Microvascular oxygen distribution in awake hamster window chamber model during hyperoxia

The microvascular effects and hemodynamic events following exposure to normobaric hyperoxia (because of inspiration of 100% O2) were studied in the awake hamster window chamber model and compared with normoxia. Hyperoxia increased arterial blood Po2 to 477.9 +/- 19.9 from 60.0 +/- 1.2 mmHg (P < 0.05). Heart rate and blood pressure were unaltered, whereas cardiac index was reduced from 196 +/- 13 to 144 +/- 31 ml.min-1.kg-1 (P < 0.05) in hyperoxia. Direct measurements in the microcirculation showed there was arteriolar vasoconstriction, reduction of microvascular flow (83% of control, P < 0.05), and functional capillary density (FCD, 74 +/- 16% of control), the latter change being significant (P < 0.05). Calculations of oxygen delivery and oxygen consumption based on the measured changes in microvascular blood flow velocity and diameter and estimates of oxygen saturation corrected for the Bohr effect due to the lowered pH and increased Pco2 showed that oxygen transport in the microvascular network did not change between normal and hyperoxic condition. The congruence of systemic and microvascular hemodynamics events found with hyperoxia suggests that the microvascular findings are common to most tissues in the organism, and that hyperoxia, due to vasoconstriction and the decrease of FCD, causes a maldistribution of perfusion in the microcirculation.     

Site-specific PEGylation of hemoglobin at Cys-93(beta): correlation between the colligative properties of the PEGylated protein and the length of the conjugated PEG chain

Increasing the molecular size of acellular hemoglobin (Hb) has been proposed as an approach to reduce its undesirable vasoactive properties. The finding that bovine Hb surface decorated with about 10 copies of PEG5K per tetramer is vasoactive provides support for this concept. The PEGylated bovine Hb has a strikingly larger molecular radius than HbA (1). The colligative properties of the PEGylated bovine Hb are distinct from those of HbA and even polymerized Hb, suggesting a role for the colligative properties of PEGylated Hb in neutralizing the vasoactivity of acellular Hb. To correlate the colligative properties of surface-decorated Hb with the mass of the PEG attached and also its vasoactivity, we have developed a new maleimide-based protocol for the site-specific conjugation of PEG to Hb, taking advantage of the unusually high reactivity of Cys-93(beta) of oxy HbA and the high reactivity of the maleimide to protein thiols. PEG chains of 5, 10, and 20 kDa have been functionalized at one of their hydroxyl groups with a maleidophenyl moiety through a carbamate linkage and used to conjugate the PEG chains at the beta-93 Cys of HbA to generate PEGylated Hbs carrying two copies of PEG (of varying chain length) per tetramer. Homogeneous preparations of (SP-PEG5K)(2)-HbA, (SP-PEG10K)(2)-HbA, and (SP-PEG20K)(2)-HbA have been isolated by ion exchange chromatography. The oxygen affinity of Hb is increased slightly on PEGylation, but the length of the PEG-chain had very little additional influence on the O(2) affinity. Both the hydrodynamic volume and the molecular radius of the Hb increased on surface decoration with PEG and exhibited a linear correlation with the mass of the PEG chain attached. On the other hand, both the viscosity and the colloidal osmotic pressure (COP) of the PEGylated Hbs exhibited an exponential increase with the increase in PEG chain length. In contrast to the molecular volume, viscosity, and COP, the vasoactivity of the PEGylated Hbs was not a direct correlate of the PEG chain length. There appeared to be a threshold for the PEG chain length beyond which the protection against vasoactivity is decreased. These results suggest that the modulation of the vasoactivity of Hb by PEG could be a function of the surface shielding afforded by the PEG, the latter being a function of the disposition of the PEG chain on the protein surface, which in turn is a function of the length of the PEG chain. Thus, the biochemically homogeneous PEGylated Hbs described in the present study, surface-decorated with PEG chains of appropriate size, could serve as potential candidates for Hb-based oxygen carriers.     

Microvascular pressure and functional capillary density in extreme hemodilution with low- and high-viscosity dextran and a low-viscosity Hb-based O2 carrier

Blood losses are usually corrected initially by the restitution of volume with plasma expanders and subsequently by the restoration of oxygen-carrying capacity using either a blood transfusion or possibly, in the near future, oxygen-carrying plasma expanders. The present study was carried out to test the hypothesis that high-plasma viscosity hemodilution maintains perfused functional capillary density (FCD) by preserving capillary pressure. Microvascular pressure responses to extreme hemodilution with low- (LV) and high-viscosity (HV) plasma expanders and an exchange transfusion with a polymerized bovine cell-free Hb (PBH) solution were analyzed in the awake hamster window chamber model (n = 26). Systemic hematocrit was reduced from 50% to 11%. PBH produced a greater mean arterial blood pressure than the nonoxygen carriers. FCD was higher after a HV plasma expander (70 +/- 15%) vs. PBH (47 +/- 12%). Microvascular pressure spanning the capillary network was higher after a HV plasma expander (16-19 mmHg) compared with PBH (12-16 mmHg) and a LV plasma expander (11-14 mmHg) but lower than control (22-26 mmHg). FCD was found to be directly proportional to capillary pressure. The use of a HV plasma expander in extreme hemodilution maintained the number of perfused capillaries and tissue perfusion by comparison with a LV plasma expander due to increased mean arterial blood pressure and capillary pressure. The use of PBH increased mean arterial pressure but reduced capillary pressure due to vasoconstriction and did not maintain FCD.     

Properties of a recombinant human hemoglobin double mutant: sickle hemoglobin with Leu-88(beta) at the primary aggregation site substituted by Ala.

A recombinant double mutant of hemoglobin (Hb), E6V/L88A(beta), was constructed to study the strength of the primary hydrophobic interaction in the gelation of sickle Hb, i.e., that between the mutant Val-6(beta) of one tetramer and the hydrophobic region between Phe-85(beta) and Leu-88(beta) on an adjacent tetramer. Thus, a construct encoding the donor Val-6(beta) of the expressed recombinant HbS and a second mutation encoding an Ala in place of Leu-88(beta) was assembled. The doubly mutated beta-globin gene was expressed in yeast together with the normal human alpha-chain, which is on the same plasmid, to produce a soluble Hb tetramer. Characterizations of the Hb double mutant by mass spectrometry, by HPLC, and by peptide mapping of tryptic digests of the mutant beta-chain were consistent with the desired mutations. The absorption spectra in the visible and the ultraviolet regions were practically superimposable for the recombinant Hb and the natural Hb purified from human red cells. Circular dichroism studies on the overall structure of the recombinant Hb double mutant and the recombinant single mutant, HbS, showed that both were correctly folded. Functional studies on the recombinant double mutant indicated that it was fully cooperative. However, its gelation concentration was significantly higher than that of either recombinant or natural sickle Hb, indicating that the strength of the interaction in this important donor-acceptor region in sickle Hb was considerably reduced even with such a conservative hydrophobic mutation.     

Microvascular and interstitial PO(2) measurements in rat skeletal muscle by phosphorescence quenching.

To clarify the transport of O(2) across the microvessels in skeletal muscle, we designed an intravital laser microscope that utilizes a phosphorescence quenching technique to determine both the microvascular and tissue PO(2). After we injected the phosphorescent probe into systemic blood, phosphorescence excited by a N(2)-dye pulse laser was detected with a photomultiplier over a 10 microm in diameter area. In vitro and in vivo calibrations confirmed that the present method is accurate for PO(2) measurements in the range of 7-90 Torr (r = 0.958) and has a rapid response time. This method was then used to measure the PO(2) of microvessels with different diameters (40-130 microm) and of interstitial spaces in rat cremaster muscle. These measurements showed a significant drop in PO(2) in the arterioles after branching (from 74.6 to 46.6 Torr) and the presence of a large PO(2) gradient at the blood-tissue interface of arterioles (15-20 Torr). These findings suggest that capillaries are not the sole source of oxygen supply to surrounding tissue.     

Distal His----Arg mutation in bovine myoglobin results in a ligand binding site similar to the abnormal beta site of hemoglobin Zurich (beta 63 His----Arg)

Carbon monoxide binding to a myoglobin mutant with distal arginine in place of histidine has been examined. The mutant is derived from a cDNA clone for Mb mRNA from fetal bovine skeletal muscle. The mutation only slightly perturbs visible/Soret spectra whereas the infrared spectrum of liganded CO is greatly modified to become nearly identical to Hb Zurich beta-subunit spectrum. The mutant IR spectra differ substantially from spectra of wild-type MbCO and normal HbCO beta-subunit. For both the Mb and the Hb the distal His----Arg mutation increases the affinity for CO and reduces the number of observed conformers. These results demonstrate that this mutation greatly reduces the differences between Mb and Hb in the structure and properties of its ligand binding sites.     

Structure and function of hemoglobin variants at an internal hydrophobic site: consequences of mutations at the beta 27 (B9) position

We have studied the structure-function relationships in newly discovered hemoglobin (Hb) mutants with substitutions occurring at the tight and highly hydrophobic cluster between the B and G helices in the beta chains, namely, Hb Knossos or beta A27S and Hb Grange-Blanche or beta A27V. The beta A27S mutant has a 50% decrease in oxygen affinity relative to native human Hb A, while the beta A27V mutant has an increased oxygen affinity. We have also engineered the artificial beta A27T mutation through site-directed mutagenesis. This new mutant exhibits functional properties similar to those of Hb A. None of these mutants is unstable. X-ray analyses show that the substitution of Val for Ala may reduce the relative stability of the T structure of the molecule through packing effects in the beta chains; for the beta A27S mutant a new hydrogen bond between serine and the carbonyl O at beta 23 (B5) Val is observed and is likely to increase the relative stability of the T structure in the mutant hemoglobin. However, no significant changes in the crystals were observed for these mutants between the quaternary R and T structures relative to native Hb A. We conclude that small tertiary structural changes in the tight hydrophobic B-G helix interface are sufficient to induce functional abnormalities resulting in either low or high intrinsic oxygen affinities.     

Microvascular flow and tissue PO(2) in skeletal muscle of chronic reduced renal mass hypertensive rats

This study determined whether arteriolar blood flow, capillary red blood cell (RBC) velocity, capillary hematocrit (Hct(cap)), and tissue PO(2) are altered in cremaster muscles of rats with chronic reduced renal mass hypertension (RRM-HT) relative to normotensive rats on high- or low-salt (NT-HS vs. NT-LS) diet. The blood flow in first- through third-order arterioles was not different between NT and HT rats, either at rest or during maximal relaxation of the vessels with 10(-4) M adenosine. Capillary RBC velocity was similar between the groups at rest but was elevated in RRM-HT and NT-HS rats during adenosine superfusion. Hct(cap) was reduced at rest in RRM-HT and NT-HS rats compared with NT-LS and was reduced in RRM-HT rats during adenosine-induced dilation. Tissue PO(2) was reduced in RRM-HT and NT-HS rats compared with NT-LS rats during control conditions and was lower in RRM-HT than in NT-LS rats during adenosine-induced dilation. These results indicate that both RRM-HT and chronic exposure of normotensive rats to a high-salt diet lead to reduced tissue oxygenation, despite the maintenance of normal arteriolar blood flow.     

Hemoglobin Fannin-Lubbock [alpha2 beta 2 119 (GH2) Gly replaced by Asp]. A new hemoglobin variant at the alpha1 beta 1 contact.

Hemoglobin Fannin-Lubbock was found in a 9-year-old Mexican-American female. The abnormal hemoglobin was detected as a fast-moving variant by electrophoresis on cellulose acetate at pH 8.4. Structural analysis indicated a substitution in the beta-chain of aspartic acid for glycine at position 119, a position involved in the alpha1beta1 contact of the hemoglobin tetramer. This contact between unlike chains is larger and undergoes a smaller shift during the process of oxygenation and deoxygenation that the alpha1beta2 contact (Perutz, M.F., Muirhead, H., Cox, J.M. and Goaman, L.C.G. (1968) Nature 219, 131-139). Mutations in this contact tend to cause slight or no changes in functional behavior. Apart from a mild anemia, the propositus did not exhibit any obvious clinical symptoms.     

Hemoglobin New Mexico: beta 100 (G2) Pro----Arg. A variant hemoglobin associated with erythrocytosis

Hemoglobin New Mexico beta 100 Pro----Arg was found in a 4-year-old black male and represents a new mutation. The propositus is also heterozygous for Hb S. The variant shows high oxygen affinity, reduced cooperatively, and a lowered alkaline Bohr effect. Addition of allosteric effectors leads to improved cooperativity and a Bohr effect that is similar to that of Hb A. The high percentage of the variant (53.5%) and its increased oxygen affinity result in erythrocytosis in this patient. The hemoglobin level and packed cell volume values are elevated. In spite of these factors the patient appears healthy and shows no discomfort. The altered oxygen-linked properties of this variant can be related to the fact that the substituted residue contributes to the alpha 2 beta 1/alpha 1 beta 2 subunit interface, an area that is critical not only to the allosteric transitions between the oxy and deoxy states but also to stabilizing the hemoglobin tetrameer.     

Mitochondrial respiratory control can compensate for intracellular O(2) gradients in cardiomyocytes at low PO(2)

In isolated single cardiomyocytes with moderately elevated mitochondrial respiration, direct evidence for intracellular radial gradients of oxygen concentration was obtained by subcellular spectrophotometry of myoglobin (Mb). When oxygen consumption was increased by carbonyl cyanide m-chlorophenylhydrazone (CCCP) during superfusion of cells with 4% oxygen, PO(2) at the cell core dropped to 2.3 mmHg, whereas Mb near the plasma membrane was almost fully saturated with oxygen. Subcellular NADH fluorometry demonstrated corresponding intracellular heterogeneities of NADH, indicating suppression of mitochondrial oxidative metabolism due to relatively slow intracellular oxygen diffusion. When oxygen consumption was increased by electrical pacing in 2% oxygen, radial oxygen gradients of similar magnitude were demonstrated (cell core PO(2) = 2.6 mmHg). However, an increase in NADH fluorescence at the cell core was not detected. Because CCCP abolished mitochondrial respiratory control while it was intact in electrically paced cardiomyocytes, we conclude that mitochondria with intact respiratory control can sustain electron transfer with reduced oxygen supply. Thus mitochondrial intrinsic regulation can compensate for relatively slow oxygen diffusion within cardiomyocytes.     

Selective oxidation of methionine beta(55)D6 at the alpha 1 beta 1 interface in hemoglobin completely destabilizes the T-state

When methionine beta(55)D6 in human hemoglobin is oxidized to its sulfoxide derivative, the modified protein appears to maintain most of the chemical and structural properties typical of the native protein. On the contrary, the functional behavior is drastically changed, being characterized (like that of the isolated chains) by high oxygen affinity (p50 = 0.47 torr in 0.1 M Tris (pH 7.3) + 0.1 M NaCl at 25 degrees C), absence of cooperativity (n = 1), and lack of Bohr effect. The complete destabilization of the T-state as a result of this modification is related to a perturbation of the alpha 1 beta 1 subunit interface, which in native hemoglobin remains static during the quaternary ligand-linked transition. Results also suggest that methionyl sulfoxide-containing hemoglobin, obtained under different conditions, assumes functionally different R-states, none of which is exactly comparable with that typical of the native protein.     

Identification of the site of photocross-linking formed in the absence of magnesium nucleotide from SH2 (Cys-697) in myosin subfragment 1 labeled with 4'-maleimidylbenzophenone

The site of photocross-linking between Cys-697 (SH2), prelabeled with 4'-[14C]maleimidylbenzophenone, and the 50-kDa segment of myosin S1 on irradiation in the absence of nucleotide has been determined by isolation of the 20-50-kDa adduct and subsequent tryptic proteolysis. Isolation and partial sequencing of the radioactively labeled peptide indicate that the site of cross-linking is Arg-239. This result indicates that, in the absence of nucleotide, Arg-239 resides at about 1.0 nm from SH2 and, on the basis of the recent work of Sutoh and Hiratsuka (Sutoh, K. and Hiratsuka T. (1988) Biochemistry 27, 2964-2969) places Arg-239 at no more than 1.45 nm from either Lys-184 or Lys-189 of the nucleotide-binding "glycine-rich" loop prior to the binding of nucleotide.     

Transition of hemoglobin between two tertiary conformations: determination of equilibrium and thermodynamic parameters from the reaction of 5,5'-dithiobis(2-nitrobenzoate) with the CysF9[93]beta sulfhydryl group

The equilibrium constant of the reaction of 5,5'-dithiobis(2-nitrobenzoate) with the CysF9[93]beta sulfhydryl group of hemoglobin decreases by 2 to 3 orders of magnitude between pH 5.6 and 9. The reaction is coupled to the ionizations of two groups on the protein. At 25 degrees C one group has a pK(a) of 5.31+/-0.2 when hemoglobin is in its (tertiary) r conformation, typified by the thiolate anion form of CysF9[93]beta; this changes to 7.73+/-0.4 in the (tertiary) t conformation, typified by the mixed disulfide form of the sulfhydryl. The second group ionizes with a pK(a) of 7.11+/-0.4 in the r conformation; this changes to 8.38+/-0.2 in the t conformation. K(rt), the equilibrium constant for the r<-->t isomerization process, is 0.22+/-0.06. The standard enthalpy and entropy changes for the isomerization are DeltaH(o)(rt)=24.2 kJ mol(-1) and DeltaS(o)(rt)=68.8 JK(-1)mol(-1), respectively.     

Hemoglobin Roseau-Pointe a Pitre alpha 2 beta 2(90) (F6) Glu----Gly: a new hemoglobin variant with slight instability and low oxygen affinity

A Dominican neonate carrying a new abnormal hemoglobin, hemoglobin Roseau Pointe-à-Pitre alpha 2 beta 2(90)(F6) Glu----Gly, was detected in Guadeloupe during application of a cord blood screening program. This variant behaved in isoelectrofocusing as an Hb D, and displayed instability and low whole blood oxygen affinity. In the affected family it was present, either isolated, or in association with a beta+ thalassemia trait.     

Additional acetylcholine (ACh) binding site at alpha4/alpha4 interface of (alpha4beta2)2alpha4 nicotinic receptor influences agonist sensitivity

Nicotinic acetylcholine receptor (nAChR) α4 and β2 subunits assemble in two alternate stoichiometries to produce (α4β2)(2)α4 and (α4β2)(2)β2, which display different agonist sensitivities. Functionally relevant agonist binding sites are thought to be located at α4(+)/β2(-) subunit interfaces, but because these interfaces are present in both receptor isoforms, it is unlikely that they account for differences in agonist sensitivities. In contrast, incorporation of either α4 or β2 as auxiliary subunits produces isoform-specific α4(+)/α4(-) or β2(+)/β2(-) interfaces. Using fully concatenated (α4β2)(2)α4 nAChRs in conjunction with structural modeling, chimeric receptors, and functional mutagenesis, we have identified an additional site at the α4(+)/α4(-) interface that accounts for isoform-specific agonist sensitivity of the (α4β2)(2)α4 nAChR. The additional site resides in a region that also contains a potentiating Zn(2+) site but is engaged by agonists to contribute to receptor activation. By engineering α4 subunits to provide a free cysteine in loop C at the α4(+)α4(-) interface, we demonstrated that the acetylcholine responses of the mutated receptors are attenuated or enhanced, respectively, following treatment with the sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate or aminoethyl methanethiosulfonate. The findings suggest that agonist occupation of the site at the α4(+)/(α4(-) interface leads to channel gating through a coupling mechanism involving loop C. Overall, we propose that the additional agonist site at the α4(+)/α4(-) interface, when occupied by agonist, contributes to receptor activation and that this additional contribution underlies the agonist sensitivity signature of (α4β2)(2)α4 nAChRs.     

Arteriolar wall PO(2) and nitric oxide release during sympathetic vasoconstriction in the rat intestine

Endothelium-derived nitric oxide (NO) attenuates arteriolar constriction in the rat small intestine during periods of increased sympathetic nerve activity. This study was undertaken to test the hypothesis that a flow-dependent fall in arteriolar wall PO(2) serves as the stimulus for endothelial NO release under these conditions. Sympathetic nerve stimulation at 3-16 Hz induced frequency-dependent arteriolar constriction, with arteriolar wall O(2) tension (PO(2)) falling from 67 +/- 3 mmHg to as low as 41 +/- 6 mmHg. Arteriolar responses to nerve stimulation were enhanced after inhibition of NO synthase with N(G)-monomethyl-L-arginine (L-NMMA). Under a high-O(2) (20%) superfusate, the fall in wall PO(2) was significantly attenuated, arteriolar constrictions were increased by 57 +/- 9 to 66 +/- 12%, and these responses were no longer sensitive to L-NMMA. The high-O(2) superfusate had no effect on vascular smooth muscle responsiveness to NO (as judged by arteriolar responses to sodium nitroprusside) or on arteriolar wall oxidant activity (as determined by the reduction of tetranitroblue tetrazolium dye). These results indicate that a flow-dependent fall in arteriolar wall PO(2) may serve as a stimulus for the release of endothelium-derived NO during periods of increased sympathetic nerve activity.     

Glycerated hemoglobin, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine. A new post-translational modification occurring in erythrocyte bisphosphoglyceromutase deficiency

A new minor Hb fraction initially designated Hbx, has been found in the hemolysate of an erythremic patient that we have previously described with a complete erythrocyte bisphosphoglycerate mutase (EC 5.4.2.4) deficiency. Hbx (3.5% of the total) was detected by isoelectric focusing and exhibited electrophoretic and chromatographic properties similar to those of several variants of the Hb central cavity. By density fractionation of red cells, it was demonstrated that Hbx was an aging hemoglobin as in the case of glycated Hb A1c. Functional studies revealed a low oxygen affinity and almost complete inhibition of the allosteric effect of the organic phosphate effectors. Structural studies demonstrated an absence of tryptic cleavage between the peptides beta T9 and beta T10 suggesting the presence of an adduct on Lys beta 82 or on a neighboring residue. Fast atom bombardment mass spectrometry and a specific enzymatic assay with glyoxylate reductase demonstrated that the beta 82 adduct was a glycerate moiety. It was concluded that Hbx was a glycerylated Hb, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine, to our knowledge the first example of glycerylated protein. The mechanism of formation of glyceryl Hb, which was found in the four studied subjects with a bisphosphoglyceromutase deficiency, remains to be determined.