A. Raymond Terepka

Fine structural changes associated with the onset of calcium, sodium and water transport by the chick chorioallantoic membrane

SummaryAn attempt is made to correlate structure and transport function in the embryonic chorioallantoic membrane. The fine structure of the endoderm and ectoderm in the membrane was examined with particular attention given to the morphological changes that occur when transport is established,in vivo. Two distinctive cells, a granule-rich cell and a mitochondria-rich cell, appear in the endoderm at the time allantoic fluid sodium, chloride and water reabsorption commences. These are indistinguishable from the cells described in toad bladder epithelium. It is suggested that the granule-rich cell is responsible for bulk water movement and the mitochondria-rich cell is specifically engaged in active sodium transport. In the ectoderm, two distinctive cell types are also found to be associated with the onset of active calcium transport. These are referred to as the capillary-covering cell and the villus-cavity cell. The preponderate capillary-covering cell is most likely responsible for transcellular calcium transport. It is postulated that the function of the villus-cavity cell is to secrete hydrogen ions which are necessary, along with carbonic anhydrase, to mobilize Ca++ from the insoluble calcium carbonate of the eggshell.

ELECTRON PROBE ANALYSIS OF THE CALCIUM DISTRIBUTION IN CELLS OF THE EMBRYONIC CHICK CHORIOALLANTOIC MEMBRANE I. A CRITICAL EVALUATION OF TECHNIQUES

The chorioallantoic membrane of the developing chick embryo is an epithelium that actively transports calcium. The methodology utilized to prepare this soft tissue for calcium localization with the electron probe x-ray microanalyzer is presented in detail. The preparative procedures are evaluated according to general histochemical principles and in relationship specifically to electron probe investigations. It is shown that the method employed in these studies preserves the normal fine structure of the tissue, prevents selective loss of calcium, permits only minor losses of total calcium and appears to maintain the distribution of calcium that existed in vivo. Examples are presented of artifacts that can be induced during tissue sectioning and mounting procedures. Problems of defining electron probe resolution in biologic specimens are discussed, and the critical importance of evaluating x-ray images in association with simultaneously generated sample current images is emphasized.

Calcium-stimulated respiration and active calcium transport in the isolated chick chorioallantoic membrane

SummaryCalcium markedly stimulates the respiration of the isolated chick chorioallantoic membrane. This stimulation of oxygen uptake appears to be closely associated with the membranes active transcellular calcium transport mechanism. In the presence of 1mm Ca++ the rate of uptake increases from 9.3±0.15 to 13.0±0.2 μliters O2/cm2/hr, an increase of about 40%. The calcium-stimulated respiration is specific for the ectodermal layer of cells, the known location of the calcium transport mechanism, and only occurs when the calcium transport mechanism is operative. Sr++ and Mn++ are transported by the tissue at a lower rate than Ca++ and cause a smaller stimulation of oxygen consumption. Mg++ and La3+ have no effect on tissue respiration. In the presence of Ca++, the organic mercurialp-chloromercuribenzene sulfonate (PCMBS) inhibits calcium transport and specifically decreases the oxygen uptake of the ectoderm to a rate identical to that obtained in a calcium-free medium. Stripping the inner shell membrane away from the chorioallantoic membrane mimics these effects. The specificity and locus of action of these two inhibitors suggest that a vital component of the active transcellular calcium transport mechanism resides on or near the outer surface of the plasma membrane of the ectodermal cells and that sulfhydryl groups are important to the normal function of this component.

The interrelationships between sodium ion, calcium transport and oxygen utilization in the isolated chick chorioallantoic membrane.

SummaryThe interrelationships between sodium ion, calcium transport and oxygen utilization have been investigated in the chick chorioallantoic membrane. The oxygen uptakes of the two surface layers of the tissue, the ectoderm and the endoderm, were separated into their basal, Na+ dependent and Ca++ dependent components. The endoderm has a basal rate of respiration of 3.6 μliters O2/cm2/hr and a Na+ dependent component of 1.4 μliters O2/cm2/hr. The ectoderm has a basal rate of respiration of about 3.5 μliters O2/cm2/hr, and Na+ and Ca++ dependent components of 1.1 and 3.6 μliters O2/cm2/hr, respectively. The rate of ectodermal calcium transport and calcium-stimulated oxygen uptake is strictly dependent on the presence of sodium in the bathing medium, and complex kinetics are observed as a function of sodium concentration. On the other hand, in 140mm Na+ the rate of calcium transport exhibits simple saturation kinetics as a function of calcium concentration. Ca++/O2 ratios determined for many different rates of transport give a ratio of about 0.5, a value much lower than similar ratios determined for other transport mechanisms. The calcium transport mechanism in the ectoderm responds to changes in transport rate very sluggishly, taking 30 to 50 min to give a maximum response. The differences between the calcium transport mechanism in this membrane and other known transport systems are discussed and it is suggested that these differences may represent the adaptations necessary for transcellular calcium transport.

Studies of the stability and extractability of vitamin D

Abstract Radioactively labelled vitamin D 3 -4-C 14 was used as a tracer to study the in vitro degradation of vitamin D. Effects were noted of various environmental conditions such as temperature, dispersion into water, exposure to air, oxygen, and ultraviolet light, with chromatography on alumina and Fluoropak 80 being employed to evaluate destruction. Procedures which avoided potentially damaging environments are described for saponifying tissues and extracting them of vitamin D and possible metabolites. They consist basically of refluxing with KOH in methanol, extraction by organic solvents in the presence of adequate concentrations of alcohol, and partition prior to chromatography or radioactivity assay.