Immunohistochemical localization of a Shaker-related voltage-gated potassium channel protein in Schistosoma mansoni (Trematoda: Digenea)

We have recently isolated a cDNA (SKV1.1) encoding a Shakei-related K+ channel from the human parasitic trematode Schistosoma mansoni. In order to better understand the functions of SKv1.1 protein, the distribution of SKv1.1 protein in adult S. mansoni was analyzed by immunohistochemistry using a region-specific antibody. SKV1.1 proteins were widely expressed in the nervous and muscular systems. The strongest immunoreactivity (IR) was observed in the nervous system of both male and female. In the nervous system, IR for SKv1.1 proteins was localized in cell bodies and nerve fibers of the anterior ganglia, the central commissure, and the main nerve cords. IR was also observed in the dorsal and the ventral peripheral nerve nets, fine nerve fibers entering into a variety of structures such as the dorsal tubercles, longitudinal and ventral muscle fibers, and oral and ventral suckers. In the muscular system, SKv1.1 proteins were localized to the longitudinal, circular, and ventral muscle fibers of male as well as in isolated muscle fibers where native A-type K+ currents were measured. Moderate IR was also seen in a large number of cell bodies in the parenchyma. These results indicate that SKv1.1 protein may play an important role in the regulation of the excitability of neurons and muscle cells of S. mansoni. (C) 1995 Academic Press, Inc.

The Paralax Infrastructure: Automatic Parallelization With a Helping Hand

Speeding up sequential programs on multicores is a challenging problem that is in urgent need of a solution. Automatic parallelization of irregular pointer-intensive codes, exempli?ed by the SPECint codes, is a very hard problem. This paper shows that, with a helping hand, such auto-parallelization is possible and fruitful. This paper makes the following contributions: (i) A compiler framework for extracting pipeline-like parallelism from outer program loops is presented. (ii) Using a light-weight programming model based on annotations, the programmer helps the compiler to ?nd thread-level parallelism. Each of the annotations speci?es only a small piece of semantic information that compiler analysis misses, e.g. stating that a variable is dead at a certain program point. The annotations are designed such that correctness is easily veri?ed. Furthermore, we present a tool for suggesting annotations to the programmer. (iii) The methodology is applied to autoparallelize several SPECint benchmarks. For the benchmark with most parallelism (hmmer), we obtain a scalable 7-fold speedup on an AMD quad-core dual processor. The annotations constitute a parallel programming model that relies extensively on a sequential program representation. Hereby, the complexity of debugging is not increased and it does not obscure the source code. These properties could prove valuable to increase the ef?ciency of parallel programming.

Glucose-induced oxidative stress in vascular contractile cells - Comparison of aortic smooth muscle cells and retinal pericytes

Free radical-mediated damage to vascular cells may be involved in the pathogenesis of diabetic vasculopathy. The aim of this study was to compare the extent of glucose-induced oxidative stress in both vascular smooth muscle cells (VSMCs) and pericytes and the effect on antioxidant enzyme gene expression and activities. Porcine aortic VSMC and retinal pericytes were cultured in either 5 or 25 mmol/l glucose for 10 days. Intracellular malondialdehyde (MDA) was measured as a marker of peroxidative damage, and mRNA expression of CuZn-SOD, MnSOD, catalase, and glutathione peroxidase (GPX) were measured by Northern analysis. Glutathione (GSH) was also measured. There was a significant increase in MDA in VSMCs in 25 mmol/l glucose (1.34 +/- 0.11 vs. 1.88 +/- 0.24 nmol/mg protein, 5 vs. 25 mmol/l D-glucose, mean +/- SE, n = 15, P

FhCaBP4: A Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains

In trematodes, there is a family of proteins which combine EF-hand-containing domains with dynein light chain (DLC)-like domains. A member of this family from the liver fluke, Fasciola hepatica-FhCaBP4-has been identified and characterised biochemically. FhCaBP4 has an N-terminal domain containing two imperfect EF-hand sequences and a C-terminal dynein light chain-like domain. Molecular modelling predicted that the two domains are joined by a flexible linker. Native gel electrophoresis demonstrated that FhCaBP4 binds to calcium, manganese, barium and strontium ions, but not to magnesium or zinc ions. The hydrophobic, fluorescent probe 8-anilinonaphthalene-1-sulphonate bound more tightly to FhCaBP4 in the presence of calcium ions. This suggests that the protein undergoes a conformational change on ion binding which increases the number of non-polar residues on the surface. FhCaBP4 was protected from limited proteolysis by the calmodulin antagonist W7, but not by trifluoperazine or praziquantel. Protein-protein cross-linking experiments showed that FhCaBP4 underwent calcium ion-dependent dimerisation. Since DLCs are commonly dimeric, it is likely that FhCaBP4 dimerises through this domain. The molecular model reveals that the calcium ion-binding site is located close to a key sequence in the DLC-like domain, suggesting a plausible mechanism for calcium-dependent dimerisation.

Pharmacological profiling of store-operated Ca(2+) entry in retinal arteriolar smooth muscle

Objective: Pharmacological profiling of store-operated Ca(2+) entry (SOCE) and molecular profiling of ORAI and TRPC expression in arterioles. 
Methods: Fura-2 based microfluorimetry was used to assess CPA-induced SOCE in rat retinal arteriolar myocytes. Arteriolar ORAI and TRP transcript expression were screened using RT-PCR. 
Results: SKF96365 and LOE908 blocked SOCE (IC(50) s of 1.2µM and 1.4µM, respectively). Gd(3+) and La(3+) potently inhibited SOCE (IC(50) s of 21nM and 42nM, respectively), but Ni(2+) showed lower potency (IC(50) = 11.6µM). 2-aminoethyldiphenyl borate (2APB) inhibited SOCE (IC(50) = 3.7µM) but enhanced basal influx (>100µM). Verapamil and nifedipine had no effect at concentrations that inhibit L-type Ca(2+) channels, but diltiazem inhibited SOCE by approximately 40% (=0.1µM). RT-PCR demonstrated transcript expression for ORAI 1, 2 and 3, and TRPC1, 3, 4 and 7. Transcripts for TRPV1 and 2, which are activated by 2APB, were also expressed. 
Conclusion: The pharmacological profile of SOCE in retinal arteriolar smooth muscle appears unique when compared to other vascular tissues. This suggests that the molecular mechanisms underlying SOCE can differ, even in closely related tissues. Taken together, the pharmacological and molecular data are most consistent with involvement of TRPC1 in SOCE, although involvement of ORAI or other TRPC channels cannot be excluded. © 2012 John Wiley & Sons Ltd.

Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle

This study assessed the contribution of L-type Ca2+ channels and other Ca2+ entry pathways to Ca2+ store refilling in choroidal arteriolar smooth muscle. Voltage-clamp recordings were made from enzymatically isolated choroidal microvascular smooth muscle cells and from cells within vessel fragments (containing <10 cells) using the whole-cell perforated patch-clamp technique. Cell Ca2+ was estimated by fura-2 microfluorimetry. After Ca2+ store depletion with caffeine (10 mM), refilling was slower in cells held at -20 mV compared to -80 mV (refilling half-time was 38 +/- 10 and 20 +/- 6 s, respectively). To attempt faster refilling via L-type Ca2+ channels, depolarising steps from -60 to -20 mV were applied during a 30 s refilling period following caffeine depletion. Each step activated L-type Ca2+ currents and [Ca2+]i transients, but failed to accelerate refilling. At -80 mV and in 20 mM TEA, prolonged caffeine exposure produced a transient Ca2+-activated Cl- current (I(Cl)(Ca)) followed by a smaller sustained current. The sustained current was resistant to anthracene-9-carboxylic acid (1 mM; an I(Cl)(Ca) blocker) and to BAPTA AM, but was abolished by 1 microM nifedipine. This nifedipine-sensitive current reversed at +29 +/- 2 mV, which shifted to +7 +/- 5 mV in Ca2+-free solution. Cyclopiazonic acid (20 microM; an inhibitor of sarcoplasmic reticulum Ca2+-ATPase) also activated the nifedipine-sensitive sustained current. At -80 mV, a 5 s caffeine exposure emptied Ca2+ stores and elicited a transient I(Cl)(Ca). After 80 s refilling, another caffeine challenge produced a similar inward current. Nifedipine (1 microM) during refilling reduced the caffeine-activated I(Cl)(Ca) by 38 +/- 5 %. The effect was concentration dependent (1-3000 nM, EC50 64 nM). In Ca2+-free solution, store refilling was similarly depressed (by 46 +/- 6 %). Endothelin-1 (10 nM) applied at -80 mV increased [Ca2+]i, which subsided to a sustained 198 +/- 28 nM above basal. Cell Ca2+ was then lowered by 1 microM nifedipine (to 135 +/- 22 nM), which reversed on washout. These results show that L-type Ca2+ channels fail to contribute to Ca2+ store refilling in choroidal arteriolar smooth muscle. Instead, they refill via a novel non-selective store-operated cation conductance that is blocked by nifedipine.

Transient Ca2+-activated Cl-currents with endothelin in isolated arteriolar smooth muscle cells of the choroid

To characterize the effects of endothelin (ET)-1 on the Ca2+-activated Cl- conductance of choroidal arteriolar smooth muscle.

Heterogeneity in cytosolic calcium regulation among different microvascular smooth muscle cells of the rat retina

Rat retinae were dissociated to yield intact microvessels 7 to 42 microm in diameter. These were loaded with fura-2 AM and single fragments anchored down in a recording bath. Intracellular Ca(2+) levels from 20- to 30-microm sections of vessel were estimated by microfluorimetry. The vessels studied were identified as metarterioles and arterioles. Only the microvascular smooth muscle cells loaded with fura-2 AM and changes in the fluorescence signal were confined to these cells: Endothelial cells did not make any contribution to the fluorescence signal nor did they contribute to the actions of the drugs. Caffeine (10 mM) or elevated K(+) (100 mM) produced a transient rise in cell Ca(2+) in the larger vessels (diameters >18 microm) but had no effect on smaller vessels (diameters 30 min) on washing out the endothelin and the vessel failed to relax. These results demonstrate heterogeneity between smaller and larger retinal vessels with regard to Ca(2+) mobilisation and homogeneity with respect to the actions of vasoactive peptides.

Diabetes downregulates large-conductance Ca2+-activated potassium beta 1 channel subunit in retinal arteriolar smooth muscle

Retinal vasoconstriction and reduced retinal blood flow precede the onset of diabetic retinopathy. The pathophysiological mechanisms that underlie increased retinal arteriolar tone during diabetes remain unclear. Normally, local Ca(2+) release events (Ca(2+)-sparks), trigger the activation of large-conductance Ca(2+)-activated K(+)(BK)-channels which hyperpolarize and relax vascular smooth muscle cells, thereby causing vasodilatation. In the present study, we examined BK channel function in retinal vascular smooth muscle cells from streptozotocin-induced diabetic rats. The BK channel inhibitor, Penitrem A, constricted nondiabetic retinal arterioles (pressurized to 70mmHg) by 28%. The BK current evoked by caffeine was dramatically reduced in retinal arterioles from diabetic animals even though caffeine-evoked [Ca(2+)](i) release was unaffected. Spontaneous BK currents were smaller in diabetic cells, but the amplitude of Ca(2+)-sparks was larger. The amplitudes of BK currents elicited by depolarizing voltage steps were similar in control and diabetic arterioles and mRNA expression of the pore-forming BKalpha subunit was unchanged. The Ca(2+)-sensitivity of single BK channels from diabetic retinal vascular smooth muscle cells was markedly reduced. The BKbeta1 subunit confers Ca(2+)-sensitivity to BK channel complexes and both transcript and protein levels for BKbeta1 were appreciably lower in diabetic retinal arterioles. The mean open times and the sensitivity of BK channels to tamoxifen were decreased in diabetic cells, consistent with a downregulation of BKbeta1 subunits. The potency of blockade by Pen A was lower for BK channels from diabetic animals. Thus, changes in the molecular composition of BK channels could account for retinal hypoperfusion in early diabetes, an idea having wider implications for the pathogenesis of diabetic hypertension.

FhCaBP3: A Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains

A DNA sequence encoding a protein with predicted EF-hand and dynein light chain binding domains was identified in a Fasciola hepatica EST library. Sequence analysis of the encoded protein revealed that the most similar known protein was the Fasciola gigantica protein FgCaBP3 and so this newly identified protein was named FhCaBP3. Molecular modelling of FhCaBP3 predicted a highly flexible N-terminal region, followed by a domain containing two EF-hand motifs the second of which is likely to be a functioning divalent ion binding site. The C-terminal domain of the protein contains a dynein light chain like region. Interestingly, molecular modelling predicts that calcium ion binding to the N-terminal domain destabilises the ß-sheet structure of the C-terminal domain. FhCaBP3 can be expressed in, and purified from, Escherichia coli. The recombinant protein dimerises and the absence of calcium ions appeared to promote dimerisation. Native gel shift assays demonstrated that the protein bound to calcium and manganese ions, but not to magnesium, barium, zinc, strontium, nickel, copper or cadmium ions. FhCaBP3 interacted with the calmodulin antagonists trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine as well as the myosin regulatory light chain-binding drug praziquantel. Despite sequence and structural similarities to other members of the same protein family from F. hepatica, FhCaBP3 has different biochemical properties to the other well characterised family members, FH22 and FhCaBP4. This suggests that each member of this trematode calcium-binding family has discrete functional roles within the organism.

Comparative capacitative calcium entry mechanisms in canine pulmonary and renal arterial smooth muscle cells.

Experiments were performed to determine whether capacitative Ca(2+) entry (CCE) can be activated in canine pulmonary and renal arterial smooth muscle cells (ASMCs) and whether activation of CCE parallels the different functional structure of the sarcoplasmic reticulum (SR) in these two cell types. The cytosolic [Ca(2+)] was measured by imaging fura-2-loaded individual cells. Increases in the cytosolic [Ca(2+)] due to store depletion in pulmonary ASMCs required simultaneous depletion of both the inositol 1,4,5-trisphosphate (InsP(3))- and ryanodine (RY)-sensitive SR Ca(2+) stores. In contrast, the cytosolic [Ca(2+)] rises in renal ASMCs occurred when the SR stores were depleted through either the InsP(3) or RY pathways. The increase in the cytosolic [Ca(2+)] due to store depletion in both pulmonary and renal ASMCs was present in cells that were voltage clamped and was abolished when cells were perfused with a Ca(2+)-free bathing solution. Rapid quenching of the fura-2 signal by 100 microM Mn(2+) following SR store depletion indicated that extracellular Ca(2+) entry increased in both cell types and also verified that activation of CCE in pulmonary ASMCs required the simultaneous depletion of the InsP(3)- and RY-sensitive SR Ca(2+) stores, while CCE could be activated in renal ASMCs by the depletion of either of the InsP(3)- or RY-sensitive SR stores. Store depletion Ca(2+) entry in both pulmonary and renal ASMCs was strongly inhibited by Ni(2+) (0.1-10 mM), slightly inhibited by Cd(2+) (200-500 microM), but was not significantly affected by the voltage-gated Ca(2+) channel (VGCC) blocker nisoldipine (10 microM). The non-selective cation channel blocker Gd(3+) (100 microM) inhibited a portion of the Ca(2+) entry in 6 of 18 renal but not pulmonary ASMCs. These results provide evidence that SR Ca(2+) store depletion activates CCE in parallel with the organization of intracellular Ca(2+) stores in canine pulmonary and renal ASMCs.