Identification of a novel murine organic anion transporter like protein 1 (OATLP1) expressed in the kidney

The organic anion transporters (OATs) are expressed in various tissues, primarily in the kidney and liver, but they are also expressed in the placenta, small intestine, and the choroid plexus, which are all epithelial tissues that transport xenobiotics. Six isoforms of OATs are currently known. Considering the variety of organic anionic compounds, other OATs isoforms can be assumed. In this connection, we have searched for a new isoform in the expressed sequence tag (EST) database. We found the new candidate clone AK052752 in the mouse kidney cDNA library and we named it mouse organic anion transporter like protein 1 (mOATLP1). The mOATLP1 cDNA consisted of 2221 base pairs that encoded a 552 amino acid residue protein with 12 putative transmembrane domains. The deduced amino acid sequence of mOATLP1 showed 37 to 63% identity to other members of the OAT family. According to the tissue distribution based on Northern blot analysis, 2.7 kb and 2.9 kb mOATLP1 transcripts (approximate sizes) were observed in the kidney and liver. An 85-kDa band (approximate) was detected using Western blot analysis of mouse kidney performed with a synthesized oligopeptide-induced mOATLP1 antibody. Immunohistochemical results showed mOATLP1 was stained in the blood vessels, glomeruli (the parietal epithelial cells and podocytes), distal convoluted tubules, connecting tubules, and inner medullary collecting tubules. mOATLP1 appears to be a novel candidate for an organic anion transporter isoform identified in the kidney.

Introduction of Organic Anion Transporters (SLC22A) and a Regulatory Mechanism by Caveolins

The kidney is an important organ for controlling the volume of body fluids, electrolytic balance and excretion/reabsorption of endogenous and exogenous compounds. Among these renal functions, excretion/reabsorption of endogenous and exogenous substance is very important for the maintenance of physiological homeostasis in the body. Recently discovered organic anion transporters (OAT or SLC22A) have important roles for renal functions. It is well known as drug transporter. Several isoforms belong to SLC22A family. They showed different transport substrate spectrums and different localizations within the kidney. Their gene expressions are changed by some stimulus. The functional transport properties are regulated by protein kinase C. In addition, the function of organic anion transporters are also regulated by protein-protein interaction, such as caveolin which is compositional protein of caveolae structure. In this review, we will give an introduction of organic anion transporters and its regulatory mechanisms.

Evidence for Cyclooxygenase-2 Association with Caveolin-3 in Primary Cultured Rat Chondrocytes

The purpose of this study was to demonstrate the cellular localization of cyclooxygenase-2 (COX-2) and caveolin-3 (Cav-3) in primarily cultured rat chondrocytes. In normal rat chondrocytes, we observed relatively high levels of Cav-3 and a very low level of COX-2 mRNA and protein. Upon treating the chondrocytes with 5 microM of CdCl2 (Cd) for 6 hr, the expressions of COX-2 mRNA and protein were increased with the decreased Cav-3 mRNA and protein expressions. The detergent insoluble caveolae-rich membranous fractions that were isolated from the rat chondrocytes and treated with Cd contained the both proteins of both COX-2 and Cav-3 in a same fraction. The immuno-precipitation experiments showed complex formation between the COX-2 and Cav-3 in the rat chondrocytes. Purified COX-2 with glutathione S-transferase-fused COX-2 also showed complex formation with Cav-3. Confocal and electron microscopy also demonstrated the co-localization of COX-2 and Cav-3 in the plasma membrane. The results from our current study show that COX-2 and Cav-3 are co-localized in the caveolae of the plasma membrane, and they form a protein-protein complex. The co-localization of COX-2 with Cav-3 in the caveolae suggests that the caveolins might play an important role for regulating the function of COX-2.

Calcium sensing receptor forms complex with and is up-regulated by caveolin-1 in cultured human osteosarcoma (Saos-2) cells

The calcium sensing receptor (CaSR) plays an important role for sensing local changes in the extracellular calcium concentration ([Ca2+]o) in bone remodeling. Although the function of CaSR is known, the regulatory mechanism of CaSR remains controversial. We report here the regulatory effect of caveolin on CaSR function as a process of CaSR regulation by using the human osteosarcoma cell line (Saos-2). The intracellular calcium concentration ([Ca2+]i) was increased by an increment of [Ca2+]o. This [Ca2+]i increment was inhibited by the pretreatment with NPS 2390, an antagonist of CaSR. RT-PCR and Western blot analysis of Saos-2 cells revealed the presence of CaSR, caveolin (Cav)-1 and -2 in both mRNA and protein expressions, but there was no expression of Cav-3 mRNA and protein in the cells. In the isolated caveolae-rich membrane fraction from Saos-2 cells, the CaSR, Cav-1 and Cav-2 proteins were localized in same fractions (fraction number 4 and 5). The immuno-precipitation experiment using the respective antibodies showed complex formation between the CaSR and Cav-1, but no complex formation of CaSR and Cav-2. Confocal microscopy also supported the co-localization of CaSR and Cav-1 at the plasma membrane. Functionally, the [Ca2+]o- induced [Ca2+]i increment was attenuated by the introduction of Cav-1 antisense oligodeoxynucleotide (ODN). From these results, in Saos-2 cells, the function of CaSR might be regulated by binding with Cav-1. Considering the decrement of CaSR activity by antisense ODN, Cav-1 up-regulates the function of CaSR under normal physiological conditions, and it may play an important role in the diverse pathophysiological processes of bone remodeling or in the CaSR- related disorders in the body.

Co-localization and interaction of organic anion transporter 1 with caveolin-2 in rat kidney

The organic anion transporters (OAT) have recently been identified. Although the some transport properties of OATs in the kidney have been verified, the regulatory mechanisms for OAT's functions are still not fully understood. The rat OAT1 (rOAT1) transports a number of negatively charged organic compounds between the cells and their extracellular milieu. Caveolin (Cav) also plays a role in membrane transport. Therefore, we investigated the protein-protein interactions between rOAT1 and caveolin-2. In the rat kidney, the expressions of rOAT1 mRNA and protein were observed in both the cortex and the outer medulla. With respect to Cav-2, the expressions of mRNA and protein were observed in all portions of the kidney (cortex < outer medulla = inner medulla). The results of Western blot analysis using the isolated caveolae-enriched membrane fractions or the immunoprecipitates by respective antibodies from the rat kidney showed that rOAT1 and Cav-2 co-localized in the same fractions and they formed complexes each other. These results were confirmed by performing confocal microscopy with immunocytochemistry using the primary cultured renal proximal tubular cells. When the synthesized cRNA of rOAT1 along with the antisense oligodeoxynucleotides of Xenopus Cav-2 were co-injected into Xenopus oocytes, the [14C]p-aminohippurate and [3H]methotrexate uptake was slightly, but significantly decreased. The similar results were also observed in rOAT1 over-expressed Chinese hamster ovary cells. These findings suggest that rOAT1 and caveolin-2 are co-expressed in the plasma membrane and rOAT1's function for organic compound transport is upregulated by Cav-2 in the normal physiological condition.

Rat Organic Anion Transporter 3 Co-localized with Caveolin-2 in Rat Kidney / 대한신장학회잡지

BACKGROUND: The recently identified organic anion transporter 3 (rOAT3) was mainly expressed in kidney, liver and brain tissue, and it contributes the movement of endogenous or exogenous substances across the cell membrane. Although the properties of rOAT3 are gradually accumulated, the regulatory mechanism of rOAT3 remains to be elucidated. Caveolin (Cav) also plays a role as a membrane transporter and as a modulating protein for some functional proteins. Therefore, we investigated the protein-protein interaction between rOAT3 and Cav-2 in rat kidney. METHODS: The expressions of rOAT3 and Cav-2 (mRNA and protein) were observed using RT-PCR and Western blot analysis. The localization of rOAT3 and Cav-2 was determined in the caveolae-rich membrane fraction isolated by sucrose gradient ultra-centrifugation. For the direct binding between the rOAT3 and Cav-2 proteins, the immuno-precipitation method and confocal microscopy were employed. In order to perform functional analysis, a Xenopus oocytes expression system with the antisense oligodeoxynucleotides (ODN) technique was used. RESULTS: The expressions of rOAT3 and Cav-2 (mRNA and protein) were detected in the kidney. The caveolae-rich membranous fractions from the kidney contained both rOAT3 and Cav-2 in the same fractions. The immuno-precipitation experiments showed the formation of a complex between the rOAT3 and Cav-2 in the kidney. The confocal microscopic results using primary cultured renal proximal epithelial cells also supported the co-localization of rOAT3 and Cav-2 at the plasma membrane. The uptake function of rOAT3, as tested for by using a Xenopus oocytes expression system was slightly inhibited (with statistical significance) by the Xenopus Cav-2 antisense ODN. CONCLUSION: rOAT3 co-localizes with caveolin-2 in the caveolae, and caveolin-2 plays an important role in regulating the function of rOAT3.

The extracellular calcium sensing receptor is expressed in mouse mesangial cells and modulates cell proliferation

The extracellular calcium sensing receptor (CaSR) belongs to the type III family of G-protein-coupled receptors, a family that comprises the metabotropic glutamate receptor and the putative vomeronasal organ receptors. The CaSR plays an important role for calcium homeostasis in parathyroid cells, kidney cells and other cells to directly 'sense' changes in the extracellular calcium ion concentration ((Ca2+)o). The mesangial cells are known to be involved in many pathologic sequences through the mediation of altered glomerular hemodynamics, cell proliferation, and matrix production. In this study, we examined the expression of the CaSR in the mouse mesangial cell lines (MMC, ATCC number CRL-1927). Reverse transcription- polymerase chain reaction (RT-PCR) was perform with CaSR-specific primers, and this was followed by nucleotide sequencing of the amplified product; this process identified the CaSR transcript in the MMCs. Moreover, CaSR protein was present in the MMCs as assessed by Western blot and immunocytochemical analysis using a polyclonal antibody specific for the CaSR. Functionally, (Ca2+)o induced the increment of the intracellular calcium concentration ((Ca2+)i) in a dose-dependent manner. This (Ca2+)i increment by (Ca2+)o was attenuated by the pretreatment with a phospholipase C inhibitor (U73122) and also by a pretreatment with a CaSR antagonist (NPS 2390). The similar results were also obtained in IP3 accumulation by (Ca2+)o. To investigate the physiological effect of the CaSR, the effect of the (Ca2+)o on cell proliferation was studied. The increased (Ca2+)o (up to 10 mM) produced a significant increase in the cell numbers. This mitogenic effect of (Ca2+)o was inhibited by the co-treatment with a CaSR antagonist. From these results, the (Ca2+)o-induced (Ca2+)i elevation in the MMC is coupled with the extracellular calcium sensing receptor. Furthermore, (Ca2+)o produces a mitogenic effect in MMCs.

Comparison of ANG II-mediated Ca2+i, IP3 Production and ATPi in Isolated Renal Proximal Convoluted Tubules of Adult SHR and WKY / 대한신장학회잡지

BACKGROUND: The renin angiotensin syaimstem plays an important role in hypertension. Therefore, the purpose of this study was to investigate the comparison of responsiveness to angiotensin II (ANG II) in isolated renal proximal convoluted tubules of spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats. METHODS: Intracellualr calcium concentration ([Ca2+i) was measured using Fura- 2/AM, inositol trisphosphate (IP3) accumulation was determined by radioimmuno assay and cellular ATP content measured using the microchemilunescene method in renal proximal tubule suspension or isolated renal proximal tubules. RESULTS: When measured the ANG II-induced [Ca2+i, the young rats showed a greater response to ANG II than adult rats in both strains. The ANG II (10-7 M)-induced [Ca2+i transient in the cortical tubule suspension from adult SHR was significantly lower than that in age-matched WKY. In isolated proximal tubule segments, ANG II-induced [Ca2+i increment was only observed in S1 segments. Comparing responsiveness to ANG II in SHR and WKY, similar phenomenon was observed as experiment using tubule suspension. IP3 accumulation by ANG II also attenuated in adult SHR. The 20-minutes incubation without any exogenous substrate in proximal convoluted tubule (S1) significantly decreased cellular ATP content and ANG II (10-7 M) inhibited decrement of cellular ATP level. The effect of ANG II on cellular ATP restoration was disappeared by the treatment with losartan. CONCLUSION: From these results, the responsiveness of ANG II to AT1A receptor is attenuated in the proximal convoluted tubules of adult SHR comparing the age- mached WKY.

Regional Differences in Dietary Supplement Use and Related Factors among College Students Participating in Nutritional Education Programs via the Internet / 대한지역사회영양학회지

The purpose of this study was to investigate the regional differences in dietary supplement use and related factors among college students participating in nutritional education programs via the internet. The subjects in this study were 797 college students (male: 518, female: 279). A cross-sectional survey was conducted using a self-administered questionnaire about dietary supplement use, demographic characteristics, health-related lifestyles, nutrient intake, and data were collected via the internet and by mail. Dietary supplements were taken by 82.2% of subjects (males: 76.3%, females: 85.3%). There was a significant regional difference in supplement use (p < 0.01). The supplements, used most frequent by male students in the Seoul and Incheon areas were lactic acid beverages and gagogi, in that order. Male students in the Kyunggi area used gagogi and lactic acid beverages in that order. Lactic acid beverages and Chinese medicine were used most frequently by male students in the Chungcheong area. Male students in the Kyungsang area used lactic acid beverages and water-soluble vitamins, in that order. Male students in Seoul and Kyungsang areas showed significantly higher percentage of keep on taking supplements compared to other areas (p < 0.05). Both male and female students in the Seoul area had more frequent medical examinations, in comparison to students in other areas (p < 0.05). Male students in the Kyungsang and Seoul areas took significantly more calcium (p < 0.05) and iron (p < 0.05), in comparison to students in other areas. Female students in the Kyunsang area took significantly more Vitamin A, phosphorous and iron (p < 0.05), while those in the Seoul area took significantly more Vitamin C (p < 0.01) and calcium (p < 0.05). Male students in the Chungcheong area took significantly less seaweed, in comparison to students in other areas (p < 0.05). Male students in the Seoul area had the highest amount of animal fat and oil, while those in the Incheon area ate the lowest amount of animal oil and fat (p < 0.05). Female students in the Kyunggi area ate the highest amount of fruit, while those in the Incheon area ate the lowest amount of fruit (p < 0.05). Female students in the Incheon area ate the highest amount of seaweed, while those in the Kyunggi area ate the lowest amount of seaweed (p < 0.01). Therefore, it can be concluded that it is necessary to develop dietary supplements to optimize the nutritional status of college students in different areas of Korea.

The Dietary Behavior, Dietary Intakes and Internet Searches Reagarding by Disease in College Students Participating in a Nutrition Education Web Class / 대한지역사회영양학회지

The purpose of this study was to investigate the dietary behavior, dietary intakes and internet searches regarding diseas by college students participating in a nutrition education web class. The study subject were 1184 college students and crosssectional survey was conducted by a self-administered questionnaire. The questionnaire deal with anthropometric data, diseas, dietary behaviors, dietary intakes, and the use of nutrition information via the internet. The data was analyzed using a SPSS PC program. Disease group was 19.6% (male: 17.1%, female: 21.9%) and non-disease group was 80.4% (male: 82.9%, female: 78.1%) of subjects. As for the kinds of diseas, the male disease group had stomach diseas (15.7%), ozena (15.7%), hepatitis (10.8%) and allergic disease (7.8%), etc, and the female disease group had stomach diseas (32.8%), allergic diseas (12.0%), bronchitis and bronchial asthma (10.4%), anemia (7.2%), and constipation (1.6%), etc. Non-disease group showed significantly higher irregularity of meal time compared to disease group. Also, Disease group showed significantly higher concern about nutrition and health, worse recognition of theirown nutrition and health satus, and made greater efforts to prevention diseas compared to the non-diseas group. In the male subjects, the average intake of Calories, Vitamin B2 and calcium was lower than the Korean RDA and in the female subjects, those of Calories, calcium and Fe were lower than the Korean RDA. Most of the diseas (97%) and the non-disease (98.2%) groups had positive attituds toward the usefulness of internet nutrition information. The disease group showed a higher confidence in the internet nutrition information and recognition of internet information on poisons compared to the non-diseas group. However, this difference was not significant. Therefore. it is necessary to provide sientifically proven nutrition of information via the internet for college students in order to promote healthful lifestyles and to prevent diseas.