J. Chayen

EFFECT OF SODIUM INTAKE ON ABILITY OF HUMAN PLASMA TO INHIBIT RENAL Na+-K+-ADENOSINE TRIPHOSPHATASE IN VITRO

Abstract An inhibitor of renal sodium-potassium-dependent ATPase was demonstrated in human plasma by a cytochemical technique. Circulating levels of this activity were 25 times greater in plasma taken when five healthy subjects were on a high-salt diet than in plasma obtained when they were on a low-salt diet. These results are consistent with the proposal that urinary sodium excretion is in part controlled by a circulating natriuretic substance.

A SENSITIVE BIOASSAY OF PARATHYROID HORMONE IN PLASMA

Understanding of calcium metabolism in health and disease has been retarded by the lack of an adequately sensitive bioassay of parathyroid hormone. The problem of dissociation of bioactivity and immunoactivity, well recognized for other polypeptide hormones, is exaggerated in the case of parathyroid hormone by the disproportionately long half‐time in the circulation of the immunoreactive fragments. A new method of assaying the biological activity of parathyroid hormone in plasma has been developed, based on the cytochemical methods which have yielded highly sensitive bioassays of other polypeptide hormones. It depends on the stimulation of glucose 6‐phosphate dehydrogenase activity in the distal convoluted tubules of segments of guinea‐pig kidney maintained in vitro, and measured by microdensitometry. The limit of sensitivity of the assay is 5 fg/ml (bPTH); the index of precision is 0.09 ± 0.04 (mean ± SEM; n= 11).

STUDIES ON THYROID STIMULATING HORMONE AND THE LONG-ACTING THYROID STIMULATING HORMONE

The basis of cytochemical bioassays is that the selected hormone will markedly alter some chemical activity of its target cells. In the case of thyroid stimulating hormone, it is well known that the hormone induces endocytosis of colloid by the thyroid follicle cells, and the endocytotic vesicles (containing the colloid) fuse with lysosomes (Wollman, 1969 ; Dumont, 1971). There is also some evidence that the stability of lysosomal membranes is diminished when changes occur at the cell membrane (Bitensky, 1963); from studies on the lysosomes of macrophages it is generally believed that the secondary lysosomes, namely the complex formed when primary lysosomes fuse with endocytotic vesicles, have less stable membranes than do the uninfluenced primary lysosomes. It follows, therefore, that one effect of thyroid stimulating hormone (TSH) and of the long acting thyroid stimulating hormone (LATS) could well be reflected in a change in the stability of the lysosomal membranes in the cells of the follicles. Recently a number of studies have been made in which the stability of lysosomes in particular cells has been measured (e.g. Bitensky el al., 1973); these depend on the rate at which a chromogenic substrate, leucine 2-naphthylamide, penetrates the lysosomal membranes, the colour being measured by microdensitornetry. It therefore seemed reasonable to see whether this quantitative cytochemical procedure for measuring Iysosomal stability could be used to monitor the effect of TSH and of LATS acting on thyroid tissue maintained in vitro.

A sensitive bioassay for adrenocorticotrophic hormone in human plasma.

A technique for the bioassay of adrenocorticotrophic hormone (ACTH) in human plasma is described, and its experimental validation discussed.

The use of a hidden metal-capture reagent for the measurement of Na+−K+-ATPase activity: A new concept in cytochemistry

SummaryThe original lead-trapping method for demonstrating Na+−K+-ATPase activity was discredited because of the effect that lead ions can have on the substrate and on the enzyme. Current methods, that measure this activity by the related K+-dependent phosphatase activity, do not appear to measure activity that is known, from microchemistry, to occur in proximal convoluted tubules. The disadvantages of using lead appear to have been overcome by the use of a new reagent in which the lead is complexed with ammonium citrate ions; phosphate, liberated enzymatically, successfully competes with these ions. The activities of total ATPase and of the ouabain sensitive Na+−K+-ATPase have been measured in three regions of the nephron in the guinea-pig and in the rat. The relative activities found, by this method, in the different regions of the latter, appear to be comparable with results found by others, using microchemical methods applied to isolated regions of the nephron.

The cytochemical bioassay of hormones.

Publisher Summary The evidence summarized in this chapter shows that it is possible to precisely measure the biochemical changes induced in target cells when the specific hormone acts on these cells with the modern techniques of cellular biology and cytochemistry. By measuring such effects, it is possible to assay the biological activity of the hormone with a degree of sensitivity that exceeds that of most currently available radioimmunoassays by two to three orders of magnitude. The increase in the use of cytochemical bioassays have resulted in the increase in their validity tests and applicability scope. The development of a cytochemical section assay for adrenocoticotropin (ACTH) has resulted in extension of these assays to routine purposes. It is a model for similar assays for other hormones. But the principle that the effect of bioreactive molecules on their target tissues can be measured very sensitively opens further vistas. These techniques can be applied to the detection, and assay, of other biologically active substances, such as pharmacologically active substances or toxic substances. These techniques can be readily used to study the analysis of end-organ sensitivity and the effects of hormones or their products on the other tissues in the body can be readily studied by these new techniques.

Synthesis of arachidonate cyclo-oxygenase products by rheumatoid and nonrheumatoid synovial lining in nonproliferative organ culture

Specimens of human rheumatoid and nonrheumatoid synovial lining were maintained in nonproliferative organ culture for 20 hours. The culture fluids were then assayed for prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and 6-keto-prostaglandin F1α (6-keto-PGF1α) by specific radioimmunoassay. The presence of each of these substances was confirmed by gas chromatography and mass spectrometry. Rheumatoid tissue produced significantly more of each cyclo-oxygenase product than nonrheumatoid tissue.

Effects on fracture healing of an antagonist of the vitamin K cycle

SummaryThe anticoagulant, dicumarol, inhibits the vitamin K cycle by blocking the conversion of the vitamin K epoxide. The effects of dicumarol on ossification have been tested by feeding it to rats in which a closed fracture of the metatarsals had been induced; the effects were studied up to 12 days postfracture. At 12 days, treatment with dicumarol caused a highly significant decrease in the amount of bone produced, without affecting the total size of the callus. Quantitative cytochemistry of unfixed, undemineralized sections showed that dicumarol also markedly affected the periosteal activities of glucose 6-phosphate dehydrogenase and of alkaline phosphatase in the first 2 mm from the fracture measured at 3 and 5 days postfracture when normally, new bone is first formed. In contrast, dicumarol had little effect on these activities in the fully formed callus.

THE HISTOCHEMICAL DEMONSTRATION OF ADENOSINE TRIPHOSPHATASE ACTIVITY IN MYOCARDIUM

Adenosine triphosphatase has been demonstrated histochemically in rat and human myocardium. To obtain its precise localization in discrete bands, apparently corresponding to the concentration of myosin, it was necessary to modify the existing technique to obtain better preservation of unfixed tissue and maximal enzymatic activity. Thus it was necessary to increase the concentration of calcium and to effect the reaction at pH 9.4 after treatment with 2:4-dinitrophenol. The specificity of the reaction was shown by these factors, by testing with phosphate esters other than adenosine triphosphate, and by the inhibitory effect of magnesium.

Altered organization of non-collagenous bone matrix in osteoporosis

In osteoporosis it is postulated that while the amount of bone is diminished, the quality of the bone is unaltered. Recently relatively novel methods of analysis have shown that, at the fracture site of osteoporotic subcapital fractures, there is a marked change in the molecular orientation of components of the non-collagenous bone matrix. These procedures, now applied to iliac crest biopsies, confirm earlier findings of altered orientation of the proteoglycans at the subcapital fracture site but show that very similar changes occur even in the iliac crests from patients with both types of osteoporotic proximal femoral fracture. Thus, whereas the amount of these acidic moieties of the non-collagenous bone matrix was unchanged, the molecular orientation was markedly altered, albeit not to the same extent as that found at the fracture site. These results imply that the quality of the bone, as well as the quantity, may be generally affected in osteoporosis.