American journal of physiology. Cell physiology
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Am. J. Physiol., Cell Physiol. · Jun 2006
Comparative StudyGlutamine's protection against cellular injury is dependent on heat shock factor-1.
Glutamine (GLN) has been shown to protect cells, tissues, and whole organisms from stress and injury. Enhanced expression of heat shock protein (HSP) has been hypothesized to be responsible for this protection. To date, there are no clear mechanistic data confirming this relationship. ⋯ This was accompanied by a GLN-mediated increase in HSF-1/HSE binding and nuclear content of phosphorylated HSF-1 after heat stress. This is the first demonstration that GLN-mediated cellular protection after heat-stress injury is related to HSF-1 expression and cellular capacity to activate an HSP response. Furthermore, the mechanism of GLN-mediated protection against injury appears to involve an increase in nuclear HSF-1 content before stress and increased HSF-1 promoter binding and phosphorylation.
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Am. J. Physiol., Cell Physiol. · Apr 2006
Hypertonic saline enhances neutrophil elastase release through activation of P2 and A3 receptors.
Hypertonic saline (HS) holds promise as a novel resuscitation fluid for the treatment of trauma patients because HS inhibits polymorphonuclear neutrophil (PMN) activation and thereby prevents host tissue damage and associated posttraumatic complications. However, depending on conditions of cell activation, HS can increase PMN degranulation, which could exacerbate tissue damage in trauma victims. The cellular mechanism by which HS increases degranulation is unknown. ⋯ A1 adenosine receptor antagonists increased the enhancing effect of HS, whereas A1 receptor agonists inhibited elastase release. These data suggest that HS upregulates degranulation via ATP release and positive feedback through P2 and A3 receptors. We propose that these feedback mechanisms can serve as potential pharmacological targets to fine-tune the clinical effectiveness of HS resuscitation.
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Am. J. Physiol., Cell Physiol. · Jan 2006
Volume sensitivity of cation-Cl- cotransporters is modulated by the interaction of two kinases: Ste20-related proline-alanine-rich kinase and WNK4.
In the present study, we have demonstrated functional interaction between Ste20-related proline-alanine-rich kinase (SPAK), WNK4 [with no lysine (K)], and the widely expressed Na+-K+-2Cl- cotransporter type 1 (NKCC1). NKCC1 function, which we measured in Xenopus laevis oocytes under both isosmotic (basal) and hyperosmotic (stimulated) conditions, was unaffected when SPAK and WNK4 were expressed alone. In contrast, expression of both kinases with NKCC1 resulted in a significant increase in cotransporter activity and an insensitivity to external osmolarity or cell volume. ⋯ Functional experiments demonstrated that the binding of SPAK to WNK4 was also required because a SPAK-interaction-deficient WNK4 mutant (Phe997Ala) did not increase NKCC1 activity. We also have shown that the transport function of K+-Cl- cotransporter type 2 (KCC2), a neuron-specific KCl cotransporter, was diminished by the expression of both kinases under both isosmotic and hyposmotic conditions. Our data are consistent with WNK4 interacting with SPAK, which in turn phosphorylates and activates NKCC1 and phosphorylates and deactivates KCC2.
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Am. J. Physiol., Cell Physiol. · Oct 2004
Functional analysis of the R1086H malignant hyperthermia mutation in the DHPR reveals an unexpected influence of the III-IV loop on skeletal muscle EC coupling.
Malignant hyperthermia (MH) is an inherited pharmacogenetic disorder caused by mutations in the skeletal muscle ryanodine receptor (RyR1) and the dihydropyridine receptor (DHPR) alpha(1S)-subunit. We characterized the effects of an MH mutation in the DHPR cytoplasmic III-IV loop of alpha(1S) (R1086H) on DHPR-RyR1 coupling after reconstitution in dysgenic (alpha(1S) null) myotubes. Compared with wild-type alpha(1S), caffeine-activated Ca(2+) release occurred at approximately fivefold lower concentrations in nonexpressing and R1086H-expressing myotubes. ⋯ The decrease in G(max) did not result from a change in retrograde coupling with RyR1 as maximal conductance-charge movement ratio (G(max)/Q(max)) was similar in alpha(1S)- and R1086H-expressing myotubes and a similar decrease in G(max) was observed for an analogous mutation engineered into the cardiac L channel (R1217H). In addition, both R1086H and R1217H DHPRs targeted normally and colocalized with RyR1 in sarcoplasmic reticulum (SR)-sarcolemmal junctions. These results indicate that the R1086H MH mutation in alpha(1S) enhances RyR1 sensitivity to activation by both endogenous (voltage sensor) and exogenous (caffeine) activators.
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Am. J. Physiol., Cell Physiol. · Aug 2004
Platelet-induced enhancement of LS174T colon carcinoma and THP-1 monocytoid cell adhesion to vascular endothelium under flow.
This study was undertaken to characterize the adhesion of LS174T colon adenocarcinoma cells to 4-h TNF-alpha-stimulated human umbilical vein endothelial cells (HUVECs) under flow in the presence and absence of platelets and erythrocytes. Cell binding to HUVECs was significantly enhanced by simultaneous perfusion of thrombin-activated, but not resting, platelets. This increase was achieved via a platelet bridging mechanism whereby a previously tethered LS174T cell (primary tether) captures a free-flowing cell (secondary tether) that subsequently attaches to the endothelium downstream of the already adherent cell. ⋯ Secondary tethering was dependent on both platelet P-selectin and alpha(IIb)beta(3)-integrin for LS174T cells and P-selectin alone for THP-1 cells. Furthermore, platelet-mediated secondary tethering of both cell types occurred in the presence of red blood cells. Altogether, these results reveal a novel role for platelets in promoting cell binding to endothelium through a secondary tethering mechanism.