An in vivo binding assay using radioautography was employed to visualize calcitonin receptors in rat tissues . At 2 min after intravenous injection of biologically active '25 I-salmon calcitonin, free hormone was separated from bound hormone by intracardiac perfusion with lactated Ringer's followed by fixation with 2 .5% glutaraldehyde . Various tissues were removed and processed for light and electron microscope radioautography . These were compared to tissues removed from animals that received identical amounts of labeled hormone with a large excess of unlabeled calcitonin. Among the tissues investigated, kidney and bone demonstrated labeling . In kidney, most silver grains were located over vesicles below the brush border of cells of the proximal convoluted tubules . These grains were still present after simultaneous injection of excess unlabeled hormone and most likely represented binding to sites involved with ingestion and degradation of hormone from the urinary filtrate . In contrast, grains localized to the basal surfaces of distal convoluted tubule cells were significantly reduced in number in control animals and represented sites of saturable, specific hormone binding . In bone, specific binding sites were found only at the periphery of osteoclasts. These labeled cells were located at resorption sites examined in tibia, humerus, and alveolar bone . This demonstration of the localization of ' 25 I-calcitonin in situ provides a new approach for studying the interaction of calciumregulating hormones with their target cells .

Previous studies in vitro and in vivo have demonstrated the presence of receptor sites for PTH on cells of the osteoblast phenotype. Nevertheless, it is unclear whether the diverse functions of this hormone in bone can all be attributed to its interaction with a single cell type. In this study, we have used a radioautographic method to examine the competitive binding of 125I-labeled rat PTH-(1-34) to the long bones of rats in vivo. Our studies confirm the presence of competitive binding to mature osteoblasts and the absence of significant competitive binding to multinucleated osteoclasts. However, by light and electron microscopic radioautographic analysis, the majority of specific competitive PTH binding was present over a cell in the intertrabecular space of the metaphyseal region, which was distinct from the mature osteoblast. This large mononuclear cell with multiple cytoplasmic extensions appeared to interface with both the bone matrix and the microvascular osseous circulation and may provide an additional target to mediate hormonal effects on the skeleton.

We have examined in vivo binding of bovine (b) PTH-(1-84) and the analog [Nle8,18, Tyr34]bPTH-(1-34) amide to hepatic, skeletal, and renal rat tissues. Bioactive 125I-labeled bPTH-(1-84) or the 125I-labeled bPTH-(1-34) analog was injected alone into experimental animals or with either unlabeled PTH or unlabeled unrelated hormone into control animals. Corresponding tissues from experimental and control animals were then processed for light and electron microscope radioautography and analyzed quantitatively and qualitatively. Binding of both PTH forms occurred on hepatocytes and sinusoidal cells in liver, and hepatocyte binding was clearly specific and competitive. Intact PTH-(1-84), but not the amino-terminal fragment, bound to Kupffer cells, indicating a sequence-specific interaction. In bone, specific competitive PTH binding was seen over osteoblasts, but not osteoclasts. Skeletal PTH binding was also seen over connective tissue mononuclear cells, and sinusoidal endothelial cells. In kidney, specific competitive PTH binding was seen over glomerular podocytes, and over the antiluminal surface of cells of the proximal tubule, the thick ascending limb of Henle's loop, and the distal tubule. Noncompetitive binding was seen on the luminal surface of proximal convoluted tubule cells. We have, therefore, distinguished specific competitive binding sites, probably related to hormone action, from noncompetitive binding sites, presumably associated with hormone metabolism, on discrete cell types or cell regions within several tissues. Our approach provides morphological correlates to the biochemical interaction of PTH with its target tissues and should enhance our understanding of the relationship of target cell structure and function.

For more than a week prior to the emergence of a hind limb, a steady electric current is driven out of the ventrolateral flank in the immature axolotl, returning through the integument in adjacent regions of the body. A marked peak in the density of this outcurrent could be observed over the exact area of hind limb formation 4 to 6 days prior to its appearance. After a bud projected from the flank, current densities were observed to decrease in magnitude yet localize about the early limb. In about one-half of the animals observed, current reversed its polarity and entered the apex of large buds, 0.4 to 0.5 mm in length. We discuss the possible role such endogenous currents and their associated fields may play in limb development and compare it to similar current flow associated with the regeneration of amphibian limbs.