Persistent polyclonal B-cell lymphocytosis is an expansion of functional IgD(+)CD27(+) memory B cells.

Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare disorder of unknown aetiology affecting predominantly young to middle-aged women. It is characterized by a polyclonal expansion of B cells, including typical binucleated lymphocytes, and is associated with the presence of the translocation t(14;18), involving the bcl-2 oncogene. The stage of differentiation of the B cells expanded in PPBL is not known. We analysed the immunophenotype of the expanded B-cell subset in five new patients with PPBL and found a large uniform expansion of a recently defined human memory B-cell population, IgD(+)CD27(+) memory B cells. After in vitro stimulation with interleukin 2 (IL-2) and Staphylococcus aureus Cowan strain I, B cells from PPBL patients produced high levels of IgM immunoglobulins, which is a characteristic feature of IgD(+)CD27(+) memory B cells. Using a quantitative real-time polymerase chain reaction method, we found a high frequency of the translocation t(14;18) in the range of 1000-3000 per 106 B cells in PPBL patients. In contrast, a much smaller number of cells with a t(14;18) was found in B cells from healthy individuals. Our finding that PPBL is an accumulation of memory B cells further suggests that chronic antigeneic stimulation plays an important part in the pathogenesis of this disorder. This IgD(+)CD27(+) memory B-cell population might harbour a certain number of 'physiological' t(14;18) translocations that increases as this population expands in PPBL patients and constitutes the majority of peripheral blood lymphocytes.

Evidence of 5-lipoxygenase overexpression in the skin of patients with systemic sclerosis: a newly identified pathway to skin inflammation in systemic sclerosis.

OBJECTIVE: Leukotrienes are a family of arachidonic acid derivatives with potent proinflammatory and profibrotic properties, and 5-lipoxygenase (5-LOX) catalyzes two key steps in the leukotriene biosynthetic pathway. Since inflammatory cell infiltrates and excessive fibrosis are hallmarks of systemic sclerosis (SSc) skin lesions, we undertook the present study to investigate the expression of 5-LOX in skin biopsy specimens from patients with SSc. METHODS: Expression of 5-LOX in skin sections from 10 SSc patients and 8 healthy controls was examined by in situ hybridization with specific riboprobes and by immunohistochemistry analysis with 5-LOX monoclonal antibodies. Synthesis of 5-LOX by cultured dermal fibroblasts from 7 patients with SSc and 4 controls was measured by fluorescence-activated cell sorter analysis. In addition, concentrations of leukotriene B4 (LTB4) and LTE4 in fibroblast supernatants after stimulation were determined using enzyme immunoassays. RESULTS: Expression of 5-LOX was found in all skin sections from SSc patients as well as from controls. However, the number and percentage of 5-LOX-positive cells were significantly higher in SSc skin sections compared with control sections. Expression of 5-LOX was seen in cells within perivascular inflammatory infiltrates as well as in fibroblasts throughout the skin. The experiments with cultured skin fibroblasts revealed that 5-LOX was constitutively expressed in these cells, which resulted in the production of leukotrienes after cell stimulation. Whereas no difference was found for LTE4, SSc fibroblasts produced significantly higher amounts of LTB4 after stimulation, compared with healthy control fibroblasts. CONCLUSION: The results of this study suggest that the 5-LOX pathway may be of significance in the pathogenesis of SSc and may represent a target for new treatment strategies.

Synergistic effect of a combined DNA and peptide vaccine against gp100 in a malignant melanoma mouse model.

Vaccination against tumors relies on tumor-associated antigens, and has been quite successful with synthetic peptides used as immunogens. Gp100 is a human melanoma-associated antigen (hgp100) with a highly homologous mouse counterpart, pmel17/gp100 (mgp100), that is expressed in melanocytes and highly tumorigenic B16 melanoma cells. Since mgp100 is poorly immunogenic in mice, we used a xenoimmunization approach and vaccinated with the hgp100 immunogene. To that end, plasmid DNA encoding hgp100 was applied as a vaccine in combination with three synthetic peptides corresponding to putative cytotoxic T cell epitopes of hgp100. Immunization with DNA and peptide-pulsed spleen cells had a synergistic effect and provided significant protection against a challenge with poorly immunogenic B16-F0 malignant melanoma cells in the syngeneic C57BL/6 mouse model. Vaccination with either plasmid DNA or peptides alone delayed the onset of tumor formation, and reduced tumor growth 2-fold and 30-fold, respectively. However, while all animals vaccinated with DNA encoding hgp100 or with peptides eventually developed tumors, 30% of the animals treated with both vaccines remained tumor free and survived for the entire observation period of 150 days. Depletion of T cell subsets revealed that the protective effect observed after vaccination with plasmid DNA was mediated by CD4+ and CD8+ T cells, while protection following vaccination with DNA encoding hgp100 in combination with peptides appears to depend on CD4+ T cells only. Furthermore, we could also demonstrate a therapeutic effect of the combined DNA/peptide regime. A single treatment cycle consisting of injections of plasmid DNA and peptide-pulsed spleen cells led to a fourfold reduction in the growth rate of preexisting tumors. The data presented demonstrate that immunization with xenoantigens induces cross-species priming leading to an immunological response against the tumor-specific antigens.

Inhibition of human hematopoietic tumor formation by targeting a repressor Myb-KRAB to DNA.

In many hematopoietic malignancies, c-Myb, a nuclear transcription factor of hematopoietic cells, is an activated oncogene. To achieve a specific inhibition of hematopoietic tumor growth, an inducible fusion protein consisting of the Myb DNA binding domain (DBD) and the active repressor domain KRAB, the Krüppel-associated box of the developmental zinc-finger protein KOX-1, was generated. The MybDBD-KRAB fusion protein is a potent repressor of Myb-induced gene expression from Myb-responsive reporter genes containing several Myb binding sites. MybDBD-KRAB expressed in the human hematopoietic promyelocytic cell line HL60 significantly reduces cell proliferation by inducing apoptosis. Expression of MybDBD-KRAB in subcutaneously injected HL60 cells leads to inhibition of tumor formation in nude mice. The MybDBD-KRAB effect is specific to cell lines expressing c-Myb. It is conceivable to fuse the KRAB domain to other DBDs of oncogenic transcription factors and target them to their respective DNA response elements to selectively drive tumor cells into apoptosis.

Retroviral gene transfer of dominant negative raf-1 mutants suppresses ha-ras-induced transformation and delays tumor formation.

Activating mutants of ras are among the most frequently found genetic alterations in human cancers. Therefore, Ras appears to be an attractive target for therapeutic intervention using gene transfer. The protein kinase Raf-1 acts as a direct downstream effector of Ras and is involved in Ras-induced cellular transformation. Using the NIH3T3 fibroblast-derived tumor cell line PEJ, which expresses oncogenic Ha-rasG12V, we analyzed whether dominant negative mutants of Raf-1 can inhibit Ras-mediated transformation. Retroviral gene transfer was used to stably transduce PEJ cells with three different dominant negative mutants of Raf-1. This resulted in reversion of the transformed phenotype in vitro as evidenced by an increase in contact inhibition and reduced anchorage-independent growth. However, tumor formation in nude mice was significantly delayed only by one of these mutants. Therefore, dominant negative mutants of the oncoprotein Myc, which is known to synergize with Raf-1 in tumor formation, were transduced into PEJ cells expressing a dominant negative Raf mutant. This leads to killing of the cells. These results indicate that although interference with Ras-induced transformation using dominant negative mutants of Raf is feasible and effective in vitro using retroviral vectors, an additional block (e.g., that of Myc) is necessary to kill PEJ cells. These results also indicate that interference with Ras-dependent signaling is not sufficient for inhibition of tumor formation of PEJ cells in vivo.

Tetracycline-induced expression of an anti-c-Myb single-chain antibody and its inhibitory effect on proliferation of the human leukemia cell line K562.

Ablation of c-Myb function might be an effective approach for the therapy of chronic myelogenous leukemia or other c-myb-dependent malignancies. To this end, we have previously used an intracellular anti-c-Myb single-chain antibody (sFv) to achieve the functional knockout of the c-Myb oncoprotein. In this study, we have employed a tetracycline-inducible system to control the expression of the sFv. A nuclear-localizing form of an anti-c-Myb sFv was cloned into a tet-regulated plasmid vector. Using a transient expression system in COS-1 cells, we observed that doxycycline (Dox) induced expression of the sFv in a dose-dependent manner, and that the sFv was localized mainly in the nucleus. The Dox-induced anti-c-Myb sFv also inhibited the transactivating activity of c-Myb in a dose-dependent manner. We subsequently confirmed the Dox-induced expression of the sFv in the leukemia cell line K562. Proliferation of the target leukemia cells was also inhibited. These results suggest that the anti-c-Myb sFv may represent a viable method for gene therapy of c-myb-dependent hematopoietic malignancies.

Expression of sentrin, a novel antiapoptotic molecule, at sites of synovial invasion in rheumatoid arthritis.

OBJECTIVE: Sentrin, a novel antiapoptotic molecule, has been shown to interact with the signal-competent form of Fas/APO-1 and tumor necrosis factor receptor I (TNFRI), and thereby, to protect cells against anti-Fas/APO-1- and TNF-induced cell death. Since reduced apoptosis in the synovial lining is supposed to contribute to synovial hyperplasia in rheumatoid arthritis (RA), we searched for the expression of sentrin-1 messenger RNA (mRNA) in synovium from patients with RA. METHODS: The expression of sentrin-1 mRNA was examined by in situ hybridization on snap-frozen sections of normal and RA synovial tissues as well as on paraffin-embedded RA synovial specimens, including the interface of cartilage-bone and invading synovium. Immunohistochemical double labeling after in situ hybridization was performed to further characterize sentrin-1 mRNA-expressing cells. In addition, quantitative analysis of sentrin-1 mRNA expression in RA synovial fibroblasts (RASF), osteoarthritis synovial fibroblasts (OASF), and normal fibroblasts was performed by quantitative real-time polymerase chain reaction. Expression levels were standardized to the expression of GAPDH. The in vivo maintenance of sentrin expression in RASF aggressively invading human cartilage was explored in the SCID mouse model of RA. RESULTS: A marked expression of sentrin-1 mRNA could be seen in all RA synovial specimens, predominantly in SF of the lining layer and at sites of invasion of RA synovium into cartilage. In normal synovial tissues, no sentrin-1 mRNA was detectable. RASF showed a maximum 32.5-fold (mean +/- SD 14.9 +/- 11.6) increase of sentrin-1 mRNA expression compared with normal fibroblasts and a maximum 31.4-fold (mean +/-SD 14.3 +/- 10.9) increase compared with OASF. When coimplanted with normal human cartilage in the SCID mouse model, invading RASF maintained their sentrin-1 mRNA expression for at least 60 days in vivo. CONCLUSION: The marked expression of sentrin in rheumatoid synovial tissue, but not in normal or OA synovial tissue, may contribute to the modulation of Fas- and TNFR-mediated apoptosis in RA synovium, and thereby extend the lifespan of invasive, cartilage-destructive SF.

Mechanism of protection against influenza A virus by DNA vaccine encoding the hemagglutinin gene.

Influenza A virus with its two major antigenic surface proteins hemagglutinin (HA) and neuraminidase (NA) is a widely used model to study DNA immunizations in mice and other animals. Natural protection against influenza A virus infection is mediated by antibodies, which mostly are not protective against antigenic shift or drift variants of the original virus. Therefore, it would be a major task to induce a protective cellular immune response to more conserved proteins or epitopes. Injection of plasmid encoding a viral antigen is known to induce cellular as well as humoral immunity. In this study we investigate the mechanism of protection after intramuscular vaccination of C57Bl/6 mice with a DNA vaccine encoding HA of influenza A/PR/8/34. After a single injection, only a small percentage of mice survive the lethal challenge with homologous virus. The amount of protection can be doubled by applying a booster injection. Furthermore, by coinjection of plasmids encoding cytokines GM-CSF and IL-12, respectively, nearly all of the mice are protected. Mice with specific defects in the cellular immune response [perforin knockout (P-/-) mice] and in the humoral immune response [IgD/IgM knockout (muMT) mice], respectively, have been immunized with HA DNA with or without cytokine DNA. Protection could only be induced in P-/- mice, whereas muMT mice succumbed to the infection. Moreover, when muMT mice were infected with only 0.75 x50% lethal dose they died all the same, whereby mice that had been depleted of CD8+ T cells before infection showed an even greater progression of illness. Altogether these results demonstrate that antibodies mediate protection after immunization with plasmid coding for HA of influenza A virus, and that booster immunizations and coinjection of plasmids encoding GM-CSF or IL-12 can improve this protection.

Immunization with Naked DNA Coexpressing Antigen and Cytokine via IRES.

Inoculation of plasmid DNA vectors encoding immunogenic proteins induce humoral as well as cell-mediated immune responses. Protection against challenge with pathogens has provided protective immunity in several instances in animal models. (1,2). DNA vaccines allow the simultaneous expression of antigens and immune-stimulatory cytokines via an internal ribosomal entry site (IRES). Here we describe the construction of a DNA vaccine against malignant melanomas using: (i) the tumor-associated antigen gp100 (or pmel17), known to be over-expressed in many malignant melanomas (3,4), and (ii) the granulocyte macrophage stimulating factor (GM-CSF) which has been shown to have a stimulatory effect on humoral and cellular immune responses (5).

Reduced melanoma tumor formation in mice immunized with DNA expressing the melanoma-specific antigen gp100/pmel17.

Plasmid DNA encoding gene products of viruses or other pathogens has recently been applied by intramuscular injection as a novel type of vaccine. It can induce cytotoxic T cell response in small animals and protect against challenge with influenza A viruses. Combinations with cytokines or DNA-encoding cytokines have been applied in order to increase the efficiency of protection. A DNA vaccine has been analyzed here against malignant melanoma encoding gp100/pmel17, a melanoma-associated antigen. A small animal model was used by injection of B16 melanoma cells to syngeneic C57Bl/6 mice. DNA vaccination before tumor cell challenge leads to about 50% reduction of tumor size. The cytokine gene coding for GM-CSF did not increase the efficiency but also led to tumor size reduction when applied alone.

Long-lasting anti-metastatic efficiency of interleukin 12-encoding plasmid DNA.

Intramuscular injection of plasmid DNA encoding both subunits of the cytokine interleukin 12 (IL-12) exhibits strong antimetastatic activity against lung metastases induced by the malignant melanoma cell line B16-F10. The protective effect of IL-12 DNA is long-lasting, since administration of tumor cells 9 days after IL-12 DNA treatment prevented metastasis formation. No effects were observed with empty plasmid controls, DNA encoding the melanoma-associated antigen pmel17/gp100, the granulocyte-macrophage colony-stimulating factor GM-CSF, B7.1, or CpG-containing oligodeoxynucleotides. IL-12 DNA is required during early phases of metastasis formation and is ineffective when administered later. Its efficiency is dose dependent. The cytotoxic T cell response contributes to the antimetastatic effect as evidenced by genetically modified CD8- or perforin knockout mice. Depletion of natural killer (NK) cells by antibodies completely abrogated the effect. In contrast, the IL-12-induced antimetastatic effect was not mediated by interferon gamma (IFN-gamma) or tumor necrosis factor alpha (TNF-alpha) as shown with IFN-gamma receptor and TNF-alpha knockout mice, respectively. Toxic side effects by IL-12 were low. Our results suggest that plasmid DNA encoding IL-12 might have potential value as gene medicine against the initiation of metastasis formation.

Functional knock-out of c-myb by an intracellular anti-c-Myb single-chain antibody.

Aberrant expression of the c-myb proto-oncogene is a key factor in the development of the neoplastic phenotype in a variety of contexts. On this basis, it has been proposed that ablation of c-myb function might be an effective approach for therapy. To this end, we have employed an intracellular single-chain antibody (sFv) approach to achieve the functional knock-out of the c-Myb onco-protein. We derived an anti-c-Myb sFv, which was configured into eukaryotic expression plasmids. We confirmed the expression of the cytoplasmic and nuclear forms of the sFvs in the correct subcellular compartments by immunofluorescent staining. Importantly, the anti-c-Myb sFvs strongly inhibited the transactivation activity of c-Myb. Furthermore, cytotoxic effect of the sFv was observed only in the c-Myb positive cell line K562. These results suggest that anti-c-Myb sFv is a valuable tool for understanding the molecular mechanisms of c-myb induced transformation. In addition, this approach may have potential utility in the gene therapy for c-myb-dependent malignant diseases.

Redundant functions of B-Myb and c-Myb in differentiating myeloid cells.

We show in this report that the human myeloid leukemia cell line GFD8 is a useful model to compare the biological function of the structurally related c-Myb and B-Myb proto-oncogenes and to investigate the c-myb domains required for this function. GFD8 cells are dependent for growth on granulocyte-macrophage colony-stimulating factor and differentiate in response to phorbol myristate acetate (PMA). We have stably transfected this cell line with constructs constitutively expressing c-Myb or B-Myb. Deregulated expression of both c-Myb and B-Myb inhibited the differentiation observed in response to PMA and, in particular, the induction of the CD11b and CD11c antigens on the cell surface, and the induction of adherence. Furthermore, c-Myb and B-Myb enhanced expression of CD13 upon PMA treatment. Although deregulated Myb expression did not alter the growth factor dependence of the cells, it led to an increase in G2 relative to G1 arrest in cells induced to differentiate in response to PMA, whereas control vector-transfected cells were blocked mostly in G1. This decrease in G1 block took place despite normal induction of the cyclin-dependent kinase inhibitor protein p21 (CIP1/WAF1). Thus, GFD8 cells stably expressing the human B-Myb protein behaved in a manner indistinguishable from those stably expressing C-Myb for both differentiation and cell cycle parameters. In agreement with these findings and differently from most previous reports, transactivation assays show that B-myb can indeed act as a strong activator of transcription. Finally, we demonstrated that although the DNA-binding domain of c-myb is required for both the differentiation block and the shift in cell cycle after PMA treatment, phosphorylation by casein kinase II and mitogen-activated protein kinase at positions 11 and 12 or 532 of c-myb, respectively, are not. We conclude that c-Myb and B-Myb may activate a common cellular program in the GFD8 cell line involved in both differentiation and cell cycle control.

Anti-tumor immunity is involved in the thymidine kinase-mediated killing of tumors induced by activated Ki-ras(G12V).

We have established a syngeneic mouse tumor model to test the efficacy of the drug-sensitizing enzyme thymidine kinase from herpes simplex virus (HSVtk) in vivo. Activated mutant Ki-ras(G12V) is frequently found in human colon cancer and adenocarcinomas of the lung and pancreas. We have transformed BALB/c-3T3 cells by stable transfection of a plasmid directing the expression of the mutant Ki-ras cDNA. To transfer the HSVtk gene into tumor cells we used a Moloney murine leukemia virus (MoMLV)-based retroviral vector that carries the HSVtk gene. In this study we show that the activity of HSV-TK inhibits tumor growth in immune-compromised nude mice following GCV treatment for up to 50 days but is not sufficient to completely eliminate all tumor cells in these mice as evidenced by the occurrence of tumors between 40 and 50 days after tumor cell implantation. By contrast, immune-competent BALB/c mice develop a long-lasting antitumor immunity in response to HSVtk transduction and GCV treatment, indicating that the immune system is important for the long-term tumor suppression in vivo. In the presence of GCV co-culturing of tumor cells with HSVtk transfected cells leads to the efficient killing of HSVtk negative tumor cells. While this retroviral vector independent HSV-TK/GCV-mediated bystander effect is not sufficient to inhibit tumor formation in athymic animals it is very efficient in immune-competent syngeneic mice. Taken together the data indicate that the antitumor activity of HSV-TK is enhanced by an intact immune system.

Human c-myb is expressed in cervical carcinomas and transactivates the HPV-16 promoter.

Human c-myb is normally involved in the regulation of proliferation and differentiation of hematopoietic cells. Until now, only a few reports have described elevated c-myb gene expression in epithelial tissue, suggesting that under certain circumstances, c-Myb protein might play a role during the process of malignant transformation of epithelial cells. To investigate a possible role of c-myb during papillomavirus-associated carcinogenesis, we investigated the c-myb mRNA expression in human papillomavirus (HPV)-associated tumors and tumor cell lines. Seven of nine cervical carcinomas and two of three carcinoma cell lines exhibited elevated c-myb transcriptional activity. In contrast to malignant cervical neoplasias, only 3 of 15 condylomata acuminata expressed a sparse signal for c-myb mRNA. Since the c-Myb protein has been described as a potent transcriptional regulator, we investigated the transactivating properties of c-Myb on the HPV-16 promoter/enhancer. Cotransfection of a chloramphenicol acetyltransferase-reporter plasmid containing the HPV-16 enhancer/promoter element with a full-length c-Myb-expressing plasmid resulted in a significant induction (4.3-fold) of the HPV-16 promoter, whereas expression of a carboxy-terminally deleted c-Myb protein led to no effects. Gel shift experiments showed a specific binding of recombinant c-Myb protein on the HPV-16 P97 enhancer. These data indicate that elevated c-myb expression occurs with HPV-associated cell transformation. Since c-Myb has been shown to stimulate the HPV-derived oncoprotein expression via transcriptional activation, it may play a role in the process of HPV-associated cervical carcinogenesis.

Interaction of the v-and c-Myb proteins with regulatory sequences of the human c-myc gene.

Eight c-Myb-binding sites have been identified in the regulatory region of the human c-myc gene using gel retardation and DNAase I footprint assays with purified bacterially expressed full-length and carboxy-terminally truncated c-Myb proteins. These binding sites exhibit different affinities whereby strong binding correlates better with conservation of the palindromic sequences, AACXGTT or AACGTT, than the previously described consensus sequence. Flanking AT-rich sequences further increase the binding affinity. The c-Myb-binding sites are arranged in pairs consisting of one high- and one low-affinity binding site. Binding of the Myb proteins to these sites is non-cooperative. The v-Myb protein protects two nucleotides fewer than the c-Myb protein. Co-transfection of reporter CAT genes, containing upstream human c-myc sequences including exon 1, with c-Myb-expressing constructs resulted in positive transactivation, which was eightfold with full-length Myb and 14-fold with the truncated Myb. This result suggests that the Myb protein could participate in regulation of human c-myc gene expression.

Cleavage of the HIV-1 p66 reverse transcriptase/RNase H by the p9 protease in vitro generates active p15 RNase H.

The reverse transcriptase/RNase H of HIV-1 is composed of a p66/p51 heterodimer when analyzed from virus particles. A recombinant reverse transcriptase (RT)/RNase H which after purification consisted mainly of p66 was analyzed as substrate of the purified recombinant HIV-1 protease p9 in vitro. The p66 protein if treated with the protease is processed to a stable p66/p51 heterodimer. A p15 protein is a prominent cleavage product which was identified as the carboxyterminal portion of p66 by means of a monoclonal antibody. It exhibits RNase H activity when tested by activated gel analysis. Presence of SDS during the incubation allowed complete degradation of p66 depending on the conditions, which indicates that conformation of a substrate is relevant for cleavage by the HIV-1 protease. A synthetic heptapeptide AET-FYVD derived from the region between RT and RNase H is cleaved efficiently in vitro by the HIV-1 protease at the F'Y junction, and may mimick a natural cleavage site. P66/p51 heterodimers exhibit higher RT and RNase H activities than p66 when renatured from polyacrylamide gels.

Coupling of 22Na and 36Cl uptake in cultured pigmented ciliary epithelial cells: a proposed role for the isoenzymes of carbonic anhydrase.

Uptake studies with 22Na and 36Cl were performed in cultured bovine pigmented ciliary epithelial cells (PE) to investigate interdependence of Na+ and Cl- transport. (1) 22Na uptake into NaCl depleted cells was stimulated by Cl-. This stimulation was abolished by the simultaneous application of amiloride (1 mM) and bumetanide (0.1 mM), indicating two independent mechanism for Cl- stimulated Na+ uptake: loop diuretic sensitive Na+/Cl- symport and an indirect stimulation of Na+/H+ exchange by Cl-. The latter component of Cl- stimulated Na+ uptake was HCO3- dependent. (2) 36Cl uptake was increased by extracellular Na+. Na+-stimulated Cl- uptake also consisted of two components. One was bumetanide sensitive and the other was blockable by amiloride and partly inhibited by the carbonic anhydrase (CA) inhibitor methazolamide (0.1 mM). (3) Homogenized PE cells were tested for biochemical CA activity using an electrometric method. The cytoplasmic as well as the membrane fraction contained specific CA activity. (4) A model is presented for Na+ and Cl- transport into PE: in addition to Na+/Cl- symport, Na+/H+ and Cl-/HCO3- double exchange may operate in the ciliary epithelium. The latter mechanism provides NaCl uptake into the cell in exchange for H+ and HCO3-, which recycle as CO2 across the membrane. This recycling of CO2 and HCO3-/H+ (and hence indirectly NaCl uptake) is facilitated by the cooperation between membrane bound and cytoplasmic CA.

Sodium bicarbonate cotransport in cultured pigmented ciliary epithelial cells.

Uptake of 22Na+ was studied in cultured bovine pigmented ciliary epithelial cells (PE) in HCO3-containing media. Two components of Na+-uptake were stimulated by intracellular acidification (NH4+-prepulse): One was amiloride-sensitive, the other DIDS-sensitive. The amiloride-sensitive component of Na+-uptake probably represents Na+/H+-exchange, which has previously been characterized in PE. The second, DIDS-sensitive component stimulated by intracellular acidification, was Cl- and HCO3--dependent. We conclude that a stilbene-sensitive, Cl--dependent Na+-HCO3--cotransport is present in PE. This transport could play an important role in aqueous humor formation.