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Whole transcriptome sequencing has revealed the existence of mRNAs for multiple membrane transporters in photoreceptors. Except for ATP binding cassette (ABC) member A4, involved in the retinoid cycle, an understanding of the function of most transport proteins in photoreceptors is lacking. In this research paper, extrusion of fluo-4, a Ca 2+ fluorescent probe, from 661W cells, a cone photoreceptor murine cell line was studied with online fluorometry and immunocytochemistry. Fluo-4 efflux was temperature dependent, required ATP but not extracellular Na + , was not affected by pH in the range 5.4-8.4, and followed saturating kinetics with a Km of nearly 4 μM, suggesting it was effected by ABC type transporters. A panel of antagonists showed an inhibitory profile typical of the C subfamily of ABC transporters. Immunofluorescence staining was positive for ABCC3, ABCC4 and ABCC5. These experimental results are compatible with fluo-4 being extruded from 661W cones by one or a combination of C-type ABC transporters. Examination of physicochemical descriptors related to drug membrane permeability and ABC substrate binding region further suggested efflux of fluo-4 by C-type ABC transporters. Possible functions of this transport mechanism in photoreceptors are discussed., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: It has been demonstrated that the accurate positioning of the graft is key to restoring shoulder stability and preventing future arthrosis development. Preoperative anteroinferior glenoid bone loss is frequently encountered when performing a Latarjet, and it has not been determined yet if the amount of bony defect can influence graft positioning. The aim of the study was to determine if a preoperative glenoid bony defect has an influence on the final coracoid graft position in the arthroscopic Latarjet procedure., Methods: Fifty-five patients who underwent the arthroscopic Latarjet procedure were included, with a minimum follow-up of 2 years. There were 51 men (92.7%). Mean age was 29.1 (SD 7.63). Western Ontario Shoulder Instability Index, Rowe, and Single Assessment Numeric Evaluation scores were fulfilled. All measurements were performed by a musculoskeletal radiologist based on a multiplanar bidimensional CT scan. Dimensions of the glenoid, glenoid defect, and glenoid track were calculated. Position of the graft was evaluated in the axial (distance to glenoid surface, angulation of the graft and screws) and sagittal planes (percentage of the coracoid graft below the equator) as described by Kany et al and Barth et al respectively., Results: There was a glenoid defect in 41 patients (74.5 %). Mean width of the defect was 4.32 mm (SD 3.08) which represented 15.3% of the native glenoid surface (SD 10.8). 78.2% of the patients were offtrack preoperatively, and 11.9% remained offtrack postoperatively. The final glenoid diameter with the graft was 32.1 mm (SD 4.34). Mean distance from the graft to the glenoid at 50% height was 1.1 mm (SD 2.19 mm) and at 25% height was 1.31 mm (SD 2.05). Mean angulation of the superior and inferior screws were 26.9° (SD 8.2°) and 27.1° (SD 7.35°), respectively. In 81.8% of the cases, the graft was deemed to be flush with the glenoid. The percentage of the coracoid graft under the equator of the glenoid was 71.2 % (SD 21.8). There was not a statistically significant difference in screw angulation or graft positioning in the axial plane when comparing patients who had a glenoid defect with those who did not, or depending on the size ( P  > .05). Percentage of graft below the equator was, however, lower in patients without bony defect ( P  = .04)., Conclusion: This study showed that accurate position of the coracoid graft is achieved in the presence of a glenoid bony defect. In the cases of intact glenoid, the height of the graft should be carefully evaluated., (© 2023 The Authors.)

Pre-blockade of the sarco-endoplasmic reticulum (ER) calcium ATPase (SERCA) with irreversible thapsigargin depresses exocytosis in adrenal bovine chromaffin cells (BCCs). Distinct expression of voltage-dependent Ca 2+ -channel subtypes and of the Ca 2+ -induced Ca 2+ release (CICR) mechanism in BCCs versus mouse chromaffin cells (MCCs) has been described. We present a parallel study on the effects of the acute SERCA blockade with reversible cyclopizonic acid (CPA), to repeated pulsing with acetylcholine (ACh) at short (15 s) and long intervals (60 s) at 37 °C, allowing the monitoring of the initial size of a ready-release vesicle pool (RRP) and its depletion and recovery in subsequent stimuli. We found (i) strong depression of exocytosis upon ACh pulsing at 15-s intervals and slower depression at 60-s intervals in both cell types; (ii) facilitation of exocytosis upon acute SERCA inhibition, with back to depression upon CPA washout in MCCs; (iii) blockade of exocytosis upon acute SERCA inhibition and pronounced rebound of exocytosis upon CPA washout in BCCs; (iv) basal [Ca 2+ ] c elevation upon stimulation with ACh at short intervals (but not at long intervals) in both cell types; and (v) augmentation of basal [Ca 2+ ] c and inhibition of peak [Ca 2+ ] c amplitude upon CPA treatment in both cell types, with milder effects upon stimulation at 60-s intervals. These results are compatible with the view that while in MCCs the uptake of Ca 2+ via SERCA contributes to the mitigation of physiological ACh triggered secretion, in BCCs the uptake of Ca 2+ into the ER facilitates such responses likely potentiating a Ca 2+ -induced Ca 2+ release mechanism. These drastic differences in the regulation of ACh-triggered secretion at 37 °C may help to understand different patterns of the regulation of exocytosis by the circulation of Ca 2+ at a functional ER Ca 2+ store.

We evaluated the activity of (1) amphotericin-B (AMB), combined with rifampicin (RIF), clarithromycin (CLA), N -acetylcysteine (NAC), ethylenediaminetetraacetic acid (EDTA), and farnesol (FAR) (1000, 1000, 1000, 4000, and 30,000 mg/L, and 300 µM, respectively), against Candida tropicalis biofilms formed on polytetrafluoroethylene (PTFE) and (2) anidulafungin (ANF) combined with the same compounds at 8, 10, 5, 40, and 30 mg/L, and 30 µM, respectively, against biofilms formed on titanium. Biofilm growth kinetics were performed in a CDC Biofilm Reactor (CBR). PTFE or titanium disks were removed from the CBR at 24, 48, 72, and 96 h to determine the Log 10 CFU/cm². Killing kinetics were performed by adding the drugs to 24-h-mature biofilms (time 0). Disks were removed after 24, 48, and 72 h of drug exposure to determine Log 10 CFU/cm². Viable cells in biofilms were 4.73 and 4.29 Log 10 CFU/cm² on PTFE and titanium, respectively. Maximum Log 10 decreases in CFU/cm² depend on the combination and were: 3.53 (AMB + EDTA), 2.65 (AMB + RIF), 3.07 (AMB + NAC), 2.52 (AMB + CLA), 1.49 (AMB + FAR), 2.26 (ANF + EDTA), 2.45 (ANF + RIF), 2.47 (ANF + NAC), 1.52 (ANF + CLA), and 0.44 (ANF + FAR). In conclusion, EDTA, NAC, RIF, and CLA improve the activity of AMB and ANF against biofilms developed on both surfaces, which could be an effective strategy against C. tropicalis biofilm-related infections.

Calcium buffering by mitochondria plays a relevant physiological function in the regulation of Ca(2+) and exocytotic signals in mature chromaffin cells (CCs) from various adult mammals. Whether a similar or different role of mitochondrial Ca(2+) buffering is present in immature CCs at early life has not been explored. Here we present a comparative study in rat embryonic CCs and rat mother CCs, of various physiological parameters that are known to be affected by mitochondrial Ca(2+) buffering during cell activation. We found that the clearance of cytosolic Ca(2+) transients ([Ca(2+)]c) elicited by high K(+) was 7-fold faster in embryo CCs compared to mother CCs. This strongly suggests that at embryonic life, the mitochondria play a more significant role in the clearance of [Ca(2+)]c loads compared to adult life. Consistent with this view are the following results concerning the transient suppression of mitochondrial Ca(2+) buffering by protonophore FCCP, in embryonic CCs compared to mother CCs: (i) faster and greater inactivation of inward calcium currents, (ii) higher K(+)-elicited [Ca(2+)]c transients with 25-fold faster clearance, (iii) higher increase of basal catecholamine release and (iv) higher potentiation of K(+)-evoked secretion. These pronounced differences could be explained by two additional features (embryo versus mother CCs): (a) slower recovery of mitochondrial resting membrane potential after the application of a transient FCCP pulse and (b) greater relative density of the mitochondria in the cytosol. This tighter control by the mitochondria of Ca(2+) and exocytotic signals may be relevant to secure a healthy catecholamine secretory response at early life.

Adrenal chromaffin cells (CCs) express high-voltage activated calcium channels (high-VACCs) of the L, N and PQ subtypes; in addition, T-type low-VACCs are also expressed during embryo and neonatal life. Effects of the more frequently used T channel blockers NNC 55-0396 (NNC), mibefradil, and Ni2+ on the whole-cell Ba2+ current (IBa), the K+-elicited [Ca2+]c transients and catecholamine secretion have been studied in adult bovine CCs (BCCs) and rat embryo CCs (RECCs). NNC, mibefradil, and Ni2+ blocked BCC IBa with IC50 of 1.8, 4.9 and 70 μM, while IC50 to block IBa in RECCs were 2.1, 4.4 and 41 μM. Pronounced blockade of K+-elicited [Ca2+]c transients and secretion was also elicited by the three agents. However, the hypoxia-induced secretion (HIS) of catecholamine in RECCs was blocked substantially (75%) with thresholds concentrations of NCC (IC20 to block IBa); this was not the case for mibefradil and Ni2+ that required higher concentrations to block the HIS response. Thus, out of the three compounds, NNC seemed to be an adequate pharmacological tool to discern the contribution of T channels to the HIS response, without a contamination with high-VACC blockade., (Copyright © 2015. Published by Elsevier B.V.)

Here we review the contribution of the various subtypes of voltage-activated calcium channels (VACCs) to the regulation of catecholamine release from chromaffin cells (CCs) at early life. Patch-clamp recording of inward currents through VACCs has revealed the expression of high-threshold VACCs (high-VACCs) of the L, N, and PQ subtypes in rat embryo CCs and ovine embryo CCs. Low-threshold VACC (low-VACC) currents (T-type) have also been recorded in rat embryo CCs and rat neonatal slices of adrenal medullae. Near full blockade by nifedipine and nimodipine of the K(+)-elicited secretion as well as the hypoxia induced secretion (HIS) supports the dominant role of L-VACC subtypes to the regulation of exocytosis at early life. Partial blockade by ω-conotoxin GVIA and ω-agatoxin IVA suggests a transient participation of N and PQ high-VACCs to the regulation of the HIS response at early stages of CC exposure to hypoxia. T-type low-VACC current did not elicit exocytosis triggered by electrical depolarising pulses applied to rat embryo CCs in one study, but largely contributed to the HIS response in neonatal rat adrenal slices in another. In spite of scarce available data, the sequence of events driving the HIS response in CCs at early life could be established as follows: (i) hypoxia blocks one or more K(+) channels; (ii) as a consequence, mild membrane depolarisation occurs; (iii) T-type low-VACCs open at membrane potentials more hyperpolarised than those required to recruit the high-VACCs; (iv) firing of action potentials then occurs; (v) fast-inactivating N and PQ high-VACCs transiently open and low-inactivating L high-VACCs remain open along the hypoxia stimulus; (vi) increase of cytosolic Ca(2+) takes place; and (vii) the exocytotic release of catecholamine occurs in two phases, an explosive initial phase, driven by Ca(2+) entry through L, N and PQ channels, followed by a more sustained catecholamine release at a slower rate driven by L-type channels.

Altered synaptic transmission with excess glutamate release has been implicated in the loss of motoneurons occurring in amyotrophic lateral sclerosis (ALS). Hyperexcitability or hypoexcitability of motoneurons from mice carrying the ALS mutation SOD1(G93A) (mSOD1) has also been reported. Here we have investigated the excitability, the ion currents, and the kinetics of the exocytotic fusion pore in chromaffin cells from postnatal day 90 to postnatal day 130 mSOD1 mice, when motor deficits are already established. With respect to wild-type (WT), mSOD1 chromaffin cells had a decrease in the following parameters: 95% in spontaneous action potentials, 70% in nicotinic current for acetylcholine (ACh), 35% in Na(+) current, 40% in Ca(2+)-dependent K(+) current, and 53% in voltage-dependent K(+) current. Ca(2+) current was increased by 37%, but the ACh-evoked elevation of cytosolic Ca(2+) was unchanged. Single exocytotic spike events triggered by ACh had the following differences (mSOD1 vs. WT): 36% lower rise rate, 60% higher decay time, 51% higher half-width, 13% lower amplitude, and 61% higher quantal size. The expression of the α3-subtype of nicotinic receptors and proteins of the exocytotic machinery was unchanged in the brain and adrenal medulla of mSOD1, with respect to WT mice. A slower fusion pore opening, expansion, and closure are likely linked to the pronounced reduction in cell excitability and in the ion currents driving action potentials in mSOD1, compared with WT chromaffin cells., (Copyright © 2015 the American Physiological Society.)