ϩ is coupled to Ca 2ϩ transport across the sarcoplasmic reticulum (SR) membrane. We propose that SR carbonic anhydrase (CA) accelerates the CO2-HCO 3 Ϫ reaction so that H ϩ ions, which are exchanged for Ca 2ϩ ions, are produced or buffered in the SR at sufficient rates. Inhibition of this SR-CA is expected to reduce the rate of H ϩ fluxes, which then will retard the kinetics of Ca 2ϩ transport. Fura 2 signals and isometric force were simultaneously recorded in fiber bundles of the soleus (SOL) and extensor digitorum longus (EDL) from rats in the absence and presence of the lipophilic CA inhibitors L-645151, chlorzolamide (CLZ), and ethoxzolamide (ETZ), as well as the hydrophilic inhibitor acetazolamide (ACTZ). Fura 2 and force signals were analyzed for time to peak (TTP), 50% decay time (t 50), and their amplitudes. L-645151, CLZ, and ETZ significantly increased TTP of fura 2 by 10-25 ms in SOL and by 5-7 ms in EDL and TTP of force by 6-30 ms in both muscles. L-645151 and ETZ significantly prolonged t 50 of fura 2 and force by 20-55 and 40-160 ms, respectively, in SOL and EDL. L-645151, CLZ, and ETZ also increased peak force of single twitches and amplitudes of fura fluorescence ratio (R 340/380) at an excitation wavelength of 340 to 380 nm. All effects of CA inhibitors on fura 2 and force signals could be reversed. ACTZ did not affect TTP, t 50, and amplitudes of fura 2 signals or force. L-645151, CLZ, and ETZ had no effects on myosin-, Ca 2ϩ -, and Na ϩ -K ϩ -ATPase activities, nor did they affect the amplitude and half-width of action potentials. We conclude that inhibition of SR-CA by impairing H ϩ countertransport is responsible for deceleration of intracellular Ca 2ϩ transients and contraction times. sarcoplasmic reticulum; H ϩ countertransport; fura 2 transients; single twitches AN EXTRACELLULAR, SARCOLEMMAL (SL) carbonic anhydrase (CA), which is GPI anchored, is present in fastand slow-twitch skeletal muscles, and CAIII occurs in the cytoplasm of slow-twitch muscles (see Ref. 43 for an overview). Several studies have provided evidence for an additional muscle CA bound to the membrane of the sarcoplasmic reticulum (SR). Bruns et al. (3) were the first to report CA activity in isolated SR vesicles from rabbit muscles. By Triton X-114 phase separation experiments, it could be shown that this CA activity originated from a membrane-bound isozyme, rather than from a cytosolic CA. Estimations of inhibition and catalysis constants revealed different properties of the CA of SR vesicle fractions and CA of SL vesicles and confirmed the existence of two membrane-bound CAs in muscle (41). Histochemical studies with the fluorescent CA inhibitor dimethylaminonaphthalene-5-sulfonamide (3, 7) and immunoelectron microscopic studies with ultrathin sections (6) demonstrated an intracellular staining pattern, which is compatible with a CA associated with the SR membrane. In a previous study, we reported that inhibition of this SR-CA leads to significant changes in single twitches of fiber bundles of the soleus (SOL) and e...

Early interactions between pathogens and host cells are often decisive for the subsequent course of infection. Here we investigated early events during infection by Listeria monocytogenes, a ubiquitously occurring facultative intracellular microorganism that exhibits severe pathogenicity, mainly in immunocompromised individuals. We show that the inflammatory chemokine CCL2 is highly up-regulated early after Listeria infection in spleens of BALB/c mice. ERTR-9؉ macrophages of the marginal zone were identified as the only infected cells and exclusive producers of CCL2 at the early time point. Consequently, clusters of different cell types were formed around infected ERTR-9 ؉ cells. Metallophilic MOMA-1 ؉ marginal zone macrophages were, however, excluded from the clusters and migrated into the B-cell follicles. Depletion of CCL2 during infection resulted in a different composition of cell clusters in the spleen and increased the mortality rate of treated mice. Interestingly, ERTR-9؉ macrophages no longer were part of clusters in such mice but remained at their original location in the marginal zone.

Transgenic (Tg) L2 mice expressing high levels of the λ2 (315) L chain contain only B cell populations involved in the first line of defense, i.e., B-1 and marginal zone (MZ) B cells. The strongly oligoclonal IgH chain repertoire of Tg B-1a cells in such mice was attributed to strong positive selection by autoantigens. In this study, we show that the MZ B cells of L2 mice correspond very closely to MZ B cells of normal mice, as revealed by surface marker expression and gene expression profiling. We demonstrate that the IgH chain repertoire of these Tg MZ B cells is extremely heterogeneous. This is in sharp contrast to the oligoclonality found in B-1a cells of the same mice, which was attributed to strong positive selection mediated by autoantigens. Therefore, the strong positive selection of the IgH chain repertoire in L2 mice is B-1a specific. Thus, our data demonstrate that despite common functional properties, MZ B and B-1a cells exhibit striking differences in their selection and/or maintenance requirements.

SUMMARY We have collected gastrointestinal, mainly colonic, mucus from humans,guinea pigs, rats, and normal and carbonic anhydrase II (CAII)-deficient mice. In the mucus of all species, substantial CA activity was present. Using antibodies against human CA isoforms we found that the human mucus CA differs from cytosolic CAI and CAII, membrane-bound CAIV, and the secreted CAVI of saliva. The high sensitivity of mucus CA to acetazolamide rules out its identity with cytosolic CAIII. Partial sequences obtained from the purified human mucus CA show similarity, but not identity, with human CAI, and clear differences from the other known CAs. Additional evidence concerning the CA isoform present in mucus was obtained for the mucus CA of other species and was derived from: (1) the mucus of CAII-deficient mice, whose high CA activity confirms that it is not CAII that is responsible; (2) the inhibitory effect of iodide, which shows that mucus CA from mice, guinea pig and humans does not have the high anion sensitivity of CAI; (3) the inactivating effect of 0.2%SDS on guinea pig, mouse and human mucus CA, ruling out the SDS-resistant CAIV; and (4) the partitioning of guinea-pig mucus CA into the water phase in Triton X114 phase separation experiments, which also argues against its identity with membrane-bound CAs, such as CAIV. A comparison of colonic mucus CA activity in normal and germ-free rats indicates that the mucus CA is not of bacterial origin but is produced by the gastrointestinal tissues. We conclude(from its immunoreactivity, from iodide inhibition and from partial amino acid sequences) that mucus CA of human origin probably represents an isozyme, which is specific for mucus and is not identical with the known CA isozymes. The results obtained for mucus CA of other species collectively point in the same direction.