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
Hypothermia prolongs activation of NF-kappaB and augments generation of inflammatory cytokines.
While moderate hypothermia is protective against ischemic cardiac and brain injury, it is associated with much higher mortality in patients with sepsis. We previously showed that in vitro exposure to moderate hypothermia (32 degrees C) delays the induction and prolongs the duration of TNF-alpha and IL-1beta secretion by lipopolysaccharide (LPS)-stimulated human mononuclear phagocytes. In the present study, we extended these observations by showing that moderate hypothermia exerts effects on TNF-alpha and IL-1beta generation in the human THP-1 monocyte cell line that are similar to those that we previously found in primary cultured monocytes; that hypothermia causes comparable changes in cytokine generation stimulated by zymosan, toxic shock syndrome toxin-1, and LPS; and that hypothermia causes similar changes in TNF-alpha and IL-1beta mRNA accumulation. ⋯ Electrophoretic mobility shift assay revealed that hypothermia prolonged NF-kappaBeta activation, identifying a potential role for this transcription factor in mediating the effects of hypothermia on TNF-alpha and IL-1beta production. Delayed reexpression of the inhibitor IkappaBalpha, shown by Northern blotting and immunoblotting, may account in part for the prolonged NF-kappaBeta activation at 32 degrees C. Augmentation of NF-kappaBeta-dependent gene expression during prolonged exposure to hypothermia may be a common mechanism leading to increased lethality in sepsis, late-onset systemic inflammatory response syndrome after accidental hypothermia, and neuroprotection after ischemia.