Organic cation transporters.

Over the last 15 years, a number of transporters that translocate organic cations were characterized functionally and also identified on the molecular level. Organic cations include endogenous compounds such as monoamine neurotransmitters, choline, and coenzymes, but also numerous drugs and xenobiotics. Some of the cloned organic cation transporters accept one main substrate or structurally similar compounds (oligospecific transporters), while others translocate a variety of structurally diverse organic cations (polyspecific transporters). This review provides a survey of cloned organic cation transporters and tentative models that illustrate how different types of organic cation transporters, expressed at specific subcellular sites in hepatocytes and renal proximal tubular cells, are assembled into an integrated functional framework. We briefly describe oligospecific Na(+)- and Cl(-)-dependent monoamine neurotransmitter transporters ( SLC6-family), high-affinity choline transporters ( SLC5-family), and high-affinity thiamine transporters ( SLC19-family), as well as polyspecific transporters that translocate some organic cations next to their preferred, noncationic substrates. The polyspecific cation transporters of the SLC22 family including the subtypes OCT1-3 and OCTN1-2 are presented in detail, covering the current knowledge about distribution, substrate specificity, and recent data on their electrical properties and regulation. Moreover, we discuss artificial and spontaneous mutations of transporters of the SLC22 family that provide novel insight as to the function of specific protein domains. Finally, we discuss the clinical potential of the increasing knowledge about polymorphisms and mutations in polyspecific organic cation transporters.

Downregulation of the Na(+)- D-glucose cotransporter SGLT1 by protein RS1 (RSC1A1) is dependent on dynamin and protein kinase C.

We have previously shown that the regulatory protein RS1, cloned from pig, rabbit and human (RSC1A1), is localized intracellularly and inhibits the transcription of the Na(+)- D-glucose cotransporter SGLT1 in LLC-PK(1) cells. We also reported that transport activities of human SGLT1 (hSGLT1) and human organic cation transporter hOCT2 expressed in Xenopus oocytes were decreased upon co-expression of human RS1 (hRS1). The present paper indicates that the glucose transporter GLUT1 and the peptide transporter PEPT1 are not influenced by hRS1. Voltage-clamp experiments in oocytes expressing hSGLT1 demonstrated that hRS1 reduced the maximal substrate-induced currents but did not change substrate activation, membrane potential dependence, Na(+) dependence or substrate selectivity of hSGLT1. Co-expression experiments with a dominant-negative dynamin mutant showed that the posttranslational inhibition of hSGLT1 by hRS1 was dependent on the function of dynamin. Finally, we observed that hRS1 changed the short-term effect of protein kinase C (PKC) on hSGLT1. Whereas the PKC activators phorbol-12-myristate-13-acetate (PMA) and sn-1,2-dioctanoyl glycerol (DOG) increased alpha-methyl glucose (AMG) uptake expressed by hSGLT1 alone as described earlier, PMA and DOG decreased AMG uptake mediated by hSGLT1 when hRS1 was co-expressed. Taken together, these data indicate that hRS1 modulates dynamin-dependent trafficking of intracellular vesicles containing hSGLT1 in Xenopus oocytes, and modulates PKC-dependent short-term regulation of this transporter.

Electrophysiological estimates of semantic and syntactic information access during tacit picture naming and listening to words.

We investigated the relative time courses of the accessibility of semantic and syntactic information in speaking and comprehension via event-related brain potentials (ERPs). Native German speakers either viewed a series of pictures (tacit picture naming experiment) or heard a series of nouns (listening experiment) and made dual choice go/nogo decisions based on each item's semantic and syntactic features. N200 peak latency results indicate that access to meaning has temporal precedence over access to syntactic information in both speaking (approximately 80 ms) and comprehension (approximately 70 ms), and are discussed in the context of current psycholinguistic theories.

An electrophysiological analysis of the time course of conceptual and syntactic encoding during tacit picture naming.

A central question in psycholinguistic research is when various types of information involved in speaking (conceptual/semantic, syntactic, and phonological information) become available during the speech planning process. Competing theories attempt to distinguish between parallel and serial models. Here, we investigated the relative time courses of conceptual and syntactic encoding in a tacit picture-naming task via event-related brain potential (ERP) recordings. Participants viewed pictures and made dual-choice go/no-go decisions based on conceptual features (whether the depicted item was heavier or lighter than 500 g) and syntactic features (whether the picture's German name had feminine or masculine syntactic gender). In support of serial models of speech production, both the lateralized readiness potential, or LRP (related to response preparation), and the N200 (related to response inhibition) measures indicated that conceptual processing began approximately 80 msec earlier than syntactic processing.

Sodium-hydrogen exchangers and sodium-bicarbonate co-transporters: ontogeny of protein expression in the rat brain.

We used western blotting to examine the developmental profiles (at embryonic day 16 and postnatal days 1, 13, 23, 33 and 105) of protein expression for three sodium-hydrogen exchanger isoforms (1, 2 and 4) and for a sodium-bicarbonate co-transporter in three CNS regions (cortex, cerebellum and brainstem-diencephalon). In microsomal preparations, sodium-hydrogen exchanger isoform 1 and sodium-bicarbonate co-transporter protein expression in the CNS increases gradually from embryonic day 16 (25-40% of the adult level) to postnatal day 105. In contrast, sodium-hydrogen exchanger isoform 2 and 4 expression reaches a maximum (three to 20 times the adult level) at around three to four weeks of age. There is significant regional heterogeneity in the expression of sodium-hydrogen exchanger and sodium-bicarbonate co-transporter proteins in the rat CNS. Sodium-hydrogen exchanger isoform 1 was highly expressed in the brainstem-diencephalon, whereas the sodium-bicarbonate co-transporter was robustly expressed in the cerebellum and brainstem-diencephalon. These data indicate that the expression of sodium-hydrogen exchanger and sodium-bicarbonate co-transporter proteins varies as a function of both development and specific brain region.

Na/HCO3 cotransporters in rat brain: expression in glia, neurons, and choroid plexus.

We studied the expression and distribution of Na/HCO(3) cotransporters in rat brain using polynucleotide probes and polyclonal antibodies derived from the electrogenic rat kidney Na/HCO(3) cotransporter (rkNBC). In whole brain, we observed a single mRNA ( approximately 7.5 kb) by Northern hybridization and a major approximately 130 kDa protein by immunoblotting with a polyclonal antiserum directed against the C terminus of rkNBC. NBC mRNA and protein were present in cortex, brainstem-diencephalon, and cerebellum. In situ hybridization revealed NBC mRNA expression throughout the CNS, with particularly high levels in olfactory bulb, hippocampal dentate gyrus, and cerebellum. NBC mRNA was present in glial cells (e.g., Bergmann glia of cerebellum and hippocampal astrocytes) and neurons (e.g., granule cells of dentate gyrus and neurons of cortex or striatum). Double hybridization of mRNA encoding NBC and glutamate transporter 1 (glial marker) confirmed that both glia and neurons express NBC. Indirect immunofluorescence microscopy demonstrated NBC protein throughout the CNS, particularly in hippocampus and cerebellum. Although NBC mRNA was restricted to cell bodies, NBC protein was distributed diffusely, compatible with a localization in cell processes and perhaps cell bodies. Double labeling with glial fibrillary acidic protein (astrocytic marker), microtubule-associated protein 2 (neuronal marker), or 2',3'-cyclic mononucleotide 3'-phosphodiesterase (oligodendrocytic marker) demonstrated expression of NBC protein in specific subpopulations of both glia and neurons. Moreover, NBC protein was present in both cultured hippocampal astrocytes and cortical neurons. NBC mRNA and protein were also present in epithelial cells of choroid plexus, ependyma, and meninges. Our results are thus consistent with multiple novel roles for Na/HCO(3) cotransport in CNS physiology.

Electrophysiological estimates of the time course of semantic and phonological encoding during implicit picture naming.

Two different event-related potential (ERP) components were used to investigate the temporal processing of semantic and phonological encoding during implicit picture naming. Participants were shown pictures and carried out a dual choice go/nogo decision based on semantic information (i.e., whether the picture was of an object or an animal) and phonological information (i.e., whether the picture's name starts with a vowel or a consonant). In addition to the already established lateralized readiness potential (LRP; related to response preparation), we introduce the N200 (presumably related to response inhibition) as a tool for measuring online language processing. Both, the LRP and the N200 data indicated that semantic processing began earlier than phonological processing. The data are discussed in the context of language production models. Therein, the LRP and N200 results, taken together, favor a serial or cascaded processing model of language production in contrast to a parallel processing account.

An electrogenic Na(+)-HCO(-)(3) cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain.

We screened rat brain cDNA libraries and used 5' rapid amplification of cDNA ends to clone two electrogenic Na(+)-HCO(-)(3) cotransporter (NBC) isoforms from rat brain (rb1NBC and rb2NBC). At the amino acid level, one clone (rb1NBC) is 96% identical to human pancreas NBC. The other clone (rb2NBC) is identical to rb1NBC except for 61 unique COOH-terminal amino acids, the result of a 97-bp deletion near the 3' end of the open-reading frame. Using RT-PCR, we confirmed that mRNA from rat brain contains this 97-bp deletion. Furthermore, we generated rabbit polyclonal antibodies that distinguish between the unique COOH-termini of rb1NBC (alpharb1NBC) and rb2NBC (alpharb2NBC). alpharb1NBC labels an approximately 130-kDa protein predominantly from kidney, and alpharb2NBC labels an approximately 130-kDa protein predominantly from brain. alpharb2NBC labels a protein that is more highly expressed in cortical neurons than astrocytes cultured from rat brain; alpharb1NBC exhibits the opposite pattern. In expression studies, applying 1.5% CO(2)/10 mM HCO(-)(3) to Xenopus oocytes injected with rb2NBC cRNA causes 1) pH(i) to recover from the initial CO(2)-induced acidification and 2) the cell to hyperpolarize. Subsequently, removing external Na(+) reverses the pH(i) increase and elicits a rapid depolarization. In the presence of 450 microM DIDS, removing external Na(+) has no effect on pH(i) and elicits a small hyperpolarization. The rate of the pH(i) decrease elicited by removing Na(+) is insensitive to removing external Cl(-). Thus rb2NBC is a DIDS-sensitive, electrogenic NBC that is predominantly expressed in brain of at least rat.

Immunolocalization of anion exchanger AE2 and Na(+)-HCO(-)(3) cotransporter in rat parotid and submandibular glands.

Salivary glands secrete K(+) and HCO(-)(3) and reabsorb Na(+) and Cl(-), but the identity of transporters involved in HCO(-)(3) transport remains unclear. We investigated localization of Cl(-)/HCO(-)(3) exchanger isoform AE2 and of Na(+)-HCO(-)(3) cotransporter (NBC) in rat parotid gland (PAR) and submandibular gland (SMG) by immunoblot and immunocytochemical techniques. Immunoblotting of PAR and SMG plasma membranes with specific antibodies against mouse kidney AE2 and rat kidney NBC revealed protein bands at approximately 160 and 180 kDa for AE2 and approximately 130 kDa for NBC, as expected for the AE2 full-length protein and consistent with the apparent molecular mass of NBC in several tissues other than kidney. Immunostaining of fixed PAR and SMG tissue sections revealed specific basolateral staining of PAR acinar cells for AE2 and NBC, but in SMG acinar cells only basolateral AE2 labeling was observed. No AE2 expression was detected in any ducts. Striated, intralobular, and main duct cells of both glands showed NBC expression predominantly at basolateral membranes, with some cells being apically stained. In SMG duct cells, NBC staining exhibited a gradient of distribution from basolateral localization in more proximal parts of the ductal tree to apical localization toward distal parts of the ductal tree. Both immunoblotting signals and immunostaining were abolished in preabsorption experiments with the respective antigens. Thus the mechanisms of fluid and anion secretion in salivary acinar cells may be different between PAR and SMG, and, because NBC was detected in acinar and duct cells, it may play a more important role in transport of HCO(-)(3) by rat salivary duct cells than previously believed.

Cloning and immunolocalization of a rat pancreatic Na(+) bicarbonate cotransporter.

In the rat, pancreatic HCO(-)(3) secretion is believed to be mediated by duct cells with an apical Cl(-)/HCO(-)(3) exchanger acting in parallel with a cAMP-activated Cl(-) channel and protons being extruded through a basolateral Na(+)/H(+) exchanger. However, this may not be the only mechanism for HCO(-)(3) secretion by the rat pancreas. Recently, several members of electrogenic Na(+)/HCO(-)(3) cotransporters (NBC) have been cloned. Here we report the cloning of a NBC from rat pancreas (rpNBC). This rpNBC is 99% identical to the longer, more common form of NBC [pNBC; 1079 amino acids (aa); 122 kDa in human heart, pancreas, prostate, and a minor clone in kidney]. The longer NBC isoforms are identical to the rat and human kidney-specific forms (kNBC; 1035 aa; 116 kDa) at the approximately 980 C-terminal aa's and are unique (with different lengths) at the initial N-terminus. Using polyclonal antibodies to the common N- and C-termini of rat kidney NBC, a approximately 130-kDa protein band was labeled by immunoblotting of rat pancreas homogenate and was enriched in the plasma membrane fraction. Immunofluorescence and immunoperoxidase light microscopy of rat pancreatic tissue with both antibodies revealed basolateral labeling of acinar cells. Labeling of both apical and basolateral membranes was found in centroacinar cells, intra- and extralobular duct, and main duct cells. The specificity of the antibody labeling was confirmed by antibody preabsorption experiments with the fusion protein used for immunization. The data suggest that rpNBC likely plays a more important role in the transport of HCO(-)(3) by rat pancreatic acinar and duct cells than previously believed.

Expression and distribution of the Na(+)-HCO(-)(3) cotransporter in human pancreas.

The cellular mechanisms of HCO(-)(3) secretion in the human pancreas are unclear. Expression of a Na(+)-HCO(-)(3) cotransporter (NBC) mRNA has been observed recently, but the distribution and physiological role of the NBC protein are not known. Here we examined the expression and localization of NBC in human pancreas by Northern blot, immunoblot, and immunofluorescence microscopy. Rat kidney NBC probes detected a single 9.5-kb band by Northern blot. On immunoblots, two polyclonal antisera directed against different epitopes of rat kidney NBC identified a single approximately 130-kDa protein. In cryosections of normal human pancreas, both antisera labeled basolateral membranes of large, morphologically identifiable ducts and produced a distinct labeling pattern in the remainder of the parenchyma. In double-labeling experiments, NBC immunoreactivity in the parenchyma colocalized with the Na(+)-K(+) pump, a basolateral marker. In contrast, NBC and cystic fibrosis transmembrane conductance regulator, an apical membrane marker, were detected within the same histological structures but at different subcellular localizations. The NBC antisera did not label acinar or islet cells. Our observations suggest that secretion of HCO(-)(3) by human pancreatic duct cells involves the basolateral uptake of Na(+) and HCO(-)(3) via NBC, an electrogenic Na(+)-HCO(-)(3) cotransporter.

Lexical access in the production of pronouns.

Speakers can use pronouns when their conceptual referents are accessible from the preceding discourse, as in 'The flower is red. It turns blue'. Theories of language production agree that in order to produce a noun semantic, syntactic, and phonological information must be accessed. However, little is known about lexical access to pronouns. In this paper, we propose a model of pronoun access in German. Since the forms of German pronouns depend on the grammatical gender of the nouns they replace, the model claims that speakers must access the syntactic representation of the replaced noun (its lemma) to select a pronoun. In two experiments using the lexical decision during naming paradigm [Levelt, W.J.M., Schriefers, H., Vorberg, D., Meyer, A.S., Pechmann, T., Havinga, J., 1991a. The time course of lexical access in speech production: a study of picture naming. Psychological Review 98, 122-142], we investigated whether lemma access automatically entails the activation of the corresponding word form or whether a word form is only activated when the noun itself is produced, but not when it is replaced by a pronoun. Experiment 1 showed that during pronoun production the phonological form of the replaced noun is activated. Experiment 2 demonstrated that this phonological activation was not a residual of the use of the noun in the preceding sentence. Thus, when a pronoun is produced, the lemma and the phonological form of the replaced noun become reactivated.

Localization of sodium bicarbonate cotransporter (NBC) protein and messenger ribonucleic acid in rat epididymis.

An acidic environment is important for sperm maturation in the epididymis and also helps to maintain mature sperm in an immotile state during storage in this organ. Both an Na+/H+ exchanger and an H+ATPase have been implicated in this process. The H+ATPase is concentrated in specialized apical (and/or narrow) and clear cells of the epididymis, while the Na+/H+ exchanger has not yet been localized in situ. As in other proton-secreting epithelia, bicarbonate transport occurs in the epididymis, where it is implicated in luminal acidification. In this study we used an antibody raised against a fusion protein (maltose-binding protein: MBP-NBC-5) from the C-terminus of the recently cloned rat kidney Na+/HCO3- cotransporter (NBC) to localize this protein in the epididymis and vas deferens of the rat. The distribution of the respective mRNA was mapped by in situ hybridization. NBC message was strongly expressed in the initial segment and the intermediate zone of the epididymis, and the NBC-5 antibody gave a strong basolateral staining in both principal cells and apical/narrow cells in this region. Western blotting revealed a single band at about 160 kDa in the epididymis. The intensity of staining as well as mRNA levels decreased in the cauda epididymidis and in the vas deferens, where only weak staining was seen. Basolateral NBC may function in parallel with apical proton secretion to regulate luminal acidification and/or bicarbonate reabsorption in the excurrent duct system.

Immunolocalization of the electrogenic Na+-HCO-3 cotransporter in mammalian and amphibian kidney.

Electrogenic cotransport of Na+ and HCO-3 is a crucial element of HCO-3 reabsorption in the renal proximal tubule (PT). An electrogenic Na+-HCO-3 cotransporter (NBC) has recently been cloned from salamander and rat kidney. In the present study, we generated polyclonal antibodies (pAbs) to NBC and used them to characterize NBC on the protein level by immunochemical methods. We generated pAbs in guinea pigs and rabbits by immunizing with a fusion protein containing the carboxy-terminal 108 amino acids (amino acids 928-1035) of rat kidney NBC (rkNBC). By indirect immunofluorescence microscopy, the pAbs strongly labeled HEK-293 cells transiently expressing NBC, but not in untransfected cells. By immunoblotting, the pAbs recognized a approximately 130-kDa band in Xenopus laevis oocytes expressing rkNBC, but not in control oocytes injected with water or cRNA for the Cl-/HCO-3 exchanger AE2. In immunoblotting experiments on renal microsomes, the pAbs specifically labeled a major band at approximately 130 kDa in both rat and rabbit, as well as a single approximately 160-kDa band in salamander kidney. By indirect immunofluorescence microscopy on 0.5-micrometer cryosections of rat and rabbit kidneys fixed in paraformaldehyde-lysine-periodate (PLP), the pAbs produced a strong and exclusively basolateral staining of the PT. In the salamander kidney, the pAbs labeled only weakly the basolateral membrane of the PT. In contrast, we observed strong basolateral labeling in the late distal tubule, but not in the early distal tubule. The specificity of the pAbs for both immunoblotting and immunohistochemistry was confirmed in antibody preabsorption experiments using either the fusion protein used for immunization or similarly prepared control fusion proteins. In summary, we have developed antibodies specific for NBC, determined the apparent molecular weights of rat, rabbit, and salamander kidney NBC proteins, and described the localization of NBC within the kidney of these mammalian and amphibian species.

Circulating Na+/K+-ATPase inhibitors: effects of neuropeptides, volume expansion and salt loading in conscious rats.

1. In mammalian plasma, many different inhibitors of Na+/K(+)-ATPase are present, but it is not clear whether their net effect on NA+/K(+)-ATPase activity changes during the regulation of electrolyte and fluid balance. We studied Na+/K(+)-ATPase inhibition by plasma extracts in conscious rats during short- and long-term body fluid regulation. 2. Male, adult, conscious, freely moving Wistar rats were subjected to one of the following protocols: (i) intracerebro-ventricular (i.c.v.) injections of angiotension II (AngII; 1, 10 and 100 ng), the AngII receptor antagonist losartan (1 microgram), atrial natriuretic peptide (ANP-III; 1 microgram) or isotonic saline (IS); (ii) intra-arterial (i.a.) injections of IS (6 or 10 mL), hypertonic saline (HS; 1.2% NaCl, 5 mL) or hypertonic plasma expander (HPS; 3.5% hetastarch in HS, 5 mL); or (iii) a low salt-high salt-low salt diet sequence (0.18/1.8/0.18% NaCl chow for 5 days each with controls receiving 0.18% NaCl on all days). Bodyweight, the intake of food and water, urine volume and Na+ concentration and weight of faeces were determined daily. Plasma samples were withdrawn repeatedly throughout the respective protocols, extracted on C18-reversed phase columns and assayed for their effect on the activity of different Na+/K(+)-ATPase preparations. 3. The inhibition of rat brain Na+/K(+)-ATPase by plasma extracts was not significantly changed by i.c.v. injection of AngII, losartan, ANP-III and IS within the observation period (30 min from respective stimuli). Similarly, no significant changes occurred after acute volume expansion by i.a. injection of IS or HS within 120 min; upon HPS, however, Na+/K(+)-ATPase inhibition was decreased by approximately 20% (P < 0.05), probably due to passive dilution. During the high-salt diet, fluid retention was effectively counteracted by an adaptive increase of urinary sodium excretion. Throughout the protocol, inhibition of pig brain Na+/K(+)-ATPase by plasma extracts did not differ significantly between groups. 4. It is concluded from these results that the short- or long-term control of body fluids in conscious rats is not associated with systematic changes in Na+/K(+)-ATPase inhibition by plasma factors.