Pancreatic polypeptide: identification of target tissues using an in vivo radioreceptor assay.

The definitive function of pancreatic polypeptide in mammalian physiology remains unknown. The identification of specific PP target tissues should be helpful to further investigations into the possible regulatory actions of this peptide. An in vivo radioreceptor assay was used in the rat to locate potential binding sites of I(125) bovine PP. In vitro, high concentrations of unlabeled hormone competitively inhibit binding to receptors by low concentrations of labeled hormone. In vivo studies showed that, in the presence of concentrated unlabeled pancreatic polypeptide, labeled PP distributes between the plasma and interstitial fluid. When excess unlabeled PP is replaced with saline in the companion animals, the labeled peptide appears to distribute in a volume that exceeds the combined plasma volume and interstitial fluid volume of the tissue. Using this in vivo receptor assay, the distribution volume that exceeds the anatomic extracellular volume has been identified as the receptor compartment. With this assay we demonstrated in the rat specific and displaceable PP binding to the ductus choledochus, duodenum, ileum, and adrenal gland. The NVV determined in the adrenal gland of experimental animals was 3.9 times greater than that found in the control group. Binding was rapid and was displaced only by excess unlabeled pancreatic polypeptide. Neither excess insulin nor excess neuropeptide Y significantly reduced this binding.

Lifetime changes in aspartate carbamyltransferase of reproductive tissue of male rats.

Aspartate carbamyltransferase (ACTase) of the testis, epididymis, and prostate gland was determined throughout the lifetime of rats. Testicular ACTase concentration (nmoles carbamyl aspartate formed per microgram protein) peaked at about 45 g body weight, then declined. Epididymal concentration increased up to 100 g body weight and around 155 g plateaued in the caput and declined slightly in the cauda. A lifetime decline followed the maximal prostatic concentration found in the youngest test rats. Maximal total testicular activity, which occurred at 350 g, was undiminished in the oldest rats, while maximal epididymal activity occurred in the latter group. Total prostatic activity peaked at about 450 g body weight. Castration and testosterone replacement showed prostatic ACTase levels to be hormone dependent.

Pyrimidine synthetic enzymes of the rat testis and epididymis following unilateral vasectomy.

The de novo pyrimidine synthetic enzyme, aspartate carbamyltransferase, and the two pyrimidine salvage enzymes, uridine and thymidine kinases, of the rat testis and epididymis were measured 1, 2, 4, 6, 8, and 10 wk following unilateral vasectomy. Vasectomy had no effect on organ wet weights and on testicular and epididymal asparate carbamyltransferase and thymidine kinase activities. Increases in the uridine kinase activity of the caput epididymidis at 2 wk and of the cauda epididymidis from the second to the sixth weeks were the only significant enzymatic changes observed.

Distribution of pyrimidine synthetic enzymes in the rat testis.

The distribution of the de novo and the salvage pathway enzymes for pyrimidine synthesis in the rat testis was investigated in separated seminiferous tubules and interstitial tissue. Aspartate carbamyltransferase (de novo synthesis) was localized primarily in the seminiferous tubules while the uridine and thymidine kinase activities were associated mainly with the interstitial tissue. To further delineate this distribution these enzymes were studied in testes from rats treated with Busulfan (the methane sulfonic acid diester of 1,4-butanediol), which causes degeneration of spermatogonia but not Sertoli cells. The results indicate that a part of the low tubular thymidine kinase activity resides in the germinal elements while the tubular uridine kinase activity is localized in the Sertoli cells.

Activity of enzymes involved in pyrimidine nucleotide synthesis during uterine growth in cyclic rats.

The pyrimidine nucleotide salvage enzymes, uridine and thymidine kinases, and the enzyme, aspartate carbamyltranferase, involved in de novo synthesis, were investigated to assess their relative contributions to the RNA and DNA precursor requirements of uterine growth in cyclic rats. Only aspartate carbamyltransferase reflected the known fluctuations in plasma oestradiol-17beta and uterine mitotic activity, being minimal at dioestrus 1 and maximal at pro-oestrus. Treatment of ovariectomized rats with oestradiol-17beta stimulated carbamyltransferase activity, but cycloheximide prevented this response, indicating the association between the enzyme and this hormone. Uridine kinase activity did not vary during the oestrous cycle, but there were peaks of thymidine kinase activity on the days of pro-oestrus and the 1st day of dioestrus.

Theoretical basis for a new in vivo radioreceptor assay for polypeptide hormones.

To date the in vivo identification and quantitation of specific hormone receptors has been difficult, time consuming, and lacking in sensitivity. We present the theory underlying a new in vivo radioreceptor assay for polypeptide hormones based on receptor theory derived from in vitro investigations and in vivo kinetic and autoradiographic studies. The assay was developed from a tissue model and a theory of hormone distribution. Measuring the labeled hormone distributed between the plasma and interstitial space in the presence or absence of excess unlabeled hormone permits the accurate determination of hormone specifically bound to receptors. This approach eliminates the problem of nonspecific binding due to free tracer, hormone degradation products, or labeled non-hormone molecules. A receptor compartment and specific binding of hormone are calculated from only four measured parameters: total tissue labeled hormone, tissue albumin, plasma labeled hormone, and plasma albumin. The method is applicable to most tissues and hormones under a variety of conditions and permits simultaneous comparison of multiple tissues in the same animal under identical conditions.

Identification of tissue insulin receptors: use of a unique in vivo radioreceptor assay.

An in vivo radioreceptor assay for polypeptide hormones has been developed and applied to the identification of tissue insulin receptors. The theoretical basis for this assay is presented elsewhere in this issue. 125I-insulin and 131I-albumin were infused into male rats with increasing amounts of unlabeled insulin. Plasma samples were taken at 1-min intervals until the animals were killed at 5 min. Tissue samples were excised and weighed and the activity due to each isotope counted. By comparing the differential distribution of the labeled tracers and applying the results to a compartment model, the specific, displaceable binding of insulin to tissue receptors could be demonstrated. Binding was detected in the liver, muscle, fat, adrenal glands, pancreas, small intestines, and spleen.