Inositol (1,4,5)-trisphosphate dynamics and intracellular calcium oscillations in pancreatic beta-cells

Glucose-stimulated insulin secretion is associated with transients of intracellular calcium concentration ([Ca2+]i) in the pancreatic beta-cell. We tested the hypothesis that inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] [Ca2+]i release is incorporated in glucose-induced [Ca2+]i oscillations in mouse islets and MIN6 cells. We found that depletion of intracellular Ca2+ stores with thapsigargin increased the oscillation frequency by twofold and inhibited the slow recovery phase of [Ca2+]i oscillations. We employed a pleckstrin homology domain-containing fluorescent biosensor, phospholipase C partial differential pleckstrin homology domain-enhanced green fluorescent protein, to visualize Ins(1,4,5)P3 dynamics in insulin-secreting MIN6 cells and mouse islets in real time using a video-rate confocal system. In both types of cells, stimulation with carbamoylcholine (CCh) and depolarization with KCl results in an increase in Ins(1,4,5)P3 accumulation in the cytoplasm. When stimulated with glucose, the Ins(1,4,5)P3 concentration in the cytoplasm oscillates in parallel with oscillations of [Ca2+]i. Maximal accumulation of Ins(1,4,5)P3 in these oscillations coincides with the peak of [Ca2+]i and tracks changes in frequencies induced by the voltage-gated K+ channel blockade. We show that Ins(1,4,5)P3 release in insulin-secreting cells can be stimulated by depolarization-induced Ca2+ flux. We conclude that Ins(1,4,5)P3 concentration oscillates in parallel with [Ca2+]i in response to glucose stimulation, but it is not the driving force for [Ca2+]i oscillations.     

Influence of three-piece and single-piece designs of two sharp-edge optic hydrophobic acrylic intraocular lenses on the prevention of posterior capsule opacification: a prospective, randomised, long-term clinical trial

BACKGROUND: Posterior capsule opacification (PCO) is still a major long-term complication of modern cataract surgery. We evaluated the impact of sharp-edged intraocular lenses (IOLs) with different haptic designs made from the same hydrophobic acrylic material on posterior and anterior lens capsule opacification. SETTING: Eye clinic of Kaunas University of Medicine, Lithuania. Prospective randomised clinical study. METHODS: Seventy-four eyes of 74 patients scheduled for cataract surgery were included in a prospective randomised clinical study. Thirty-seven eyes of 37 patients received a three-piece acrylic hydrophobic (AcrySof, MA3OBA, Alcon) IOL; and thirty-seven eyes of 37 patients received a one-piece acrylic hydrophobic (AcrySof, SA3OAL, Alcon) IOL. Visual acuity, anterior capsule opacification (ACO), capsular folds, capsulorrhexis/optic overlapping and posterior capsule opacification (PCO) were evaluated. ACO was assessed subjectively. PCO values in the entire IOL optic area and in the central 3 mm optic zone were assessed using a photographic image-analysis system (EPCO2000). Follow-ups were performed postoperatively at 1 day, 6 months, 1 year and 2 years. RESULTS: There were no significant differences in best corrected visual acuity, grade of ACO and capsulorrhexis/optic overlapping between IOL types during the follow-up period. Patients in the one-piece acrylic hydrophobic IOL group more frequently presented with capsular folds behind the IOL optic area than those in the three-piece IOL group. In the three-piece acrylic hydrophobic IOL group, PCO values (mean (SD)) of the entire IOL optic area were significantly lower six months postoperative (three-piece: 0.002 (0.009); one-piece: 0.007 (0.017); p=0.04), one year postoperative (three-piece: 0.004 (0.016); one-piece: 0.026 (0.041); p=0.001) as well as one year postoperative in the central 3 mm optic zone (three-piece: 0.000 (0.0002); one-piece: 0.019 (0.049); p=0.001). However, two years postoperative, the PCO values of the groups did not show significant differences (entire IOL optic area: three-piece, 0.136 (0.223); one-piece, 0.154 (0.190); p=0.18; central zone: three-piece, 0.023 (0.065); one-piece: 0.020 (0.039); p=0.44). CONCLUSION: The 2 year follow-up after cataract surgery showed no significant difference in ACO and PCO development between three-piece and one-piece acrylic hydrophobic intraocular lenses.     

Endoscopic vs open saphenous vein harvest for coronary artery bypass grafting: a prospective randomized trial

OBJECTIVE: Endoscopic saphenous vein harvesting (EVH) for coronary artery bypass grafting (CABG) has been developed to reduce leg wound morbidity and improve patient satisfaction. Choosing between EVH of a short vein segment from the thigh and open venous harvesting (OVH) of a short segment from the calf represents a clinical dilemma as EVH is easiest to perform from the thigh and OVH is easiest to perform from the calf. The purpose of this study was to investigate whether leg wound morbidity was reduced after EVH of a short vein segment from the thigh compared with OVH from the calf. Secondly we investigated whether EVH would reduce length of hospital stay and improve cosmetic results. METHODS: From April 2004 to June 2007, 132 patients undergoing elective isolated CABG were randomized to have a short segment of saphenous vein harvested either by the EVH or OVH technique. Clinical follow-up was scheduled at day 5 and at 1 month. Primary end-points included wound morbidity. Secondary end-points included harvest time, length of hospital stay, cosmetic results and need for additional wound care after discharge. RESULTS: The groups were preoperative similar. Three patients in the OVH group were excluded from the study as it became apparent that it was necessary to extend the incision beyond the knee. Harvest time was longer for the EVH group, but these patients suffered from significantly fewer cases of infectious and non-infective wound complications, with a substantial reduction in the need for post-discharge leg wound care. The purulent infection rates in the EVH and OVH groups were 0% and 11%, respectively. The overall leg wound morbidity rates regarding cellulitis, purulent infection, dehiscence and skin necrosis were 3% and 27% in the EVH and OVH groups, respectively (p<0.001). The length of hospital stay was similar. The conversion rate from EVH to OVH was 14%. The EVH group experienced less pain and better cosmetic results. CONCLUSIONS: EVH of a short vein segment from the thigh results in less wound morbidity and better cosmetic results compared with OVH of a short vein segment from the calf.     

The Time-Course of Voluntary and Electrically Evoked Muscle Performance During and After Stretch-Shortening Exercise is Different

The aim of the study was to establish the dynamics of maximal voluntary contraction force (MVCF), height of drop jump (DJ) and electrically evoked quadriceps muscle force at different stimulation frequencies during and after 100 DJs (stretch-shortening exercise, SSE). Healthy untrained men (n = 11; age = 21.8 ± 1.7 years) participated in the study. DJs were performed with 30 s intervals between jumps from the height of 0.5 m with counter-movement to 90 degrees angle in the knee and immediate maximal rebound. The force of the quadriceps muscle, evoked by electrical stimulation at 1 Hz (Pt), 20 Hz (P20) and 100 Hz (P100) frequencies (electrically evoked performance, EEP), MVCF and height of DJ (voluntary evoked performance, VEP) were established during SSE (after 10, 50, 100 DJ) as well as at 1, 4, 8, 24, 48 and 72 h after SSE. Time-course of P20 and P100 during and after SSE was time (ANOVA: p < 0.001) and frequency dependent (ANOVA: p < 0.001) The Pt, P20 and P100 decreased significantly (p < 0.01) more than MVCF and H of DJ during SSE. At the beginning of SSE (during 1-10 DJs) P20 and P100 decreased significantly (p < 0.001) more than during 11-50 and 51-100 DJs. There was a significant (p < 0.05) increase in Pt, P20 and P100 from 8 h to 48 h, whereas height of DJ and MVCF significantly decreased at that time. In conclusion, the differences in time course of VEP and EEP are most evident at beginning of SSE, where VEP does not change as EEP decreases, and within 8-48 hours after SSE, where VEP decreases as EEP increases.

Key points

• There was no change in voluntary muscle performance while electrically evoked performance decreased significantly during first 10 drop jumps.
• There was a significant increase in electrically evoked muscle performance from 8 h to 48 h after 100 drop jumps, whereas voluntary contraction force, decreased significantly.
• The secondary decrease in the height of drop jump as well as in maximal voluntary contraction force correlated significantly with muscle soreness within 24-48 h after exercise.

Key words: Drop jump, muscle damage, electrical stimulation, low frequency fatigue     

TRPC6 channel translocation into phagosomal membrane augments phagosomal function

Defects in the innate immune system in the lung with attendant bacterial infections contribute to lung tissue damage, respiratory insufficiency, and ultimately death in the pathogenesis of cystic fibrosis (CF). Professional phagocytes, including alveolar macrophages (AMs), have specialized pathways that ensure efficient killing of pathogens in phagosomes. Phagosomal acidification facilitates the optimal functioning of degradative enzymes, ultimately contributing to bacterial killing. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H⁺ into the organelle. Critical to phagosomal acidification are various channels derived from the plasma membrane, including the anion channel cystic fibrosis transmembrane conductance regulator, which shunt the transmembrane potential generated by movement of protons. Here we show that the transient receptor potential canonical-6 (TRPC6) calcium-permeable channel in the AM also functions to shunt the transmembrane potential generated by proton pumping and is capable of restoring microbicidal function to compromised AMs in CF and enhancement of function in non-CF cells. TRPC6 channel activity is enhanced via translocation to the cell surface (and then ultimately to the phagosome during phagocytosis) in response to G-protein signaling activated by the small molecule (R)-roscovitine and its derivatives. These data show that enhancing vesicular insertion of the TRPC6 channel to the plasma membrane may represent a general mechanism for restoring phagosome activity in conditions, where it is lost or impaired.Chronic infection and inflammation in the airways in cystic fibrosis (CF), as well as chronic obstructive pulmonary disease (COPD), tuberculosis, and asthma are now among the most common chronic diseases. Pulmonary infection associated with these diseases has historically been treated with antibiotics that kill bacteria but also select for development of resistance in the pathogen in the chronically infected lung (1, 2). One solution to antimicrobial drug resistance is to target the host rather than the pathogen. This strategy necessitates finding alternative targets or signaling strategies amplifying or restoring bactericidal capacity in the cells charged with the task of resolving chronic infection.Mononuclear phagocytes orchestrate the innate immune response in the lung through the combinatorial interplay between the phagocytic uptake and killing of bacterial invaders, clearance of apoptotic cells, antigen presentation, and secretion of vesicle-bound signaling molecules to recruit help in the resolution of infection. Ionic fluxes across the phagosomal membrane that encloses the pathogen produce a hostile acidic environment developed through the action of a V-ATPase proton translocation. However, a positive intraphagosomal membrane potential generated by proton translocation minimizes the proton content of the phagosome. An influx of Cl⁻ via Cl⁻ channels reduces the membrane potential generated by the proton pump, thereby, allowing maximal acidification of the phagolysosomal compartment, which in turn maximizes the activation of lysosomal degradative enzymes, generation of hypochlorous acid, and consequent bacterial killing (3, 4). We have previously demonstrated that murine alveolar macrophages (AMs) use the anion channel cystic fibrosis transmembrane conductance regulator (CFTR) as a Cl⁻ permeation pathway in the phagosomal membrane. In CF, loss of functional CFTR in the AM alkalinizes the phagosomal lumen and allows antimicrobial-resistant bacterial pathogens to survive macrophage surveillance.Not all tissue macrophages use CFTR as a charge compensation pathway in phagolysosomal acidification (4). In fact, recent data suggest that multiple V-ATPase charge-shunt pathways can exist in diverse macrophage lineages (5) via lysosomal recruitment and membrane insertion upon particle uptake. This observation led us to search for possible alternative charge-shunt pathways in pulmonary macrophages and how they might be activated or targeted to the maturing phagosome. Could a pharmacological tool circumvent the defect in CF AMs and activate alternative pathways to rescue both organellar acidification and bactericidal activity in cells expressing mutations in CFTR? Such a tool might activate parallel charge-shunt pathways used in peritoneal macrophages for maximum acidification, thereby allowing them to clear bacteria independently of CFTR expression as well as amplify the microbicidal response in the presence of functional CFTR. The transport proteins and channels active in peritoneal macrophage bacterial clearance have not been described but may involve a K⁺/H⁺ exchanger important in promoting excitatory synaptic vesicle filling (6) or a cation channel moving positive charge out of the phagolysosomal compartment, as has been suggested for macrophage cell lines (7).We began our investigations pursuing a novel pharmacological strategy to identify compounds that would resolve bacterial infection in the CF lung without the use of antimicrobials. We picked a cellular defect in CF because of the availability of animal models and extended our observations to non-CF human pulmonary cells. We designed screening assays of phagosome function, which could be used in a clinical setting as both diagnostic and investigational tools. We interrogated host function by studying surface receptor-mediated mechanisms that might provide parallel signaling pathways in subcellular organellar function in the resolution of disease.We report that a series of small molecules first identified in chemotherapeutics, (R)-roscovitine [2-(R)-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine] and its derivatives, restore microbicidal function to compromised AMs in CF and enhance function in non-CF cells. The compounds use a G protein-mediated signaling pathway, which results in the mobilization of transient receptor potential canonical-6 (TRPC6) calcium-permeable, nonselective cation channels to the plasma membrane and subsequently to the phagosomal membrane. Members of the TRP channel family have been implicated in a number of critical phagocytic functions, including particle uptake, migration, and reactive oxygen species (ROS) production (5, 8–11). Numerous studies (12–15) have suggested TRP channels as potential targets for the development of therapies in pulmonary inflammation because of their abundant and diverse cellular expression throughout the respiratory tree. Although TRPC6 channels have been previously identified in lung macrophages and shown to be up-regulated in COPD, their precise role in the pathophysiology of the disease is yet to be determined (16). Perhaps more relevant to our study, a TRPC6-mediated Ca²⁺ influx is increased in human CF airway epithelial cells, possibly because of a functional association between CFTR and TRPC6 that is lost in CF (17), with unknown consequences. To our knowledge, this study is the first to associate TRPC6 channels a specific drug-targeting strategy for the resolution chronic pulmonary infection.