Hypoxic pulmonary vasoconstriction.

It has been known for more than 60 years, and suspected for over 100, that alveolar hypoxia causes pulmonary vasoconstriction by means of mechanisms local to the lung. For the last 20 years, it has been clear that the essential sensor, transduction, and effector mechanisms responsible for hypoxic pulmonary vasoconstriction (HPV) reside in the pulmonary arterial smooth muscle cell. The main focus of this review is the cellular and molecular work performed to clarify these intrinsic mechanisms and to determine how they are facilitated and inhibited by the extrinsic influences of other cells. Because the interaction of intrinsic and extrinsic mechanisms is likely to shape expression of HPV in vivo, we relate results obtained in cells to HPV in more intact preparations, such as intact and isolated lungs and isolated pulmonary vessels. Finally, we evaluate evidence regarding the contribution of HPV to the physiological and pathophysiological processes involved in the transition from fetal to neonatal life, pulmonary gas exchange, high-altitude pulmonary edema, and pulmonary hypertension. Although understanding of HPV has advanced significantly, major areas of ignorance and uncertainty await resolution.

Digoxin inhibits development of hypoxic pulmonary hypertension in mice.

Chronic hypoxia is an inciting factor for the development of pulmonary arterial hypertension. The mechanisms involved in the development of hypoxic pulmonary hypertension (HPH) include hypoxia-inducible factor 1 (HIF-1)-dependent transactivation of genes controlling pulmonary arterial smooth muscle cell (PASMC) intracellular calcium concentration ([Ca(2+)](i)) and pH. Recently, digoxin was shown to inhibit HIF-1 transcriptional activity. In this study, we tested the hypothesis that digoxin could prevent and reverse the development of HPH. Mice were injected daily with saline or digoxin and exposed to room air or ambient hypoxia for 3 wk. Treatment with digoxin attenuated the development of right ventricle (RV) hypertrophy and prevented the pulmonary vascular remodeling and increases in PASMC [Ca(2+)](i), pH, and RV pressure that occur in mice exposed to chronic hypoxia. When started after pulmonary hypertension was established, digoxin attenuated the hypoxia-induced increases in RV pressure and PASMC pH and [Ca(2+)](i). These preclinical data support a role for HIF-1 inhibitors in the treatment of HPH.

Activation of hypoxia-inducible factor-1 in pulmonary arterial smooth muscle cells by endothelin-1.

Numerous cellular responses to hypoxia are mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 plays a central role in the pathogenesis of hypoxic pulmonary hypertension. Under certain conditions, HIF-1 may utilize feedforward mechanisms to amplify its activity. Since hypoxia increases endothelin-1 (ET-1) levels in the lung, we hypothesized that during moderate, prolonged hypoxia ET-1 might contribute to HIF-1 signaling in pulmonary arterial smooth muscle cells (PASMCs). Primary cultures of rat PASMCs were treated with ET-1 or exposed to moderate, prolonged hypoxia (4% O(2) for 60 h). Levels of the oxygen-sensitive HIF-1α subunit and expression of HIF target genes were increased in both hypoxic cells and cells treated with ET-1. Both hypoxia and ET-1 also increased HIF-1α mRNA expression and decreased mRNA and protein expression of prolyl hydroxylase 2 (PHD2), which is the protein responsible for targeting HIF-1α for O(2)-dependent degradation. The induction of HIF-1α by moderate, prolonged hypoxia was blocked by BQ-123, an antagonist of ET-1 receptor subtype A. The effects of ET-1 were mediated by increased intracellular calcium, generation of reactive oxygen species, and ERK1/2 activation. Neither ET-1 nor moderate hypoxia induced the expression of HIF-1α or HIF target genes in aortic smooth muscle cells. These results suggest that ET-1 induces a PASMC-specific increase in HIF-1α levels by upregulation of HIF-1α synthesis and downregulation of PHD2-mediated degradation, thereby amplifying the induction of HIF-1α in PASMCs during moderate, prolonged hypoxia.

Ca2+ responses of pulmonary arterial myocytes to acute hypoxia require release from ryanodine and inositol trisphosphate receptors in sarcoplasmic reticulum.

In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing Ca(2+) release from the sarcoplasmic reticulum (SR) and Ca(2+) influx through store- and voltage-operated Ca(2+) channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca(2+) release, we measured [Ca(2+)](i) with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca(2+)-free Krebs Ringer bicarbonate solution (KRBS), brief exposures to caffeine (30 mM) and norepinephrine (300 µM), which activate SR ryanodine and inositol trisphosphate receptors (RyR, IP(3)R), respectively, or 4% O(2) caused rapid transient increases in [Ca(2+)](i), indicating intracellular Ca(2+) release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA; 10 µM) blocked subsequent Ca(2+) release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 µM) blocked Ca(2+) release to caffeine and hypoxia but not norepinephrine. The IP(3)R antagonist xestospongin C (XeC, 0.1 µM) blocked Ca(2+) release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca(2+)](i) that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca(2+)](i) induced by hypoxia, as well as the subsequent Ca(2+) influx that sustained this increase, required release of Ca(2+) from both RyR and IP(3)R, and 2) the SR Ca(2+) stores accessed by RyR, IP(3)R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca(2+)-ATPase.

Kinase-dependent activation of voltage-gated Ca2+ channels by ET-1 in pulmonary arterial myocytes during chronic hypoxia.

Exposure to chronic hypoxia (CH) causes pulmonary hypertension. The vasoconstrictor endothelin-1 (ET-1) is thought to play a role in the development of hypoxic pulmonary hypertension. In pulmonary arterial smooth muscle cells (PASMCs) from chronically hypoxic rats, ET-1 signaling is altered, with the ET-1-induced change in intracellular calcium concentration (Δ[Ca(2+)](i)) occurring through activation of voltage-dependent Ca(2+) channels (VDCC) even though ET-1-induced depolarization via inhibition of K(+) channels is lost. The mechanism underlying this response is unclear. We hypothesized that activation of VDCCs by ET-1 following CH might be mediated by protein kinase C (PKC) and/or Rho kinase, both of which have been shown to phosphorylate and activate VDCCs. To test this hypothesis, we examined the effects of PKC and Rho kinase inhibitors on the ET-1-induced Δ[Ca(2+)](i) in PASMCs from rats exposed to CH (10% O(2), 3 wk) using the Ca(2+)-sensitive dye fura 2-AM and fluorescent microscopy techniques. We found that staurosporine and GF109203X, inhibitors of PKC, and Y-27632 and HA 1077, Rho kinase inhibitors, reduced the ET-1-induced Δ[Ca(2+)](i) by >70%. Inhibition of tyrosine kinases (TKs) with genistein or tyrphostin A23, or combined inhibition of PKC, TKs, and Rho kinase, reduced the Δ[Ca(2+)](i) to a similar extent as inhibition of either PKC or Rho kinase alone. The ability of PKC or Rho kinase to activate VDCCs in our cells was verified using phorbol 12-myristate 13-acetate and GTP-γ-S. These results suggest that following CH, the ET-1-induced Δ[Ca(2+)](i) in PASMCs occurs via Ca(2+) influx through VDCCs mediated primarily by PKC, TKs, and Rho kinase.

Hypoxia-induced migration in pulmonary arterial smooth muscle cells requires calcium-dependent upregulation of aquaporin 1.

Pulmonary arterial smooth muscle cell (PASMC) migration is a key component of the vascular remodeling that occurs during the development of hypoxic pulmonary hypertension, although the mechanisms governing this phenomenon remain poorly understood. Aquaporin-1 (AQP1), an integral membrane water channel protein, has recently been shown to aid in migration of endothelial cells. Since AQP1 is expressed in certain types of vascular smooth muscle, we hypothesized that AQP1 would be expressed in PASMCs and would be required for migration in response to hypoxia. Using PCR and immunoblot techniques, we determined the expression of AQPs in pulmonary vascular smooth muscle and the effect of hypoxia on AQP levels, and we examined the role of AQP1 in hypoxia-induced migration in rat PASMCs using Transwell filter assays. Moreover, since the cytoplasmic tail of AQP1 contains a putative calcium binding site and an increase in intracellular calcium concentration ([Ca(2+)](i)) is a hallmark of hypoxic exposure in PASMCs, we also determined whether the responses were Ca(2+) dependent. Results were compared with those obtained in aortic smooth muscle cells (AoSMCs). We found that although AQP1 was abundant in both PASMCs and AoSMCs, hypoxia selectively increased AQP1 protein levels, [Ca(2+)](i), and migration in PASMCs. Blockade of Ca(2+) entry through voltage-dependent Ca(2+) or nonselective cation channels prevented the hypoxia-induced increase in PASMC [Ca(2+)](i), AQP1 levels, and migration. Silencing AQP1 via siRNA also prevented hypoxia-induced migration of PASMCs. Our results suggest that hypoxia induces a PASMC-specific increase in [Ca(2+)](i) that results in increased AQP1 protein levels and cell migration.

Enhancement of myofilament calcium sensitivity by acute hypoxia in rat distal pulmonary arteries.

Hypoxic contraction of pulmonary arterial smooth muscle is thought to require increases in both intracellular Ca(2+) concentration ([Ca(2+)](i)) and myofilament Ca(2+) sensitivity, which may or may not be endothelium-dependent. To examine the effects of hypoxia and endothelium on Ca(2+) sensitivity in pulmonary arterial smooth muscle, we measured the relation between [Ca(2+)](i) and isometric force at 37°C during normoxia (21% O(2)-5% CO(2)) and after 30 min of hypoxia (1% O(2)-5% CO(2)) in endothelium-intact (E+) and -denuded (E-) rat distal intrapulmonary arteries (IPA) permeabilized with staphylococcal α-toxin. Endothelial denudation enhanced Ca(2+) sensitivity during normoxia but did not alter the effects of hypoxia, which shifted the [Ca(2+)](i)-force relation to higher force in E+ and E- IPA. Neither hypoxia nor endothelial denudation altered Ca(2+) sensitivity in mesenteric arteries. In E+ and E- IPA, hypoxic enhancement of Ca(2+) sensitivity was abolished by the nitric oxide synthase inhibitor N(ω)-nitro-l-arginine methyl ester (30 µM), which shifted normoxic [Ca(2+)](i)-force relations to higher force. In E- IPA, the Rho kinase antagonist Y-27632 (10 µM) shifted the normoxic [Ca(2+)](i)-force relation to lower force but did not alter the effects of hypoxia. These results suggest that acute hypoxia enhanced myofilament Ca(2+) sensitivity in rat IPA by decreasing nitric oxide production and/or activity in smooth muscle, thereby revealing a high basal level of Ca(2+) sensitivity, due in part to Rho kinase, which otherwise did not contribute to Ca(2+) sensitization by hypoxia.

Acetazolamide prevents hypoxia-induced reactive oxygen species generation and calcium release in pulmonary arterial smooth muscle.

Upon sensing a reduction in local oxygen partial pressure, pulmonary vessels constrict, a phenomenon known as hypoxic pulmonary vasoconstriction. Excessive hypoxic pulmonary vasoconstriction can occur with ascent to high altitude and is a contributing factor to the development of high-altitude pulmonary edema. The carbonic anhydrase inhibitor, acetazolamide, attenuates hypoxic pulmonary vasoconstriction through stimulation of alveolar ventilation via modulation of acid-base homeostasis and by direct effects on pulmonary vascular smooth muscle. In pulmonary arterial smooth muscle cells (PASMCs), acetazolamide prevents hypoxia-induced increases in intracellular calcium concentration ([Ca2+]i), although the exact mechanism by which this occurs is unknown. In this study, we explored the effect of acetazolamide on various calcium-handling pathways in PASMCs. Using fluorescent microscopy, we tested whether acetazolamide directly inhibited store-operated calcium entry or calcium release from the sarcoplasmic reticulum, two well-documented sources of hypoxia-induced increases in [Ca2+]i in PASMCs. Acetazolamide had no effect on calcium entry stimulated by store-depletion, nor on calcium release from the sarcoplasmic reticulum induced by either phenylephrine to activate inositol triphosphate receptors or caffeine to activate ryanodine receptors. In contrast, acetazolamide completely prevented Ca2+-release from the sarcoplasmic reticulum induced by hypoxia (4% O2). Since these results suggest the acetazolamide interferes with a mechanism upstream of the inositol triphosphate and ryanodine receptors, we also determined whether acetazolamide might prevent hypoxia-induced changes in reactive oxygen species production. Using roGFP, a ratiometric reactive oxygen species-sensitive fluorescent probe, we found that hypoxia caused a significant increase in reactive oxygen species in PASMCs that was prevented by 100 µM acetazolamide. Together, these results suggest that acetazolamide prevents hypoxia-induced changes in [Ca2+]i by attenuating reactive oxygen species production and subsequent activation of Ca2+-release from sarcoplasmic reticulum stores.

Knockdown of stromal interaction molecule 1 attenuates store-operated Ca2+ entry and Ca2+ responses to acute hypoxia in pulmonary arterial smooth muscle.

Stromal interaction molecule 1 (STIM1) is a recently discovered membrane-spanning protein thought to sense lumenal Ca(2+) in sarco(endo)plasmic reticulum (SR/ER) and transduce activation of Ca(2+)-permeable store-operated channels (SOC) in plasmalemma in response to SR/ER Ca(2+) depletion. To evaluate the role of STIM1 and a closely related protein, STIM2, in Ca(2+) signaling of rat distal pulmonary arterial smooth muscle cells (PASMC) during hypoxia, we used fluorescent microscopy and the Ca(2+)-sensitive dye, fura 2, to measure basal intracellular Ca(2+) concentration ([Ca(2+)](i)), store-operated Ca(2+) entry (SOCE), and increases in [Ca(2+)](i) caused by acute hypoxia (4% O(2)) or depolarization (60 mmol/l KCl) in cells treated with small interfering RNA targeted to STIM1 (siSTIM1) or STIM2 (siSTIM2). As determined by real-time quantitative PCR analysis (qPCR), STIM1 mRNA was 200-fold more abundant than STIM2 in untreated control PASMC. siSTIM1 and siSTIM2 caused specific and significant knockdown of both mRNA measured by qPCR and protein measured by Western blotting. siSTIM1 markedly inhibited SOCE and abolished the sustained [Ca(2+)](i) response to hypoxia but did not alter the initial transient [Ca(2+)](i) response to hypoxia, the [Ca(2+)](i) response to depolarization, or basal [Ca(2+)](i). The only effect of siSTIM2 was a smaller inhibition of SOCE. We conclude that STIM1 was quantitatively more important than STIM2 in activation of SOC in rat distal PASMC and that the increase in [Ca(2+)](i) induced by acute hypoxia in these cells required SR Ca(2+) release and STIM1-dependent activation of SOC.

Investigating unsaturated fat, monensin, or bromoethanesulfonate in continuous cultures retaining ruminal protozoa. I. Fermentation, biohydrogenation, and microbial protein synthesis.

Methane is an end product of ruminal fermentation that is energetically wasteful and contributes to global climate change. Bromoethanesulfonate, animal-vegetable fat, and monensin were compared with a control treatment to suppress different functional groups of ruminal prokaryotes in the presence or absence of protozoa to evaluate changes in fermentation, digestibility, and microbial N outflow. Four dual-flow continuous culture fermenter systems were used in 4 periods in a 4 x 4 Latin square design split into 2 subperiods. In subperiod 1, a multistage filter system (50-microm smallest pore size) retained most protozoa. At the start of subperiod 2, conventional filters (300-microm pore size) were substituted to efflux protozoa via filtrate pumps over 3 d; after a further 7 d of adaptation, the fermenters were sampled for 3 d. Treatments were retained during both subperiods. Flow of total N and digestibilities of NDF and OM were 18, 16, and 9% higher, respectively, for the defaunated subperiod but were not different among treatments. Ammonia concentration was 33% higher in the faunated fermenters but was not affected by treatment. Defaunation increased the flow of nonammonia N and bacterial N from the fermenters. Protozoal counts were not different among treatments, but bromoethanesulfonate increased the generation time from 43.2 to 55.6 h. Methanogenesis was unaffected by defaunation but tended to be increased by unsaturated fat. Defaunation did not affect total volatile fatty acid production but decreased the acetate:propionate ratio; monensin increased production of isovalerate and valerate. Biohydrogenation of unsaturated fatty acids was impaired in the defaunated fermenters because effluent flows of oleic, linoleic, and linolenic acids were 60, 77, and 69% higher, and the ratio of vaccenic acid:unsaturated FA ratio was decreased by 34% in the effluent. This ratio was increased in both subperiods with the added fat diet, indicating an accumulation of intermediates of biohydrogenation. However, the flow of 18:2 conjugated linoleic acid was unaffected by defaunation or by treatments other than added fat. The flows of trans-10, trans-11, and total trans-18:1 fatty acids were not affected by monensin or faunation status.

Rumen ciliated protozoa decrease generation time and adjust 18S ribosomal DNA copies to adapt to decreased transfer interval, starvation, and monensin.

Defaunation studies have documented decreased ammonia concentrations associated with reduced microbial protein recycling and wastage of dietary protein, whereas many methods to suppress protozoa can reduce feed intake or depress ruminal organic matter or fiber digestibility. Therefore, more research is needed to optimize dietary conditions that improve protozoal growth and ruminal outflow relative to autolysis and recycling. Response in growth rate to ruminal outflow was simulated by abrupt changes in transfer interval of batch cultures, and substrate availability was evaluated by feeding without or with abrupt addition of monensin, which was postulated to inhibit digestive vacuole function. In experiment 1, Entodinium caudatum, a mix of Entodinium species, Epidinium caudatum, or Ophryoscolex caudatus cultures rapidly adjusted their generation times to approach respective changes in transfer interval from 3 to 2 or 1 d (cultures were always fed at 24-h intervals). Monensin (0.25 microM) consistently delayed this response. To evaluate a metabolic upshift associated with feeding or a downshift associated with substrate depletion, experiment 2 used real-time PCR to quantify protozoal 18S rRNA gene (rDNA) copies that were expressed relative to cell numbers or to the cellular constituents N and nucleic acids after feeding without or with monensin (0.5 microM). The 18S rDNA copies per milligram of nucleic acids were least for Ophryoscolex compared with the other cultures. When averaged over cultures (no culture x treatment interaction), 18S rDNA copies per unit of nucleic acids decreased at 16 h when cultures were starved but increased with feeding unless monensin uncoupled availability of consumed substrate. Rumen protozoal growth increased in response to decreased transfer interval in experiment 1. Substrate availability appeared to initiate metabolic responses preparing for cell growth, explaining how cultures could rapidly adjust to decreasing transfer interval in experiment 2. Because feeding was not coupled with transfer in experiment 2, however, a metabolic control probably arrested cell division to prevent overgrowth relative to substrate availability.

Hypoxia inducible factor 1 mediates hypoxia-induced TRPC expression and elevated intracellular Ca2+ in pulmonary arterial smooth muscle cells.

Chronic hypoxia (CH) causes pulmonary vasoconstriction because of increased pulmonary arterial smooth muscle cell (PASMC) contraction and proliferation. We previously demonstrated that intracellular Ca(2+) concentration ([Ca(2+)](i)) was elevated in PASMCs from chronically hypoxic rats because of Ca(2+) influx through pathways other than L-type Ca(2+) channels and that development of hypoxic pulmonary hypertension required full expression of the transcription factor hypoxia inducible factor 1 (HIF-1). In this study, we examined the effect of CH on the activity and expression of store-operated Ca(2+) channels (SOCCs) and the regulation of these channels by HIF-1. Capacitative Ca(2+) entry (CCE) was enhanced in PASMCs from intrapulmonary arteries of rats exposed to CH (10% O(2); 21 days), and exposure to Ca(2+)-free extracellular solution or SOCC antagonists (SKF96365 or NiCl(2)) decreased resting [Ca(2+)](i) in these cells. Expression of TRPC1 and TRPC6, but not TRPC4, mRNA and protein was increased in PASMCs from rats and wild-type mice exposed to CH, in PASMCs from normoxic animals cultured under hypoxic conditions (4% O(2); 60 hours), and in PASMCs in which HIF-1 was overexpressed under nonhypoxic conditions. Hypoxia-induced increases in basal [Ca(2+)](i) and TRPC expression were absent in mice partially deficient for HIF-1. These results suggest that increased TRPC expression, leading to enhanced CCE through SOCCs, may contribute to hypoxic pulmonary hypertension by facilitating Ca(2+) influx and increasing basal [Ca(2+)](i) in PASMCs and that this response is mediated by HIF-1.

ATP-dependent K+ channels modulate vasoconstrictor responses to severe hypoxia in isolated ferret lungs.

In normo- and hypoglycemic ferret lungs, the pulmonary vascular response to severe hypoxia (PiO2 less than or equal to 10 mmHg) is characterized by an initial intense vasoconstriction followed by marked vasodilation, whereas in hyperglycemic lungs, vasodilation is minimal, causing vasoconstriction to be sustained. In contrast, the response to moderate hypoxia is characterized by a slowly developing sustained vasoconstriction which is unaffected by glucose concentration. To determine the role of ATP-dependent K+ (KATP) channels in these responses, we examined the effects of cromakalim, which opens KATP channels, and glibenclamide, which closes them. During steady-state vasoconstriction induced in isolated ferret lungs by moderate hypoxia, cromakalim caused dose-dependent vasodilation (EC50 = 7 x 10(-7) M) which was reversed by glibenclamide (IC50 = 8 x 10(-7) M), indicating that KATP channels were present and capable of modulating vascular tone. During severe hypoxia in hypoglycemic lungs [( glucose] less than 1 mM), glibenclamide markedly inhibited the secondary vasodilation. Raising perfusate glucose concentration to 14 +/- 0.4 mM had the same effect. As a result, initial vasoconstrictor responses were well sustained. However, neither glibenclamide nor hyperglycemia affected vasoconstrictor responses to moderate hypoxia or KCl, indicating that effects during severe hypoxia were not due to nonspecific potentiation of vasoconstriction. These findings suggest that in the ferret lung (a) severe hypoxia decreased ATP concentration and thereby opened KATP channels, resulting in increased K+ efflux, hyperpolarization, vasodilation, and reversal of the initial vasoconstrictor response; and (b) hyperglycemia prevented this sequence of events.

Hyperoxic sheep pulmonary microvascular endothelial cells generate free radicals via mitochondrial electron transport.

Free radical generation by hyperoxic endothelial cells was studied using electron paramagnetic resonance (EPR) spectroscopy and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the radical species produced, whether mitochondrial electron transport was involved, and the effect of the radical generation on cell mortality. Sheep pulmonary microvascular endothelial cell suspensions exposed to 100% O2 for 30 min exhibited prominent DMPO-OH and, occasionally, additional smaller DMPO-R signals thought to arise from the trapping of superoxide anion (O2-.), hydroxyl (.OH), and alkyl (.R) radicals. Superoxide dismutase (SOD) quenched both signals suggesting that the observed radicals were derived from O2-.. Studies with deferoxamine suggested that the generation of .R occurred secondary to the formation of .OH from O2-. via an iron-mediated Fenton reaction. Blocking mitochondrial electron transport with rotenone (20 microM) markedly decreased radical generation. Cell mortality increased slightly in oxygen-exposed cells. This increase was not significantly altered by SOD or deferoxamine, nor was it different from the mortality observed in air-exposed cells. These results suggest that endothelial cells exposed to hyperoxia for 30 min produce free radicals via mitochondrial electron transport, but under the conditions of these experiments, this radical generation did not appear cause cell death.

Impaired physiological responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1alpha.

Chronic hypoxia induces polycythemia, pulmonary hypertension, right ventricular hypertrophy, and weight loss. Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding proteins that mediate adaptive responses to hypoxia, including erythropoietin, vascular endothelial growth factor, and glycolytic enzymes. Expression of the HIF-1alpha subunit increases exponentially as O2 concentration is decreased. Hif1a-/- mouse embryos with complete deficiency of HIF-1alpha due to homozygosity for a null allele at the Hif1a locus die at midgestation, with multiple cardiovascular malformations and mesenchymal cell death. Hif1a+/- heterozygotes develop normally and are indistinguishable from Hif1a+/+ wild-type littermates when maintained under normoxic conditions. In this study, the physiological responses of Hif1a+/- and Hif1a+/+ mice exposed to 10% O2 for one to six weeks were analyzed. Hif1a+/- mice demonstrated significantly delayed development of polycythemia, right ventricular hypertrophy, pulmonary hypertension, and pulmonary vascular remodeling and significantly greater weight loss compared with wild-type littermates. These results indicate that partial HIF-1alpha deficiency has significant effects on multiple systemic responses to chronic hypoxia.

Assessment of ruminal bacterial populations and protozoal generation time in cows fed different methionine sources.

Methionine supplemented as 2-hydroxy-4-(methylthio)-butanoic acid (HMB) has been suggested to alter bacterial or protozoal populations in the rumen. Our objective was to determine if source of Met would change microbial populations in the rumen and to compare those results to samples from the omasum. The ruminal and omasal samples were collected from cows fed control (no Met), dl-Met, HMB, or the isopropyl ester of HMB (HMBi; estimated 50% rumen protection) in a replicated 4 x 4 Latin square design. In one square, changes in protozoal populations were determined using microscopic counts and denaturing gradient gel electrophoresis (DGGE), whereas changes in bacterial populations were determined using DGGE and ribosomal intergenic spacer length polymorphism (RIS-LP). Neither the protozoal counts nor the DGGE banding patterns derived from protozoa were different among the dietary treatments or for ruminal vs. omasal samples. As revealed by both DGGE and RIS-LP, bacterial populations clustered by treatments in ruminal and especially in omasal samples. Using cows from both Latin squares, the flow of protozoal cells from the rumen was quantified by multiplying protozoal cell count in omasal fluid by the omasal fluid flow (using CoEDTA as a liquid flow marker) or was estimated by rumen pool size of cells multiplied by either the ruminal dilution rate of CoEDTA (after termination of CoEDTA dosing) or the passage rate of Yb-marked particles. Compared with the omasal fluid flow measurement (16.4 h), protozoal generation time was approximated much more closely using the particulate than the fluid passage rate from the rumen (generation times of 15.7 and 7.5 h, respectively). There seems to be minimal selective retention of protozoal genera in the rumen in dairy cattle fed every 2 h. Data support the validity of the omasal sampling technique under our conditions.

Effects of tidal volume and respiratory frequency on lung lymph flow.

Ventilation (V) increases lung lymph flow (Ql), but the separate effects of tidal volume (Vt) and frequency (f) and the role of V-induced changes in edema formation are poorly understood. An isolated, in situ sheep lung preparation was used to examine these effects. In eight sheep with f = 10 min(-1), results obtained during 30-min periods with Vt = 5 or 20 ml/kg were compared with values obtained during bracketed 30-min control periods (Vt = 12.5 ml/kg). Eight other sheep with constant Vt (12.5 ml/kg) were studied at f = 5 or 20 min(-1) and compared with f = 10 min(-1). Three additional groups of six sheep were perfused for 100 min with control V (10 ml/kg, 10 min(-1)). Vt was then kept constant or changed to 20 or 3 ml/kg during a second 100-min period. Increases in Vt or f increased Ql and vice versa, without corresponding effects on the rate of edema formation. For the same change in V, changing Vt had a greater effect on Ql than changing f. The change in Ql caused by an increase in Vt was significantly greater after the accumulation of interstitial edema. The change in Ql caused by a sustained increase in Vt was transient and did not correlate with the rate of edema formation, suggesting that V altered Ql through direct mechanical effects on edema-filled compartments and lymphatic vessels rather than through V-induced changes in fluid filtration.

Effects of 2-hydroxy-4-(methylthio) butanoic acid (HMB) and its isopropyl ester on milk production and composition by Holstein cows.

The esterification of 2-hydroxy-4-(methylthio)-butanoic acid (HMB) to isopropanol (HMBi) decreases the rate and extent of its ruminal breakdown. The modes of action of HMB and HMBi appear to be different. The quantification of the production response to HMBi has not been done. The objectives of this study were (1) to determine the lactation response to HMB, (2) to determine the lactation response to HMBi, and (3) to evaluate whether the response to HMBi is affected by HMB in the diet. Sixty-one Holstein cows (24 primiparous, 37 multiparous) were assigned to 1 of 4 dietary treatments 21 to 28 d after calving. The base diet consisted of [on a dry matter (DM) basis] 32.5% corn silage, 17.5% alfalfa hay, 10% whole cottonseed, and 40% of a pelleted concentrate made primarily of ground corn, soybean meal, and blood meal, and was fed for 16 wk as a control diet. To prepare the dietary treatments, the base diet was supplemented with 0.1% of diet DM with HMB (treatment 2), with 0.15% HMBi (treatment 3), or with 0.045% HMB and 0.15% HMBi (treatment 4). Results showed a significant increase in milk yield (2.9 kg/d), protein content (0.15%), protein yield (115 g/d), fat yield (165 g/d), and lactose yield (182 g/d) from HMBi. Supplementation of HMB had small and nonsignificant effects on milk yield and composition. There were no significant interaction effects of HMB with HMBi on any of the production traits measured in this experiment. Plasma free Met as a proportion of essential amino acids was increased by HMBi, but not by HMB. Dietary supplementation of HMBi increased gross N efficiency expressed as the proportion of ingested N secreted in milk. Consequently, HMBi significantly improved N efficiency.

Evaluation of a real-time PCR assay quantifying the ruminal pool size and duodenal flow of protozoal nitrogen.

We have recently developed a real-time polymerase chain reaction (PCR) assay to quantify copies of the genes encoding protozoal 18S rRNA. The assay includes procedures for isolating and concentrating protozoal cells from the rumen for use as a standard to convert 18S rRNA gene copies to a biomass basis. The current objectives were to 1) determine the degree of reduction of bacterial contamination in the protozoal standard, 2) determine if protozoal standards derived from ruminal fluid are appropriate for predicting duodenal flows, and 3) evaluate the assay's determined values for protozoal N in the rumen and flowing to the duodenum compared with independent measurements. Our protozoal collection method reduced non-associated bacterial contamination by 33-fold, the contamination of which could otherwise significantly bias RNA (microbial marker) and N percentages of concentrated protozoal fractions. Based on denaturing gradient gel electrophoresis, the use of protozoal cells isolated from ruminal fluid appears appropriate for use in quantitative assays determining protozoal N flow postruminally. Using real-time PCR, protozoal N was determined to be 4.8 and 12.7% of the rumen microbial N pool and 5.9 and 11.9% of the duodenal flow of microbial N on diets containing low (16%) or high (21%) forage neutral detergent fiber, respectively, which were comparable with independent measures and expectations.