Selective evaluation of high density lipoprotein from mouse small intestine by an in situ perfusion technique.

The small intestine (SI) is the second-greatest source of HDL in mice. However, the selective evaluation of SI-derived HDL (SI-HDL) has been difficult because even the origin of HDL obtained in vivo from the intestinal lymph duct of anesthetized rodents is doubtful. To shed light on this question, we have developed a novel in situ perfusion technique using surgically isolated mouse SI, with which the possible filtration of plasma HDL into the SI lymph duct can be prevented. With the developed method, we studied the characteristics of and mechanism for the production and regulation of SI-HDL. Nascent HDL particles were detected in SI lymph perfusates in WT mice, but not in ABCA1 KO mice. SI-HDL had a high protein content and was smaller than plasma HDL. SI-HDL was rich in TG and apo AIV compared with HDL in liver perfusates. SI-HDL was increased by high-fat diets and reduced in apo E KO mice. In conclusion, with our in situ perfusion model that enables the selective evaluation of SI-HDL, we demonstrated that ABCA1 plays an important role in intestinal HDL production, and SI-HDL is small, dense, rich in apo AIV, and regulated by nutritional and genetic factors.

Ins(1,4,5)P(3) facilitates ATP accumulation via phosphocreatine/creatine kinase in the endoplasmic reticulum extracted from MDCK cells.

So far, the content and accumulation of ATP in isolated endoplasmic reticulum (ER) are little understood. First, we confirmed using electron microscopic and Western blotting techniques that the samples extracted from MDCK cells are endoplasmic reticulum (ER). The amounts of ATP in the extracted ER were measured from the filtrate after a spinning down of ultrafiltration spin column packed with ER. When the ER sample (5mug) after 3days freezing was suspended in intracellular medium (ICM), 0.1% Triton X and ultrapure water (UPW), ATP amounts from the ER with UPW were the highest and over 10 times compared with that from the control with ICM, indicating that UPW is the most effective tool in destroying the ER membrane. After a 10-min-incubation with ICM containing phosphocreatine (PCr)/creatine kinase (CK) of the fresh ER. ATP amounts in the filtrate obtained by spinning down were not changed from that in the control (no PCr/CK). However, ATP amounts in the filtrate from the second spinning down of the ER (treated with PCr/CK) suspended in UPW became over 10-fold compared with the control. When 1muM inositol(1,4,5)trisphosphate (Ins(1,4,5)P(3)) was added in the incubation medium (ICM with PCr/CK), ATP amounts from the filtrate after the second spinning down were further enhanced around three times. This enhancement was almost canceled by Ca(2+)-removal from ICM and by adding thapsigargin, a Ca(2+)-ATPase inhibitor, but not by 2-APB and heparin, Ins(1,4,5)P(3) receptor antagonists. Administration of 500muM adenosine to the incubation medium (with PCr/CK) failed to enhance the accumulation of ATP in the ER. These findings suggest that the ER originally contains ATP and ATP accumulation in the ER is promoted by PCr/CK and Ins(1,4,5)P(3).

Caffeine-inducible ATP release is mediated by Ca2+-signal transducing system from the endoplasmic reticulum to mitochondria.

Adenosine triphosphate (ATP) is released as an autocrine/paracrine signal from a variety of cells. The present study was undertaken to clarify the Ca2+-signal pathway involved in the caffeine-inducible release of ATP from cultured smooth muscle cells (SMC). The release of ATP induced by caffeine (3 mM) was almost completely inhibited by ryanodine and tetracaine, but not by 2-APB, thus being mediated by ryanodine receptors (RyR). The expression of messenger RNA from only RyR-2 was detected in the cells. Furthermore, the induced release was attenuated by mitochondrial inhibitors, rotenone and oligomycin and by Cl- channel blockers, niflumic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid. Increase in Ca2+-signals with fluo 4 and rhod-2 caused by caffeine were reduced by tetracaine and oligomycin plus carbonyl cyanide m-chlorophenylhydrazone, respectively. A close spatial relation between the endoplasmic reticulum (ER) and mitochondria was electromicroscopically observed in the SMC, supporting the existence of a Ca2+-signaling bridge on both the organelli. These results suggest that caffeine stimulates ryanodine receptor (RyR-2) and facilitates a Ca2+-signal transducing system from ER to mitochondria, and then, the signal appears to accelerate the ATP synthesis in mitochondria. In addition, the mitochondrial event may lead further cell signaling to the cell membrane and activates Cl- channels, resulting in the extracellular release of cytosolic ATP.

Effects of reconstituted HDL on charge-based LDL subfractions as characterized by capillary isotachophoresis.

Modified LDL in human plasma including small, dense LDL (sdLDL) and oxidized LDL carries a more negative charge than unmodified LDL and is atherogenic. We examined the effects of apolipoprotein A-I (apoA-I)/POPC discs on charge-based LDL subfractions as determined by capillary isotachophoresis (cITP). Three normal healthy subjects and seven patients with metabolic disorders were included in the study. LDL in human plasma was separated into two major subfractions, fast- and slow-migrating LDL (fLDL and sLDL), by cITP. Normal LDL was characterized by low fLDL, and mildly oxidized LDL in vitro and mildly modified LDL in human plasma were characterized by increased fLDL. Moderately oxidized LDL in vitro and moderately modified LDL in a patient with hypertriglyceridemia and HDL deficiency were characterized by both increased fLDL and a new LDL subfraction with a faster mobility than fLDL [very-fast-migrating LDL as determined by cITP (vfLDL)]. cITP LDL subfractions with faster electrophoretic mobility (fLDL vs. sLDL, vfLDL vs. fLDL) were associated with an increased content of sdLDL. Incubation of a plasma fraction with d>1.019 g/ml (depleted of triglyceride-rich lipoproteins) in the presence of apoA-I/POPC discs at 37 degrees C greatly decreased vfLDL and fLDL but increased sLDL. Incubation of whole plasma from patients with an altered distribution of cITP LDL subfractions in the presence of apoA-I/POPC discs also greatly decreased fLDL but increased sLDL. ApoA-I/POPC discs decreased the cITP fLDL level, the free cholesterol concentration, and platelet-activating factor acetylhydrolase activity in the sdLDL subclasses (d=1.040-1.063 g/ml) and increased the size of LDL. ApoA-I/POPC discs reduced charge-modified LDL in human plasma by remodeling cITP fLDL into sLDL subfractions.

Long-term cadmium exposure accelerates age-related mitochondrial changes in renal epithelial cells.

Long-term cadmium exposure leads to mitochondrial dysfunction in the proximal tubular epithelial cells. Mitochondrial DNA deletion may contribute to the pathogenesis of cadmium-induced nephropathy. The aim of our study is to clarify the accumulation of mitochondrial DNA deletion and mitochondrial dysfunction in the renal cortex of rats injected three times/week with 1 ml of 1 mM CdCl2 or saline for 80 weeks. After 40-week cadmium injection, mitochondrial number diminished, and cadmium in the renal cortex reached a saturation level. At this time interval, nearly 30% of cadmium in the whole cell fraction was found in the mitochondria. Cytochrome c oxidase (COX) activity in the proximal tubular epithelial cells decreased after 40-week exposure of cadmium. Oxidized phosphatidylcholine (oxPC) started to accumulate in the cytochrome c-positive mitochondria in some tubular epithelial cells after 80-week exposure. After 40 weeks, accumulation of the 4834-bp deletion in mitochondrial DNA was evident in both control and cadmium-treated groups. However, the amount of accumulated mitochondrial DNA deletion tended to increase after 40-week exposure, and was significantly greater after 80 weeks of exposure, compared to the control. Our results indicate that long-term cadmium exposure in rats accelerates accumulation of 4834-bp mitochondrial DNA deletions and impairment of mitochondrial function associated with accumulation of oxidized product.

Cadmium-induced nephropathy in rats is mediated by expression of senescence-associated beta-galactosidase and accumulation of mitochondrial DNA deletion.

Long-term exposure to cadmium (Cd) induces perturbation of kidney proximal tubular epithelial cells. Mitochondrial dysfunction in renal cortical cells may contribute to the pathogenesis of Cd-induced nephropathy. In this study, we examined the accumulation of mitochondrial DNA (mtDNA) with a large deletion and cellular senescence in the renal cortex. Wistar rats at 8 weeks of age were intraperitoneally injected with 1 mL of 1 mM CdCl(2) or saline, 3 times/week for 5, 20, 40, or 80 weeks. Mitochondrial Cd content in the renal cortex was quantified by atomic absorption analysis. Cytochrome c oxidase (CCO) and senescence-associated beta-galactosidase (SA-beta-gal) activity were determined in renal cortex by enzyme-histochemistry. mtDNA in total DNA extracted from the renal cortex was amplified by PCR, and mtDNA deletions, including 4,834-bp (nt8118-nt12937) deletion, were determined and semiquantified. After 40 weeks of Cd injection, Cd levels in the renal cortex reached a saturation level, and 30% of the level of the whole-cell fraction was found in the mitochondria. CCO activity in the renal cortex, which was predominantly found in proximal tubular cells, decreased after 40 weeks of Cd exposure. Expression of SA-beta-gal was detected primarily in the proximal tubular cells and significantly increased after 80 weeks of Cd exposure. After 40 weeks of study, accumulation of 4,834-bp deletion in mtDNA was evident in both groups of rats; however, the amount of the deletion was significantly greater in Cd-treated rats than in control rats. Our results indicate that long-term Cd exposure induced a post-regenerative state of proximal tubular cells, which accelerated accumulation of 4,834-bp mtDNA deletions in the renal cortex, suggesting that Cd may be a senescence acceleration factor for kidney proximal tubular epithelial cells, which results in Cd-induced nephropathy.

Mitochondrial DNA deletion of proximal tubules is the result of itai-itai disease.

BACKGROUND: The pathogenesis of itai-itai disease continues to be controversial, although cadmium (Cd) poisoning which arises via polluted water and rice in Japan is likely involved. Until recently, however, a well-defined animal model for Cd intoxication was not available. An animal model for itai-itai disease was produced in rats by low-dose Cd treatment, intraperitoneally for a period of 70-80 weeks. Osteomalacia followed the renal damage. RESULTS: A gene deletion in the mitochondrial DNA was found in the mitochondria of the proximal tubule cells of rats with chronic Cd intoxication, as was shown by the increased smaller PCR product seen by gel electrophoresis in one DNA region, where ATPase and cytochrome oxidase genes are located. However, the PCR product was different from that seen with a gene deletion associated with aging: del4834bp. Renal damage from Cd intoxication initially caused mitochondrial dysfunction indicated by the disturbance in reabsorption in the proximal tubules and decreased amounts of ATP, ATPase, and cytochrome oxidase with gradually progressing tubular proteinuria, and, finally, chronic renal failure with tubulointerstitial damage throughout the renal cortex. These gave rise to osteomalacia, subsequently. CONCLUSION: We concluded that in Cd poisoning, a mitochondrial gene deletion in the mitochondria of the proximal tubule cells was the primary event for the pathogenesis of osteomalacia in itai-itai disease.