Regulation of human CLC-3 channels by multifunctional Ca2+/calmodulin-dependent protein kinase

The multifunctional calcium/calmodulin-dependent protein kinase II, CaMKII, has been shown to regulate chloride movement and cellular function in both excitable and non-excitable cells. We show that the plasma membrane expression of a member of the ClC family of Cl(-) channels, human CLC-3 (hCLC-3), a 90-kDa protein, is regulated by CaMKII. We cloned the full-length hCLC-3 gene from the human colonic tumor cell line T84, previously shown to express a CaMKII-activated Cl(-) conductance (I(Cl,CaMKII)), and transfected this gene into the mammalian epithelial cell line tsA, which lacks endogenous expression of I(Cl,CaMKII). Biotinylation experiments demonstrated plasma membrane expression of hCLC-3 in the stably transfected cells. In whole cell patch clamp experiments, autonomously active CaMKII was introduced into tsA cells stably transfected with hCLC-3 via the patch pipette. Cells transfected with the hCLC-3 gene showed a 22-fold increase in current density over cells expressing the vector alone. Kinase-dependent current expression was abolished in the presence of the autocamtide-2-related inhibitory peptide, a specific inhibitor of CaMKII. A mutation of glycine 280 to glutamic acid in the conserved motif in the putative pore region of the channel changed anion selectivity from I(-) > Cl(-) to Cl(-) > I(-). These results indicate that hCLC-3 encodes a Cl(-) channel that is regulated by CaMKII-dependent phosphorylation.     

Synaptotagmin I binds intestinal epithelial NHE3 and mediates cAMP- and Ca2+-induced endocytosis by recruitment of AP2 and clathrin

Apical membrane sodium hydrogen exchanger 3 (NHE3), a major pathway for non-nutrient-dependent intestinal Na(+) absorption, is tightly regulated by second messenger systems that affect its functional activity and membrane trafficking. However, the events and components involved in NHE3 regulation are only partially understood. We report that the adaptor protein synaptotagmin I (Syt I) plays a pivotal role in cAMP- and Ca(2+)-induced cargo recognition of NHE3 and initiation of its endocytosis. Both mouse small intestine (jejunum) and Caco-2BBe Syt I coimmunoprecipitated with NHE3, particularly following increases in cellular cAMP or Ca(2+). Following short interfering RNA (siRNA) suppression of Syt I expression, cAMP- and Ca(2+)-induced inhibition of NHE3 activity were still observed but NHE3 endocytosis was blocked, as assessed by (22)Na influx and apical membrane biotin labeling, respectively. Similar effects on NHE3 inhibition and endocytosis were observed by siRNA suppression of either the mu-subunit of the adaptor protein 2 (AP2) complex or the heavy chain of clathrin. Coimmunoprecipitation analyses of NHE3 with these adaptor proteins revealed that cAMP- and Ca(2+)-induced NHE3-Syt I interaction preceded and was required for recruitment of AP2 and the clathrin complex. Confocal microscopy confirmed both the time sequence and protein associations of these events. We conclude that Syt I plays a pivotal role in mediating cAMP- and Ca(2+)-induced endocytosis of NHE3 (but not in inhibition of activity) through cargo recognition of NHE3 and subsequent recruitment of AP2-clathrin assembly required for membrane endocytosis.     

Neuroplastic adaptations to exercise: neuronal remodeling in cardiorespiratory and locomotor areas.

Neuronal activity has been shown to be attenuated in cardiorespiratory and locomotor centers of the brain in response to a single bout of exercise in trained (TR) vs. untrained (UN) animals, but the mechanisms remain obscure. Based on this finding, dendritic branching patterns of seven brain areas associated with cardiorespiratory and locomotor activity were examined in TR and UN animals. Twenty-eight male Sprague-Dawley rats were kept in individual cages and divided into TR and UN. TR were provided with a running wheel and exercised spontaneously. After 85 or 120 days, exercise training indexes were obtained, including maximal oxygen consumption, percent body fat, resting heart rate, and heart weight-to-body weight ratios. The brain was removed and processed according to a modified Golgi-Cox procedure. Impregnated neurons from seven brain areas were examined in coronal sections: the periaqueductal gray, posterior hypothalamic area, nucleus of the tractus solitarius, rostral ventrolateral medulla, cuneiform nucleus, nucleus cuneatus, and cerebral cortex. Neurons were traced using a camera lucida technique and analyzed using the Sholl analysis of dendritic branching. t-tests were conducted to compare the mean number of intersections per neuron by grouping inner rings and outer rings and also comparing the total number of intersections per animal. There were significant differences between groups in the posterior hypothalamic area, periaqueductal gray, cuneiform nucleus, and nucleus of the tractus solitarius in the inner rings, outer rings, and the total number of intersections per animal. Our results show that dendritic fields of neurons in important cardiorespiratory and locomotor centers of the brain are attenuated in TR animals.     

Sprint training shortens prolonged action potential duration in postinfarction rat myocyte: mechanisms.

Two electrophysiological manifestations of myocardial infarction (MI)-induced myocyte hypertrophy are prolongation of action potential duration (APD) and reduction of transient outward current (I(to)) density. Because high-intensity sprint training (HIST) ameliorated myocyte hypertrophy and improved myocyte Ca(2+) homeostasis and contractility after MI, the present study evaluated whether 6-8 wk of HIST would shorten the prolonged APD and improve the depressed I(to) in post-MI myocytes. There were no differences in resting membrane potential and action potential amplitude (APA) measured in myocytes isolated from sham-sedentary (Sed), MI-Sed, and MI-HIST groups. Times required for repolarization to 50 and 90% APA were significantly (P < 0.001) prolonged in MI-Sed myocytes. HIST reduced times required for repolarization to 50 and 90% APA to values observed in Sham-Sed myocytes. The fast and slow components of I(to) were significantly (P < 0.0001) reduced in MI-Sed myocytes. HIST significantly (P < 0.001) enhanced the fast and slow components of I(to) in MI myocytes, although not to levels observed in Sham-Sed myocytes. There were no significant differences in steady-state I(to) inactivation and activation parameters among Sham-Sed, MI-Sed, and MI-HIST myocytes. Likewise, recovery from time-dependent inactivation was also similar among the three groups. We suggest that normalization of APD after MI by HIST may be mediated by restoration of I(to) toward normal levels.     

Novel role of the vitamin D receptor in maintaining the integrity of the intestinal mucosal barrier

Emerging evidence supports a pathological link between vitamin D deficiency and the risk of inflammatory bowel disease (IBD). To explore the mechanism we used the dextran sulfate sodium (DSS)-induced colitis model to investigate the role of the vitamin D receptor (VDR) in mucosal barrier homeostasis. While VDR(+/+) mice were mostly resistant to 2.5% DSS, VDR(-/-) mice developed severe diarrhea, rectal bleeding, and marked body weight loss, leading to death in 2 wk. Histological examination revealed extensive ulceration and impaired wound healing in the colonic epithelium of DSS-treated VDR(-/-) mice. Severe ulceration in VDR(-/-) mice was preceded by a greater loss of intestinal transepithelial electric resistance (TER) compared with VDR(+/+) mice. Confocal and electron microscopy (EM) revealed severe disruption in epithelial junctions in VDR(-/-) mice after 3-day DSS treatment. Therefore, VDR(-/-) mice were much more susceptible to DSS-induced mucosal injury than VDR(+/+) mice. In cell cultures, 1,25-dihydroxy-vitamin D(3) [1,25(OH)(2)D(3)] markedly enhanced tight junctions formed by Caco-2 monolayers by increasing junction protein expression and TER and preserved the structural integrity of tight junctions in the presence of DSS. VDR knockdown with small interfering (si)RNA reduced the junction proteins and TER in Caco-2 monolayers. 1,25(OH)(2)D(3) can also stimulate epithelial cell migration in vitro. These observations suggest that VDR plays a critical role in mucosal barrier homeostasis by preserving the integrity of junction complexes and the healing capacity of the colonic epithelium. Therefore, vitamin D deficiency may compromise the mucosal barrier, leading to increased susceptibility to mucosal damage and increased risk of IBD.     

Small Intestine Microbiota Regulate Host Digestive and Absorptive Adaptive Responses to Dietary Lipids

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Effects of Type II diabetes on capillary hemodynamics in skeletal muscle

Microcirculatory red blood cell (RBC) hemodynamics are impaired within skeletal muscle of Type I diabetic rats (Kindig CA, Sexton WL, Fedde MR, and Poole DC. Respir Physiol 111: 163-175, 1998). Whether muscle microcirculatory dysfunction occurs in Type II diabetes, the more prevalent form of the disease, is unknown. We hypothesized that Type II diabetes would reduce the proportion of capillaries supporting continuous RBC flow and RBC hemodynamics within the spinotrapezius muscle of the Goto-Kakizaki Type II diabetic rat (GK). With the use of intravital microscopy, muscle capillary diameter (d(c)), capillary lineal density, capillary tube hematocrit (Hct(cap)), RBC flux (F(RBC)), and velocity (V(RBC)) were measured in healthy male Wistar (control: n = 5, blood glucose, 105 +/- 5 mg/dl) and male GK (n = 7, blood glucose, 263 +/- 34 mg/dl) rats under resting conditions. Mean arterial pressure did not differ between groups (P > 0.05). Sarcomere length was set to a physiological length ( approximately 2.7 mum) to ensure that muscle stretching did not alter capillary hemodynamics; d(c) was not different between control and GK rats (P > 0.05), but the percentage of RBC-perfused capillaries (control: 93 +/- 3; GK: 66 +/- 5 %), Hct(cap), V(RBC), F(RBC), and O(2) delivery per unit of muscle were all decreased in GK rats (P < 0.05). This study indicates that Type II diabetes reduces both convective O(2) delivery and diffusive O(2) transport properties within muscle microcirculation. If these microcirculatory deficits are present during exercise, it may provide a basis for the reduced O(2) exchange characteristic of Type II diabetic patients.