Urea and ethylene glycol-facilitated transport systems in the human red cell membrane. Saturation, competition, and asymmetry.

The equilibrium exchange of [14C]urea and ethylene glycol was measured using a new type of fast flow system. Approximately equal volumes of saline and air were mixed to form a segmented fluid stream into which 14C-loaded red cells are injected. The stream flows through three filter chambers which allow sampling of the 14C in the extracellular fluid at three time points. The chambers are designed so that they do not disrupt the segmented bubble pattern. The alternating air and saline segments prevent laminar dispersion in the flowing stream and ensure good mixing at the injection and sampling sites. The equilibrium exchange of both urea and ethylene glycol showed saturation kinetics. The maximum permeability (Po) measured in the limit of zero solute concentration is 1.6 X 10(-3) cm/s for urea and 4.8 X 10(-4) cm/s for ethylene glycol (T = 23 degrees C). The apparent dissociation constant (Km) was 218 mM for urea and 175 mM for ethylene glycol. The Po for thiourea is 2.3 X 10(-6) cm/s and the Km is 19 mM. Urea and thiourea inhibit the transport of each other and the inhibition constant (KI) is approximately equal to the Km for both compounds. 53 other analogues of urea were screened for their inhibition of urea or thiourea transport. Several analogues [e.g., 1-(3,4-dichloro-phenyl)-2-thiourea] had a KI in the range of 0.03 mM. The affinity of the inhibitor increased as it was made more hydrophobic. The urea analogues did not significantly inhibit the ethylene glycol or osmotic permeability. Glycerol inhibited ethylene glycol permeability with a KI of 1,200 mM.

Is the erythrocyte sodium pump altered in human obesity?

Ouabain binding and electrolyte concentrations of erythrocytes, and Na+, K+-ATPase activity of red cell ghosts were measured in normal and obese subjects, ranging from 88-257% of their ideal body weight. All three independent measurements were virtually the same in obese and nonobese groups, and no correlations were found between these three variables and the percentage of ideal body weight. These results differ from previous reports of either increased or decreased sodium pump function and suggest that Na+, K+-ATPase does not directly influence human obesity.

Simple method for visualization of the islets in fixed but otherwise intact pancreas.

A simple, inexpensive, reproducible method is described for prominently displaying the islets of Langerhans. The method consists of sequential arterial perfusion of the organ of the sacrificed animal with saline, formalin, hematoxylin, and water, followed by clearing in methyl salicylate. The procedure should be useful whenever islet tissue needs to be quantitatively distinquished from non islet tissue and fixation is allowable.

A simple method for the purification of rat brain Na+,K+-adenosine triphosphatase (ATPase).

Several methods of purification of Na+,K+-adenosine triphosphatase (ATPase) have been previously described for a wide variety of tissues. In general, highest activity preparations have necessitated large amounts of tissue and many purification steps. This article describes a technique that allows partial purification of Na+,K+-ATPase from as few as 15 rat brains and should be of interest to investigators of the pharmacology of this particular enzyme system. In this modified version of the Jorgensen procedure (Biochim Biophys Acta 356:36--52, 1974) we purified the Na+,K+-ATPase from 15--90 rat brains, and obtained enzyme preparations with a mean specific activity of 552 +/- 37.6 mumol Pi/mg of protein/hr (95.5% ouabain sensitive). This "purified" enzyme had an activity ratio (Mg2+ + Na+ + K+)/(Mg2+ + Na+) of 47.4 +/- 12.3 SEM, compared to 3.29 +/- 0.17 SEM for the untreated microsomes. Ouabain inhibited the "purified" enzyme with an I50 of 6 X 10(-9) M. Ouabain binding (644 pmol/mg of protein) yielded a turnover number of 13,700 min-1. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of the enzyme revealed predominantly the alpha and beta subunits with some minor contaminant bands. Previous methods of purification of rat brain Na+,K+-ATPase have employed sodium deoxycholate and high concentrations of NaI; the reported specific activity obtained was generally 150--350 mumol Pi/mg of protein/hr. We have employed higher SDS concentrations than in Jorgensen's technique for rabbit kidney but the procedure is simpler because sucrose gradients are not used. Final wash steps also include 10--20% glycerol in the media. These modifications have yielded Na+,K+-ATPase of significantly higher specific activity than previously reported for rat brain.

Blood flow to the rabbit pancreas with special reference to the islets of Langerhans.

Nonradioactive microspheres of various sized (mean batch diameters ranging from 6 microns ato 26 microns) were administered to unfasted rabbits under sodium pentobarbital anesthesia by a brief injection into the left ventricle. Flow rate per bead was determined by the reference organ method. After prompt death of the animal, the spheres were located and counted miroscopically (islet vs. nonislet) in fixed, stained, and cleared portions of the pancreas. According to an analysis of the distribution of spheres as a function of bead diameter, 11-23% of the total pancreatic blood flow went directly to the islets and 77-89% to the "acini" (nonislet tissues). After retrograde postmortem injection of spheres 6 microns, 9 microns and 11 microns in diameter, practically none reached the islets, whereas after orthograde postmortem injection, they did so in the same proportions as in vivo. These results, supplemented by certain control experiments, support the view that all, or nearly all, efferent islet blood flow goes to the acinar capillaries before leaving the organ. We conclude that the arterial supplies to the rabbit exocrine and endocrine pancreas are in parallel, with most of the flow going to the exocrine portion. However, the flow to the islets is large enough to permit significant local actions of the islet hormones on the exocrine pancreas, in confirmation of the existence of an insuloacinar portal system.