Fluoxetine Inhibits K+ Transport Pathways (K+ Efflux, Na+-K+-2Cl− Cotransport, and Na+ Pump) Underlying Volume Regulation in Corneal Endothelial Cells

We have studied regulatory volume responses of cultured bovine corneal endothelial cells (CBCEC) using light scattering. We assessed the contributions of fluoxetine (Prozac) and bumetanide-sensitive membrane ion transport pathways to such responses by determining K⁺ efflux and influx. Cells swollen by a 20% hypo-osmotic solution underwent a regulatory volume decrease (RVD) response, which after 6 min restored relative cell volume by 98%. Fluoxetine inhibited RVD recovery; 20 μm by 26%, and 50 μm totally. Fluoxetine had a triphasic effect on K⁺ efflux; from 20 to 100 μm it inhibited efflux 2-fold, whereas at higher concentrations the efflux first increased to 1.5-fold above the control value, and then decreased again. Cells shrunk by a 20% hyperosmotic solution underwent a regulatory volume increase (RVI) which also after 6 min restored the cell volume by 99%. Fluoxetine inhibited RVI; 20 μm by 25%, and 50 μm completely. Bumetanide (1 μm) inhibited RVI by 43%. In a Cl⁻-free medium, fluoxetine (50–500 μm) progressively inhibited bumetanide-insensitive K⁺ influx. The inhibitions of RVI and K⁺ influx induced by fluoxetine 20 to 50 μm were similar to those induced by 1 μm bumetanide and by Cl⁻-free medium. A computer simulation suggests that fluoxetine can interact with the selectivity filter of K⁺ channels. The data suggest that CBCEC can mediate RVD and RVI in part through increases in K⁺ efflux and Na-K-2Cl cotransport (NKCC) activity. Interestingly, the data also suggest that fluoxetine at 20 to 50 μm inhibits NKCC, and at 100–1000 μm inhibits the Na⁺ pump. One possible explanation for these findings is that fluoxetine could interact with K⁺-selective sites in K⁺ channels, the NKC cotransporter and the Na⁺ pump.     

Mammalian target of rapamycin inhibition in macrophages of asymptomatic HIV+ persons reverses the decrease in TLR-4-mediated TNF-α release through prolongation of MAPK pathway activation

TLR-4-mediated signaling is significantly impaired in macrophages from HIV(+) persons, predominantly owing to altered MyD88-dependent pathway signaling caused in part by constitutive activation of PI3K. In this study we assessed in these macrophages if the blunted increase in TLR-4-mediated TNF-α release induced by lipid A (LA) is associated with PI3K-induced upregulation of mammalian target of rapamycin (mTOR) activity. mTOR inhibition with rapamycin enhanced TLR-4-mediated TNF-α release, but suppressed anti-inflammatory IL-10 release. Targeted gene silencing of mTOR in macrophages resulted in LA-induced TNF-α and IL-10 release patterns similar to those induced by rapamycin. Rapamycin restored MyD88/IL-1R-associated kinase interaction in a dose-dependent manner. Targeted gene silencing of MyD88 (short hairpin RNA) and mTOR (RNA interference) inhibition resulted in TLR-4-mediated 70-kDa ribosomal protein S6 kinase activation and enhanced TNF-α release, whereas IL-10 release was inhibited in both silenced and nonsilenced HIV(+) macrophages. Furthermore, mTOR inhibition augmented LA-induced TNF-α release through enhanced and prolonged phosphorylation of ERK1/2 and JNK1/2 MAPK, which was associated with time-dependent MKP-1 destabilization. Taken together, impaired TLR-4-mediated TNF-α release in HIV(+) macrophages is attributable in part to mTOR activation by constitutive PI3K expression in a MyD88-dependent signaling pathway. These changes result in MAPK phosphatase 1 stabilization, which shortens and blunts MAPK activation. mTOR inhibition may serve as a potential therapeutic target to upregulate macrophage innate immune host defense responsiveness in HIV(+) persons.     

Cytotoxic alpha-bromoacrylic derivatives of low molecular weight

In vitro and in vivo activities of a small series of alpha-bromoacrylic derivatives of low molecular weight (MW) are described and compared with those of alpha-bromoacrylic derivatives of distamycin-like frames. Low MW compounds, when lacking of a strong basic moiety, are potent cytotoxics, while analogues bearing a strong basic moiety are not. This suggests the existence of an active transport mechanism for distamycin-derived cytotoxics characterized by strong basic amidino or guanidino moieties. Low MW compounds are inactive in vivo, possibly because of the metabolic lability of alpha-bromoacrylic moiety. The same moiety is however present in a series of potent anticancer distamycin-like minor groove binders, for example, PNU-166196 (brostallicin), a fact that underlines the features of the latter.     

Cell signaling pathways mediating epidermal growth factor stimulation of Na:K:2Cl cotransport activity in rabbit corneal epithelial cells

We characterized the signaling and ion transport pathways that mediate epidermal growth factor receptor physiological control in SV40-immortalized rabbit corneal epithelial cells (tRCEC). Our evaluation employed single-cell fluorescence imaging to measure the intracellular [Na⁺]_i in these cells loaded with the Na⁺ sensitive dye, SBFI. EGF (1 to 5 ng/ml) transiently increased [Na⁺]_i from 10 mm to as much as 35 mm after 25 min, which was followed by a decline towards its control value. These increases waned at higher EGF concentrations up to 50 ng/ml. Both inhibition of EGF receptor-linked tyrosine kinase activity (50 μm RG-13022) and cPLA₂ activity (10 μm AACOCF₃) obviated EGF-induced increases in [Na⁺]_i. In contrast, PGE₂ (10 μg/ml) and cAMP (2 mm) increased [Na⁺]_i by 25 mm. Inhibition of NKCC activity through exposure to either Cl-free Ringers or 300 μm furosemide in NaCl Ringers eliminated EGF-induced increases in [Na⁺]_i. Similarly, EGF failed to increase [Na⁺]_i following inhibition of: 1) PKA activity (10 μm H-89); 2) Erk1/2 (15 μm PD98059) or 3) p38 (15 μm SB203580) activity. Stimulation protein kinase C activity (0.1 μm PMA) transiently increased [Na⁺]_i followed by a decline towards its baseline value. EGF-induced increases in [Na⁺]_i were unaltered by inhibition of K⁺ conductance (100 μm 4-AP). Taken together, EGF stimulates Erk1/2; p38 and cPLA₂ activity. Their stimulation increases PGE₂ and cAMP levels resulting in PKA and NKCC activation. Received: 18 December 2000/Revised: 24 May 2001     

Renal weights in the southern African black population

Renal weights of 430 adult black subjects coming to medicolegal autopsy at the Diepkloof State Mortuary, a large urban area southwest of Johannesburg, South Africa, were analyzed. The subjects were from 10 southern African black ethnic groups-Zulu, Sotho, Tswana, Xhosa, Shangaan, Swazi, Venda, Ndebele, Kalanga, and Malawi. The aims of the study were: 1) to ascertain the anatomical "norm" as it pertains to renal weights in this diverse population group; 2) to formulate standard reference tables that might be of use to the practicing pathologist in the southern African arena; 3) to provide a range of values that take into account the variables of age, sex, race, body weight, and body height; and 4) to provide a standard of comparison with anthropological and anatomical studies conducted on North American black, North American Caucasian, Indian subcontinent, Burmese, and Jamaican population groups. In each of the 430 subjects, age, sex, ethnic group, supine body length, body weight, individual left and right renal weights, and causes of death were noted. The latter were divided into 6 categories: 1) penetrating incised wounds; 2) multiple injuries; 3) gunshot wounds; 4) craniocerebral injuries; 5) various miscellaneous nonnatural causes of death; and 6) natural causes of death. The above variables were analyzed by computer and compared with respect to renal weights. No statistically significant differences were observed between the sexes or the age groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane transport parameters in frog corneal epithelium measured using impedance analysis techniques

Summary Active Cl^– transport in bullfrog corneal epithelium was studied using transepithelial impendance analysis methods, and direct-current (DC) measurements of membrane voltages and resistance ratios. The technique allows the estimation of the apical and basolateral membrane conductances, and the paracellular conductance, and does not rely on the use of membrane conductance-altering agents to obtain these measurements as was requisite in earlier DC equivalent-circuit analysis studies. In addition, the analysis results in estimates of the apical and basolateral membrane capacitances, and allows resolution of the paracellular conductance into properties of the tight junctions and lateral spaces. Membrane capacitances (proportional to areas) were used to estimate the specific conductances of the apical and basolateral membranes, as well as to evaluate coupling between the cell layers. We confirm results obtained from earlier studies: (1) apical membrane conductance is proportional to the rate of active Cl^– transport and is, highly Cl^– selective; (2) intracellular Cl^– activity is above electrochemical equilibrium, thereby providing a net driving force for apical membrane Cl^– exit; (3) the paracellular conductance is comparable to the transcellular conductance. We also found that: (1) the paracellular conductance is composed of the series combination of the junctional conductance and a nonnegligible lateral space resistance; (2) a small K⁺ conductance reported in the apical membrane may result from Cl^– channels possessing a finite permeability to K⁺; (3) the basolateral membrane areas is 36 times greater than the apical membrane area which is consistent with the notion of electrical coupling between the five to six cell layers of the epithelium; (4) the specific conductance of the basolateral membrane is many times lower than that of the apical membrane; (5) the net transport of Cl^– is modulated primarily by changes in the conductance of the apical membrane and not by changes in the net electrochemical gradient resulting from opposite changes in the electrical and chemical gradients; (6) the conductance of the basolateral membrane does not change with transport which implies that the net driving force for K⁺ exit increases with transport, possibly due to an increase in the intracellular K⁺ activity.     

Localization of C-protein isoforms in chicken skeletal muscle: ultrastructural detection using monoclonal antibodies

Monoclonal antibodies (McAbs) specific for the fast (MF-1) and slow (ALD-66) isoforms of C-protein from chicken skeletal muscle have been produced and characterized. Using these antibodies it was possible to demonstrate that skeletal muscles of varying fiber type express different isoforms of this protein and that in the posterior latissimus dorsi muscle both isoforms are co-expressed in the same myofiber (17, 18). Since we had shown that both isoforms were present in all sarcomeres, it was feasible to test whether the two isoforms co- distributed in the same 43-nm repeat within the A-band, thereby establishing a minimum number of C-proteins per repeat in the thick filaments. Here we describe the ultrastructural localization of C- protein in myofibers from three muscle types of the chicken using these same McAbs. We observed that although C-protein was present in a 43-nm repeat along the filaments in all three muscles, there were marked differences in the absolute number and position occupied by the different isoforms. Since McAbs MF-1 and ALD-66 decorated the same 43- nm repeats in the A-bands of the posterior latissimus dorsal muscle, we suggest that at least two C-proteins can co-localize at binding sites 43 nm apart along thick filaments of this muscle.     

The complete sequence of a chicken-muscle cDNA encoding the acidic ribosomal protein P1

We have isolated and sequenced a complete cDNA encoding the acidic phosphoprotein P1 from chicken. The analysis of the deduced protein sequence and its comparison with the known sequence of P proteins from human, rat and Artemia salina indicates that the central, alanine-rich region of these proteins was probably generated by internal duplications of the gene followed by modifications within each repeat. This observation explains the length heterogeneity and sequence divergence of this particular region when compared with the highly conserved structure of the remaining segments of the protein.