Electrogenic bicarbonate secretion in the turtle bladder: Apical membrane conductance characteristics

Summary We have recently shown that stimulation of electrogenic HCO ₃ ^– secretion is accompanied by a simultaneous increase in short-circuit current (I _sc, equivalent to HCO ₃ ^– secretion rate under these conditions), apical membrane capacitance (C _a , proportional to membrane area), and apical membrane conductance (G _a , proportional to membrane ionic permeability). The current experiments were undertaken to explore the ionic basis for the increase inG _a and the possibility that the rate of electrogenic HCO ₃ ^– secretion is regulated by changes inG _a . Membrane electrical parameters were measured using impedance-analysis techniques before and after stimulation of electrogenic HCO ₃ ^– secretion with cAMP in three solutions which contained different chloride concentrations. In another series of experiments, the effects of an anion channel blocker, anthracene-9-carboxylic acid (9-AA), were measured after stimulation of electrogenic HCO ₃ ^– secretion with cAMP. The major conclusions are: (i) a measurable apical Cl^– conductance exists in control hemibladders; (ii) the transport-associated increase inG _a includes a Cl^–-conductive component; (iii)G _a also appears to reflect a HCO ₃ ^– conductance; (iv) the relative magnitudes of the apical membrane conductances to Cl^– and HCO ₃ ^– are similar; (v) 9-AA reducesG _a andI _sc appear cAMP-stimulated hemibladders; and (vi) alterations inI _sc appear to be mediated by changes inG _a .     

Vasopressin alters the mechanism of apical Cl− entry from Na+:Cl− to Na+:K+:2Cl− cotransport in mouse medullary thick ascending limb

Summary Experiments were performed usingin vitro perfused medullary thick ascending limbs of Henle (MTAL) and in suspensions of MTAL tubules isolated from mouse kidney to evaluate the effects of arginine vasopressin (AVP) on the K⁺ dependence of the apical, furosemide-sensitive Na⁺:Cl^– cotransporter and on transport-related oxygen consumption (QO₂). In isolated perfused MTAL segments, the rate of cell swelling induced by removing K⁺ from, and adding onemm ouabain to, the basolateral solution [ouabain(zero-K⁺)] provided an index to apical cotransporter activity and was used to evaluated the ionic requirements of the apical cotransporter in the presence and absence of AVP. In the absence of AVP cotransporter activity required Na⁺ and Cl^–, but not K⁺, while in the presence of AVP the apical cotransporter required all three ions.⁸⁶Rb⁺ uptake into MTAL tubules in suspension was significant only after exposure of tubules to AVP. Moreover,²²Na⁺ uptake was unaffected by extracellular K⁺ in the absence of AVP while after AVP exposure²²Na⁺ uptake was strictly K⁺-dependent. The AVP-induced coupling of K⁺ to the Na⁺:Cl^– cotransporter resulted in a doubling in the rate of NaCl absorption without a parallel increase in the rate of cellular²²Na⁺ uptake or transport-related oxygen consumption. These results indicate that arginine vasopressin alters the mode of a loop diuretic-sensitive transporter from Na⁺:Cl^– cotransport to Na⁺:K⁺:2Cl^– cotransport in the mouse MTAL with the latter providing a distinct metabolic advantage for sodium transport. A model for AVP action on NaCl absorption by the MTAL is presented and the physiological significance of the coupling of K⁺ to the apical Na⁺:Cl^– cotransporter in the MTAL and of the enhanced metabolic efficiency are discussed.     

The biology of Giardia spp.

Gardia spp. are flagellated protozoans that parasitize the small intestines of mammals, birds, reptiles, and amphibians. The infectious cysts begin excysting in the acidic environment of the stomach and become trophozoites (the vegetative form). The trophozoites attach to the intestinal mucosa through the suction generated by a ventral disk and cause diarrhea and malabsorption by mechanisms that are not well understood. Giardia spp. have a number of unique features, including a predominantly anaerobic metabolism, complete dependence on salvage of exogenous nucleotides, a limited ability to synthesize and degrade carbohydrates and lipids, and two nuclei that are equal by all criteria that have been tested. The small size and unique sequence of G. lamblia rRNA molecules have led to the proposal that Giardia is the most primitive eukaryotic organism. Three Giardia spp. have been identified by light lamblia, G. muris, and G. agilis, but electron microscopy has allowed further species to be described within the G. lamblia group, some of which have been substantiated by differences in the rDNA. Animal models and human infections have led to the conclusion that intestinal infection is controlled primarily through the humoral immune system (T-cell dependent in the mouse model). A major immunogenic cysteine-rich surface antigen is able to vary in vitro and in vivo in the course of an infection and may provide a means of evading the host immune response or perhaps a means of adapting to different intestinal environments.     

Structural and functional properties of the p60 proteins from different Listeria species.

The major extracellular protein p60 of Listeria monocytogenes seems to be required for this microorganism's adherence to and invasion of 3T6 mouse fibroblasts but not for adherence to human epithelial Caco-2 cells. Western blot analysis with polyclonal antibodies against p60 of L. monocytogenes indicated the presence of cross-reacting proteins in the culture supernatants of all Listeria species. Protein p60 of L. monocytogenes could restore adhesion of the L. monocytogenes mutant RIII (impaired in the synthesis of p60) to mouse fibroblasts more efficiently than that of Listeria grayi. The amino acid sequences of the p60-related proteins of L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi indicated highly conserved regions of about 120 amino acids at both the N-terminal and the C-terminal ends. The middle portions of these proteins, consisting of about 240 amino acids, varied considerably. These parts include the repeat domain consisting of repetitions of Thr (T) and Asn (N) which was present only, albeit in different arrangements, in the p60 proteins of L. monocytogenes and L. innocua. The p60-related proteins of L. grayi, L. ivanovii, L. seeligeri, and L. welshimeri each contained an insertion of 54 amino acids which was absent in the p60 proteins of L. monocytogenes and L. innocua.     

The association of insulin-elicited phosphotyrosine proteins with src homology 2 domains.

The interactions of the phosphotyrosine (Tyr(P))-containing proteins in basal and insulin-stimulated 3T3-L1 adipocytes with src homology 2 (SH2) domains from phosphatidylinositol 3-kinase (PI3K), ras GTPase-activating protein (GAP), and phospholipase C gamma have been examined. The Tyr(P) forms of the insulin receptor and its 160-kDa substrate protein (pp160) associated with fusion proteins containing either or both the SH2 domains of PI3K, but not with fusion proteins containing the two SH2 domains of GAP or phospholipase C gamma. These results demonstrate a specificity for the association of the Tyr(P) form of the insulin receptor and pp160 with SH2 domains that parallels the reported effects of insulin on PI3K, GAP, and phospholipase C gamma in vivo. Immunoprecipitates of pp160 from the cytosol of insulin-treated, but not basal, 3T3-L1 adipocytes contained PI3K activity. Moreover, the Tyr(P) form of pp160 with associated PI3K activity migrated at 10 S on a sucrose velocity gradient, whereas the Tyr(P) form without associated activity migrated at 6 S. These findings indicate that the Tyr(P) form of pp160 associates directly with PI3K in vivo.     

Distinct domains of an oligotopic membrane protein are Sec-dependent and Sec-independent for membrane insertion.

Leader peptidase of Escherichia coli spans the plasma membrane twice with its amino terminus on the periplasmic surface of the membrane and its large carboxyl-terminal domain protruding into the periplasm. To monitor the transfer of the amino terminus of leader peptidase to the periplasm, we have constructed a fusion protein between the 18-residue amino-terminal periplasmic domain of Pf3 bacteriophage coat protein and the beginning of leader peptidase. We find that neither the SecA or SecY proteins nor a transmembrane electrochemical potential is required for insertion of the amino terminus, while the transfer of the carboxyl-terminal domain of leader peptidase has these requirements. The first 35 residues of leader peptidase, which include the first hydrophobic domain and the carboxyl-terminal positively charged cluster, are sufficient to insert the amino terminus. When positively charged residues are introduced before the first transmembrane segment, translocation of the amino terminus is abolished. These studies in protein membrane topogenesis, showing that there are different requirements for amino and carboxyl termini insertion, indicate that multiple mechanisms exist even within the same protein.     

Retinoic acid-induced cartilage degradation is caused by cartilage cells

Summary Cartilage cubes, prepared from the proximal epiphyses of neonatal rat humeri and consisting of cartilage tissue only, were cultured in the presence of retinoic acid. The retinoid induced the loss of metachromatic staining with toluidine blue, which correlates with the loss of proteoglycan, followed by tissue degradation processes resulting in a distinct reduction of the cartilage mass. Histologically, fibroblast-like cells appeared within chondrones, indicating a transformation of chondroblasts. Focal tissue degradation was observed after only 2 days. Electron microscopically, the clustered cells within the zone of tissue degradation were rich in various lysosomal structures indicating their lytic activity. Cycloheximide and EDTA completely blocked the retinoic acid effects suggesting that protein synthesis was required and that metalloproteinases may be involved in the degradation processes. In conclusion, with the new test system described here we demonstrated that cartilage cells themselves performed the tissue degradation induced by retinoic acid.     

Retroviral-mediated gene transfer of the leukocyte integrin CD18 subunit

Children with leukocyte adherence deficiency (LAD) exhibit heterogeneous defects in the leukocyte integrin CD18 subunit that prevent surface expression of functional CD11/CD18 leukocyte integrin adherence complexes. We used a retroviral vector, designated LCD18SN, to transfer the CD18 cDNA into K562 human myeloid leukemia cells and into EBV B-cells from a child with LAD. Transfer of the LCD18SN retroviral construct, which expresses the CD18 cDNA from the Moloney Murine leukemia virus (MoMLV) long terminal repeat (LTR), into K562 cells resulted in relatively high levels of CD18 mRNA and intracellular protein. Retroviral-mediated gene transfer of CD18 into LAD EBV B-cells resulted in low, but readily measurable, levels of surface expression of the CD11a/CD18 complex in these previously deficient lymphocytes. The reconstitution of surface expression of the CD11a/CD18 complex by gene transfer of the CD18 cDNA into LAD EBV B-cells indicates that this syndrome represents a candidate disorder for gene therapy.