In the present experiments in HEL cells, we have investigated the requirement for a hyperpolarised resting membrane potential for the initial activation of the Ca(2+) activated K(+) channel, KCa3.1, following activation of the Ca(2+) release activated Ca(2+) (CRAC) entry pathway. In intact cells, fluorimetric measurements of [Ca(2+)]i following thapsigargin-mediated activation of CRAC entry revealed a sustained increase in [Ca(2+)]i. Block of KCa3.1 by application of charybdotoxin resulted in a 50% reduction in the steady-state [Ca(2+)]i, consistent with the well established role for KCa3.1-mediated hyperpolarisation in augmenting CRAC entry. Interestingly, subsequent depolarisation to 0mV by application of gramicidin resulted in a fall in steady-state Ca(2+) levels to values theoretically below that required for activation of KCa3.1. Whole cell patch clamp experiments confirmed the lack of KCa3.1 activation at 0mV following activation of the CRAC entry pathway, indicating an absolute requirement for a hyperpolarised resting membrane potential for the initial activation of KCa3.1 leading to hyperpolarisation and augmented Ca(2+) entry. Current clamp experiments confirmed the requirement for a hyperpolarised resting membrane potential in KCa3.1 activation by CRAC entry. Given the critical role played by KCa3.1 and membrane potential in general in the control of CRAC-mediated [Ca(2+)]i changes, we investigated the hypothesis that inhibition of the CRAC-mediated changes in [Ca(2+)]i observed following 2-APB addition may in part arise from direct inhibition of KCa3.1 by 2-APB. Under whole cell patch clamp, 2-APB, at concentrations typically used to block the CRAC channel, potently inhibited KCa3.1 in a reversible manner (half maximal inhibition 14.2 μM). This block was accompanied by a marked shift in the reversal potential to depolarised values approaching that set by endogenous membrane conductances. At the single channel level, 2-APB applied to the cytosolic face resulted in a significant reduction in open channel probability and a fall in the mean open time of the residual channel activity. Our data highlight the absolute requirement for a hyperpolarising resting membrane conductance for the initial activation of KCa3.1 by CRAC entry. Additionally, our results document direct inhibition of KCa3.1 by 2-APB, thus highlighting the need for caution when ascribing the site of inhibition of 2-APB exclusively to the CRAC entry pathway in experiments where membrane potential is not controlled.
Various surgical methods to prevent postoperative cerebrospinal fluid (CSF) leaks during transsphenoidal surgery have been reported. However, comparative studies are scarce. We aimed to compare the efficacy of a fibrin-coated collagen fleece (TachoSil) versus a dural sealant (DuraSeal) to prevent postoperative CSF leakage. We perform a retrospective study comparing two methods of sellar closure during endoscopic endonasal transsphenoidal surgery (EETS) for pituitary adenoma resection: TachoSil patching versus DuraSeal packing. Data concerning diagnosis, reconstruction technique, and surgical outcomes were analyzed. The primary endpoint was postoperative CSF leak rate. We reviewed 198 consecutive patients who underwent 219 EETS for pituitary adenoma from February 2007 and July 2018. Intraoperative CSF leak occurred in 47 cases (21.5%). A total of 33 postoperative CSF leaks were observed (15.1%). A reduction of postoperative CSF leaks in the TachoSil application group compared to the conventional technique using Duraseal was observed (7.7% and 18.2%, respectively; p = 0.062; Pearson exact test) although non-statistically significant. Two patients required lumbar drainage, and no revision repair was necessary to treat postoperative CSF rhinorrhea in Tachosil group. Fibrin-coated collagen fleece patching may be a valuable method to prevent postoperative cerebrospinal fluid (CSF) leaks during EETS for pituitary adenoma resection.
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