The ultrastructure of human secretory ameloblasts

Summary The ultrastructure of secreting ameloblasts of deciduous teeth from a human foetus (crown-rump length 195 mm) was investigated. The ameloblasts demonstrate a formation of granules in a juxtanuclear Golgi complex. In the Tomes' process the granules are released either through the lateral plasma membrane into the intercellular space between the Tomes' processes or directly through the apical plasma membrane into the enamel.The human ameloblasts differ from non-human ameloblasts in having a non-oriented vesicular granular endoplasmic reticulum. Further, the majority of mitochondria are situated in the apical part of the ameloblast adjacent to the Tomes' process.We would like to thank chief-surgeon A. Christensen, Bispebjerg Hospital, Copenhagen for his help in acquiring foetal material. For technical assistance we would like to thank M. Balslev and U. Eberth, Anatomy Department A.     

Fine structure of the secretory and nonsecretory ameloblasts in the frog. I. Fine structure of the secretory ameloblasts.

Amelogenesis in the tooth germs of the frog Rana pipiens was examined by electron microscopy at different stages of tooth development. Cellular changes in secretory ameloblasts during this process showed many basic similarities to those in mammalian amelogenesis. Amelogenesis can be divided into three stages based on histological criteria such as thickness of enamel and the relative position of the tooth germ within the continuous succession of teeth. These stages are early, transitional and late. The fine structure of the enamel-secreting cells reflects the functional role of these ameloblasts as primarily secretory in the early stage, possibly transporting in the late stage and reorganizing between the two functions in the transitional stage. In early amelogenesis the cell exhibits well-developed granular endoplasmic reticulum, Golgi complex, microtubules, dense granules, smooth and coated vesicles, lysosome-like bodies in supranuclear and distal portions of the cell and mitochondria initially concentrated in the basal part of the cell. Numerous autophagic vacuoles are observed concomitant with the loss of some cell organelles at the transitional stage. During late amelogenesis the ameloblasts exhibit numerous vesicles, granules, convoluted cell membranes, junctional complexes and widely distributed mitochondria. Toward the end of amelogenesis, cells become oriented parallel to the enamel surface and the number of organelles is reduced. Amelogenesis in the frog is an extracellular process and mineralization seems to occur simultaneously with matrix formation.     

Qualitative electron probe analysis of secretory ameloblasts and odontoblasts in the rat incisor

Summary Rapidly frozen growing rat incisors were freeze fractured and freeze dried in preparation for energy dispersive X-ray emission microanalysis in a scanning electron microscope. Ca levels were found to be elevated in the distal cell body of odontoblasts, whereas Ca was uniformly low over all parts of the cell body of secretory ameloblasts. The results suggest fundamental differences in the mechanisms by which these two cell types process Ca, and that Ca possibly diffuses through the secretory ameloblast layer on its way to the enamel.     

Co-distribution of annexin VI and actin in secretory ameloblasts and odontoblasts of rat incisor

Summary Annexin VI and actin were detected by immunoblot analysis in the enamel- and dentin-related portions of dental tissues. Annexin VI was found mainly in the particulate fraction whereas actin was detected in both the soluble and particulate fractions. By immunoelectron microscopy, annexin VI antibodies conjugated with colloidal gold were seen to label the mitochondria, the cytosol and the nucleus of secretory ameloblasts and odontoblasts of rat incisor. In the processes of these cell, the plasmalemmal undercoat was labeled. Antiactin antibodies labeled the desmosome-like junctions, the cytosol, and the mitochondria of the cell bodies. Extensive labeling was seen at the periphery of the Tomes' processes and odontoblast processes. These results suggest that annexin VI may play a role in Ca²⁺-regulation in the cell bodies, especially as a calcium receptor protein in the mitochondria. Moreover, annexin VI and actin seem to be co-distributed in secretory processes. Thus, these proteins might be both involved in exocytotic and endocytotic events.     

Planar cell polarity protein localization in the secretory ameloblasts of rat incisors

The localization of the planar cell polarity proteins Vang12, frizzled-3, Vang11, and Celsr1 in the rat incisors was examined using immunocytochemistry. The results showed that Vang12 was localized at two regions of the Tomes' processes of inner enamel-secretory ameloblasts in rat incisors: a proximal and a distal region. In contrast, frizzled-3 was localized at adherens junctions of the proximal and distal areas of inner enamel- and outer enamel-secretory ameloblasts, where N-cadherin and β-catenin were localized. frizzled-3 was also localized in differentiating inner enamel epithelial cells. Vang11 was localized sparsely in differentiating preameloblasts and extensively at the cell boundary of stratum intermedium. Celsr1 was not localized in ameloblasts but localized in odontoblasts extensively. These results suggest the involvement of planar cell polarity proteins in odontogenesis.     

Characterization of putative secretory sites on ameloblasts of the rat incisor

The distribution and structure of the putative sites where enamel matrix is secreted from the ameloblast were studied by correlating the external topography with the distribution of organelles in Tomes' process cut in various planes of section. Both the interrod and rod secretion sites are associated with deep membrane infoldings. It was found that the interrod secretion site completely surrounds each ameloblast, and the marked interdigitation of adjacent cells results in a cooperative growth front for interrod enamel. In contrast, the rod secretion site is present on only one surface of the interdigitating portion of Tomes' process. Numerous granules were observed adjacent to the membrane infoldings associated with both sites, and granules were seen fused to membrane infoldings suggesting that the matrix of enamel is a merocrine secretion product.     

Histochemical and electron probe analysis of secretory ameloblasts of developing rat molar teeth

Summary Calcium was not found in secretory ameloblasts and stratum intermedium cells when treated with OsO₄-pyroantimonate or when surfaces prepared by fracturing fresh, rapidly frozen, developing molar tooth germs were subject to electron probe X-ray analysis.Pyroantimonate reaction product, considered to be calcium, was found in mitochondria of enamel organ cells which were first placed in a bath containing calcium and potassium. The plasma membrane was disrupted in cells which showed mitochondrial localization of reaction product.The results provide no data which indicates that enamel organ cells have a direct, active role in the movement of calcium into the enamel. Rather, it is suggested that the secretory enamel organ might serve as a selective barrier in regulating the initial mineralization of enamel.     

Histochemical and electron probe analysis of secretory ameloblasts of developing rat molar teeth

Calcium was not found in secretory ameloblasts and stratum intermedium cells when treated with OsO4-pyroantimonate or when surfaces prepared by fracturing fresh, rapidly frozen, developing molar tooth germs were subject to electron probe X-ray analysis. Pyroantimonate reaction product, considered to be calcium, was found in mitochondria of enamel organ cells which were first placed in a bath containing calcium and potassium. The plasma membrane was disrupted in cells ehich showed mitochondrial localization of reaction product. The results provide no data which indicates that enamel organ cells have a direct, active role in the movement of calcium into the enamel. Rather, it is suggested that the secretory enamel organ might serve as a selective barrier in regulating the initial mineralization of enamel.     

Cytochemical calcium distribution in secretory ameloblasts of the rat in relation to enamel mineralization

Calcium distribution in secretory ameloblasts was studied in rat incisor enamel in which mineralization was temporarily disturbed by injection of either fluoride or cobalt. Pyroantimonate precipitates of calcium were analysed morphometrically in regions of the cell membranes, mitochondria and secretory granules. The disturbances in mineralization were characterized by accumulations of unmineralized enamel matrix at the secretory regions of Tomes' process within 1 h after injection. Fluoride-induced disturbances in mineralization were not accompanied by marked changes in calcium concentration and distribution. It may be that fluoride causes alterations in the synthesis and secretion of the organic matrix which affects its ability to mineralize. Secretory ameloblasts treated with cobalt showed a broad basis for interference with calcium, in particular that which is associated with cell membranes and secretory granules. Secretory ameloblasts may be actively controlling the availability of calcium to enamel by mechanisms involving the cell membrane as well as the secretory granules.     

Ultrastructure of secretory ameloblasts in the house musk shrew, Suncus murinus, Insectivora

Tooth germs from neonatal house musk shrews, Suncus murinus, were used for the study. The tooth morphogenesis was compared electron microscopically to that of Primates. In the tooth germ at the bell stage, the ameloblast was 3 x 50 microns in size, columnar in shape and had several tubular-type Golgi apparatus which were at the distal end of the cell. Most mitochondria were noted at the proximal end of ameloblasts. Tomes' processes were 1 micron in width, protruded 10 microns from the ameloblast and had many dense bodies and two kinds of vesicles. They were morphologically different from human ameloblasts and enamel rods.     

Enamel protein biosynthesis and secretion in mouse incisor secretory ameloblasts as revealed by high-resolution immunocytochemistry

Mouse secretory ameloblasts express a number of enamel proteins, which have been divided into amelogenin and enamelin subfamilies. We have used polyclonal antibodies to murine amelogenins to reveal enamel proteins in mouse ameloblasts using the protein A-gold immunocytochemical technique. Specific immunolabeling was detected over the extracellular enamel matrix and over the rough endoplasmic reticulum, the saccules of the Golgi apparatus, and the secretory granules of the ameloblasts. In addition, some lysosome-like granules were also labeled. Only background labeling was obtained over mitochondria, nuclei, cytosol, adjacent odontoblasts, and dentin. Quantitation of the intensity of labeling showed the presence of an increasing gradient along the secretory pathway, which may correspond to the concentration or the maturation of these proteins as they are processed by the cell. These findings indicate that the ameloblast displays an intracellular distribution of its secretory products similar to that of other merocrine secreting cells. The presence of enamel proteins in lysosomes suggests that crinophagy and/or resorption occurs in these cells.     

Brown fat activity in fasted and refed rats

Four days of fasting in the rat reduced brown-adipose-tissue (BAT) mass, mitochondrial protein, and tissue protein content. Specific binding of guanosine diposphate (GDP) to BAT mitochondria was depressed by 55% in 4d-fasted rats. Rats fasted for 3 d, and then refed a single carbohydrate meal (40 kJ), showed a significant increase in specific GDP-binding (27% above fasted) 24 h later, and a large increase in total binding. Specific activities of cytochrome oxidase and -glycerophosphate dehydrogenase in BAT mitochondria were not significantly affected by fasting or refeeding. These results suggest that BAT may be partly responsible for the fall in metabolic rate associated with fasting and the delayed increase after carbo-hydrate refeeding. These effects may be due to changes in the mitochondrial proton-conductance pathway in brown fat.     

The modulation of Ca-ATPase activity and protein-lipid interactions in the sarcoplasmic reticulum by ATP

The time-course of ATP hydrolysis by Ca-ATPase of purified sarcoplasmic reticulum is biphasic with an initial rate over 1 to 2 min exceeding the subsequent rate. Hydrolysis of GTP and p-nitrophenylphosphate (pNPP) occurs at a slower but constant rate. Arrhenius plots of GTP, p-nitrophenylphosphate and initial rates of ATP hydrolysis all exhibit a discontinuity at about 20-24 degrees C; no breaks are observed in plots of the slower phase of ATP hydrolysis. The effect of substrate hydrolysis on the disposition of the enzyme in the membrane was examined by monitoring the quenching of tryptophan fluorescence by pyrene present in the hydrophobic domain of the membrane. The presence of ATP, but not GTP, prevents a temperature-dependent decrease in fluorescence quenching suggesting that ATP binding causes a change in the protein domain in contact with the membrane lipids.     

Increased sensitivity to neurotensin in fasted rats

The present study was designed to investigate the influence of fasting on the ability of neurotensin (NT) to produce hypotension in anesthetized rats, to constrict the coronary vessels of perfused rat hearts and to stimulate isolated rat stomach strips and spontaneously beating atria of rats. The hypotensive effects of NT, in contrast to those elicited by bradykinin and isoproterenol, were markedly increased in 24h-fasted and 72h-fasted rats compared to control fed rats. The increase in perfusion pressure provoked by NT in perfused hearts derived from 24h-fasted and 72h-fasted rats was much larger than in hearts derived from control fed animals. On the other hand, the constrictor action of angiotensin in perfused rat hearts was not altered by fasting. The stimulatory effect of NT in stomach strips and atrial preparations derived from fasted rats was not increased when compared to tissues from control fed animals. It is suggested that prolonged period of fasting in rats may contribute to sensitize the cardiovascular system of these animals to the action of NT.     

Alteration of peripheral beta-adrenergic responsiveness in fasted rats

Total food deprivation for 72 hrs (3 day fast) in female rats resulted in a reduction in serum thyroid hormones as well as a reduced peripheral beta-adrenergic responsiveness to isoproterenol. Food deprivation for 48 or 72 hrs significantly decreased both serum T3 and T4 values as compared to non-fasted controls. There were no significant differences in either T3 or T4 levels as a result of a 24 hr fast. Rats deprived of food for 72 hr had significantly smaller increases in oxygen consumption, colonic and tail skin temperatures following administration of isoproterenol (100 micrograms/kg b.w., s.c.) when compared to non-fasted control rats. Arterial blood pressure and heart rates were measured in unrestrained, unanesthetized, chronically cannulated rats. Food deprivation for 72 hrs significantly attenuated the decrease in blood pressure and the increase in heart rate associated with administration of isoproterenol (10 micrograms/kg b.w., s.c.). Possible mechanisms for the reduced beta-adrenergic responsiveness associated fasting are discussed.     

Phosphoinositide metabolism in adipocytes from fed and fasted rats

Phosphoinositide turnover was investigated in adipocytes from fed and 48 hour fasted rats. Insulin stimulated phosphoinositide synthesis both in adipocytes from fed and fasted rats. Fasting enhanced this effect of insulin 2-fold. Hydrolysis of phosphoinositides to inositol phosphates was not activated by insulin, neither transient after 2 minutes nor after 60 minutes stimulation. Under similar conditions, alpha 1-adrenergic receptor stimulation induced a pronounced inositol phosphate production. Thus, it is suggested that phosphoinositide hydrolysis is not involved in insulin action. The alpha 1-adrenergic effect was similar in adipocytes from fed and fasting rats.     

Insulin-induced hypoglycemia in fed and fasted exercising rats.

To determine running performance and hormonal and metabolic responses during insulin-induced hypoglycemia, fed and fasted male rats (315 +/- 3 g) were infused with insulin (100 mU/ml, 1.5 ml/h) or saline (1.5 ml/h) for 60 min and then killed at rest or after running on the treadmill (21 m/min, 15% grade). Insulin-infused fed rats ran poorly during the second 10 min of a 20-min exercise test. They were capable of running a total of 43 +/- 5 min, compared with 138 +/- 6 min for saline-infused fed rats. Fasted insulin-infused rats were able to run only 12.8 +/- 0.8 min, compared with 122 +/- 15 min for fasted saline-infused rats. In fasted rats, blood glucose was 1.6 +/- 0.1 mM after 60 min of insulin infusion and 1.2 +/- 0.1 mM after running to exhaustion. Artificial increase of plasma free fatty acids had no effect on performance. Intravenous infusion of glucose at the time of fatigue produced an immediate recovery, allowing the formerly fatigued rats to run 20 min without development of fatigue. These results provide evidence that severe hypoglycemia can be a significant cause of fatigue, even if it occurs early in the course of an exercise bout.