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The Na(+)/H(+) exchanger 3 (NHE3) is expressed in the brush border membrane (BBM) of proximal tubules (PT). Its activity is down-regulated on increases in intracellular cAMP levels. The aim of this study was to investigate the contribution of the protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC) dependent pathways in the regulation of NHE3 by adenosine 3',5'-cyclic monophosphate (cAMP). Opossum kidney cells and murine kidney slices were treated with cAMP analogs, which selectively activate either PKA or EPAC. Activation of either pathway resulted in an inhibition of NHE3 activity. The EPAC-induced effect was independent of PKA as indicated by the lack of activation of the kinase and the insensitivity to the PKA inhibitor H89. Both PKA and EPAC inhibited NHE3 activity without inducing changes in the expression of the transporter in BBM. Activation of PKA, but not of EPAC, led to an increase of NHE3 phosphorylation. In contrast, activation of PKA, but not of EPAC, inhibited renal type IIa Na(+)-coupled inorganic phosphate cotransporter (NaPi-IIa), another Na-dependent transporter expressed in proximal BBM. PKA, but not EPAC, induced the retrieval of NaPi-IIa from BBM. Our results suggest that EPAC activation may represent a previously unrecognized mechanism involved in the cAMP regulation of NHE3, whereas regulation of NaPi-IIa is mediated by PKA but not by EPAC.

Adult dragonflies augment their compound eyes with three simple eyes known as the dorsal ocelli. While the ocellar system is known to mediate stabilizing head reflexes during flight, the ability of the ocellar retina to dynamically resolve the environment is unknown. For the first time, we directly measured the angular sensitivities of the photoreceptors of the dragonfly median (middle) ocellus. We performed a second-order Wiener Kernel analysis of intracellular recordings of light-adapted photoreceptors. These were stimulated with one-dimensional horizontal or vertical patterns of concurrent UV and green light with different contrast levels and at different ambient temperatures. The photoreceptors were found to have anisotropic receptive fields with vertical and horizontal acceptance angles of 15 degrees and 28 degrees, respectively. The first-order (linear) temporal kernels contained significant undershoots whose amplitudes are invariant under changes in the contrast of the stimulus but significantly reduced at higher temperatures. The second-order kernels showed evidence of two distinct nonlinear components: a fast acting self-facilitation, which is dominant in the UV, followed by delayed self- and cross-inhibition of UV and green light responses. No facilitatory interactions between the UV and green light were found, indicating that facilitation of the green and UV responses occurs in isolated compartments. Inhibition between UV and green stimuli was present, indicating that inhibition occurs at a common point in the UV and green response pathways. We present a nonlinear cascade model (NLN) with initial stages consisting of separate UV and green pathways. Each pathway contains a fast facilitating nonlinearity coupled to a linear response. The linear response is described by an extended log-normal model, accounting for the phasic component. The final nonlinearity is composed of self-inhibition in the UV and green pathways and inhibition between these pathways. The model can largely predict the response of the photoreceptors to UV and green light.

The Na(+)/phosphate cotransporter NaPi-IIa (SLC34A1) is the major transporter mediating the reabsorption of P(i) in the proximal tubule. Expression and activity of NaPi-IIa is regulated by several factors, including parathyroid hormone, dopamine, metabolic acidosis, and dietary P(i) intake. Dopamine induces natriuresis and phosphaturia in vivo, and its actions on several Na(+)-transporting systems such as NHE3 and Na(+)-K(+)-ATPase have been investigated in detail. Using freshly isolated mouse kidney slices, perfused proximal tubules, and cultured renal epithelial cells, we examined the acute effects of dopamine on NaPi-IIa expression and localization. Incubation of isolated kidney slices with the selective D(1)-like receptor agonists fenoldopam (10 microM) and SKF-38393 (10 microM) for 1 h induced NaPi-IIa internalization and reduced expression of NaPi-IIa in the brush border membrane (BBM). The D(2)-like selective agonist quinpirole (1 microM) had no effect. The D(1) and D(2) agonists did not affect the renal Na(+)/sulfate cotransporter NaSi in the BBM of the proximal tubule. Studies with isolated perfused proximal tubules demonstrated that activation of luminal, but not basolateral, D(1)-like receptors caused NaPi-IIa internalization. In kidney slices, inhibition of PKC (1 microM chelerythrine) or ERK1/2 (20 microM PD-098089) pathways did not prevent the fenoldopam-induced internalization. Inhibition with the PKA blocker H-89 (10 microM) abolished the effect of fenoldopam. Immunoblot demonstrated a reduction of NaPi-IIa protein in BBMs from kidney slices treated with fenoldopam. Incubation of opossum kidney cells transfected with NaPi-IIa-green fluorescent protein chimera shifted fluorescence from the apical membrane to an intracellular pool. In summary, dopamine induces internalization of NaPi-IIa by activation of luminal D(1)-like receptors, an effect that is mediated by PKA.

During metabolic acidosis, P(i) serves as an important buffer to remove protons from the body. P(i) is released from bone together with carbonate buffering protons in blood. In addition, in the kidney, the fractional excretion of phosphate is increased allowing for the excretion of more acid equivalents in urine. The role of intestinal P(i) absorption in providing P(i) to buffer protons and compensating for loss from bone during metabolic acidosis has not been clarified yet. Inducing metabolic acidosis (NH(4)Cl in drinking water) for 2 or 7 days in mice increased urinary fractional P(i) excretion twofold, whereas serum P(i) levels were not altered. Na(+)-dependent P(i) transport in the small intestine, however, was stimulated from 1.89 +/- 3.22 to 40.72 +/- 11.98 pmol/mg protein (2 days of NH(4)Cl) in brush-border membrane vesicles prepared from total small intestine. Similarly, the protein abundance of the Na(+)-dependent phosphate cotransporter NaPi-IIb in the brush-border membrane was increased 5.3-fold, whereas mRNA levels remained stable. According to immunohistochemistry and real-time PCR NaPi-IIb expression was found to be mainly confined to the ileum in the small intestine, and this distribution was not altered during metabolic acidosis. These results suggest that the stimulation of intestinal P(i) absorption during metabolic acidosis may contribute to the buffering of acid equivalents by providing phosphate and may also help to prevent excessive liberation of phosphate from bone.

Inorganic phosphate (P(i)) is reabsorbed in the renal proximal tubule mainly via the type-IIa sodium-phosphate cotransporter (NaPi-IIa). This protein is regulated tightly by different factors, among them dietary P(i) intake and parathyroid hormone (PTH). A number of PDZ-domain-containing proteins have been shown to interact with NaPi-IIa in vitro, such as Na(+)/H(+) exchanger-3 regulatory factor-1 (NHERF1) and PDZK1. PDZK1 is highly abundant in kidney and co-localizes with NaPi-IIa in the brush border membrane of proximal tubules. Recently, a knock-out mouse model for PDZK1 (Pdzk1(-/-)) has been generated, allowing the role of PDZK1 in the expression and regulation of the NaPi-IIa cotransporter to be examined in in vivo and in ex vivo preparations. The localization of NaPi-IIa and other proteins interacting with PDZK1 in vitro [Na(+)/H(+) exchanger (NHE3), chloride-formate exchanger (CFEX)/putative anion transporter-1 (PAT1), NHERF1] was not altered in Pdzk1(-/-) mice. The abundance of NaPi-IIa adapted to acute and chronic changes in dietary P(i) intake, but steady-state levels of NaPi-IIa were reduced in Pdzk1(-/-) under a P(i) rich diet. This was paralleled by a higher urinary fractional P(i) excretion. The abundance of the anion exchanger CFEX/PAT1 (SLC26A6) was also reduced. In contrast, NHERF1 abundance increased in the brush border membrane of Pdzk1(-/-) mice fed a high-P(i) diet. Acute regulation of NaPi-IIa by PTH in vivo and by PTH and activators of protein kinases A, C and G (PKA, PKC and PKG) in vitro (kidney slice preparation) was not altered in Pdzk1(-/-) mice. In conclusion, loss of PDZK1 did not result in major changes in proximal tubule function or NaPi-IIa regulation. However, under a P(i)-rich diet, loss of PDZK1 reduced NaPi-IIa abundance indicating that PDZK1 may play a role in the trafficking or stability of NaPi-IIa under these conditions.

The morphological sensillum types on the antennae of male and female Cactoblastis cactorum were visualized by scanning electron microscopy. Electrophysiological recordings were performed for the first time on single olfactory sensilla of C. cactorum. The male sensilla trichodea house a receptor cell responding to the putative pheromone component (9Z,12E)-tetradecadienyl acetate. The sensilla trichodea of the females were much shorter than those of the males and contained specialized receptor cells responding to certain terpenoids, the most frequent being the nerolidol-sensitive cell. The sensilla auricillica and sensilla basiconica of both sexes contained cells responding less specifically to terpenoid compounds as well as to green leaf volatiles. Cells of the sensilla coeloconica responded to aliphatic aldehydes and acids. Eight volatile organic compounds emitted by Opuntia stricta, a host plant of C. cactorum, were identified using gas chromatography-mass spectrometry, beta-caryophyllene being the major compound. Five compounds identified by gas chromatography in the headspace of O. stricta elicited responses in olfactory receptor cells of C. cactorum, nonanal being the most active compound and therefore a candidate attractant of C. cactorum.

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693-705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V < or = -80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

The rat renal Na(+)/P(i) cotransporter (NaP(i)-IIa) contains 12 native cysteines. When individually replaced by a serine, none appears essential for proper expression and function. Nevertheless, the formation of one essential cysteine bridge (C5/C6), together with a postulated second bridge, is necessary. To determine the minimum cysteine residues required for functional NaP(i)-IIa, with the goal of generating a Cys-less backbone for structure-function studies, mutants were constructed in which multiple endogenous cysteines were replaced by serines in different combinations. In Xenopus oocytes, most mutants were functional, except those where cysteine pairs C4/C9, C4/C12 or C9/C12 were simultaneously deleted. This suggested that one of these pairs could form the second cysteine bridge essential for expression and/or protein function. Up to eight cysteines could therefore be removed to give a functional Cys-reduced NaP(i)-IIa with activity and kinetics comparable to the wild-type (WT). This construct, like all intermediate mutants and the WT, was insensitive to cysteine-modifying methanethiosulfonate (MTS) reagents. Moreover, by introducing a novel cysteine into the Cys-reduced NaP(i)-IIa at a site functionally important in the WT (Ser-460), the loss of transport function reported for mutant S460C, after exposure to MTS reagents, was recapitulated. This confirmed that the MTS reagent site of action was Cys-460 and that modification of native cysteines does not contribute to S460C behavior.

Two highly similar regions in the predicted first intracellular (ICL-1) and third extracellular loop (ECL-3) of the type IIa Na+/P(i) cotransporter (NaPi-IIa) have been shown previously to contain functionally important sites by applying the substituted cysteine accessibility method (SCAM). Incubation in methanethiosulfonate (MTS) reagents of mutants that contain novel cysteines in both loops led to full inhibition of cotransport activity. To elucidate further the role these regions play in defining the transport mechanism, a double mutant (A203C-S460C) was constructed with novel cysteines in each region. The effect of cysteine modification by different MTS reagents on two electrogenic transport modes (leak and cotransport) was investigated. MTSEA (2-aminoethyl MTS hydrobromide) and MTSES (MTS ethylsulfonate) led to full inhibition of cotransport and increased the leak, whereas incubation in MTSET (2-[trimethylammonium]ethyl MTS bromide) inhibited only cotransport. The behavior of other double mutants with a cysteine retained at one site and hydrophobic or hydrophilic residues substituted at the other site, indicated that most likely only Cys-460 was modifiable, but the residue at Ala-203 was critical for conferring the leak and cotransport mode behavior. Substrate interaction with the double mutant was unaffected by MTS exposure as the apparent P(i) and Na+ affinities for P(i)-induced currents and respective activation functions were unchanged after cysteine modification. This suggested that the modified site did not interfere with substrate recognition/binding, but prevents translocation of the fully loaded carrier. The time-dependency of cotransport loss and leak growth during modification of the double cysteine mutant was reciprocal, which suggested that the modified site is a kinetic codeterminant of both transport modes. The behavior is consistent with a kinetic model for NaPi-IIa that predicts mutual exclusiveness of both transport modes. Together, these findings suggest that parts of the opposing linker regions are associated with the NaPi-IIa transport pathway.

It is suggested that the dragonfly median ocellus is specifically adapted to detect horizontally extended features rather than merely changes in overall intensity. Evidence is presented from the optics, tapetal reflections and retinal ultrastructure. The underfocused ocelli of adult insects are generally incapable of resolving images. However, in the dragonfly median ocellus the geometry of the lens indicates that some image detail is present at the retina in the vertical dimension. Details in the horizontal dimension are blurred by the strongly astigmatic lens. In the excised eye the image of a point source forms a horizontal streak at the level of the retina. Tapetal reflections from the intact eye show that the field of view is not circular as in most other insects but elliptical with the major axis horizontal, and that resolution in the vertical direction is better than in the horizontal. Measurements of tapetal reflections in locust ocelli confirm their visual fields are wide and circular and their optics strongly underfocused. The ultrastructure suggests adaptation for resolution, sensitivity and a high metabolic rate, with long, widely separated rhabdoms, retinulae cupped by reflecting pigment, abundant tracheoles and mitochondria, and convoluted, amplified retinula cell plasma membranes.

The effects of the arginine-modifying reagent phenylglyoxal on the kinetics of the type IIa Na + /Pi cotransporter expressed in Xenopus, oocytes were studied by means of 32Pi uptake and electrophysiology. Phenylglyoxal incubation induced up to 60% loss of cotransport function but only marginally altered the Na+-leak. Substrate activation and pH dependency remained essentially unaltered, whereas the voltage dependency of Pi-induced change in electrogenic response was significantly reduced. Presteady-state charge movements were suppressed and the equilibrium charge distribution was shifted slightly towards hyperpolarizing potentials. Charge movements in the absence of external Na+ were also suppressed, which indicated that the empty-carrier kinetics were modified. These effects were incorporated into an ordered alternating access model for NaPi-IIa, whereby the arginine modification by phenylglyoxal was modeled as altered apparent electrical distances moved by mobile charges, together with a slower rate of translocation of the electroneutral, fully loaded carrier.

Intrasequence comparison of the type IIa Na(+)-P(i) cotransport protein revealed two regions with high similarity in the first intracellular (ICL-1) and third extracellular (ECL-3) loops. Because the ECL-3 loop contains functionally important sites that have been identified by cysteine scanning, we applied this method to corresponding sites in the ICL-1 loop. The accessibility of novel cysteines by methanethiosulfonate reagents was assayed electrophysiologically. Mutants N199C and V202C were fully inhibited after methanethiosulfonate ethylammonium exposure, whereas other mutants showed marginal reductions in cotransport function. None showed significant functional loss after exposure to impermeant methanethiosulfonate ethyltrimethylammonium, which suggested a sidedness of Cys modification. Compared with the wild-type (WT), mutant A203C showed altered Na(+) leak kinetics, whereas N199C exhibited decreased apparent substrate affinities. To delineate the role of residue N199 in conferring substrate affinity, other mutations at this site were made. Only two mutants yielded significant (32)P(i) uptake and inward P(i)-induced currents with decreased P(i) affinity; for the others, P(i) application suppressed only the Na(+) leak. We suggest that ICL-1 and ECL-3 sites contribute to the transport pathway and that site N199 is implicated in defining the transport mode.

The type IIa Na/Pi cotransporter mediates proximal tubular brush-border membrane secondary active phosphate (Pi) flux. It is rate limiting in tubular Pi reabsorption and, thus, a final target in many physiological and pathophysiological situations of altered renal Pi handling. In the present short review, we will briefly summarize our current knowledge about the transport mechanism (cycle) as well as particular regions of the transporter protein ("molecular domains") that potentially determine transport characteristics.

Purpose: The introduction of the FDT perimeter prompted the comparison of three tests employing frequency doubling (FD) stimuli. These measures compared different visual field locations and contrast ranges. Frequency of seeing curves were examined for the method most similar to FDT., Methods: For 146 eyes the following were obtained: (i) contrast matches to two suprathreshold FD stimuli (normal subjects, ocular hypertensve suspects, primary open angle glaucoma subjects); (ii) two alternative forced choice (2AFC) thresholds for horizontally versus vertically orientated FD gratings: and (iii) contrast thresholds determined by method of adjustment (MOA) for five different stimulus types., Results: A model based on the worst of the MOA hemifield thresholds performed best. The suprathreshold contrast matching tests performed worst. Frequency of seeing curves were fitted for the 146 eyes of the 2AFC tests. Although the MOA thresholds were higher than the 2AFC thresholds (for normals mean +/- SE, 8.47 +/- 0.43 dL, P < 0.0000), the best diagnostic concordance was at lower limens (75% or 80% correct) of the fitted frequency of seeing curves., Conclusions: There was good diagnostic concordance between the MOA and 2AFC methods although the thresholds were 1.8-fold different on a log-scale. This suggests that the same neural mechanism mediates both thresholds for rapidly flickering, spatially coarse, patterns.

The transport function of the rat type IIa Na(+)/P(i) cotransporter is inhibited after binding the cysteine modifying reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) to a cysteine residue substituted for a serine at position 460 (S460C) in the predicted third extracellular loop. This suggests that Ser-460 lies in a functionally important region of the protein. To establish a "structure-function" profile for the regions that flank Ser-460, the substituted cysteine accessibility method was employed. 18 mutants were constructed in which selected amino acids from Arg-437 through Leu-465 were substituted one by one for a cysteine. Mutants were expressed in Xenopus oocytes and transport function (cotransport and slippage) and kinetics were assayed by electrophysiology with or without prior treatment with cysteine modifying (methanethiosulfonate, MTS) reagents. Except for mutant I447C, mutants with cysteines at sites from Arg-437 through Thr-449, as well as Pro-461, were inactive. Cotransport function of mutants with Cys substitutions at sites Arg-462 through Leu-465 showed low sensitivity to MTS reagents. The preceding mutants (Cys substitution at Thr-451 to Ser-460) showed a periodic accessibility pattern that would be expected for an alpha-helix motif. Apart from loss of transport function, exposure of mutants A453C and A455C to MTSEA or 2-(triethylammonium)ethyl MTS bromide (MTSET) increased the uncoupled slippage current, which implicated the mutated sites in the leak pathway. Mutants from Ala-453 through Ala-459 showed less pH dependency, but generally stronger voltage dependency compared with the wild type, whereas those flanking this group were more sensitive to pH and showed weaker voltage dependence of cotransport mode kinetics. Our data indicate that parts of the third extracellular loop are involved in the translocation of the fully loaded carrier and show a membrane-associated alpha-helical structure.

Sensory structures that detect atmospheric carbon dioxide have been identified and described to the subcellular level in adults of Lepidoptera, Diptera, Hymenoptera, Isoptera, Chilopoda, and Ixodidae, as well as in lepidopteran larvae. The structures are usually composed of clusters of wall-pore type sensilla that may form distinct sensory organs, often recessed in pits or capsules. In insects, they are located on either the palps or the antennae, in chilopods on the head capsule, and in ixodids on the forelegs. In the two cases where the central projections have been examined (Lepidoptera and mosquitoes), the clustering is preserved to the level of second order neurons, which are located in the deutocerebrum. Individual sensilla usually contain a single receptor neuron that is sensitive to CO(2); it may be accompanied by other neurons that respond to other olfactory qualities. The distal dendritic processes of CO(2)-sensitive neurons invariably show an increased surface area, dividing into many cylindrical branches or into lamellar structures. Lamellar membranes are often closely linked to arrays of microtubules. Fine pore canal tubules are usually associated with the cuticular pores., (Copyright 1999 Wiley-Liss, Inc.)

The substituted cysteine accessibility approach, combined with chemical modification using membrane-impermeant alkylating reagents, was used to identify functionally important structural elements of the rat type IIa Na(+)/P(i) cotransporter protein. Single point mutants with different amino acids replaced by cysteines were made and the constructs expressed in Xenopus oocytes were tested for function by electrophysiology. Of the 15 mutants with substituted cysteines located at or near predicted membrane-spanning domains and associated linker regions, 6 displayed measurable transport function comparable to wild-type (WT) protein. Transport function of oocytes expressing WT protein was unchanged after exposure to the alkylating reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA, 100 microM), which indicated that native cysteines were inaccessible. However, for one of the mutants (S460C) that showed kinetic properties comparable with the WT, alkylation led to a complete suppression of P(i) transport. Alkylation in 100 mM Na(+) by either cationic ([2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), MTSEA) or anionic [sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES)] reagents suppressed the P(i) response equally well, whereas exposure to methanethiosulfonate (MTS) reagents in 0 mM Na(+) resulted in protection from the MTS effect at depolarized potentials. This indicated that accessibility to site 460 was dependent on the conformational state of the empty carrier. The slippage current remained after alkylation. Moreover, after alkylation, phosphonoformic acid and saturating P(i) suppressed the slippage current equally, which indicated that P(i) binding could occur without cotransport. Pre-steady state relaxations were partially suppressed and their kinetics were significantly faster after alkylation; nevertheless, the remaining charge movement was Na(+) dependent, consistent with an intact slippage pathway. Based on an alternating access model for type IIa Na(+)/P(i) cotransport, these results suggest that site 460 is located in a region involved in conformational changes of the empty carrier.

The importance of phosphatidylinositide 3- kinase(s) [PI 3-kinase(s)] in membrane trafficking processes led us to examine its/their possible role in parathyroid-hormone- (PTH-) induced endocytosis and lysosomal degradation of the type IIa Na/Pi-cotransporter in opossum kidney cells (OK-cells). We used wortmannin, a potent inhibitor of several mammalian PI 3-kinase isoforms, and measured Na/Pi-cotransporter activity and type IIa Na/Pi-cotransporter protein expression; also the induction of a negative dominant subunit (Deltap85) was used to reduce PI 3-kinase activity. Wortmannin and Deltap85 led to a reduction of Na/Pi-cotransport activity but were unable to prevent its inhibition by PTH. Wortmannin led in a dose- and time-dependent manner to a reduction of Na/Pi-cotransport activity and transporter protein expression, and retarded their recovery from PTH-induced inhibition/degradation. The data suggest that a PI 3-kinase "controlled" mechanism is involved in the synthesis (and/or routing) of the apical type IIa Na/Pi-cotransporter in OK-cells.

1. The rate of inorganic phosphate (Pi) reabsorption in the mammalian kidney is determined by the amount of type II sodium-coupled inorganic phosphate (Na+-Pi) cotransport protein present in the brush border membrane. Under physiological conditions, parathyroid hormone (PTH) leads to an inhibition of Na+-Pi cotransport activity, most probably mediated by the protein kinase A (PKA) and/or C (PKC) pathways. 2. In this study, PKC-induced inhibition of type II Na+-Pi cotransport activity was characterized in Xenopus laevis oocytes using electrophysiological and immunodetection techniques. Transport function was quantified in terms of Pi-activated current. 3. Oocytes expressing the type IIa rat renal, type IIb flounder renal or type IIb mouse intestinal Na+-Pi cotransporters lost > 50 % of Pi-activated transport function when exposed to the PKC activators DOG (1,2-dioctanoyl-sn-glycerol) or PMA (phorbol 12-myristate 13-acetate). DOG-induced inhibition was partially reduced with the PKC inhibitors staurosporine and bisindolylmaleimide I. Oocytes exposed to the inactive phorbol ester 4alpha-PDD (4alpha-phorbol 12,13-didecanoate) showed no significant loss of cotransporter function. 4. Oocytes expressing the rat renal Na+-SO42- cotransporter alone, or coexpressing this with the type IIa rat renal Na+-Pi cotransporter, showed no downregulation of SO42--activated cotransport activity by DOG. 5. Steady-state and presteady-state voltage-dependent kinetics of type II Na+-Pi cotransporter function were unaffected by DOG. 6. DOG induced a decrease in membrane capacitance which indicated a reduction in membrane area, thereby providing evidence for PKC-mediated endocytosis. 7. Immunocytochemical studies showed a redistribution of type II Na+-Pi cotransporters from the oolemma to the submembrane region after DOG treatment. Surface biotinylation confirmed a DOG-induced internalization of the transport protein. 8. These findings document a specific retrieval of exogenous type II Na+-Pi cotransporters induced by activation of a PKC pathway in the Xenopus oocyte.

Although noise in general can induce hearing loss, environmental noise represents an important risk for children, teenagers and young adults. Epidemiological investigations now support the occurrence of an increasing number of irreversible hearing losses in these groups. Major causes of hearing loss are toys (guns), explosives and electroacoustically amplified music delivered by head sets or heard in discotheques and open air concerts. Clinical indications are discussed.

The type II Na/Pi-cotransporter is expressed preferentially in renal proximal tubular epithelial cells. Comparison of the 5′ flanking region of the human NPT-2 gene with the opossum cell line (OK cell) and the murine Npt2 promoters revealed two conserved regions, one representing a putative C/EBP alpha site, the other a consensus TATA-box. In contrast to the OK cell and murine Npt-2 gene, the human exon 1 is flanked by two Alu-repeats, a short 90-bp inverted Alu element which is located within the promoter region and a full-length forward repeat present in intron 1. A 497-bp human promoter fragment including the inverted Alu-repeat was cloned in front of a luciferase reporter gene. The construct was active in OK and HeLa-S3 cells but no activity could be detected in the human monocyte cell line U937, the murine renal cortex cell line MCT and the dog kidney cell line MDCK. A twofold increase in promoter activity was observed in HeLa-S3 cells for a 5′ truncated fragment of 253 bp missing the inverted Alu-repeat. In the OK cell system the absence of the Alu-repeat was unable to modify promoter activity. In electrophoretic mobility shift assays (EMSAs) with a 31-bp oligonucleotide representing the conserved region with homology to C/EBP alpha we could provide evidence for specific DNA/protein interactions with nuclear extracts derived from kidney and liver cell lines but not for HeLa-S3 and U937 nuclear extracts. Specific interactions could also be observed with nuclear extracts from renal cortex, medulla and rat liver but not from rat spleen, intestine and heart. Southern-Western blotting techniques suggest that a 31-kDa nuclear protein from kidney-derived cells binds to the C/EBP-like region of the NPT2 promoter.

We have studied the involvement of proteolytic pathways in the regulation of the Na/Pi cotransporter type II by parathyroid hormone (PTH) in opossum kidney cells. Inhibition of lysosomal degradation (by leupeptin, ammonium chloride, methylamine, chloroquine, L-methionine methyl ester) prevented the PTH-mediated degradation of the transporter, whereas inhibition of the proteasomal pathway (by lactacystin) did not. Moreover it was found (i) that whereas lysosomal inhibitors prevented the PTH-mediated degradation of the transporter they did not prevent the PTH-mediated inhibition of the Na/Pi cotransport and (ii) that treating opossum kidney cells with lysosomal inhibitors led to an increased expression of the transporter without any concomitant increase in the Na/Pi cotransport. Further analysis by subcellular fractionation and morphological techniques showed (i) that the Na/Pi cotransporter is constitutively transported to and degraded within late endosomes/lysosomes and (ii) that PTH leads to the increased degradation of the transporter in late endosomes/lysosomes.

In this study, we present a new method to obtain pure, viable, freshly isolated hepatic stellate cells. Stellate cells were purified by cell sorting using their high side scatter (SSC) of incident light. Purity of the cells was established by light and transmission electron microscopy (TEM). Starting from stellate cells that were 50% to 70% enriched by centrifugation in 11% Nycodenz, the cell purity after sorting was found to be 96.6% +/- 2.9%. Viability of the sorted cells was 90.8% +/- 2.2% as measured by the Trypan blue exclusion test and was confirmed by cell culturing. Per hour of sorting, 1.4 +/- 0.4 million stellate cells were obtained. Sorting runs of up to 4 hours were practically feasible, resulting in yields of 5 to 6 million cells per rat liver. Cells attached to plastic substratum within 24 hours. Subsequently, they spread and underwent spontaneous transition into myofibroblast-like cells. The purity of sorted cells was documented by reverse-transcriptase polymerase chain reaction (RT-PCR) experiments using specific primer pairs for messenger RNA (mRNA) species that were only present in parenchymal (preproalbumin), endothelial (endothelial cell nitric oxide synthase [eNOS]), stellate (desmin), or Kupffer cells (77- to 88-kd fucose receptor). Contaminating mRNA species were absent in sorted stellate cells. Next, we examined freshly sorted stellate cells by Western blotting to confirm the presence of relevant cytoskeletal proteins. Cells were positive for vimentin, desmin, and glial fibrillary acidic protein (GFAP), but negative for alpha-smooth muscle actin (alpha-SMA). Sorted and cultured cells were immunophenotyped for the presence of collagen types I, III, and IV, laminin, and the cytoskeletal proteins, alpha-SMA, desmin, vimentin, and GFAP. At 90 hours in culture, cells expressed all the investigated extracellular matrix proteins. Desmin was present in 82% +/- 1%, vimentin in 96% +/- 2.5%, and GFAP in 91% +/- 4.5% of cells. Alpha-SMA was present in 91% +/- 2% of cultured cells. We conclude that cell sorting based on SSC of incident light is a convenient method to obtain virtually pure stellate cells that can be used for direct analysis or for culturing. Although the yields obtained with this method are lower than with standard methods, and additional equipment is required, SSC-activated sorting offers the possibility of very pure cells when essential for analyses based on sensitive detection methods such as RT-PCR.

The renal type II Na-Pi cotransport is the rate-limiting step in proximal tubular phosphate (Pi) reabsorption. Among the different "proximal tubular" cell lines, this transporter seem only to be expressed in opossum kidney cells (OK cells). We have isolated the 5'-flanking region of the ok-Npt2 gene (OK cell type II Na-Pi cotransporter) including exons 1-3 and containing a TFIID site (TATA box), a GCCAAT site, an AP1 site, and two microsatellite GGAA repeats. Major transcription initiation sites were determined by primer extension and rapid amplification of 5' cDNA ends (5'-RACE). A 327-bp fragment containing the TFIID and GCAAT element was driving the downstream luciferase reporter gene in homologous transfection assays. Slightly reduced promoter activity was observed with a 198-bp fragment containing the GCAAT element; shorter fragments were without activity. Promoter activity (327-bp fragment) could also be observed in transfections into HeLa cells but not in U937 human macrophage cells, MCT mouse kidney cortex cells, and MDCK cells. Different "physiological" stimuli known to be associated with altered proximal tubular Na-Pi cotransport activity are without effect on transcriptional activity in above homologous transfection experiments.

Sensory organs that detect CO 2 are common in herbivorous moths and butterflies, but their function has been unclear until now. As the CO 2 gradients in the vicinity of a host plant depend on its physiological condition, CO 2 could provide a sensory cue for the suitability of the plant as a larval food source. This study investigated whether changing the atmospheric CO 2 concentration affected oviposition by Cactoblastis cactorum on its host, the cactus Opuntia stricta. On host plants exposed to rapid fluctuations in CO 2 concentration, the frequency of oviposition was reduced by a factor of 3.2 compared to the control. As the fluctuations mask the much smaller CO 2 signals generated by the plants, this suggests that those signals constitute an important component of the host identification process. On host plants exposed to a constant background of doubled CO 2 , oviposition was also reduced, by a factor of 1.8. An increased background reduces host signal detectability, partially as a consequence of a general principle of sensory physiology (Weber-Fechner's law), and partially due to other factors specific to CO 2 -receptor neurons.

The effect of volatile anaesthetics such as N2O, Xe, short-chain alkanes and cyclopropane, at pharmacologically relevant concentrations, on olfactory receptor neurons of insects was tested in electrophysiological recordings. CO2-receptor neurons in moths and flies respond with increased action potential activity, whereas in honeybees the effect is inhibitory. With increasing chain length of the alkanes, the effectiveness increases initially, in adherence to the Meyer-Overton rule; alkanes of a chain length of 5 and above are less effective or evoke suppression of action potentials. In olfactory receptor neurons sensitive to benzoic acid in female moths of Bombyx mori and in pheromone receptor neurons of male moths of Antheraea polyphemus, anaesthetics are ineffective if applied alone; if superimposed on an excitatory olfactory stimulus, an inhibitory effect occurs. Local stimulation of only part of a sensory dendrite reveals that the anaesthetics are effective only if applied at the same location as the excitatory stimulus. This indicates that the anaesthetics reversibly block the reception of pheromone or its effect on the conductance of the receptor cell membrane. The observed interactions are consistent with the hypothesis that the anaesthetics do not interact with the primary transduction process, but rather affect a later stage such as the activation of ion channels.

The interaction between the moth, Cactoblastis cactorum, and the cactus, Opuntia stricta, is used as a model to examine the question of whether the CO 2 sense of a herbivorous insect can detect the CO 2 gradients associated with a plant's metabolic activity. Both the anatomical and the electrophysiological characteristics of CO 2 -sensitive receptor neurons in C. cactorum indicate an adaptation to the detection of small fluctuations around the atmospheric background. Evidence is provided that further rises in background will impair the function of the sensory organ. In the habitat of the plant, during the diurnal window of the moth's activity, two types of CO 2 gradients occur that are detectable by the moth's sensors. The first gradient, associated with soil respiration, is vertical and extends from the soil surface to an altitude of approximately 1 m. Its magnitude is well above the detectability limit of the sensors. The notion that this gradient provides, to a flying insect, a cue for the maintenance of a flight altitude favourable for host detection is supported by field observations of behaviour. The second gradient, associated with CO 2 fixation by the plant, extends from the surfaces of photosynthetic organs (cladodes) over a boundary layer distance of approximately 5 mm. Again, its magnitude is well above the detectability limit. The notion that this gradient provides, to a walking insect, a cue to the physiological condition of the plant is supported by the observation that females of C. cactorum, prior to oviposition, actively probe the plant surface with their CO 2 sensors. In a simulation of probing, pronounced responses of the sensors to the CO 2 -fixing capacity of O. stricta are observed. We propose that by probing the boundary layer, females of C. cactorum can detect the healthiest, most active O. stricta cladodes, accounting for earlier observations that the most vigorous plants attract the greatest density of egg sticks.

We have isolated a cDNA (ileal NaSi-1) from rat small intestine by homology screening with a cDNA (renal NaSi-1) encoding rat kidney cortex Na(+)-SO4(2-) cotransport. Ileal NaSi-1 cRNA specifically stimulates Na(+)-dependent SO4(2-) uptake in a time- and dose-dependent manner in Xenopus laevis oocytes, with kinetic parameters almost identical to those of the renal NaSi-1. Ileal NaSi-1 cDNA contains 2722 base pairs (bp), almost 500 bp more than the renal NaSi-1 cDNA; however, it encodes a protein of 595 amino acids identical to the renal NaSi-1 protein. Northern blot analysis shows strong signals in rat lower small intestine and kidney cortex (2.9 x 10(3) and 2.3 x 10(3) bases), with the ileal NaSi-1 corresponding to the longer transcript. We conclude that we have identified a rat ileal cDNA that encodes a membrane protein most likely involved in brush-border Na(+)-SO4(2-) cotransport. It differs to the renal NaSi-1 only in the length of the 3' untranslated region, suggesting that the major difference lies in the differential use of polyadenylation signals.

Recently, we have isolated a complementary DNA most likely related to rabbit kidney cortex brush border membrane sodium/phosphate (Na/Pi) cotransport activity [NaPi-1 (1)]. To further elucidate the cellular mechanisms involved in dietary 'adaptation' of renal Na/Pi cotransport, we have exposed young rabbits for 2 weeks to either a low phosphate (Pi) diet (LPD) or a high Pi diet (HPD). Initial linear uptake of Na/Pi cotransport in isolated brush border membrane vesicles was increased in rabbits on a LPD compared with those on a HPD. Injection of equal amounts of total mRNA isolated from kidney cortex of LPD or HPD rabbits into Xenopus laevis oocytes resulted in a higher stimulation of Na-dependent oocyte Pi uptake in LPD than HPD preparations. No difference in the content of 'specific' mRNA (NaPi-1 cDNA probe, Northern blots) and of the content of the 'specific' brush border membrane protein (NaPi-1 antipeptide antibody, Western blots) between LPD and HPD preparations was observed. We conclude that 'chronic' dietary Pi deprivation leads to a protein synthesis-dependent alteration of Na/Pi cotransport activity which does not involve a change in the total amount of a protein related to the recently cloned NaPi-1 protein.

A technique is described and tested for mapping the sensitivities and preferred directions of motion at different locations within the receptive fields of direction-selective motion-detecting visual neurons. The procedure is to record the responses to a number of visual stimuli, each stimulus presentation consisting of a set of short, randomly-oriented, moving bars arranged in a square grid. Each bar moves perpendicularly to its long axis. The vector describing the sensitivity and preferred direction of motion at each grid location is obtained as a sum of the unit vectors defining the directions of motion of the bars in each of the stimuli at that location, weighted by the strengths of the corresponding responses. The resulting vector field specifies the optimum flow field for the neuron. The advantage of this technique over the conventional approach of probing the receptive field sequentially at each grid location is that the parallel nature of the stimulus is sensitive to nonlinear interactions (such as shunting inhibition for mutual facilitation) between different regions of the visual field. The technique is used to determine accurately the motion receptive fields of direction-selective motion detecting neurons in the optic lobes of insects. It is potentially applicable to motion-sensitive neurons with highly structured receptive fields, such as those in the optic tectum of the pigeon or in area MST of the monkey.

An algorithm is derived which enables the range of an object to be measured by passive optical means. It is shown that the distance of an object from an observation point is given by the ratio of the angular intensity gradient to the translational intensity gradient of a segment of the object's intensity profile, as seen from the observation point. The algorithm is implemented by two rows of photodetectors with angular sensitivity functions g(alpha) and g'(alpha), positioned in pairs along a baseline, at equal distances and with parallel optical axes. Weighted sums of the outputs are formed for each row, the weights being defined by a pair of functions of position on the baseline h'(T) and h(T). The ratio of the two sums is proportional to distance, independent of background intensity and contrast. A device based on this principle, using simple analog electronics and commercially available photodetectors, is described, and its performance is evaluated.

Parathyroid hormone (PTH) controls two proximal tubular brush border membrane transport systems, Na+/phosphate co-transport and Na+/H+ exchange. In OK cells, a cell line with proximal tubular transport characteristics, PTH acts via kinase C and kinase A activation to inhibit Na+/phosphate co-transport [6, 8, 9, 19, 22]. In the present study, we show that PTH inhibits Na+/H+ exchange and that this effect can be mimicked by pharmacological activation of kinase A and kinase C. Ionomycin-dependent increases in cytoplasmic Ca2+ concentration do not induce inhibition of Na+/H+ exchange; PTH-dependent inhibition of Na+/H+ exchange is not prevented by ionomycin or by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (Ca2+ clamping). Detailed dose-response curves for the different agonists, given either alone or in combination, suggest that the two regulatory cascades (kinase A and kinase C) are operating independent of each other and reach a common final target, resulting in 40-50% inhibition of Na+/H+ exchange. An analysis of intracellular pH sensitivity of Na+/H+ exchange suggests that inhibition is not related to a shift in set point, but is rather explained by a reduced Vmax of Na+/H+ exchange and/or reduced affinity for protons at the internal membrane surface. It is suggested that kinase A as well as kinase C can mediate PTH inhibition of renal proximal tubular Na+/H+ exchange and that the relative importance of a particular regulatory cascade is determined by the PTH-concentration-dependent rates in the liberation of diacylglycerol (phospholipase C/kinase C) and cAMP (adenylate cyclase/kinase A).

The inclusion of ultra-sound in the routine diagnosis of sinus disease reduces the margin of error by about 6%, compared to that using clinical records and x-ray. The investigator should have sufficient experience in operating the ultrasonic equipment. Especially valuable is the ultrasonic diagnosis in cases of discharge, mucosal swelling or tumor in the sinus, as well as in cases of sinus examination of children and for control of patients having had conservative treatment or operations.

L-3H-lysine uptake into brush border membrane vesicles was measured by a rapid filtration technique. A significant binding of L-lysine at the vesicle interior was observed. Extrapolating initial linear uptake to zero incubation time did not indicate binding of the amino acid to the external membrane surface. Sodium stimulated the L-lysine uptake specifically. Experiments in the presence of potassium/valinomycin induced diffusion potentials, and experiments with a potential sensitive fluorescent dye documented an electrogenic uptake mechanism for L-lysine only in the presence of sodium. Sodium independent uptake proceeds via an electroneutral pathway. Transstimulation experiments show carrier mediated uptake in the presence and absence of sodium. An outwardly directed proton-gradient stimulated L-lysine uptake in the presence and absence of sodium. Saturation of L-lysine uptake was observed in the presence and absence of sodium. In the absence of sodium, L-lysine uptake was inhibited by L-arginine, L-cystine, L-phenylalanine and L-methionine. The sodium dependent uptake was inhibited by L-arginine and L-cysteine; small inhibition by L-phenylalanine was observed. In the presence or absence of sodium, L-lysine uptake was inhibited neither by D-lysine nor by L-glutamic acid. These results document carrier mediated transport of L-lysine via (a) transport mechanism(s) not obligatory requiring sodium.

Atrophy of the small intestinal mucosa is functionally characterized by a reduction in non-electrolyte transport in vivo. In order to elucidate the cellular defect being responsible for this malabsorption, we have studied the Na+-dependent D-glucose accumulation as well as the activities of aminopeptidase M and maltase in brush border membrane vesicles prepared from jejunal self-emptying blind loops and corresponding intestinal segments of sham-operated control rats. Membrane vesicles from atrophic mucosa did not show any differences in D-glucose uptake or in enzyme activities when compared with those derived from normal intestine. Thus it is unlikely that the impaired non-electrolyte absorption in the atrophic mucosa in vivo is due to a defect in cellular transport processes. It is more probable that the functional impairment is the result of the diminished absorptive surface in this pathophysiological condition.

The activity of single motor units of the splenius-capitis muscle of healthy human subjects and patients with unilateral vestibular lesions was recorded by means of microelectrodes. The major part of these units reacted to vestibular stimulation in the same way as primary and secondary vestibular neurones of other vertebrates. The reactions of these units to trapezoidale stimuli is identical to that of vestibular neurones. An angular acceleration of more than 0.6 degrees/s2 is for these units a stimulus which is above threshold, this too corresponds to findings in the vestibular nerve. In patients with unilateral vestibular lesions the threshold for rotatory stimuli is markedly increased on the side of the lesion, whereas the threshold on the other side remains constant. This method is very sensitive and vestibular lesions can be detected which cannot be seen using the common technics for vestibular examination.

At twenty-three 20-30-year-old normal-hearing subjects the characteristic curves of amplitudes and latencies of the five early potentials, appearing within the first 8 ms after stimulus onset, were obtained. The potentials were registered with the earlobe-vertex pick-up, as described by Sohmer and with click stumulus rates of 5/sec, 10/sec and 100/sec; It could be seen: All five potentials depend in amplitude and latency on intensity; all potentials show with increasing click rate an adaptation and a delayed latency, when increasing the click rate from 10/sec to 50/sec; the adaptation phenomenon of the 1st, 2nd, 3rd and 4th potential is the most prominent, the 4th potential shows the smallest adaptation effect; the characteristic curve at 10/sec of the 1st and 3rd potential demonstrates a break at 70 dB SPL; the latencies of the five potentials begin to increase at click rates higher than 10/sec; the 4th potential seems to be the most suitable one for measuring the acoustical threshold; only the fifth potential diminishes in amplitude and increases in latency after administration of an anaesthetic, but an additional relaxant does not alter it any more.

The tinnitus on moist tubotympanical catarrh and otosclerosis had been registered before and after the operation in relation to the frequence and the audibility. 222 children ears and 55 adult with a moist tubotympanical catarrh had been studied. Of these 28 children 26 adult ears had a tinnitus. After the operation and after using a "Donaldson silicone Drain Tube" without relation of the tinnitus there had been reached a very good win of hearing and excepted for 6 adults, whose tinnitus only had been lower, and elimination of the tinnitus. In 65 patients from 71 patients with stapedectomy it was able to look out for the tinnitus before and after the operation. Before the operation 44 patients had no tinnitus and 21 patients had a very disagreeable tinnitus; 17 patients from these had after the stapedectomy no longer a tinnitus. The audibility of the tinnitus from 4 patients had been reduced very much. The result of hearing the management of the inner ear after the operation haven't had any relation to the tinnitus before the operation.

At a female patient suffering from multiple sclerosis with demyelinisation mainly in the pontine and mesencephalic tegmentum, the five early potentials consisting of the compound action potential of the acoustic nerve (Pot. I) and the four brainstem potentials (Pot. II-V) as well as the slow evoked vertex-potential were registered. The threshold behaviour, the characteristic curve and the latency curve of all potentials were obtained, and thereby damages of the central acoustic pathways found out. A topodiagnostic is tried by means of the alterations of the peripheral and central ERA, and by means of the informations described in the literature. A control registration, carried out four weeks later, demonstrated an improvement of the diagnosis.

Uptake of SO(4) (2-) into brush-border membrane vesicles isolated from rat kindey cortex by a Ca(2+)-precipitation method was investigated by using a rapid-filtration technique. Uptake of SO(4) (2-) by the vesicles was osmotically sensitive and represented transport into an intra-vesicular space. Transport of SO(4) (2-) by brush-border membranes was stimulated in the presence of Na(+), compared with the presence of K(+) or other univalent cations. A typical ;overshoot' phenomenon was observed in the presence of an NaCl gradient (100mm-Na(+) outside/zero mm-Na(+) inside). Radioactive-SO(4) (2-) exchange was faster in the presence of Na(+) than in the presence of K(+). Addition of gramicidin-D, an ionophore for univalent cations, decreased the Na(+)-gradient-driven SO(4) (2-) uptake. SO(4) (2-) uptake was only saturable in the presence of Na(+). Counter-transport of Na(+)-dependent SO(4) (2-) transport was shown with MoO(4) (2-) and S(2)O(3) (2-), but not with PO(4) (2-). Changing the electrical potential difference across the vesicle membrane by establishing different diffusion potentials (anion replacement; K(+) gradient+/-valinomycin) was not able to alter Na(+)-dependent SO(4) (2-) uptake. The experiments indicate the presence of an electroneutral Na(+)/SO(4) (2-)-co-transport system in brush-border membrane vesicles isolated from rat kidney cortex.

By employing ultrasound in the routine diagnosis of sinus diseases, the margin of error in the accuracy of diagnosis can be reduced by 10% compared to that using clinical records and x-rays, provided however, that the investigator has sufficient experience in operating the ultrasonic equipment. The inclusion of ultrasonic diagnostics has proven itself particularly valuable in the diagnosis if both discharges and tumors in children and for progress control of patients having had conservative treatment or operations.

Brush border membranes were isolated from rat renal cortex by a divalent cation precipitation method. L-35S-cystine uptake into the vesicles was measured by a rapid filtration method. Covalent incorporation of tracer into membrane proteins was observed after prolonged incubations. At short incubation periods (1 min) binding was small and allowed an analysis of transmembrane transport. To guarantee transport of L-cystine, the experiments were performed in the presence of the oxidant diamide. Sodium stimulated L-cystine uptake specifically. A potassium/valinomycin induced inside negative diffusion potential stimulated sodium dependent L-cystine transport. Thus, transport is potential sensitive in the presence of sodium. At low substrate and inhibitor concentrations, L-cystine transport was inhibited by L-lysine, L-ornithine and L-arginine but not by D-lysine in the presence and absence of sodium. At higher inhibitor concentration, the neutral amino acids L-phenylalanine and L-leucine also inhibited L-cystine uptake, but only the sodium dependent uptake. These inhibition experiments suggest that L-cystine is transported by the brush border membrane by a transport system for basic amino acids not necessarily requiring sodium. In addition, transport of L-cystine can also proceed via sodium dependent transport pathways for neutral amino acids. In the concentration range tested (up to 0.225 mmoles/l), no saturation of L-cystine transport was observed in the presence and absence of sodium.

Uptake of taurocholate into brush-border membrane vesicles isolated from rat small intestine by a Ca(2+) -precipitation method was investigated by using a rapid-filtration technique. Uptake of taurocholate by ileal brush-border membranes consisted of three phenomena: binding to the outside of the vesicles, transfer across the vesicle membrane and binding to the intravesicular compartment. The transport of taurocholate across the brush-border membranes was stimulated in the presence of Na(+) compared with the presence of K(+); stimulation was about 11-fold in the presence of a NaCl gradient (Na(o)>Na(i)), where the subscripts refer to ;outside' and ;inside' respectively, and 4-fold under equilibrium conditions for Na(+) (Na(o)=Na(i)). In the presence of a Na(+) gradient a typical ;overshoot' phenomenon was observed. Membranes preloaded with unlabelled taurocholate showed an accelerated entry of labelled taurocholate (tracer exchange) in the presence of Na(+) compared with the presence of K(+). The stimulation by Na(+) was observed only in membrane preparations from the ileum. Addition of monactin, an ionophore for univalent cations, decreased the Na(+)-gradient-driven taurocholate uptake. The Na(+)-dependent taurocholate transport showed saturation kinetics and the phenomenon of counterflow and was inhibited by glycocholate. Other cations such as Li(+), Rb(+) and Cs(+) could not replace Na(+) in its stimulatory action. When the electrical potential difference across the vesicle membrane was altered by establishing different diffusion potentials (anion replacement; K(+) gradient+/-valinomycin) a more-negative potential inside stimulated Na(+)-dependent taurocholate transport. These data demonstrate the presence of a rheogenic (potential sensitive) Na(+)-taurocholate co-transport system in ileal brush-border membranes and support the hypothesis that the reabsorption of bile acids in the ileum is a secondary active uptake.

The effects of intravesicular NAD on Na+-dependent 32Pi uptake were investigated in isolated rat kidney brush border membrane vesicles (BBMV). NAD was introduced into the vesicles by osmotic shock, and extravesicular NAD was removed by passing the vesicles through a anion exchange column. The effectiveness of the osmotic shock procedure and the hydrolysis of extra- and intravesicular NAD were controlled by enzymatic analysis and thin layer chromatography. ADP-ribosylation of the membrane proteins was analyzed in vesicles osmotically shocked in the presence of either [adenylate-32P]-NAD or [adenine-2,8-3H]-NAD by SDS-polyacrylamide gel electrophoresis. It was found that the Na+-dependent Pi uptake was inhibited when the BBMV were incubated with NAD at alkaline pH, which resulted in rapid NAD hydrolysis. When NAD was present in the intravesicular space only, the Na+-dependent Pi uptake was not inhibited. 32P from NAD was rapidly incorporated into a number of brush border membrane proteins, but no incorporation of 3H-adenine could be detected. The results provide evidence that NAD does not inhibit Pi transport by a direct interaction with the cytoplasmic side of the brush border membrane. No evidence of ADP-ribosylation of the brush border membrane protein(s) was found.

The Na+/H+ exchange system was studied in brush border membrane vesicles isolated from cortical and medullary regions of the proximal tubule of rabbit kidney. The activity of the exchanger was assessed by measuring hydrogen influx (monitored by acridine orange fluorescence), 22 Na influx and the sensitivity of these fluxes to amiloride and its analogue ethylisopropyl amiloride. In contrast to previously published data (indicating the absence of pH-gradient driven and amiloride sensitive 22Na-influx in medullary site vesicles (13, 15], Na+/H+ exchange activity could be detected in both membrane preparations by sodium tracer and fluorescence detection of hydrogen influx. Amiloride inhibition of 22Na influx was more effectively protected by increasing sodium concentration in cortical than in medullary vesicles, suggesting differences in the action of amiloride in these preparations.

Infections of the middle and external ear caused by the problem-micro-organism Pseudomonas aeruginosa can be cured by local therapy with Ciprofloxacin and Tutofusin very quickly and without any complications. Drum ruptures caused by ear secretions close up again spontaneously. Tympanon tubes can be left in situ. Function disturbances of the middle and internal ear clear up and the functions return to normal.

Spike potentials were recorded from single, afferent fibres in the pigeon auditory nerve. Pure-tone stimuli were presented in quiet and in combination with wide band noise. Presented alone, tones produced tuned response areas; noise generally drove spike rate to well above the spontaneous rate measured in quiet. When presented in combination with noise, tones up to 75 dB SPL at frequencies far from the fibre's response area had no effect on the noise-driven spike rate. As the tone frequency was shifted towards the response area, from above or below CF, suppression of the noise-driven spike rate became stronger until the tone reached the edge of the response area. Suppression of the noise-driven rate was directly proportional to the level of the tone. Within the area of response to the tone, tone-driven spike rates generally were unchanged or variably decreased (occasionally slightly increased) by tone-on-noise stimulation, depending on the relation of the tone frequency to CF and the level of the tone relative to that of the noise. Tuning properties were unaffected. It is suggested that in the pigeon, the suppression of driven spike rate during presentation of combination stimuli, which is common to all fibres, depends on the same mechanism as the suppression of spontaneous firing by tones that is observed in a proportion of fibres (Temchin, A.N. (1988), J. Comp. Physiol. A 163, 99-115; Hill et al., (1989) Hear. Res. 39, 37-48).