[Complication of self-injection with petroleum jelly for penis augmentation]. / Komplikation durch Selbstinjektion mit Vaseline zur Penisaugmentation.

An increasing number of complications after penis enlargement procedures, sometimes self-performed, are being observed in Germany and in the other countries. This report presents a case of a 43-year-old patient who presented with multiple fistulas, paraffinomas and bacterial superinfection after having injected petroleum jelly into his penis. In order to remove the foreign bodies as well as the infected and necrotic tissue the complete epithelium had to be radically excised. After further local and surgical wound treatment penis reconstruction with a full thickness skin graft was performed which later led to a functional and aesthetical complete restoration to the original condition.

Bevacizumab in combination with sequential high-dose chemotherapy in solid cancer, a feasibility study.

We evaluated the feasibility and toxicity of bevacizumab in combination with sequential high-dose (HD) ifosfamide, carboplatin and etoposide refractory to standard chemotherapy in patients with sarcoma and germ cell cancer (GCC). Sixteen patients (13 sarcomas, 3 GCC) received SD-ICE followed by 4 cycles of HD-ICE, qd22 with stem cell support in combination with bevacizumab. All 16 patients were evaluable for toxicity and efficacy, and received 51 cycles (median 3.3). There was no increase in toxicity except of a relatively high incidence of ifosfamide encephalopathy in 17 cycles when compared with previous HD-ICE protocols. One almost complete response in the patient with GCC, previously progressive with three preceding protocols, was observed. Six patients had a partial response (sarcoma 4/13 patients; GCC 2/3 patients), and five patients stable disease (sarcoma 5/13 patients). The median PFS/OS for sarcoma was 5 months (confidence interval (CI): 3.1-6.9) and 13 months (CI: 3.6-24.4), respectively. To our knowledge, this is the first report of the addition of bevacizumab to HD-ICE. This combination did not show new unexpected toxicities except for a relatively high rate of ifosfamide encephalopathy. The efficacy in these heavily pretreated patients including possible reversal of chemotherapy resistance by the addition of bevacizumab indicates a possible potential of bevacizumab in this combination.

In vitro differentiation of human dental follicle cells with dexamethasone and insulin.

The dental follicle is an ectomesenchymally derived connective tissue harboring precursor cells for the tooth supporting apparatus. In this study, we examined gene expression of freshly isolated human dental follicle cells during osteogenic differentiation in vitro. These plastic adherent fibroblastic cells express Notch-1, nestin and vimentin. We differentiated dental follicle cells with dexamethasone or insulin-based protocols into membrane-like structures containing mineralizing foci. An analysis of mineralized tissue with atomic force microscopy illustrated a bone and cementum-like structure. A real-time RT-PCR analysis was developed to investigate expression of typical osteoblast or cementoblast related genes during differentiation. Gene expressions of osteocalcin (OCN), bone morphogenic protein (BMP)-2 and nestin were increased during the both differentiation approaches. Our work demonstrates differentiation of dental follicle cells with an insulin-based protocol for the first time.

Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth.

The dental follicle is an ectomesenchymal tissue surrounding the developing tooth germ. It is believed that this tissue contains stem cells and lineage committed progenitor cells or precursor cells (PCs) for cementoblasts, periodontal ligament cells, and osteoblasts. In this study, we report the isolation of PCs derived from dental follicle of human third molar teeth. These fibroblast-like, colony forming and plastic adherent cells expressed putative stem cell markers Notch-1 and Nestin. We compared gene expressions of PCs, human mesenchymal stem cells (hMSCs), periodontal ligament cells (PDL-cells) and osteoblasts (MG63) for delimitation of PCs. Interestingly, PCs expressed higher amounts of insulin-like growth factor-2 (IGF-2) transcripts than hMSCs. Differentiation capacity was demonstrated under in vitro conditions for PCs. Long-term cultures with dexamethasone produced compact calcified nodules or appeared as plain membrane structures of different dimensions consisting of a connective tissue like matrix encapsulated by a mesothelium-like cellular structure. PCs differentially express osteocalcin (OCN) and bone sialoprotein (BS) after transplantation in immunocompromised mice but without any sign of cementum or bone formation. Therefore, our results demonstrate that cultured PCs are unique undifferentiated lineage committed cells residing in the periodontium prior or during tooth eruption.

[Limits in cementless hip revision total hip arthroplasty. Midterm experience with an oblong revision cup]. / Grenzen der zementfreien Revisionsarthroplastik. Mittelfristige Resultate mit der längsovalen Revisionspfanne.

UNLABELLED: Revision of an acetabular component in a patient who has severe periacetabular bone loss is a complex problem, particularly when there is not enough bone stock to allow placement of an acetabular component near the normal anatomical hip center. To fill the defect, a valuable option for revision arthroplasty is the cementless oblong revision cup (LOR). METHODS: 50 consecutive revisions of the acetabular component were performed in 48 patients. The mean age at the time of revision was sixty-one years (range, thirty-three to seventy-eight years). Forty-eight hips were available for follow-up, at a mean of thirty-two months (range, eighteen to sixty-one months). The acetabular defect classified according to Paprosky, the migration and the radiolucencies were followed radiologically. RESULTS: 8 hips (16 %) were revised again: two because of infection (4 %) and six because of instability (12 %). The revised hips are not associated to the preoperative degree of acetabular defect (34 % defect type III) (P > 0.05). The mean Harris Hip score was corrected from 36.5 (range, 7.5 to 92.5) to 78.2 points (range, 47.6 to 97.6) (P < 0.01). The mean d'Aubigné Score was corrected from 8.3 (range, 4 to 6) to 15 points (range, 10 to 18) (P < 0.01). Neither pre- nor postoperative results were associated to the degree of acetabular defect (P > 0.05). However, patients with multiple revisions had a significantly reduced clinical outcome than patients with the first revision (P < 0.05). The hip center of rotation, cranially placed to the contralateral side (0.92 cm) was corrected by the revision to a more normal anatomic rotation center (0.27 cm). Partial zonal radiolucencies, always smaller than 1.5 mm were seen in 30 % of the patients. The mean migration of the acetabular component was not significant (P > 0.05). CONCLUSION: The authors support the use of the cementless oblong revision cup if contact can be made with host bone to more than 50 %. If this is not possible, acetabular bone reconstruction combined with a roof ring and a cemented cup is the component of choice.

[Objective measures of gait following revision hip arthroplasty. First medium-term results 2.6 years after surgery]. / Ganganalyse bei Patienten nach Hüftpfannenwechsel. Erste mittelfristige Ergebnisse 2,6 Jahre postoperativ.

AIM: The purpose of the study was to identify the functional impairments after revision arthroplasty by gait analysis. METHODS: This retrospective study compared 33 patients (mean age 58.5 years) who have undergone revision of an acetabular component (mean follow-up 2.6 years) with a group of normal control subjects. Gait analysis including recording of the three dimensional kinetics and kinematics was performed in all patients. Surface electromyography of seven leg and trunk muscles were registered bilaterally. The vertical ground reaction forces were determined by two force plates. These data were correlated with the Harris Hip Score, the d'Aubigné Score and the radiographic analysis (centre of rotation). RESULTS: The analysis revealed a decreased hip range of motion during gait (p < 0.0001). In the sagittal plane there was a significant decrease in the hip extension at the end of the stance phase (p < 0.0001). The control group reached a mean extension of - 7.6 degrees, the operated patients were limited by the extension deficit (+ 9.1) in step length (p < 0.0016) and velocity (p < 0.0001). Kinetic parameters indicated a reduced hip abductor moment (p < 0.0001). Compensation of gait instability was observed in an extended stance phase (p = 0.0389). The hip muscle activity was increased to stabilize the impaired hip. The changed kinematic parameters are observed with secondary impairments in knee extension and reduced dorsiflexion in ankle motion (p < 0.0001). Neither the Harris Hip score (77.8 points) nor the d'Aubigné score (14.9 points) were associated with the motion analysis (p > 0.05). Deterioration in kinematics are indicated by cranialisation of the centre of rotation (p = 0.18). However, medial movement of the centre of rotation does not influence the kinematic data (p > 0.05). CONCLUSION: Despite sufficient satisfactory clinical data the gait analysis confirmed objective impairments of the operated hip and neighboring joints. Gait instability is revealed in a decreased hip extension and deficient hip abduction.

Reconstitution of bile acid transport in a heterologous cell by cotransfection of transporters for bile acid uptake and efflux.

The rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 (CBATP) is a 110-kDa transmembrane phosphoglycoprotein that is thought to have bile acid efflux, ecto-ATPase, and cell adhesion properties. Its extracellular amino-terminal domain is highly homologous to carcinoembryonic antigen (CEA), a glycophosphatidyl inositol-anchored membrane protein with cell adhesion properties and a marker for adenocarcinoma. In the current study, we examined the possibility of more clearly defining the role of CBATP in bile acid efflux by cotransfecting a heterologous cell, the COS cell, with cDNAs for a bile acid importer, the ileal bile acid transporter (IBAT), as well as for CBATP. The results show that when IBAT mediates uptake of [3H]taurocholate to a level 20-fold higher than that achieved previously by nonspecific pinocytosis, CBATP mediates time-, temperature- and concentration-dependent efflux. Efflux of [3H]taurocholate mediated by CBATP in the cotransfected COS cells is saturable and has curvilinear kinetic characteristics (Vmax = 400 pmol/mg protein/min, Km = 70 microM). It is inhibited by 4,4'-diisothiocyanostilbene-2,2-disulfonic acid and dependent on ATP but not dependent on membrane potential. Although CEA could not mediate bile acid efflux in COS cells cotransfected with IBAT and CEA, efflux of [3H]taurocholate was detected in COS cells cotransfected with IBAT and a chimeric molecule having the carboxyl-terminal tail and membrane spanning domain of CBATP and the amino-terminal extracellular tail of CEA. Taken together, these data provide further evidence that CBATP confers bile acid efflux properties on heterologous cells and that its cytoplasmic tail and membrane spanning segment are integral to this property. The data also establish a model system for more clearly defining the molecular determinants of bile acid transport mediated by this molecule.

Bile acid transport across the hepatocyte canalicular membrane.

Transport of bile acids across the canalicular membrane of the hepatocyte provides the primary motive force for generation of bile flow and is rate limiting in the vectorial movement of bile acids from blood to bile. Several distinct carriers for bile acids have been defined based on physiological studies in isolated hepatocytes, membrane vesicles, hepatocyte couples, and the perfused rat liver including membrane potential-driven and ATP-dependent mechanisms. Several groups have isolated and functionally reconstituted a canalicular bile acid transport protein of M(r) approximately 110 kDa. The ATP-dependent mechanism for secretion of monovalent bile acids appears to be mediated by a yet to be identified protein of the ATP binding cassette family of transporters. However, it remains conjectural whether the ATP-dependent and membrane potential-driven components of canalicular bile acid transport are mediated by one or more transport proteins. Bile acid sulfates and glucuronides are substrates for the canalicular multispecific organic anion transporter whose activity has recently been associated with the multidrug resistance-associated protein.

Ventilation scintigraphy of the middle ear.

UNLABELLED: In this study, an attempt was made to administer radioactive gas into the tympanic cavity to measure initial gas trappings as well as clearance from the middle ear to evaluate eustachian tube function. METHODS: Twenty-eight patients were administered 50 MBq 133Xe gas. Three different methods for gas application were tested: (a) direct injection through a tympanostomy tube in two patients, (b) administration through a nasopharyngeal catheter combined with Valsalva maneuvers in six subjects without middle ear dysfunction and (c) insufflation into the pharyngeal space through a nose olive performed in 12 patients with normal eustachian tube function and in eight patients with one-sided tube dysfunction. RESULTS: All three approaches were successful in visualizing middle ear ventilation, demonstrating tracer trapping within the tympanic cavities in 20 of 28 patients. Semiquantitative evaluation by region of interest techniques revealed a left-to-right uptake ratio of 48.4%-51.6% in 13 patients without tube dysfunction. Five patients with one-sided tube dysfunction showed a significantly lower median uptake of 31.6% (p = 0.01). The clearance half-lives ranged from 9 to 283 min in normal subjects and 37-64 min in patients with one-sided tube malfunction, demonstrating no statistically significant difference between the two groups and a trend towards increased washout in patients with tympanic dysfunction. CONCLUSION: Middle ear ventilation scintigraphy with 133Xe through a nose olive is an easy-to-perform test to evaluate eustachian tube function and has a success rate of about 70%, thus, reflecting the complex physiological mechanisms involved.

Site-directed mutagenesis within an ectoplasmic ATPase consensus sequence abrogates the cell aggregating properties of the rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 and carcinoembryonic antigen.

Recent studies of the rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 (CBATP), a member of the carcinoembryonic antigen (CEA) supergene family, indicate that it is a multifunctional protein possessing bile acid efflux, ecto-ATPase, and intercellular aggregating properties. Cheung et al. (Cheung, P. H., Luo, W., Qiu, Y., Zhang, K. E., Millron, P., Lin, S. H. (1993) J. Biol. Chem. 268, 24303-24310) have shown that the amino-terminal Ig V-like domain of this protein is required for its aggregating properties, much like the homologous amino-terminal domain of CEA is required for its aggregating properties. The amino-terminal domains of both CBATP and CEA include a consensus ATPase sequence. Site-directed mutagenesis within this ATPase consensus sequence completely eliminates the ecto-ATPase activity of CBATP (Sippel, C. J., McCollum, M., Perlmutter, D. H. (1994) J. Biol. Chem. 269, 2820-2826). In this study we examined the possibility that it is this ATPase consensus sequence which is required for the cell aggregating properties of CBATP and CEA and whether there is a relationship between ATPase, aggregating, and bile acid efflux activities. For this we used a baculovirus vector to express in Sf9 cells wild type as well as mutant and chimeric CBATP and CEA molecules. The results indicate that Arg-98 in the ATPase consensus sequence of CBATP and the corresponding residue of CEA are essential for the aggregating properties of these molecules. Moreover Arg-98 is essential for CBATP to interact with itself, CEA to interact with itself, and CBATP to interact with CEA. However, the role of Arg-98 in aggregation is distinct from its role in ecto-ATPase activity and the aggregating properties cannot be attributed to a change in ATP metabolism in the pericellular milieu.

Quantitative salivary gland scintigraphy in the diagnosis of parenchymal damage after treatment with radioiodine.

This study was undertaken to quantify salivary gland parenchymal damage after radioiodine treatment with a standard protective regimen of ascorbic acid. Altogether, 106 patients underwent quantitative salivary gland scintigraphy with 99Tcm-pertechnetate prior to and 3 months after radioiodine therapy. Parenchymal function was quantified by calculating 99Tcm-pertechnetate uptake 13 min post-injection. Patients received 131I doses ranging from 400 MBq to 24 GBq (cumulative). Among the patients who received large doses of 131I, severe parenchymal destruction could be visually analysed as well as quantitatively evaluated. In contrast, after low-dose radioiodine treatment, mild parenchymal impairment was demonstrated by quantitative evaluation only. In conclusion, standardized quantitative salivary gland scintigraphy is essential for the reliable detection of mild parenchymal malfunction. Despite the standard protection regimen using ascorbic acid as a sialogogue, radioiodine therapy induces loss of salivary gland parenchymal function even with low doses of 131I.

The value of bone scanning in pre-operative decision-making in patients with progressive facial asymmetry.

Hemimandibular elongation is characterized by persistent unilateral growth, resulting in unilateral overgrowth of the mandible. The surgical treatment strategy depends on condylar growth activity, which cannot be assessed by conventional radiological procedures. Therefore, this study was undertaken to evaluate the usefulness of bone scanning in hemimandibular elongation. Twenty-seven patients underwent bone scanning prior to surgery. Growth activity was quantified by calculating the L/R ratio. In the case of more pronounced right-sided growth producing a L/R ratio of less than 1, inverse values were used. Corrective osteotomy was performed in the patients with a L/R < 1.10, whereas patients with a L/R > 1.10 underwent condylectomy. Twenty-three patients had a L/R ratio < 1.10 and were followed up for 3 years. In 16 patients, a corrective osteotomy was performed without any relapse post-operatively. Four patients showed marked unilateral increased uptake. In one patient, a pre-operative bone scan was not considered, and corrective osteotomy was performed with subsequent recurrence of unilateral overgrowth of the mandible. The final patient underwent condylectomy without relapse. In two patients, it was decided to repeat the bone scan after a follow-up period of 12 months. In conclusion, bone scanning has significant clinical value in pre-operative decision-making in hemimandibular elongation by guiding surgical strategy with respect to condylar growth activity.

Somatostatin receptor scintigraphy in the differential diagnosis of meningioma.

The aim of this study was to evaluate somatostatin receptor scintigraphy (SRS) in patients with meningioma proven or suspected on magnetic resonance imaging (MRI). Prior to surgery, 47 patients were investigated up to 24 h following the injection of 200 MBq 111In-octreotide. Tracer uptake was compared with the histological presence of meningioma. Histology revealed 43 meningiomas, 3 neurinomas and 1 ependymoma. A true-positive SRS result was obtained in 36 patients, in 13 of whom a tumour volume of < 10 ml was noted. A false-negative SRS result was obtained in seven patients, all of whom had a tumour volume of < 10 ml. Whereas MRI alone was decisive in 38 of 47 patients, it could only provide a differential diagnosis in the remaining 9 patients. A positive SRS result confirmed meningioma in five of these patients, and a negative SRS result excluded meningioma in the other four. Therefore, cases of SRS-negative meningioma do exist. Nevertheless, significant clinical benefit can be obtained from functional imaging with 111In-octreotide in patients with an inconclusive MRI result, as large meningiomas can be excluded by scintigraphy alone, whereas meningiomas of any size may be confirmed in combination with specific MRI results.

Somatostatin receptor scintigraphy in the staging of small cell lung cancer.

The aim of this study was to evaluate somatostatin receptor scintigraphy (SRS) in the staging of patients with small cell lung cancer. Prior to chemotherapy, 20 patients were investigated up to 24 h following an injection of 200 MBq 111In-octreotide. Following chemotherapy and restaging, four patients were re-evaluated. Primary tumour was detected in 18 of 23 studies, which exhibited increasing target-to-back-ground ratios over time. Lymph node metastases and distant metastases were detected in 7 of 27 and 8 of 31 sites, respectively. Thus, the overall sensitivity for detecting metastases was less than 26%. SRS did not result in any upstaging of patients. We conclude that in patients with small cell lung cancer, functional imaging by SRS has no impact on clinical decision making.

Radiotherapy with iodine-131 in recurrent malignant struma ovarii.

Malignant struma ovarii is a very rare disease and, therefore, there is neither common agreement on treatment regimens nor sufficient follow-up experience. We present a case of a 49-year-old woman with malignant struma ovarii of the follicular type, who received ablative radioiodine treatment after thyroidectomy and surgical removal of the primary tumour. During follow-up examinations an increasing thyroglobulin level was found, caused by a tumour relapse with suspected urinary bladder infiltration on CT and proven uptake of radioiodine on whole-body scanning with iodine-131. After administration of 6GBq 131I, complete tumour regression was achieved with no evidence of a new relapse during a 30-month follow-up period. Correspondingly, repeated thyroglobulin measurements were all negative. This case demonstrates the benefit of combined surgical and radioiodine treatment of malignant struma ovarii for both monitoring and therapy of relapse or metastases; thus, the same therapeutic regimen as is employed in primary differentiated thyroid carcinoma may be recommended.

111In-octreotide imaging in patients with long-standing Graves' ophthalmopathy.

The aim of this study was to examine patients with long-standing Graves' ophthalmopathy using 111In-octreotide scintigraphy. Sixteen patients with inactive ophthalmopathy of up to 114 months duration and 14 normals were investigated for 48 h following an injection of 200 MBq 111In-octreotide. No significant tracer accumulation in the orbital region could be identified in any of the patients with long-standing Graves' ophthalmopathy. The orbit to brain (O/B) ratios after 24 and 48 h were 2.39 +/- 0.36 and 2.15 +/- 0.44 versus 2.17 +/- 0.33 and 2.20 +/- 0.37 for the patients and normals, respectively (N.S.). 111In-octreotide accumulation in ophthalmopathy described in the literature may thus be a passing event limited to its active stage, which is consistent with the concept of imaging a lymphocytic infiltration. In this study, the lack of accumulation of 111In-octreotide in the orbital region during the inactive stage demonstrates an absence of somatostatin receptors in orbital tissue itself. Thus, in patients with inactive Graves' ophthalmopathy, there is no basis for a diagnostic approach with somatostatin.

Value of quantitative salivary gland scintigraphy in the early stage of Sjögren's syndrome.

The aim of this study was to test the impact of quantitative salivary gland scintigraphy in patients with suspected Sjögren's syndrome. Thirteen patients with suspected Sjögren's syndrome were investigated. During clinical work-up, three had severe and four had mild Sjögren's syndrome, while six were normal. Quantitative salivary gland scintigraphy was performed using a standardized method. The normal data-base consisted of 172 patients without any evidence of salivary gland malfunction. Visual and quantitative comparisons of the patients' scintigrams were made. In the patients with severe Sjögren's syndrome, uptake was 0.10 +/- 0.04% and 0.09 +/- 0.03% in the parotid and submandibular glands respectively, confirming the visual diagnosis. In the patients without Sjögren's syndrome, concordance between the visual and quantitative evaluations could also be shown. In contrast, among the patients with mild Sjögren's syndrome, uptake was diminished (P < 0.05), amounting to 0.21 +/- 0.05% and 0.16 +/- 0.02% in the parotid and submandibular glands respectively, while visual analysis indicated normal parenchymatous function. In conclusion, quantitative salivary gland scintigraphy is essential for the reliable detection of parenchymatous malfunction at an early stage of Sjögren's syndrome, which may be missed by visual analysis alone.

Bile acid efflux mediated by the rat liver canalicular bile acid transport/ecto-ATPase protein requires serine 503 phosphorylation and is regulated by tyrosine 488 phosphorylation.

Transfection of cDNA for a hepatocyte canalicular phosphoprotein, the rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105, confers bile acid efflux and ecto-ATPase activities on heterologous cells (Sippel, C. J., Suchy, F. J., Ananthanarayanan, M., and Perlmutter D. H. (1993) J. Biol. Chem. 268, 2083-2091). Our previous studies have also indicated that there is a positive correlation between the degree of phosphorylation of this transporter and its bile acid efflux activity. In this study, we introduced site-specific mutations of amino acid residues within a protein kinase C-dependent (T502A, S503A) and a tyrosine kinase-dependent (Y488F) phosphorylation consensus sequence in the cytoplasmic tail of this transporter in order to map the sites that are phosphorylated in vivo and to examine the functional significance of each. COS cells were transfected with mutant and wild type constructs using the pCDM8 expression vector. Metabolic labeling and cell surface labeling showed that the mutant proteins were synthesized and delivered to the cell surface as efficiently as the wild type. Phosphoamino acid analysis using lysates of transfected cells showed that the T502A, S503A mutant contained [32P]phosphotyrosine, the Y488F mutant contained [32P]phosphoserine, and the wild type contained both 32P-labeled amino acids, proving that Ser503 and Tyr488 are the only amino acids phosphorylated in this system under control conditions. Bile acid transport activity was completely abrogated in cells transfected with the T502A, S503A mutant cDNA and was retained but altered in kinetic characteristics in cells transfected with the Y488F mutant cDNA, even though both of these constructs conferred ecto-ATPase activity to the same extent as the wild type cDNA. Taken together, these data show that the bile acid efflux activity of this transporter requires site-specific phosphorylation of Ser503 and is regulated by site-specific phosphorylation of Tyr488.

Bile acid transport by the rat liver canalicular bile acid transport/ecto-ATPase protein is dependent on ATP but not on its own ecto-ATPase activity.

We have shown that bile acid efflux and ecto-ATPase activities are two distinct properties of a single rat liver hepatocyte canalicular membrane protein (Sippel, C. J., Suchy, F. J., Ananthanarayanan, M., and Perlmutter, D. H. (1993) J. Biol. Chem. 268, 2083-2091). Bile acid efflux in COS cells transfected with this rat hepatocyte canalicular bile acid transport/ectoATPase cDNA is stimulated by ATP and inhibited by nonhydrolyzable ATP analogs. In this study, we depleted transfected COS cells of ATP to examine whether bile acid efflux mediated by this transporter was dependent on ATP or just stimulated by ATP. We also used mutagenesis of an ATPase consensus sequence in the ectoplasmic domain to examine the relationship of ATPase activity to bile acid efflux mediated by the same polypeptide. The results indicate that bile acid transport is abrogated by ATP depletion and reconstituted by exogenous ATP in a concentration-dependent and saturable manner. Introduction of mutations at amino acids Gly97 and Arg98 in the ATPase consensus sequence abrogated ATPase activity but did not affect synthesis or cell surface delivery of the transporter and did not affect its bile acid transport activity. Taken together, the data indicate that bile acid efflux mediated by the rat hepatocyte canalicular bile acid transport/ecto-ATPase protein is dependent on ATP but not on its own ATPase activity. The data, therefore, imply that 1) ATP affects its bile acid transport activity through an entirely distinct mechanism; and 2) if there is any functional relationship between the ecto-ATPase and bile acid transport properties, it is mediated indirectly through regulation of net ATP concentrations in the canalicular space by the ecto-ATPase.

The rat liver ecto-ATPase is also a canalicular bile acid transport protein.

A approximately 110-kDa glycoprotein purified from canalicular vesicles by bile acid affinity chromatography has been identified as the canalicular bile acid transport protein. Internal amino acid sequence and chemical and immunochemical characteristics of this protein were found to be identical to a rat liver canalicular ecto-ATPase. In order to definitively determine whether these were two activities of a single polypeptide, we examined the possibility that transfection of cDNA for the ecto-ATPase would confer bile acid transport characteristics, as well as ecto-ATPase activity, on heterologous cells. The results show that transfection of the ecto-ATPase cDNA conferred on COS cells de novo synthesis of a approximately 110-kDa polypeptide, as immunoprecipitated by antibody to the purified canalicular bile acid transport protein and conferred on COS cells the capacity to pump out [3H]taurocholate with efflux characteristics comparable with those previously determined in canalicular membrane vesicles (Km = 100 microM; Vmax = 200 pmol/mg of protein/20 s). A truncated ecto-ATPase cDNA, missing the cytoplasmic tail, was targeted correctly to the cell surface but did not confer bile acid transport activity on COS cells. The results of this study also show that the canalicular ecto-ATPase/bile acid transport protein is phosphorylated on its cytoplasmic tail and that its phosphorylation is stimulated by activation of protein kinase C and inhibited by inhibitors of protein kinase C activation. Moreover, inhibition of protein kinase C activation by staurosporine completely abrogates bile acid transport but does not affect ATPase activity. This study, therefore, demonstrates that the rat liver canalicular ecto-ATPase is also a bile acid transport protein, that the capacity to pump out bile acid can be conferred on a heterologous cell by DNA-mediated gene transfer, and that phosphorylation within the cytoplasmic tail of the transporter is essential for bile acid efflux activity but not for ATPase activity.