Peripheral chemoreflex responsiveness is increased at elevated levels of carbon dioxide after episodic hypoxia in awake humans.

We hypothesized that the acute ventilatory response to hypoxia is enhanced after exposure to episodic hypoxia in awake humans. Eleven subjects completed a series of rebreathing trials before and after exposure to eight 4-min episodes of hypoxia. During the rebreathing trials, subjects initially hyperventilated to reduce the partial pressure of carbon dioxide (Pet(CO(2))) below 25 Torr. Subjects then breathed from a bag containing normocapnic (42 Torr), low (50 Torr), or high oxygen (140 Torr) gas mixtures. During the trials, Pet(CO(2)) increased while a constant oxygen level was maintained. The point at which ventilation began to rise in a linear fashion as Pet(CO(2)) increased was considered to be the ventilatory recruitment threshold. The ventilatory response below and above the recruitment threshold was determined. Ventilation did not persist above baseline values immediately after exposure to episodic hypoxia; however, Pet(CO(2)) levels were reduced compared with baseline. In contrast, compared with baseline, the ventilatory response to progressive increases in carbon dioxide during rebreathing trials in the presence of low but not high oxygen levels was increased after exposure to episodic hypoxia. This increase occurred when carbon dioxide levels were above but not below the ventilatory recruitment threshold. We conclude that long-term facilitation of ventilation (i.e., increases in ventilation that persist when normoxia is restored after episodic hypoxia) is not expressed in awake humans in the presence of hypocapnia. Nevertheless, despite this lack of expression, the acute ventilatory response to hypoxia in the presence of hypercapnia is increased after exposure to episodic hypoxia.     

Location of a dicyclohexylcarbodiimide-reactive glutamate residue in the Neurospora crassa plasma membrane H+-ATPase

The proton pump (H+-ATPase) found in the plasma membrane of the fungus Neurospora crassa is inactivated by dicyclohexylcarbodiimide (DCCD). Kinetic and labeling experiments have suggested that inactivation at 0 degrees C results from the covalent attachment of DCCD to a single site in the Mr = 100,000 catalytic subunit (Sussman, M. R., and Slayman, C. W. (1983) J. Biol. Chem. 258, 1839-1843). In the present study, when [14C]DCCD-labeled enzyme was treated with the cleavage reagent, N-bromosuccinimide, a single major radioactive peptide fragment migrating at about Mr = 5,300 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was produced. The fragment was coupled to glass beads and partially sequenced by automated solid-phase Edman degradation at the amino terminus and at an internal tryptic cleavage site. By comparison to the DNA-derived amino acid sequence for the entire Mr = 100,000 polypeptide (Hager, K., and Slayman, C. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7693-7697), the fragment has been identified as arising by cleavage at tyrosine 100 and tryptophan 141. Covalently incorporated [14C]DCCD was released at a position corresponding to glutamate 129. The DCCD-reactive glutamate is located in the middle of the first of eight predicted transmembrane sequences. When the sequence surrounding the DCCD site is compared to that surrounding the DCCD-reactive residue of two other proton pumps, the F0F1-ATPase and cytochrome c oxidase, no homology is apparent apart from an abundance of hydrophobic amino acids.     

A potassium-proton symport in Neurospora crassa

Combined ion flux and electrophysiological measurements have been used to characterized active transport of potassium by cells of Neurospora crassa that have been moderately starved of K+ and then maintained in the presence of millimolar free calcium ions. These conditions elicit a high-affinity (K1/2 = 1-10 microM) potassium uptake system that is strongly depolarizing. Current-voltage measurements have demonstrated a K+-associated inward current exceeding (at saturation) half the total current normally driven outward through the plasma membrane proton pump. Potassium activity ratios and fluxes have been compared quantitatively with electrophysiological parameters, by using small (approximately 15 micron diam) spherical cells of Neurospora grown in ethylene glycol. All data are consistent with a transport mechanism that carries K ions inward by cotransport with H ions, which move down the electrochemical gradient created by the primary proton pump. The stoichiometry of entry is 1 K ion with 1 H ion; overall charge balance is maintained by pumped extrusion of two protons, to yield a net flux stoichiometry of 1 K+ exchanging for 1 H+. The mechanism is competent to sustain the largest stable K+ gradients that have been measured in Neurospora, with no direct contribution from phosphate hydrolysis or redox processes. Such a potassium-proton symport mechanism could account for many observations reported on K+ movement in other fungi, in algae, and in higher plants.     

Furosemide-sensitive potassium efflux in cultured mouse fibroblasts

Transfer of LM(TK-) cells from normal growth medium to medium lacking K+ leads to a rapid loss of intracellular K+, which is 50-70% inhibited by furosemide or bumetanide. The diuretic-sensitive component of K+ efflux requires both Na+ and Cl-, and is presumably mediated by a K+, Na+, Cl- cotransport system of the kind described in avian erythrocytes and Ehrlich ascites cells. It can be calculated that such a system should be near equilibrium under normal growth conditions but should mediate net efflux (as observed) when the driving force is altered by reducing extracellular K+. The diuretic-sensitive component of net K+ efflux is also sensitive to amiloride. This effect is probably indirect, however, with amiloride acting to block the Na+ influx that supplies Na+ to the cotransport system. At the low extracellular K+ concentrations employed in these studies, the diuretic-sensitive system is a physiologically important pathway of K+ loss. The rate of growth in low-K+ medium can be increased (or the rate of cell lysis decreased) by adding diuretic or by reducing external Na+ or Cl-.     

Identification of sphingomyelin turnover as an effector mechanism for the action of tumor necrosis factor alpha and gamma-interferon. Specific role in cell differentiation.

The biochemical signaling mechanisms involved in transducing the effects of tumor necrosis factor alpha (TNF alpha) and gamma-interferon (gamma-IFN) on leukemia cell differentiation are poorly defined. Recent studies established the existence of a sphingomyelin cycle that operates in response to the action of vitamin D3 on HL-60 cells and that may transduce the effects of vitamin D3 on cell differentiation (Okazaki, T., Bell, R., and Hannun, Y. (1989) J. Biol. Chem. 264, 19076-19080). The effects of TNF alpha and gamma-IFN on sphingomyelin turnover were determined, and the specificity and role of sphingomyelin hydrolysis in HL-60 human promyelocytic leukemia cells with 20% hydrolysis of sphingomyelin at 15 min, 40% hydrolysis at 30-60 min, and return to base line at 2 h. The hydrolyzed sphingomyelin (18 pmol/nmol total phospholipid) was accompanied by the concomitant generation of ceramide (11.2 pmol/nmol total phospholipid). gamma-IFN also caused reversible hydrolysis of sphingomyelin with onset at 1 h and peak effect at 2 h. This sphingomyelin cycle appeared to be specific to the monocytic pathway of HL-60 differentiation, since it was not activated by retinoic acid or dibutyryl cAMP, inducers of granulocytic differentiation, nor with phorbol myristate acetate, an inducer of macrophage-like differentiation. Addition of synthetic ceramide or bacterial sphingomyelinase induced monocytic differentiation of HL-60 cells. Cell-permeable ceramide also caused prompt down-regulation of mRNA for the c-myc protooncogene. The time course of c-myc down-regulation was consistent with the action of ceramide as the mediator of TNF alpha action. These results suggest that sphingomyelin turnover may be an important signaling mechanism transducing the actions of TNF alpha and gamma-IFN with specific function in cell differentiation.     

Permeability measurements on mitochondria from wild-type and poky strains of Neurospora crassa.

The permeability properties of isolated Neurospora mitochondria were determined by measuring the rate at which the mitochondria swell in isotonic solutions of various organic and inorganic molecules. Like mammalian mitochondria, wild-type Neurospora mitochondria were impermeable to sucrose and only slightly more permeable to most inorganic ions (K, Na, Cl). Their permeability to K was greatly increased by valinomycin and by monensin. In addition, the mitochondria contain specific systems mediating PO4 uptake and PO4- malate, fumarate, and succinate exchange. Mitochondria from the maternally inherited poky strain of Neurospora, previously demonstrated to possess defective ribosomes and a grossly cytochrome chain, showed a slight but significant increase in permeability to inorganic ions. They contained, however, the specific uptake and exchange systems for phosphate and dicarboxylate anions, a result suggesting that these systems do not depend upon mitochondrially synthesized polypeptides.     

Agonal phase,ischaemic times,and renal vascular abnormalities and outcome of cadaver kidney transplants.

A retrospective study of 250 cadaver kidney transplants was carried out to determine the effects of the agonal period, the warm and cold ischaemic times, and the use of kidneys with vascular anomalies on the primary success and failure and the subsequent level of function of the transplants. Kidneys with vascular anomalies or from non-ventilated donors had a primary failure rate of over 30%, whereas those with normal vasculature or from ventilated donors had a rate of 17%. An initial warm ischaemic time of more than 60 minutes was associated with a primary failure rate of 57% and a cold ischaemic time of over 550 minutes with a primary failure rate of 47%. The interrelationship between the warm and cold ischaemic times in the primary success or failure of the transplants was examined and criteria defined for selecting potentially viable cadaver kidneys for transplantation, as follows: (1) The donor should be (a) ventilated, (b) aged 6-50 years, and (c) have normal ante-mortem renal function and have secreted more than 1-5 1 of urine in the 24 hours before death (or an equivalent volume if the urinary output was recorded for less than 24 hours before death); (2) the kidney should have normal renal vasculature enabling single arterial and venous anastomoses to be performed; (3) kidneys with I.W.I.T.s of longer than 60 minutes should not be used; (4) for kidneys with I.W.I.T.s of less than 20 minutes the C.I.T. is not critical but should not exceed 12 hours; (5) for kidneys with I.W.I.T.s of 20-60 minutes the C.I.T. should not exceed 450 minutes.     

Sphingosine Kinase 1 Is Regulated by Peroxisome Proliferator-activated Receptor α in Response to Free Fatty Acids and Is Essential for Skeletal Muscle Interleukin-6 Production and Signaling in Diet-induced Obesity

We previously demonstrated that sphingosine kinase 1 (Sphk1) expression and activity are up-regulated by exogenous palmitate (PAL) in a skeletal muscle model system and in diet-induced obesity in mice; however, potential functions and in vivo relevance of this have not been addressed. Here, we aimed to determine the mechanism by which PAL regulates SphK1 in muscle, and to determine potential roles for its product, sphingosine-1-phosphate (S1P), in muscle biology in the context of obesity. Cloning and analysis of the mouse Sphk1 promoter revealed a peroxisome proliferator-activated receptor (PPAR) α cis-element that mediated activation of a reporter under control of the Sphk1 promoter; direct interaction of PPARα was demonstrated by chromatin immunoprecipitation. PAL treatment induced the proinflammatory cytokine interleukin (IL)-6 in a manner dependent on SphK1, and this was attenuated by inhibition of the sphingosine-1-phosphate receptor 3 (S1PR3)_. Diet-induced obesity in mice demonstrated that IL-6 expression in muscle, but not adipose tissue, increased in obesity, but this was attenuated in Sphk1^−/− mice. Moreover, plasma IL-6 levels were significantly decreased in obese Sphk1^−/− mice relative to obese wild type mice, and muscle, but not adipose tissue IL-6 signaling was activated. These data indicate that PPARα regulates Sphk1 expression in the context of fatty acid oversupply and links PAL to muscle IL-6 production. Moreover, this function of SphK1 in diet-induced obesity suggests a potential role for SphK1 in obesity-associated pathological outcomes.     

Inhibition of Sphingosine Kinase-2 in a Murine Model of Lupus Nephritis

Sphingosine-1-phosphate (S1P), a potent bioactive lipid, is emerging as a central mediator in inflammation and immune responses. We have previously implicated S1P and its synthetic enzyme sphingosine kinase (SK) in inflammatory and autoimmune disorders, including inflammatory bowel disease and rheumatoid arthritis. Generation of S1P requires phosphorylation of sphingosine by SK, of which there are two isoforms. Numerous studies have implicated SK1 in immune cell trafficking, inflammation and autoimmune disorders. In this study, we set out to determine the role of SK and S1P in lupus nephritis (LN). To this end, we examined S1P and dihydro-S1P (dh-S1P) levels in serum and kidney tissues from a mouse model of LN. Interestingly dh-S1P was significantly elevated in serum and kidney tissue from LN mice, which is more readily phosphorylated by SK2. Therefore, we employed the use of the specific SK2 inhibitor, ABC294640 in our murine model of LN. Treatment with ABC294640 did not improve vascular or interstitial pathology associated with LN. However, mice treated with the SK2 inhibitor did demonstrate decreases in glomerular pathology and accumulation of B and T cells in the spleen these were not statistically different from lpr mice treated with vehicle. LN mice treated with ABC294640 did not have improved urine thromboxane levels or urine proteinuria measurements. Both S1P and dh-S1P levels in circulation were significantly reduced with ABC294640 treatment; however, dh-S1P was actually elevated in kidneys from LN mice treated with ABC294640. Together these data demonstrate a role for SKs in LN; however, they suggest that inhibition of SK1 or perhaps both SK isoforms would better prevent elevations in S1P and dh-S1P and potentially better protect against LN.     

Inhibition of caspases inhibits the release of apoptotic bodies: Bcl-2 inhibits the initiation of formation of apoptotic bodies in chemotherapeutic agent-induced apoptosis

During apoptosis, the cell actively dismantles itself and reduces cell size by the formation and pinching off of portions of cytoplasm and nucleus as "apoptotic bodies." We have combined our previously established quantitative assay relating the amount of release of [3H]-membrane lipid to the degree of apoptosis with electron microscopy (EM) at a series of timepoints to study apoptosis of lymphoid cells exposed to vincristine or etoposide. We find that the [3H]-membrane lipid release assay correlates well with EM studies showing the formation and release of apoptotic bodies and cell death, and both processes are regulated in parallel by inducers or inhibitors of apoptosis. Overexpression of Bcl-2 or inhibition of caspases by DEVD inhibited equally well the activation of caspases as indicated by PARP cleavage. They also inhibited [3H]-membrane lipid release and release of apoptotic bodies. EM showed that cells overexpressing Bcl-2 displayed near-normal morphology and viability in response to vincristine or etoposide. In contrast, DEVD did not prevent cell death. Although DEVD inhibited the chromatin condensation, PARP cleavage, release of apoptotic bodies, and release of labeled lipid, DEVD-treated cells showed accumulation of heterogeneous vesicles trapped in the condensed cytoplasm. These results suggest that inhibition of caspases arrested the maturation and release of apoptotic bodies. Our results also imply that Bcl-2 regulates processes in addition to caspase activation.