When the editor of the Indian Journal of Dermatology (IJD) approached me with a request to be the editor of a symposium on ‘uriticaria’, to be published in this esteemed journal, I felt delighted and grateful to him for rightly selecting an ever-growing, contemporary, and challenging subject. Entrusted with this daunting task, I decided to directly approach the most legendary persons on this subject from different parts of the globe, namely, Drs. H Maibach, C L Goh, and A K Bajaj from the United States, Singapore, and India, respectively. All of them responded eagerly to my request and kept their promise by sending their articles in time, in spite of their extremely hectic schedules. On behalf of the IJD, I must express our sincere thanks and gratitude to all of them.The symposium comprises four articles on as many important aspects of this important condition: contact urticaria, chronic autoimmune urticaria, management of difficult difficult urticaria, and the updates on the disease.Keeping in mind his monumental contribution to the world of dermatology, we are dedicating this symposium on ‘Urticaria’ in IJD to Prof. H Maibach as a tribute to him on behalf of all the dermatologists in India.In conclusion, I must congratulate the editor, the executive editors, the associate editors and the whole team of IJD for publishing such a symposium on urticaria.


However, p2xA was found to have an exclusively intracellular localisation making it unclear whether Dictyostelium utilise P2 receptors in a manner analogous to vertebrates. Using an apoaequorin expressing strain we show here that Dictyostelium do possess cell surface P2 receptors that facilitate Ca2+ influx in response to extracellular ATP and ADP . Indicative of P2X receptor activation, responses were rapid reaching peak within 2.91 ± 0.04 s, required extracellular Ca2+, were inhibited by Gd3+, modified by extracellular pH and were not affected by deletion of either the single Gβ or iplA genes. Responses also remained unaffected by disruption of p2xA or p2xE showing that these genes are not involved. Cu2+ and Zn2+ inhibited purine-evoked Ca2+ influx with IC50 values of 0.9 and 6.3 μM, respectively. 300 μM Zn2+ completely abolished the initial large rapid rise in intracellular Ca2+ revealing the presence of an additional smaller, slower P2Y-like response. The existence of P2 receptors in Dictyostelium makes this organism a valuable model to explore fundamental aspects of purinergic signalling.The presence of five P2X-like genes ( Dictyostelium genome Dictyostelium genes predicted to code for proteins with weak homology to vertebrate P2X receptors .Sequencing of the 1−7), which assemble as functional homo- or hetero-trimeric channels. Vertebrate P2X receptors play a fundamental role in a wide array of physiological processes including neurotransmission, smooth muscle contraction, immune cell function, bone formation and platelet aggregation Vertebrate P2X receptors are cell surface cation selective ion channels gated by extracellular ATP and consist of seven distinct subtypes  q/G11 to phospholipase C-β , via Gi/Go to inhibit adenyl cyclase  and to Gs to stimulate adenyl cyclase (P2Y11). Whilst the literature has tended not to classify the Dictyostelium cAMP receptors as P2 receptors, it could be argued that these correspond to Dictyostelium P2Y receptors since they are 7TM receptors activated by an extracellular purine ligand (cAMP). It is therefore possible that some of the orphan Dictyostelium 7TM receptors are activated by extracellular nucleotides other than cAMP.In addition to ionotropic P2X receptors, vertebrate purinergic signalling is also mediated by metabotropic P2Y receptors. Unlike the P2X channels, P2Y receptors are 7TM G-protein coupled receptors and have a wider agonist profile responding to purines, pyrimidines and UDP–glucose Dictyostelium45Ca2+per se making interpretation of the results with respect to P2 receptor function unclear.Early studies performed prior to the discovery of P2 receptors demonstrated that extracellular ATP could enhance chemoattractant-mediated cell aggregation in Dictyostelium p2xA gene codes for an intracellular protein Dictyostelium possess cell surface P2 receptors for extracellular nucleotides. Vertebrate P2X receptors are permeable to Ca2+Schistosoma mansoni P2X Dictyostelium p2xA1,2,4,6 and 11 receptors also result in an increase in intracellular Ca2+ via activation of inositol 1,3,5-trisphosphate receptors leading to release of Ca2+ from intracellular stores. We therefore assessed P2 receptor function in Dictyostelium by utilising an apoaequorin expressing strain 2+ levels. Using this technique, we clearly demonstrate the presence of cell surface P2 receptors for extracellular ATP and ADP in Dictyostelium.In light of these early studies and the more recent finding that the O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP), 2-(methylthio)adenosine 5′-triphosphate (2-MeSATP), suramin, pyridoxal phosphate-6-azo (PPADS), apyrase and ATP bioluminescent assay kit were from Sigma. Pluronic F-127 was from Invitrogen. α-[32P]-dCTP was from Amersham.Benzyl coelenterazine (h-CTZ) was from Lux Biotechnology. Adenosine 5′-triphosphate (ATP) magnesium salt, adenosine 5′-diphosphate (ADP), adenosine monophosphate (AMP), uridine 5′-triphosphate (UTP), adenosine, α,β-methyleneadenosine 5′-triphoshate (αβ-MeATP), 2′(3′)-50 buffer , resuspended at 5 × 107 cells/ml in H50 buffer and 105 μl electroporated  with 15 μg of pPROF120 plasmid Cells were harvested from axenic medium, washed twice in ice-cold Hp2xA− and p2xE− strains were produced in Ax2 by homologous recombination using disruption vectors based on pLPBLP p2xA 5′-flanking region (1846 bp) forward primer: 5′-TAAAGGGCC CAACTTCATTGTCTCATTCTAGTCATAGTAATCCAATTG-3′; reverse primer: 5′-TGAAGTCGACCTAAACGTCTGTCTCTAATTCTTACAATCTTTACTGTAG-3′. p2xA 3′-flanking region (1678 bp) forward primer: 5′-ATGAACTAGTGAATAACAACTG ATGAAGGTGAAGATGTTTTATATAC-3′; reverse primer: 5′-GGGGCGGCCGCTACAAAATAAAATCAAAAATGACAATAACAACTATTTAAC-3′. p2xE 5′-flanking region (990 bp) forward primer: 5′-ACCTGGGCCCAAAGACTCATTATTTTCC-3′; reverse primer: 5′-ACCTAAGCTTTCTATTTTTGCAATTAATAATTAC-3′. p2xE 3′-flanking region (1526 bp) forward primer: 5′-AGTATGCGGCCGCTATGCCCCAAAGAAGTAG-3′ reverse primer: 5′-TCACCCCGCGGTCACCATTAACGAAACC-3′). Flanking homology regions were cloned on either side of the blasticidin S resistance cassette via the exogenous restriction enodnuclease sites introduced by the primers during amplification . Disruption cassettes were liberated as ApaI/NotI and ApaI/SacII fragments, for p2xA− and p2xE−, respectively, and 15 μg used to transform Ax2 amoebae. Two independent null strains were isolated from 96-well tissue culture plates in axenic medium 12, 0.02 mg/l biotin and 0.2 mg/l riboflavin) plus 10 μg/ml blasticidin S as the selective agent. p2xA and p2xE gene disruptants among the many blasticidin resistant clones were identified by PCR, using oligonucleotides located outside the disruption cassette and within the blasticidin resistance cassette, with Southern blot analysis performed to verify a single site of integration.The 32P]-dCTP labelled XhoI–EcoRV restricted BSR cassette. The membrane was pre-hybridised and hybridised in Church buffer  at 65 °C then washed (5 × 20 min) with 0.2 × SSC , 0.1% SDS at 65 °C. Hybridised bands were visualised by autoradiography.NcoI or XbaI digested genomic DNA was probed with α-[Dictyostelium Ax2 and DH1 and transformants derived from these strains were grown axenically in HL-5 media in shaken suspension (180 rpm) at 22 °C. Strains expressing apoaequorin and iplA/p2xA/p2xE null mutants were grown in media supplemented with 30 μg/ml G418 and 10 μg/ml blasticidin S, respectively. Minimal growth medium for DH1 was supplemented with 20 μg/ml uracil.In vivo reconstitution of aequorin was achieved by incubating apoaequorin expressing strains in the presence of 5 μM h-CTZ, added from a 1000× suspension in methanol containing 20% (w/v) Pluronic 127. Cells (1 × 107 per ml) were incubated for 6 h in HL-5 or for 7 h in MES–DB buffer  to permit measurement of responses in vegetative and aggregation competent cells, respectively. Cells were then washed twice and resuspended in MES–DB buffer at 2.2–3.3 × 106 cells/ml prior to measurements. Desensitisation of P2 receptors during the preparatory steps necessitated allocating a recovery period of 1 h prior to measurement of ATP/ADP responses. Variations in the amount of desensitisation and rate of recovery prevented direct comparison of magnitude between experiments and strains by percentage aequorin consumption. Data were therefore normalised to a common response between experiments. Luminescence signals were recorded using a Lumat LB 9507 luminometer (Berthold Technologies), with 3 ml of cell suspension. Agonists (300 μl) were delivered using the auto-injector of the luminometer so as to standardise the speed and force of injection between experiments. Dependence on extracellular calcium was determined by suspending cells in nominally calcium-free MES–DB, or in MES–DB containing 50 μM CaCl2 with subsequent injection of ATP or buffer only control solutions containing increasing concentrations of CaCl2 (50 μM to 21.5 mM). PPADS, suramin and di/trivalent ions were added 10 min prior to measurement of ATP-induced responses. To overcome problems with solubility the inhibitory effect of copper ions was investigated using MES–DB buffer at pH 5.2.6 cells/ml) or apyrase (0.01–1 U/ml) in MES–DB buffer were incubated at 22 °C for 20–60 min, the reaction was terminated with 2 V of 2% H2SO4, 0.5% SDS, 0.5% ammonium molybdate and the phosphomolydbate complex was reduced in 0.1% ascorbic acid.Phosphate released from the hydrolysis of 500 μM ATP or ADP was determined by optical absorbance of the phosphomolybdate complex at 700 nm . Ax2 wild type cells ) according to the manufacturer's instructions.50 values using GraphPad Prism software . Responses are expressed as means with standard errors and the indicated sample sizes.Peak areas and response kinetics were determined using WinWCP software . The area of the mean mechanosensitive “buffer” response for each experiment was subtracted from the area of agonist evoked responses prior to determination of EC2+ upon exposure of both vegetative  and a mean decay of 6.13 ± 0.12 s (peak to 90% decay). Cells also remained refractory to subsequent applications of ATP   D. After cubation D showingcubation F.2+ in Dictyostelium and determined the suitability of the aequorin system for analysing nucleotide-evoked responses, we next tested a range of potential nucleotide agonists (O-(4-benzoylbenzoyl)-ATP (BzATP) (12.7 ± 3.5% ATP response). No significant response was observed with AMP, adenosine, UTP, UDP or 2-MeSATP . The response to both ATP and ADP was concentration dependent with pEC50 values of 5.13 ± 0.05 (7.5 μM) and 5.21 ± 0.05 (6.1 μM), respectively (Having established that extracellular ATP evoked a rise in intracellular Caagonists A. ADP prectively B and C. ectively D.Dictyostelium intracellular Ca2+ response to extracellular ATP and ADP is mediated by an ionotropic (P2X) or metabotropic (P2Y) mechanism. As an initial indicator we directly compared the time course of the response to ATP to that of cAMP, an agonist known to act via a family of four seven transmembrane receptors 2+ differs between these two agonists. The response to ATP required the presence of extracellular Ca2+ since responses were severely attenuated when experiments were carried out in nominally free Ca2+ buffer  ). However there was no significant difference between ATP concentration response curves for wild type DH1 and the mutant Gβ− strain (Gβ− pEC50 = 4.84 ± 0.03 (14.3 μM)) (Gβ gene (Gβ.Disruption of the  iplA−AQ B. The re(7.9 μM) A. The tiild type E. Having4.3 μM)) C. Simila-mediated mechanism of Ca2+ influx rather than a metabotropic (P2Y)-mediated mechanism. Dictyostelium possesses a family of five genes predicted to encode proteins with homology to vertebrate P2X ion channels and one member of this family (p2xA) has recently been shown to be localised to intracellular membranes on the contractile vacuole system where it plays a role in osmoregulation p2xA gene in mediating the Dictyostelium response to extracellular nucleotides along with that of another member of the Dictyostelium P2X-like gene family, p2xE. Mutant strains were generated using homologous recombination to disrupt either the p2xA or p2xE genes  for p2xA− cells and 4.77 ± 0.06 (16.9 μM) for p2xE− cells . Responses in the presence of 10 μM PPADS were reduced by ∼25%   by ∼25% A. HoweveDictyostelium ATP response by 66.7 ± 6.0% and decreased the pEC50 from 5.13 ± 0.05 (7.5 μM) at pH 6.2 to 5.56 ± 0.08 (2.7 μM) at pH 7.2. Conversely, decreasing pH to 5.2 reduced the maximum response by 44.1 ± 2.2% and increased the pEC50 to 4.75 ± 0.05 (17.6 μM)  B and C. 17.6 μM) D.Dictyostelium p2xA when expressed in HEK293 cells Dictyostelium response to extracellular ATP. Copper inhibited the response with a pIC50 value of 6.05 ± 0.02 (0.9 μM)  E and F.2+ also blocked the Dictyostelium response to ATP with a pIC50 value of 5.20 ± 0.06 (6.3 μM) (50 concentration of Zn2+ displayed a pEC50 similar to that in the absence of Zn2+ (4.95 ± 0.05 (11.2 μM) but with a reduced maximal response  A. The coresponse C. 300 μMlar Ca2+ B. Removalication D.Dictyostelium raised the possibility that nucleotides other than cAMP may act as extracellular signalling molecules in this model eukaryotic organism. In this study we have utilised an apoaequorin expressing strain to determine whether extracellular nucleotides can elicit purinergic signalling. We demonstrate that Dictyostelium do indeed possess cell surface purinergic receptors that facilitate a rapid increase in intracellular Ca2+ in response to extracellular ATP and ADP. This rapid purine evoked increase in intracellular Ca2+ likely represents a different phenomenon to the ATP-induced increase in basal 45Ca2+ uptake described in 1980 45Ca2+ were observed 5 and 10 min after ATP application with the rate of 45Ca2+ uptake declining to below baseline at 15 and 20 min post-stimulation. In our study, we describe a far more rapid calcium influx that begins immediately after ATP addition, peaks within 3 s and returns to baseline within 20 s after agonist application. This rapid purinergic response required extracellular Ca2+  compared to P2XA (EC50 > 200 μM) 50 ∼ 6.1 μM), whereas it is not an agonist of P2XA p2xA gene by homologous recombination had no effect on the endogenous purinergic response. Similarly we also mutated the Dictyostelium p2xE gene and again the purinergic response remained. Three other P2X-like genes are present in the Dictyostelium genome  and it is therefore possible that one or a combination of these are involved in mediating the response to extracellular purines.The endogenous 2+ a slower component became apparent  raising the intriguing possibility that direct, G-protein independent, activation of a Ca2+ channel by a P2Y-like receptor could be involved.Whilst the dominant response to extracellular ATP/ADP showed characteristics indicative of P2X receptor activation, there was also a very small component of the response with a much slower onset. Normally this small slower component was masked by decay of the large P2X-like response. However when the P2X-like component was blocked with 300 μM Znapparent D. Apart Dictyostelium is not known. Using the under-agar chemotactic assay we were unable to detect any chemotactic response to ATP or ADP (data not shown). However, whilst not directly chemotactic in itself ATP has been shown to reduce the time to onset of cell aggregation, in response to starvation, in several species of Dictyostelium2+ calcium was observed in both aggregation competent and axenically growing cells, which are unresponsive to cAMP. Also, in contrast to the ADP response observed in this study, the enhancement of aggregation by ATP is not mimicked by ADP The physiological function of purinergic signalling to extracellular ATP and ADP in Dictyostelium also possess cell surface purinergic receptors for extracellular nucleotides, which upon activation facilitate a rapid increase in intracellular Ca2+ concentration. Dictyostelium therefore represent a valuable model system to explore fundamental aspects of purinergic signalling in a eukaryotic organism with a relatively simple and well-annotated genome that is readily amenable to genetic manipulation and has the potential for powerful functional screens.In this study we demonstrate that, in addition to the previously described intracellular P2XA receptor, None.


Remodeling of the extracellular matrix (ECM) has been implicated in ovarian cancer, and we hypothesize that these alterations may provide a better optical marker of early disease than currently available imaging/screening methods and that understanding their physical manifestations will provide insight into invasion.For this investigation we use Second Harmonic Generation (SHG) imaging microcopy to study changes in the structure of the ovarian ECM in human normal and malignant ex vivo biopsies. This method directly visualizes the type I collagen in the ECM and provides quantitative metrics of the fibrillar assembly. To quantify these changes in collagen morphology we utilized an integrated approach combining 3D SHG imaging measurements and bulk optical parameter measurements in conjunction with Monte Carlo simulations of the experimental data to extract tissue structural properties.We find the SHG emission attributes  and bulk optical parameters, both of which are related to the tissue structure, are significantly different in the tumors in a manner that is consistent with the change in collagen assembly. The normal and malignant tissues have highly different collagen fiber assemblies, where collectively, our findings show that the malignant ovaries are characterized by lower cell density, denser collagen, as well as higher regularity at both the fibril and fiber levels. This further suggests that the assembly in cancer may be comprised of newly synthesized collagen as opposed to modification of existing collagen.Due to the large structural changes in tissue assembly and the SHG sensitivity to these collagen alterations, quantitative discrimination is achieved using small patient data sets. Ultimately these measurements may be developed as intrinsic biomarkers for use in clinical applications. In 2008, there were an estimated 21,650 new cases of ovarian cancer in the United States and 15,520 deaths . Little is currently known about markers of premalignancy, early malignancy or early pathways for malignancy that could be potentially manipulated for prevention or early detection of ovarian cancer. Additionally, it is recognized that malignant cells with a specific and identifying molecular fingerprint are not always histologically identifiable in seemingly normal epithelium adjacent to tumor, such thThus there remains a compelling need for new technologies that have both sufficient resolution and specificity to detect microscopic tumors or precursor lesions. Probing alterations in the ECM composition and structure may be a promising approach in this regard, as these changes are thought to be critical for tumor initiation and progression for several epithelial carcinomas -7. For eTo investigate this possibility, we have explored the use of high resolution (~0.5 microns) Second Harmonic Generation (SHG) imaging microscopy to objec(2) is the second order nonlinear susceptibility tensor of the collagen, and whose magnitude determines the contrast level. Due to the second order symmetry constraints imposed by Eq 1, the SHG contrast vanishes for assemblies with mirror symmetry (i.e. centro-symmetric environment) and increases for well-ordered structures[(2) which is experimentally manifested in the SHG intensity. This tensor further contains information on the alignment of the collagen molecules in the fibrils/fibers[where P is the induced polarization, E is the electric field vector of the laser, and χtructures. Thus thls/fibers. Additiols/fibers-27. In tSHG has already been shown to have potential applicability for cancer diagnosis by revealing changes in the ECM in tumors relative to normal tissues. For example, the Dong and Pavo(2)) as the SHG creation attributes. These, together with the bulk optical parameters, are used in a comparative manner to quantify differences in tissue structure in normal and diseased ovary.Here we take a more general approach of quantifying differences in the ECM of ex vivo normal and malignant human ovaries by using SHG to measure or extract changes in the respective fibril/fiber assemblies. The quantitative approaches to be shown in this paper avoid the pitfalls of empirical morphological assessments, and additionally avoid the problems with pure signal intensity based-measurements which can be confounded by scattering. Indeed, the magnitude and directionality of the scattering contains information on the tissue structure, which is encoded in the 3D SHG measurements. TogetheThe ovarian biopsies were removed using the University of Connecticut Health Center IRB approved protocol with patient consent prior to the procedure. The biopsies were removed and immediately fixed in 4% formalin and refrigerated for 24 hours. These specimens were then switched to PBS and kept at 4 degrees. For SHG imaging and bulk optical parameter measurements, un-stained sections of 100 and 50 microns, respectively, were cut with a vibratome. Independent classification of the ovary was based on histologic diagnosis of cancer (n = 3) or no known risk factors for normal ovaries (n = 5). All of the cancers were high grade and late stage (III or IV), although the tissue was harvested from the primary tumor in the ovary.s, and scattering anisotropy, g, are part of the metric to characterize the changes in tissue structure in normal and malignant ovaries. The inverse of μs is the mean free path (MFP), and is the distance a photon will propagate before undergoing a scattering collision and changing direction. This anisotropy, g, is related to the directionality of the scattering, and varies from 0 to 1. The upper limit corresponds to highly forward-directed scattering and is reflective of very highly ordered tissues, and 0 represents the isotropic scattering associated with randomly organized structures. These bulk optical parameters (plus the absorption coefficient μa) were determined at the 890 nm laser excitation wavelength using the titanium sapphire laser (see below) and the approximate SHG (457 nm Argon ion line) wavelengths using a dual integrating sphere approach and angular resolved scattering measurements as previously reported[a and reduced scattering coefficient μs' where:The scattering coefficient μ reported. The ref reported. These p reported,35,36 toThe SHG imaging system has been described in detail elsewhere and is only briefly described here. The insSHG/BSHG) and subsequent SHG propagation through the tissue which is based on μs and g at 445 nm. The FSHG/BSHG is highly dependent upon the fibril diameter, the packing density and regularity relative to the size-scale of the SHG wavelength[s) and organization (primarily g) of the fibrillar assembly. The measured SHG directional (F/B ratios) values were determined by integration of the intensity of 5-10 frames per optical section every few microns of depth using ImageJ software http://rsb.info.nih.gov/ij/.The measured depth dependence of the forward-backward intensity ratio (F/B) of the SHG signal is one means to characterize structural changes in the ECM between normal and malignant ovarian biopsies. This axial response arises from a convolution between the initial SHG directional emission ratio  and primary filter effect (loss of laser intensity due to scattering) and secondary filter effect . Since biological tissues have intrinsic heterogeneity in concentration, we have found a normalized approach necessary to account for local variability in SHG intensities in the same tissue (different fields) and to make relative comparisons between tissues[The measured attenuation, i.e. rate of intensity decrease with increasing depth into tissue, of the forward SHG signal is also used to characterize structural changes in the ECM. The attenuation results from a compounded mechanism of SHG creation attributes (the Fn tissues. To this(2) values for the different tissues, determines the initial SHG intensity. An initial emission directionality, FSHG/BSHG, is also assumed. The propagation of the SHG signal (intensity and direction) is then calculated using the measured bulk optical parameters at the SHG wavelength. Collectively, by analyzing the trajectories of 50,000 photons at each focal depth, these steps simulate the detected directionality (F/B) and attenuation as a function of depth into the tissue.The 3D SHG directional and attenuation measurements are a convolution of the SHG creation attributes and the bulk optical parameters. These contributing factors are not directly determinable but can be decoupled by using Monte Carlo simulations. To this end, we have adapted the MCML framework of photoex vivo biopsies. The left panels in Figure We first present qualitative evidence that SHG imaging has the sensitivity and specificity to reveal distinct collagen fibrillar assemblies in un-stained normal and malignant en face optical sections from thicker tissue slices. Still, different fibrillar assemblies are observed in cross section as well. The TPEF channel primarily arises from eosin which stains essentially all proteins, which in the stroma is predominantly type I collagen. Thus for both the normal and cancer cases the TPEF from eosin and SHG reveal the same respective overall image features. However, the eosin contrast is less well-defined, which is likely a consequence of the sensitivity of SHG to fibril/fiber assembly, where, in contrast, there are no symmetry constraints on the fluorescence. This overlap gives us confidence that we can look at similar features by SHG as by histology, and, as we will show below, with enhanced information content. In addition, the ability to image intact thicker tissues in situ (100-200 microns) by SHG allows for the acquisition over more fields of view than is practical by conventional histology.To place these images in context, we compared them to standard H&E histopathology cross sections (5 microns thick) from the same respective biopsy. The middle and right panels of Figure Next, we compare 3D renderings of SHG images with histological cross sections from the same biopsies  to examine the respective cell content and corresponding spatial distribution in the ECM Figure . This iss, μs', μa, and g) for the normal and malignant tissues at the laser (890 nm) and SHG (457 nm) wavelengths are given in Table a) is negligible  compared to scattering (μs) and we therefore we have focused our attention on the latter. The malignant tissues are more highly scattering at the SHG wavelength compared to the normal (267 vs 172 cm-1) where this difference was significant (p = 0.008). We interpret the higher scattering of the cancer to be indicative that the matrix is more densely packed than the normal tissue, resulting in a smaller MFP. This conclusion is also borne out by inspection of the SHG renderings and H&E staining , thus the actual values might be somewhat higher. However, as we have not extracted different values for ovarian tissues of 50 or 100 μm in thickness, we do not believe this to be a significant effect. This was also shown to be valid in prior work on dermis[The scattering anisotropy values for the normal and malignant tissues at 890 nm and 457 nm and corresponding t-tests are shown in Tables on dermis. Moreoves and g values for the normal and malignant tissues were different at the laser and SHG wavelengths, we first note that the redcued scattering coefficient μs' was different at both wavelengths . Additionally, the spectral dependence or spectral slope[s' was different where the malignant and normal tissues were characterized by a 1.5 and 3 fold respective increase in μs' between the laser wavelength and SHG wavelengths. This flatter spectral slope for the more ordered malignant tissue  is predicted by the recent theoretical treatment by Backman [s' provides one piece of quantitative evidence of the change in tissue structure that occurs during ovarian cancer. These bulk optical parameters are also incorporated in Monte Carlo simulations of the depth dependent SHG directionality and attenuation shown below.While not all the respective μral slope of μs' w Backman . We have Backman . Thus thSHG/BSHG) and subsequent signal propagation through the tissue based on μs and g at the SHG wavelength. The averaged experimentally measured F/B vs depth plots for normal (n = 5) and malignant ovaries (n = 3) are shown in Figure -1), such that photons that are initially forward directed have a higher probability of continuing to propagate in this direction. To validate the distinction between these tissues t-tests were performed at 10 micron depth intervals, and the differences were statistically significant .The measured forward-backward intensity ratio (F/B) arises from a convolution between the SHG directional emission creation ratio  from the SHG propagation (based on μs and g). To estimate this creation attribute we ran simulations varying the forward emitted fraction (FSHG) from 50-100%. Representative simulations for the normal and malignant biopsies are shown in Figure 2 function yielded %FSHG of 93% and 77%, for the normal and cancer, respectively, where the uncertainly in each case is approximately ± 3%. The corresponding Monte Carlo simulation generated from the best fit for each tissue type  are overlapped with the experimental SHG data in Figure SHG values.We utilized Monte Carlo simulations of these plots using the measured bulk optical parameters at inputs to decouple the initial emission directionality (i.e. FSHG), as was extracted from the simulation of the data.The SHG emission directionalities between the tissues are significantly different and can be interpreted by the difference in fibril assembly. Using a space filling analysis of TEM images we found that the packing of the fibrils in the malignant tissue to be more regular relative to the normal (10% vs 15% inter-fibrillar space respectively). Based on our mathematical model of SHG in fibrillar tissues, the fibrSHG/BSHG emission directionality, the relative SHG intensities governed by the relative magnitudes of the χ(2) nonlinear susceptibility tensors, and primary and secondary filter effects from the respective bulk optical properties. As we have previously reported,[(2) values in intact tissues as the measured signal is convolved with scattering when the tissues are thicker than one MFP or ~50 microns. However this can be achieved using much thinner histological sections (~5 microns). We note that the eosin staining does not contribute to the observed SHG. Measurement of the relative SHG intensities from these sections yields a factor of 3.9 ± 0.1 (p < 0.005) increased brightness for the cancer over that of normal tissue.The averaged normalized forward attenuation data with standard errors are shown in Figure reported, it is no(2) values are shown in Figure (2)) and larger μs for the cancers, as these separately would result in slower and faster normalized attenuations, respectively. We point out if the relative SHG conversion efficiencies were not independently known, this approach would allow this determination once the respective bulk optical parameters were measured at the laser excitation and SHG wavelengths. This would be accomplished by running simulations varying the relative conversion efficiency and then comparing the results to the experimental data to achieve the best fit .We now use all our measured factors as inputs into Monte Carlo simulations of the SHG attenuation. The simulated data for the normal (open squares) and malignant tumors (open circles) based on the bulk optical parameters (see Table (2) values) and initial emission directionality (FSHG/BSHG). For example, these creation attributes depend on packing density and order of the inter-fibril structure, both of which change in cancer  values, which are related to collagen concentration and assembly, which was also different for both tissues. The directly measured or extracted SHG attributes or bulk optical properties that are different for the normal and malignant ovaries along with the physical interpretation are summarized in Table Our goal in this work was to develop SHG imaging and analysis metrics that can be used to understand changes in ECM structure during carcinogenesis and eventually be utilized in clinical applications. The enabling aspects of SHG for tissue characterization lie not solely in the ability to visualize the fibrillar morphology, but also in the contrast being reflective of the supramolecular and fibrillar structure of collagen ,18. Thess'  and it is possible there could be discrimination in earlier stage disease, where both the SHG and bulk optical parameters could likely be different due to different levels of ECM remodeling. Specifically, we predict that earlier stage tumors would be characterized by decreased scattering and SHG brightness relative to the latter stage tumors imaged here. Moreover, even for these tissues, the attenuation response at other wavelengths may provide discrimination due to the differences in the spectral slope of μCollectively, the SHG creation attributes, bulk optical parameters, and cell density (by H&E histology) suggest that a profound alteration of the ECM occurs in ovarian cancer. An unanswered and important question is if this structure represents remodeled existing collagen or new synthesis by the stromal cells, or both. There is significant translational importance to this question as it addresses the temporal evolution of invasion. To begin to address this question, we first note that the SHG intensity was nearly 4 fold greater in the malignant ovary, suggesting the collagen was both higher in abundance and more organized. We have investigated the fibril size and packing using TEM and found similar size distributions of fibril diameter in the normal and malignant biopsies, where the most probable diameter was ~80 nm. However, the fibrils in the tumor are more closely packed, which would result in a greater SHG intensity.We can next assess the regularity of the collagen fiber alignment by measuring the SHG anisotropy. This anisotropy, β, is calculated by:par and Iperp correspond to the SHG intensity detected after a polarizer oriented parallel and perpendicularly to the laser polarization, respectively. Here the linear polarization of the laser is fixed at 45 degrees relative to the fiber axis (on a fiber by fiber basis) and then the SHG parallel and perpendicular components are measured relative to this excitation polarization. We chose this angle because this orientation yields the strongest SHG intensity for collagen[where I collagen. Values s' between the laser excitation and SHG wavelengths. Using the formalism of Backman,[While this issue warrants much further investigation, the existing data based on similar fibril diameter (based on TEM) and increased fibril/fiber organization supports the argument that the collagen beneath the surface epithelium is more likely to be newly synthesized as opposed to existing collagen fibrils being remodeled by proteases. This is also indicated by the spectral dependence of μ Backman, we find  Backman,. The norWe have achieved statistical discrimination in the measured F/B directional data based on a small data set of normal and malignant ovaries because the optical signatures are profoundly different in these tissues but homogeneous within each type. Moreover, the SHG creation attributes and bulk optical properties are consistent with the differences in fibrillar assembly between the tissues based on the morphology observed by H&E, and EM. By contrast, inherent variability and non-specificity in modalities such as fluorescence precludes this definitive distinction. For example, a previous paper on ovarian cancer found significant heterogeneity between diagnostic groups arising from innate tissue variations. The autThrough this exploratory study derived from a basic science perspective we have identified a collection of physical/structural properties of the ECM that change when the ovary becomes malignant. This characterization was enabled by the high sensitivity/specificity of SHG to collagen assembly. Our longer term goals are to use these structural changes in the ECM as a specific biomarker in vivo. While at the 890 nm excitation wavelength there was no discrimination between normal and diseased ovary in the forward attenuation data, this may be different at other wavelengths due to the spectral slope differences (i.e. from the different refractive index correlation functions) as well as different stages of disease. Moreover, performing these measurements in the backward collection geometry would yield all the same data as shown here in the forward case. With our current technology, the directional measurements cannot be performed in vivo, however given the rapid advances in fiber-optics and miniaturized scanning devices we envision that a multiple probe scheme could be constructed where the same information could be extracted. We note that the directional measurements could be applied to ex vivo biopsies of more accessible epithelial tissues such as skin and breast, where ECM remodeling also accompanies carcinogenesis.We have performed an exploratory study to understand and quantify changes in tissue structure that occur during ovarian carcinogenesis. In this effort we have identified optical signatures  that are different between normal and malignant ovarian tissues and have established their relative sensitivities. We found the depth-dependent directional and attenuation responses are consistent with the differences in tissue structure at both the fibril and fiber levels of assembly. The increased order of the fibril/fiber assembly suggests that in the malignant tissues that the collagen has been newly synthesized. We find using surprisingly small patient data sets that our methods provide statistical discrimination between normal and malignant ovarian biopsies in terms of the SHG creation attributes as well the bulk optical parameters, pointing to the specificity of the approach. We anticipate that the methods will prove valuable in terms of better investigating changes in the ECM structure which will impact our understanding of early disease. We lastly suggest that SHG imaging may become a powerful tool for future diagnostic applications as an early biomarker for the ovary and potentially other epithelial cancers.SHG/BSHG: emitted forward to backward ratio; g: scattering anisotropy; MCML: Monte Carlo multilayer; MFP: mean free path; NA: numerical aperture; SHG: Second Harmonic Generation; H&E: hematoxylin and eosin; TEM: transmission electron microscopy; TPEF: two-photon excited fluorescence; μs': reduced scattering coefficient; μs: scattering coefficient.ECM: extracellular matrix; EM: electron microscopy; F/B: measured forward to backward ratio; FThe authors declare that they have no competing interests.ON prepared the specimens, acquired, process and analyzed the spectroscopic and image data, RBL performed the Monte Carlo simulations, MAB co-conceived the project, acquired the tissue specimens, and helped draft the manuscript, PJC co-conceived the project and primarily drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/94/prepub


With advancing age (8–11 mo), some mice demonstrate advanced hypertrophy but are not in congestive heart failure (NFTG), while others evolve to frank dilated congestive heart failure (FTG). We demonstrate that older NFTG myocytes exhibit a hypercontractile state over a wide range of stimulation frequencies, but maintain a normal SR Ca2+ load compared to age matched non-transgenic (NTG) myocytes. However, at high stimulation rates (2–4 Hz) signs of diastolic contractile failure appear in NFTG cells. The evolution of frank congestive failure in FTG is accompanied by a further increase in heart mass and myocyte size, and phospholamban and ryanodine receptor protein levels and phosphorylation become reduced. In FTG, the SR Ca2+ load increases and Ca2+ release following excitation, increases further. An enhanced NCX function in FTG, as reflected by an accelerated relaxation of the caffeine-induced Ca2+ transient, is insufficient to maintain a normal diastolic Ca2+ during high rates of stimulation. Although a high SR Ca2+ release following excitation is maintained, the hypercontractile state is not maintained at high rates of stimulation, and signs of both systolic and diastolic contractile failure appear. Thus, the dilated cardiomyopathy that evolves in this mouse model exhibits signs of both systolic and diastolic failure, but not a deficient SR Ca2+ loading or release, as occurs in some other cardiomyopathic models.Mice over-expressing the α Although multiple alterations in various aspects of myocyte excitation-contraction coupling have been observed in HF, the central mechanism of the reduced Ca2+ transient amplitude has been emphasized and has been attributed to a reduction of sarcoplasmic reticulum (SR) Ca2+ content Much evidence has accumulated since the first cytosolic Ca1C-subunit of the L-type Ca2+ channel (α1CTG) indicates that like droves of other “boutique mice” (2+ regulatory mechanisms are mobilized. The remarkable orchestration among cardiomyocyte Ca2+ regulatory proteins in this model at 4months (mo) is instructive because it provides clues with respect to coordinated, adaptive remodeling of Ca2+ regulation to maintain a normal SR Ca2+ load at 4 mo of age 2+ release from the SR to produce a whole cell Ca2+ transient of increased amplitude, neither the SR Ca2+ load, as assessed by caffeine induced Ca2+ release, nor diastolic cytosolic Ca2+ levels, nor Ca2+ spark characteristics, are altered 2+ efflux to balance the enhanced Ca2+ influx via the overexpressed L-type Ca2+ channel prevents excess cytosolic calcium loading Longitudinal assessment of cardiac structure and function in a novel transgenic mouse that over expresses the αue mice” Table S11CTG mouse maladapts into a dilated, lethal cardiomyopathy 2+ regulation observed at a younger age (4 mo) in NTG cells wane with the evolution of more advanced hypertrophy and HF that accompany advancing age. Specifically, we hypothesized that these adaptations become compromised in the non-failing advanced hypertrophic stage (NFTG), and “fail” outright as the end-stage dilated congestive failure (FTG) evolves.Between 8–11 mo of age, however, the adapted, hypertrophic heart of the αHeart weight in FTG was over two-fold greater than in NTG . Body weICa density in NFTG and FTG was increased compared to NTG, despite a significant increase in cell capacitance . No statistically significant difference was observed between NFTG and FTG  elicited by electrical stimulation declined sharply between the first stimulation following rest and steady state stimulation at 0.5 Hz. There was little further decline thereafter as the stimulation rate was increased . FollowiMAX) of the Indo fluorescent transient , like that of the Indo fluorescence transient  and 73% and 54% decrease in its phosphorylation levels of PS16 and PT17, respectively normalized to total PLNp; by at 48% decrease in RyR2 levels but 115% hyperphosphorylation of RyRs at Ser2089 (Quantitative Western Blot techniques comparing NFTG and NTG (8–11 mo) showed: a 30% decrease in SERCA2a levels; 50% increase in NCX; 120% increase in PLN Ser2089 .m); a 57% decrease in the PLNp; a 36% decrease in PLN-PS16; a 60% decrease in PLNp-PT17; a 5% decrease in SERCA2a; a 20% increase in NCX; a 43% increase in PKCα; and surprisingly, a 70% decrease in RyR2 and 25% decrease in RyR2-PS2809  showed: an 80% decrease in the PLN monomer . Thus, although NCX function, as reflected in a reduced T50 of the caffeine transient relaxation, is enhanced in FTG, this is not sufficient to maintain a normal diastolic Ca2+ during high rates of stimulation.There are several novel results of the present study. In NFTG at 8–11 mo of age, heart mass and average myocyte sizes are increased by about 25% relative to age-matched NTG as is the case in 4 mo α1CTG myocytes have not been previously measured. Over a wide range of stimulation rates, the NFTG exhibit a hypercontractile state accompanied by diastolic failure due to an inability to maintain normal diastolic length. But in FTG, despite increased Ca2+ transient amplitude, the contraction amplitude across a broad range of stimulation rates FTG is not greater than in NFTG. This indicates that a relative systolic contractile failure has occurred with evolution from the NFTG to FTG state. At high rates of stimulation, the contraction amplitude of FTG declines below that of NFTG to the level in NTG without a decline in SR Ca2+ release and is accompanied by marked diastolic abnormalities. Thus NFTG and FTG differ in that the latter exhibit frank contractile systolic failure and more severe Ca2+ and contractile diastolic failure than do the former.Contractile characteristics of α2+ transient and contraction amplitudes. A common clinical correlate of NFTG may be hypertrophic non-dilated cardiomyopathy Diastolic abnormalities accompany hypercontractile function in other animal experimental models 2+]i transient following excitation and may involve myofilaments, Ca2+ energetic mechanisms, matrix properties or interactions of all.A hypercontractile state is attributable, at least in part, to an increased cytosolic [Ca2+ influx, reuptake and extrusion from the cell 2+ handling determine negative staircase in a cellular heart failure model It has recently been suggested that, like NFTG myocytes in the present study, an abnormal frequency-dependent response of human heart failure is a pathophysiologic signature of the cardiomyopathic state 2+ load afforded to older NFTG mice fails as the dilated, congested cardiomyopathic state evolves. In marked contrast to most other cardiomyopathies, the excitation-induced Ca2+ transient amplitude of FTG is increased. Also in contrast to most other experimental cardiomyopathies,   Table S32+ load increases during the evolution from NFTG to FTG, since ICa,L density does not increase, and Ca2+ efflux via NCX is augmented, it might be expected that mechanisms to reduce Ca2+ pumping into the SR might prevail: in many other HF models, SERCA2a becomes reduced, PLN increases, and its phosphorylation decreases 2+ load in FTG is apparently linked to an increased Ca2+ availability for SR pumping, as evidenced by an increase in diastolic Indo-1 fluorescence during pacing. The latter may also reflect an increase in the net cell Ca2+ load between the NFTG and FTG states.It is perplexing that the SR Ca2+ leak. Unlike some other heart failure models, RyR2 and RyR2-P decrease, in the FTG , another conundrum. Some reports suggest that PKA phosphorylation of RyR2 has little functional relevance for diastolic Ca2+ release if SR Ca2+ levels remain constant 2+ leak as causal of arrhythmias and sensitivities to heart failure 2+ leak theory, as well as the decreased SR Ca2+ content concept, as some studies have not been confirmed by others reduced total RyR2, phosphorylated RyR2 at Serine 2809 and increased SR Ca2+ loading Alterations in RyR 2+ remains elevated does not represent a “good” model of heart failure. But what, then, is a “good model” of heart failure? A “good model” of heart failure should represent what happens in the human condition. The presently in vogue dictum is that a reduced SR Ca2+ load in end stage human HF is central to contractile dysfunction of human cardiomyocytes 2+ accumulation demonstrated slow uptake kinetics and a complete lack of Ca2+ release compared to non-failing samples, in which Ca2+ release was vigorous 2+ load, muscles or myocytes under study conditions that have been employed exhibit an increase in the diastolic Ca2+ at higher pacing rates i.e. similar patterns observed in the present study in FTG.Some might argue that the FTG in which SR Ca2+ channel. Channel availability and single channel activity were markedly altered, the culprit being an up regulation of the β2-subunit of the L-VDCC complex. Further, the ensemble single channel activities of the terminally failing mouse heart and human heart were identical. Thus, quantitative measurements of single L-type channel function are vital when considering mechanisms involved in frank heart failure in all models of HF, regardless of species.A recent study on failing human and mouse frank heart failure 1CTG, heart e.g., those analyzed in the present study, have no preload or afterload, two factors that, not only regulate contraction, but also regulate myofilament Ca2+ binding and release characteristics. It is noteworthy, though, that regardless of the SR Ca2+ load, contractile failure in cells or muscles from end stage, dilated heart failure hearts is exacerbated with increasing pacing rates in virtually all studies employing different pacing rates 2+ binding or energetic fatigue, especially the activity of the β2-subunit of the L-VDCC, should continue to be recognized as mechanisms that underlie HF Other possible mechanisms in the conundrum of the end stage HF phenotype include heart rate responses Heart remodeling (dilatation), itself, leads to increased afterload, fibrosis, myofilament disarray and increased cell death 1C-TG model, overexpression of the β2a subunit of the L-VDCC leads to enhanced ICa,L resulting in Ca2+ dependent apoptosis in adult myocytes Of possible relevance to the α2+ load and reduced Ca2+ amplitude, at least under conditions that have been employed in these studies  littermates of the same age were used in all of the experiments. The animal protocols used in these studies were approved by the Animal Care Committees of each institution involved. At 9 mo of age some α1CTG mice begin to manifest a dilated cardiomyopathy with markedly impaired contractile function in vivo1CTG -mice at this age exhibited two phenotypes: the absence of gross cardiac dilatation and congestion, evidenced by the absence of pericardial and pleural effusions and atrial thrombi (n = 4), and gross cardiac dilatation with both pericardial and pleural effusions and left atrial thrombi (n = 4). We designated these two groups of mice at sacrifice as non-failing α1CTG (NFTG) and failing α1CTG (FTG), respectively, and structured our data analysis to compare some aspects of Ca2+ regulation and contraction characteristics of myocytes isolated from these hearts.FBV/n in-house bred 8–11 mo old α2+ transients in Indo-1-AM loaded myocytes at frequencies of stimulation ranging from 0.5–4 Hz. The same stimulation protocol was used to measure myocyte mechanics in both Indo-1-loaded and unloaded cells. The SR Ca2+ load was determined measuring caffeine induced Ca2+ transients in Indo-1-AM loaded cells. Inward Ca2+ current through the L-type channel was measured under step-voltage protocols. We carried out quantitative and traditional Western blotting for the Ca2+ handling proteins. FTG and NFTG subgroup comparisons were stratified prospectively and investigators were blinded when doing their experiments. All methods have been previously published and are detailed in the We measured CaResults are reported as mean ±SEM. Differences among NTG and NFTG and FTG cells were compared by ANOVA. A value of p<0.05 was considered significant.Table S1(0.13 MB DOC)Click here for additional data file.Table S2(0.04 MB DOC)Click here for additional data file.Table S3(0.05 MB DOC)Click here for additional data file.Methods S1(0.06 MB DOC)Click here for additional data file.


Work from our group has shown that cytosolic phosphorylation of calnexin (CLNX) controls Ca2+ uptake into the ER via the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) 2b.The accumulation of misfolded proteins within the endoplasmic reticulum (ER) triggers a cellular process known as the Unfolded Protein Response (UPR). One of the earliest responses is the attenuation of protein translation. Little is known about the role that Ca2+ dependent phosphatase, associates with the (PKR)-like ER kinase (PERK), and promotes PERK auto-phosphorylation. This association, in turn, increases the phosphorylation level of eukaryotic initiation factor-2 α (eIF2-α) and attenuates protein translation. Data supporting these conclusions were obtained from co-immunoprecipitations, pull-down assays, in-vitro kinase assays, siRNA treatments and [35S]-methionine incorporation measurements. The interaction of CN with PERK was facilitated at elevated cytosolic Ca2+ concentrations and involved the cytosolic domain of PERK. CN levels were rapidly increased by ER stressors, which could be blocked by siRNA treatments for CN-Aα in cultured astrocytes. Downregulation of CN blocked subsequent ER-stress-induced increases in phosphorylated elF2-α. CN knockdown in Xenopus oocytes predisposed them to induction of apoptosis. Here, we demonstrate that calcineurin (CN), a Ca2+ increased. These data were obtained from [γ32P]-CLNX immunoprecipitations and Ca2+ imaging measurements. CLNX was dephosphorylated when Xenopus oocytes were treated with ER stressors. Dephosphorylation was pharmacologically blocked by treatment with CN inhibitors. We also found that CLNX was dephosphorylated by CN when Ca2+. We further show that phosphorylated CN-A exhibits decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is re-established.Finally, evidence is presented that PERK phosphorylates CN-A at low resting levels of Ca2+ homeostasis.Our data suggest two new complementary roles for CN in the regulation of the early UPR. First, CN binding to PERK enhances inhibition of protein translation to allow the cell time to recover. The induction of the early UPR, as indicated by increased P-elF2α, is critically dependent on a translational increase in CN-Aα. Second, CN dephosphorylates CLNX and likely removes inhibition of SERCA2b activity, which would aid the rapid restoration of ER Ca These processes of signaling and biosynthesis are deeply inter-connected The ER is a dynamic organelle that plays a critical role in a variety of processes, including CaUnfolded Protein Response (UPR) that attempts to restore homeostasis in the ER When the load of newly synthesized proteins exceeds the folding and/or processing capacity of the organelle, the ER enters into a stress condition. This activates a signal transduction pathway called the 2+ levels in the ER is primarily attained by the activity of SERCAs 2+ into the ER. These Ca2+-ATPases counteract the loss of Ca2+ via leaks and the opening of Ca2+ release channels 2+ in the ER is a balance between Ca2+ release, uptake and buffering by Ca2+-binding proteins in the lumen. Calreticulin (CRT) and CLNX are Ca2+ -binding chaperones that reside in the ER 2+ store refilling Maintenance of Ca2+ and calmodulin dependent serine/threonine phosphatase. This heterodimer phosphatase is composed of a catalytic subunit, calcineurin A (CN-A) and a regulatory subunit, calcineurin B (CN-B) 2+ levels, the phosphatase is relatively inactive. An increase in intracellular Ca2+ activates CN-A through Ca2+/CaM binding, which dissociates AI from the catalytic domain 2+ signaling in a multitude of cellular pathways has been well documented 2+ signaling in restoring ER homeostasis, once ER stress has been triggered. Here we reveal that CN plays key roles in restoring ER homeostasis during stress. CN activity boosts the refilling of Ca2+ stores so that optimal conditions for protein processing/folding are rapidly reached. CN also directly interacts with PERK to increase its auto-phosphorylation, which helps to attenuate protein translation while homeostasis is being restored. Finally, we show that a knockdown of CN levels in Xenopus oocytes results in a decrease of protein synthesis inhibition and a rapid acceleration of apoptosis. Taken together, these data underscore the importance of CN activity in the rescue of cells from ER stress.Calcineurin is a Ca2+-ATPases 2+ stores with a concomitant increase in cytosolic Ca2+, causing accumulation of malfolded proteins within the ER 2+ store refilling while dephosphorylation increased SERCA 2b activity Xenopus oocytes as previously described . After 3 days of protein expression, oocytes were labelled with [γ32P]ATP for 20 minutes, the microsomal fraction was extracted and an anti-CLNX antibody was used to immunoprecipitate the protein. We observed a significant level of phosphorylation of CLNX under normal resting conditions  and FK506. Preincubation of oocytes with these inhibitors completely reversed the dephosphorylation of CLNX in response to Tg (Thapsigargin (Tg) is an irreversible inhibitor of the ER Canditions . To detenditions . These rse to Tg  and treaXenopus oocytes overexpressing CLNXs and labeled with [γ32P]ATP as described above were used. As with Tg, Tm treatment of overexpressing oocytes significantly (p<0.05) induced dephosphorylation of CLNX. Similarly, CN inhibitors cyclosporin A and FK506 completely reversed this stress induced dephosphorylation (Tunicamycin (Tm) has a different mechanism of action than Tg to induce ER stress. It inhibits glycosylation of nascent proteins thereby causing accumulation of malfolded proteins in this organelle rylation .2+ was being released into the cytosol during Tm-induced ER stress. To test this hypothesis, we measured cytosolic Ca2+ in single oocytes using fluorescence microscopy. Xenopus oocytes were injected with the ratiometric Ca2+ indicator dye Fura 2 . After a 20–30 minutes, oocytes were imaged. Ca2+ levels were expressed as the ratio of fluorescence for 340 and 380 excitation (R340/380). When Fura-2 loaded oocytes were exposed to Tm (2.5 µg/ml), we observed a slow rise in cytosolic Ca2+  increased to 1.09±0.03 corresponding to 174±9 nM Ca2+. In parallel experiments, we also found that treatment with Tg (1 µM) for 15 minutes increased the Fura-2 ratio to 1.01±0.04 (n = 11 oocytes), corresponding to 150±10 nM Ca2+. These data suggest that Tm treatment releases Ca2+ from intracellular stores.The dependence of CLNX phosphorylation on CN activity suggested that Calic Ca2+ . The aveXenopus oocytes were treated with Tg (1 µM) for either 15, 30 or 60 minutes. A second group of oocytes were initially treated with DTT (1 mM) for 60 minutes and then washed for either 0, 20 or 60 minutes. Like Tg and Tm, DTT is an ER stress inducer, but its effects are reported to be reversible Our data revealed that CN dephosphorylation of CLNX occurred rapidly in response to ER stress. Since PERK is presently considered the most proximal luminal sensor of the UPR 2+ and calmodulin (CaM) affected this interaction given the known dependence of this phosphatase on Ca2+ and CaM. To this end, in vitro GST pull-down experiments were performed between PERK and CN-A, using two Ca2+ concentrations that were chosen to mimic high  and low  cytosol levels. A GST fusion protein was created with only the cytosolic domain of PERK (GST-cPERK), which was then used to pull down recombinant human CN-A α and CN-B. We found that the interaction of CN-A with GST-cPERK was significantly (p<0.01) stronger in high Ca2+ concentration  or CN combined with PERK (CN-PERK) and P-CN enzyme activity was further diminished in low Ca2+ , an in vitro model of ischemia. We observed a significant increase in the levels of CN-A . siRNA (CN-Aα) treated astrocytes showed no significant increase in P-elF2α in response to Tg treatment. Control astrocytes that were mock-transfected exhibited expected increase in P-elF2α . We concXenopus oocytes Given the fact that cells commit to cell death if they are unable to reduce or recover from ER stress, we wanted to test the physiological impact of CN activity on apoptosis. To accomplish this, we took advantage of an assay originally pioneered by Newmeyer and co-workers 2+ dependent manner. We previously established that when the ER is optimally loaded with Ca2+, the most favorable condition necessary for protein processing and folding, CLNX is phosphorylated and physically interacts with SERCA 2b to inhibit its activity 3-mediated Ca2+ release caused a Ca2+ dependent dephosphorylation of serine residue (S562) in the cytosolic domain of CLNX. This removed the functional interaction of CLNX with the pump, removing inhibition and maximizing SERCA 2b-mediated Ca2+ store refilling. CLNX phosphorylation had already been shown to regulate its association with the ribosome, which facilitated the presentation and binding of newly synthesized glycoproteins to the chaperone 2+ increase in the cytosol. These data are consistent with Tm-induced Ca2+ mobilization in fibroblast and CHO cells 2+/CaM dependent mechanism. We suggest that CN phosphatase activity provides the cell with additional time to restore ER homeostasis while the organelle is being refilled with Ca2+.In this study, we have shown that CN works to restore ER homeostasis immediately after ER stress has been initiated. CN performs this important function with the aid of two ER transmembrane proteins: CLNX and PERK. Consequently, CN can now be viewed as an active participant in the UPR by virtue of its ability to couple the cytoplasmic side to the ER lumen in a CaXenopus oocytes within 30–60 minutes of being stressed by Tg or DTT. We also found that CN-A interacted with PERK during stress, and that the kinetics of this association were correlated with the increase in CN-A levels. This suggested to us that the rise in CN levels could cause the subsequent interaction and activation of PERK. We confirmed this hypothesis in an independent model system, cultured mouse astrocytes. We found that ER-stress induced by OGD or thapsigargin treatment in cultured astrocytes rapidly increases CN-Aα levels. Because we were able to block this increase by siRNA treatments in astrocytes, it appears that stress-induced increases in CN-Aα are translationally dependent. Remarkably, the induction of the early UPR in astrocytes, as indicated by increased P-elF2α, was critically dependent on this rapid increase in CN-Aα. Specifically, when astrocytes were treated with siRNA specific for CN-Aα, we observed no significant increase in P-elF2α in response to Tg treatment. In vitro experiments with recombinant proteins also support this model. We demonstrated that the presence of CN-Aα/B significantly increased the autophosphorylation of GST-cPERK. A residual level of GST-cPERK phosphorylation in the absence of CN-Aα/B was likely due to dimerization of the GST-portion of GST-cPERK as reported by Surprisingly, we discovered that CN-A levels were significantly increased in the cytosol of 2+ dependent manner. Association was significantly increased in conditions that mimic high cytosolic Ca2+. The significance of this finding is that this association should occur immediately after ER stress has been triggered when the cytosolic Ca2+ concentration initially increases. It is worth noting that in contrast to the CaM dependence of CN phosphatase activity, the association of CN with PERK appears to be inhibited by CaM. This interaction does not appear to be mediated by another protein, since no other protein was added to the in vitro assay. Moreover, we did not detect an interaction between CN-A and the catalytically inactive mutant GST-cPERK K/A. One explanation for this result is that lysine-618 is a critical residue in the CN binding site of PERK. Alternatively, CN may only be able to interact with PERK after a conformational change occurs in response to autophosphorylation. In this light, lysine 618 is critical to either PERK autophosphorylation and to the subsequent conformational change Another important observation from our pull-down experiments was that CN-A interacted with cPERK in a Ca2+. Interestingly, a ligand for PERK with the properties that we have described has been previously sought after Our model is consistent with the current of view of stress activated PERK. BiP is normally bound to the luminal domain of PERK and acts as negative regulator of activation 2+. Phosphorylation decreased the Vmax of CN to 70%, without changing its affinity (Km) for substrate .The Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich Corp. . Stock solutions of Tg were resuspended in DMSO (2 mM) and stock solutions of Tm were resuspended in methanol at 45°C .CLNX mRNA was prepared as described previously 5 per well in a 6 well format prior to transfection. CN-Aα siRNA (PPP3CA)  was used at 100 nM and transfected with Dharmafect #4 . Transfections were carried out following the manufactuers protocol. Astrocytes were transfected for 17 hours and observed 48 hours post transfection. At 48 hrs, astrocytes were subjected to 1 µM final DMSO vehicle or 1 µM thapsigargin treatment for 1 hour at 37°C. Astrocytes were then rinsed twice with PBS and scrapped into 100 µl of SDS sample buffer  and supplemented with 1 mM sodium orthovanadate , 10 units/ml Leupeptin , and 10 units/ml aprotinin  and used for Western blotting.C8D1A cells  were plated at 1×1032]P-labeled CLNX was performed as described previously Oocytes extracts were prepared as described in 3VO4, 1 mMNaPPi, 0.1 mM ZnCl2, 1 mM NaMOb) and protease inhibitors . Actin was detected with a mouse monoclonal antibody . PERK antibody was obtained from ABGENT .Cultured C8D1A astrocytes  were rinsed twice with PBS and scraped down with a rubber policeman in the presence of 100 µl of lysis buffer per 35 mm dish used. Lysis Buffer was prepared as 15 mM Tris HCl, pH 7.6, 150 mM NaCl, 10% Glycerol, 1% TritonX 100, 1 mM EDTA supplemented with phosphatase inhibitors . Oocytes were injected with 50 nl Fura-2 salt  for 20–30 min at 18°C until equilibration was reached. Measurements were performed in ND96 low Ca2+  at 18–20°C using a Nikon Eclipse TE 300 microscope with a 20×0.75 NA multi-immersion (water for our experiments) lens. Fura-2 was excited at a wavelength of 340 and 380 nm, and emitted fluorescence was collected via a 510 nm long pass filter, using a ORCA-ER charge-coupled video camera device . Frames were collected every 2 s and the 340/380 ratio was analyzed using Open Lab software  following background subtraction. Ca2+ calibration of Fura-2 fluorescence was performed in vitro using the Ca2+ calibration Buffer Kit #2 . An affinity constant of 200 nM was obtained and used to convert Fura-2 ratios into Ca2+ concentrations according to max, Rmin and Sb/Sf were measured as 8.0785, 0.40001 and 8.7369, respectively.CaThe GST fusion protein purification was performed as described in 2+ concentrations. The proteins were eluted by boiling in reducing Laemmli Buffer and were resolved through 12% SDS-PAGE using a mouse anti CN-A antibody and visualized by enhanced chemiluminescence. Ca2+ concentrations were calculated according to existing algorithms Binding of human recombinant CN-Aα/B (2 pmoles)  to GST-cPERK (0.40 nmoles) was performed at 4°C during 1 hour with over-end rotation. As controls, either GST alone or GST-cPERK K/A (0.40 nmoles), and when indicated CaM (2 pmoles), were used. Proteins were incubated and the washes were performed as previously described 2, 1.5 mM DTT, 0.5 mM EGTA, 1 mM NaOAc, 6% glycerol and 0.1 mM ATP, 50 µCi [γ32P]ATP  in the presence of 2 free Ca2+ concentrations (30 nM or 1.4 µM). CaM at 1.6 µM was added as indicated. After 3 min, CN-Aα/B (1.7 µM) was added and incubated for 30 min. The reactions were stopped by boiling in reducing Laemmli Buffer and the proteins were resolved through 10% SDS-PAGE. The gel was fixed, dried, and proteins were visualized by autoradiography.CN-Aα/B phosphorylation and autophosphorylation of cPERK were performed as follows. GST-cPERK, GST-cPERK K/A or GST alone (0.1 µM), were incubated for 3 min at 30°C in a 30 µl reaction mixture  ATP-labeled CLNX from Xenopus oocytes extracts (top). Immunoprecipitated proteins from control oocytes (injected with ddH20) (Cntrl) or from oocytes overexpressing CLNX (mRNA CLNX) were resolved through a 10% SDS-PAGE. Phosphorylated CLNX was visualized by autoradiography. Each lane corresponds to immunoprecipitated CLNX from 15 oocytes per group. (bottom) Western blots of CLNX were performed from the same oocyte extracts before IP. Note that endogenous levels of CLNX phosphorylation are relatively minor and obscured by exogenous phosphorylated CLNX.(0.07 MB TIF)Click here for additional data file.Figure S2Calcineurin inhibitors CsA and FK506 do not induce ER Stress. (A) Western blot showing phosphorylation levels of eIF2α and BiP from vehicle control oocytes (lane 1) or oocytes treated with CsA (200 nM) and FK506 (20 nM) for 16 hours (lane 2). Two oocyte equivalents were loaded per lane and proteins were resolved through 12% SDS-PAGE (P-elF2α) or 7% SDS-PAGE (BiP). P-eIF2α antibody used from Assay Designs (cat# KAP-CP131E). Actin Western blot is shown as loading control. BiP antibody used from Assay Designs (cat# SPA-826).(B) Histograms from 5 independent Western blots pooled from 3 different frogs with n = 15 oocytes per group. Intensity values were normalized with Actin and are represented as the mean ± SEM. ns indicates no statistical significance.(0.08 MB TIF)Click here for additional data file.Figure S3Levels of PERK and CN-A immunoprecipitated with anti-CN-A from oocyte cell extracts. (A) CN-A immunoprecipitation (IP) followed by PERK Western blot from control oocyte extracts (lane 1) or oocytes treated with 1 uM Tg for 15, 30 or 60 minutes , or oocytes treated with DTT for 60 minutes and washed for 0, 20 or 60 minutes . Immunoprecipitated proteins were resolved through 7% SDS-PAGE, transferred to nitrocellulose and probed for PERK with an antibody from ABGENT(cat# AP8054b). Note the presence of two dark bands around 150 kD (above and below 150 kD) corresponding possibly to P-PERK and PERK respectively in lanes from ER stress-induced treatment (lanes 2–6). Interestingly, the band corresponding to P-PERK is reduced in control oocyte extracts (lane 1) or in extracts from DTT treated/washed oocytes for 60 minutes (lane 7). (B) Nitrocellulose membrane shown in A was stripped and probed for CN-A with an antibody from Assay Designs (cat# SPA-610). Note the increase in CN immunoreactivity detected in extracts from ER-stress-induced oocytes (lanes 2–6), which is practically undetectable in control oocytes (lane 1) or DTT treated/washed oocytes for 60 minutes (lane 7). The darker band running at around 50 kD corresponds to detection of IgG from the immunoprecipitation.(0.27 MB TIF)Click here for additional data file.Figure S4Specificity of PERK binding to anti-PERK labeled beads and characterization of anti-PERK antibodies. (A) Commassie blue stained 7% SDS-PAGE gel loaded with Xenopus oocyte extracts that were obtained from a co-imunoprecipitation with an antibody against CN-A from Assay Designs (cat# SPA-610) (lanes 2 and 4) or incubated with protein A/G agarose beads alone (lanes 1 and 3). After Protein A/G agarose pellet was obtained, proteins were resolved through a 7% SDS-PAGE. (B) A fraction of the immunoprecipitated sample was loaded on the gel and transferred to nitrocellulose, probed with PERK antibody (ABGENT cat# AP8054b) and developed by autoradiography. Bands around the molecular weight of PERK (dashed area around 150 kD) were obtained from the immunoprecipitated samples (lanes 2 and 4). These bands are absent in the agarose beads controls (lanes 1 and 3) indicating no specific binding of PERK to the agarose beads. The bottom gel in B shows that protein was loaded in lanes 1 and 3, but did not contain an PERK immunoreactivity. The darker spots in lanes 2 and 4 most likely correspond to the immunoglobulin from the immunoprecipitates. Oo extr 1× and Ooc extr 2× corresponds to extracts from 15 and 30 oocytes, respectively. (C) Western Blot from mouse cultured astrocyte extracts  at rest (Cntrl) or after 60 minutes of ER stress induction by oxygen glucose deprivation (OGD). Protein extracts were run on 7% SDS-PAGE and transferred to nitrocellulose membranes. Membrane on the left panel was probed with a PERK antibody generated in house (anti-PERKUT). Notice the presence of distinct bands around the molecular weight of PERK (dashed area around 150 kD). Right panel corresponds to a similar membrane probed with the anti-PERKUT antibody, in combination with the antigenic peptide used to generate the antibody. Notice the disappearance of bands at the molecular weight of PERK (dashed area around 150 kD) indicating competition of the antigenic peptide for PERK proteins present on the right membrane. The other bands remain unchanged in both membranes on top and bottom panels and are considered non-specific. (D) Western Blot probed with the Pre-immune serum and compared to the anti-PERKUT for primary mouse astrocyte extracts. Proteins were resolved through 7% SDS-PAGE and transferred to nitrocellulose membranes. Membrane probed with rabbit serum before immunization is non-reactive in comparison with middle panel membrane that was probed with anti-PERKUT antibody. Note again that the bands around 150 kD are competed off with the peptide used to immunize the rabbits. (E) Immunoprecipitation from primary mouse astrocytes using different concentrations of the generated anti-PERKUT antibody  or pre-immune serum (lanes 1 and 3) followed by Western Blot with a commercial antibody from Abgent (cat# AP8054b), anti-PERKCM. Note the top band in the dashed rectangle is darker than the lower band at 150 kD and likely corresponds to P-PERK and PERK, respectively. P-PERK is known to migrate higher than PERK. (F) Western Blot from primary mouse astrocytes using pre immune serum (left panel) or anti-PERKUT antibody (middle panels) or commercial antibody anti-PERKCM . Extracts were obtained from cultured astrocytes at rest  and after 60 minutes OGD were used (lanes 2 and 4). Extracts from astrocytes were also treated with Vehicle  and 300 nM thapsigargin . Note the stress induced increase in PERK-P using the anti-PERKUT, but not the anti-PERKCM antibody.(0.42 MB TIF)Click here for additional data file.Figure S5Quantification of PERK loading for GST pull-down assay. Molar amounts of protein for GST-pull-down assays were calibrated by loading known volumes of proteins  side by side on the same gel with known volumes of Albumin (1 µg/µl) as a standard. Proteins were resolved through a 10% SDS-PAGE and stained with Coomassie blue. The concentration of GST, GST-cPERK and GST-cPERK K/A was converted to µmoles using the Albumin standard curve and apparent molecular weight of GST = 25 kD; GST-cPERK = 90 kD and GST-cPERKK/A = 75 kD.(0.37 MB TIF)Click here for additional data file.Figure S6Coomassie blue stained gel shown as loading control for the kinase assay on (0.12 MB TIF)Click here for additional data file.Figure S7Phosphatase activity of CN-A. The specific activity of human recombinant CN-A was measured spectrophotometrically (O.D.410 nm) using p-NPP as substrate varying concentrations from 5 to 150 mM. The assay was initiated by addition of p-NPP, incubated at 30°C for 20 min and stopped by addition of 200 µl of 13% K2HPO4 and immediately chilled on ice. Specific activity was based on a pKa of 7.17 obtaining a measured molar extinction coefficient of 17,300 M-1 cm-1 at 410 nm at pH 8.58 for p-NPP. (A) The specific activity of CN-A without ATP (CN w/o ATP), without PERK (CN w/o PERK) compared to phosphorylated CN-A (P-CN) in high (3.2 µM) Ca2+. (B) Specific activity of CN-ATP, CN-PERK and P-CN in low (46 nM) Ca2+. Data are an average of 5 independent experiments. Error bars are within the size of the symbols see .(0.06 MB TIF)Click here for additional data file.Figure S8[35S]-Methionine-Cysteine incorporation in oocytes injected with CN morpholinos and treated with Thapsigargin. (A) Autoradiography of total protein of total protein synthesized in control oocytes or injected with morpholinos as was described (0.16 MB TIF)Click here for additional data file.Figure S9Temporal progression of apoptotic-like morphology in isolated liver nuclei incubated with oocyte cytosolic extract. The percentage of apoptotic nuclei increases by 2 hours (first panels) and peaks by 4 hours (middle panels) for control extracts. In comparison, no change in the morphology of apoptotic nuclei is observed with buffer alone at 4 hours (right panels). Nuclei are stained with Hoechst dye (100 µg/µl) for visualization. Upper panels are high magnification of lower images for the white-framed regions.(0.27 MB TIF)Click here for additional data file.Table S1CN-A phosphatase activity.(0.03 MB DOC)Click here for additional data file.


To further investigate the cell-penetrating capabilities of the toxin, we prepared thiolated, fluorescent derivatives of IpTxa. Biological activity and spectroscopic properties indicate that these derivatives retain high affinity for RyRs and are only 5- to 10-fold less active than native IpTxa. Our results demonstrate that IpTxa is capable of crossing cell membranes to alter the release of Ca2+in vivo, and has the capacity to carry a large, membrane-impermeable cargo across the plasma membrane, a finding with exciting implications for novel drug delivery. Scorpion venoms are rich in ion channel-modifying peptides, which have proven to be invaluable probes of ion channel structure-function relationship. We previously isolated imperatoxin A (IpTxa), a 3.7 kDa peptide activator of Cas (RyRs) ,2,3 and  Furthermore, IpTxa’s high sequence identity with MCa suggests that the toxin may also be a cell-penetrating peptide. We therefore designed experiments to both investigate the effects of IpTxa on calcium signaling in intact cardiomyocytes, and to demonstrate its cell-penetrating capabilities. Ventricular cardiomyocytes were enzymatically digested, loaded with the Ca2+ indicator Fluo-3, and placed into the perfusion chamber of a Zeiss confocal microscope . Cells were field-stimulated under Normal Tyrode (NT) solution, and Ca2+ transients were recorded in line-scan mode (control). Perfusion was then changed to NT + IpTxa (in the range of 100 nM-30 µM), and transients were recorded in the presence of toxin (+IpTxa). We hypothesized that if IpTxa penetrates cardiomyocytes to reach its intracellular target, it would alter the shape of [Ca2+]i transients elicited with field-stimulation. As expected, changes in [Ca2+]i transients were evident within seconds of perfusion with the toxin. Perfusion of IpTxa at high concentrations (30 µM) resulted in the complete cessation of [Ca2+]i transients , IpTxa perfusion elicited several discernible stimulatory effects in most cells. The most commonly observed response to perfusion with these IpTxa concentrations was a significant increase in the amplitude of the [Cady-state . In simiansients . They atgertschi .2+]i in some cells perfused with 100 nM to 3 µM IpTxa , which is known to induce delayed afterdepolarizations (DADs). However, the exact mechanism by which IpTxa elicits cessation of contractions, subtle Ca2+ leak or discrete Ca2+ waves requires further investigation.In addition to the above response, we observed elevated diastolic ryanodine, although higher concentrations were needed to obtain the same half-maximal effective concentration (EC50). As reported previously, EC50 was 10 nM for native IpTxa, whereas Alexa-IpTxa exhibited an EC50 of 75 nM. Thus, modified IpTxa shows excellent activation of RyRs, which is only 7.5-fold lower than the native toxin. This result is impressive in light of previous chemical modification studies using scorpion toxins, which have indicated that modifications of amino groups lead to a loss of toxic activity Ryanodine was purchased from Dupont NEN . Phosphatidylethanolamine and phosphatidylserine were obtained from Avanti Polar Lipids, Inc. . Type II collagenase was acquired from Worthington Biochemical . Heparin was obtained from Wedgewood Pharmacy . Fluo-3 and Alexa-Fluor-546-maleimide was purchased from Invitrogen . All other chemicals were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA). Indicator dye was first dissolved in dimethyl sulfoxide, then diluted further in buffer solution.[3H]Ryanodine binding to rabbit skeletal SR was carried out as previously described  to 10 μM. Ca2+/EGTA ratio was calculated using the stability constants of Fabiato [3H]ryanodine  was diluted directly in the incubation medium to a final concentration of 7 nM. Protein concentration was in the range of 0.2-0.4 mg/ml and was determined by Bradford method. Incubation lasted 90 min at 36°C. Samples (0.1 ml) were always run in duplicate, filtered on Whatman GF/C glass filters, and washed twice with 5 ml of distilled water using a Brandel M24-R cell harvester . Nonspecific binding was determined in the presence of 10 μM unlabeled ryanodine and has been subtracted from each sample. Heavy sarcoplasmic reticulum (SR) was prepared from rabbit white back and leg muscle using the procedure described by Meissner . [3H]Ryaescribed ,19. Brie Fabiato . [3H]rya2PO4, 0.5 MgCl2, 25 HEPES, 22 glucose, 0.01 μg/mL insulin, pH7.4 with NaOH) and kept at room temperature until used within 6 h. Mouse ventricular myocytes were isolated by collagenase digestion of perfused mouse hearts using the method of Mitra and Morad  with mod2+] using the Ca2+-dependent fluorescent dye Fluo-3 AMChanges in cytosolic i transients, whereas permeation of Alexa-IpTxa is slower (minutes), possibly due to the insertion of the membrane-impermeable fluorophore. The latter, however, shows that IpTxa is capable of delivering cargo to intracellular compartments, which opens an exciting field of intracellular drug delivery by RyR-targeting peptides. In this study, we have demonstrated that IpTxa, both in its native state and as a thiolated derivative, has the ability to cross the plasma membrane of intact cardiomyocytes to modulate its intracellular target, the sarcoplasmic reticulum Ca2+ signaling as evidenced by 1) an acute increase in the amplitude of the [Ca2+]i transient, indicative of enhanced Ca2+ release from the SR, followed by a gradual diminution in transient amplitude until reaching a new steady state at a lower amplitude than in control; 2) an elevation in diastolic [Ca2+]i indicative of increased Ca2+ leak through RyRs; 3) cessation of [Ca2+]i transients at high toxin concentrations; 4) unsolicited contractions. Except for 3, which is discussed below, these effects are similar to those observed after perfusion of hadrucalcin, a closely-related scorpion toxin [2+ signaling are attributed to depletion of Ca2+ from the SR [2+ transients would be incompatible with an exclusive effect of IpTxa on SR Ca depletion, inasmuch as Ca2+ entry via L-type Ca2+ channels is a small, but measurable component of the intracellular Ca2+ transient [2+ transients, but we cannot rule out that high micromolar concentrations of IpTxa may totally abrogate Ca2+ transients by “upsetting” the external membrane and preventing membrane depolarization, thus exerting a less specific effect . Although no such concentrations of IpTxa are ever reached in native environments and they rather represent experimenter-generated conditions, the effect warrants further investigation. Perfusion of IpTxa on intact cells results in extremely rapid effects (lag time 2-3 sec) on intracellular Caon toxin , and to m the SR . However2+ signaling in ventricular myocytes holds promise for future investigation into diseases associated with dysfunctional Ca2+ handling such as catecholaminergic polymorphic ventricular tachycardia (CPVT), a genetic syndrome caused by dysfunctional Ca2+ release. CPVT results from various mutations of RyR2 that may go undetected until physical or emotional stressors augment pre-existing Ca2+ handling dysfunction, causing acute responses characterized by arrhythmias with the potential of degenerating into ventricular fibrillation. Though various animal models have been developed for the myriad of mutations associated with this disease, such models can be problematic due to compensatory Ca2+ removal mechanisms that could mask the mutant RyR phenotype. IpTxa, at low to moderate concentrations (as defined above), holds potential for a useful tool as a RyR modulator with the capability of rapidly inducing RyR hyperactivity unencumbered from effects on other channels or Ca2+ transport systems, allowing researchers to observe acute changes in Ca2+ and subsequent compensatory changes as they occur in real time. Future research will be devoted to unraveling the mechanism by which IpTxa evokes these changes in Ca2+ signaling.The fact that IpTxa can acutely perturb CaIn addition, we have described a method for the preparation of scorpion toxin derivatives containing fluorescent probes which are biologically active and retain high binding affinity for RyRs. The usefulness of these derivatives is two-fold, in that they can provide valuable information on the molecular environment of the toxin’s intracellular target and serve as a site-specific probe for RyRs. We have also demonstrated the capacity of IpTxa to permeate the external membrane, although the exact mechanism by which this translocation occurs remains to be explained. This characteristic makes IpTxa an excellent tool for the study of RyR in intact cells. Furthermore, the toxin’s ability to carry non-permeable cargo across the plasma membrane suggests the possibility that calcium channel-modifying drugs could be targeted specifically to RyRs, providing a novel therapeutic agent for the treatment of RyR channelopathies.


Lysophosphatidylcholine (LPC), an atherogenic component of oxidized low-density lipoprotein, has been shown to induce the attenuation of endothelium-dependent vascular relaxation. Although benidipine, a dihydropyridine-calcium channel blocker, is known to have endothelial protective effects, the effects of benidipine on LPC-induced endothelial dysfunction remain unknown. We examined the effects of benidipine on the impairment of endothelium-dependent relaxation induced by LPC.2+]i) and membrane fluidity altered by LPC in primary cultured rat aortic endothelial cells were examined. [Ca2+]i was measured using the fluorescent calcium indicator fura-2. Membrane fluidity was monitored by measuring fluorescence recovery after photobleaching.Benidipine was administered orally to rats and aortas were then isolated. Aortic rings were treated with LPC and endothelial functions were then evaluated. Additionally, the effects of benidipine on intracellular calcium concentration ([Ca2+]i in fura-2 loaded endothelial cells. The increase in [Ca2+]i was suppressed after exposure to LPC. Plasma membrane fluidity increased following incubation with LPC. Benidipine inhibited the LPC-induced increase in membrane fluidity and impairment of increase in [Ca2+]i.Treatment with LPC impaired endothelial function. Benidipine prevents the impairment of relaxation induced by LPC. Acetylcholine elicited an increase in [Ca2+]i. Benidipine possesses membrane stabilization properties in LPC-treated endothelial cells. It is speculated that the preservation of membrane fluidity by benidipine may play a role in the retainment of calcium mobilization. The present findings may provide new insights into the endothelial protective effects of benidipine.These results suggest that benidipine inhibited LPC-induced endothelial dysfunction by maintaining increase in [Ca Oxidize2+]i) ,4. The m C (PKC) . Activatal cells . On the al cells . LPC hasal cells ,8. It is2+]i attenuated by LPC were examined. Finally, the inhibitory potency of benidipine on LPC-induced membrane perturbation was assessed.Benidipine hydrochloride (benidipine), a dihydropyridine-calcium channel blocker, has potent and long-acting antihypertensive effects . We prevMale SD rats 7–8 weeks  were used. All animals were kept at 19–25°C in a 12 hr light/dark cycle. Food and water were available ad libitum to all animals. This study was conducted in accordance with the Standards for Proper Conduct of Animal Experiments of Kyowa Hakko Kirin.in vitro assay, benidipine, fura-2 acetoxy methylester, PMA, PDB, Ro-31-8220, calphostin C, PDTC, ascorbic acid and BHT were dissolved in dimethylsulfoxide (DMSO). LPC was dissolved in ethanol. Other chemicals were dissolved in distilled water.Benidipine and amlodipine besilate (amlodipine) were produced by Kyowa Hakko Kirin. Nifedipine, L-α-lysophosphatidylcholine , L-phenylephrine hydrochloride (PE), acetylcholine chloride (ACh), pluronic F-127, phorbol 12-myristate 13-acetate (PMA), calphostin C and pyrrolidine dithiocarbamate (PDTC) were purchased from Sigma-Aldrich . Fura-2 acetoxy methylester was purchased from Wako Pure Chemical Industries . Ro-31-8220 and phorbol 12, 13-dibutyrate (PDB) were purchased from Nacalai Tesque . Butylated hydroxytoluene (BHT) was purchased from LKT Laboratories . 1-Acyl-2-[6-[N-amino]hexanoyl]-sn-glycero-3-phosphocholine (NBD-PC) was purchased from Avanti Polar Lipids . Ascorbic acid was purchased from Kanto Kagaku . Benidipine, amlodipine and nifedipine were suspended in 0.5% w/v methylcellulose 400 cP  and administered orally. For the 2+5%CO2) Krebs-Henseleit solution comprising 119 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgSO4, 1.8 mmol/L CaCl2, 1.2 mmol/L KH2PO4, 24.9 mmol/L NaHCO3 and 11.1 mmol/L glucose, and then cut into 2–3 mm length rings. Aortic rings were attached to holders and then placed in an organ bath filled with 20 mL Krebs-Henseleit solution. Following 60 min equilibration, aortic rings were incubated with 5 μmol/L LPC or 0.05% ethanol for 60 min. Tissues were washed three times, and vasorelaxant responses were measured as previously described i in endothelial cells [2+]iresponses to ACh, we measured [Ca2+]i in rat aortic endothelial cells. [Ca2+]i was monitored by measuring the fluorescence (F340/F380) of fura-2. Cells were treated with 5 μmol/L fura-2 acetoxy methylester and 0.04% pluronic F-127 in Hanks solution  for 15 min at 37°C. Cells were washed three times and fluorescence signals were monitored using an Argus-50 fluorescence spectrophotometer  at room temperature. Response to 3 μmol/L ACh was obtained and was then followed by washing. Cells were incubated with 3 μmol/L LPC or 0.03% ethanol for 30 min followed by washing. After washing, cells were treated with 3 μmol/L ACh. Changes in responses to ACh were expressed as percent values. Benidipine (1 or 10 nmol/L) or 0.1% DMSO was simultaneously added with LPC or ethanol for 30 min to evaluate the effects on ACh-induced increases in [Ca2+]i. The PKC activators PMA and PDB, and PKC inhibitors Ro-31-8220 and calphostin C were also examined in an effort to delineate the involvement of PKC upregulation in LPC-induced endothelial dysfunction. It has been reported that anti-oxidants can mimic the action of benidipine as manifested by the inhibition of LPC-induced VCAM-1 expression and ROS production i  resulting in the formation of peroxynitrite (ONOO-) [2+]i. The lack of effects of the antioxidants suggested that the effects of benidipine did not emanate from its anti-oxidant activity.In the present study, we demonstrated that LPC inhibited receptor-mediated EDR and increases in i [2+]i in human umbilical vein endothelial cells [2+]i suppression. The direct PKC activators PMA and PDB had no effect on ACh-induced increases in [Ca2+]i. These findings suggest that PKC activation does not account for the inhibition of ACh-induced increases in [Ca2+]i in rat aortic endothelial cells. We showed that LPC induced significant increases in membrane fluidity. It was speculated that LPC probably induces the suppression of ACh-mediated increases in [Ca2+]i by increasing membrane fluidity. However, the mechanism underlying ACh-induced increases in [Ca2+]i remain unclear. Further studies are needed to rule out a causal relationship between membrane fluidity and increases in [Ca2+]i.The precise mechanisms responsible for the inhibitory effect of LPC on EDR have not been elucidated -8. There2+]i . The inh2+]i ,8. An in2+]i ,8. This 2+]i . On the al cells . In our 2+]i at 10 nmol/L. LPC significantly increased relative membrane fluidity. In the presence of LPC, benidipine preserved an adequate level of membrane fluidity at 10 nmol/L. Benidipine possesses high liposolubility and affinity for cell membranes compared with amlodipine and nifedipine [2+]i. In the present study, benidipine exclusively inhibited LPC-induced attenuation of EDR. It seems that the unique effects of benidipine are due to its high affinity for the cell membrane. It has been reported that the maximum plasma drug concentration of benidipine after oral administration to rats at a dose of 3 mg/kg, which is an antihypertensive dosage of spontaneously hypertensive rats, is 36 nmol/L (19.5 ng/mL) i: intracellular calcium concentration; EDR: endothelium-dependent relaxation; eNOS: endothelial nitric oxide synthase; DMSO: dimethylsulfoxide; LPC: L-α-lysophosphatidylcholine (C16:0); NBD-PC: 1-Acyl-2-[6-[N-amino]hexanoyl]-sn-glycero-3-phosphocholine; NO: nitric oxide; ox-LDL: oxidized low-density lipoprotein; PDB: phorbol 12, 13-dibutyrate; PDTC: pyrrolidine dithiocarbamate; PE: L-phenylephrine hydrochloride; PMA: phorbol 12-myristate 13-acetate; ROS: reactive oxygen species; VCAM-1: vascular cell adhesion molecule-1.ACh: acetylcholine chloride; BHT: Butylated hydroxytoluene; [CaThe authors declare that they have no competing interests.MT designed and performed experiments, and prepared a draft of the manuscript. KY and MW revised the drafted manuscript.