D-lactate increases pulmonary apoptosis by restricting phosphorylation of bad and eNOS in a rat model of hemorrhagic shock.
Resuscitation with racemic lactated Ringer's solution (containing equal amounts of D and L isomers of lactate) has been shown to induce pulmonary apoptosis. Substitution of DL-isomer lactate with ketone bodies (beta-hydroxybutyrate, BHB), sodium pyruvate, or L-isomer of lactate decrease this injury without changing the energy status of the tissues or the expression of apoptotic genes. These modified solutions however alter the function of apoptotic proteins through an unknown mechanism. We postulated that DL-LR induces apoptosis by restricting the phosphorylation of key apoptotic proteins.


METHODS
Male Sprague Dawley rats (n = 30, 5/group) were subjected to a three stage, volume-controlled hemorrhage and randomized to the following groups. 1) No hemorrhage (Sham); 2) Hemorrhage and no resuscitation (NR); 3) Resuscitation with 3x shed blood volume of racemic LR (DL-LR); 4) Resuscitation with 3x shed blood volume of LR containing only the L-isomer of lactate (L-LR); 5) Resuscitation with 3s shed blood volume of pyruvate Ringer's (PR); 6) Resuscitation with 3s shed blood volume of ketone Ringer's (KR). The modified Ringer's solutions were identical to racemic LR except for equimolar substitution of DL-lactate for L-lactate, pyruvate and BHB respectively. Lung tissue was obtained 2 hours later and subjected to Western Blotting. The levels of Akt, Bad, and eNOS (total and phosphorylated) proteins were measured. Finally, the expression of gene coding for protein 14-3-3 was measured using RT-PCR.


RESULTS
Resuscitation with DL-LR caused a significant (p < 0.05) increase in the total Bad and a decrease in phosphorylated Bad protein expression in the lung. It also caused an increase in the phosphorylated Akt levels and a decrease in gene coding for protein 14-3-3. These changes were consistent with signaling imbalances that favor apoptosis. Modified LR solutions, on the other hand, did not cause these alterations. Phosphorylation pattern of eNOS supported the involvement of PI3K/Akt pathway in this process.


CONCLUSIONS
Racemic lactate plays a role in the induction of pulmonary apoptosis by restricting phosphorylation of Bad and eNOS proteins.
Jaskille
Amín
A
Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA.
Alam
Hasan B
HB
Rhee
Peter
P
Hanes
William
W
Kirkpatrick
John R
JR
Koustova
Elena
E
D048948:14-3-3 Proteins; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D015153:Blotting, Western; D002352:Carrier Proteins / Q000187:drug effects* / Q000378:metabolism; D002626:Chemistry, Pharmaceutical; D004195:Disease Models, Animal; D004353:Drug Evaluation, Preclinical; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D007262:Infusions, Intravenous; D007552:Isotonic Solutions / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use*; D008168:Lung / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008297:Male; D019001:Nitric Oxide Synthase / Q000187:drug effects* / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012151:Resuscitation / Q000379:methods; D000077325:Ringer's Lactate; D000077331:Ringer's Solution; D012771:Shock, Hemorrhagic / Q000188:drug therapy / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D014446:Tyrosine 3-Monooxygenase / Q000187:drug effects / Q000235:genetics; D051025:bcl-Associated Death Protein
D048948:14-3-3 Proteins; C494821:Bad protein, rat; D002352:Carrier Proteins; D007552:Isotonic Solutions; D011518:Proto-Oncogene Proteins; D000077325:Ringer's Lactate; C439503:Ringer's ethyl pyruvate; D051025:bcl-Associated Death Protein; D000077331:Ringer's Solution; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D014446:Tyrosine 3-Monooxygenase; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt