Alcohol disrupts levels and function of the cystic fibrosis transmembrane conductance regulator to promote development of pancreatitis.

BACKGROUND & AIMS: Excessive consumption of ethanol is one of the most common causes of acute and chronic pancreatitis. Alterations to the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) also cause pancreatitis. However, little is known about the role of CFTR in the pathogenesis of alcohol-induced pancreatitis. METHODS: We measured CFTR activity based on chloride concentrations in sweat from patients with cystic fibrosis, patients admitted to the emergency department because of excessive alcohol consumption, and healthy volunteers. We measured CFTR levels and localization in pancreatic tissues and in patients with acute or chronic pancreatitis induced by alcohol. We studied the effects of ethanol, fatty acids, and fatty acid ethyl esters on secretion of pancreatic fluid and HCO3(-), levels and function of CFTR, and exchange of Cl(-) for HCO3(-) in pancreatic cell lines as well as in tissues from guinea pigs and CFTR knockout mice after administration of alcohol. RESULTS: Chloride concentrations increased in sweat samples from patients who acutely abused alcohol but not in samples from healthy volunteers, indicating that alcohol affects CFTR function. Pancreatic tissues from patients with acute or chronic pancreatitis had lower levels of CFTR than tissues from healthy volunteers. Alcohol and fatty acids inhibited secretion of fluid and HCO3(-), as well as CFTR activity, in pancreatic ductal epithelial cells. These effects were mediated by sustained increases in concentrations of intracellular calcium and adenosine 3',5'-cyclic monophosphate, depletion of adenosine triphosphate, and depolarization of mitochondrial membranes. In pancreatic cell lines and pancreatic tissues of mice and guinea pigs, administration of ethanol reduced expression of CFTR messenger RNA, reduced the stability of CFTR at the cell surface, and disrupted folding of CFTR at the endoplasmic reticulum. CFTR knockout mice given ethanol or fatty acids developed more severe pancreatitis than mice not given ethanol or fatty acids. CONCLUSIONS: Based on studies of human, mouse, and guinea pig pancreata, alcohol disrupts expression and localization of the CFTR. This appears to contribute to development of pancreatitis. Strategies to increase CFTR levels or function might be used to treat alcohol-associated pancreatitis.

[Fast three-dimensional two-photon scanning methods for studying neuronal physiology on cellular and network level]. / Háromdimenziós, gyors, kétfoton-pásztázó eljárások sejt- és hálózatszintu idegsejtvizsgálatokhoz.

Erratum to the article published on December 27th 2015 in Issue 52 of Orvosi Hetilap [Orv. Hetil., 2015, 156(52), 2120-2126, DOI: 10.1556/650.2015.30329]. The name of Dávid Mezey was not correctly typed. The corresponding author asked for the following correction to be published.

[Fast three-dimensional two-photon scanning methods for studying neuronal physiology on cellular and network level]. / Háromdimenziós, gyors, kétfoton-pásztázó eljárások sejt- és hálózatszintu idegsejtvizsgálatokhoz.

INTRODUCTION: Two-photon microscopy is the ideal tool to study how signals are processed in the functional brain tissue. However, early raster scanning strategies were inadequate to record fast 3D events like action potentials. AIM: The aim of the authors was to record various neuronal activity patterns with high signal-to-noise ratio in an optical manner. METHOD: Authors developed new data acquisition methods and microscope hardware. RESULTS: Multiple Line Scanning enables the experimenter to select multiple regions of interests, doing this not just increases repetition speed, but also the signal-to-noise ratio of the fluorescence transients. On the same principle, an acousto-optical deflector based 3D scanning microscope has been developed with a sub-millisecond temporal resolution and a millimeter z-scanning range. Its usability is demonstrated by obtaining 3D optical recordings of action potential backpropagation in several hundred micrometers long neuronal processes of single neurons and by 3D random-access scanning of Ca(2+) transients in hundreds of neurons in the mouse visual cortex. CONCLUSIONS: Region of interest scanning enables high signal-to-noise ratio and repetition speed, while keeping good depth penetration of the two-photon microscopes.

Na+/Ca2+ exchangers regulate the migration and proliferation of human gastric myofibroblasts.

Gastrointestinal myofibroblasts are contractile, electrically nonexcitable, transitional cells that play a role in extracellular matrix production, in ulcer healing, and in pathophysiological conditions they contribute to chronic inflammation and tumor development. Na+/Ca2+ exchangers (NCX) are known to have a crucial role in Ca2+ homeostasis of contractile cells, however, no information is available concerning the role of NCX in the proliferation and migration of gastrointestinal myofibroblasts. In this study, our aim was to investigate the role of NCX in the Ca2+ homeostasis, migration, and proliferation of human gastrointestinal myofibroblasts, focusing on human gastric myofibroblasts (HGMs). We used microfluorometric measurements to investigate the intracellular Ca2+ and Na+ concentrations, PCR analysis and immunostaining to show the presence of the NCX, patch clamp for measuring NCX activity, and proliferation and migration assays to investigate the functional role of the exchanger. We showed that 53.0±8.1% of the HGMs present Ca2+ oscillations, which depend on extracellular Ca2+ and Na+, and can be inhibited by NCX inhibitors. NCX1, NCX2, and NCX3 were expressed at both mRNA and protein levels in HGMs, and they contribute to the intracellular Ca2+ and Na+ homeostasis as well, regardless of the oscillatory activity. NCX inhibitors significantly blocked the basal and insulin-like growth factor II-stimulated migration and proliferation rates of HGMs. In conclusion, we showed that NCX plays a pivotal role in regulating the Ca2+ homeostasis, migration, and proliferation of HGMs. The inhibition of NCX activity may be a potential therapeutic target in hyperproliferative gastric diseases.

Trypsin reduces pancreatic ductal bicarbonate secretion by inhibiting CFTR Cl⁻ channels and luminal anion exchangers.

BACKGROUND & AIMS: The effects of trypsin on pancreatic ductal epithelial cells (PDECs) vary among species and depend on the localization of proteinase-activated receptor 2 (PAR-2). We compared PAR-2 localization in human and guinea-pig PDECs, and used isolated guinea pig ducts to study the effects of trypsin and a PAR-2 agonist on bicarbonate secretion. METHODS: PAR-2 localization was analyzed by immunohistochemistry in guinea pig and human pancreatic tissue samples (from 15 patients with chronic pancreatitis and 15 without pancreatic disease). Functionally, guinea pig PDECs were studied by microperfusion of isolated ducts, measurements of intracellular pH and intracellular Ca(2+) concentration, and patch clamp analysis. The effect of pH on trypsinogen autoactivation was assessed using recombinant human cationic trypsinogen. RESULTS: PAR-2 localized to the apical membrane of human and guinea pig PDECs. Trypsin increased intracellular Ca(2+) concentration and intracellular pH and inhibited secretion of bicarbonate by the luminal anion exchanger and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. Autoactivation of human cationic trypsinogen accelerated when the pH was reduced from 8.5 to 6.0. PAR-2 expression was strongly down-regulated, at transcriptional and protein levels, in the ducts of patients with chronic pancreatitis, consistent with increased activity of intraductal trypsin. Importantly, in PAR-2 knockout mice, the effects of trypsin were markedly reduced. CONCLUSIONS: Trypsin reduces pancreatic ductal bicarbonate secretion via PAR-2-dependent inhibition of the apical anion exchanger and the CFTR Cl(-) channel. This could contribute to the development of chronic pancreatitis by decreasing luminal pH and promoting premature activation of trypsinogen in the pancreatic ducts.

Sharp-wave ripple doublets induce complex dendritic spikes in parvalbumin interneurons in vivo.

Neuronal plasticity has been shown to be causally linked to coincidence detection through dendritic spikes (dSpikes). We demonstrate the existence of SPW-R-associated, branch-specific, local dSpikes and their computational role in basal dendrites of hippocampal PV+ interneurons in awake animals. To measure the entire dendritic arbor of long thin dendrites during SPW-Rs, we used fast 3D acousto-optical imaging through an eccentric deep-brain adapter and ipsilateral local field potential recording. The regenerative calcium spike started at variable, NMDA-AMPA-dependent, hot spots and propagated in both direction with a high amplitude beyond a critical distance threshold (~150 µm) involving voltage-gated calcium channels. A supralinear dendritic summation emerged during SPW-R doublets when two successive SPW-R events coincide within a short temporal window (~150 ms), e.g., during more complex association tasks, and generated large dSpikes with an about 2.5-3-fold amplitude increase which propagated down to the soma. Our results suggest that these doublet-associated dSpikes can work as a dendritic-level temporal and spatial coincidence detector during SPW-R-related network computation in awake mice.

Fast 3D Imaging of Spine, Dendritic, and Neuronal Assemblies in Behaving Animals.

Understanding neural computation requires methods such as 3D acousto-optical (AO) scanning that can simultaneously read out neural activity on both the somatic and dendritic scales. AO point scanning can increase measurement speed and signal-to-noise ratio (SNR) by several orders of magnitude, but high optical resolution requires long point-to-point switching time, which limits imaging capability. Here we present a novel technology, 3D DRIFT AO scanning, which can extend each scanning point to small 3D lines, surfaces, or volume elements for flexible and fast imaging of complex structures simultaneously in multiple locations. Our method was demonstrated by fast 3D recording of over 150 dendritic spines with 3D lines, over 100 somata with squares and cubes, or multiple spiny dendritic segments with surface and volume elements, including in behaving animals. Finally, a 4-fold improvement in total excitation efficiency resulted in about 500 × 500 × 650 µm scanning volume with genetically encoded calcium indicators (GECIs).

Microglia protect against brain injury and their selective elimination dysregulates neuronal network activity after stroke.

Microglia are the main immune cells of the brain and contribute to common brain diseases. However, it is unclear how microglia influence neuronal activity and survival in the injured brain in vivo. Here we develop a precisely controlled model of brain injury induced by cerebral ischaemia combined with fast in vivo two-photon calcium imaging and selective microglial manipulation. We show that selective elimination of microglia leads to a striking, 60% increase in infarct size, which is reversed by microglial repopulation. Microglia-mediated protection includes reduction of excitotoxic injury, since an absence of microglia leads to dysregulated neuronal calcium responses, calcium overload and increased neuronal death. Furthermore, the incidence of spreading depolarization (SD) is markedly reduced in the absence of microglia. Thus, microglia are involved in changes in neuronal network activity and SD after brain injury in vivo that could have important implications for common brain diseases.

The effects of acute blood loss on blood pressure recovery from the Valsalva maneuver.

Human baroreflex regulation plays an important role in stabilising blood pressure. Though we have several indices to quantify cardiovagal responses, sympathetic baroreflex gain remains difficult to assess. We investigated how the recently validated pressure recovery time (PRT) and sympathetic baroreflex gain (SBRS) derived from the Valsalva maneuver was influenced by acute blood loss. 26 healthy blood donors were included in the study (age 35 ± 15 years; 20 men). SBRS was derived from the blood pressure drop (SAP delta) and pressure recovery time during the Valsalva maneuver. Besides we calculated cardiovagal baroreflex parameters, the Valsalva ratio (VR) and a simplified baroreflex gain (VBRS). We compared these parameters before and after the withdrawal of 350-400 ml blood. The baseline systolic blood pressure was the same before and after blood donation (123 ± 17 vs 126 ± 23 mm Hg, NS). The minimum systolic pressure (SAP min) during phase III was significantly lower, and the SAP delta significantly greater after blood withdrawal (SAP min 83 ± 24 mm Hg vs 69 ± 27 mm Hg, p<0.001; SAP delta 41 ± 15 mm Hg vs 57 ± 16 mm Hg, p<0.001). PRT increased significantly (from 2.0 to 3.6s, p<0.006). SBRS did not change between the study conditions (24 ± 12 mm Hg/s vs 22 ± 10 mm Hg/s, NS), nor did the VR and the VBRS: In conclusion, after the acute loss of approximately 350-400 ml blood there was a greater blood pressure drop in phase II and III and a slower blood pressure recovery in phase IV of the Valsalva maneuver that resulted in an unchanged SBRS.