Comparison of laparoscopic and open colonic resection within fast-track and traditional perioperative care pathways: Clinical outcomes and in-hospital costs.

BACKGROUND: This study examined short-term clinical outcomes and in-hospital costs of laparoscopic and open colonic resection within fast-track and traditional care pathways. MATERIAL AND METHODS: A case-control study was performed. From 2007 to 2009, 116 patients underwent laparoscopic or open colonic resection for benign or malignant disease within fast-track care pathway. The control group consisted of 116 age-, sex-, comorbidity-, type of surgery-, and diagnosis-matched patients who received a traditional perioperative care from 2000 to 2007. The main measures of outcome were postoperative hospital stay and in-hospital costs, with 30-day mortality, morbidity, reoperation, and readmission rates as secondary outcomes. RESULTS: The study groups were well balanced for baseline characteristics. Postoperative hospital stay was shorter in the fast-track than in the control group: laparoscopic resection median 3 versus 5 days (p < 0.001) and open resection 4 versus 7 days (p < 0.001). In multivariate analysis fast-track care, laparoscopic surgery and complications were independent determinants affecting the length of hospital stay. Overall, there was a trend toward lower in-hospital costs in the fast-track group compared with the traditional care group, but the difference was not statistically significant. Open surgery within fast-track care was the least costly option compared to laparoscopic or open surgery within traditional care but not significantly so when compared with laparoscopy within fast-track care. Intake of solid food and bowel function recovered 1 day earlier in the fast-track group than in the control group (p < 0.001). Complications were more frequent after open surgery than after laparoscopic surgery (23.3% vs 11.0%, p = 0.012). Reoperation and readmission rates were similar between the study groups. CONCLUSION: Laparoscopy improves the efficiency of fast-track perioperative care without significantly increasing in-hospital costs.

The anion exchanger Ae2 is required for enamel maturation in mouse teeth.

One of the mechanisms by which epithelial cells regulate intracellular pH is exchanging bicarbonate for Cl(-). We tested the hypothesis that in ameloblasts the anion exchanger-2 (Ae2) is involved in pH regulation during maturation stage amelogenesis. Quantitative X-ray microprobe mineral content analysis, scanning electron microscopy, histology, micro-computed tomography and Ae2 immuno-localisation analyses were applied to Ae2-deficient and wild-type mouse mandibles. Immuno-localisation of Ae2 in wild-type mouse incisors showed a very strong expression of Ae2 in the basolateral membranes of the maturation stage ameloblasts. Strikingly, zones of contiguous ameloblasts were found within the maturation stage in which Ae2 expression was extremely low as opposed to neighbouring cells. Maturation stage ameloblasts of the Ae2(a,b)(-/-) mice failed to stain for Ae2 and showed progressive disorganisation as enamel development advanced. Maturation stage enamel of the Ae2(a,b)(-/-) mice contained substantially less mineral and more protein than wild-type enamel as determined by quantitative X-ray microanalysis. Incisor enamel was more severely affected than molar enamel. Scanning electron microscopy revealed that the rod-inter-rod structures of the Ae2(a,b)(-/-) mice incisor enamel were absent. Mineral content of dentine and bone of Ae2(a,b)(-/-) mice was not significantly different from wild-type mice. The enamel from knockout mouse teeth wore down much faster than that from wild-type litter mates. Basolateral bicarbonate secretion via the anionic exchanger Ae2 is essential for mineral growth in the maturation stage enamel. The observed zonal expression of Ae2 in the maturation stage ameloblasts is in line with a model for cyclic proton secretion during maturation stage amelogenesis.

Comparison of a peanut agglutinin test and an immunochemical faecal occult blood test in detecting colorectal neoplasia in symptomatic patients.

BACKGROUND: Currently available methods for detection of early-stage colorectal cancer are reliant on faecal occult blood (FOB) tests. Bleeding, however, is not specific for colorectal neoplasia. Enzymatically detected or peanut agglutinin (PNA)-detectable galactose-beta1-3-N-acetyl-galactosamine residues found in rectal mucus have been used to detect colorectal cancer. METHODS: The sensitivity and specificity of the PNA rectal mucus test were compared with those of an immunological test for faecal occult blood (Hemolex) in 199 symptomatic patients referred for colorectal investigations. All patients also underwent a colonoscopy. SDS-PAGE and PNA-overlay were used to characterize PNA-binding proteins in normal and malignant colorectal tissue. RESULTS: The PNA test had a similar sensitivity to that of Hemolex for colorectal carcinoma (83% vs. 72%), adenomas (55% vs. 50%), inflammatory bowel disease (52% vs. 48%) and hyperplastic polyps (48% vs. 25%). The sensitivity of the PNA test and Hemolex for colorectal neoplasia was 69% vs. 59% and specificity 68% vs. 86% (p=0.002). SDS-PAGE and PNA-overlay showed some commonly expressed PNA-binding proteins in both normal mucosa and colorectal cancer and a higher and even selective expression of 160 kD PNA-binding protein in colorectal cancer. CONCLUSIONS: A single PNA test in its present form is as sensitive an indicator of colorectal neoplasia as Hemolex completed over three days, but lacks specificity. The 160 kD cancer-associated antigen we have identified is under further characterization for development of a more specific PNA test.

Expression of transmembrane serine protease TMPRSS2 in mouse and human tissues.

The purpose of this study was to clarify the expression of TMPRSS2 in mice during development and to compare the tissue distribution of the transcripts in adult mouse and human tissues. Mouse TMPRSS2 cDNA was cloned; the predicted amino acid sequence contains 490 residues sharing 81.4% similarity with human TMPRSS2. According to northern blots, mouse TMPRSS2 is expressed mainly in the prostate and kidney, while human TMPRSS2 is expressed in the prostate, colon, stomach, and salivary gland. In situ hybridization analyses of mouse embryos and adult tissues revealed that TMPRSS2 was expressed in the epithelia of the gastrointestinal, urogenital, and respiratory tracts. Expression was very selective and constant after the gene was turned on during development. Expression of TMPRSS2 was localized in the luminal epithelial cells of the mouse and human prostate. The information presented here will be useful in further studies regarding the function and physiological significance of TMPRSS2.

Identification of the full-length AE2 (AE2a) isoform as the Golgi-associated anion exchanger in fibroblasts.

Na(+)-independent Cl(-)/HCO(3)(-) exchangers (AE1, AE2, AE3) are generally known as ubiquitous, multispanning plasma membrane proteins that regulate intracellular pH and transepithelial acid-base balance in animal tissues. However, previous immunological evidence has suggested that anion exchanger (AE) proteins may also be present in intracellular membranes, including membranes of the Golgi complex and mitochondria. Here we provide several lines of evidence to show that an AE protein is indeed a resident of the Golgi membranes and that this protein corresponds to the full-length AE2a isoform in fibroblasts. First, both the N- and C-terminal antibodies to AE2 (but not to AE1) detected an AE protein in the Golgi membranes. Golgi localization of this AE2 antigen was evident also in cycloheximide-treated cells, indicating that it is a true Golgi-resident protein. Second, our Northern blotting and RT-PCR analyses demonstrated the presence of only the full-length AE2a mRNA in cells that show prominent Golgi staining with antibodies to AE2. Third, antisense oligonucleotides directed against the translational initiation site of the AE2a mRNA markedly inhibited the expression of the endogenous AE2 protein in the Golgi. Finally, transient expression of the GFP-tagged full-length AE2a protein resulted in predominant accumulation of the fusion protein in the Golgi membranes in COS-7 and CHO-K1 cells. Golgi localization of the AE2a probably involves its oligomerization and/or association with the recently identified Golgi membrane skeleton, because a substantial portion of both the endogenous AE2a and the GFP-tagged fusion protein resisted detergent extraction in cold. (J Histochem Cytochem 49:259-269, 2001)

A single C-terminal peptide segment mediates both membrane association and localization of lysyl hydroxylase in the endoplasmic reticulum.

Hydroxylation of lysyl residues is crucial for the unique glycosylation pattern found in collagens and for the mechanical strength of fully assembled extracellular collagen fibers. Hydroxylation is catalyzed in the lumen of the endoplasmic reticulum (ER) by a specific enzyme, lysyl hydroxylase (LH). The absence of the known ER-specific retrieval motifs in its primary structure and its association with the ER membranes in vivo have suggested that the enzyme is localized in the ER via a novel retention/retrieval mechanism. We have identified here a 40-amino acid C-terminal peptide segment of LH that is able to convert cathepsin D, normally a soluble lysosomal protease, into a membrane-associated protein. The same segment also markedly slows down the transport of the reporter protein from the ER into post-ER compartments, as assessed by our pulse-chase experiments. The retardation efficiency mediated by this C-terminal peptide segment is comparable with that of the intact LH but lower than that of the KDEL receptor-based retrieval mechanism. Within this 40-amino acid segment, the first 25 amino acids appear to be the most crucial ones in terms of membrane association and ER localization, because the last 15 C-terminal amino acids did not possess substantial retardation activity alone. Our findings thus define a short peptide segment very close to the extreme C terminus of LH as the only necessary determinant both for its membrane association and localization in the ER.

Primary structure of a sperm cell anion exchanger and its messenger ribonucleic acid expression during spermatogenesis.

Chloride/bicarbonate (Cl-/HCO(3)-) exchangers are a family of proteins (anion exchanger [AE] gene family) that regulate many vital cellular processes such as intracellular pH, cell volume, and Cl- concentration. They may also be involved in the regulation of sperm cell motility and acrosome reaction during fertilization, as these two phenomena are bicarbonate dependent, and we have previously shown that a polypeptide immunologically related to erythrocyte band 3 is expressed in mammalian sperm cells. We have now identified this putative sperm cell anion exchanger as the AE2 isoform of this gene family. First, we determined its complete primary structure from the human testis lambda gt 11 cDNA library. The cloned sequence was found to consist of 3896 base pairs (bp) with an open reading frame of 3726 bp, and to be almost identical to the previously published human genomic AE2 sequence. Only four amino acid disparities were found between these two sequences. Second, our in situ hybridization analyses showed that AE2 mRNA is expressed in developing sperm cells, indicating that the cloned sequence corresponds to the sperm cell AE. Our reverse transcription-polymerase chain reaction analyses suggested further that the expression of AE2 mRNA was variable to some extent during the epithelial cell cycle. Strongest expression was observed at stages VII-XIV except for stage X, i.e., when major structural and morphological changes take place. These results suggest that the full-length AE2 isoform regulates HCO(3)- transport in mature sperm cells and thus their motility in vivo.

EAST, an epidermal growth factor receptor- and Eps15-associated protein with Src homology 3 and tyrosine-based activation motif domains.

We describe the cloning and characterization of a new cytoplasmic protein designated epidermal growth factor receptor-associated protein with SH3- and TAM domains (EAST). It contains an Src homology 3 domain in its midregion and a tyrosine-based activation motif in its COOH terminus. Antibodies to EAST recognize a 68-kDa protein that is present in most chicken tissues. An epidermal growth factor (EGF)-dependent association between the EGF receptor (EGFR) and EAST was shown by reciprocal immunoprecipitation/immunoblotting studies with specific antibodies. Activated EGFR catalyzed the tyrosine phosphorylation of EAST, as judged by an in vitro kinase assay with both immunoprecipitated and purified EGFR. Immunoprecipitation/immunoblotting experiments also demonstrated an association between EAST and eps15, an EGFR substrate associated with clathrin-coated pits and vesicles, which is essential in the endocytotic pathway. The association between EAST and eps15 was not affected by EGF treatment. In immunofluorescence microscopy, EAST was shown to partially colocalize with clathrin. The sequence of the NH2-terminal portion of EAST shows a high degree of similarity with a group of proteins involved in endocytosis or vesicle trafficking. Thus, EAST is a novel signal transduction component probably involved in EGF signaling and in the endocytotic machinery.

A splice-site mutation that induces exon skipping and reduction in lysyl hydroxylase mRNA levels but does not create a nonsense codon in Ehlers-Danlos syndrome type VI.

The type VI variant of Ehlers-Danlos syndrome (EDS) is a heritable connective tissue disorder caused by a deficiency in the activity of lysyl hydroxylase, an enzyme required for the post-translational processing of collagens. We have characterized a novel type of mutation in a young female patient with type VI EDS, in which cells possess only 12% of the lysyl hydroxylase activity that is detected in unaffected cells. The syndrome was found to be caused by a homozygous insertion of two thymidines at the 5' splice site consensus sequence of intron 9 in the lysyl hydroxylase gene. The insertion interfered with normal splicing of the primary RNA transcript and resulted in an inframe deletion of the 132 nucleotides coded by exon 9 from the lysyl hydroxylase mRNA. In addition, the mutation caused a marked reduction in the steady-state level of the truncated mRNA, which was less than 15% of the level found in unaffected cells. The mutation also reduced the amount of the enzyme protein produced, which was estimated to be about 20% of that in control cells. However, the mutation did not affect the stability of the abnormally spliced mRNA nor the normal localization of the enzyme protein in the endoplasmic reticulum. According to our results, the reduction in enzymatic activity observed in this patient is caused by low levels of both lysyl hydroxylase mRNA and enzyme protein. The primary cellular defect associated with this mutation, therefore, appears to be at the level of nuclear mRNA metabolism even though the mutation did not create a premature translation termination codon.

Mouse 17 beta-hydroxysteroid dehydrogenase type 2 mRNA is predominantly expressed in hepatocytes and in surface epithelial cells of the gastrointestinal and urinary tracts.

17 beta-Hydroxysteroid dehydrogenase (17HSD) type 2 efficiently catalyzes the conversion of the high activity 17 beta-hydroxy forms of sex steroids into less potent 17-ketosteroids. In the present study in situ hybridization was utilized to analyze the cellular localization of 17HSD type 2 expression in adult male and female mice. The data indicate that 17HSD type 2 mRNA is expressed in several epithelial cell layers, including both absorptive and secretory epithelia as well as protective epithelium. In both males and females, strong expression of 17HSD type 2 was particularly detected in epithelial cells of the gastrointestinal and urinary tracts. The mRNA was expressed in the stratified squamous epithelium of the esophagus, and surface epithelial cells of the stomach, small intestine and colon. The hepatocytes of the liver and the thick limbs of the loops of Henle in the kidneys, as well as the epithelium of the urinary bladder, also showed strong expression of 17HSD type 2 mRNA in both male and female mice. In the genital tracts, low 17HSD type 2 expression was detected in the seminiferous tubules, the uterine epithelial cells and the surface epithelium of the ovary. Expression of the mRNA was also detected in the sebaceous glands of the skin. The results indicate that in both male and female mice, 17HSD type 2 is expressed mainly in the various epithelial cell types of the gastrointestinal and urinary tracts, and therefore suggest a role for the enzyme in steroid inactivation in a range of tissues and cell types not considered as classical sex steroid target tissues.

Protein disulfide isomerase and newly synthesized procollagen chains form higher-order structures in the lumen of the endoplasmic reticulum.

A number of proteins that act as necessary catalysts for correct protein folding and oligomerization in the endoplasmic reticulum (ER) are known to be retained in the organelle via the KDEL-receptor mediated retrieval mechanism. However, a complementary system that may help to retain these proteins in the organelle lumen has been suggested to exist and likely involves physical protein-protein interactions at the level of endoplasmic reticulum (ER) itself. In this report, we provide both morphological and biochemical evidence in support of this proposal. We show that in collagen-secreting human skin fibroblasts, protein disulfide isomerase and newly synthesized procollagen chains exist predominantly in an "aggregated" state, and form a reticular-like matrix in the ER lumen in vivo. The size of the aggregates was found to be variable, and may exceed 1.5 million Da. Aggregate formation appeared to be transient and to involve multiple types of protein-protein interactions, including formation of aberrant disulfide bonds. Association of protein disulfide isomerase, on the other hand, was found to require at least partly function-related disulfide bonds. These results support the existence of a reticular-like matrix in the ER lumen, and suggest that aggregation may be part of the normal maturation pathway during collagen biosynthesis.

Detection of colorectal neoplasia with peanut-agglutinin (PNA)-reactive carbohydrate structures in rectal mucus.

In contrast to normal colorectal mucosa, peanut-agglutinin-(PNA)-reactive glycoconjugates are commonly expressed in most colorectal carcinomas and in some pre-malignant conditions such as adenomas and ulcerative colitis. Since enzymatically detectable galactose-beta1-3-N-acetyl-galactosamine residues are found in rectal mucus obtained from patients with carcinoma of the large bowel, it was investigated here whether PNA-reactive carbohydrate structures in rectal mucus can be exploited in the detection of colorectal neoplasia. Samples of rectal mucus obtained from 261 randomly selected patients with colorectal symptoms were applied on nitrocellulose filters. The presence of PNA-reactive glycoconjugates in mucus samples was determined by a peroxidase-conjugated PNA-overlay procedure. The results were correlated to findings from total colonoscopy/surgery and histopathology. PNA-reactive carbohydrate structures were detected in 76% of patients with carcinoma (p < 0.005), in 62% of patients with adenoma (p < 0.005), in 69% of patients with inflammatory bowel disease (p < 0.005), and in 38% of patients with hyperplastic polyps (NS), in contrast to 21% of the control subjects with macroscopically normal colorectal mucosa. These results show that PNA-reactive carbohydrate alterations in rectal mucus correlates with neoplastic and hyperproliferative conditions of the colorectal mucosa. The specificity of the PNA test for colorectal neoplasia was 76%. Therefore the use of more discriminate carbohydrate probes are needed for the pre-symptomatic detection of colorectal neoplasia.

Membrane-bound carbonic anhydrase IV is expressed in the luminal plasma membrane of the human gallbladder epithelium.

Alkaline hepatic bile is acidified in the gallbladder to prevent calcium precipitation and gallstone formation. Because membrane-bound carbonic anhydrase (CA) isoenzyme IV participates with cytoplasmic CA II in the acidification of urine in the kidney, we studied its expression in different regions of the human biliary tract using immunohistochemical techniques. The enzyme was expressed in the apical plasma membrane of the gallbladder epithelial cells and in the endothelium of the subepithelial capillaries. In the liver, some epithelial cells of the large bile ducts showed positive staining. Its presence in the gallbladder epithelium could be confirmed by Western blotting, which showed a single 35-kd polypeptide band, corresponding in molecular weight to the intact enzyme. The majority of the enzyme was phased to Triton X-114 detergent phase. A small amount of 35-kd polypeptide was also seen in the water phase. Smaller proteolytic fragments of the enzyme were not seen, suggesting that the tissue sample was well preserved. The results show that CA IV is expressed in abundance in the human gallbladder epithelium, where it may participate together with cytoplasmic CA II and ion transporters in acidification of the gallbladder bile via bicarbonate reabsorption.

Defective maturation of a viral glycoprotein and partial loss of the Golgi stack structure during in vitro myogenesis.

In the present study, the functional and structural reorganization of the Golgi compartment shortly after the fusion of rat L6 myoblasts into multinucleated muscle cells was examined. When we followed the maturation and the transport of a bulk flow marker protein, the vesicular stomatitis virus G glycoprotein, in the fused cells, we found that only about half of the newly synthesized G protein acquired endo H resistance within the Golgi prior to its transport to the cell surface. The other half of the G protein remained endo H-sensitive and was retarded intracellularly. Our immunofluorescence and cell fractionation data indicated that this maturation defect did not result from the inefficient transport of the G protein into the Golgi, but rather from the functional impairment of the Golgi compartment in the fused cells. In accordance with this view, electron microscopy revealed that the majority of the Golgi-derived elements in the fused cells were structurally abnormal and consisted of large tubulovesicular "Golgi clusters." Our results support the view that reorganization of the Golgi complex during myogenesis involves at least a partial loss of both Golgi structure and function.

A normal rabbit serum containing Golgi-specific autoantibodies identifies a novel 74-kDa trans-Golgi resident protein.

A normal rabbit serum has been identified which contains Golgi-specific autoantibodies. In indirect immunofluorescence experiments the serum was found to stain the juxtanuclear Golgi complex in a variety of cell lines, including human skin fibroblasts, rat osteoblasts, rat myoblasts (L6), baby hamster kidney epithelial cells, and human embryonic kidney cells (293). Thus, the antigen(s) recognized by this serum seems to be well conserved and universally expressed in various mammalian cell types. Immunoelectron microscopy revealed that the epitope resides in the luminal side of the Golgi membranes, and that the antigen is concentrated in the trans-face of the Golgi stacks. In agreement with these results, brefeldin A treatment did not release the antigen from the membranes, but caused its redistribution partly into the endoplasmic reticulum but also into the juxtanuclear area, similarly as with other proteins known to be present in the trans-Golgi cisternae or trans-Golgi network. Our immunoprecipitation studies in human skin fibroblasts demonstrated that the serum recognizes specifically only a single protein with a molecular size of 74 kDa. This protein also cosedimented with a known trans-Golgi-specific marker protein, galactosyltransferase, after fractionation of subcellular organelles by Nycodenz gradient centrifugation. The widespread and polarized expression of this 74-kDa trans-Golgi resident protein suggests that it is required for the late Golgi functions in different mammalian cell types.

Lysyl hydroxylase, a collagen processing enzyme, exemplifies a novel class of luminally-oriented peripheral membrane proteins in the endoplasmic reticulum.

Lysyl hydroxylase (LH), an enzyme required early during collagen biosynthesis, appears to be exceptional among proteins that are thought to be residents of the endoplasmic reticulum (ER). It is a homodimer and does not contain either of the two previously characterized ER-specific retention motifs (KDEL or the double lysine motif) in its primary structure. We now show that LH, nevertheless, resides in the lumen of the ER. In immunofluorescence experiments, LH co-localizes with a KDEL-containing protein, protein disulfide isomerase (PDI), and also co-sediments with it after fractionation of subcellular organelles by sucrose density gradient centrifugation. In addition, LH seems to be stress-inducible. In one respect, however, LH differs from PDI and other known luminal proteins in the organelle. It is found in situ only in association with the ER membranes. Our cell fractionation and Triton X-114 phase separation experiments suggest that it binds to the membranes via weak electrostatic interactions. LH can thus be regarded as a first luminally-oriented "peripheral membrane" protein which has been characterized in the ER. The results suggest a novel possibility by which ER lumen can acquire its specific protein components from the bulk flow.

Polarized expression of a band 3-related protein in mammalian sperm cells.

Extracellular bicarbonate is known to be essential for sperm motility. This finding indirectly suggests that sperm cells possess a specific carrier protein, allowing this impermeant anion to cross the cell's plasma membrane. In this study, we have identified a protein in both human and rat sperm cells that is immunologically related to erythrocyte bicarbonate/Cl exchanger (AE1) and its close homolog (AE2). We used antibodies raised against synthetic C-terminal peptides of either AE1 (band 3) or a related protein (AE2) expressed mainly in the stomach. Indirect immunofluorescence experiments revealed that both antibodies recognized a protein that is expressed in a highly polarized fashion in the sperm cells, being present only in the equatorial segment of the sperm head. Confocal laser scanning microscopy further showed that the human protein is arranged circularly close to or at the plasma membrane and that it forms a ring-like structure around the sperm head. In the human testis, the seminiferous tubules were also stained with the anti-AE2 antiserum, indicating that the protein is also expressed in developing sperm cells. This observation was also supported by Northern blot analysis, which confirmed the presence of a 4.5-kb AE2 mRNA in the rat testis tissue. We suggest that the sperm band 3-related protein could be the transport protein that mediates the effects of extracellular bicarbonate on sperm motility.

The osteoclast proton pump differs in its pharmacology and catalytic subunits from other vacuolar H(+)-ATPases.

Osteoclasts are multinucleated cells derived from the mononuclear phagocyte system in the hematopoietic bone marrow. Their function is to resorb bone during skeletal growth and remodeling. They perform this function by acidifying an enclosed extracellular space, the bone resorbing compartment. Analysis of proton transport by inside-out vesicles derived from highly purified chicken osteoclast membranes has revealed the presence of a novel type of multisubunit vacuolar-like H(+)-ATPase. Unlike H(+)-ATPases derived from any other cell type or organelle, proton transport and ATPase activity in osteoclast vesicles are sensitive to two classes of inhibitors, namely V-ATPase inhibitors [N-ethyl-maleimide (NEM) and bafilomycin A1] and vanadate (IC50 100 mumol l-1), an inhibitor previously found to affect only P-ATPases. The osteoclast V-ATPase morphologically resembles vacuolar proton pumps and contains several vacuolar-like subunits (115 x 10(3), 39 x 10(3) and 16 x 10(3)M(r)), demonstrated by Western blot analysis. Subunits A and B of the catalytic domain of the enzyme, however, differ from that of other V-ATPases. In osteoclasts, subunit A has an M(r) of 63 x 10(3) instead of 67 x 10(3)-70 x 10(3); in contrast, monocytes, macrophages and kidney microsomes, which contain a vanadate-insensitive H(+)-ATPase, express the classical subunit A (70 x 10(3)M(r)). Moreover, two types of 57 x 10(3)-60 x 10(3)M(r) B subunits are also found: they are differentially recognized by antibodies and one is expressed predominantly in osteoclasts and the other in bone marrow cells and in kidney microsomes. Preliminary cloning data have indicated that the B subunit expressed in osteoclasts may be similar to the brain isoform. The osteoclast proton pump may, therefore, constitute a novel class of V-ATPase, with a unique pharmacology and specific isoforms of two subunits in the catalytic portion of the enzyme.

Sensitivity to vanadate and isoforms of subunits A and B distinguish the osteoclast proton pump from other vacuolar H+ ATPases.

Analysis of proton (H+) transport by inside-out vesicles derived from highly purified chicken osteoclast (OC) membranes has revealed the presence of a newly discovered type of vacuolar H+ ATPase (V-ATPase). Unlike vesicles derived from any other cell type or organelle, H+ transport in OC-derived vesicles is sensitive to V-ATPase inhibitors (N-ethylmaleimide and Bafilomycin A1) and vanadate (IC50, 100 microM), an inhibitor previously found to affect only P-type ATPases. The OC H+ ATPase contains several V-like subunits (115, 39, and 16 kDa) but subunits A and B of the catalytic domain of the enzyme differ from that of other V-ATPases. In OCs, subunit A has a mass of 63 kDa instead of the 67-70 kDa expressed in monocytes, macrophages, and kidney microsomes, which contain a vanadate-insensitive H+ ATPase. Moreover, two types of 57- to 60-kDa B subunits are also found: one is expressed predominantly in OCs and the other is expressed in kidney microsomes. The OC H+ pump may therefore constitute a class of H+ ATPase with a unique pharmacology and specific isoforms of two subunits in the catalytic portion of the enzyme. This H+ ATPase is involved in resorption of bone and may be expressed in a cell-specific manner, thereby opening possibilities for therapeutic intervention.

Cell cycle-dependent coupling of the calcitonin receptor to different G proteins.

Calcitonin is a calcium regulating peptide hormone with binding sites in kidney and bone as well as in the central nervous system. The mechanisms of signal transduction by calcitonin receptors were studied in a pig kidney cell line where the hormone was found to regulate sodium pumps. Calcitonin receptors activated the cyclic adenosine monophosphate (cAMP) or the protein kinase C (PKC) pathways. The two transduction pathways required guanosine triphosphate (GTP)-binding proteins (G proteins) (the choleratoxin sensitive Gs and the pertussis toxin sensitive Gi, respectively) and led to opposite biological responses. Moreover, selective activation of one or the other pathway was cell cycle-dependent. Therefore, calcitonin may induce different biological responses in target cells depending on their positions in the cell cycle. Such a modulation of ligand-induced responses could be of importance in rapidly growing cell populations such as during embryogenesis, growth, and tumor formation.