Akt2 phosphorylates ezrin to trigger NHE3 translocation and activation

Initiation of Na(+)-glucose cotransport in intestinal absorptive epithelia causes NHE3 to be translocated to the apical plasma membrane, leading to cytoplasmic alkalinization. We reported recently that this NHE3 translocation requires ezrin phosphorylation. However, the kinase that phosphorylates ezrin in this process has not been identified. Because Akt has also been implicated in NHE3 translocation, we investigated the hypothesis that Akt phosphorylates ezrin. After initiation of Na(+)-glucose cotransport, Akt is activated with kinetics that parallel those of ezrin phosphorylation. Inhibition of p38 MAP kinase, which blocks ezrin phosphorylation, also prevents Akt activation. Purified Akt directly phosphorylates recombinant ezrin at threonine 567 in vitro in an ATP-dependent manner. This in vitro phosphorylation can be prevented by Akt inhibitors. In intact cells, inhibition of either phosphoinositide 3-kinase, an upstream regulator of Akt, or inhibition of Akt itself using inhibitors validated in vitro prevents ezrin phosphorylation after initiation of Na(+)-glucose cotransport. Specific small interfering RNA knockdown of Akt2 prevented ezrin phosphorylation in intact cells. Pharmacological Akt inhibition or Akt2 knockdown also prevented NHE3 translocation and activation after initiation of Na(+)-glucose cotransport, confirming the functional role of Akt2. These studies therefore identify Akt2 as a critical kinase that regulates ezrin phosphorylation and activation. This Akt2-dependent ezrin phosphorylation leads to NHE3 translocation and activation.     

New microsatellite markers isolated from mungbean (Vigna radiata (L.) Wilczek)

In this study, we reported the isolation and analysis of new polymorphic microsatellites in mungbean (Vigna radiata (L.) Wilczek). Twelve out of 210 primer pairs screened in 30 mungbean accessions gave polymorphism. The polymorphic markers detected two to three alleles per locus with an average of 2.08. Observed heterozygosity varied from 0 to 0.133, while expected heterozygosity ranged from 0.095 to 0.498. Tests for Hardy-Weinberg equilibrium (HWE) and pairwise linkage disequilibrium of the polymorphic loci revealed that all loci except MB-SSR14 significantly departed from HWE and four pairwise combinations, viz. MB-SSR14 vs. MB-SSR42, MB-SSR42 vs. MB-SSR87, MB-SSR114 vs. MB-SSR121, and MB-SSR175 vs. MB-SSR231 significantly deviated from linkage disequilibrium. The markers are being used to study genetic diversity and genome mapping of mungbean.     

Effects of arterial hypotension on microvascular oxygen exchange in contracting skeletal muscle.

In healthy animals under normotensive conditions (N), contracting skeletal muscle perfusion is regulated to maintain microvascular O2 pressures (PmvO2) at levels commensurate with O2 demands. Hypovolemic hypotension (H) impairs muscle contractile function; we tested whether this condition would alter the matching of O2 delivery (Qo2) to O2 utilization (Vo2), as determined by PmvO2 at the onset of muscle contractions. PmvO2 in the spinotrapezius muscles of seven female Sprague-Dawley rats (280+/-6 g) was measured every 2 s across the transition from rest to 1-Hz twitch contractions. Measurements were made under N (mean arterial pressure, 97+/-4 mmHg) and H (induced by arterial section; mean arterial pressure, 58+/-3 mmHg, P<0.05) conditions; PmvO2 profiles were modeled using a multicomponent exponential fitted with independent time delays. Hypotension reduced muscle blood flow at rest (24+/-8 vs. 6+/-1 ml-1.min-1.100 g-1 for N and H, respectively; P<0.05) and during contractions (74+/-20 vs. 22+/-4 ml-1.min-1.100 g-1 for N and H, respectively; P<0.05). H significantly decreased resting PmvO2 and steady-state contracting PmvO2(19.4+/-2.4 vs. 8.7+/-1.6 Torr for N and H, respectively, P<0.05). At the onset of contractions, H reduced the time delay (11.8+/-1.7 vs. 5.9+/-0.9 s for N and H, respectively, P<0.05) before the fall in PmvO2 and accelerated the rate of PmvO2 decrease (time constant, 12.6+/-1.4 vs. 7.3+/-0.9 s for N and H, respectively, P<0.05). Muscle Vo2 was reduced by 71% at rest and 64% with contractions in H vs. N, and O2 extraction during H averaged 78% at rest and 94% during contractions vs. 51 and 78% in N. These results demonstrate that H constrains the increase of skeletal muscle Qo2 relative to that of Vo2 at the onset of contractions, leading to a decreased PmvO2. According to Fick's law, this scenario will decrease blood-myocyte O2 flux, thereby slowing Vo2 kinetics and exacerbating the O2 deficit generated at exercise onset.     

Sprint training attenuates myocyte hypertrophy and improves Ca2+ homeostasis in postinfarction myocytes

Zhang, Xue-Qian, Yuk-Chow Ng, Timothy I. Musch, Russell L. Moore, R. Zelis, and Joseph Y. Cheung. Sprint training attenuates myocyte hypertrophy and improvesCa²⁺ homeostasis in postinfarctionmyocytes. J. Appl. Physiol. 84(2): 544-552, 1998.Myocytes isolated from rat hearts 3 wk aftermyocardial infarction (MI) had decreasedNa⁺/Ca²⁺exchange currents(I_Na/Ca; 3 Na⁺ out:1Ca²⁺ in) and sarcoplasmicreticulum (SR)-releasable Ca²⁺contents. These defects in Ca²⁺regulation may contribute to abnormal contractility in MI myocytes. Because exercise training elicits positive adaptations in cardiac contractile function and myocardialCa²⁺ regulation, thepresent study examined whether 6-8 wk ofhigh-intensity sprint training (HIST) would ameliorate some of thecellular maladaptations observed in post-MI rats with limited exerciseactivity (Sed). In MI rats, HIST did not affect citrate synthaseactivities of plantaris muscles but significantly increased thepercentage of cardiac -myosin heavy chain (MHC) isoforms (57.2 ± 1.9 vs. 49.3 ± 3.5 in MI-HIST vs. MI-Sed, respectively;P  0.05). At the single myocytelevel, HIST attenuated cellular hypertrophy observed post-MI, asevidenced by reductions in cell lengths (112 ± 4 vs. 130 ± 5 µm in MI-HIST vs. MI-Sed, respectively;P  0.005) and cell capacitances (212 ± 8 vs. 242 ± 9 pF in MI-HIST vs. MI-Sed, respectively; P  0.015). ReverseI_Na/Ca wassignificantly lower (P  0.0001) inmyocytes from MI-Sed rats compared with those from rats that were shamoperated and sedentary. HIST significantly increased reverseI_Na/Ca(P  0.05) without affecting theamount ofNa⁺/Ca²⁺exchangers (detected by immunoblotting) in MI myocytes. SR-releasable Ca²⁺ content, as estimated byintegrating forwardI_Na/Ca duringcaffeine-induced SR Ca²⁺ release,was also significantly increased (P  0.02) by HIST in MI myocytes. We conclude that the enhanced cardiacoutput and stroke volume in post-MI rats subjected to HIST aremediated, at least in part, by reversal of cellular maladaptationspost-MI.

     

Acute effects of hydrogen peroxide on skeletal muscle microvascular oxygenation from rest to contractions

Reactive oxygen species, such as hydrogen peroxide (H(2)O(2)), exert a critical regulatory role on skeletal muscle function. Whether acute increases in H(2)O(2) modulate muscle microvascular O(2) delivery-utilization (Qo(2)/Vo(2)) matching [i.e., microvascular partial pressure of O(2) (Pmv(O(2)))] at rest and following the onset of contractions is unknown. The hypothesis was tested that H(2)O(2) treatment (exogenous H(2)O(2)) would enhance Pmv(O(2)) and slow Pmv(O(2)) kinetics during contractions compared with control. Anesthetized, healthy young Sprague-Dawley rats had their spinotrapezius muscles either exposed for measurement of blood flow (and therefore QO(2)), VO(2), and Pmv(O(2)), or exteriorized for measurement of force production. Electrically stimulated twitch contractions (1 Hz, ~7 V, 2-ms pulse duration, 3 min) were evoked following acute superfusion with Krebs-Henseleit (control) and H(2)O(2) (100 μM). Relative to control, H(2)O(2) treatment elicited disproportionate increases in QO(2) and VO(2) that elevated Pmv(O(2)) at rest and throughout contractions and slowed overall Pmv(O(2)) kinetics (i.e., ~85% slower mean response time; P < 0.05). Accordingly, H(2)O(2) resulted in ~33% greater overall Pmv(O(2)), as assessed by the area under the Pmv(O(2)) curve (P < 0.05). Muscle force production was not altered with H(2)O(2) treatment (P > 0.05), evidencing reduced economy during contractions (~40% decrease in the force/VO(2) relationship; P < 0.05). These findings indicate that, although increasing the driving force for blood-myocyte O(2) flux (i.e., Pmv(O(2))), transient elevations in H(2)O(2) impair skeletal muscle function (i.e., reduced economy during contractions), which mechanistically may underlie, in part, the reduced exercise tolerance in conditions associated with oxidative stress.     

Laser capture microdissection and metagenomic analysis of intact mucosa-associated microbial communities of human colon

Metagenomic analysis of colonic mucosa-associated microbes has been complicated by technical challenges that disrupt or alter community structure and function. In the present study, we determined the feasibility of laser capture microdissection (LCM) of intact regional human colonic mucosa-associated microbes followed by phi29 multiple displacement amplification (MDA) and massively parallel sequencing for metagenomic analysis. Samples were obtained from the healthy human subject without bowel preparation and frozen sections immediately prepared. Regional mucosa-associated microbes were successfully dissected using LCM with minimal contamination by host cells, their DNA extracted and subjected to phi29 MDA with a high fidelity, prior to shotgun sequencing using the GS-FLX DNA sequencer. Metagenomic analysis of approximately 67 million base pairs of DNA sequences from two samples revealed that the metabolic functional profiles in mucosa-associated microbes were as diverse as those reported in feces, specifically the representation of functional genes associated with carbohydrate, protein, and nucleic acid utilization. In summary, these studies demonstrate the feasibility of the approach to study the structure and metagenomic profiles of human intestinal mucosa-associated microbial communities at small spatial scales.     

Hypotonicity stimulates phosphatidylcholine hydrolysis and generates diacylglycerol in erythrocytes

Exposure of skate erythrocytes to hypotonic medium stimulates a rapid increase in levels of 1,2-diacylglycerol. Other treatments which produce cell swelling such as replacement of a portion of medium NaCl with the permeant solutes ethylene glycol or ammonium chloride also stimulate increases in diacylglycerol. Whereas the reduction of medium osmolarity to 460 mosm (from 940) stimulated a persistent diacylglycerol increase, the increase after reduction to 660 mosm was transient, peaking at 2.5 min and then slowly declining. This decline could be prevented by preincubation with the diacylglycerol kinase inhibitor R59022. To investigate the source of the increased diacylglycerol, the rate of incorporation of [32P]PO4 into each major phospholipid was measured. Reduction of osmolarity to 660 mosm stimulated the incorporation of phosphate into phosphatidylcholine markedly, with a smaller increase observed into phosphatidylinositol. To demonstrate phosphatidylcholine hydrolysis, erythrocytes were prelabeled with [32P]PO4. Subsequent exposure to hypotonic (660 mosm) medium stimulated a decrease in radioactivity in phosphatidylcholine and a large increase in radioactivity in phosphatidic acid. When stimulated in the presence of ethanol, 32PO4-labeled phosphatidylethanol was formed, suggesting activation of phospholipase D. In addition, the initial formation of 32PO4-labeled phosphatidic acid was not sensitive to inhibition of diacylglycerol kinase, supporting the role of direct activation of phospholipase D. These results indicate that hypotonicity and the accompanying cell swelling induce cell membrane phospholipid turnover, predominantly phosphatidylcholine, and production of the protein kinase C activator, diacylglycerol, which appears to occur via activation of phospholipase D.     

Peptide fragments of AMP-18, a novel secreted gastric antrum mucosal protein,are mitogenic and motogenic

Antrum mucosal protein (AMP)-18 is a novel 18-kDa protein synthesized by cells of the gastric antrum mucosa. The protein is present in secretion granules of murine gastric antrum epithelial cells and is a component of canine antrum mucus, suggesting that it is secreted into the viscoelastic gel layer on the mucosal surface. Release of the protein appears to be regulated because forskolin decreased the amount of immunoreactive AMP-18 in primary cultures of canine antrum mucosal epithelial cells, and indomethacin gavaged into the stomach of mice reduced AMP-18 content in antrum mucosal tissue before inducing histological injury. A functional domain of the protein was identified by preparing peptides derived from the center of human AMP-18. A 21-mer peptide stimulated growth of gastric and intestinal epithelial cells, but not fibroblasts, and increased restitution of scrape-wounded gastric epithelial monolayers. These functions of AMP-18 suggest that its release onto the apical cell surface is regulated and that the protein and/or peptide fragments may protect the antral mucosa and promote healing by facilitating restitution and proliferation after injury.