"Fast excitation" CID in a quadrupole ion trap mass spectrometer.

Collision-induced dissociation (CID) in a quadrupole ion trap mass spectrometer is usually performed by applying a small amplitude excitation voltage at the same secular frequency as the ion of interest. Here we disclose studies examining the use of large amplitude voltage excitations (applied for short periods of time) to cause fragmentation of the ions of interest. This process has been examined using leucine enkephalin as the model compound and the motion of the ions within the ion trap simulated using ITSIM. The resulting fragmentation information obtained is identical with that observed by conventional resonance excitation CID. "Fast excitation" CID deposits (as determined by the intensity ratio of the a(4)/b(4) ion of leucine enkephalin) approximately the same amount of internal energy into an ion as conventional resonance excitation CID where the excitation signal is applied for much longer periods of time. The major difference between the two excitation techniques is the higher rate of excitation (gain in kinetic energy) between successive collisions with helium atoms with "fast excitation" CID as opposed to the conventional resonance excitation CID. With conventional resonance excitation CID ions fragment while the excitation voltage is still being applied whereas for "fast excitation" CID a higher proportion of the ions fragment in the ion cooling time following the excitation pulse. The fragmentation of the (M + 17H)(17+) of horse heart myoglobin is also shown to illustrate the application of "fast excitation" CID to proteins.

Identification of bacteriophage MS2 coat protein from E. coli lysates via ion trap collisional activation of intact protein ions.

Collisional activation of the intact MS2 viral capsid protein with subsequent ion/ion reactions has been used to identify the presence of this virus in E. coli lysates. Tandem ion trap mass spectrometry experiments on the +7, +8, and +9 charge states, followed by ion/ion reactions, provided the necessary sequence tag information (and molecular weight data) needed for protein identification via database searching. The most directly informative structural information is obtained from those charge states that produce a series of product ions arising from fragmentation at adjacent residues. The formation of these product ions via dissociation at adjacent amino acid residues depends greatly on the charge state of the parent ion. Database searching of the charge-state-specific sequence tags was performed by two different search engines: the ProteinInfo program from the Protein information Retrieval On-line World Wide Web Lab or PROWL and the TagIdent program from the ExPASy molecular biology server. These search engines were used in conjunction with the sequence tag information generated via collisional activation of the intact viral coat protein. These programs were used to evaluate the feasibility of generating sequence tags from collisional activation of intact multiply charged protein ions in a quadrupole ion trap.

Ion trap collisional activation of the (M + 2H)2+ - (M + 17H)17+ ions of human hemoglobin beta-chain.

The parent ions of human hemoglobin beta-chain ranging in charge from 2+ to 17+ have been subjected to ion trap collisional activation. The highest charge-state ions (17+ to 13+) yielded series of products arising from dissociation of adjacent residues. The intermediate charge-state ions (12+ to 5+) tended to fragment preferentially at the N-terminal sides of proline residues and the C-terminal sides of acidic residues. Many, but not all, of the possible cleavages at proline, aspartic acid, and glutamic acid residues were represented in the spectra. The lowest charge-state ions were difficult to dissociate with high efficiency and yielded spectra with poorly defined product ion signals. This observation is attributed to sequential fragmentations arising from losses of small molecules such as water and/or ammonia. The poor fragmentation efficiency observed for the low charge states is due at least in part to the low trapping wells used to store the ions. Higher ion stabilities due to lower Coulombic repulsion and charges being sequestered at highly basic sites may also play an important role. Ion/ion proton-transfer reactions involving protein parent ions allows for the formation of a wide range of parent ion charge states. In addition, the ion/ion proton-transfer reactions involving protein dissociation products simplify interpretation of the product ion spectra.

Gas phase H/D exchange kinetics: DI versus D2O.

The gas phase H/D exchange reactions of bradykinin (M + 3H)3+ ions with D2O and DI were monitored in a quadrupole ion trap mass spectrometer. The H/D exchange kinetics of both chemical probes (D2O and DI) indicate the presence of two noninterconverting reactive gas phase ion populations of bradykinin (M + 3H)3+ at room temperature. The H/D exchange involving DI, however, generally proceeds faster than that involving D2O. The rate observations described here can be rationalized on the basis of the "relay mechanism" (see Campbell et al. J. Am. Chem. Soc. 1995, 117, 12840-12854) recently proposed to account for H/D exchange between D2O and gaseous protonated polypeptides. The higher exchange rate with DI is believed to arise primarily as a result of its lower gas-phase acidity relative to that of D2O and, secondarily, as a result of the longer bond length of DI relative to that of OD in D2O.

Ion trap collisional activation of disulfide linkage intact and reduced multiply protonated polypeptides.

The presence of disulfide linkages in multiply charged polypeptide ions tends to inhibit the formation of structurally informative product ions under conventional quadrupole ion trap collisional activation conditions. In particular, fragmentation that requires two cleavages (i.e., cleavage of a disulfide linkage and a peptide linkage) is strongly suppressed. Reduction of the disulfide linkage(s) by use of dithiothreitol yields parent ions upon electrospray without this complication. Far richer structural information is revealed by ion trap collisional activation of the disulfide-reduced species than from the native species. These observations are illustrated with doubly protonated native and reduced somatosin, the [M + 5H](5+) ion of native bovine insulin and the [M + 4H](4+) and [M + 3H](3+) ions of the B-chain of bovine insulin produced by reduction of the disulfide linkages in insulin, and the [M + 11H](11+) ion of native chicken lysozyme and the [M + 11H](11+) and [M + 14H](14+) ions of reduced lysozyme. In each case, the product ions produced by ion trap collisional activation were subjected to ion/ion proton transfer reactions to facilitate interpretation of the product ion spectra. These studies clearly suggest that the identification of polypeptides with one or more disulfide linkages via application of ion trap collisional activation to the multiply charged parent ions formed directly by electrospray could be problematic. Means for cleaving the disulfide linkage, such as reduction by dithiothreitol prior to electrospray, are therefore desirable in these cases.

Influences of zinc on fertilisation and development of bovine oocytes in vitro.

The effects of zinc (as ZnCl2) on in vitro production of bovine embryos (IVMFC) and components of the procedure, that is in vitro oocyte maturation (IVM), fertilisation (IVF) and embryo development in culture (IVC), and the effect of added zinc on sperm motility were studied. Immature cumulus oocyte complexes (COCs) were aspirated from ovarian follicles (2-5 mm diameter) at slaughter, and matured, fertilised and cultured in chemically defined conditions. The presence of zinc (10, 100 or 1000 micrograms added per millilitre) throughout IVMFC inhibited fertilisation. After addition of 10 micrograms zinc per millilitre separately to media for IVM and IVF, fertilisation was inhibited only when zinc was present for IVM. When present for IVF, 80% of oocytes selected for IVM reached 2- to 4-cell stages by 46 h after insemination whereas 67% of control oocytes (inseminated without added zinc) cleaved. Higher zinc concentrations (100 and 1000 micrograms added per millilitre) for IVF inhibited fertilisation. Sperm motility was reduced with addition of 10 micrograms per millilitre of zinc for sperm preparation (i.e. capacitation interval). Addition of 1.0 microgram zinc per millilitre to media used through IVMFC, or to the IVC medium alone, resulted in inhibition of development after 2- to 4-cell stages. When added to IVM or to both IVM and IVF media 1.0 microgram/ml of zinc compromised development to the morula stage and beyond. Maturing bovine oocytes may be more sensitive to 1.0 microgram ml of zinc in vitro than in vivo because a concentration of 3.0 micrograms/ml has been reported for bovine follicular fluid. Fertilisation was not adversely affected by 10 micrograms/ml of zinc; however, higher concentrations were inhibitory.

Hydroiodic acid attachment kinetics as a chemical probe of gaseous protein ion structure: bovine pancreatic trypsin inhibitor.

The kinetics of attachment of hydroiodic acid (HI) to the (M + 6H)6+ ions of native and reduced forms of bovine pancreatic trypsin inhibitor (BPTI) in the quadrupole ion trap environment are reported. Distinctly nonlinear (pseudo first-order) reaction kinetics are observed for reaction of the native ions, indicating two or more noninterconverting structures in the parent ion population. The reduced form, on the other hand, shows very nearly linear reaction kinetics. Both forms of the parent ion attach a maximum of five molecules of hydroiodic acid. This number is expected based on the amino acid composition of the protein. There is a total of 11 strongly basic sites in the protein (i.e., six arginines, four lysines, and one N-terminus). An ion with protons occupying six of the basic sites has five available for hydroiodic acid attachment. The kinetics of successive attachment of HI to the native and reduced forms of BPTI also differ, particularly for the addition of the fourth and fifth HI molecules. A very simple kinetic model describes the behavior of the reduced form reasonably well, suggesting that all of the neutral basic sites in the reduced BPTI ions have roughly equal reactivity. However, the behavior of the native ion is not well-described by this simple model. The results are discussed within the context of differences in the three-dimensional structures of the ions that result from the presence or absence of the three disulfide linkages found in native BPTI. The HI reaction kinetics appears to have potential as a chemical probe of protein ion three-dimensional structure in the gas phase. Hydroiodic acid attachment chemistry is significantly different from other chemistries used to probe three-dimensional structure and hence, promises to yield complementary information.

Simplification of product ion spectra derived from multiply charged parent ions via ion/ion chemistry.

High-mass multiply charged ions fragment to yield a mixture of products of varying mass and charge. When the measurement of mass-to-charge ratio is used to determine products ion mass, product ion charge must first be established. To minimize charge-state ambiguity in product ion spectra derived from multiply charged parent ions, product ions have been subjected to proton-transfer reactions with oppositely charged ions to reduce product ion charge states largely to +1. This procedure greatly simplifies the interpretation of product ion spectra derived from multiply charged ions. Illustrative data are presented for the +4 and +3 parent ions derived from electrospray of melittin and the +12 to +4 parent ions of bovine ubiquitin, whereby product ions were formed in a conventional quadrupole ion trap tandem mass spectrometry experiment. Data are also shown for product ion mixtures derived from interface-induced dissociation of multiply charged ions derived from bovine ubiquitin, tuna cytochrome c, bovine cytochrome c, and equine cytochrome c. The use of ion/ion chemistry to simplify product ion spectra derived from multiply charged parent ions significantly extends the size range of macromolecules for which the quadrupole ion trap can derive structural information.

Charge manipulation for improved mass determination of high-mass species and mixture components by electrospray mass spectrometry.

The manipulation of the charge states of high-mass ions can facilitate mass determination in electrospray (ES) mass spectrometry. Specifically, the reduction of charge (which leads to ions of higher mass-to-charge ratios) can significantly reduce peak overlap. Signals associated with various charge states of high-mass ions are more easily resolved at low charge states and chemical noise tends to be significantly lower at high mass-to-charge ratios than in the normal mass-to-charge window typically associated with electrospray. Algorithms that transform ES mass spectra to zero-charge spectra are most likely to yield unambiguous results when charge states are clearly resolved and when signal-to-noise ratios are relatively high. Charge manipulation can enhance the value of the transformation algorithms in cases in which compromises their utility. Such situations include ES mass spectra of high-mass species that yield charge states that are not baseline resolved, mixtures with many components and mixtures in which the signals from major components overwhelm signals from minor components. Examples of improved mass determination are illustrated for proteins using ion-ion chemistry as the means for charge state manipulation and the quadrupole ion trap as the mass analyzer.

Ion/ion proton-transfer kinetics: implications for analysis of ions derived from electrospray of protein mixtures.

Protein ions of different mass and charge but similar mass-to-charge ratios are shown to undergo significantly different rates of differential neutralization, defined as the rate of change of charge with time, upon initiation of reactions with oppositely charged ions in the quadrupole ion trap. Overlapping charge state distributions arising from mixtures of ions of dissimilar charge are separated on the mass-to-charge scale at short reactions times. It is also demonstrated that the time frame for near total neutralization, defined as charge reduction to the 1+ ion, is relatively insensitive to initial charge state. It is shown, for example, that the (M + 11H)(11+)-(M + 22H)22+ ions derived from horse skeletal muscle apomyoglobin yield the (M + H)+ ion as the major ion/ion reaction product over the same reaction period that largely converts doubly protonated bradykinin to the singly protonated species. Less than 25% of the bradykinin ions are expected to be totally neutralized when roughly 7% of the myoglobin ions are expected to be totally neutralized. The phenomenon of significantly different initial differential neutralization rates for ions of dissimilar charge, and the relative insensitivity to ion charge for total neutralization, can be used to advantage in strategies for protein ion mixture analysis.

Ion/ion chemistry of high-mass multiply charged ions.

Electrospray ionization has enabled the establishment of a new area of ion chemistry research based on the study of the reactions of high-mass multiply charged ions with ions of opposite polarity. The multiple-charging phenomenon associated with electrospray makes possible the generation of multiply charged reactant ions that yield charged products as a result of partial neutralization due to ion/ion chemistry. The charged products can be readily studied with mass spectrometric methods, providing useful insights into reaction mechanisms. This review presents the research done in this area, all of which has been performed within the past decade. Ion/ion chemistry has been studied at near-atmospheric pressure in a reaction region that leads to the atmospheric/vacuum interface of a mass spectrometer, and within a quadrupole ion trap operated with a bath gas at a pressure of 1 mtorr. Proton transfer has been the most common reaction type for high-mass ions, but other forms of "charge transfer," such as electron transfer and fluoride transfer, have also been observed. For some ion/ion reactions, attachment of the two reactants has been observed. Multiply charged ion/ion reactions are fast, due to the long-range Coulombic attraction, and they are universal in that any pair of oppositely charged ions is expected to react due to the high exothermicity associated with mutual neutralization. The kinetics of reaction for multiply charged ions, derived from the same molecule with a given singly charged reactant ion, follow a charge-squared dependence, at least under normal quadrupole ion trap conditions. This dependence suggests that reaction rates are determined by the long-range Coulomb attraction, and that the ions react with constant efficiency as a function of charge state. In the case of proton transfer reactions from polypeptides to even-electron perfluorocarbon anions, no fragmentation of the polypeptide product ions has, as yet, been observed. Electron transfer from small oligonucleotide anions to rare gas cations, on the other hand, results in extensive fragmentation of the nucleic acid product ions. The extent of fragmentation decreases as the size of the oligonucleotide anions increases, reflecting a decrease in fragmentation rates associated with an increase in the number of internal degrees of freedom of the oligonucleotide. When ion-cooling rates become competitive with dissociation rates, the initially formed product ions are stabilized and fragmentation is avoided. Collisional cooling, therefore, likely plays an important role in the relative lack of dissociation observed thus far as a result of ion/ion reactions for most high-mass ions. The observed dependence of ion/ion reaction rates on the square of the ion charge, the universal nature of mutual neutralization, and the relative lack of fragmentation that arises from ion/ion reactions, makes ion/ion chemistry a particularly useful means for manipulating charge states. This review emphasizes applications that take advantage of the unique characteristics of ion/ion proton transfer chemistry for manipulating charge states. These applications include mixture analysis by electrospray, precursor ion charge state manipulation for tandem mass spectrometry studies, and simplified interpretation of product ion spectra.

Ion/ion reactions for oligopeptide mixture analysis: application to mixtures comprised of 0.5-100 kDa components.

Oligopeptide mixtures have been subjected to electrospray ionization, accumulated within a quadrupole ion trap, and subjected to ion/ion proton transfer reactions with anions derived from perfluoro-1,3-dimethylcyclohexane. Various mixtures were studied with approximate molecular weight ranges of 0.5-8.5, 12-30, 45-100, and 0.5-100 kDa. Mixtures of known composition were studied to evaluate the mixture complexity amenable to electrospray combined with ion/ion reactions to reduce spectral complexity associated with multiple charging. Mixture analysis with at least 40 components of low and medium molecular weight and roughly comparable solution concentrations appears to be straightforward. No matrix effects upon ionization were implicated in the data for the low and medium molecular weight mixtures but bovine albumin appeared to inhibit signals from bovine transferrin and chicken conalbumin in the high molecular weight mix. Furthermore, the presence of abundant low mass-to-charge ions appeared to inhibit signals from high molecular weight proteins (> 40 kDa) in the 0.5-100 kDa mix. Such an observation is consistent with dynamic range limitations that can arise from discrimination based on ion space charge effects, although an ionization matrix effect could not be precluded from the data reported here. The results reported here indicate that the limitation to mixture complexity amenable to electrospray mass spectrometry imposed by spectral congestion associated with multiple charging can be significantly reduced via ion/ion reactions. The use of ion/ion reactions can therefore facilitate the study of other factors that can impose limitations to mixture analysis, such as matrix effects upon ionization and differences in ion transmission, accumulation, storage, and detection efficiencies.

Counting basic sites in oligopeptides via gas-phase ion chemistry.

Cations derived from oligopeptides ranging from laminin fragment (5 residues) to beta-lactoglobulin (162 residues) have been subjected to gas-phase ion/molecule reactions with hydroiodic acid. The sum of the ion charge state and the maximum number of molecules of hydroiodic acid that attach to the ion is equal to the total number of lysines, arginines, histidines, and N-termini consisting of a primary amine for ions derived from all 21 oligopeptides studied. These results suggest that ion/molecule reactions can provide useful information regarding oligopeptide basic site number, which might be used as a criterion for searching protein data bases instead of, or in conjunction with, use of proteolytic digestion or gas-phase ion dissociation procedures.

Charge reduction of oligonucleotide anions via gas-phase electron transfer to xenon cations.

Multiply-charged anions derived from electrospray (ES) ionization of the oligonucleotide 5'-d(GTCTTAGCGCTAAGAC)-3' have been subjected to electron transfer reactions with ionized xenon in a quadrupole ion trap and found to undergo minimal fragmentation. This observation stands in contrast with electron transfer to rare gases from anions of smaller oligonucleotides which have been shown to undergo extensive fragmentation. The present results are attributed to longer anion lifetimes for the larger oligonucleotide anions (following highly exothermic electron transfer) which then allow for collisional cooling by the helium bath gas. Reduction to singly charged anions with minimal fragmentation is noted, indicating that xenon cations can be considered as a reagent for simplifying ES mass spectra of moderately sized oligonucleotides. The relative merits of the use of xenon cations in the quadropole ion trap for this purpose is discussed.

Ion/ion proton transfer reactions for protein mixture analysis.

Ion/ion proton transfer reactions are shown to be an effective means to facilitate the resolution of ions in electrospray mass spectrometry that differ in mass and charge but are similar in mass-to-charge ratio. Examples are shown in which a minor contaminant protein in a ribonuclease B solution is clearly apparent after ion/ion proton transfer but not in the conventional electrospray mass spectrum. A further example involving a mixture of bovine serum albumin and bovine transferrin also showed the identification of previously unnoticed "contaminant" polymer. The latter mixture also illustrated important issues in the use of the quadrupole ion trap as a reaction vessel and mass analyzer for high mass-to-charge ratio ions. The results suggest that the use of ion trap operating parameters specifically tailored for storage, ejection, detection, and mass-to-charge analysis of high mass-to-charge ratio ions can have attractive analytical figures of merit for determining mixtures of relatively high-mass proteins and, by extension, other types of high-mass biopolymers.

Cation attachment to multiply charged anions of oxidized bovine insulin A-chain.

Multiply charged anions of oxidized bovine insulin A-chain react with protonated quinoline exclusively by proton transfer in a Paul trap operated with helium bath gas at a pressure of 10(-3) Torr. The isomeric [C9H8N]+ ions formed from the reaction of [C4H4]+ with pyridine, on the other hand, react largely by attachment to the multiply charged anions of oxidized bovine insulin A-chain. This observation can be rationalized on the basis of competition between unimolecular decomposition versus cooling of the ion-ion collision complex. In the case of protonated quinoline, no significant barriers are expected along the reaction coordinate for proton transfer. However, the [C9H8N]+ ion-molecule reaction product is not expected to transfer a proton without undergoing rearrangement, as is consistent with ion trap collisional activation results. The rearrangement reaction introduces a significant barrier along the reaction coordinate, thereby increasing the lifetime of the ion-ion collision complex. RRKM modeling for a polypeptide of comparable size suggests that a barrier of 0.6 eV or greater will allow for the observation of cation attachment whereas the lifetimes of collision complexes with well depths less than approximately 0.6 eV are too short for collisional cooling by the bath gas to be effective.

Calcium dynamics and homeostasis in a mathematical model of the principal cell of the cortical collecting tubule.

Calcium (Ca) dynamics are incorporated into a mathematical model of the principal cell in the cortical collecting tubule developed earlier in Strieter et al. (1992a. Am. J Physiol. 263:F1063-1075). The Ca components are modeled after the Othmer-Tang model for IP(3)-sensitive calcium channels (1993, in Experimental and Theoretical Advances in Biological Pattern Formation, 295-319). There are IP(3)-sensitive Ca channels and ATP-driven pumps on the membrane of the endoplasmic reticulum. Calcium enters the cell passively down its electrochemical gradient. A Ca pump and Na/Ca exchange in the basolateral membrane are responsible for the extrusion of cytoplasmic calcium. Na/Ca exchange can also operate in reverse mode to transport Ca into the cell. Regulatory effects of cytoplasmic Ca on the apical Na channels are modeled after experimental data that indicate apical Na permeability varies inversely with cytoplasmic Ca concentration. Numerical results on changes in intracellular Ca caused by decreasing NaCl in the bath and the lumen are similar to those from experiments in Bourdeau and Lau (1990. Am. J Physiol. 258:F1497-1503). This match of simulation and experiment requires the synergistic action of the Na/Ca exchanger and the Ca regulated apical Na permeability. In a homogeneous medium, cytoplasmic Ca becomes oscillatory when extracellular Na is severely decreased, as observed in experiments of cultured principal cells (Koster, H., C. van Os and R. Bindels. 1993. Kidney Int.43:828-836). This essentially pathological situation arises because the hyperpolarization of membrane potential caused by Na-free medium increases Ca influx into the cell, while the Na/Ca exchanger is inactivated by the low extracellular Na and can no longer move Ca out of the cell effectively. The raising of the total amount of intracellular Ca induces oscillatory Ca movement between the cytoplasm and the endoplasmic reticulum. Ca homeostasis is investigated under the condition of severe extracellular Ca variations. As extracellular Ca is decreased, Ca regulation is greatly impaired if Ca does not regulate apical ionic transport. The simulations indicate that the Na/Ca exchanger alone has only limited regulatory capacity. The Ca regulated apical sodium or potassium permeability are essential for regulation of cytoplasmic Ca in the principal cell of the cortical collecting tubule.

Simplification and analysis of models of calcium dynamics based on IP3-sensitive calcium channel kinetics.

We study the models for calcium (Ca) dynamics developed in earlier studies, in each of which the key component is the kinetics of intracellular inositol-1,4,5-trisphosphate-sensitive Ca channels. After rapidly equilibrating steps are eliminated, the channel kinetics in these models are represented by a single differential equation that is linear in the state of the channel. In the reduced kinetic model, the graph of the steady-state fraction of conducting channels as a function of log10(Ca) is a bell-shaped curve. Dynamically, a step increase in inositol-1,4,5-trisphosphate induces an incremental increase in the fraction of conducting channels, whereas a step increase in Ca can either potentiate or inhibit channel activation, depending on the Ca level before and after the increase. The relationships among these models are discussed, and experimental tests to distinguish between them are given. Under certain conditions the models for intracellular calcium dynamics are reduced to the singular perturbed form epsilon dx/d tau = f(x, y, p), dy/d tau = g(x, y, p). Phase-plane analysis is applied to a generic form of these simplified models to show how different types of Ca response, such as excitability, oscillations, and a sustained elevation of Ca, can arise. The generic model can also be used to study frequency encoding of hormonal stimuli, to determine the conditions for stable traveling Ca waves, and to understand the effect of channel properties on the wave speed.

Comparison of central core and radially separated models of renal inner medulla.

In this paper we describe the effect of partitioning exchange of ascending thin limb (ATL) and collecting duct (CD) between a central vascular space (CORE) and a radially separated capillary node (NODE) in a mathematical model of the concentrating mechanism of the renal inner medulla. A detailed description of the model has been provided [J. L. Stephenson, J. F. Jen, H. Wang, and R. P. Tewarson. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F680-F692, 1995]. We define a partition coefficient theta, which denotes the fractional exchange of CD and ATL with the NODE. Thus with theta = 0 we have a central core model, in which the ATL and CD exchange with the CORE only, and with theta = 1 we have a totally radially separated model, in which the ATL and CD exchange with the NODE only. Decreasing the partition coefficient from 1 to 0 effects a continuous transition from a totally radially separated model to a central core model. As this transition progresses with increasing exchange with the CORE, the osmolalities in all structures become nearly the same at the papilla, and the ability to transport salt uphill is lost. This is true even with no radial diffusion. However, radial diffusion and direct exchange with the CORE act synergistically in decreasing osmolality differences at the papilla and the capacity for convective uphill transport. These are lost in a more or less parallel way. There is, however, no significant concomitant change in concentrating ability. These results indicate that models with radial mixing of the interstitial vascular space are probably reasonably good approximations for the inner medulla.