PtdSer as a signaling lipid determined by privileged localization of ORP5 and ORP8 at ER/PM junctional foci to determine PM and ER PtdSer/PI(4)P ratio and cell function.

The membrane contact site ER/PM junctions are hubs for signaling pathways, including Ca2+ signaling. Phosphatidylserine (PtdSer) mediates various physiological functions; however, junctional PtdSer composition and the role of PtdSer in Ca2+ signaling and Ca2+-dependent gene regulation are not understood. Here, we show that STIM1-formed junctions are required for PI(4)P/PtdSer exchange by ORP5 and ORP8, which have reciprocal lipid exchange modes and function as a rheostat that sets the junctional PtdSer/PI(4)P ratio. Targeting the ORP5 and ORP8 and their lipid transfer ORD domains to PM subdomains revealed that ORP5 sets low and ORP8 high junctional PI(4)P/PtdSer ratio that controls STIM1-STIM1 and STIM1-Orai1 interaction and the activity of the SERCA pump to determine the pattern of receptor-evoked Ca2+ oscillations, and consequently translocation of NFAT to the nucleus. Significantly, targeting the ORP5 and ORP8 ORDs to the STIM1 ER subdomain reversed their function. Notably, changing PI(4)P/PtdSer ratio by hydrolysis of PM or ER PtdSer with targeted PtdSer-specific PLA1a1 reproduced the ORPs function. The function of the ORPs is determined both by their differential lipid exchange modes and by privileged localization at the ER/PM subdomains. These findings reveal a role of PtdSer as a signaling lipid that controls the available PM PI(4)P, the unappreciated role of ER PtdSer in cell function, and the diversity of the ER/PM junctions. The effect of PtdSer on the junctional PI(4)P level should have multiple implications in cellular signaling and functions.

Diurnal properties of voltage-gated Ca2+ currents in suprachiasmatic nucleus and roles in action potential firing.

KEY POINTS: Circadian oscillations in spontaneous action potential firing in the suprachiasmatic nucleus (SCN) translate time-of-day throughout the mammalian brain. The ion channels that regulate the circadian pattern of SCN firing have not been comprehensively identified. Ca2+ channels regulate action potential activity across many types of excitable cells, and the activity of L-, N-, P/Q- and R-type channels are required for normal daytime firing frequency in SCN neurons and circuit rhythms. Only the L-type Ca2+ current exhibits a day versus night difference in current magnitude, providing insight into the mechanism that produces rhythmic action potential firing in SCN. ABSTRACT: The mammalian circadian clock encodes time via rhythmic action potential activity in the suprachiasmatic nucleus (SCN) of the hypothalamus, which governs daily rhythms in physiology and behaviour. SCN neurons exhibit 24 h oscillations in spontaneous firing, with higher firing during day compared to night. Several ionic currents have been identified that regulate SCN firing, including voltage-gated Ca2+ currents, but the circadian regulation of distinct voltage-gated Ca2+ channel (VGCC) components has not been comprehensively addressed. In this study, whole-cell L- (nimodipine-sensitive), N- and P/Q- (ω-agatoxin IVA, ω-conotoxin GVIA, ω-conotoxin MVIIC-sensitive), R- (Ni2+ -sensitive) and T-type (TTA-P2-sensitive) currents were recorded from day and night SCN slices. Using standard voltage protocols, Ni2+ -sensitive currents comprised the largest proportion of total VGCC current, followed by nimodipine-, ω-agatoxin IVA-, ω-conotoxin GVIA- and TTA-P2-sensitive currents. Only the nimodipine-sensitive current exhibited a diurnal difference in magnitude, with daytime current larger than night. No diurnal variation was observed for the other Ca2+ current subtypes. The difference in nimodipine-sensitive current was due to larger peak current activated during the day, not differences in inactivation, and was eliminated by Bay K8644. Blocking L-type channels decreased firing selectively during the day, consistent with higher current magnitudes, and reduced SCN circuit rhythmicity recorded by multi-electrode arrays. Yet blocking N-, P/Q- and R-type channels also decreased daytime firing, with little effect at night, and decreased circuit rhythmicity. These data identify a unique diurnal regulation of L-type current among the major VGCC subtypes in SCN neurons, but also reveal that diurnal modulation is not required for time-of-day-specific effects on firing and circuit rhythmicity.

hERG1a and hERG1b potassium channel subunits directly interact and preferentially form heteromeric channels.

Voltage-activated human ether-á-go-go-related gene (hERG) potassium channels are critical for the repolarization of cardiac action potentials and tune-spike frequency adaptation in neurons. Two isoforms of mammalian ERG1 channel subunits, ERG1a and ERG1b, are the principal subunits that conduct the IKr current in the heart and are also broadly expressed in the nervous system. However, there is little direct evidence that ERG1a and ERG1b form heteromeric channels. Here, using electrophysiology, biochemistry, and fluorescence approaches, we systematically tested for direct interactions between hERG1a and hERG1b subunits. We report 1) that hERG1a dominant-negative subunits suppress hERG1b currents (and vice versa), 2) that disulfide bonds form between single cysteine residues experimentally introduced into an extracellular loop of hERG1a and hERG1b subunits and produce hERG1a-hERG1b dimers, and 3) that hERG1a and hERG1b subunits tagged with fluorescent proteins that are FRET pairs exhibit robust energy transfer at the plasma membrane. Thus, multiple lines of evidence indicated a physical interaction between hERG1a and hERG1b, consistent with them forming heteromeric channels. Moreover, co-expression of variable ratios of hERG1a and hERG1b RNA yielded channels with deactivation kinetics that reached a plateau and were different from those of hERG1b channels, consistent with a preference of hERG1b subunits for hERG1a subunits. Cross-linking studies revealed that an equal input of hERG1a and hERG1b yields more hERG1a-hERG1a or hERG1a-hERG1b dimers than hERG1b-hERG1b dimers, also suggesting that hERG1b preferentially interacts with hERG1a. We conclude that hERG1b preferentially forms heteromeric ion channels with hERG1a at the plasma membrane.

Gated regulation of CRAC channel ion selectivity by STIM1.

Two defining functional features of ion channels are ion selectivity and channel gating. Ion selectivity is generally considered an immutable property of the open channel structure, whereas gating involves transitions between open and closed channel states, typically without changes in ion selectivity. In store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels, the molecular mechanism of channel gating by the CRAC channel activator, stromal interaction molecule 1 (STIM1), remains unknown. CRAC channels are distinguished by a very high Ca(2+) selectivity and are instrumental in generating sustained intracellular calcium concentration elevations that are necessary for gene expression and effector function in many eukaryotic cells. Here we probe the central features of the STIM1 gating mechanism in the human CRAC channel protein, ORAI1, and identify V102, a residue located in the extracellular region of the pore, as a candidate for the channel gate. Mutations at V102 produce constitutively active CRAC channels that are open even in the absence of STIM1. Unexpectedly, although STIM1-free V102 mutant channels are not Ca(2+)-selective, their Ca(2+) selectivity is dose-dependently boosted by interactions with STIM1. Similar enhancement of Ca(2+) selectivity is also seen in wild-type ORAI1 channels by increasing the number of STIM1 activation domains that are directly tethered to ORAI1 channels, or by increasing the relative expression of full-length STIM1. Thus, exquisite Ca(2+) selectivity is not an intrinsic property of CRAC channels but rather a tuneable feature that is bestowed on otherwise non-selective ORAI1 channels by STIM1. Our results demonstrate that STIM1-mediated gating of CRAC channels occurs through an unusual mechanism in which permeation and gating are closely coupled.

Respiratory Syncytial Virus Uses CX3CR1 as a Receptor on Primary Human Airway Epithelial Cultures.

Respiratory syncytial virus (RSV) is the most frequent cause of lower respiratory disease in infants, but no vaccine or effective therapy is available. The initiation of RSV infection of immortalized cells is largely dependent on cell surface heparan sulfate (HS), a receptor for the RSV attachment (G) glycoprotein in immortalized cells. However, RSV infects the ciliated cells in primary well differentiated human airway epithelial (HAE) cultures via the apical surface, but HS is not detectable on this surface. Here we show that soluble HS inhibits infection of immortalized cells, but not HAE cultures, confirming that HS is not the receptor on HAE cultures. Conversely, a "non-neutralizing" monoclonal antibody against the G protein that does not block RSV infection of immortalized cells, does inhibit infection of HAE cultures. This antibody was previously shown to block the interaction between the G protein and the chemokine receptor CX3CR1 and we have mapped the binding site for this antibody to the CX3C motif and its surrounding region in the G protein. We show that CX3CR1 is present on the apical surface of ciliated cells in HAE cultures and especially on the cilia. RSV infection of HAE cultures is reduced by an antibody against CX3CR1 and by mutations in the G protein CX3C motif. Additionally, mice lacking CX3CR1 are less susceptible to RSV infection. These findings demonstrate that RSV uses CX3CR1 as a cellular receptor on HAE cultures and highlight the importance of using a physiologically relevant model to study virus entry and antibody neutralization.

The C- and N-terminal STIM1 binding sites on Orai1 are required for both trapping and gating CRAC channels.

Ca(2+) release-activated Ca(2+) (CRAC) channels are activated through a mechanism wherein depletion of intracellular calcium stores results in the aggregation of stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca(2+) sensor, and Orai1, the CRAC channel protein, at overlapping sites in the ER and plasma membranes (PMs). The redistribution of CRAC channels is driven through direct STIM1-Orai1 binding, an important event that not only controls gating, but also regulates Orai1 ion selectivity. Orai1 harbours two STIM1 binding sites, one each on the intracellular C- and N-termini. Previous studies have proposed modular functions for these sites, with the C-terminal site thought to regulate STIM1-Orai1 binding and trapping of Orai1 at the ER-PM junctions, and the N-terminal site mediating gating. However, here we find that a variety of mutations in the N-terminal site impair the binding of Orai1 to STIM1 and to the soluble CRAC activation domain (CAD). Gating could be restored in several N- and C-terminal point mutants by directly tethering the minimal STIM1 activation domain (S) to Orai1 (Orai1-SS channels), indicating that loss of gating in these mutants by full-length STIM1 results from insufficient ligand binding. By contrast, gating could not be restored in mutant Orai1-SS channels carrying more drastic deletions that removed the STIM1 binding sites (1-85, 73-85, or 272-279 Orai1), suggesting that STIM1 binding to both sites is essential for channel activation. Moreover, analysis of ion selectivity indicated that the molecular requirements for gating and modulation of ion selectivity are similar, yet substantively different from those for Orai1 puncta formation, suggesting that ion selectivity and gating are mechanistically coupled in CRAC channels. Our results indicate that the C- and N-terminal STIM1 binding sites are both essential for multiple aspects of Orai1 function including STIM1-Orai1 association, Orai1 trapping, and channel activation.

Omega-3 Index improves after increased intake of foods with omega-3 polyunsaturated fatty acids among US service academy cadets.

The inclusion of omega-3 fatty acids in our dietary intake is important for performance and recovery and may reduce the risk of various health issues. Studies have shown the omega-3 fatty acid status of US service members is low. The purpose of this study was to evaluate whether offering fish and omega-3-enhanced foods would increase the Omega-3 Index (O3I). We hypothesize cadets will increase O3I with enhanced omega-3 options more than fish alone. Food service venues at 3 US service academies offered fish and other omega-3 foods to cadets for 12 weeks. Questionnaires were used to collect information on the dietary habits and omega-3 food intake of participants. The O3I of each participant was measured at baseline, mid- (6 weeks), and after data collection (12 weeks) time points. Following the 12 weeks, we found a significant increase in O3I. More specifically, the intake of other omega-3 foods, smoothies (3 per week) and toppings (3 per week), increased O3I in cadets. This study identified a strategy encouraging omega-3 food intake and improving O3I among cadets. These results help us understand how we can more effectively impact military service member nutrition for optimal health and performance.

Permeation, selectivity and gating in store-operated CRAC channels.

Store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels are a widespread mechanism for generating cellular Ca(2+) signals and regulate many Ca(2+)-dependent functions, including transcription, motility and proliferation. The opening of CRAC channels in response to depletion of intracellular Ca(2+) stores involves a cascade of cellular events that culminate in direct interactions between STIM1, the endoplasmic reticulum Ca(2+) sensor, and the channels composed of Orai proteins. Evidence gathered over the last two decades indicates that CRAC channels display a unique functional pore fingerprint characterized by exquisite Ca(2+) selectivity, low unitary conductance, and low permeability to large cations. Here, we review the key pore properties of CRAC channels and discuss recent progress in addressing the molecular foundations of these properties. Structure-function and cysteine-scanning studies have revealed the identity and organization of pore-lining residues, including those that form the selectivity filter, providing a structural framework for understanding CRAC channel pore properties. Recent studies in pore mutants that produce STIM1-independent constitutive channel activation indicate that exquisite Ca(2+) selectivity in CRAC channels is not hardwired into Orai proteins, but is instead manifested only following the binding of STIM1 to the intrinsically poorly Ca(2+)-selective Orai channels. These findings reveal new functional aspects of CRAC channels and suggest that the selectivity filter of the CRAC channel is a dynamic structure whose conformation and functional properties are powerfully regulated by the channel activation stimulus.

Structural determinants of ion permeation in CRAC channels.

CRAC channels generate Ca(2+) signals critical for the activation of immune cells and exhibit an intriguing pore profile distinguished by extremely high Ca(2+) selectivity, low Cs(+) permeability, and small unitary conductance. To identify the ion conduction pathway and gain insight into the structural bases of these permeation characteristics, we introduced cysteine residues in the CRAC channel pore subunit, Orai1, and probed their accessibility to various thiol-reactive reagents. Our results indicate that the architecture of the ion conduction pathway is characterized by a flexible outer vestibule formed by the TM1-TM2 loop, which leads to a narrow pore flanked by residues of a helical TM1 segment. Residues in TM3, and specifically, E190, a residue considered important for ion selectivity, are not close to the pore. Moreover, the outer vestibule does not significantly contribute to ion selectivity, implying that Ca(2+) selectivity is conferred mainly by E106. The ion conduction pathway is sufficiently narrow along much of its length to permit stable coordination of Cd(2+) by several TM1 residues, which likely explains the slow flux of ions within the restrained geometry of the pore. These results provide a structural framework to understand the unique permeation properties of CRAC channels.

Targeting lymphotoxin-mediated negative selection to prevent prostate cancer in mice with genetic predisposition.

The identification of individuals genetically susceptible to cancer calls for preventive measures to minimize the cancer risk in these high-risk populations. Immune prevention is made necessary by the anticipated health threat, but lack of enough high-affinity T cells against tumor-associated antigens and the unpredictability of tumor antigens make antigen-based immune prevention untenable for cancer. To address this issue, we explored a non-antigen-based cancer immune prevention strategy using the transgenic adenocarcinoma of mouse prostate model that spontaneously develops prostate cancer with 100% penetrance. We show that targeted mutation of the lymphotoxin alpha (LTalpha) gene efficiently rescued tumor-reactive T cells, drastically reduced cancer incidence, and almost completely ablated metastasis. Remarkably, short-term treatments with the fusion protein consisting of constant region of IgG and extracellular domain of lymphotoxin beta receptor (LTbetaRIg) interrupted clonal deletion, reduced the size of the primary cancer, and completely prevented metastasis later in life. Our data demonstrated the value of non-antigen-based immune prevention for those with a genetic predisposition to cancer.

Contributions of CaV1.3 Channels to Ca2+ Current and Ca2+-Activated BK Current in the Suprachiasmatic Nucleus.

Daily regulation of Ca2+ - and voltage-activated BK K+ channel activity is required for action potential rhythmicity in the suprachiasmatic nucleus (SCN) of the hypothalamus, the brain's circadian clock. In SCN neurons, BK activation is dependent upon multiple types of Ca2+ channels in a circadian manner. Daytime BK current predominantly requires Ca2+ influx through L-type Ca2+ channels (LTCCs), a time when BK channels are closely coupled with their Ca2+ source. Here we show that daytime BK current is resistant to the Ca2+ chelator BAPTA. However, at night when LTCCs contribute little to BK activation, BK current decreases by a third in BAPTA compared to control EGTA conditions. In phase with this time-of-day specific effect on BK current activation, LTCC current is larger during the day. The specific Ca2+ channel subtypes underlying the LTCC current in SCN, as well as the subtypes contributing the Ca2+ influx relevant for BK current activation, have not been identified. SCN neurons express two LTCC subtypes, CaV1.2 and CaV1.3. While a role for CaV1.2 channels has been identified during the night, CaV1.3 channel modulation has also been suggested to contribute to daytime SCN action potential activity, as well as subthreshold Ca2+ oscillations. Here we characterize the role of CaV1.3 channels in LTCC and BK current activation in SCN neurons using a global deletion of CACNA1D in mouse (CaV1.3 KO). CaV1.3 KO SCN neurons had a 50% reduction in the daytime LTCC current, but not total Ca2+ current, with no difference in Ca2+ current levels at night. During the day, CaV1.3 KO neurons exhibited oscillations in membrane potential, and most neurons, although not all, also had BK currents. Changes in BK current activation were only detectable at the highest voltage tested. These data show that while CaV1.3 channels contribute to the daytime Ca2+ current, this does not translate into a major effect on the daytime BK current. These data suggest that BK current activation does not absolutely require CaV1.3 channels and may therefore also depend on other LTCC subtypes, such as CaV1.2.

Ca2+ Signaling in Exocrine Cells.

Calcium (Ca2+) and cyclic AMP (cAMP) signaling cross talk and synergize to stimulate the cardinal functions of exocrine cells, regulated exocytosis, and fluid and electrolyte secretion. This physiological process requires the organization of the two signaling pathways into complexes at defined cellular domains and close placement. Such domains are formed by membrane contact sites (MCS). This review discusses the basic properties of Ca2+ signaling in exocrine cells, the role of MCS in the organization of cell signaling and in cross talk and synergism between the Ca2+ and cAMP signaling pathways and, finally, the mechanism by which the Ca2+ and cAMP pathways synergize to stimulate epithelial fluid and electrolyte secretion.

Membrane transporters for anions that use a relay mechanism.

A new type of synthetic membrane transporter is described and shown to operate in vesicles by a relay mechanism. The transporter structure is a phosphatidylcholine derivative with a urea group appended to the end of its sn-2 acyl chain. The urea can bind a chloride ion at the membrane surface via hydrogen bonds and then relay it through the bilayer interior to an acceptor molecule located in the opposite membrane leaflet. Three phosphatidylcholine derivatives were studied and transport rates increased with transporter affinity for chloride. The results of various controls studies are consistent with an anion countertransport process using a relay mechanism and a kinetically active aggregate of two or four transporter molecules. Transport is inhibited if the transporter resides in only one leaflet of the membrane, if the bilayer is too thick, and if the counteranion is sulfate dianion. The expected favorable formulation properties of these amphiphilic compounds should facilitate efforts to transform them into tools for biomedical research and perhaps as therapeutic agents.

Orai1 mutations alter ion permeation and Ca2+-dependent fast inactivation of CRAC channels: evidence for coupling of permeation and gating.

Ca(2+) entry through store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels is an essential trigger for lymphocyte activation and proliferation. The recent identification of Orai1 as a key CRAC channel pore subunit paves the way for understanding the molecular basis of Ca(2+) selectivity, ion permeation, and regulation of CRAC channels. Previous Orai1 mutagenesis studies have indicated that a set of conserved acidic amino acids in trans membrane domains I and III and in the I-II loop (E106, E190, D110, D112, D114) are essential for the CRAC channel's high Ca(2+) selectivity. To further dissect the contribution of Orai1 domains important for ion permeation and channel gating, we examined the role of these conserved acidic residues on pore geometry, properties of Ca(2+) block, and channel regulation by Ca(2+). We find that alteration of the acidic residues lowers Ca(2+) selectivity and results in striking increases in Cs(+) permeation. This is likely the result of enlargement of the unusually narrow pore of the CRAC channel, thus relieving steric hindrance for Cs(+) permeation. Ca(2+) binding to the selectivity filter appears to be primarily affected by changes in the apparent on-rate, consistent with a rate-limiting barrier for Ca(2+) binding. Unexpectedly, the mutations diminish Ca(2+)-mediated fast inactivation, a key mode of CRAC channel regulation. The decrease in fast inactivation in the mutant channels correlates with the decrease in Ca(2+) selectivity, increase in Cs(+) permeability, and enlargement of the pore. We propose that the structural elements involved in ion permeation overlap with those involved in the gating of CRAC channels.

Differential contribution of Ca2+ sources to day and night BK current activation in the circadian clock.

Large conductance K+ (BK) channels are expressed widely in neurons, where their activation is regulated by membrane depolarization and intracellular Ca2+ (Ca2+i). To enable this regulation, BK channels functionally couple to both voltage-gated Ca2+ channels (VGCCs) and channels mediating Ca2+ release from intracellular stores. However, the relationship between BK channels and their specific Ca2+ source for particular patterns of excitability is not well understood. In neurons within the suprachiasmatic nucleus (SCN)-the brain's circadian clock-BK current, VGCC current, and Ca2+i are diurnally regulated, but paradoxically, BK current is greatest at night when VGCC current and Ca2+i are reduced. Here, to determine whether diurnal regulation of Ca2+ is relevant for BK channel activation, we combine pharmacology with day and night patch-clamp recordings in acute slices of SCN. We find that activation of BK current depends primarily on three types of channels but that the relative contribution changes between day and night. BK current can be abrogated with nimodipine during the day but not at night, establishing that L-type Ca2+ channels (LTCCs) are the primary daytime Ca2+ source for BK activation. In contrast, dantrolene causes a significant decrease in BK current at night, suggesting that nighttime BK activation is driven by ryanodine receptor (RyR)-mediated Ca2+i release. The N- and P/Q-type Ca2+ channel blocker ω-conotoxin MVIIC causes a smaller reduction of BK current that does not differ between day and night. Finally, inhibition of LTCCs, but not RyRs, eliminates BK inactivation, but the BK ß2 subunit was not required for activation of BK current by LTCCs. These data reveal a dynamic coupling strategy between BK channels and their Ca2+ sources in the SCN, contributing to diurnal regulation of SCN excitability.

Conformational control of HCl co-transporter: imidazole functionalised isophthalamide vs. 2,6-dicarboxamidopyridine.

Replacement of the central isophthalamide core in a synthetic HCl receptor, with a 2,6-dicarboxamidopyridine, leads to a more preorganised molecular structure that exhibits higher chloride affinity and membrane transport flux.

Recent Advances in Synthetic Membrane Transporters.

It is 25 years since the first report of a synthetic ion channel transporter. Today, dozens of molecular and supramolecular designs have been developed to facilitate ion and small molecule transport across a bilayer membrane. Presented here is a concise summary of the advances made over the past four years. The transporters are grouped into three mechanistic classes: mobile carrier, monomeric channel, and self-assembled pore. Common building blocks are crown ethers, steroids, cyclodextrins, peptides, curcubiturils, and calixarenes. The eventual goal is to produce functional supramolecular devices such as sensors, enzyme assays, and lead candidates for pharmaceutical development.

STIM1 activates CRAC channels through rotation of the pore helix to open a hydrophobic gate.

Store-operated Ca2+ release-activated Ca2+ (CRAC) channels constitute a major pathway for Ca2+ influx and mediate many essential signalling functions in animal cells, yet how they open remains elusive. Here, we investigate the gating mechanism of the human CRAC channel Orai1 by its activator, stromal interacting molecule 1 (STIM1). We find that two rings of pore-lining residues, V102 and F99, work together to form a hydrophobic gate. Mutations of these residues to polar amino acids produce channels with leaky gates that conduct ions in the resting state. STIM1-mediated channel activation occurs through rotation of the pore helix, which displaces the F99 residues away from the pore axis to increase pore hydration, allowing ions to flow through the V102-F99 hydrophobic band. Pore helix rotation by STIM1 also explains the dynamic coupling between CRAC channel gating and ion selectivity. This hydrophobic gating mechanism has implications for CRAC channel function, pharmacology and disease-causing mutations.

Clinical Utility and Economic Impact of CYP2D6 Genotyping.

Pharmacogenetics examines an individual's genetic makeup to help predict the safety and efficacy of medications. Practical application optimizes treatment selection to decrease the failure rate of medications and improve clinical outcomes. Lack of efficacy is costly due to adverse drug reactions and increased hospital stays. Cytochrome P450 2D6 (CYP2D6) metabolizes roughly 25% of all drugs. Detecting variants that cause altered CYP2D6 enzymatic activity identifies patients at risk of adverse drug reactions or therapeutic failure with standard dosages of medications metabolized by CYP2D6. This article discusses the clinical application of pharmacogenetics to improve care and decrease costs.

Effects of erythropoietin on systemic hematocrit and oxygen transport in the splenectomized horse.

To test the hypotheses that erythropoietin (rhuEPO) treatment increases systemic hematocrit, maximal O2 uptake (VO2max, by elevated perfusive and diffusive O2 conductances) and performance five female horses (4-13 years) received 15 IU/kg rhuEPO (erythropoietin) three times per week for three weeks. These horses had been splenectomized over 1 year previously to avoid confounding effects from the mobilization of splenic red blood cell reserves. Each horse performed three maximal exercise tests (one per month) on an inclined (4°) treadmill to the limit of tolerance; two control trials and one following EPO treatment. Measurements of hemoglobin concentration ([Hb] and hematocrit), plasma and blood volume, VO2, cardiac output as well as arterial and mixed venous blood gases were made at rest and during maximal exercise. EPO increased resting [Hb] by 18% from 13.3 ± 0.6 to 15.7 ± 0.8 g/dL (mean ± SD) corresponding to an increased hematocrit from 36 ± 2 to 46 ± 2% concurrent with 23 and 10% reductions in plasma and blood volume, respectively (all P<0.05). EPO elevated VO2max by 20% from 25.7 ± 1.7 to 30.9 ± 3.4 L/min (P<0.05) via a 17% increase in arterial O2 content and 18% greater arteriovenous O2 difference in the face of an unchanged cardiac output. To achieve the greater VO2max after EPO, diffusive O2 conductance increased ∼ 30% (from 580 ± 76 to 752 ± 166 mL O2/mmHg/min, P<0.05) which was substantially greater than the elevation of perfusive O2 conductance. These effects of EPO were associated with an increased exercise performance (total running time: control, 216 ± 72; EPO, 264 ± 48 s, P<0.05). We conclude that EPO substantially increases VO2max and performance in the splenectomized horse via improved perfusive and diffusive O2 transport.