Lysophosphatidic acid inhibits cholera toxin-induced secretory diarrhea through CFTR-dependent protein interactions.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells that line the airway, gut, and exocrine glands; it is well established that CFTR plays a pivotal role in cholera toxin (CTX)-induced secretory diarrhea. Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in blood and foods, has been reported to play a vital role in a variety of conditions involving gastrointestinal wound repair, apoptosis, inflammatory bowel disease, and diarrhea. Here we show, for the first time, that type 2 LPA receptors (LPA2) are expressed at the apical surface of intestinal epithelial cells, where they form a macromolecular complex with Na+/H+ exchanger regulatory factor-2 and CFTR through a PSD95/Dlg/ZO-1-based interaction. LPA inhibited CFTR-dependent iodide efflux through LPA2-mediated Gi pathway, and LPA inhibited CFTR-mediated short-circuit currents in a compartmentalized fashion. CFTR-dependent intestinal fluid secretion induced by CTX in mice was reduced substantially by LPA administration; disruption of this complex using a cell-permeant LPA2-specific peptide reversed LPA2-mediated inhibition. Thus, LPA-rich foods may represent an alternative method of treating certain forms of diarrhea.

The early- and late stages in phenotypic modulation of vascular smooth muscle cells: differential roles for lysophosphatidic acid.

Lysophosphatidic acid (LPA) has been implicated as causative in phenotypic modulation (PM) of cultured vascular smooth muscle cells (VSMC) in their transition to the dedifferentiated phenotype. We evaluated the contribution of the three major LPA receptors, LPA1 and LPA2 GPCR and PPARgamma, on PM of VSMC. Expression of differentiated VSMC-specific marker genes, including smooth muscle alpha-actin, smooth muscle myosin heavy chain, calponin, SM-22alpha, and h-caldesmon, was measured by quantitative real-time PCR in VSMC cultures and aortic rings kept in serum-free chemically defined medium or serum- or LPA-containing medium using wild-type C57BL/6, LPA1, LPA2, and LPA1&2 receptor knockout mice. Within hours after cells were deprived of physiological cues, the expression of VSMC marker genes, regardless of genotype, rapidly decreased. This early PM was neither prevented by IGF-I, inhibitors of p38, ERK1/2, or PPARgamma nor significantly accelerated by LPA or serum. To elucidate the mechanism of PM in vivo, carotid artery ligation with/without replacement of blood with Krebs solution was used to evaluate contributions of blood flow and pressure. Early PM in the common carotid was induced by depressurization regardless of the presence/absence of blood, but eliminating blood flow while maintaining blood pressure or after sham surgery elicited no early PM. The present results indicate that LPA, serum, dissociation of VSMC, IGF-I, p38, ERK1/2, LPA1, and LPA2 are not causative factors of early PM of VSMC. Tensile stress generated by blood pressure may be the fundamental signal maintaining the fully differentiated phenotype of VSMC.

Mitochondrial DNA mutations activate the mitochondrial apoptotic pathway and cause dilated cardiomyopathy.

OBJECTIVE: To determine whether low frequency mitochondrial DNA (mtDNA) mutations are pathogenic. METHODS: We studied mice that express a proofreading-deficient mitochondrial DNA polymerase in the heart and develop cardiac mtDNA mutations. RESULTS: At 4 weeks of age, when point mutation levels had risen to on average two per mitochondrial genome, these mice developed severe dilated cardiomyopathy. Interstitial fibrosis first became apparent at 4 weeks of age and progressed with age. Sporadic myocytic death occurred in all regions of the heart, apparently due to apoptosis as assessed by histological analysis and TUNEL staining. The frequency of TUNEL-positive cells peaked at 4-5 weeks of age and then gradually declined. While mitochondrial respiratory function, ultrastructure, and number remained normal, cytochrome c was released from mitochondria, a known apoptotic signal. CONCLUSION: mtDNA mutations therefore are pathogenic, and seem to trigger apoptosis through the mitochondrial pathway.

Lysophosphatidic acid-induced arterial wall remodeling: requirement of PPARgamma but not LPA1 or LPA2 GPCR.

Lysophosphatidic acid (LPA) and its ether analog alkyl-glycerophosphate (AGP) elicit arterial wall remodeling when applied intralumenally into the uninjured carotid artery. LPA is the ligand of eight GPCRs and the peroxisome proliferator-activated receptor gamma (PPARgamma). We pursued a gene knockout strategy to identify the LPA receptor subtypes necessary for the neointimal response in a non-injury model of carotid remodeling and also compared the effects of AGP and the PPARgamma agonist rosiglitazone (ROSI) on balloon injury-elicited neointima development. In the balloon injury model AGP significantly increased neointima; however, rosiglitazone application attenuated it. AGP and ROSI were also applied intralumenally for 1h without injury into the carotid arteries of LPA(1), LPA(2), LPA(1&2) double knockout, and Mx1Cre-inducible conditional PPARgamma knockout mice targeted to vascular smooth muscle cells, macrophages, and endothelial cells. The neointima was quantified and also stained for CD31, CD68, CD11b, and alpha-smooth muscle actin markers. In LPA(1), LPA(2), LPA(1&2) GPCR knockout, Mx1Cre transgenic, PPARgamma(fl/-), and uninduced Mx1CrexPPARgamma(fl/-) mice AGP- and ROSI-elicited neointima was indistinguishable in its progression and cytological features from that of WT C57BL/6 mice. In PPARgamma(-/-) knockout mice, generated by activation of Mx1Cre-mediated recombination, AGP and ROSI failed to elicit neointima and vascular wall remodeling. Our findings point to a difference in the effects of AGP and ROSI between the balloon injury- and the non-injury chemically-induced neointima. The present data provide genetic evidence for the requirement of PPARgamma in AGP- and ROSI-elicited neointimal thickening in the non-injury model and reveal that the overwhelming majority of the cells in the neointimal layer express alpha-smooth muscle actin.

Posterior paresis and osteolysis in guinea pigs (Cavia porcellus) secondary to Freund's adjuvant immunization.

Bilateral hindlimb paresis occurred in 3 guinea pigs after immunization with an adjuvant-antigen mixture containing complete Freund's adjuvant. Doses were injected into unanaesthetized animals, divided among 3 or 4 sites, and given slightly off midline in the subcutaneous tissues of the back. Neurologic examination of affected animals revealed intact flexor and panniculus responses and limited voluntary movement of the hindlimbs. Histopathologic interpretation of 2 affected animals showed fibrogranulomatous material effacing the skeletal muscle and vertebral bone, with marked bone lysis and infiltration into the marrow space and spinal canal. In addition, multiple granulomas in the pulmonary parenchyma were noted. A postmortem radiograph of the excised thoracolumbar spine of 1 animal revealed a soft tissue swelling and "moth-eaten" and geographic osteolysis of 2 spinous processes. Hindlimb paresis and osteolysis likely resulted from accidental injection of the adjuvant-antigen mixture into the epaxial musculature and subsequent extension of injection site granulomas into the spinal canal.