Towards outcome-based programme evaluation: using student comparative self-assessments to determine teaching effectiveness.

BACKGROUND: Programme evaluation of medical education should be multi-dimensional. While structural and organisational aspects of teaching are frequently assessed, programme evaluation tools are rarely matched to specific learning objectives. AIMS: This study used one medical school's catalogue of specific learning objectives to implement and critically appraise a novel programme evaluation tool based on comparative student self-assessments. METHOD: Medical students enrolled in the clinical phase of the undergraduate curriculum in Göttingen were invited to self-rate their knowledge, skills and attitudes before and after each course. A newly developed formula controlling for student performance levels when entering a course was used to compute a percentage gain in knowledge, skills and attitudes. Data derived from a prospective, longitudinal intervention study on the development of electrocardiogram interpretation skills including 636 students from four consecutive cohorts were used to provide validity evidence of the new approach. RESULTS: The novel tool appeared superior to plain mean differences and effect sizes in detecting outstanding teaching as well as shortcomings of the curriculum. In addition, it adequately reflected objectively measured performance levels and was responsive to curriculum change. CONCLUSIONS: Comparative student self-assessment is a valid tool to appraise undergraduate medical curricula at the level of specific learning objectives.

Membrane translocation of glutaric acid and its derivatives.

The neurodegenerative disorder glutaric aciduria type I (GA I) is characterized by increased levels of cytotoxic metabolites such as glutaric acid (GA) and 3-hydroxyglutaric (3OHGA). The present report summarizes recent investigations providing insights into mechanisms of intra- and intercellular translocation of these metabolites. Initiated by microarray analyses in a mouse model of GA I, the sodium-dependent dicarboxylate cotransporter 3 (NaC3) was the first molecule identified to mediate the translocation of GA and 3OHGA with high and low affinity, respectively. More recently, organic anion transporters (OAT) 1 and 4 have been reported to be high-affinity transporters for GA and 3OHGA as well as D-2- and L-2-hydroxyglutaric acid (D2OHGA, L2OHGA). The concerted action of NaC3 and OATs may be important for the directed uptake and excretion of GA, 3OHGA, D2OHGA and L2OHGA in kidney proximal tubule cells. In addition, experimental data on cultured neuronal and glial cells isolated from mouse brain demonstrated that GA rather than 3OHGA may competitively inhibit the anaplerotic supply of tricarboxylic acid cycle intermediates from astrocytes to neurons. The identification of GA and GA derivative transporters may represent targets for new approaches to treat patients with GA I and related disorders.

Molecular evidence of organic ion transporters in the rat adrenal cortex with adrenocorticotropin-regulated zonal expression.

Experimental evidence suggested that secretion of steroid hormones from adrenocortical cells involves carrier-mediated transport: Cortisol release from, and uptake of p-[3H]aminohippurate into, bovine adrenocortical cells showed properties of the renal p-[3H]aminohippurate/anion exchanger OAT1. Other poly-specific transporters such as organic anion-transporting polypeptides (oatps) and organic cation transporters (OCTs) could also be involved in steroid hormone release. A homology-cloning procedure was established to detect these transporters in rat adrenal gland cDNA. PCR revealed the presence of OAT1, oatp1, oatp2, and oatp3. In situ hybridization localized OAT1 in the outer zona fasciculata, oatp3 in the zona glomerulosa, and oatp1 and oatp2 in the inner zona fasciculata and outer zona reticularis. An OCT2-specific probe produced signals in the zona glomerulosa and outer zona fasciculata. Pretreatment of rats with ACTH increased the expression of OAT1 mRNA that spread to all zones, and hypophysectomy strongly decreased it. A less pronounced regulation was detected for OCT2 and oatp3. Specific antibodies confirmed the localization of OAT1 in the outer zona fasciculata, supporting a possible role of OAT1 in cortisol release. The zonated distribution of transporters furthermore suggest that oatp1-3 and OCT2 may be important for the endocrine function of rat adrenocortical cells.

Transport of organic anions across the basolateral membrane of proximal tubule cells.

Renal proximal tubules secrete diverse organic anions (OA) including widely prescribed anionic drugs. Here, we review the molecular properties of cloned transporters involved in uptake of OA from blood into proximal tubule cells and provide extensive lists of substrates handled by these transport systems. Where tested, transporters have been immunolocalized to the basolateral cell membrane. The sulfate anion transporter 1 (sat-1) cloned from human, rat and mouse, transported oxalate and sulfate. Drugs found earlier to interact with sulfate transport in vivo have not yet been tested with sat-1. The Na(+)-dicarboxylate cotransporter 3 (NaDC-3) was cloned from human, rat, mouse and flounder, and transported three Na(+) with one divalent di- or tricarboxylate, such as citric acid cycle intermediates and the heavy metal chelator 2,3-dimercaptosuccinate (succimer). The organic anion transporter 1 (OAT1) cloned from several species was shown to exchange extracellular OA against intracellular alpha-ketoglutarate. OAT1 translocated, e.g., anti-inflammatory drugs, antiviral drugs, beta-lactam antibiotics, loop diuretics, ochratoxin A, and p-aminohippurate. Several OA, including probenecid, inhibited OAT1. Human, rat and mouse OAT2 transported selected anti-inflammatory and antiviral drugs, methotrexate, ochratoxin A, and, with high affinities, prostaglandins E(2) and F(2alpha). OAT3 cloned from human, rat and mouse showed a substrate specificity overlapping with that of OAT1. In addition, OAT3 interacted with sulfated steroid hormones such as estrone-3-sulfate. The driving forces for OAT2 and OAT3, the relative contributions of all OA transporters to, and the impact of transporter regulation by protein kinases on renal drug excretion in vivo must be determined in future experiments.

Interaction of the metal chelator 2,3-dimercapto-1-propanesulfonate with the rabbit multispecific organic anion transporter 1 (rbOAT1).

The metal chelator DMPS (2,3-dimercapto-1-propanesulfonate) is used to treat heavy metal intoxication because it increases renal excretion of these toxins, which are accumulated in proximal tubule cells. To evaluate the involvement of the organic anion transporter 1 (OAT1) in the renal flux of DMPS, we examined the effect of DMPS on transport mediated by the rabbit ortholog of OAT1 and compared these characteristics with those observed in intact isolated rabbit proximal tubules. The rabbit OAT1 (rbOAT1) cDNA consisted of 2124 base pairs encoding a protein of 551 amino acids. Heterologous expression in COS-7 cells revealed rbOAT1-mediated transport of p-aminohippurate (PAH; K(t) = 16 microM). A 1 mM concentration of unlabeled PAH, alpha-ketoglutarate, urate, or probenecid inhibited [(3)H]PAH uptake by 70 to 90%. cis-Inhibition and trans-stimulation experiments using several Krebs cycle intermediates implicated alpha-ketoglutarate as the main intracellular exchange anion. Reduced DMPS inhibited rbOAT1-mediated fluorescein transport with an apparent K(i) of 102 microM. These characteristics paralleled those observed in isolated rabbit proximal tubules. PAH was transported into nonperfused single proximal tubule S(2) segments with a K(t) of 76 microM. DMPS inhibited FL uptake into single tubule segments with a K(i-app) of 71 microM. Fluorescein efflux from preloaded tubules was trans-stimulated by 1 mM PAH and 1 mM DMPS, consistent with DMPS entry into tubule cells by rbOAT1. In summary, rbOAT1 mediates basolateral uptake of DMPS into proximal tubule cells, implicating this process in the detoxification process of heavy metals in the kidneys.

DNA sequence recognition of thiazole-containing cross-linked polyamides can be favored.

The binding ability of cross-linked thiazolated polyamides (containing the base sequence-reading elements thiazole(Th)-pyrrole(Py)-pyr-role(Py) and thiazole(Th)-imidazole(Im)-pyrrol(Py) to various DNA dodecamers has been investigated. CD titration experiments at high salt concentration demonstrate that the dimers with a heptanediyl linker (C7 dimer) show a significantly higher sequence specificity than their corresponding monomers. The dimer of Th-Py-Py primarily prefers binding to pure AT sequences and that of Th-Im-Py to the dodecamer sequences containing a GC pair within the central sequence (e.g. AACGTT). Surprisingly, the sequence binding ability is strongly influenced by the presence of a T-A step: e.g. Th-Py-Py has a similar affinity to the sequences TTTAAA and ATCGTA; likewise Th-Im-Py shows a preference for these sequences. The CD results correlate with footprinting data. Related biochemical studies on the effect of polyamides on DNA gyrase activity in vitro show that the C7 dimers most effectively inhibit the enzyme activity compared with the monomers and the natural reference minor groove binder distamycin. The highest inhibitory potency is observed for the Th-Py-Py-dimer. The role of the T-A step in binding of the cross-linked dimer to the minor groove is discussed in light of the sequence recognition of the TATA box binding protein.

Molecular physiology of renal p-aminohippurate secretion.

Renal proximal tubules secrete various organic anions, including drugs and p-aminohippurate (PAH). Uptake of PAH from blood into tubule cells occurs by exchange with intracellular alpha-ketoglutarate and is mediated by the organic anion transporter 1. PAH exit into tubule lumen is species specific and may involve ATP-independent and -dependent transporters.

Binding of bis-linked netropsin derivatives in the parallel-stranded hairpin form to DNA.

Cis-diammine Pt(II)- bridged bis-netropsin and oligomethylene-bridged bis-netropsin in which two monomers are linked in a tail-to-tail manner bind to the DNA oligomer with the sequence 5'-CCTATATCC-3' in a parallel-stranded hairpin form with a stoichiometry 1:1. The difference circular dichroism (CD) spectra characteristic of binding of these ligands in the hairpin form are similar. They differ from CD patterns obtained for binding to the same duplex of another bis-netropsin in which two netropsin moieties were linked in a head-to-tail manner. This reflects the fact that tail-to-tail and head-to-tail bis-netropsins use parallel and antiparallel side-by-side motifs, respectively, for binding to DNA in the hairpin forms. The binding affinity of cis-diammine Pt(II)-bridged bis-netropsin in the hairpin form to DNA oligomers with nucleotide sequences 5'-CCTATATCC-3' (I), 5'-CCTTAATCC-3' (II), 5'-CCTTATTCC-3' (III), 5'-CCTTTTTCC-3' (IV) and 5'-CCAATTTCC-3' (V) decreases in the order I = II > III > IV > V . The binding of oligomethylene-bridged bis-netropsin in the hairpin form follows a similar hierarchy. An opposite order of sequence preferences is observed for partially bonded monodentate binding mode of the synthetic ligand.

Genomic structure and in vivo expression of the human organic anion transporter 1 (hOAT1) gene.

The human organic anion transporter 1 (hOAT1) plays a key role in the secretion of an array of potentially toxic organic anions including many clinically important drugs. Here we report on the genomic cloning of hOAT1. A human genomic library was used for screening of a PAC (P1 artificial chromosome) clone applying PCR techniques. Sequencing of several restriction subclones and of a PCR-generated clone revealed that the hOAT1 gene spans 8.2 kb and is composed of 10 exons divided by 9 introns. RT-PCR studies in a human kidney specimen led to the detection of two new splice variants, hOAT1-3 and hOAT1-4, showing a 132-bp in-frame deletion. Using fluorescence in situ hybridization (FISH) we mapped the hOAT1 gene as a single signal to chromosome 11q13.1-q13.2. Additionally, 600 bp of the 5' flanking region was analyzed, illustrating the probable transcription start site at nt -280, a NF-kappaB-site at nt -397 and several putative transcription factor binding sites.

Functional and molecular biological evidence of SGLT-1 in the ruminal epithelium of sheep.

Because of the effective catabolism of D-glucose to short-chain fatty acids by intraruminal microorganisms, the absorption of D-glucose from the rumen was thought to be of minor importance. However, clinical studies suggested that significant quantities of D-glucose are transported from the ruminal contents to the blood. We therefore tested the ruminal epithelium of sheep for the presence of Na(+)-glucose cotransporter 1 (SGLT-1) on both the functional and mRNA levels. In the absence of an electrochemical gradient, 3-O-methylglucose (3-OMG) was net absorbed across isolated ruminal epithelia mounted in Ussing chambers. The net transport of 3-OMG followed Michaelis-Menten kinetics and was sensitive to phlorizin or decreasing Na(+) concentrations. The mucosal addition of 10 mM D-glucose induced an immediate, phlorizin-sensitive increase in short-circuit current (I(sc)). I(sc) could also be increased by serosal addition of D-glucose or D-mannose, but electrogenic uptake of D-glucose or 3-OMG added on the mucosal side was still detectable after serosal stimulation of I(sc). RT-PCR using primers specific for the ovine intestinal SGLT-1 with subsequent TA cloning and sequencing revealed 100% identity between the cloned cDNA and mRNA fragment 187-621 of ovine intestinal SGLT-1. In conclusion, the ruminal epithelium has a high-affinity SGLT-1, which indicates that it maintains the capacity for D-glucose absorption.

Structure of renal organic anion and cation transporters.

Here we review the structural and functional properties of organic anion transporters (OAT1, OAT2, OAT3) and organic cation transporters (OCTN1, OCTN2, OCT1, OCT2, OCT3), some of which are involved in renal proximal tubular organic anion and cation secretion. These transporters share a predicted 12-transmembrane domain (TMD) structure with a large extracellular loop between TMD1 and TMD2, carrying potential N-glycosylation sites. Conserved amino acid motifs revealed a relationship to the sugar transporter family within the major facilitator superfamily. Following heterologous expression, most OATs transported the model anion p-aminohippurate (PAH). OAT1, but not OAT2, exhibited PAH-alpha-ketoglutarate exchange. OCT1-3 transported the model cations tetraethylammonium (TEA), N(1)-methylnicotinamide, and 1-methyl-4-phenylpyridinium. OCTNs exhibited transport of TEA and/or preferably the zwitterionic carnitine. Substrate substitution as well as cis-inhibition experiments demonstrated polyspecificity of the OATs, OCTs, and OCTN1. On the basis of comparison of the structurally closely related OATs and OCTs, it may be possible to delineate the binding sites for organic anions and cations in future experiments.

Potential-dependent steady-state kinetics of a dicarboxylate transporter cloned from winter flounder kidney.

The two-electrode voltage-clamp technique in combination with tracer uptake experiments was used to investigate the dependence of dicarboxylate transport kinetics on membrane potential in Xenopus laevis oocytes expressing the flounder renal high-affinity-type sodium dicarboxylate cotransporter (fNaDC-3). Steady-state succinate-dependent currents in the presence of Na+ were saturable with an apparent affinity constant for succinate, K0.5,succ, of 60 microM. K0.5,succ was independent of membrane potential, suggesting succinate binding at the surface of the fNaDC-3 protein. The maximal succinate-dependent current, deltaImax, increased with hyperpolarization, suggesting that the empty carrier may translocate net charge. Succinate-induced currents showed sigmoidal dependence on Na+ concentration, and K0.5,Na+ decreased with hyperpolarization, suggesting Na+ binding in an ion well. Lowering the external Na+ concentration to 20 mM increased K0.5,succ approximately threefold. Succinate-induced currents were inhibited by Li+ with an Ki,Li+ of approximately 0.5 mM, and a Hill coefficient of below unity indicating the interaction of one Li+ ion with an inhibitory site at fNaDC-3.

Voltage-driven p-aminohippurate, chloride, and urate transport in porcine renal brush-border membrane vesicles.

p-Aminohippurate (PAH) and urate are secreted into the proximal tubule lumen across the brush-border membrane. Here we used brush-border membrane vesicles from pig kidney to study PAH and urate transport. Efflux and influx of [3H]PAH were influenced by K+-diffusion potentials indicating electrogenic PAH transport. An outside>inside PAH concentration difference accelerated voltage-sensitive, Na+-coupled D-glucose uptake as efficiently as did an outside>inside Cl- concentration difference, suggesting comparable conductances for PAH and Cl- in brush-border membrane vesicles. Up to 1 mM of the uricosurics indacrinone, tienilic acid, losartan and probenecid, as well as of the stilbenes, DIDS and SITS, and of the loop diuretics furosemide and bumetanide inhibited voltage-driven PAH uptake, but not, or only slightly, voltage-driven Cl- uptake. Voltage-driven [14C]urate uptake, however, was inhibited by 0.1 mM DIDS, 0.2 mM losartan and 0.5 mM probenecid to a similar extent as [3H]PAH uptake. One millimolar pyrazinoic acid, oxonate, xanthine and adenosine inhibited neither [3H]PAH nor [14C]urate uptake. These results suggest that PAH and urate share an anion conductance which is distinct from the Cl- conductance and is probably not the same as a recently identified urate channel (Leal-Pinto E et a]. J Biol Chem 272:617-625, 1997).

DNA Sequence Recognition of a Cross-Linked Polyamide: CD Studies, Footprinting and Effects on the Activity of DNA Gyrase.

Abstract Using circular dichroism the binding ability of a cross-linked thiazole-lexitropsin, composed of two polyamide strands (with the base binding residues thiazole-imidazole-pyrrole) to a series of dodecamer duplexes containing different central sequences, has been examined. The binding of the dimer with a heptanediyl linker (C7 dimer) was compared with that of the lexitropsin monomer at 200 mM NaCl and 2 M NaCl. The C7 dimer exhibits a clear-cut different binding tendency to various dodecamers at 2 M NaCl indicating that sequence specificity becomes apparent at high salt concentration. The highest binding preference occurs to the dodecamers with the central sequences: AACGTT, AAGTTT and ATCGTA but almost no affinity was observed at 2 M NaCl for AGCGCT, ATCGAT and AAATTT. From the results it appears that the sequence selectivity of the dimer can be ascribed to the side-by-side binding mode of the cross-linked polyamide strands in the minor groove. In contrast no similar variation was found in the binding behavior for the lexitropsin monomer. Modification of the leading residue on the thiazoles of the dimer significantly lowers (or even abolishs) the binding ability, e.g. if the amino group is replaced by formyl or an acetyl residue. Footprinting and melting temperature data are in agreement with the CD results. Comparative in vitro studies on the influence of the lexitropsins on DNA gyrase demonstrate that the dimer has a higher inhibitory potency on the enzymatic activity compared to the monomer in accord with the observed DNA binding differences. A scheme of a possible side- by-side alignment of the C7 dimer in the minor groove is proposed.

Demonstration of a Na(+)-dicarboxylate cotransporter in bovine adrenocortical cells.

Our study found the uptake of [14C]succinate into bovine adrenocortical cells to be sodium-dependent, inhibited by lithium, and to have an apparent K(m) of 146 mumol/l. Succinate uptake was inhibited by glutarate, fumarate, alpha-ketoglutarate, and maleate but not by 2,3-dimethylsuccinate or cis-aconitate, specific inhibitors of the basolateral Na(+)-dicarboxylate transporter of renal proximal tubule cells. Succinate uptake was highest at pH 6.0 and decreased with increasing pH. Transport of succinate was not significantly inhibited by citrate at pH 7.4 whereas at pH 6.0 inhibition of succinate uptake by citrate was small but significant. The affinity of the adrenal dicarboxylate transporter towards succinate ranges in between the low affinity of the renal luminal dicarboxylate transporter and the high affinity of the respective basolateral transporter. The pH dependency of succinate uptake and the missing inhibition by citrate at pH 7.4 differ from both the luminal and from the basolateral dicarboxylate transporters in kidney, liver, intestine, and placenta. These functional characteristics provide evidence for the existence of a Na(+)-dicarboxylate cotransporter in adrenocortical cells which may supply cholesterol metabolism with reducing substrates.

Demonstration of a probenecid-inhibitable anion exchanger involved in the release of cortisol and cAMP and in the uptake of p-aminohippurate in bovine adrenocortical cells.

Recently we provided evidence for the involvement of a probenecid-inhibitable anion exchanger in cortisol release from primary cultures of bovine adrenocortical cells. In the present study, we further characterized this exchange transporter. Adrenocorticotropic hormone stimulated 3H-p-aminohippurate (3H-PAH) uptake into as well as cortisol release from the cells about two- and tenfold, respectively. Probenecid inhibited both 3H-PAH uptake and cortisol release by about 55 and 63%. Preincubation of the cells with 1 mM PAH trans-stimulated 3H-PAH uptake by 30%, whereas cortisol release was inhibited by 30%. 3H-PAH uptake was cis-inhibited by 1 mM glutarate or by 1 mM cortisol in the medium, while cortisol release was trans-stimulated by glutarate. PAH in the incubation medium showed saturable cis-inhibition of 3H-PAH uptake. The release of cyclic adenosine monophosphate, a substrate of the renal PAH exchanger, was also inhibited by probenecid and trans-stimulated by glutarate. In summary, the trans-stimulation and cis-inhibition experiments support the concept of an anion exchanger involved in cortisol and cyclic adenosine monophosphate release from and PAH uptake into adrenocortical cells.

Expression cloning and characterization of a novel sodium-dicarboxylate cotransporter from winter flounder kidney.

A cDNA coding for a Na+-dicarboxylate cotransporter, fNaDC-3, from winter flounder (Pseudopleuronectes americanus) kidney was isolated by functional expression in Xenopus laevis oocytes. The fNaDC-3 cDNA is 2384 nucleotides long and encodes a protein of 601 amino acids with a calculated molecular mass of 66.4 kDa. Secondary structure analysis predicts at least eight membrane-spanning domains. Transport of succinate by fNaDC-3 was sodium-dependent, could be inhibited by lithium, and evoked an inward current. The apparent affinity constant (Km) of fNaDC-3 for succinate of 30 microM resembles that of Na+-dicarboxylate transport in the basolateral membrane of mammalian renal proximal tubules. The substrates specific for the basolateral transporter, 2,3-dimethylsuccinate and cis-aconitate, not only inhibited succinate uptake but also evoked inward currents, proving that they are transported by fNaDC-3. Succinate transport via fNaDC-3 decreased by lowering pH, as did citrate transport, although much more moderately. These characteristics suggest that fNaDC-3 is a new type of Na+-dicarboxylate transporter that most likely corresponds to the Na+-dicarboxylate cotransporter in the basolateral membrane of mammalian renal proximal tubules.

Chloride conductance in HT29 cells: investigations with apical membrane vesicles and RT-PCR.

Vesicles enriched in a marker enzyme for apical membranes were isolated from HT29 cells. These vesicles contain an anion conductance with the selectivity gluconate approximately sulphate4, 4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS)>glibenclamide. The Cl- conductance was insensitive to Ca2+ and to extravesicular cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and inosine 5'-triphosphate (ITP). Using the reverse transcription polymerase chain reaction (RT-PCR) technique and sequencing of the amplified products we detected messenger ribonucleic acid (mRNA) for the cystic fibrosis transmembrane conductance regulator (CFTR), the putative Cl- channel or Cl- channel regulator pICln, and the Cl- channels ClC-2, ClC-3, ClC-5 and ClC-6 in HT29 cells. The properties of the vesicles' Cl- conductance resemble those of the intermediate conductance outwardly rectifying Cl- channel and tentatively exclude contributions of CFTR, pICln and ClC-2. Whether ClC-3, ClC-5, ClC-6 are involved in Cl- conductance remains to be determined.

Adenosine inhibits the transfected Na+-H+ exchanger NHE3 in Xenopus laevis renal epithelial cells (A6/C1).

1. Adenosine influences the vectorial transport of Na+ and HCO3- across kidney epithelial cells. However, its action on effector proteins, such as the Na+-H+ exchanger NHE3, an epithelial brush border isoform of the Na+-H+ exchanger (NHE) gene family, is not yet defined. 2. The present study was conducted in Xenopus laevis distal nephron A6 epithelia which express both an apical adenosine receptor of the A1 type (coupled to protein kinase C (PKC)) and a basolateral receptor of the A2 type (coupled to protein kinase A (PKA)). The untransfected A6 cell line expresses a single NHE type (XNHE) which is restricted to the basolateral membrane and which is activated by PKA. 3. A6 cell lines were generated which express exogenous rat NHE3. Measurements of side-specific pHi recovery from acid loads in the presence of HOE694 (an inhibitor with differential potency towards individual NHE isoforms) detected an apical resistant Na+-H+ exchange only in transfected cell lines. The sensitivity of the basolateral NHE to HOE694 was unchanged, suggesting that exogenous NHE3 was restricted to the apical membrane. 4. Stimulation of the apical A1 receptor with N 6-cyclopentyladenosine (CPA) inhibited both apical NHE3 and basolateral XNHE. These effects were mimicked by the addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and partially prevented by the PKC inhibitor calphostin C which also blocked the effect of PMA. 5. Stimulation of the basolateral A2 receptor with CPA inhibited apical NHE3 and stimulated basolateral XNHE. These effects were mimicked by 8-bromo-cAMP and partially prevented by the PKA inhibitor H89 which entirely blocked the effect of 8-bromo-cAMP. 6. In conclusion, CPA inhibits rat NHE3 expressed apically in A6 epithelia via both the apical PKC-coupled A1 and the basolateral PKA-coupled A2 adenosine receptors.

NH4+ conductance in Xenopus laevis oocytes.II. Effect Of hypoosmolality.

Superfusing Xenopus laevis oocytes with NH4Cl (10 mmol/l, pH 7.5) resulted in an inward current at a clamp potential of -70 mV. In paired experiments (n=22), the NH4Cl-induced peak current was -293+/-94 nA, under control conditions (osmolality: 240 mosmol/kg), and rose to -523+/-196 nA when osmolality was reduced to 144 mosmol/kg. In parallel with the rise in NH4Cl-induced inward current, membrane conductance at -70 mV doubled and the zero-current potential changed from +3.3+/-9.4 mV to -22.0+/-8.0 mV (n=22) in the presence of NH4Cl during exposure to a hypoosmolar solution. In the absence of NH4Cl, oocytes responded to hypoosmolality with a shift in zero-current potential to more negative values and an increased conductance which became partially sensitive to isosorbiddinitrate (ISDN), suggesting the activation of a volume-sensitive K+ channel. Membrane conductance in the presence of NH4Cl was decreased by ISDN to similar extents under isoosmolal and hypoosmolal conditions, indicating that NH4+ enters the oocytes through a volume-sensitive conductance separate from the ISDN-sensitive K+ channel.