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PubMed2006

Calbindin-D28K dynamically controls TRPV5-mediated Ca2+ transport.

In Ca(2+)-transporting epithelia, calbindin-D(28K) (CaBP(28K)) facilitates Ca(2+) diffusion from the luminal Ca(2+) entry side of the cell to the basolateral side, where Ca(2+) is extruded into the extracellular compartment. Simultaneously, CaBP(28K) provides protection against toxic high Ca(2+) levels by buffering the cytosolic Ca(2+) concentration ([Ca(2+)](i)) during high Ca(2+) influx. CaBP(28K) consistently colocalizes with the epithelial Ca(2+) channel TRPV5, which constitutes the apical entry step in renal Ca(2+)-transporting epithelial cells. Here, we demonstrate using protein-binding analysis, subcellular fractionation and evanescent-field microscopy that CaBP(28K) translocates towards the plasma membrane and directly associates with TRPV5 at a low [Ca(2+)](i). (45)Ca(2+) uptake measurements, electrophysiological recordings and transcellular Ca(2+) transport assays of lentivirus-infected primary rabbit connecting tubule/distal convolute tubule cells revealed that associated CaBP(28K) tightly buffers the flux of Ca(2+) entering the cell via TRPV5, facilitating high Ca(2+) transport rates by preventing channel inactivation. In summary, CaBP(28K) acts in Ca(2+)-transporting epithelia as a dynamic Ca(2+) buffer, regulating [Ca(2+)] in close vicinity to the TRPV5 pore by direct association with the channel.

Animals↗
PubMed2006

Positively charged C-terminal subdomains of EcoRV endonuclease: contributions to DNA binding, bending, and cleavage.

The carboxy-terminal subdomains of the homodimeric EcoRV restriction endonuclease each bear a net charge of +4 and are positioned on the inner concave surface of the 50 degree DNA bend that is induced by the enzyme. A complete kinetic and structural analysis of a truncated EcoRV mutant lacking these domains was performed to assess the importance of this diffuse charge in facilitating DNA binding, bending, and cleavage. At the level of formation of an enzyme-DNA complex, the association rate for the dimeric mutant enzyme was sharply decreased by 10(3)-fold, while the equilibrium dissociation constant was weakened by nearly 10(6)-fold compared with that of wild-type EcoRV. Thus, the C-terminal subdomains strongly stabilize the enzyme-DNA ground-state complex in which the DNA is known to be bent. Further, the extent of DNA bending as observed by fluorescence resonance energy transfer was also significantly decreased. The crystal structure of the truncated enzyme bound to DNA and calcium ions at 2.4 A resolution reveals that the global fold is preserved and suggests that a divalent metal ion crucial to catalysis is destabilized in the active site. This may explain the 100-fold decrease in the rate of metal-dependent phosphoryl transfer observed for the mutant. These results show that diffuse positive charge associated with the C-terminal subdomains of EcoRV plays a key role in DNA association, bending, and cleavage.

Binding Sites↗
PubMed2005

Submergence-induced morphological, anatomical, and biochemical responses in a terrestrial species affect gas diffusion resistance and photosynthetic performance.

Gas exchange between the plant and the environment is severely hampered when plants are submerged, leading to oxygen and energy deficits. A straightforward way to reduce these shortages of oxygen and carbohydrates would be continued photosynthesis under water, but this possibility has received only little attention. Here, we combine several techniques to investigate the consequences of anatomical and biochemical responses of the terrestrial species Rumex palustris to submergence for different aspects of photosynthesis under water. The orientation of the chloroplasts in submergence-acclimated leaves was toward the epidermis instead of the intercellular spaces, indicating that underwater CO(2) diffuses through the cuticle and epidermis. Interestingly, both the cuticle thickness and the epidermal cell wall thickness were significantly reduced upon submergence, suggesting a considerable decrease in diffusion resistance. This decrease in diffusion resistance greatly facilitated underwater photosynthesis, as indicated by higher underwater photosynthesis rates in submergence-acclimated leaves at all CO(2) concentrations investigated. The increased availability of internal CO(2) in these "aquatic" leaves reduced photorespiration, and furthermore reduced excitation pressure of the electron transport system and, thus, the risk of photodamage. Acclimation to submergence also altered photosynthesis biochemistry as reduced Rubisco contents were observed in aquatic leaves, indicating a lower carboxylation capacity. Electron transport capacity was also reduced in these leaves but not as strongly as the reduction in Rubisco, indicating a substantial increase of the ratio between electron transport and carboxylation capacity upon submergence. This novel finding suggests that this ratio may be less conservative than previously thought.

Acclimatization↗
PubMed2004

N-hexanoyl-sphingomyelin potentiates in vitro doxorubicin cytotoxicity by enhancing its cellular influx.

Anticancer drugs generally have intracellular targets, implicating transport over the plasma membrane. For amphiphilic agents, such as the anthracycline doxorubicin, this occurs by passive diffusion. We investigated whether exogenous membrane-permeable lipid analogues improve this drug influx. Combinations of drugs and lipid analogues were coadministered to cultured endothelial cells and various tumour cell lines, and subsequent drug accumulation in cells was quantified. We identified N-hexanoyl-sphingomyelin (SM) as a potent enhancer of drug uptake. Low micromolar amounts of this short-chain sphingolipid, being not toxic itself, enhanced the uptake of doxorubicin up to 300% and decreased its EC(50) toxicity values seven- to 14-fold. N-hexanoyl SM acts at the level of the plasma membrane, but was found not incorporated in (isolated) lipid rafts, and artificial disruption or elimination of raft constituents did not affect its drug uptake-enhancing effect. Further, any mechanistic role of the endocytic machinery, membrane leakage or ABC-transporter-mediated efflux could be excluded. Finally, a correlation was established between the degree of drug lipophilicity, as defined by partitioning in a two-phase octanol-water system, and the susceptibility of the drug towards the uptake-enhancing effect of the sphingolipid. A clear optimum was found for amphiphilic drugs, such as doxorubicin, epirubicin and topotecan, indicating that N-hexanoyl-SM might act by modulating the average degree of plasma membrane lipophilicity, in turn facilitating transbilayer drug diffusion. The concept of short-chain sphingolipids as amphiphilic drug potentiators provides novel opportunities for improving drug delivery technologies.

Adenocarcinoma↗
PubMed1998

Cytologic evidence for mechanisms of K+ transport and genesis of Hensen bodies and subsurface cisternae in outer hair cells.

UNLABELLED: A system commonly termed the tubulocisternal endoplasmic reticulum (TCER), but designated here the canalicular reticulum (CR), occurs selectively in ion-transporting epithelia, in which it is interpreted as facilitating the transcellular diffusion of ions. Mechanoelectrical transduction in the cochlear outer hair cells (OHCs) depends on the apical influx and the subsequent basolateral efflux of K+. Cytologic structures that possibly mediate K+ transport in gerbil OHCs were investigated here. METHODS: Cochleas were fixed primarily with glutaraldehyde and secondarily with reagents for demonstrating TCER or were fixed with a ferrocyanide-osmium tetroxide solution to preserve intracellular membranes. The distribution of membranous structures retained with these techniques was examined by using electron microscopy. RESULTS: Secondary fixation with osmium tetroxide-ferrocyanide permitted ultrastructural demonstration in OHCs of increased numbers of Hensen bodies and newly detected membranous systems, including CR, linear cisternae, small clusters of cytosolic vesicles and complexes of canaliculi, segmented cisternae, and mitochondria. CR filling an apical stratum beside and below the cuticular plate and contacting laterally the uppermost subsurface cisternae (SSC) was situated to sequester and transport the apical K+ influx that attends the acoustically generated receptor potential and the silent current. The close association of CR with numerous, highly developed Golgi bodies exclusively in the apex of the cell suggested genesis of CR from Golgi cisternae. Nonbranching, linear cisternae occupied a lower cell stratum and spread from CR laterally to a more inferior region of the SSC. Small clusters of vesicles in the central cytosol resembled Hensen bodies in their envelopment by branching canaliculi and segmented cisternae in close association with mitochondria. Viewing the vesicles in Hensen bodies and the small clusters as functioning like most other cytoplasmic vesicles in transport of cell membrane permitted the interpretation that these vesicles move nascent membrane from the canalicular-mitochondrial complex to the SSC. Other small clusters of vesicles contacted the innermost layer of the SSC, often at cisterna-depleted foci in which the vesicles appeared to either replenish the SSC or arise in the course of its turnover. Proximity of multivesicular bodies and lysosomes to small vesicle clusters in foci of depleted SSC implicated the lysosomes in digesting vesicles released from the SCC. Populations of unique, large, lysosome-like bodies and of small, dense bodies in the upper cytosol of OHCs appeared to be involved in different catabolic pathways mediating the turnover ofSSC, CR, and other structures. CONCLUSIONS: Cochlear OHCs contain previously unrecognized membranous organelles that facilitate ion transport and presumably contribute thereby to mechanoelectrical transduction. Vesicles in small clusters and Hensen bodies arise from complexes of canaliculi, cisternae, and mitochondria and contribute membrane to the genesis of the SSC.

Animals↗
PubMed2004

Agonist-induced internalization of leukotriene B(4) receptor 1 requires G-protein-coupled receptor kinase 2 but not arrestins.

The leukotriene B(4) (LTB(4)) receptor (BLT1) becomes desensitized upon repeated agonist stimulation. Little is known, however, about BLT1 internalization, which follows desensitization in most G-protein-coupled receptors (GPCR). In the current study, transiently expressed BLT1 readily internalized, after LTB(4) stimulation, in RBL-2H3 cells that express high levels of endogenous GPCR kinase 2 (GRK2) but did not in COS-7 or human embryonic kidney (HEK) 293 cells, which do not overexpress GRK. The internalization of BLT1 could be blocked in RBL-2H3 cells by coexpressing dominant-negative (DN) GRK2 K220R and could be promoted in HEK293 cells by coexpressing wild-type (WT) GRK2. Coexpression of WT or DN nonvisual arrestins had no effect on BLT1 internalization. Moreover, upon stimulation with LTB(4), BLT1 did not induce arrestin-green fluorescence protein redistribution in either cell type, even in the presence of overexpressed GRK2. Coimmunoprecipitation experiments confirmed that BLT1 could associate with GRK2 but not with arrestins. A C-tail-truncated mutant of BLT1 lost the capacity to internalize and associate with GRK2 upon exposure to LTB(4), suggesting that the C-tail was required for receptor internalization and association with GRK2. Taken together, our results indicate that the C terminus of BLT1 plays a pivotal role in receptor internalization and GRK2 association. Moreover, ligand-induced BLT1 internalization is dependent on GRK2 but independent of arrestins. This may allow differential, cell-type-specific signaling in response to LTB(4), depending on GRK expression levels.

Animals↗
PubMedDate not supplied

Carbonic anhydrase activity in different placenta types: a comparative study of pig, horse, cow, mink, rat, and human.

The placenta has multiple functions, being the organ which provides oxygen and nutrients to the developing conceptus. In the placenta, the enzyme carbonic anhydrase (CA) may provide ions for exchange with Na+, K+, and Cl- in transepithelial movement of ions and fluid, as well as facilitating carbon dioxide diffusion. It can also be active in intermediary metabolism, such as gluconeogenesis, urea, and fatty acid synthesis. Placental material from pig, horse, cow, mink, rat, and human was therefore investigated, representing placenta types with variations in shape, internal architecture, and nature of the interhemal barrier. After glutaraldehyde fixation, sections were stained by a histochemical CA-method demonstrating all active isozymes. The most striking feature in common was a positive reaction in the maternal capillaries, when present, as in pig, horse, cow, and mink. In the maternal epithelium, the activation of CA was only observed in the pig, which also exhibited the strongest activity at the maternal interface, which reacted moderately in rat, weakly in horse, and was not visible in cow and human. The trophoblast was positive in pig and rat, whereas it was negative in horse, cow, human, and mink placentae except for few scattered trophoblast cells in pig, horse, and cow, which showed very intense activity. In the fetal capillaries, a positive reactivity was only observed in mink and human. The utilization of CA in placental transfer and metabolism is thus highest in the pig, rat, and mink, compared with horse, cow, and human. It can therefore be concluded that the activation and localization of CA in the placental interhemal barrier varies considerably among species.

Animals↗
PubMed2000

Polymorphism of Saccharomyces cerevisiae aquaporins.

Aquaporin water channels facilitate the transmembrane diffusion of water and higher organisms possess a large number of isoforms. The genome of the yeast Saccharomyces cerevisiae contains two highly similar aquaporin genes, AQY1 and AQY2. AQY1 has been shown to encode a functional water channel but only in certain laboratory strains. Here we show that the AQY2 gene is interrupted by an 11 bp deletion in 23 of the 27 laboratory strains tested, with the exception of strains from the sigma 1278b background, which also exhibit a functional Aqy1p. However, although the AQY2 gene from sigma 1278b is highly homologous to functional aquaporins, we did not observe Aqy2p-mediated water transport in Xenopus oocytes. A survey of 52 yeast strains revealed that all industrial and wild yeasts carry the allele encoding a functional Aqy1p, while none of these strains appear to have a functional Aqy2p. We conclude that natural and industrial conditions provide selective pressure to maintain AQY1 but apparently not AQY2.

Amino Acid Sequence↗
PubMed2003

Vitamin D target proteins: function and regulation.

Recent findings have indicated that calbindin-D(28k), the first known target of vitamin D action, is present in osteoblasts and protects against TNF and glucocorticoid induced apoptosis of osteoblastic cells. Cytokine mediated destruction of pancreatic beta cells, a cause of insulin dependent diabetes, is also inhibited by calbindin-D(28k). In calbindin-D(28k) transfected pancreatic beta cells free radical formation by cytokines is inhibited by calbindin. Thus, besides its role as a facilitator of calcium diffusion, calbindin has a major role in protecting against cellular degeneration in different cell types. Besides calbindin, the other known pronounced effect of 1,25(OH)(2)D(3) in intestine and kidney is increased synthesis of 25(OH)D(3) 24-hydroxylase (24(OH)ase) which is involved in the catabolism of 1,25(OH)(2)D(3). We have noted that CCAAT enhancer binding protein beta (C/EBPbeta) is induced by 1,25(OH)(2)D(3) in kidney and osteoblastic cells and can enhance the transcriptional response of 24(OH)ase to 1,25(OH)(2)D(3). These studies establish C/EBPbeta as a novel 1,25(OH)(2)D(3) target gene and indicate a role for C/EBPbeta in 24(OH)ase transcription. These studies extend our previous studies related to factors that affect vitamin D receptor (VDR) mediated 24(OH)ase transcription (YY1, TFIIB, CBP) and the effect of signaling pathways on 24(OH)ase transcription and cofactor recruitment.

Animals↗
PubMed1980

The role of the Escherichia coli lambda receptor in the transport of maltose and maltodextrins.

The lambda receptor is a peptidoglycan-associated integral protein that spans the outer membrane. Beside its function in phage lambda adsorption it participates in transport. The latter function can be summarized as follows: 1) Receptor allows the nonspecific permeation of small molecules other than maltose and maltodextrins (in close analogy to a molecular sieve). Here the only criterion for selectivity is size and it has the properties of an unspecific pore. In this respect, it is similar to the outer membrane proteins Ia, Ib, and Ic, the porins. 2) It is a binding protein for maltodextrins. Binding affinity is low but increases by a factor of 500 as the chain length of the maltodextrins increases. In contrast, the affinity of the periplasmic maltose-binding protein for maltose and maltodextrins is similarly high (in the microM range). 3) In the in vitro system of liposomes, the lambda receptor facilitates specifically the diffusion of maltodextrins that exceed the size limit given by its porin function. This clearly demonstrates that the lambda receptor alone is able to specifically overcome the permeability barrier of the outer membrane for maltodextrins. 4) From the genetic and kinetic analysis of maltose and maltodextrin transport, it can be concluded that the lambda receptor interacts with the periplasmic maltose-binding protein. 5) Electron microscopic studies indicate a location for the maltose-binding protein in the outer cell envelope. This location is dependent on the presence of the lambda receptor.

ATP-Binding Cassette Transporters↗
PubMed2002

Serotonergic sensory-motor neurons mediate a behavioral response to hypoxia in pond snail embryos.

Oxygen (O(2)) is one of the most important environmental factors that affects both physiological processes and development of aerobic animals, yet little is known about the neural mechanism of O(2) sensing and adaptive responses to low O(2) (hypoxia) during development. In the pond snail, Helisoma trivolvis, the first embryonic neurons (ENC1s) to develop are a pair of serotonergic sensory-motor cells that regulate a cilia-driven rotational behavior. Here, we report that the ENC1-ciliary cell circuit mediates an adaptive behavioral response to hypoxia. Exposure of egg masses to hypoxia elicited a dose-dependent and reversible acceleration of embryonic rotation that mixed capsular fluid, thereby facilitating O(2) diffusion to the embryo. The O(2) partial pressures (Po(2)) for threshold, half-maximal, and maximal rotational response were 60, 28, and 13 mm Hg, respectively. During hypoxia, embryos relocated to the periphery of the egg masses where higher Po(2) levels occurred. Furthermore, intermittent hypoxia treatments induced a sensitization of the rotational response. In isolated ciliary cells, ciliary beating was unaffected by hypoxia, suggesting that in the embryo, O(2) sensing occurs upstream of the motile cilia. The rotational response of embryos to hypoxia was attenuated by application of the serotonin receptor antagonist, mianserin, correlated to the development of ENC1-ciliary cell circuit, and abolished by laser-ablation of ENC1s. Together, these data suggest that ENC1s are unique oxygen sensors that may provide a good single cell model for the examination of mechanistic, developmental, and evolutionary aspects of O(2) sensing.

Animals↗
PubMed1989

On the molecular mechanism of intestinal calcium transport.

The intestinal absorption of calcium is certainly a complex process, dependent on several factors of which vitamin D, via 1,25(OH)2D3, is the major controlling hormone. The efficiency of calcium absorption is a function of calcium status and calcium need. As the body's demand for calcium increases, the process commonly termed, adaptation, is activated in which the synthesis of 1,25(OH)2D3 from precursor is increased, resulting in the stimulation of the rate of calcium absorption. The increased demand for calcium might result from the ingestion of a diet deficient in calcium, from growth, pregnancy, lactation and egg shell formation in the laying hen. Accomapanying the change in calcium absorptive efficiency are molecular modifications of the transporting enterocytes, some mentioned herein and elsewhere (Wasserman & Chandler, 1985; Wasserman, 1980; Wasserman et al., 1984). Highly correlated with the rate of calcium absorption under a wide variety of conditions is the concentration of the vitamin D-induced calcium-binding protein, calbindin-D28K (avian type) and calbindin-D9K (mammalian intestinal type). The role of calbindin-D in this transport process is not precisely known but is considered to act at the present time as a cytosolic facilitator of Ca2+ diffusion from the brush border membrane to the basolateral membrane. In addition to the induction of calbindin-D synthesis, 1,25(OH)2D3 exerts other effects on the intestinal epithelium that can have consequences on the calcium absorptive process. Some of these effects are summarized in Figure 14. Vitamin D-dependent reactions might be either direct effects of 1,25(OH)2D3 or indirect effects due to elevated intracellular Ca2+ concentrations. These include changes in the fluidity of the brush border membrane, an increase in microvillar alkaline phosphatase-low affinity Ca-activated ATPase activity, an association of calmodulin with the 105 kD brush border cytoskeletal protein and, following calbindin D synthesis, the binding of calbindin D to a 60 kD brush border protein and to microtubules. The latter has been suggested to be related to the proposed transfer of Ca2+ by an endocytotic-exocytotic mechanism. In addition, a vitamin D-dependent intestinal membrane calcium-binding protein has been identified (Kowarski & Schachter, 1980). Playing into this multi-component system is a stimulation of cyclic nucleotide synthesis by 1,25(OH)2D3 which, through activation of cyclic nucleotide-dependent protein kinases, might modify membrane Ca2+ "channels" by phosphorylation reactions.4+ Intracellular organelles, i.e., the endoplasmic reticulum, mitochondria, the Golgi apparatus, are potent sequesters of Ca2+ and could contribute to the protection of the cell from excessively high Ca2+ concentrations by transiently storing absorbed Ca2+.

Animals↗
PubMed1988

Oxygen binding properties, capillary densities and heart weights in high altitude camelids.

The oxygen binding properties of the blood of the camelid species vicuna, llama, alpaca and dromedary camel were measured and evaluated with respect to interspecific differences. The highest blood oxygen affinity, not only among camelids but of all mammals investigated so far, was found in the vicuna (P50 = 17.6 Torr compared to 20.3-21.6 Torr in the other species). Low hematocrits (23-34%) and small red blood cells (21-30 microns 3) are common features of all camelids, but the lowest values are found in the Lama species. Capillary densities were determined in heart and soleus muscle of vicuna and llama. Again, the vicuna shows exceptional values (3720 cap/mm2 on average in the heart) for a mammal of this body size. Finally, heart weight as percent of body weight is higher in the vicuna (0.7-0.9%) than in the other camelids studied (0.5-0.7%). The possibility that these parameters, measured in New World tylopodes at sea level, are not likely to change considerably with transfer to high altitude, is discussed. In the vicuna, a unique combination of the following features seems to be responsible for an outstanding physical capability at high altitude: saturation of blood with oxygen in the lung is favored by a high blood oxygen affinity, oxygen supply being facilitated by low diffusion distances in the muscle tissue. Loading, as well as unloading, of oxygen is improved by a relatively high oxygen transfer conductance of the red blood cells, which is due to their small size and which compensates the negative effect of a low hematocrit on the oxygen conductance of blood.(ABSTRACT TRUNCATED AT 250 WORDS)

2,3-Diphosphoglycerate↗
PubMed1994

Capsaicin-sensitive sensory neurons in healing gastric lesions induced by HCl in rats.

The role of capsaicin-sensitive sensory neurons in the healing of HCl-induced gastric lesions was investigated in rats. Rats fasted for 18 hr were given 0.6 N HCl orally for induction of gastric lesions, and they were fed normally from 1 hr later. On various days after HCl treatment, area of lesions, acid secretion, mucosal H+ permeability, and blood flow responses were measured. Functional ablation of capsaicin-sensitive sensory neurons was performed two weeks before the experiment by subcutaneous injections of high-dose capsaicin. Sensory deafferentation did not affect the development of gastric damage in response to HCl but significantly delayed the healing of these lesions. The mucosa damaged by HCl secreted less acid but showed significant rise in H+ permeability, resulting in acid back-diffusion accompanied by an increase of mucosal blood flow. Sensory deafferentation had no effect on acid secretion and mucosal permeability changes in the damaged stomach but completely blocked the hyperemic response caused by acid back-diffusion. Capsaicin-sensitive sensory neurons may contribute to healing of gastric lesions, probably by mediating the mucosal hyperemic responses associated with acid back-diffusion and by facilitating acid disposal in the mucosa.

Animals↗
PubMed1985

Incorporation of (35S)sulfate and (3H)glucosamine into glycoaminoglycans in rat incisor predentine and dentine: comparison by autoradiography of fixation by rapid-freezing, freeze-substitution, and aldehyde fixation.

At 4 and 24 hours after injection of (35S)sulfate, there were 22-67% fewer silver grains on conventionally fixed sections than on sections fixed by cryotechniques. Differences were smaller when (3H) glucosamine was chosen as precursor. The number of silver grains increased between 4 and 24 hours in predentine and in a 30-micron band of dentine that had already undergone mineralization. Only a few grains were observed, however, in the 5-micron dentine band located at the mineralization front. This suggests that glycoaminoglycans, which in predentine have a space-filling role, facilitate transport and diffusion and inhibit mineralization, may limit crystal growth in dentine once a certain degree of apatite formation has been reached. All these properties are correlated with the structural and functional properties of the tissues.

Aldehydes↗
PubMed2002

Structural microdomains in the lateral plasma membrane of cochlear outer hair cells.

The basal and lateral regions of the plasma membrane of cochlear outer hair cells are structurally and functionally distinct. The lateral region contains thousands of motor proteins but few voltage-gated channels. The basal region, conversely, contains a high number of voltage-gated channels but is devoid of motor proteins. It has been suggested that the cortical cytoskeleton is responsible for maintaining this regional distinction. Toward elucidating the structure of the outer hair cell's electromotile mechanism, we investigated the physical organization of the lateral plasma membrane in living guinea pig outer hair cells by analyzing the distribution pattern of the anionic long-chain carbocyanine SP-DiIC18(3) within this area, before and after electrical stimulation and with an intact and a disrupted cytoskeleton. We observed punctate, intensely fluorescent patches as well as areas of weaker fluorescence, with clear local maxima and minima, upon labeling the cells with this membrane-soluble probe. This discrete distribution of SP-DilC18(3) suggests that the lateral plasma membrane of guinea pig outer hair cells may be composed of small structural domains (microdomains). Disrupting the cytoskeleton with either trypsin or toxin B from Clostridium difficile did not change this pattern of distribution, thus indicating that this treatment did not facilitate the lateral diffusion of the probes. Electrical stimulation using whole-cell patch-clamp techniques, on the other hand, induced two responses: fast motility and reversible displacement of the fluorescent probes. Both responses were inhibited by internal perfusion with salicylate, while disruption of the cytoskeleton did not inhibit OHC fast motility but affected the electrically induced redistribution of fluorescent probes. Together, these results suggest that the lateral plasma membrane of guinea pig outer hair cells contains structural microdomains and that the cytoskeleton does not appear to be playing a major role in maintaining the lateral separation of these distinct molecular regions.

Animals↗
PubMed1984

Mechanism of pyridoxine uptake by isolated rat liver cells.

Uptake of [3H]pyridoxine by isolated rat hepatocytes was detected at substrate concentrations as low as 0.5 microM. At this concentration, an initial uptake rate was 1.87 +/- 0.17 pmol/10(6) cells X min at 37 degrees C. Both the initial and a subsequent much slower pyridoxine accumulation were strongly inhibited by low temperature as well as by 10 mM ethionine, which competes for available ATP, or 1 microM carbonylcyanide-p-trifluoromethoxyphenylhydrazone, which inhibits oxidative phosphorylation and the supply of ATP. The uptake process is apparently insensitive to 1 mM ouabain and is Na+ independent. The initial uptake rate was saturable at higher concentrations of [3H]pyridoxine with an apparent Km of 28 +/- 8 microM and Vmax of 106 +/- 27 pmol/10(6) cells X min. The Km value corresponds to that reported for pyridoxine as a substrate of pyridoxal kinase. Moreover, the transport process was inhibited by structural analogs of [3H]pyridoxine even at concentrations equimolar to the normal substrate. The established order of inhibitory effectiveness, 4'-deoxypyridoxine greater than unlabeled pyridoxine greater than 5'-deoxypyridoxine, is in agreement with known properties of the kinase. It is concluded that pyridoxine uptake probably occurs by diffusion, simple or facilitated, followed by metabolic trapping due to pyridoxal kinase-catalyzed phosphorylation. Pyridoxine deficiency had no significant effect on uptake of this B6 vitamer by hepatocytes.

Adenosine Triphosphate↗
PubMed1993

Effects of exogenous phospholipids on platelet activation.

Intercalation of amphipaths into the plasma membrane of platelets has a marked effect on their morphology. Incubation of platelets with phosphatidylcholines (PC) results in rounding of the platelet body and speculation, while incubation with aminophospholipids such as dilauroylphosphatidylserine (DLPS) results in a biphasic shape change consistent with the bilayer couple model (Sheetz, M.P. and Singer, S.J. (1982) Proc. Natl. Acad. Sci. USA 71, 4457-4461) and with the activity of an aminophospholipid translocator facilitating transverse bilayer diffusion (Daleke, D.L. and Huestis, W.H. (1985) Biochemistry 24, 5406-5416). The present study extends this work to investigate the effects of PC and PS on platelet responses to a natural agonist, thrombin. PC incorporation produces a concentration-dependent progression of shape changes, beginning with surface ruffling and development of fine spicules, followed by sphering of the cell body, and ending with the apparent loss of spicules. PC reduces platelet responses to thrombin only under conditions that promote membrane vesiculation, seen morphologically as a loss of spicules and biochemically as a loss of 14C-PC labeled membrane. PS homologues of varying acyl chain composition induce concentration- and time-dependent platelet sphering. Incorporation of PS inhibits thrombin-induced platelet shape change, granule secretion, and protein phosphorylation. Inhibition of these responses requires transit of the exogenous PS to the cytofacial leaflet of the membrane bilayer.

Blood Platelets↗