Mohammed M. Sayeed

Cellular Alterations With Shock and Ischemia

From the Department of Surgery, Washington University School of Medicine, and The Jewish Hospital of St. Louis, St. Louis, Missouri 63110. Presented at the nineteenth annual meeting of the American College of Angiology, San Juan, Puerto Rico, January 21-25, 1973. Supported by Army contract DADA-17-69-9165 and U.S. Public Health Service Grant 2 RO 1 HL 12278-04. Ultimately, an inadequate circulation will adversely affect the functions of cell populations of various organs. Such changes could be important factors in the problems of unresponsive shock, prolonged extracorporeal circulation, ischemia and preservation of organs by perfusion techniques for transplantation. Haldane once said that anoxia not only stops the machine, but also wrecks the machinery. In order to evaluate such changes and to try to determine how the

(Na+ + K+)-ATPase Activity in the Liver with Hemorrhagic Shock

Summary (Na+ + K+)-ATPase activity was measured in the liver of rats subjected to an early stage of hemorrhagic shock (25% of shed blood taken back spontaneously) and a late stage (50-70% of shed blood taken back). In unbled control animals the activity was 10.6 ± 1.04 (mean ± SEM) nmoles H+/(mg protein X min). An approximately twofold increase in activity in early shock and a threefold increase in late shock was found. When animals were treated by return of the shed blood plus 5-6 ml of Ringers lactate solution in the early and late stages of shock and allowed 1 hr for recovery, the (Na+ + K+)-ATPase activity decreased to near control levels. These results also indicate that the increased liver (Na+ + K+)-ATPase activity in shock is reversible with treatment.

The Effect of Low ATP on Glucose Uptake in Soleus Muscle During Hemorrhagic Shock

Summary Subclavian arteries of rats were cannulated and the animals were bled rapidly to a pressure of 40 mm Hg and maintained at that level for 2 hr. Glucose uptake by isolated soleus muscles from normal rats and rats which were subjected to severe hemorrhagic shock (“shock muscles”) was the same although ATP levels were significantly lowered in muscles of the latter rats. Whereas shock muscles did not show increased glucose uptake, these muscles still responded to the in vitro effect of anoxia by showing increased glucose uptake, further depletion of ATP and increased lactic acid production. The present experiments indicate that: (a) during severe hemorrhagic shock, there is no cause-effect relationship between glucose uptake and ATP depletion and (b) whatever the mechanism of action of anoxia in promoting glucose uptake in vitro might be, it is quite different from that of in vivo. The studies described in this paper were largely inspired by experiments undertaken by I. H. Chaudry, during the course of his PhD. For this the authors thank Dr. M. K. Gould of Monash University, Australia. We are also thankful to Mr. G. J. Planer for technical assistance and for her skill and assistance in typing of this manuscript, we thank Ms. Gail Perry. This investigation was supported by U.S. Public Health Service Grant 2 RO1 HL 12278-04 and Army Contract DADA-17-69-9165.

Failure of nicotinamide in the treatment of hemorrhagic shock

Abstract Hemorrhagic shock in rats was produced by bleeding the animals to a mean arterial pressure of 40 mm Hg which was maintained for 1.5 hr. ATP, ADP, and NAD (nicotinamide adenine dinucleotide) levels decreased and NADH (reduced pyridine nucleotide) levels increased significantly in liver and kidney of such animals. Infusion of NAD, nicotinamide, or nicotinic acid following shock increased the tissue NAD and decreased the NADH levels. Under ideal conditions, the oxidation of NADH to NAD should result in phosphorylation of ADP. Thus more ATP would be expected to be present. However, infusion of the above compounds failed to affect the tissue ATP levels after shock. Failure of animals to survive shock despite increases in tissue NAD levels following NAD, nicotinamide, or nicotinic acid infusion suggests that these infusions have no salutary effect in hemorrhagic shock.

Intestinal Enzyme Activities Following Whole-Body Irradiation in Hamsters

Summary The phosphomonoesterase and Mg- (Na + K) -adenosine triphosphatase (ATPase) activities were measured in the mucosa of the small intestine of hamsters (Mesocrkelus auratus) exposed to 1,000 rads 60Co whole-body irradiation. The enzyme assays were made in irradiated and sham-irradiated hamsters sacrificed at intervals through Day 4 postirradiation. The two enzy-mic activities expressed per unit of tissue weight were significantly higher in the irradiated animals as compared to the sham-irradiated group at Day 2 postirradiation. No changes were found in the protein contents of jejunal mucosa between irradiated and sham-irracliated animals. While the ATPase activity was significantly lowered at Day 4, the phosphomonoesterase activity was observed to be higher at that time. The increase in enzyme activities may represent increased synthesis of these enzymes in the crypt cells that gave origin to the mature cells of the intestinal villi of animals sacrificed at Day 2 postirradiation. The increase in ATPase activity at Day 2 followed by a decrease at Day 4 corresponds to the previously observed alterations in the intestinal glucose absorption by irradiated hamsters.

Age difference in the intestinal phosphomonoesterase activity of mice.

The intestinal mucosal cells have been shown to contain specific phosphatases along with a group of nonspecific phosphomonoesterases. Ockerman (1) has shown the presence of a glucose 6-phosphatase in the microsomal fraction of the jejunal mucosal cells. A Na+, K+, activated adenosine triphosphatase activity has been shown to reside in the cell membrane fraction (2, 3). Whereas the adenosine triphosphatase has been implicated in the action of the intestinal sugar pump (4), the metabolic function of glucose-6-phosphatase awaits elucidation. While the precise roles of the intestinal phosphatases remain unknown, their involvement in the processes of mucosal transport and metabolism is a good possibility. The present report is concerned with changes in the intestinal phosphomonoesterase activity in relation to the age of the animal. This study was undertaken in view of the possibility that the process of aging may have an influence on the behavior of the enzyme apparatus which in turn may suggest changes in the intestinal metabolic and absorptive functions. Materials and Methods. The homogenate and the cell membrane fraction used in this study as the sources of enzyme were prepared from two groups of female mice of B6D2F1/J strain (The Jackson Laboratory, Bar Harbor, Maine). One of these, consisting of 11-month-old mice weighing 34.0 + 2.0 g, is referred to in this paper as the adult group. The other group was comprised of 34-month-old animals weighing 26 + 1.5 g and is referred to as the aged group. 1 The animals were sacrificed after a 24-hr fast. The entire small intestine was removed, everted, and scraped on a chilled glass surface by means of a glass slide. Two-tenths percent (w/v) homogenates of mucosal scraping were prepared, in a diluent of one part Krebs-Ringer-bicarbonate and three parts tris (hydroxymethyl) aminomethane-HCl (Tris) buffer adjusted to pH 8.00. The homogenate was centrifuged at 1000g for 40 min, and the resulting sediment was suspended in the Krebs-Ringer-bicarbonate and Tris mixture to a final volume equal to that of the homogenate used in obtaining that amount of sediment.

Effect of hydrocortisone and dexamethasone on xylose uptake by isolated rat soleus muscle.

To determine the effects of glucocorticoids on sugar uptake, xylose uptake by isolated rat soleus muscle of bilaterally adrenalectomized animals was studied. The results indicate that in vitro addition of 10-4 M hydrocortisone, dexamethasone or hydrocortisone sodium succinate had no inhibitory effect on basal xylose uptake. In the presence of both low and high medium insulin, the above steroids failed to inhibit insulin-stimulated uptake. When the concentration of hydrocortisone sodium succinate was increased to 10-2 M, insulinstimulated uptake was decreased. The results thus indicate that glucocorticoids at concentrations observed under physiological or pathological conditions do not inhibit basal or insulin-stimulated sugar uptake.

Nicotinamide Adenine Dinucleotide (NAD) Content of Liver with Hemorrhagic Shock

Summary Nicotinamide adenine dinucleotides were determined in the liver of rats subjected to three stages of hemorrhagic shock and treatment. Total nucleotide content was decreased in all stages. The oxidized form, NAD+, appeared to decrease more rapidly than the reduced form, NADH; however this was likely the result of a shift from oxidized to reduced state due to decreased tissue perfusion. Increased enzymic degradation of NAD did not appear to be the mechanism whereby NAD content was lowered; NADase activity of shock liver was not different from that of control liver. These results also indicate that the decreased NAD content in shock is restored to normal by nonspecific treatment by replenishment of circulatory volume. The authors are grateful for the technical assistance of Betty Henton and the clerical assistance of Gail Perry.

3-Methyl Glucose Transport in Isolated Epithelial Cells from Rat Small Intestine

Summary The transport of nonmetaboliz-able sugar, 3-methyl glucose (3MG), was studied in the isolated intestinal epithelial cells from rat small intestine. Epithelial cells when incubated in Krebs-Ringer bicarbonate (KRB) medium for 20 min (37°) accumulated 3MG to approximately 4 times its concentration in the medium. Active transport of 3MG into epithelial cells was abolished by: (a) replacement of Na+ in the KRB with K+ or choline, (b) addition of 0.1 mM dinitrophenol (DNP) or iodoacetamide (IA), or (c) anaerobic conditions. The passive uptake of 3MG into epithelial cells, however, varied under these conditions. The net uptake values as compared to control, approximately, were: 65% with DNP, 53% with IA or anoxia, and 40% in the absence of Na+. The greater decrease in 3MG uptake in the Na+ free medium might indicate a facilitation by Na+ of the passive 3MG transport process. The technical assistance of Ms. Ann Mitchell is gratefully acknowledged. This work was supported by grants from U. S. Public Health Service Grant No. HE 12278-04, U. S. Army Contract No. DA DA 17-69-C-9165, and General Research Support Grant No. SOI RR05491 from NIH.

Kinetics of phosphoglucose ester hydrolysis by rat intestinal mucosa.

Summary The enzymatic hydrolysis of glucose 1-phosphate (G1P) and glucose 6-phosphate (G6P) by homogenates of rat intestinal mucosa was quantitated. These substrates were not utilized by enzymes other than phosphatases to any appreciable extent. The mean K m value for the hydrolysis of G6P was higher (4.3 mM) than that for G1P (1.69 mM). A variability was observed in Km values in each substrate group. However, the Km /Vmax ratio was constant for each of the two substrates. Such proportional changes in Km and Vmax values allow the approximation of the rate constant (K 1) related to the formation of the enzyme-substrate complex. The K 1 value thus estimated for G1P was approximately 1.68 times that for G6P. This work was supported by U. S. Army Research and Development Command, Department of the Army, under Research Contract No. DA-49-193-MD-2415, and by Research Grant AM 11945 from the National Institutes of Health, U. S. Public Health Service.