Biomedical subjects

P Tietz

Publications and source records attributed to P Tietz.

8 recordsLinked to original sources

PubMed2005

Membrane microdomains in hepatocytes: potential target areas for proteins involved in canalicular bile secretion.

The formation of hepatic bile requires that water be transported across liver epithelia. Rat hepatocytes express three aquaporins (AQPs): AQP8, AQP9, and AQP0. Recognizing that cholesterol and sphingolipids are thought to promote the assembly of proteins into specialized membrane microdomains, we hypothesized that canalicular bile secretion involves the trafficking of vesicles to and from localized lipid-enriched microdomains in the canalicular plasma membrane. Hepatocyte plasma membranes were sonicated in Triton and centrifuged overnight on a sucrose gradient to yield a Triton-soluble pellet and a Triton-insoluble, sphingolipid-enriched microdomain fraction at the 5%/30% sucrose interface. The detergent-insoluble portion of the hepatocyte plasma membrane was enriched in alkaline phosphatase (a microdomain-positive marker) and devoid of amino-peptidase N (a microdomain-negative marker), enriched in caveolin, both AQP8 and AQP9, but negative for clathrin. The microdomain fractions contained chloride-bicarbonate anion exchanger isoform 2 and multidrug resistance-associated protein 2. Exposure of isolated hepatocytes to glucagon increased the expression of AQP8 but not AQP9 in the microdomain fractions. Sphingolipid analysis of the insoluble fraction showed the predominant species to be sphingomyelin. These data support the presence of sphingolipid-enriched microdomains of the hepatocyte membrane that represent potential localized target areas for the clustering of AQPs and functionally related proteins involved in canalicular bile secretion.

Animals↗

PubMed2000

Alternative splicing of the rat sodium/bile acid transporter changes its cellular localization and transport properties.

Bile secretion involves the structural and functional interplay of hepatocytes and cholangiocytes, the cells lining the intrahepatic bile ducts. Hepatocytes actively secrete bile acids into the canalicular space and cholangiocytes then transport bile acids in a vectorial manner across their apical and basolateral plasma membranes. The initial step in the transepithelial transport of bile acids across rat cholangiocytes is apical uptake by a Na(+)-dependent bile acid transporter (ASBT). To date, the molecular basis of the obligate efflux mechanism for extrusion of bile acids across the cholangiocyte basolateral membrane remains unknown. We have identified an exon-2 skipped, alternatively spliced form of ASBT, designated t-ASBT, expressed in rat cholangiocytes, ileum, and kidney. Alternative splicing causes a frameshift that produces a 154-aa protein. Antipeptide antibodies detected the approximately 19 kDa t-ASBT polypeptide in rat cholangiocytes, ileum, and kidney. The t-ASBT was specifically localized to the basolateral domain of cholangiocytes. Transport studies in Xenopus oocytes revealed that t-ASBT can function as a bile acid efflux protein. Thus, alternative splicing changes the cellular targeting of ASBT, alters its functional properties, and provides a mechanism for rat cholangiocytes and other bile acid-transporting epithelia to extrude bile acids. Our work represents an example in which a single gene appears to encode via alternative splicing both uptake and obligate efflux carriers in a bile acid-transporting epithelial cell.

Alternative Splicing↗

PubMed1998

Cholangiocyte-specific rat liver proteins identified by establishment of a two-dimensional gel protein database.

The liver is composed of a variety of cells that form a functional unit involved in uptake, synthesis, metabolism, and secretion. Until recently, most studies examining liver function did not analyze the specific proteins expressed or functions performed by the multiple individual cell types that constitute the hepatic mass. In the last decade, novel isolation methods have been developed that allow the purification of liver cell populations highly enriched in one type of liver cell. Here, we present a detailed two-dimensional (2-D) protein map of rat bile duct epithelial cells (i.e., cholangiocytes) using a recently developed isolation procedure. In addition, we identify 27 major cholangiocyte proteins either by comparison to maps of known rat liver proteins (based on pI and Mr) or by tryptic digestion and microsequencing. Finally, we compare the relative abundance of individual proteins present in cholangiocytes to whole liver as well as hepatocyte-specific proteins. Our results show that cholangiocytes express a unique array of individual proteins. The cholangiocyte 2-D protein pattern is markedly different from that of isolated rat hepatocytes or whole rat liver, with high levels of proteins previously known to be expressed by cholangiocytes (e.g., cytokeratins, actins) as well as protein not previously demonstrated to be expressed at high levels (e.g., annexin V, selenium binding protein). We conclude that this cholangiocyte-derived, 2-D protein map will be a crucial resource for studies directed at our understanding of cholangiocyte physiology and pathobiology.

Animals↗

PubMed1998

Cryptosporidium parvum is cytopathic for cultured human biliary epithelia via an apoptotic mechanism.

While the clinical features of sclerosing cholangitis secondary to opportunistic infections of the biliary tree in patients with acquired immunodeficiency syndrome (AIDS) are well known, the mechanisms by which microbial pathogens such as Cryptosporidium parvum associated with this syndrome actually cause disease are obscure. We established an in vitro model of biliary cryptosporidiosis employing a human biliary epithelial cell line. Using morphological and biochemical techniques, we examined the interaction of C. parvum with cultured human cholangiocytes. When the apical plasma membrane of polarized, confluent monolayers of human biliary epithelial cells was exposed to C. parvum oocysts that had been excysted in vitro, sporozoites attached to and invaded the cells in a time-, dose-, temperature-, and pH-dependent manner. The infectious process was both plasma membrane domain- and cell-specific, because no attachment or invasion occurred when the basolateral membrane of cholangiocytes was exposed to the parasite, or when a human hepatocyte cell line (HepG2) was used. Time-lapse video microscopy and scanning electron microscopy (SEM) showed that sporozoite attachment was rapid, involved extensive cholangiocyte membrane ruffling, and culminated in parasite penetration into a tight-fitting vacuole formed by invagination of the plasma membrane similar to those found in naturally occurring infection in vivo. Transmission electron microscopy (TEM) showed that C. parvum organisms formed parasitophorus vacuoles and were able to undergo a complete reproductive cycle, forming both asexual and sexual reproductive stages. Unexpectedly, direct cytopathic effects were noted in infected monolayers, with widespread programmed cell death (i.e., apoptosis) of biliary epithelial cells as assessed both morphologically and biochemically beginning within hours after exposure to the organism. The novel finding of specific cytopathic invasion of biliary epithelia by C. parvum may be relevant to the pathogenesis and possible therapy of the secondary sclerosing cholangitis seen in AIDS patients with biliary cryptosporidiosis.

Animals↗

PubMed1997

Characterization of apical and basolateral plasma membrane domains derived from cultured rat cholangiocytes.

Cholangiocytes, the epithelial cells that line intrahepatic bile ducts, are composed of plasma membranes with discrete apical (lumenal) and basolateral domains that contain different channels, transporters, and receptors. In recent work, we developed a long-term, primary culture system of normal rat cholangiocytes (NRC). Our aims here were to prepare and characterize apical and basolateral plasma membrane vesicles from NRC. Using serial isopycnic centrifugation on sucrose gradients, we generated separate apical and basolateral plasma membrane vesicles. We characterized these vesicles by transmission electron microscopy, specific marker enzyme assays, and immunoblotting; we also determined the percentage of sealed vesicles and their intravesicular volume and sidedness using biochemical approaches. Our results showed that vesicles derived from the apical and basolateral plasma membrane domains of NRC were highly purified, predominately sealed, right-side-out vesicles that differed in size and lipid and protein composition. This experimental model represents a novel tool that will be useful for additional functional studies on the channels, transporters, and receptors differentially distributed in the plasma membrane of biliary epithelia.

Animals↗

PubMed1997

Rat cholangiocytes absorb bile acids at their apical domain via the ileal sodium-dependent bile acid transporter.

Although bile acid transport by bile duct epithelial cells, or cholangiocytes, has been postulated, the details of this process remain unclear. Thus, we performed transport studies with [3H]taurocholate in confluent polarized monolayers of normal rat cholangiocytes (NRC). We observed unidirectional (i.e., apical to basolateral) Na+-dependent transcellular transport of [3H]taurocholate. Kinetic studies in purified vesicles derived from the apical domain of NRC disclosed saturable Na+-dependent uptake of [3H]taurocholate, with apparent Km and Vmax values of 209+/-45 microM and 1.23+/-0.14 nmol/mg/10 s, respectively. Reverse transcriptase PCR (RT-PCR) using degenerate primers for both the rat liver Na+-dependent taurocholate-cotransporting polypeptide and rat ileal apical Na+-dependent bile acid transporter, designated Ntcp and ASBT, respectively, revealed a 206-bp product in NRC whose sequence was identical to the ASBT. Northern blot analysis demonstrated that the size of the ASBT transcript was identical in NRC, freshly isolated cholangiocytes, and terminal ileum. In situ RT-PCR on normal rat liver showed that the message for ASBT was present only in cholangiocytes. Immunoblots using a well-characterized antibody for the ASBT demonstrated a 48-kD protein present only in apical membranes. Indirect immunohistochemistry revealed apical localization of ASBT in cholangiocytes in normal rat liver. The data provide direct evidence that conjugated bile acids are taken up at the apical domain of cholangiocytes via the ASBT, and are consistent with the notion that cholangiocyte physiology may be directly influenced by bile acids.

Animals↗

PubMed1997

[Modern fetal autopsy oriented to clinical demands using technical information methods].

Modern ultrasonic diagnostic instruments with high resolution and color Doppler sonography allow the obstetrician to describe pathological findings very precisely and at an early stage of pregnancy. The use of high-resolution ultrasound makes it possible to detect heart malformations already between the 8th and 10th week of gestation. This development has essentially changed the clinical requirements on fetal autopsy which in its traditional way no longer meets the increased demands. Unless the pathologists faces this challenge the importance of fetal autopsy will diminish. Modern image databases and communication techniques allow the pathologist to have direct access to text and images of pre-findings by other clinics. Great importance, however, must be attached to the interdisciplinary cooperation with obstetricians and geneticists. We report on routine fetal autopsy by using an interdisciplinary database an the Charité.

Autopsy↗

PubMed1981

Synthesis and biological activity of vitamin D3 3 beta-sulfate. Role of vitamin D3 sulfates in calcium homeostasis.

To determine the biological activity of vitamin D sulfates, we synthesized vitamin D3 3 beta-sulfate and tested its biological activity in vitamin D-deficient hypocalcemic rats. When vitamin D3 sulfate was administered as a single oral dose of 208,000 or 416,000 pmol (100 micrograms or 200 micrograms), it increased active calcium transport in the duodenum and was also able to mobilize calcium from bone and soft tissue. Dose levels below this failed to elicit a response. Vitamin D3 itself was active at doses as low as 260 pmol when administered in this manner. In order to test the biological activity of vitamin D3 sulfate in various doses when administered chronically, we tested the biological activity of vitamin D3 sulfate after 5 days or oral dosing: vitamin D3 sulfate was active at doses of 52,000 pmol/day (25 microgram), whereas vitamin D3 was active at doses of 65 to 260 pmol/day over a period of 5 days. When administered as a single intravenous dose, vitamin D3 sulfate exhibited no biological activity in doses as high as 52,000 pmol. Vitamin D3, however, was active at a dose of as low as 65 pmol. We conclude that vitamin D3 sulfate, a metabolite of vitamin D3 of heretofore unknown biological activity, is considerably less active than vitamin d3 itself.

Animals↗