Calcium pumping into the endoplasmic reticulum (ER) lumen is thought to be coupled to a countertransport of protons through sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) and the members of the ClC family of chloride channels. However, pH in the ER lumen remains neutral, which suggests a mechanism responsible for proton re-entry. We studied whether cation-proton exchangers could act as routes for such a re-entry. ER Ca(2+) uptake was measured in permeabilized immortalized hypothalamic neurons, primary rat cortical neurons and mouse cardiac fibers. Replacement of K(+) in the uptake solution with Na(+) or tetraethylammonium led to a strong inhibition of Ca(2+) uptake in neurons and cardiomyocytes. Furthermore, inhibitors of the potassium-proton exchanger (quinine or propranolol) but not of the sodium-proton exchanger reduced ER Ca(2+) uptake by 56-82%. Externally added nigericin, a potassium-proton exchanger, attenuated the inhibitory effect of propranolol. Inhibitors of small conductance calcium-sensitive K(+) (SK(Ca)) channels (UCL 1684, dequalinium) blocked the uptake of Ca(2+) by the ER in all preparations by 48-94%, whereas inhibitors of other K(+) channels (IK(Ca), BK(Ca) and K(ATP)) had no effect. Fluorescence microscopy and western blot analysis revealed the presence of both SK(Ca) channels and the potassium-proton exchanger leucine zipper-EF-hand-containing transmembrane protein 1 (LETM1) in ER in situ and in the purified ER fraction. The data obtained demonstrate that SK(Ca) channels and LETM1 reside in the ER membrane and that their activity is essential for ER Ca(2+) uptake.

Neuropoietin (NP) is a member of the gp130 cytokine family that is closely related to cardiotrophin-1(CT-1) and shares functional and structural features with other family members, including ciliary neurotrophic factor (CNTF) and cardiotrophin-like cytokine (CLC). Studies have shown that NP can play a role in the development of the nervous system, as well as affect adipogenesis and fat cell function. However, the signaling mechanisms utilized by NP in adipocytes have not been examined. In our present studies, we demonstrate that NP-induced activation of STAT3 tyrosine phosphorylation is independent of leukemia inhibitory factor receptor (LIFR) phosphorylation and degradation. Although it is widely accepted that NP signals via the LIFR, our studies reveal that NP results in phosphorylation of gp130, but not LIFR. These observations suggest that the profound effects that NP has on adipocytes are not mediated via LIFR signaling.

CLH-3 is a meiotic cell cycle-regulated ClC Cl- channel that is functionally expressed in oocytes of the nematode Caenorhabditis elegans. CLH-3a and CLH-3b are alternatively spliced variants that have identical intramembrane regions, but which exhibit striking differences in their N- and C-termini. Structural and functional studies indicate that N- and C-terminal domains modulate ClC channel activity. We therefore postulated that alternative splicing of CLH-3 would alter channel gating and physiological functions. To begin testing this hypothesis, we characterized the biophysical properties of CLH-3a and CLH-3b expressed heterologously in HEK293 cells. CLH-3a activates more slowly and requires stronger hyperpolarization for activation than CLH-3b. Depolarizing conditioning voltages dramatically increase CLH-3a current amplitude and induce a slow inactivation process at hyperpolarized voltages, but have no significant effect on CLH-3b activity. CLH-3a also differs significantly in its extracellular Cl- and pH sensitivity compared to CLH-3b. Immunofluorescence microscopy demonstrated that CLH-3b is translationally expressed during all stages of oocyte development, and furthermore, the biophysical properties of the native oocyte Cl- current are indistinguishable from those of heterologously expressed CLH-3b. We conclude that CLH-3b carries the oocyte Cl- current and that the channel probably functions in nonexcitable cells to depolarize membrane potential and/or mediate net Cl- transport. The unique voltage-dependent properties of CLH-3a suggest that the channel may function in muscle cells and neurones to regulate membrane excitability. We suggest that alternative splicing of CLH-3 N- and C-termini modifies the functional properties of the channel by altering the accessibility and/or function of pore-associated ion-binding sites.

Quercus pubescens Willd., a species distributed from Spain to southwest Asia, ranks high for drought tolerance among European oaks. Q. pubescens performs a role of outstanding significance in most Mediterranean forest ecosystems, but few mechanistic studies have been conducted to explore its response to environmental constrains, due to the lack of genomic resources. In our study, we performed a deep transcriptomic sequencing in Q. pubescens leaves, including de novo assembly, functional annotation and the identification of new molecular markers. Our results are a pre-requisite for undertaking molecular functional studies, and may give support in population and association genetic studies. 254,265,700 clean reads were generated by the Illumina HiSeq 2000 platform, with an average length of 98 bp. De novo assembly, using CLC Genomics, produced 96,006 contigs, having a mean length of 618 bp. Sequence similarity analyses against seven public databases (Uniprot, NR, RefSeq and KOGs at NCBI, Pfam, InterPro and KEGG) resulted in 83,065 transcripts annotated with gene descriptions, conserved protein domains, or gene ontology terms. These annotations and local BLAST allowed identify genes specifically associated with mechanisms of drought avoidance. Finally, 14,202 microsatellite markers and 18,425 single nucleotide polymorphisms (SNPs) were, in silico, discovered in assembled and annotated sequences. We completed a successful global analysis of the Q. pubescens leaf transcriptome using RNA-seq. The assembled and annotated sequences together with newly discovered molecular markers provide genomic information for functional genomic studies in Q. pubescens, with special emphasis to response mechanisms to severe constrain of the Mediterranean climate. Our tools enable comparative genomics studies on other Quercus species taking advantage of large intra-specific ecophysiological differences.

CLC-K chloride channels play a crucial role in kidney physiology and genetic mutations, affecting their function are responsible for severe renal salt loss in humans. Thus, compounds that selectively bind to CLC-Ka and/or CLC-Kb channels and modulate their activity may have a significant therapeutic potential. Here, we compare the biophysical and pharmacological behaviors of human CLC-K channels expressed either in HEK293 cells or in Xenopus oocytes and we show that CLC-K channel properties are greatly influenced by the biochemical environment surrounding the channels. Indeed, in HEK293 cells the potentiating effect of niflumic acid (NFA) on CLC-Ka/barttin and CLC-Kb/barttin channels seems to be absent while the blocking efficacy of niflumic acid and benzofuran derivatives observed in oocytes is preserved. The NFA block does not seem to involve the accessory subunit barttin on CLC-K1 channels. In addition, the sensitivity of CLC-Ks to external Ca(2+) is reduced in HEK293 cells. Based on our findings, we propose that mammalian cell lines are a suitable expression system for the pharmacological profiling of CLC-Ks.

Renal Fanconi syndrome (FS) is a generalized dysfunction of proximal tubular epithelial cells leading to the urinary leak of essential metabolites like phosphate, uric acid, glucose, amino acids and low molecular weight proteins. From inherited forms involving mutations on apparently unrelated genes to acquired forms induced by drugs, heavy metals or monoclonal immunoglobulin (Ig) light chains (LC), heterogeneous causalities of FS have complicated the understanding of this pathology for a long time. Experimental models of FS have allowed researchers to face the challenge and have helped unravel the main mechanisms disturbing proximal tubule reabsorption. Administration of cadmium to animals first demonstrated an inhibition of Na/K/ATPase activity, highlighting how a single toxic component could induce the general sodium-linked transport defect observed in FS. Today, genetically modified mice allow the development of reliable and reproducible experimental models for inherited or acquired forms of FS. One of the most exciting advances offered by these models is the unexpected major role of endocytosis in the function of the proximal tubule revealed by megalin and ClC-5 knockout mice. Using gene-targeted insertion, a transgenic mouse for LC-associated FS, the most frequent adult form of FS, has also been recently developed and represents a major step in the development of models of this pathology. Beyond deciphering molecular and cellular events at the origin of FS, these models also represent essential tools for the development of therapeutic strategies.

Resting skeletal muscle fibres have a large membrane Cl(-) conductance (G(Cl)) that dampens their excitability. Recently, however, muscle activity was shown to induce PKC-mediated reduction in G(Cl) in rat muscles of 40-90%. To examine the physiological significance of this PKC-mediated G(Cl) reduction for the function of muscles, this study explored effects of G(Cl) reductions on contractile endurance in isolated rat muscles. Contractile endurance was assessed from the ability of muscle to maintain force during prolonged stimulation under conditions when G(Cl) was manipulated by: (i) inhibition of PKC, (ii) reduction of solution Cl(-) or (iii) inhibition of ClC-1 Cl(-) channels using 9-anthracene-carboxylic acid (9-AC). Experiments showed that contractile endurance was optimally preserved by reductions in G(Cl) similar to what occurs in active muscle. Contrastingly, further G(Cl) reductions compromised the endurance. The experiments thus show a biphasic relationship between G(Cl) and contractile endurance in which partial G(Cl) reduction improves endurance while further G(Cl) reduction compromises endurance. Intracellular recordings of trains of action potentials suggest that this biphasic dependency of contractile endurance on G(Cl) reflects that lowering G(Cl) enhances muscle excitability but low G(Cl) also increases the depolarisation of muscle fibres during excitation and reduces their ability to re-accumulate K(+) lost during excitation. If G(Cl) becomes very low, the latter actions dominate causing reduced endurance. It is concluded that the PKC-mediated ClC-1 channel inhibition in active muscle reduces G(Cl) to a level that optimises contractile endurance during intense exercise.

1. ClC-3 encodes a volume-regulated Cl- channel (ICl,vol) in heart. We studied the regulation of native and recombinant cardiac ICl,vol by intracellular cyclic AMP (cAMPi). 2. Symmetrical high Cl- concentrations were used to effectively separate outwardly rectifying ICl,vol from other non-rectifying Cl- currents, such as the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+-activated Cl- currents (ICl,CFTR and ICl,Ca, respectively), which are concomitantly expressed in cardiac myocytes. 3. 8-Bromo-cyclic AMP (8-Br-cAMP) significantly inhibited ICl,vol in most guinea-pig atrial myocytes. In approximately 30 % of the atrial myocytes examined, 8-Br-cAMP increased macroscopic Cl- currents. However, the 8-Br-cAMP-stimulated difference currents exhibited a linear current-voltage (I-V ) relation, consistent with activation of ICl,CFTR, not ICl,vol. 4. In canine atrial myocytes, isoprenaline (1 microM) consistently reduced ICl,vol in Ca2+-free hypotonic bath solutions with strong intracellular Ca2+ (Ca2+i) buffering. In Ca2+-containing hypotonic bath solutions with weak Ca2+i buffering, however, isoprenaline increased net macroscopic Cl- currents. Isoprenaline-stimulated difference currents were not outwardly rectifying, consistent with activation of ICl,Ca, not ICl, vol. 5. In NIH/3T3 cells transfected with gpClC-3 (the gene encoding ICl,vol), 8-Br-cAMP consistently inhibited ICl,ClC-3. These effects were prevented by a protein kinase A (PKA) inhibitor, KT5720, or by mutation of a single consensus protein kinase C (PKC) phosphorylation site (S51A) on the N-terminus of ClC-3, which also mediates PKC inhibition of ICl,ClC-3. 6. We conclude that cAMPi causes inhibition of ICl,vol in mammalian heart due to cross-phosphorylation of the same PKC consensus site on ClC-3 by PKA. Our results suggest that contamination of macroscopic ICl,vol by ICl,CFTR and/or ICl,Ca may account for some of the inconsistent and controversial effects of cAMPi on ICl,vol previously reported in native cardiac myocytes.

ClC Cl(-) channels in endosomes, synaptosomes, lysosomes, and beta-cell insulin granules provide charge neutralization support for the functionally indispensable acidification of the luminal interior by electrogenic H(+)-ATPases (Jentsch, T. J., Stein, V., Weinreich, F., and Zdebik, A. A. (2002) Physiol. Rev. 82, 503-568). Regulation of ClC activity is, therefore, of widespread biological significance (Forgac, M. (1999) J. Biol. Chem. 274, 12951-12954). We now ascribe just such a regulatory function to the increases in cellular levels of inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P(4)) that inevitably accompany activation of the ubiquitous Ins(1,4,5)P(3) signaling pathway. We used confocal imaging to record insulin granule acidification in single mouse pancreatic beta-cells. Granule acidification was reduced by perfusion of single cells with 10 microm Ins(3,4,5,6)P(4) (the concentration following receptor activation), whereas at 1 microm ("resting" levels), Ins(3,4,5,6)P(4) was ineffective. This response to Ins(3,4,5,6)P(4) was not mimicked by 100 microm Ins(1,4,5,6)P(4) or by 100 microm Ins(1,3,4,5,6)P(5). Ins(3,4,5,6)P(4) did not affect granular H(+)-ATPase activity or H(+) leak, indicating that Ins(3,4,5,6)P(4) instead inhibited charge neutralization by ClC. The Ins(3,4,5,6)P(4)-mediated inhibition of vesicle acidification reduced exocytic release of insulin as determined by whole-cell capacitance recordings. This may impinge upon type 2 diabetes etiology. Regulatory control over vesicle acidification by this negative signaling pathway in other cell types should be considered.

BACKGROUND

Single-incision laparoscopic surgery has been proposed as a minimally invasive technique with the advantages of fewer scars and reduced pain. The aim of this study was to perform a systematic review and meta-analysis of prospective randomized clinical trials of single-access laparoscopic cholecystectomy (SALC) versus classic laparoscopic cholecystectomy (CLC).

METHODS

All randomized controlled trials were identified through electronic searches (MEDLINE, PubMed, SAGES, and Cochrane Central Register of Controlled Trials) up to October 2011. Methodologically appropriate clinical trials identified in the search process were included in a meta-analysis to provide a pooled estimate of effect.

RESULTS

Nine true randomized controlled trials were included in the analysis and reported a total of 695 patients, divided into the SALC group of 362 patients and the CLC group of 333 patients. Median operating time was longer with 57 minutes in SALC versus 45 minutes in CLC (P=0.00001). There was no significant difference in length of stay (SALC 1.36 d vs. CLC 1.15 d, P=0.18). Conversion to laparotomy in either group was similar; however, in 18 of 66 SALC patients an additional instrument was used, compared with 1 of 67 CLC patients (P=0.0003). Complications were not significant different [16% in SALC vs. 12% in the CLC group (P=0.74)]. Median postoperative pain with the visual analog scale score was 3.8 points in SALC versus 3.15 points in the CLC group (P=0.48). Cosmetic satisfaction was significantly more satisfying with 9 points favoring SALC versus 0 points favoring CLC (P=0.0005) in contrast to the quality-of-life questionnaire where there was no significant difference in patient overall satisfaction between SALC and CLC groups (P=0.0515).

CONCLUSIONS

SALC required longer operative times than CLC without significant benefits in patient overall satisfaction, postoperative pain, and hospital stay. Only satisfaction with the cosmetic result showed a significantly higher preference towards SALC.

The ability to maintain cellular volume is an important general physiological function. Swelling induced by hypotonic stress results in the opening of channels, through which ions exit with accompanying water loss (regulatory volume decrease, RVD). RVD has been shown to occur in mammalian sperm, primarily through the opening of quinine-sensitive potassium channels. However, as yet, direct evidence for the participation of anion channels in sperm RVD has been lacking. The chloride channel type ClC-3 is believed to be involved in RVD in other cell types. Using electronic cell sizing for cell volume measurement, the following results were obtained. (i) The anion channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), tamoxifen and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) increased hypotonic swelling in concentration-dependent fashion, whereas verapamil (P-glycoprotein inhibitor) had little effect. The most potent, NPPB and DIDS, blocked RVD without affecting cell membrane integrity at effective concentrations. (ii) When gramicidin was included to dissipate Na+/K+ gradients, major secondary swelling was observed under hypotonic conditions. This secondary swelling could be reduced by NPPB, and suppressed completely by replacing chloride in the medium with sulphate, an ion which does not pass through chloride channels. It was deduced that the initial hypotonic swelling activated an anion channel through which chloride ions could then enter freely down a concentration gradient, owing to the lack of a counter-gradient of potassium. (iii) Taurine, an osmolyte often involved in RVD, does not appear to play a role in sperm RVD because lengthy preincubation with taurine did not alter sperm RVD response. Our observations provide direct evidence that a chloride channel (possibly ClC-3) is involved in the process of volume regulation in mammalian sperm.

OBJECTIVE

Cholangiolocellular carcinoma (CLC) is a rare subtype of intrahepatic cholangiocarcinoma as categorized according to the World Health Organization criteria. Recent study also revealed that CLC would originate from hepatic progenitor cells, which are liver-specific stem cells. We reviewed the imaging findings of CLCs, including those of intrahepatic metastases.

METHODS

Eight patients who underwent partial hepatectomy were included in this study. Images, including dynamic computed tomographic and magnetic resonance images, were reviewed.

RESULTS

On arterial phase images, the lesions appeared as masses exhibiting early and complete enhancement or predominantly peripheral enhancement. Retention of the contrast media in the lesions was observed in half of the lesions. The metastatic nodules had indistinct borders and showed early enhancement along with delayed central enhancement.

CONCLUSIONS

Although CLC with dense central fibrosis exhibits imaging features similar to those exhibited by intrahepatic cholangiocarcinoma, early enhancement in most lesions with persistent delayed enhancement is one of the characteristic imaging findings of this rare tumor.

In the past few years, the artificial pancreas-the commonly accepted term for closed-loop control (CLC) of blood glucose in diabetes-has become a hot topic in research and technology development. In the summer of 2014, we initiated a 6-month trial evaluating the safety of 24/7 CLC during free-living conditions.

Following an initial 1-month Phase 1, 14 individuals (10 males/4 females) with type 1 diabetes at three clinical centers in the United States and one in Italy continued with a 5-month Phase 2, which included 24/7 CLC using the wireless portable Diabetes Assistant (DiAs) developed at the University of Virginia Center for Diabetes Technology. Median subject characteristics were age 45 years, duration of diabetes 27 years, total daily insulin 0.53 U/kg/day, and baseline HbA1c 7.2% (55 mmol/mol).

Compared with the baseline observation period, the frequency of hypoglycemia below 3.9 mmol/L during the last 3 months of CLC was lower: 4.1% versus 1.3%, P < 0.001. This was accompanied by a downward trend in HbA1c from 7.2% (55 mmol/mol) to 7.0% (53 mmol/mol) at 6 months. HbA1c improvement was correlated with system use (Spearman r = 0.55). The user experience was favorable with identified benefit particularly at night and overall trust in the system. There were no serious adverse events, severe hypoglycemia, or diabetic ketoacidosis.

We conclude that CLC technology has matured and is safe for prolonged use in patients' natural environment. Based on these promising results, a large randomized trial is warranted to assess long-term CLC efficacy and safety.

Human ClC-2 Cl(-) (hClC-2) channels are activated by protein kinase A (PKA) and low extracellular pH(o). Both of these effects are prevented by the PKA inhibitor, myristoylated PKI. The aims of the present study were to identify the PKA phosphorylation site(s) important for PKA activation of hClC-2 at neutral and low pH(o) and to examine the relationship between PKA and low pH(o) activation. Recombinant hClC-2 with point mutations of consensus phosphorylation sites was prepared and stably expressed in HEK-293 cells. The responses to forskolin plus isobutylmethylxanthine at neutral and acidic pH(o) were studied by whole cell patch clamp in the presence and absence of phosphatase inhibitors. The double phosphorylation site (RRAT655(A) plus RGET691(A)) mutant hClC-2 lost PKA activation and low pH(o) activation. Either RRAT or RGET was sufficient for PKA activation of hClC-2 at pH(o) 7.4, as long as phosphatase inhibitors (cyclosporin A or endothal) were present. At pH(o) 6 only RGET was needed for PKA activation of hClC-2. Low pH(o) activation of hClC-2 Cl(-) channel activity was PKA-dependent, retained in RGET(A) mutant hClC-2, but lost in RRAT(A) mutant hClC-2. RRAT655(D) mutant hClC-2 was constitutively active and was further activated by PKA at pH(o) 7.4 and 6.0, consistent with the above findings. These results show that activation of hClC-2 is differentially regulated by PKA at two sites, RRAT655 and RGET691. Either RRAT655 or RGET691 was sufficient for activation at pH(o) 7.4. RGET, but not RRAT, was sufficient for activation at pH(o) 6.0. However, in the RGET691(D) mutant, there was PKA activation at pH(o) 6.0.

Diabetic kidney disease is initially associated with hypertension and increased urinary albumin excretion. The hypertension is mediated by enhanced volume expansion due to enhanced salt and water retention by the kidney. The increased urinary albumin is not only due to increased glomerular leak, but also to a decrease in albumin reabsorption by the proximal tubule. The precise molecular mechanisms underlying these two phenomena and whether there is any link between the increase in Na(+) retention and proteinuria remain unresolved. There is significant evidence to suggest that increased Na(+) retention by the proximal tubule Na(+)/H(+) exchanger isoform 3 (NHE3) can play a role in some forms of hypertension. Increased NHE3 activity in models of diabetes mellitus may explain, in part, the enhanced salt retention observed in patients with diabetic kidney disease. The NHE3 also plays a role in receptor-mediated albumin uptake in the proximal tubule. The uptake of albumin requires the assembly of a macromolecular complex that is thought to include the megalin/cubulin receptor, NHE3, the vacuolar type H(+)-ATPase (v-H(+)-ATPase), the Cl(-) channel ClC-5 and interactions with the actin cytoskeleton. The NHE3 seems to exist in two functionally distinct membrane domains, one involved with Na(+) reabsorption and the other involved in albumin uptake. The present review focuses on the evidence derived from in vivo studies, as well as complementary studies in cell culture models, for a dual role of NHE3 in both Na(+) retention and albumin uptake. We suggest a possible mechanism by which disruption of the proximal tubule albumin uptake mechanism in diabetes mellitus may lead to both increased Na(+) retention and proteinuria.

Although the function of rhythmic contractions in the vascular wall - vasomotion - is still under debate, it has been suggested to play a significant role for tissue oxygen homeostasis and under pathological conditions where tissue perfusion is affected. Vasomotion has further been suggested to be important for blood pressure control and has been shown to be reduced in diabetes. Vasomotion is initiated by the coordinated activation of smooth muscle cells (SMCs) in the vascular wall leading to rhythmic contractions. We have suggested the model for generation of this rhythmic activity and have shown that vasomotion initiates via interaction between intracellular calcium released from the sarcoplasmic reticulum and changes in membrane potential. Rhythmic changes in intracellular calcium induce, under certain conditions (in the presence of sufficient concentration of cGMP), changes in membrane potential that lock the electrically-connected SMCs into phase. Synchronized depolarization induces synchronous calcium influx and thus produces rhythmic contraction of blood vessels. I have demonstrated and characterized a new chloride channel in vascular SMCs, which has properties necessary to coordinate SMCs in the vascular wall. Chloride channels have been investigated for many years but remained somewhat in the shadow of cation channels. We know now the molecular structures of some chloride channels, i.e. GABA receptors, "cystic fibrosis transmem-brane conductance regulator" (CFTR) and the ClC chloride channel family. There is one particular group of chloride channels, the calcium activated chloride channels (CaCCs), whose molecular structure is debated still. There are currently no pharmacological tools that activate or inhibit CaCCs with any significant selectivity. The existence of CaCCs in almost all cells in the body has been known for many years based on electrophysiological and other functional studies. CaCCs have been suggested to be important for regulation of membrane potential and cellular volume, as well as for body homeostasis. CaCCs are well characterized in vascular tissues but only at the functional level. The lack of their molecular structure makes it difficult to study the clinical significance of these channels. Based on patch clamp measurements of ion currents, I have previously characterized in SMCs a chloride current with unique properties. This chloride current activated by cGMP, has very high sensitivity to calcium and can be inhibited by low concentrations of zinc ions, while the traditional inhibitors of CaCCs affect this current only at very high concentrations. This cGMP-dependent, calcium-activated chloride current has a linear volt-age-dependence, which differs from previously characterized CaCCs, and it has characteristic anion permeability. This current has been detected in SMCs isolated from a number of different vascular beds but, importantly, it has not been detected in pulmonary arteries. Moreover, this current has been shown in SMCs isolated intestine indicating its broad distribution. Based on unique characteristics I have suggested that the cGMP-dependent calcium-activated chloride current can synchronize SMCs in the vascular wall and that bestrophin protein could be the molecular substrate for this current. Bestrophin has been characterized first as a gene in which mutations cause vitelliform macular dystrophy (VMD) or Best diseases. Based on heterologous expression it has been suggested that bestrophin is a chloride channel. This question is nevertheless controversial since caution should be taken in heterologous expression of calcium-activated chloride channel candidates. The presence of chloride channels in virtually all living cells is an essential problem as well as the dependence of ion channel properties on the complex interaction of many cellular proteins. I was the first who coupled the endogenous chloride current to one of four known bestrophin isoforms. PCR and Western blot studies on different blood vessels demonstrated the presence of bestrophin-3 protein with the exception of pulmonary arteries where the cGMP-dependent current is also absent). There was a strong indication that bestrophin-3 expression could be essential for the cGMP-dependent calcium-activated chloride current. To couple bestrophin-3 expression and this current I have used small interfering RNA (siRNA) technique to downregulate the expression of the candidate (bestrophin-3) and have studied the effect of this specific downregulation on chloride currents. I showed that bestrophin-3 expression is associated with the cGMP-dependent calcium-activated chloride current. This study does not tell us whether bestrophin-3 forms the channel or it is an essential subunit but the previous mutagenic experiments suggested the first possibility. Electrical communication between SMCs is essential for successful synchronization and depends on channels between the cells called gap junctions. The majority of cardiovascular diseases (e.g. hypertension and atherosclerosis) are associated with defects in intercellular communications or in gap junction regulation. The molecular mechanisms responsible for these defects are un-known because of lack of specific experimental tools. Our comprehensive study on the often used gap junction inhibitors heptanol and 18β-glycyrrhetinic acid demonstrated unspecific effects of these drugs at the concentrations where they have no or little gap junctions effects. Other drugs, e.g. 18α-glycyrrhetinic acid and connexin-mimetic peptides are better to inhibit gap junctions but also have demonstrated unspecific effects. Previous studies suggested that channels and transporters in the cell membrane do not function independently but interact as functional units in the spatially restricted areas of the cell. I have demonstrated a close functional interaction between gap junctions and Na+,K+-ATPase, Na+/Ca2+-exchanger and ATP-dependent K+ channels in the spatially restricted manner. I have shown that inhibition of the ouabain-sensitive Na+, K+-ATPase inhibits calcium efflux by the Na+/Ca2+-exchanger and this leads to the local elevation of intracellular calcium and inhibition of intercellular communications. This explains the inhibitory action of ouabain on vasomotion. I have also found that the ATP-dependent K+ channel is an important player in this functional unit and this interaction is reciprocal, since K+ channel supplies Na+, K+-ATPase with K+ ions while the ATP-dependent K+ channel current also regulates the Na+, K+-ATPase. This dissertation is based on nine scientific publications where I have suggested the model for generation of vasomotion and characterized the essential elements of this model.

BACKGROUND

Transvaginal video-assisted cholecystectomy (TVC) has so far not been prospectively evaluated using an internationally recognized health-related quality of life (HRQoL) assessment. We report the results of a prospectively studied cohort of patients with clinical and quality of life data.

METHODS

Prospectively controlled study of 128 patients undergoing TVC and 147 patients with conventional laparoscopic cholecystectomy (CLC). Data reported include patient demography, body mass index, anesthetic risk score (ASA), laboratory data, surgical times, length of hospital stay, pain score, analgesic medication used, complications, and quality of life scores using the combined method of SF-36 and GIQoL.

RESULTS

Ninety-five TVC and 96 CLC patients fully completed pre- and postoperative HRQoL questionnaires. Patients with incomplete or missing questionnaires were excluded as well as patients with signs of acute cholecystitis. Differences included cardiovascular comorbidity and previous surgical procedures, but there was no difference in age (p = 0.4), body mass index (p = 0.4), ASA grade (p = 0.4), or preoperative quality of life. No difference was seen in laboratory data, surgical times, or length of hospital stay. Pain score and analgesic medication showed a clear trend and significant differences in favor of TVC. There was no difference in complications. Quality of life and postoperative sexual function did not show any differences between the two groups.

CONCLUSIONS

This is the first study to report HRQoL outcomes after TVC using a recognized combined HRQoL assessment method. Although differences do exist in patient comorbidity and previous surgical experience, both groups were comparable. Less postoperative pain and no difference in HRQoL in TVC patients underlines this new procedure as a feasible standard approach in female patients. This study also is the first to differentiate between acute cholecystitis and symptomatic cholecystolithiasis in patients undergoing TVC.

We investigated the influence of small- and large-dose capsaicin in modulating systemic inflammatory responses during different stages of sepsis in rats. Rats were divided into six groups: group C, control; group S, sepsis; group CLC, small dose of capsaicin (1 mg/kg subcutaneously); group SLC, small dose of capsaicin plus sepsis; group CHC, large dose of capsaicin (150 mg/kg subcutaneously); group SHC, large dose of capsaicin plus sepsis. Rats were made septic by cecal ligation and puncture (CLP). Each group was subdivided into two subgroups. The animals were killed at 9 or 18 h after CLP. Plasma concentrations of calcitonin gene-related peptide (CGRP), tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-10, and total nitrite/nitrate (NOx) were measured. Superoxide dismutase and malondialdehyde (MDA) were determined in liver, lung, and heart tissues. CGRP was increased in groups S, CLC, and SLC when compared with the other groups. In the SLC group, plasma concentrations of TNF-alpha, IL-6, NOx, and tissue MDA levels were reduced and IL-10 level was increased when compared with groups S and SHC 18 h after CLP (P < 0.05). Small-dose capsaicin treatment increased antiinflammatory IL-10 levels and attenuated the increases in proinflammatory cytokines, NOx, and tissue MDA in septic rats.

BACKGROUND

Francisella tularensis is a category-A select agent and is responsible for tularemia in humans and animals. The surface components of F. tularensis that contribute to virulence are not well characterized. An electron-dense capsule has been postulated to be present around F. tularensis based primarily on electron microscopy, but this specific antigen has not been isolated or characterized.

RESULTS

A capsule-like complex (CLC) was effectively extracted from the cell surface of an F. tularensis live vaccine strain (LVS) lacking O-antigen with 0.5% phenol after 10 passages in defined medium broth and growth on defined medium agar for 5 days at 32°C in 7% CO₂. The large molecular size CLC was extracted by enzyme digestion, ethanol precipitation, and ultracentrifugation, and consisted of glucose, galactose, mannose, and Proteinase K-resistant protein. Quantitative reverse transcriptase PCR showed that expression of genes in a putative polysaccharide locus in the LVS genome (FTL_1432 through FTL_1421) was upregulated when CLC expression was enhanced. Open reading frames FTL_1423 and FLT_1422, which have homology to genes encoding for glycosyl transferases, were deleted by allelic exchange, and the resulting mutant after passage in broth (LVSΔ1423/1422_P10) lacked most or all of the CLC, as determined by electron microscopy, and CLC isolation and analysis. Complementation of LVSΔ1423/1422 and subsequent passage in broth restored CLC expression. LVSΔ1423/1422_P10 was attenuated in BALB/c mice inoculated intranasally (IN) and intraperitoneally with greater than 80 times and 270 times the LVS LD₅₀, respectively. Following immunization, mice challenged IN with over 700 times the LD₅₀ of LVS remained healthy and asymptomatic.

CONCLUSIONS

Our results indicated that the CLC may be a glycoprotein, FTL_1422 and -FTL_1423 were involved in CLC biosynthesis, the CLC contributed to the virulence of F. tularensis LVS, and a CLC-deficient mutant of LVS can protect mice against challenge with the parent strain.

The widely expressed chloride channel ClC-2 is stimulated by the serum and glucocorticoid inducible kinase SGK1. The SGK1-dependent regulation of several carriers involves the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments explored whether SGK1-dependent regulation of ClC-2 similarly involves PIKfyve. The conductance of Xenopus oocytes is increased more than eightfold by ClC-2 expression. In ClC-2-expressing oocytes, but not in water-injected oocytes, the current was further enhanced by coexpression of either, PIKfyve or constitutively active (S422D)SGK1. Coexpression of the inactive SGK1 mutant (K127N)SGK1 did not significantly alter the current in ClC-2-expressing oocytes and abrogated the stimulation of the current by PIKfyve-coexpression. The stimulating effect of PIKfyve was abolished by replacement of the serine with alanine in the SGK1 consensus sequence ((S318A)PIKfyve). Coexpression of (S318A)PIKfyve significantly blunted the stimulating effect of (S422D)SGK1 on ClC-2-activity. In conclusion, PIKfyve is a potent stimulator of ClC-2-activity and contributes to SGK1-dependent regulation of ClC-2.

Primary focal segmental glomerulosclerosis (FSGS) is one of the major causes of steroid-resistant nephrotic syndrome, and renal prognosis in patients with steroid-resistant FSGS is poor. It has been long speculated that a circulating permeability factor should be implicated in the pathogenesis of the disease because a substantial portion of the patients with primary FSGS experience recurrence shortly after transplantation. Although molecules such as cardiotrophin-like cytokine 1 (CLC-1) and anti-CD40 antibody have been proposed to be potential circulating permeability factors, a definitive factor remains to be discovered. Soluble urokinase-type plasminogen activator receptor (suPAR) has attracted substantial attention and garnered scrutiny by renal researchers since Reiser's group suggested that it was linked to the pathogenesis of primary FSGS and that it might be useful as a diagnostic biomarker. A number of different cohort studies have shown that serum suPAR levels are negatively associated with renal function and can scarcely differentiate FSGS from the other glomerular/renal diseases. In contrast to initial studies, several in vivo studies investigating the effects of forced suPAR upregulation could not show the induction of proteinuria or podocyte injury. Currently it is suggested that a different form of suPAR, which cannot be measured by presently available enzyme-linked immunosorbent assay, might be the culprit; however, it remains to be determined whether this is the case. Because a circulating permeability factor might be a useful biomarker for diagnosing FSGS as well as a potent therapeutic target for primary and recurrent FSGS, further dedicated work will be needed.

OBJECTIVE

Statins and fibrates can produce mild to life-threatening skeletal muscle damage. Resting chloride channel conductance (gCl), carried by the ClC-1 channel, is reduced in muscles of rats chronically treated with fluvastatin, atorvastatin or fenofibrate, along with increased resting cytosolic calcium in statin-treated rats. A high gCl, controlled by the Ca(2+)-dependent protein kinase C (PKC), maintains sarcolemma electrical stability and its reduction alters muscle function. Here, we investigated how statins and fenofibrate impaired gCl.

METHODS

In rats treated with fluvastatin, atorvastatin or fenofibrate, we examined the involvement of PKC in gCl reduction by the two intracellular microelectrodes technique and ClC-1 mRNA level by quantitative real time-polymerase chain reaction. Direct drug effects were tested by patch clamp analysis on human ClC-1 channels expressed in human embryonic kidney (HEK) 293 cells.

RESULTS

Chelerythrine, a PKC inhibitor, applied in vitro on muscle dissected from atorvastatin-treated rats fully restored gCl, suggesting the involvement of this enzyme in statin action. Chelerythrine partially restored gCl in muscles from fluvastatin-treated rats but not in those from fenofibrate-treated rats, implying additional mechanisms for gCl impairment. Accordingly, a decrease of ClC-1 channel mRNA was found in both fluvastatin- and fenofibrate-treated rat muscles. Fenofibric acid, the in vivo metabolite of fenofibrate, but not fluvastatin, rapidly reduced chloride currents in HEK 293 cells.

CONCLUSIONS

Our data suggest multiple mechanisms underlie the effect of statins and fenofibrate on ClC-1 channel conductance. While statins promote Ca(2+)-mediated PKC activation, fenofibrate directly inhibits ClC-1 channels and both fluvastatin and fenofibrate impair expression of mRNA for ClC-1.

Sertoli cell secretory activities are highly dependent on ion channel functions and critical to spermatogenesis. The steroid hormone 1alpha,25(OH)2-vitamin D3 (1,25(OH)2-D3) stimulates exocytosis in different cell systems by activating a nongenotropic vitamin D receptor (VDR). Here, we described 1,25(OH)2-D3 stimulation of secretion via Cl(-) channel activation in the mouse immature Sertoli cell line TM4. 1,25(OH)2-D3 potentiation of chloride currents was dependent on hormone concentration, and correlated with a significant increase in whole-cell capacitance within 20-40 min. In addition, Cl(-) currents were potentiated by the nongenomic VDR agonist 1alpha,25(OH)2 lumisterol D3 (JN), while 1,25(OH)2-D3 potentiation of channels was suppressed by nongenomic VDR antagonist 1beta,25(OH)2-vitamin D3 (HL). Treatment of TM4 cells with PKC and PKA activators PMA and forskolin respectively, increased Cl(-) currents significantly, while PKC and PKA inhibitors Go6983 and H-89, respectively, abolished 1,25(OH)2-D3 stimulation of Cl(-) currents, suggesting phosphorylation pathways in 1,25(OH))2-D3 mediated channel responses. RT-PCR demonstrated the expression of outwardly rectifying ClC-3 channels in TM4 cells. Taken together, our results demonstrate a PKA/PKC-dependent 1,25(OH)2-D3/VDR nongenotropic pathway leading to Cl(-) channel and exocytosis activation in Sertoli cells. We conclude that 1,25(OH)2-D3 appears to be a modulator of male reproductive functions at least in part by stimulating Sertoli cell secretory functions.

Cl- currents were observed under whole cell clamp conditions in cells of the rat cortical collecting duct (CCD), connecting tubule (CNT), and thick ascending limb of Henle's loop (TALH). These currents were much larger in intercalated cells compared with principal cells of the CCD and were also larger in the TALH and in the CNT compared with the CCD. The conductance had no strong voltage dependence, and steady-state currents were similar in inward and outward directions with similar Cl- concentrations on both sides of the membrane. Current transients were observed, particularly at low Cl- concentrations, which could be explained by solute depletion and concentration in fluid layers next to the membrane. The currents had a remarkable selectivity among anions. Among halides, Br- and F- conductances were only 15% of that of Cl-, and I- conductance was immeasurably small. SCN- and OCN- conductances were approximately 50%, and aspartate, glutamate, and methanesulfonate conductance was approximately 5% that of Cl-. No conductance could be measured for any other anion tested, including NO3-, HCO3-, formate, acetate, or isethionate; NO3- and I- appeared to block the channels weakly. Conductances were diminished by lowering the extracellular pH to 6.4. The properties of the conductance fit best with those of the cloned renal anion channel ClC-K2 and likely reflect the basolateral Cl- conductances of the cells of these nephron segments.