääketieteellisestä kehityksestä krooninen sydämen vajaatoiminta on silti edelleen vakava, elämänlaatua voimakkaasti rajoittava sairaus. Kalsiumherkistäjät ovat uusi, sydämen pumppausvoimaa lisäävä lääkeryhmä. Levosimendaani, kotimaista alkuperää oleva kalsiumherkistäjä, on kliinisessä käytössä akuutin vajaatoiminnan hoitoon suonensisäisesti ja lyhytaikaisesti annosteltavana valmisteena. Levosimendaanilla on aktiivinen metaboliitti, OR-1896, jonka oletetaan olevan vuorokauden mittaisen levosimendaani-infuusion jälkeen havaittujen useita päiviä kestävien hyödyllisisten vaikutuksisten takana. Levosimendaanin kroonisen, suun kautta tapahtuvan annostelun vaikutuksista tieto on vähäisempää, mutta sillä näyttää olevan positiivisia vaikutuksia potilaiden raportoimana. FM Marjut Louhelainen on selvittänyt väitöskirjassaan suun kautta annosteltavan levosimendaanin ja sen pitkäkestoisen aktiivisen metaboliitin vaikutuksia kroonisen vajaatoiminnan hoidossa käyttämällä sekä hypertensiivisen sydäntaudin että 2 tyypin diabeteksen komplisoimaan sydäninfarktin kokeellisia malleja. Tutkimuksessa selvitettiin lisäksi vajaatoimintaan johtavia molekyylitason tapahtumia sydänlihaksessa. Tutkimuksessa osoitettiin, että krooninen suun kautta annosteltu hoito sekä kalsiumherkistäjä levosimendaanilla että sen aktiivisella metaboliitilla estää hypertensiiviseen sydämen vajaatoiminnan aikaasaamaa sydämen uudelleenmuovaantumista ja siihen liittyvää kuolleisuutta. Nämä vaikutukset välittyivät vähentyneen sydänlihassoluhypertrofian, solukuolleisuuden ja neurohumaraalisen aktivaation kautta. Levosimendaanin ja OR-1896:n osoitettiin myös parantavan sydämen pumppausfunktiota tyyppi 2 diabeteksen komplisoimassa sydäninfarktissa. Ei-diabeettiseen tilanteeseen verrattuna diabetekseen liittyvä infarktin jälkeinen vajaatoiminnan kehitys oli yhteydessä lisääntyneeseen tulehdukseen, fibroosiin, solukuolemaan, neurohumoraaliseen aktivaatioon ja ennenaikaiseen kudoksen vanhenemiseen. Sekä levosimendaani, että OR-1869 vähensivät tulehduksen, fibroosin ja solukuoleman merkkejä ja vaimensi neurohumoraalista aktivaatiota. OR-1896 myös vähensi solujen vanhenemiseen liittyvien merkkiaineiden ilmentymistä. Väitöskirjassa todettiin, että suun kautta annosteltuna sekä levosimendaani, että sen aktiivinen metaboliitti OR-1896, omaavat terapeuttista potentiaalia sekä hypertensiivisen sydäntaudin hoitoon että sydäninfarktin jälkeisen vajaatoiminnan estoon. FM Marjut Louhelaisen farmakologian alaan kuuluva väitöskirja Effects of oral calcium sensitizers on experimental heart failure tarkastetaan Helsingin yliopiston Lääketieteellisessä tiedekunnassa perjantaina 29.01.2010 klo 12 (Biomedicum Helsinki, luentosali 2, Haartmaninkatu 8, Helsinki). Vastaväittäjänä toimii professori Raimo Tuominen, Helsingin yliopiston Farmasian tiedekunnasta ja kustoksena professori Eero Mervaala Helsingin yliopiston Lääketieteellisestä tiedekunnasta.

Molecular and Cellular Mechanisms Behind Juvenile Neuronal Ceroid Lipofuscinosis (JNCL, Batten disease)

Neurodegenerative disorders are chronic, progressive, and often fatal disorders of the nervous system caused by dysfunction, and ultimately, death of neuronal cells. The underlying mechanisms of neurodegeneration are poorly understood, and monogenic disorders can be utilised as disease models to elucidate the pathogenesis. Juvenile neuronal ceroid-lipofuscinosis (JNCL, Batten disease) is a recessively inherited lysosomal storage disorder with progressive neurodegeneration and accumulation of autofluorescent storage material in most tissues. It is caused by mutations in the CLN3 gene; however, the exact function of the corresponding CLN3 protein, as well as the molecular mechanisms of JNCL pathogenesis have remained elusive. JNCL disease exclusively affects the central nervous system leaving other organs unaffected, and therefore it is of a particular importance to conduct studies in brain tissue and neuronal cells. The aim of this thesis project was to elucidate the molecular and cell biological mechanisms underlying JNCL. This was the first study to describe the endogenous Cln3 protein, and it was shown that Cln3 localised to neuronal cells in the mouse brain. At a subcellular level, endogenous Cln3 was localised to the presynaptic terminals and to the synaptosome compartment, but not to the synaptic vesicles. Studies with the CLN3-deficient cells demonstrated an impaired endocytic membrane trafficking, and established an interconnection between CLN3, microtubulus-binding Hook1 and Rab proteins. This novel data was not only important in characterising the roles of CLN3 in cells, but also provided significant information delineating the versatile role of the Rab proteins. To identify affected cellular pathways in JNCL, global gene expression profiling of the knock-out mouse Cln3-/- neurons was performed and systematically analysed; this revealed a slight dysfunction of the mitochondria, cytoskeletal abnormality in the microtubule plus-end, and an impaired recovery from depolarizing stimulus when specific N-type Ca2+ channels were inhibited, thus leading to a prolonged time of higher intracellular calcium. All these defective pathways are interrelated, and may together be sufficient to initiate the neurodegenerative process. Results of this thesis also suggest that in neuronal cells, CLN3 most likely functions at endocytic vesicles at the presynaptic terminal, potentially involved in the regulation of the calcium-mediated synaptic transmission.

Unravelling Molecular and Cellular Disease Mechanisms in Infantile Neuronal Ceroid Lipofuscinosis (INCL)

Studying neurodegeneration provides an opportunity to gain insights into normal cell physiology, and not just pathophysiology. In this thesis work the focus is on Infantile Neuronal Ceroid Lipofuscinosis (INCL). It is a recessively inherited lysosomal storage disorder. The disease belongs to the neuronal ceroid lipofuscinoses (NCLs), a group of common progressive neurodegenerative diseases of the childhood. Characteristic accumulation of autofluorescent storage material is seen in most tissues but only neurons of the central nervous system are damaged and eventually lost during the course of the disease leaving most other cell types unaffected. The disease is caused by mutations in the CLN1 gene, but the physiological function of the corresponding protein the palmitoyl protein thioesterase (PPT1) has remained elusive. The aim of this thesis work was to shed light on the molecular and cell biological mechanisms behind INCL. This study pinpointed the localization of PPT1 in axonal presynapses of neurons. It also established the role of PPT1 in early neuronal maturation as well as importance in mature neuronal synapses. This study revealed an endocytic defect in INCL patient cells manifesting itself as delayed trafficking of receptor and non-receptor mediated endocytic markers. Furthermore, this study was the first to connect the INCL storage proteins the sphingolipid activator proteins (SAPs) A and D to pathological events on the cellular level. Abnormal endocytic processing and intracellular re-localization was demonstrated in patient cells and disease model knock-out mouse neurons. To identify early affected cellular and metabolic pathways in INCL, knock-out mouse neurons were studied by global transcript profiling and functional analysis. The gene expression analysis revealed changes in neuronal maturation and cell communication strongly associated with the regulated secretory system. Furthermore, cholesterol metabolic pathways were found to be affected. Functional studies with the knock-out mouse model revealed abnormalities in neuronal maturation as well as key neuronal functions including abnormalities in intracellular calcium homeostasis and cholesterol metabolism. Together the findings, introduced in this thesis work, support the essential role of PPT1 in developing neurons as well as synaptic sites of mature neurons. Results of this thesis also elucidate early events in INCL pathogenesis revealing defective pathways ultimately leading to the neurodegenerative process. These results contribute to the understanding of the vital physiological function of PPT1 and broader knowledge of common cellular mechanisms behind neurodegeneration. These results add to the knowledge of these severe diseases offering basis for new approaches in treatment strategies.

Redistribution of subcellular calcium in rat liver on administration of vanadate

Mitochondria isolated from the livers of rats administered with sodium meta-, ortho-, or polyvanadate, but not vanadyl sulphate, exhibited enhanced Ca2+ — stimulated respiration and uptake of calcium. These effects were shown also by mitochondria isolated from livers perfused with polyvanadate. The concentration of acid-soluble calcium decreased significantly in the mitochondrial fraction on vanadate treatment, while that in the cytosol showed a corresponding increase. Phenoxybenzamine, an antagonist to a-adrenergic receptors, effectively inhibited vanadate-induced Ca2+ mobilization, but surgical sympathectomy was without effect. This is the first demonstration of vanadate mimicking agr-adrenergic agonists in vivo.

H2O2 has a role in cellular regulation

H2O2, in addition to producing highly reactive molecules through hydroxyl radicals or peroxidase action, can exert a number of direct effects on cells, organelles and enzymes. The stimulations include glucose transport, glucose incorporation into glycogen, HMP shunt pathway, lipid synthesis, release of calcium from mitochondria and of arachidonate from phospholipids, poly ADP ribosylation, and insulin receptor tyrosine kinase and pyruvate dehydrogenase activities. The inactivations include glycolysis, lipolysis, reacylation of lysophospholipids, ATP synthesis, superoxide dismutase and protein kinase C. Damages to DNA and proteoglycan and general cytotoxicity possibly through oxygen radicals were also observed. A whole new range of effects will be opened by the finding that H2O2 can act as a signal transducer in oxidative stress by oxidizing a dithiol protein to disulphide form which then activates transcription of the stress inducible genes. Many of these direct effects seem to be obtained by dithiol-disulphide modification of proteins and their active sites, as part of adaptive responses in oxidative stress.

Interplay of Substrate Conductivity, Cellular Microenvironment, and Pulsatile Electrical Stimulation toward Osteogenesis of Human Mesenchymal Stem Cells in Vitro

The influences of physical stimuli such as surface elasticity, topography, and chemistry over mesenchymal stem cell proliferation and differentiation are well investigated. In this context, a fundamentally different approach was adopted, and we have demonstrated the interplay of inherent substrate conductivity, defined chemical composition of cellular microenvironment, and intermittent delivery of electric pulses to drive mesenchymal stem cell differentiation toward osteogenesis. For this, conducting polyaniline (PANI) substrates were coated with collagen type 1 (Coll) alone or in association with sulfated hyaluronan (sHya) to form artificial extracellular matrix (aECM), which mimics the native microenvironment of bone tissue. Further, bone marrow derived human mesenchymal stem cells (hMSCs) were cultured on these moderately conductive (10(-4)10(-3) S/cm) aECM coated PANI substrates and exposed intermittently to pulsed electric field (PEF) generated through transformer-like coupling (TLC) approach over 28 days. On the basis of critical analysis over an array of end points, it was inferred that Coll/sHya coated PANI (PANI/Coll/sHya) substrates had enhanced proliferative capacity of hMSCs up to 28 days in culture, even in the absence of PEF stimulation. On the contrary, the adopted PEF stimulation protocol (7 ms rectangular pulses, 3.6 mV/cm, 10 Hz) is shown to enhance osteogenic differentiation potential of hMSCs. Additionally, PEF stimulated hMSCs had also displayed different morphological characteristics as their nonstimulated counterparts. Concomitantly, earlier onset of ALP activity was also observed on PANI/Coll/sHya substrates and resulted in more calcium deposition. Moreover, real-time polymerase chain reaction results indicated higher mRNA levels of alkaline phosphatase and osteocalcin, whereas the expression of other osteogenic markers such as Runt-related transcription factor 2, Col1A, and osteopontin exhibited a dynamic pattern similar to control cells that are cultured in osteogenic medium. Taken together, our experimental results illustrate the interplay of multiple parameters such as substrate conductivity, electric field stimulation, and aECM coating on the modulation of hMSC proliferation and differentiation in vitro.

Fluid flow induced calcium response in bone cell network

In our previous work, bone cell networks with controlled spacing and functional intercellular gap junctions had been successfully established by using microcontact printing and self assembled monolayers technologies [Guo, X. E., E. Takai, X. Jiang, Q. Xu, G. M. Whitesides, J. T. Yardley, C. T. Hung, E. M. Chow, T. Hantschel, and K. D. Costa. Mol. Cell. Biomech. 3:95-107, 2006]. The present study investigated the calcium response and the underlying signaling pathways in patterned bone cell networks exposed to a steady fluid flow. The glass slides with cell networks were separated into eight groups for treatment with specific pharmacological agents that inhibit pathways significant in bone cell calcium signaling. The calcium transients of the network were recorded and quantitatively evaluated with a set of network parameters. The results showed that 18 alpha-GA (gap junction blocker), suramin (ATP inhibitor), and thapsigargin (depleting intracellular calcium stores) significantly reduced the occurrence of multiple calcium peaks, which were visually obvious in the untreated group. The number of responsive peaks also decreased slightly yet significantly when either the COX-2/PGE(2) or the NOS/nitric oxide pathway was disrupted. Different from all other groups, cells treated with 18 alpha-GA maintained a high concentration of intracellular calcium following the first peak. In the absence of calcium in the culture medium, the intracellular calcium concentration decreased slowly with fluid flow without any calcium transients observed. These findings have identified important factors in the flow mediated calcium signaling of bone cells within a patterned network.

A calcium-dependent interaction between calmodulin and the calponin homology domain of human IQGAP1

IQGAPs are cytoskeletal scaffolding proteins which collect information from a variety of signalling pathways and pass it on to the microfilaments and microtubules. There is a well-characterised interaction between IQGAP and calmodulin through a series of IQ-motifs towards the middle of the primary sequence. However, it has been shown previously that the calponin homology domain (CHD), located at the N-terminus of the protein, can also interact weakly with calmodulin. Using a recombinant fragment of human IQGAP1 which encompasses the CHD, we have demonstrated that the CHD undergoes a calcium ion-dependent interaction with calmodulin. The CHD can also displace the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulphonate from calcium-calmodulin, suggesting that the interaction involves non-polar residues on the surface of calmodulin. Molecular modelling identified a possible site on the CHD for calmodulin interaction. The physiological significance of this interaction remains to be discovered.

Biocompatibility of Calcium Phosphate Bone Cement with Optimised Mechanical Properties

The broad aim of this work was to investigate and optimise the properties of calcium phosphate bone cements (CPCs) for use in vertebroplasty to achieve effective primary fixation of spinal fractures. The incorporation of collagen, both bovine and from a marine sponge (Chondrosia reniformis), into a CPC was investigated. The biological properties of the CPC and collagen-CPC composites were assessed in vitro through the use of human bone marrow stromal cells. Cytotoxicity, proliferation and osteoblastic differentiation were evaluated using lactate dehydrogenase, PicoGreen and alkaline phosphatase activity assays respectively. The addition of both types of collagen resulted in an increase in cytotoxicity, albeit not to a clinically relevant level. Cellular proliferation after 1, 7 and 14 days was unchanged. The osteogenic potential of the CPC was reduced through the addition of bovine collagen but remained unchanged in the case of the marine collagen. These findings, coupled with previous work showing that incorporation of marine collagen in this way can improve the physical properties of CPCs, suggest that such a composite may offer an alternative to CPCs in applications where low setting times and higher mechanical stability are important.

Modulation of intracellular calcium homeostasis blocks autophagosome formation

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress-the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca (2+)-ATPase inhibitor thapsigargin (TG)-potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.

The sarcolemmal calcium pump inhibits the calcineurin/nuclear factor of activated T-cell pathway via interaction with the calcineurin A catalytic subunit

The calcineurin/nuclear factor of activated T-cell (NFAT) pathway represents a crucial transducer of cellular function. There is increasing evidence placing the sarcolemmal calcium pump, or plasma membrane calcium/calmodulin ATPase pump (PMCA), as a potential modulator of signal transduction pathways. We demonstrate a novel interaction between PMCA and the calcium/calmodulin-dependent phosphatase, calcineurin, in mammalian cells. The interaction domains were located to the catalytic domain of PMCA4b and the catalytic domain of the calcineurin A subunit. Endogenous calcineurin activity, assessed by measuring the transcriptional activity of its best characterized substrate, NFAT, was significantly inhibited by 60% in the presence of ectopic PMCA4b. This inhibition was notably reversed by the co-expression of the PMCA4b interaction domain, demonstrating the functional significance of this interaction. PMCA4b was, however, unable to confer its inhibitory effect in the presence of a calcium/calmodulin-independent constitutively active mutant calcineurin A suggesting a calcium/calmodulin-dependent mechanism. The modulatory function of PMCA4b is further supported by the observation that endogenous calcineurin moves from the cytoplasm to the plasma membrane when PMCA4b is overexpressed. We suggest recruitment by PMCA4b of calcineurin to a low calcium environment as a possible explanation for these findings. In summary, our results offer strong evidence for a novel functional interaction between PMCA and calcineurin, suggesting a role for PMCA as a negative modulator of calcineurin-mediated signaling pathways in mammalian cells. This study reinforces the emerging role of PMCA as a molecular organizer and regulator of signaling transduction pathways.

TRP Channels and calcium signalling in dental pulp stem cells

Introduction: Ca2+ ion is an important intracellular messenger essential for the regulation of various cellular functions including proliferation, differentiation and apoptosis. Transient Receptor Potential (TRP) channels are calcium permeable cationic channels that play important role in regulation of free intracellular calcium ([Ca2+]i) in response to thermal, physical and chemical stimuli. Ca2+ signalling in human dental pulp stem cells (hDPSCs) and the ion channels regulating Ca2+ are largely not known. Objectives: Investigate changes in [Ca2+]i and determine the ion channels that regulate calcium signalling in hDPSCs. Methods: DPSCs were derived from immature third molars and cells less than passage 6 were used in all the experiments. Changes in [Ca2+]i were studied with Fura2 calcium imaging. RNA was extracted from DPSCs and a panel of TRP channel gene expression was determined by qPCR employing custom designed FAM TRP specific primers and probes (Roche, UK) and the Light Cycler 480 Probes Master (Roche). Results: hDPSCs express gene transcripts for all TRP families including TRPV1, V2, V4, TRPA1, TRPC3, TRPC5, TRPC6, TRPM3, TRPM7 and TRPP2. Stimulation of cells with appropriate TRP channel agonist induced increase in [Ca2+]i and similar responses were obtained when cell were mechanically stimulated by membrane stretch with application of hypotonic solution. Conclusion: TRP channels mediate calcium signalling in hDPSCs that merit further investigation.

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by connexin43.

Intercellular Ca(2+) wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca(2+) wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca(2+) waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca(2+) wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca(2+) waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca(2+) waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.