Calmodulin Contributes to Gating Control in Olfactory Calcium-activated Chloride Channels

In sensory neurons of the peripheral nervous system, receptor potentials can be amplified by depolarizing Cl currents. In mammalian olfactory sensory neurons (OSNs), this anion-based signal amplification results from the sequential activation of two distinct types of transduction channels: cAMP-gated Ca channels and Ca-activated Cl channels. The Cl current increases the initial receptor current about 10-fold and leads to the excitation of the neuron. Here we examine the activation mechanism of the Ca-dependent Cl channel. We focus on calmodulin, which is known to mediate Ca effects on various ion channels. We show that the cell line Odora, which is derived from OSN precursor cells in the rat olfactory epithelium, expresses Ca-activated Cl channels. Single-channel conductance, ion selectivity, voltage dependence, sensitivity to niflumic acid, and Ca sensitivity match between Odora channels and OSN channels. Transfection of Odora cells with CaM mutants reduces the Ca sensitivity of the Cl channels. This result points to the participation of calmodulin in the gating process of Ca-ativated Cl channels, and helps to understand how signal amplification works in the olfactory sensory cilia. Calmodulin was previously shown to mediate feedback inhibition of cAMP-synthesis and of the cAMP-gated Ca channels in OSNs. Our results suggest that calmodulin may also be instrumental in the generation of the excitatory Cl current. It appears to play a pivotal role in the peripheral signal processing of olfactory sensory information. Moreover, recent results from other peripheral neurons, as well as from smooth muscle cells, indicate that the calmodulin-controlled, anion-based signal amplification operates in various cell types where it converts Ca signals into membrane depolarization

The Ca-activated Cl Channel and its Control in Rat Olfactory Receptor Neurons

Odorants activate sensory transduction in olfactory receptor neurons (ORNs) via a cAMP-signaling cascade, which results in the opening of nonselective, cyclic nucleotide–gated (CNG) channels. The consequent Ca2+ influx through CNG channels activates Cl channels, which serve to amplify the transduction signal. We investigate here some general properties of this Ca-activated Cl channel in rat, as well as its functional interplay with the CNG channel, by using inside-out membrane patches excised from ORN dendritic knobs/cilia. At physiological concentrations of external divalent cations, the maximally activated Cl current was ∼30 times as large as the CNG current. The Cl channels on an excised patch could be activated by Ca2+ flux through the CNG channels opened by cAMP. The magnitude of the Cl current depended on the strength of Ca buffering in the bath solution, suggesting that the CNG and Cl channels were probably not organized as constituents of a local transducisome complex. Likewise, Cl channels and the Na/Ca exchanger, which extrudes Ca2+, appear to be spatially segregated. Based on the theory of buffered Ca2+ diffusion, we determined the Ca2+ diffusion coefficient and calculated that the CNG and Cl channel densities on the membrane were ∼8 and 62 μm−2, respectively. These densities, together with the Ca2+ diffusion coefficient, demonstrate that a given Cl channel is activated by Ca2+ originating from multiple CNG channels, thus allowing low-noise amplification of the olfactory receptor current

Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons

<p>Abstract</p> <p>Background</p> <p>Chloride currents in peripheral nociceptive neurons have been implicated in the generation of afferent nociceptive signals, as Cl^-accumulation in sensory endings establishes the driving force for depolarizing, and even excitatory, Cl^-currents. The intracellular Cl^-concentration can, however, vary considerably between individual DRG neurons. This raises the question, whether the contribution of Cl^-currents to signal generation differs between individual afferent neurons, and whether the specific Cl^-levels in these neurons are subject to modulation. Based on the hypothesis that modulation of the peripheral Cl^-homeostasis is involved in the generation of inflammatory hyperalgesia, we examined the effects of inflammatory mediators on intracellular Cl^-concentrations and on the expression levels of Cl^-transporters in rat DRG neurons.</p> <p>Results</p> <p>We developed an <it>in vitro </it>assay for testing how inflammatory mediators influence Cl^-concentration and the expression of Cl^-transporters. Intact DRGs were treated with 100 ng/ml NGF, 1.8 μM ATP, 0.9 μM bradykinin, and 1.4 μM PGE₂for 1–3 hours. Two-photon fluorescence lifetime imaging with the Cl^--sensitive dye MQAE revealed an increase of the intracellular Cl^-concentration within 2 hours of treatment. This effect coincided with enhanced phosphorylation of the Na⁺-K⁺-2Cl^-cotransporter NKCC1, suggesting that an increased activity of that transporter caused the early rise of intracellular Cl^-levels. Immunohistochemistry of NKCC1 and KCC2, the main neuronal Cl^-importer and exporter, respectively, exposed an inverse regulation by the inflammatory mediators. While the NKCC1 immunosignal increased, that of KCC2 declined after 3 hours of treatment. In contrast, the mRNA levels of the two transporters did not change markedly during this time. These data demonstrate a fundamental transition in Cl^-homeostasis toward a state of augmented Cl^-accumulation, which is induced by a 1–3 hour treatment with inflammatory mediators.</p> <p>Conclusion</p> <p>Our findings indicate that inflammatory mediators impact on Cl^-homeostasis in DRG neurons. Inflammatory mediators raise intracellular Cl^-levels and, hence, the driving force for depolarizing Cl^-efflux. These findings corroborate current concepts for the role of Cl^-regulation in the generation of inflammatory hyperalgesia and allodynia. As the intracellular Cl^-concentration rises in DRG neurons, afferent signals can be boosted by excitatory Cl^-currents in the presynaptic terminals. Moreover, excitatory Cl^-currents in peripheral sensory endings may also contribute to the generation or modulation of afferent signals, especially in inflamed tissue.</p

Safety and Precision of Two Different Flap-morphologies Created During Low Energy Femtosecond Laser-assisted LASIK

Purpose: Currently, two major principles exist to create LASIK flaps: firstly, a strictly horizontal (2D) cut similar to the microkeratome-cut and secondly an angled cut with a “step-like” edge (3D). The strictly horizontal (2D) cut method can be performed using apparatus such as the low-energy FEMTO LDV Z8 laser and its predecessors which are specific to this type. Alternatively, the low-energy FEMTO LDV Z8 laser’s 3D flap design creates an interlocking flap-interface surface which potentially contributes toward flap stability. In addition, the FEMTO LDV Z8 offers flap-position adjustments after docking (before flap-creation). The current study analyzed precision, safety, efficacy, as well as patient self-reported pain and comfort levels after applying two different types of LASIK flap morphologies which were created with a low-energy, high-frequency femtosecond (fs) laser device. Methods: A prospective, interventional, randomized, contralateral eye, single-center comparison study was conducted from November 2019 to March 2020 at the Hamburg vision clinic/ zentrumsehstärke, Hamburg, Germany. Eleven patients and 22 eyes received low-energy fs LASIK treatment for myopia or myopic astigmatism in both eyes. Before the treatment, the eyes were randomized (one eye was treated with the 2D, the other eye with the 3D method). Results: The mean central flap thickness one month after surgery was 110.7 ± 1.6 μm (2D) and 111.2 ± 1.7 μm (3D); P = 0.365 (2D vs 3D). Flap thickness measured at 13 different points resulted in no statistically significant differences between any of the measurement points within/between both groups; demonstrating good planarity of the flap was achieved using both methods. Despite not being statistically significant, the surgeons recognized an increase in the presence of an opaque bubble layer in the 3D flap eyes during surgery and some patients reported higher, yet not statistically significant, pain scores in the 3D flap eyes during the first hours after the treatment. Overall, safety- and efficacy indices were 1.03 and 1.03, respectively. Conclusion: In this prospective, randomized, contralateral eye study, the low-energy fs laser yielded predictable lamellar flap thicknesses and geometry at one-month followup. Based on these results, efficacy and safety of the corresponding laser application, that is, 2D vs 3D, are equivalen

Rescue bedside laparotomy in the intensive care unit in patients too unstable for transport to the operating room

INTRODUCTION: The prognoses of critically ill patients with a requirement for emergency laparotomy and severe respiratory and/or hemodynamic instability precluding transport to the operating room (OR) are often fatal without surgery. Attempting emergency surgery at the bedside might equally result in an adverse outcome. However, risk factors and predictors that could support clinical decision making have not been identified so far. This study describes the clinical characteristics, indicative pathophysiology and outcomes in patients undergoing resuscitative laparotomy in the intensive care unit (ICU). METHODS: This was a retrospective observational study of all critically ill adult patients undergoing resuscitative laparotomy in the ICUs of a German university hospital from January 2005 to July 2013. Clinical characteristics, risk factors, and treatments were compared between survivors and non-survivors. The primary endpoint was 28-day survival. RESULTS: A total of 41 patients with a median age of 64 (21 to 83) were included. The most frequent reasons for ICU admission were sepsis, pneumonia, and pancreatic surgery. All patients were mechanically ventilated, receiving vasopressors, and were in multiple organ failure. Twenty-nine patients (70.7%) were on renal replacement therapy and two patients (4.9%) on extracorporeal membrane oxygenation. The main reasons for surgery were suspected intra-abdominal bleeding (39.0%), suspected intestinal ischemia (24.4%) or abdominal compartment syndrome (24.4%). Twenty-eight-day, ICU and hospital mortalities were 75.6%, 80.5%, and 82.9%, respectively. In six out of ten patients (60%) who survived surgery for more than 28 days, bedside laparotomy was rated as a life-saving procedure by an interdisciplinary group of the investigators. CONCLUSIONS: These findings suggest that in selected critically ill patients with a vital indication for emergency laparotomy and severe cardiopulmonary instability precluding transport to the OR, a bedside resuscitative laparotomy in the ICU can be considered as a rescue procedure, even though very high mortality is to be expected

Corneal Biomechanical Changes after Crosslinking for Progressive Keratoconus with the Corneal Visualization Scheimpflug Technology

Purpose. To evaluate the effect of corneal crosslinking in progressive keratoconus by applying in vivo corneal visualization Scheimpflug technology. Design. Longitudinal retrospective study. Subjects and Controls. Seventeen eyes of patients treated with corneal crosslinking for progressive keratoconus. Methods. Corneal visualization Scheimpflug technology analyses (research software version 6.07r08) of subjects with progressive keratoconus before and 3 months after corneal crosslinking (CXL) were reviewed retrospectively. t-test (for normal distribution) and Wilcoxon matched-pairs test (if not normally distributed) were used to test for statistically significant differences between pre- and post-CXL analyses. Results. We demonstrated statistically significant differences for the intraocular pressure (median: +3 mmHg, P =0.004), the central corneal pachymetry (pachy; mean: −35 µm, P<0.001), the timespan between the air impulse release and the first applanation of the cornea (A1time; median: +0.12 ms, P<0.05), and the timespan between the air impulse release and the second applanation of the cornea (A2time; median: −37 ms, P<0.05). Conclusions. With the A1time and the A2time, we identified two parameters that demonstrated a statistically significant improvement of the biomechanical properties of the cornea after CXL. Despite the known initial decrease of the pachymetry after CXL, none of the analyzed parameters indicated a progression of the keratoconus