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Intraneoplastic application of metrizamide-containing liposomes: kinetic studies with computed tomography.

Liposomes may serve as drug carriers not only for systemic chemotherapy but also for intraneoplastic drug therapy because they show a sustained drug release. In the present study, the in vivo kinetics of intraneoplastic deposits of large multilamellar vesicles containing metrizamide was followed up in a rat tumor model with computed tomography. The influence of four different lipid compositions on the retardation capacity of large multilamellar liposomes was investigated. By comparing the dynamic data of X-ray attenuation and volume of liposome deposits, a rank order for the in vivo stability of metrizamide containing multilamellar vesicles could be established: the least stable liposomes were made of pure dimyristoyl-phosphatidyl-choline, the most stable type was made of equimolar parts of stearoyl-palmitoyl-phosphatidyl-choline and cholesterol. Of intermediate stability were liposomes made of equimolar parts of dimyristoyl-phosphatidyl-choline and cholesterol, and those made of pure stearoyl-palmitoyl-phosphatidyl-choline. The addition of 50% cholesterol increased the membrane stability of both dimyristoyl-phosphatidyl-choline and stearoyl-palmitoyl-phosphatidyl-choline liposomes. No diffusion of large multilamellar liposomes away from the injection site was observed. The in vivo stability of the liposomes was considerably less than that observed in vitro, suggesting active degradation processes. It is concluded that large, multilamellar liposomes may be suitable carriers for intraneoplastic chemotherapy. The present model is easily adaptable to be transferred into clinical conditions, and may allow direct monitoring of intraneoplastic liposome-mediated chemotherapy in human brain tumors.

Animals↗

Actions of opiates, substance P, and serotonin on the excitability of primary afferent terminals and observations on interneuronal activity in the neonatal rat's dorsal horn in vitro.

Approximately 5 segments of lumbo-thoracic spinal cord together with connected dorsal root ganglia were removed from 1-11-day-old rats and maintained in vitro. Dorsal root afferents, recorded from the ganglion and stimulated at the root entry zone, had conduction velocities typical of unmyelinated fibers (less than 2 m/s). The spinal terminals of individual afferents showed increased excitability with bath application of substance P and serotonin and decreased excitability with morphine sulfate, [D-ala2]methionine-enkephalinamide, manganese ions and magnesium ions. Naloxone by itself elicited no change in excitability, although it appeared to reduce the ongoing effect of opiates. Neurons recorded extracellularly in the dorsal horn responded to afferent volleys with one or more of 3 distinct phases: an excitation roughly coincident with the volley's arrival, a 50-300 ms period of inhibition, and a late excitation of 150-300 ms latency. The excitability results are accounted for by a model in which substance P, gamma-aminobutyric acid and possibly other depolarizing agents are contained in interneurons which synapse on afferent terminals. These interneurons could receive inhibitory enkephalinergic input, and, in the neonate but not the adult, excitatory serotoninergic input. An alternate scheme would have enkephalin and serotonin acting directly on afferent terminals, although perhaps by non-synaptic diffusion since the appropriate synapses have not been seen in histochemical studies. Such an action for enkephalin might explain the existence of opiate receptor on afferent terminals. The interneuronal responses to afferent volleys are parallel in most aspects to those found in the dorsal horns of adult mammals in vivo.

Afferent Pathways↗

Cell-to-cell interactions in bone.

Bone development (modeling) occurs by migration, aggregation, and condensation of immature osteo/chondroprogenitor cells to form the cartilaginous anlage. This process requires precisely controlled cell-cell interactions. Likewise, bone remodeling in the adult skeleton is a dynamic process that requires coordinated cellular activities among osteoblasts, osteocytes, and osteoclasts to maintain bone homeostasis. The cooperative nature of both bone modeling and remodeling requires tightly regulated mechanisms of intercellular recognition and communication that permit the cells to sort and migrate, synchronize activity, equalize hormonal responses, and diffuse locally generated signals. Osteoblasts and osteocytes achieve these interactions through cadherin-based adherens junctions as well as by formation of communicating junctions, gap junctions. This review examines the current knowledge of how direct cell-to-cell interactions modulate osteoblast function.

Animals↗

Assessment of transport rates of proteins and peptides across primary human alveolar epithelial cell monolayers.

In this study, we investigated bi-directional fluxes (i.e., in absorptive and secretive directions) of human serum proteins [albumin (HSA), transferrin (TF), and immunoglobulin G (IgG)] and peptides/proteins of potential therapeutic relevance [insulin (INS), glucagon-like peptide-1 (GLP-1), growth hormone (GH), and parathyroid hormone (PTH)] across tight monolayers of human alveolar epithelial cells (hAEpC) in primary culture. Apparent permeability coefficients (P(app); x10(-7)cm/s, mean+/-S.D.) for GLP-1 (6.13+/-0.87 (absorptive) versus 1.91+/-0.51 (secretive)), HSA (2.45+/-1.02 versus 0.21+/-0.31), TF (0.88+/-0.15 versus 0.30+/-0.03), and IgG (0.36+/-0.22 versus 0.15+/-0.16) were all strongly direction-dependent, i.e., net absorptive, while PTH (2.20+/-0.30 versus 1.80+/-0.77), GH (8.33+/-1.24 versus 9.02+/-3.43), and INS (0.77+/-0.15 versus 0.72+/-0.36) showed no directionality. Trichloroacetic acid precipitation analysis of tested molecules collected from donor and receiver fluids exhibited very little degradation. This is the first study on permeability data for a range of peptides and proteins across an in vitro model of the human alveolar epithelial barrier. These data indicate that there is no apparent size-dependent transport conforming to passive restricted diffusion for the tested substances across human alveolar barrier, in part confirming net absorptive transcytosis. The obtained data differ significantly from previously published reports utilising monolayers from different species. It can be concluded that the use of homologous tissue should be preferred to avoid species differences.

Blood Proteins↗

Protein rotational motion in solution measured by polarized fluorescence depletion.

A microscope-based system is described for directly measuring protein rotational motion in viscous environments such as cell membranes by polarized fluorescence depletion (PFD). Proteins labeled with fluorophores having a high quantum yield for triplet formation, such as eosin isothiocyanate (EITC), are examined anaerobically in a fluorescence microscope. An acousto-optic modulator generates a several-microsecond pulse of linearly polarized light which produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. When the sample is then probed with light polarized parallel to the excitation pulse, fluorescence recovers over 0-1,000 microseconds as the sum of two exponentials. One exponential corresponds to triplet decay and the other to the rotational relaxation. An exciting pulse perpendicular to the probe beam is then applied. Fluorescence recovery following this pulse is the difference of the same two exponentials. Equations for fluorescence recovery kinetics to be expected in various experimentally significant cases are derived. Least-squares analysis using these equations then permits the triplet lifetime and rotational correlation time to be determined directly from PFD data. Instrumentation for PFD measurements is discussed that permits photobleaching recovery measurements of lateral diffusion coefficients using the same microscope system. With this apparatus, both rotational and translational diffusion coefficients (Dr, Dt) were measured for EITC-labeled bovine serum albumin in glycerol solutions. Values obtained for Dr and Dt are discussed in light of both the PFD models and the experimental system.

Mathematics↗

Model-independent time-resolved fluorescence depolarization from ordered biological assemblies applied to restricted motion of myosin cross-bridges in muscle fibers.

We formulate a model-independent description of time-dependent restricted molecular rotational motion and apply it to the time-resolved fluorescence depolarization signal from fluorescence-labeled molecules following polarized excitation. In this treatment, the time-dependent molecular orientation distribution is equal to the operation of a linear time-development operator on the initial orientation distribution. The formula for the time-development operator is derived from the equation of motion for the orientation distribution. The time-development operator is then expanded in time in terms of a complete set of orthonormal polynomials. When this expression for the orientation distribution is used to calculate the time-resolved fluorescence depolarization signal, properties of the orthonormal polynomials allow the signal to be quantitated uniquely in terms of matrix elements of the differential operators from the equation of motion. We demonstrate how to obtain the experimental value of the matrix elements directly from the fluorescence depolarization signal. In this paper, we also describe the application of the model-independent formalism to fluorescence-labeled myosin cross-bridges in muscle fibers when the fibers are in a variety of physiological states. We show that the analysis of time-resolved fluorescence depolarization data with the new formalism and the rotational diffusion in a potential model results in a slight revision of our previous estimate of the relaxed cross-bridge rotational diffusion constant and the determination of rank 6 order parameters that were ignored in the previous analysis [Burghardt, T. P., & Thompson, N. L. (1985) Biochemistry 24, 3731-3735]. The rank 6 order parameters are shown to make a significant contribution to the proposed steady-state angular distribution of the relaxed cross-bridges. Order parameters of rank 6 do not contribute to the steady-state fluorescence polarization signal and can only be detected with the time-resolved signal [Burghardt, T. P. (1984) Biopolymers 23, 2383-2406]. We have also applied the model-independent formalism to new data from fibers in rigor when the fibers are in a configuration such that the excitation light polarization is perpendicular to the fiber axis and the light propagates along the fiber axis, so that the fluorescence depolarization signal is sensitive to probe rotational motions about the fiber axis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Dynamics of coilin in Cajal bodies of the Xenopus germinal vesicle.

Cajal bodies (CBs) are complex organelles found in the nuclei of a wide variety of organisms, including vertebrates, invertebrates, plants, and yeast. In most cell types CBs are <1 microm in diameter, severely limiting the range of experimental observations that can be made on them. By contrast, CBs in the amphibian oocyte nucleus (also called the germinal vesicle) are 2-10 microm in diameter. We have taken advantage of this large size to carry out kinetic studies on coilin, a protein that is specifically enriched in CBs. We labeled coilin with photoactivatable green fluorescent protein and analyzed the movement of the protein by confocal microscopy in unfixed germinal vesicles isolated in oil. We showed that coilin leaves the CB relatively slowly (minutes rather than seconds) with kinetics similar to earlier measurements on its entrance. We also showed that coilin diffuses very slowly within the CB, consistent with its being in a large macromolecular complex. Finally, we found that the movement of coilin is not directly affected by the transcriptional state of the nucleus or ongoing nucleocytoplasmic exchange. These data on the kinetics of coilin reinforce the conclusion that CB components are in a constant state of flux, consistent with models that postulate an active role for CBs in nuclear physiology.

Animals↗

Role of intercellular and intracellular communication by nitric oxide in coupling of muscarinic receptors to activation of guanylate cyclase in neuronal cells.

Muscarinic receptor-mediated cyclic GMP formation and release of nitric oxide (NO) (or a precursor thereof) were compared in mouse neuroblastoma N1E-115 cells. [3H]Cyclic GMP was assayed in cells prelabeled with [3H]guanine. Release of NO upon the addition of muscarinic agonists to unlabeled neuroblastoma cells (NO donor cells) was quantitated indirectly by its ability to increase the [3H]cyclic GMP level in labeled cells whose muscarinic receptors were inactivated by irreversible alkylation (NO detector cells). Carbachol increased NO release in a concentration-dependent manner, with half-maximal stimulation at 173 microM (compared to 96 microM for direct activation of cyclic GMP formation). The maximal effect of carbachol in stimulating release of NO when measured indirectly was lower than that in elevating [3H]cyclic GMP directly in donor cells. Hemoglobin was more effective in blocking the actions of released NO than in attenuating direct stimulation of [3H]cyclic GMP synthesis. There was a good correlation between the ability of a series of muscarinic agonists to release NO or to activate [3H]cyclic GMP formation directly, and the potency of pirenzepine in inhibiting the two responses. Furthermore, there was a similar magnitude of desensitization of both responses by prolonged receptor activation or stimulation of protein kinase C. NO release was also regulated in relation to the cellular growth phase. A model is proposed in which a fraction of NO generated upon receptor activation does not diffuse extracellularly and stimulates cyclic GMP synthesis within the same cell where it is formed (locally acting NO). The remainder of NO that is extruded extracellularly might travel to neighboring cells (neurotransmitter NO) or might be taken back into the cells of origin (homing NO).

Alkylation↗

Development of a macromolecular diffusion pathway in the lens.

The mammalian lens consists of an aged core of quiescent cells enveloped by a layer of synthetically active cells. Abundant gap junctions within and between these cell populations ensure that the lens functions as an electrical syncytium and facilitates the exchange of small molecules between surface and core cells. In the present study, we utilized an in vivo mouse model to characterize the properties of an additional pathway, permeable to macromolecules, which co-exists with gap-junction-mediated communication in the lens core. The TgN(GFPU)5Nagy strain of mice carries a green fluorescent protein (GFP) transgene. In the lenses of hemizyous animals, GFP was expressed in a variegated fashion, allowing diffusion of GFP to be visualized directly. Early in development, GFP expression in scattered fiber cells resulted in a checkerboard fluorescence pattern in the lens. However, at E15 and later, the centrally located fiber cells became uniformly fluorescent. In the adult lens, a superficial layer of cells, approximately 100 microm thick, retained the original mosaic fluorescence pattern, but the remainder, and majority, of the tissue was uniformly fluorescent. We reasoned that at the border between the two distinct labeling patterns, a macromolecule-permeable intercellular pathway was established. To test this hypothesis, we microinjected 10 kDa fluorescent dextran into individual fiber cells and followed its diffusion by time-lapse microscopy. Injections at depths of >100 microm resulted in intercellular diffusion of dextran from injected cells. By contrast, when injections were made into superficial fiber cells, the injected cell invariably retained the dextran. Together, these data suggest that, in addition to being coupled by gap junctions, cells in the lens core are interconnected by a macromolecule-permeable pathway. At all ages examined, a significant proportion of the nucleated fiber cell population of the lens was located within this region of the lens.

Animals↗

Systematic errors in the measurement of emissivity caused by directional effects.

Accurate knowledge of surface emissivity is essential for applications in remote sensing (remote temperature measurement), radiative transport, and modeling of environmental energy balances. Direct measurements of surface emissivity are difficult when there is considerable background radiation at the same wavelength as the emitted radiation. This occurs, for example, when objects at temperatures near room temperature are measured in a terrestrial environment by use ofthe infrared 8-14-microm band.This problem is usually treated by assumption of a perfectly diffuse surface or of diffuse background radiation. However, real surfaces and actual background radiation are not diffuse; therefore there will be a systematic measurement error. It is demonstrated that, in some cases, the deviations from a diffuse behavior lead to large errors in the measured emissivity. Past measurements made with simplifying assumptions should therefore be reevaluated and corrected. Recommendations are presented for improving experimental procedures in emissivity measurement.

Journal Article↗

Characterization of interaction between cationic lipid-oligonucleotide complexes and cellular membrane lipids using confocal imaging and fluorescence correlation spectroscopy.

Complexes formed by cationic liposomes and single-strand oligodeoxynucleotides (CL-ODN) are promising delivery systems for antisense therapy. ODN release from the complexes is an essential step for inhibiting activity of antisense drugs. We applied fluorescence correlation spectroscopy and confocal laser scanning microscopy to monitor CL-ODN complex interaction with membrane lipids leading to ODN release. To model cellular membranes we used giant unilamellar vesicles and investigated the transport of Cy-5-labeled ODNs across DiO-labeled membranes. For the first time, we directly observed that ODN molecules are transferred across the lipid bilayers and are kept inside the giant unilamellar vesicles after release from the carriers. ODN dissociation from the carrier was assessed by comparing diffusion constants of CL-ODN complexes and ODNs before complexation and after release. Freely diffusing Cy-5-labeled ODN (16-nt) has diffusion constant D(ODN) = 1.3 +/- 0.1 x 10(-6) cm2/s. Fluorescence correlation spectroscopy curves for CL-ODN complexes were fitted with two components, which both have significantly slower diffusion in the range of D(CL-ODN) = approximately 1.5 x 10(-8) cm2/s. Released ODN has the mean diffusion constant D = 1.1 +/- 0.2 x 10(-6) cm2/s, which signifies that ODN is dissociated from cationic lipids. In contrast to earlier studies, we report that phosphatidylethanolamine can trigger ODN release from the carrier in the full absence of anionic phosphatidylserine in the target membrane and that phosphatidylethanolamine-mediated release is as extensive as in the case of phosphatidylserine. The presented methodology provides an effective tool for probing a delivery potential of newly created lipid formulations of CL-ODN complexes for optimal design of carriers.

Biological Transport↗

[Neuron membrane depolarization under the influence of cyclic-3',5'-adenosine monophosphate and its possible role in the neuronal molecular computer (MC)].

The separate fourth intracellular microelectrode was used for controlling the conditions of cyclic nucleotide injection in neurons of Helix pomatia. Ionoforetic increase in intracellular cyclic AMP concentration elicits membrane depolarization in many neurons. Phosphodiesterase inhibitors 3-isobutyl-1-methylxantine and SQ-20009 prolong this depolarization and raise its level. In cell F-1 of helix brain sometimes cAMP induces weak hyperpolarization, but this response turns to usual depolarization after 3-isobutyl-1-methylxantine application. It is suggested that cell molecular computer has an analog input, where diffusion of cAMP, cGMP and Ca++ being a modelling process. Adenylate cyclase and guanylate cyclase and ionic channels of membrane are regulated sources. Phosphodiesterases with Ca2+-binding activator proteins are molecular out flowers and protein kinases--detectors that transform the data about the concentrations of cAMP and cGMP into codes for MCC. Protein kinases control over the activity of proteins directly. The depolarization effect on neuron membrane seems to be associated with protein kinase activation or with direct action of cAMP on phospholipase.

Adenylyl Cyclases↗

[Modeling of elastic deformation and vascular resistance of arterial and venous vasa vasorum].

As in most living tissues, a network of nutritional vessels, the so-called vasa vasorum, irrigates the vessel wall under physiological conditions. An alteration or obstruction of this network can induce severe lesions. Most normal arteries and veins are irrigated by a vasa vasorum network located mainly in the adventice. They essentially supply oxygen to the outer layers of the vascular wall, the inner layer being mainly oxygenated by direct diffusion from bloodstream. Vasa vasorum responds to vasomotor stimuli and can even regress, e.g., after vascularization of arterial grafts. Their pathophysiological importance for arteries is now established. Indeed, it is known that an infusion disorder or vasa vasorum alteration may induce or promote early atherosclerotic lesions, fibrodysplasia or even media necrosis. From a mechanical point of view, and considering the three layers as a unique material, the vessel shows non-isotropic linear elastic and incompressible (v = 0.5) behaviour in the case of minimal or moderate deformation. But in the case of major deformation, the vessel displays a non-linear behaviour. The interaction between vasa vasorum supply and the mechanical properties of the arterial vascular wall can promote the occurrence of aneurysms as soon as vasa vasorum irrigation decreases. Some authors have hypothesized that these microvessels could fulfil the same function in the venous wall. It appears also that microcirculation flow rates are lower in varicose veins than in healthy ones and that partial oxygen pressure, already low in a healthy vein media, is even lower in a varicose vein. All these facts underline the importance of supply by the vasa vasorum network and its determining role in maintaining vascular wall integrity. In addition, the influence of vessel non-linear properties and their pathological changes on microcirculation would partially explain media necrosis in arteries and veins. Studying vascular wall deformation under the influence of intraluminal pressure revealed that an initially circular vasa vasorum rapidly takes on an elliptical shape which results more from crosswise circumferential stretching of the wall than from radial crushing. This induces increased hydraulic resistance. Thus permanent overpressure reduces vascular wall irrigation. Once the wall has been devascularized, it will loose its elasticity, harden and retain its maximal deformation. A vicious circle is then created. This phenomenon, noticeable in arteries, could be more serious in veins because their walls are thinner and elasticity modulus is lower. For example, for an intraluminal overpressure of 100 mmHg in an artery and 10 mmHg in a vein the ellipticity of the vasa vasorum becomes 1.2 and 3 respectively. Based on the hypothesis of a linear elastic behaviour and a periodical intraluminal overpressure, the ratio of the two axis of an arterial vasa vasorum B/A varies from 1.13 to 1.28 for Pa = 100 + 30 sin (2 pi t) mmHg, and from 1.24 to 1.44 for Pa = 160 + 40 sin (2 pi t) mmHg. In this case, the ratio of hydraulic resistances R(ellipse)/R(circle) changes little (less than 1, the ratio of the axis varies from 1.1 to 2.6 for Pa = 5 + 5 sin (2 pi t) mmHg) and from 1.8 to 5.8 for Pa = 10 + 5 sin (2 pi t) mmHg). Thus the ratio of hydraulic resistance varies from 1 to 1.5 and from 1.2 to 2.8 respectively. In practice Young's modulus increases in parallel with luminal pressure by limiting vascular wall and vasa vasorum deformation. If we consider the non-linear behaviour of the vessel wall and suppose the same conditions of intraluminal pressure, the ratio of the axis of the venous vasa vasorum in a hypertensive patient varies from 1.6 to 2.6 (instead of 1.8 to 5.8 in the case of linear model). This ratio is higher than that of the healthy subject which is less than 1.7. So the vascular structure in physiological conditions itself reacts to the pressure increases which may jeopardize vasa vasorum irrigation by delaying mural transfor

Animals↗

Direct measurement of intracellular O2 gradients; role of convection and myoglobin.

During steady phasic exercise in a red muscle the entire O2 gradient between capillary and mitochondria occurs as a step over less than 5 m. The magnitude of this step is determined by VO2 and capillary PO2, and is independent of distance from a capillary, or local capillary density. The above cannot be explained by ordinary and/or facilitated diffusion, according to a classical Krogh model. A step gradient can be produced by intracellular convection and Mb, acting in concert. A uniformly low cell PO2 maximizes the trans-capillary O2 gradient, and hence the O2 flux. Since the O2 affinity of Mb is about 50 times less than that cytochrome a, a3, mitochondria can respire maximally at tensions well below the Mb P50. It seems likely that the principal function of Mb during steady, phasic exercise is to compensate for short capillary transit times by accelerating O2 release from Hb.

Animals↗

Correction of eddy-current distortions in diffusion tensor images using the known directions and strengths of diffusion gradients.

PURPOSE: To correct eddy-current artifacts in diffusion tensor (DT) images without the need to obtain auxiliary scans for the sole purpose of correction. MATERIALS AND METHODS: DT images are susceptible to distortions caused by eddy currents induced by large diffusion gradients. We propose a new postacquisition correction algorithm that does not require any auxiliary reference scans. It also avoids the problematic procedure of cross-correlating images with significantly different contrasts. A linear model is used to describe the dependence of distortion parameters (translation, scaling, and shear) on the diffusion gradients. The model is solved numerically to provide an individual correction for every diffusion-weighted (DW) image. RESULTS: The assumptions of the linear model were successfully verified in a series of experiments on a silicon oil phantom. The correction obtained for this phantom was compared with correction obtained by a previously published method. The algorithm was then shown to markedly reduce eddy-current distortions in DT images from human subjects. CONCLUSION: The proposed algorithm can accurately correct eddy-current artifacts in DT images. Its principal advantages are that only images with comparable signals and contrasts are cross-correlated, and no additional scans are required.

Algorithms↗

Application of two-photon flash photolysis to reveal intercellular communication and intracellular Ca2+ movements.

Two-photon excitation makes it possible to excite molecules in volumes of much less than 1 fl. In two-photon flash photolysis (TPFP) this property is used to release effector molecules from caged precursors with high three-dimensional resolution. We describe and examine the benefits of using TPFP in model solutions and in a number of cell systems to study their spatial and temporal properties. Using TPFP of caged fluorescein, we determined the free diffusion coefficient of fluorescein (D=4 x 0(-6) cm(2)/s at 20 degrees C, which is in close agreement with published values). TPFP of caged fluorescein in lens tissue in situ revealed spatial nonuniformities in intercellular fiber cell coupling by gap junctions. At the lens periphery, intercellular transport was predominantly directed along rows of cells, but was nearly isotropic further from the periphery. To test an algorithm aiming to reconstruct the Ca(2+) release flux underlying physiological Ca(2+) signals in heart muscle cells, TPFP of DM-Nitrophen was utilized to generate artificial microscopic Ca(2+) signals with known underlying Ca(2+) release flux. In an experiment with mouse oocytes, the recently developed Ca(2+) cage dimethoxynitrophenyl-ethyleneglycol-bis-(beta-aminoethylether)-N,N,N('),N(') tetraacetic acid-4 (DMNPE-4) was released in the oocyte cytosol and inside a nucleolus. Analysis of the resulting fluorescence changes suggested that the effective diffusion coefficient within the nucleolus was half of that in the cytosol. These experiments demonstrate the utility of TPFP as a novel tool for the optical study of biomedical systems.

Action Potentials↗

An immortal cell line to study the role of endogenous CFTR in electrolyte absorption.

The intact human reabsorptive sweat duct (RD) has been a reliable model for investigations of the functional role of "endogenous" CFTR (cystic fibrosis transmembrane conductance regulator) in normal and abnormal electrolyte absorptive function. But to overcome the limitations imposed by the use of fresh, intact tissue, we transformed cultured RD cells using the chimeric virus Ad5/SV40 1613 ori-. The resultant cell line, RD2(NL), has remained differentiated forming a polarized epithelium that expressed two fundamental components of absorption, a cAMP activated Cl- conductance (GCl) and an amiloride-sensitive Na+ conductance (GNa). In the unstimulated state, there was a low level of transport activity; however, addition of forskolin (10(-5) M) significantly increased the Cl- diffusion potential (Vt) generated by a luminally directed Cl- gradient from -15.3 +/- 0.7 mV to -23.9 +/- 1.1 mV, n = 39; and decreased the transepithelial resistance (Rt) from 814.8 +/- 56.3 omega.cm2 to 750.5 +/- 47.5 omega.cm2, n = 39, (n = number of cultures). cAMP activation, anion selectivity (Cl- > I- > gluconate), and a dependence upon metabolic energy (metabolic poisoning inhibited GCl), all indicate that the GCl expressed in RD2(NL) is in fact CFTR-GCl. The presence of an apical amiloride-sensitive GNa was shown by the amiloride (10(-5) M) inhibition of GNa as indicated by a reduction of Vt and equivalent short circuit current by 78.0 +/- 3.1% and 77.9 +/- 2.6%, respectively, and an increase in Rt by 7.2 +/- 0.8%, n = 36. In conclusion, the RD2(NL) cell line presents the first model system in which CFTR-GCl is expressed in a purely absorptive tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗

Essential role of NHE3 in facilitating formate-dependent NaCl absorption in the proximal tubule.

The absorption of NaCl in the proximal tubule is markedly stimulated by formate. This stimulation of NaCl transport is consistent with a cell model involving Cl(-)-formate exchange in parallel with pH-coupled formate recycling due to nonionic diffusion of formic acid or H(+)-formate cotransport. The formate recycling process requires H(+) secretion. Although Na(+)-H(+) exchanger isoform NHE3 accounts for the largest component of H(+) secretion in the proximal tubule, 40-50% of the rates of HCO absorption or cellular H(+) extrusion persist in NHE3 null mice. The purpose of the present investigation is to use NHE3 null mice to directly test the role of apical membrane NHE3 in mediating NaCl absorption stimulated by formate. We demonstrate that formate stimulates NaCl absorption in the mouse proximal tubule microperfused in vivo, but the component of NaCl absorption stimulated by formate is absent in NHE3 null mice. In contrast, stimulation of NaCl absorption by oxalate is preserved in NHE3 null mice, indicating that oxalate-stimulated NaCl absorption is independent of Na(+)-H(+) exchange. The virtually complete dependence of formate-induced NaCl absorption on NHE3 activity raises the possibility that NHE3 and the formate transporters are functionally coupled in the brush border membrane.

Absorption↗