Mathias Höchli

In situ localization of the hepatocytic na+/taurocholate cotransporting polypeptide in rat liver

BACKGROUND/AIMS An Na+/taurocholate cotransporting polypeptide (Ntcp) has recently been cloned from rat liver. The aim of this study was to directly characterize the native Ntcp on the protein level and study its in situ distribution in rat liver. METHODS A rabbit antiserum was raised against a fusion protein containing the maltose-binding protein and the C terminus of Ntcp. Native Ntcp was localized in situ by immunofluorescent techniques. Expression of Ntcp was directly correlated with taurocholate uptake measurements in stably transfected Chinese hamster ovary cells. RESULTS Native Ntcp showed an apparent molecular weight of 51,000. After deglycosylation of isolated basolateral rat liver plasma membranes, the apparent molecular weight of Ntcp decreased to 33,500. In intact rat liver, Ntcp was selectively localized at the basolateral surface domain of hepatocytes. In short-term cultured hepatocytes, a positive surface immunoreaction was only obtained in detergent-permeabilized cell cultures. In stably transfected Chinese hamster ovary cells, the surface expression of immunopositive Ntcp was associated with Na(+)-dependent taurocholate uptake activity. CONCLUSIONS Native Ntcp represents a glycoprotein of the basolateral hepatocyte plasma membrane with its C-terminal end facing the intracellular compartment. Furthermore, surface expression of Ntcp is a prerequisite for Na(+)-dependent taurocholate uptake to occur, thus providing further proof for its bile acid transport function in rat liver.

Polyspecific substrate uptake by the hepatic organic anion transporter Oatp1 in stably transfected CHO cells

The rat liver organic anion transporting polypeptide (Oatp1) has been extensively characterized mainly in the Xenopus laevis expression system as a polyspecific carrier transporting organic anions (bile salts), neutral compounds, and even organic cations. In this study, we extended this characterization using a mammalian expression system and confirm the basolateral hepatic expression of Oatp1 with a new antibody. Besides sulfobromophthalein [Michaelis-Menten constant ( K m) of ∼3 μM], taurocholate ( K m of ∼32 μM), and estradiol- 17β-glucuronide ( K m of ∼4 μM), substrates previously shown to be transported by Oatp1 in transfected HeLa cells, we determined the kinetic parameters for cholate ( K m of ∼54 μM), glycocholate ( K m of ∼54 μM), estrone-3-sulfate ( K m of ∼11 μM), CRC-220 ( K m of ∼57 μM), ouabain ( K m of ∼3,000 μM), and ochratoxin A ( K m of ∼29 μM) in stably transfected Chinese hamster ovary (CHO) cells. In addition, three new substrates, taurochenodeoxycholate ( K m of ∼7 μM), tauroursodeoxycholate ( K m of ∼13 μM), and dehydroepiandrosterone sulfate ( K m of ∼5 μM), were also investigated. The results establish the polyspecific nature of Oatp1 in a mammalian expression system and definitely identify conjugated dihydroxy bile salts and steroid conjugates as high-affinity endogenous substrates of Oatp1.

Calbindin D-28k and Monoamine Oxidase A Immunoreactive Neurons in the Nucleus Basalis of Meynert in Senile Dementia of the Alzheimer Type and Parkinson's Disease

In this study, calbindin D-28k (CaBP), monoamine oxidase A (MAO A) and nerve growth factor receptor (NGFr) immunoreactivities were investigated in the nucleus basalis of Meynert (NbM) in patients with senile dementia of the Alzheimer type (SDAT), with Parkinsons disease (PD) with or without dementia, and in controls. Immunocytochemistry using specific antibodies in differing serial sections was employed, and cell counts and NbM nuclear volume measurements were made. Most of the large multipolar NbM neurons showed CaBP immunoreactivity in the cytoplasm of their somata, dendrites and axons. In adjacent, NGFr-reacted sections, the large NbM neurons were also found to be intensely immunoreactive for NGFr on their cellular surfaces. In addition, a subpopulation of large NbM neurons and glial cells were found to be immunoreactive for MAO A. The number of CaBP-immunoreactive (CaBP-i) neurons was decreased by an average of 55% in the 6 SDAT patients, 70% in the 2 nondemented PD patients and 40% in the 1 demented PD patient. The volume calculated for the compact part of the NbM formed by the CaBP-i neuronal somata decreased by an average of 47% in SDAT. On the other hand, measurements in the volume of NGFr-i neurons (including the dendritic arborization) showed an average decrease of 25% in SDAT patients compared to controls. Although all SDAT and PD patients showed a decrease of CaBP-i neurons in the NbM, a loss of MAO-A-i NbM neurons was found only in those patients with dementia. Therefore, the relative proportions of MAO-A-i to CaBP-i neurons were increased in the nondemented PD patients (14.2 and 19.6%) when compared with those in the demented PD patient (2.2%) and with the SDAT patients (0.3-5.6%). These data indicate that a balanced presence of MAO-A-i cholinergic, large NbM neurons may be necessary for the proper maintenance of cognitive function. Functionally this may be translated to mean that dementing changes may cause a decrease from the normal amount of MAO A enzyme activity. This suggests that therapeutic strategies based upon correction of MAO-A activities by MAO-A inhibitors may be important to ameliorating some of the loss in cholinergic function in dementias of SDAT and PD.

Distribution of Neuropeptide Y, C-Terminal Flanking Peptide of NPY and Galanin and Coexistence with Catecholamine in the Locus coeruleus of Normal Human, Alzheimer's Dementia and Parkinson's Disease Brains

The study demonstrates the presence of neuropeptide Y (NPY)-, C-terminal flanking peptide of NPY (C-PON)-, and galanin (GA)-immunoreactive (-i) neurons and axons in the locus coeruleus in normal adult

Raphe Serotonin Neurons in the Human Brain Stem in Normal Controls and Patients with Senile Dementia of the Alzheimer Type and Parkinson's Disease: Relationship to Monoamine Oxidase Enzyme Localization (Part 1 of 3)

The raphe serotonin system was studied in patients with senile dementia of the Alzheimer type (SDAT) or with Parkinsons disease and compared with normal controls. Postmortem brain stems were cut in c

Parvalbumin Neurons in the Hippocampus in Senile Dementia of the Alzheimer Type, Parkinson's Disease and Multi-Infarct Dementia (Part 1 of 2)

The immunogold-silver-staining method (IGSS) was used for the detection of hippocampal neurons containing the cytoplasmic calcium-binding protein parvalbumin in senile dementia of the Alzheimer type (SDAT), Parkinsons disease and multi-infarct dementia (MID). Computer-based image analysis of cell numbers in the hippocampus proper and the entorhinal cortex, measurement of total dendritic length of selected neurons and quantitative microdensitometry of immunoreacted sections was performed. In severe SDAT cases a loss of parvalbumin-immunoreactive (parv-i) neurons and a reduction of dendritic fields was detected especially in CA1 and the subiculum. In the entorhinal cortex the general atrophy exceeded the decrease of parvalbumin immunoreactivity. Mild SDAT cases showed only minor alterations of parv-i neurons and in cases of parkinsonism without cognitive disorders or in those with depression parv-i neurons were not affected. However, in cases of parkinsonism with dementia and in MID a severe cell loss and a drastic reduction of dendritic arborization in the hippocampus proper was detected, which was accompanied by additional morphological alterations due to ischemic damage. We conclude that parv-i neuronal networks in the hippocampus are damaged in some patients with dementing disorders in SDAT, Parkinsons disease and MID consecutive to Alzheimer or ischemia related pathology.

Angiotensin II immunoreactivity in the human striatum and hippocampus of elderly controls and patients with senile dementia of the Alzheimer type and multi-infarct dementia

The distribution of angiotensin II (A II) immunoreactivity in the postmortem human corpus striatum was studied in control cases and in patients with senile dementia of the Alzheimer type (SDAT) and multi-infarct dementia (MID). Additionally A II immunoreactivity was examined in the hippocampus of control and SDAT patients. A II-immunopositive cellular structures found in the striatum were the large neurons and vesiculated fibers in the globus pallidus (GP), especially in the dorsal parts, and some aspiny neurons in the putamen. Changes in the cell morphology marked the neurons in the GP of SDAT patients. In the MID cases only a few A II-immunopositive fibers and no cell somata were present in the GP. An extracellular, perivascular A II-staining pattern, which was found only in 1 control case, was common to all SDAT and MID patients. A II-immunopositive cellular structures found in the hippocampus included distorted neuronal processes and vesiculated fibers, which were partly coexisting with neuritic plaques as shown by the double labeling with thioflavin. The perivascular A II staining was also observed in the hippocampus, which was more predominant in SDAT cases. The definition of these two A II-immunopositive cell types in human corpus striatum suggests that A II may be involved in local neuronal or projection pathways. Possibly A II participates in the development of cerebrovascular pathologies in elderly control and demented brains.