April A. Whitbeck

Effect of Propranolol on Oxygen Binding to Hemoglobin In Vitro and In Vivo

We have shown that propranolol reduces oxygen binding by hemoglobin in intact red cells by increasing the selective permeability of the red cell membrane resulting in an exodus of potassium, chloride, and water. The latter effects result in a new distribution of hydrogen ion between cell and plasma, and thereby a reduction in red cell pH. The reduction in pH can fully explain the change in hemoglobins affinity for oxygen based on the Bohr effect. Either D- or DL-propranolol can produce the change in red cell pH and oxygen binding by hemoglobin. The drug action on permeability is not prevented by epinephrine, although it is by chlorbutanol. Hence, the membrane action of propranolol does not appear to be related to its activity as a beta-adrenergic receptor blocking agent.Propranolol produced a marked alteration in red cell shape as well as in hydration (hypovolumic stomatocytes). The two effects were separable since dehydration of the cell by the addition of sucrose to plasma did not result in stomatocytes and chlorbutanol blocked the enhancement of permeability of the red cell membrane by propranolol without preventing the shape change (isovolumic stomatocytes). This suggests that propranolol may have two separate sites of membrane interaction.Propranolol (10 to 360 mg) administered to human subjects did not affect hemoglobin-oxygen affinity. This is explained by the fact that the concentration in blood after such doses is nearly 4,000 to 100-fold lower than that required to achieve changes in blood in vitro.

The relationships between arterial oxygen flow rate, oxygen binding by hemoglobin, and oxygen utilization after myocardial infarction.

The interrelationships of arterial oxygen flow rate index, oxygen binding by hemoglobin, and oxygen consumption have been examined in patients with acute myocardial infarction. Proportional extraction of oxygen increased in close association with decreasing oxygen flow rate, and hence, whole body oxygen consumption was constant over nearly a three-fold variation in arterial oxygen flow rate. A reduction in hemoglobin-oxygen affinity at in vivo conditions of pH. Pco(2) and temperature also occurred in proportion to the reduction in arterial oxygen flow rate. Therefore, the increased proportional removal of oxygen from arterial blood at low oxygen flow rates, required to maintain oxygen consumption, may have been facilitated by the reduced affinity of hemoglobin for oxygen at in vivo conditions. However, the decrease in affinity did not appear to explain more than 30-40% of the increased extraction. Respiratory alkalosis was a frequent occurrence in these patients and 2,3-diphosphoglycerate was positively associated with blood pH as well as with the time-averaged proportion of deoxyhemoglobin in arterial and venous blood.Hemoglobin-oxygen affinity measured at standard conditions and the mixed venous oxygen saturation were equally good indicators of reduced arterial oxygen flow rate in patients without shock. However, Svo(2) is more easily measured and is a more useful indicator of reduced oxygen flow rate, since its relationship to oxygen flow appears to be independent of affinity changes and time.

In situ hybridization analysis of developmental stages of Pneumocystis carinii that are transcriptionally active for a major surface glycoprotein gene

An abundant glycoprotein on the surface of Pneumocystis carinii, termed gpA or gp120, is thought to play a role in the interaction of this opportunistic pathogen with its host. Using RNA:RNA hybridization techniques, the in situ expression of gpA mRNA in developmental forms of the organism was investigated in a ferret model of P. carinii pneumonia. The results suggested that the relative abundance of gpA‐specific mRNA was variable in different developmental stages of ferret P. carinii. P. carinii localized along the epithelial lining of alveoli were transcriptionally active. Immunocytochemical detection of gpA and Giemsa staining suggested that many of these organisms were trophic forms of P. carinii. While no detectable gpA mRNA signal was found in the majority of P. carinii cysts, a portion of identifiable cysts co‐localized with significant levels of gpA mRNA signal. Differential staining of the cyst wall with Gomoris methenamine silver suggested that the transcriptionally active P. carinii cysts were the intermediate or precyst forms of the organism, while the cysts with no detectable mRNA signal were either mature or empty (excysted). Alveolar macrophages were observed surrounded by transcriptionally active organisms; however, no gpA‐transcriptional activity was detected within macrophages. Taken together, the results suggest that transcription of gpA occurs in forms of P. carinii that are actively replicating, and in close proximity or contact with, alveolar epithelial cells.

Oxygen Binding to Haemoglobin in Subjects with Hypoproliferative Anaemia, with and without Chronic Renal Disease: Role of pH

Summary. The relationships of haenioglobin concentration and blood pH to red cell 2,3‐dipliosphoglycerate and oxygen binding by haenioglobin have been studicd in healthy subjects and subjects with hypoproliferative anaemia with or without severe chronic renal disease. Red cell 2,3‐DPG was inversely correlated with haemoglobin deficit and directly and equally strongly associated with blood pH in anaemic subjects without chronic renal disease. In subjects with chronic renal disease receiving regular haemodialysis, predialysis pH was not increased despite severe anaemia, and red cell 2,3‐DPG was not significantly elevated, except in subjects who had a sustained alkalosis due to the use of sodium bicarbonate.

Acidification of plasma by the red cell due to radiographic contrast materials.

The effect of water-soluble radiographic contrast material on pH when added to blood in clinical dosages in vitro or when used in vivo for diagnostic purposes was examined. Contrast material caused a reduction of blood pH. The mechanism of this occurence was found to be the balancing of the negative charge of intracellular organic anions by the extracellular anionic contrast material molecules. The normal negative potential of about 10 mV across the red cell membrane was reduced, nullified, or reversed depending on the concentration of contrast material added to blood. As the inside of the cell became more positive with respect to the outside, protons were, in effect, repelled into plasma, although the apparent exodus of protons occurs by the generation and outward diffusion of carbon dioxide. Since the acidemia is dependent on rehydration of carbon dioxide in plasma, a reaction measured in seconds, the site of injection and transit time of dye will contribute to the pH of the plasma during passage through a regional capillary bed. We speculate that an alteration in membrane potential and/or the acute acidemia may contribute to the adverse effects of contrast material, particularly on tissues dependent on membrane electrical rhythmicity such as the myocardium.

Evidence for prokaryotic transcription and translation control regions in the human factor IX gene.

A human factor IX cDNA clone isolated from a liver cDNA library constructed in phage lambda gt11 vector was shown to express factor IX protein in Escherichia coli. A factor IX immunospecific protein of 46.8 kDa was expressed, but was not a beta-galactosidase-factor IX fusion protein. Expression was seen when the factor IX cDNA was cloned into two different vector systems, lambda gt11 and pUC9, in both orientations with respect to the vector lacZ promoter. The expression of factor IX was not under control of the lacZ promoter of either vector system. In addition, when the factor IX cDNA fragment was subcloned in both orientations into a promoterless cloning vector (p CPP3), the factor IX cDNA fragment demonstrated promoter activity when inserted in only one orientation resulting in expression of chloramphenicol acetyl transferase in E. coli and Bacillus subtilis. A DNA computer search of the N-terminal sequences of the factor IX gene revealed prokaryotic-like promoter and ribosome-binding site (RBS) sequences with strong homology to the E. coli consensus sequences. The predicted sites homologous with prokaryotic promoter and RBS consensus sequences are followed by an in-frame methionine which could correspond to the translation start codon of the expressed factor IX. This report provides the first evidence that a eukaryotic gene encodes the information necessary for both transcription and translation to control gene expression in a prokaryotic host.

Factitious changes in binding of oxygen to hemoglobin when based on extracellular pH in the presence of certain blood additives like radiographic contrast media.

Construction of oxygen-hemoglobin DISSOCIATION CURVES BASED ON EXTRACELLULAR PH and using blood tonometered with 5 per cent CO2, is misleading under certain experimental conditions. These include the presence in blood of poorly penetrating non-ionic molecules like sucrose of poorly penetrating anionic aompounds like radiographic contrast materials. False conclusions regarding the position of the oxygen-hemoglobin dissociation curve can result because of the disturbance of the normal pH gradient between plasma and red cell induced by such chemicals.