Abstract. Secretory granules of sheep thyroid parafollicular cells contain serotonin, a serotonin-binding protein, and calcitonin. Parafollicular cells, isolated by affinity chromatography, were found to secrete serotonin when activated by thyrotropin (TSH) or elevated [Ca2÷]~. TSH also induced a rise in [Ca2÷]~. We studied the effect of these secretogogues on the pH difference (ApH) across the membranes of the secretory granules of isolated parafollicular cells. The trapping of the weak bases, acridine orange or 3-(2,4 dinitro anilino)-Y-amino-N-methyl dipropylamine (DAMP), within the granules was used to evaluate ApH. In contrast to lysosomes, which served as an internal control, the secretory granules of resting parafollicular cells displayed a limited and variable ability to trap either acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyldipropylamine; however, when parafollicular cells were stimulated with TSH or elevated [Ca2+],, the granules acidified. Weak base trapping was also used to evaluate the ATP-driven H ÷ translocation into isolated parafollicular granules. The isolated parafollicular granules did not acidify in response to addition of ATP unless their transmembrane potential was collapsed by the K ÷ ionophore, valinomycin. Secretory granules isolated from TSH-treated parafollicular cells had a high chloride conductance than did granules isolated similarly from untreated cells. Furthermore, ATP-driven H + translocation into parafollicular granules isolated from TSH-stimulated parafollicular cells occurred even in the absence of valinomycin. These results demonstrate that secretogogues can regulate the internal pH of the serotonin-storing secretory granules of parafollicular cells by opening a chloride channel associated with the granule membrane. This is the first demonstration that the pH of secretory vesicles may be modified by altering the conductance of a counterion for the H ÷ translocating ATPase.

A radiographic and histologic study of bat bones showed that there is a progressive loss of bone tissue during hibernation and abrupt reversal at arousal. Enhanced osteolysis accounted for the bone loss; osteoclasia was not observed.Increasing evidence strongly indicates that the parafollicular cells (light cells, C cells) of the thyroid gland synthesize calcitonin, the bone resorption inhibiting hormone (Pearse, '66; Matsuzawa and Kurosumi, '67; Ericson, '68). Although the importance of calcitonin in mammalian physiology is still enigmatic (Hirsch and Munson, '69), strong evidence that calcitonin may be physiologically important in the hibernator has been found. Parafollicular cells are sparse in the thyroid glands of most mammals (Stux, Thompson, Isler and Leblond, '63; Young, Duncan and Care, '68; Klinck, Oertel and Winship, '70), but they are widespread and abundant in the thyroid glands of hibernators (Azzali, '66; Azzali, '67; Nunez, Gould, Hamilton, Hayward and Holt, '67; Pearse and Welsch, '68; Gabe and Martoja, '69; Olivereau, '70).In the bat, parafollicular cells show rather striking seasonal variations in fine structure. During the first half of hibernation, the amount of granular endoplasmic reticulum is greatly reduced and the calcitonin-secretion granules lose their solid dense core (Nunez et al., '67; Nunez, Gould and Holt, '70) suggesting that calcitonin may be functionally inactive during the hibernation period. Cessation of calcitonin secretion during hibernation should result in increased bone resorption. However, the few reports on bone changes in hibernation are incomplete and conflict (Mayer and Bernick, '63; Bruce and Wiebers, '69). Therefore, in an effort to determine the effects of hibernation on ANAT. REC., 172: 97-108.bone and the mechanisms involved, and to establish possible interrelationships between bone changes and parafollicular cell activity, a histologic and radiographic study of the femur of active, prehibernating and hibernating bats was carried out. The results of the study are reported herein. MATERIAL AND METHODSThe bats used in this study were adult 'Myotis lucifugus, were of either sex, and were obtained from caves in Central Illinois before (September), during (November, December, January, and February) and after hibernation (late April) and are illustrated in figure 1. Bones were dissected and cleaned of soft tissues and pIaced in 10% fonnalin until they were radiographed and processed for histological examination.The bones were radiographed in a Faxitron 804 self-contained unit having a tungsten target and a 40 mil. Beryllium window. The nominal focal spot size was 0.5 mm. The tube was operated at 10 K.V.P., 3 MA, for exposure times of three minutes at a 24" focus-film distance. Kodak RPM film (having a speed value, grain size and contrast characteristics between industrial type M and industrial type A film) was used and processed through a medical X-Omat using a 90 second cycle. The radiographs were examined by light microscopy and were photographed a...

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Ultrastructural changes that occur in follicular cells of the bat thyroid gland just prior to, and immediately after arousal from hibernation are discussed in relation to the known changes which occur in thyroid function during arousal from hibernation. The most distinctive ultrastructural change that takes place just before emergence from hibernation is the occurrence, extracellularly, of concentrations of small vesicles lying in the colloid near the cell's apical plasma membrane. Similar accumulations of vesicles are absent in the apical cytoplasm of the follicular cell. Other principal changes from the early hibernating state found at this time are an increase in the number of apical vacuoles, dense granules and multivesicular bodies. These changes are followed at arousal itself by the appearance of large numbers of intracytoplasmic colloid droplets, often intimately associated with dense granules. An unusual feature of these follicular cells is that although they are rich in colloid droplets, apical pseudopods cannot be found.Numerous studies have demonstrated that the thyroid gland of mammalian hibernators exhibits an endogenous yearly cycle of activity. The thyroid is most active during the spring, when the animals are emerging from hibernation, and least active during the winter when the animals are hibernating (Kayser, '61; Hoffman, '65). In order to correlate these seasonal changes in thyroidal activity with the ultrastructure of the gland, we have periodically examined the fine structure of the bat follicular cells during the animals' yearly life cycle. In preceding papers we have already described the ultrastructural changes which occur in bat follicular cells during the homeothermic (active), preparatory and hibernating phases of the annual life cycle (Nunez and Becker, '70; Nunez, '71). In this study we describe the changes in follicular cells of the bat just prior to, and during arousal from hibernation. MATERIALS AND METHODSAdult bats of the species Myotis lucifzigzis and Pipestrilliis pipestrillus were used. All animals were captured in their natural habitat. Hibernating bats were collected in a cave in mid-April near the end of their period of hibernation. Active bats were caught in a barn in late April and in early June. The hibernating and active bats were of either sex. Hibernating animals were gently placed, in the cave where they were collected, into containers, the bottom halves of which were filled with ice in order to maintain the hibernating state. They were taken to the laboratory and kept in a cold room at 4°C. Three of the hibernating bats were killed by rapid decapitation in the cold room the following morning. The other animals were kept at 4°C in the hibernating state and killed 24, 48 and 96 hours later. Active bats were kept in the laboratory at room temperature and killed the following day. For electron microscopy, thyroid glands were removed in toto, and fixed in ice-cold 6.25% glutaraldehyde in 0.067 M cacodylate buffer, pH 7.3, for 4 hours. Next, the tissues were w...