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Morphological evidence for the involvement of calcium in neurohypophysial hormone release
278
Brain Research, 238 (1982) 278-281 Elsevier Biomedical Press
Morphological evidence for the involvement of calcium in neurohypophysial hormone release
SAROLTA KARCSO, FERENC A. LASZLO, G/~BOR JANCS0, LAJOS TOTH and ERNO B~,CSY Endocrine Unit, First Department of Medicine; (G.J.) Department of Physiology and (L.T.) Department of Anatomy, University Medical School, Szeged and (E.B.) Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest (Hungary)
(Accepted December 24th, 1981) Key words: neurohypophysis - - hyperosmoticity - - calcium - - microscopy histochemistry - - X-ray microanalysis
An elevated intracellular Ca 2+ concentration in the secretory nerve endings of the rat neurohypophysis was detected histochemically by means of light microscopy concomitant with the vasopressin secretion evoked by hypertonic saline. The electron microscopic and X-ray microanalytical results furnish morphological evidence for the function-dependent Ca 2+ storage capacity of the mitochondria, and suggest their role in the regulation of the free Ca2+ level in the neurosecretory axon terminal. A large body of biochemical evidence indicates that the release of neurohypophysial hormones is accompanied by the uptake of Ca 2+ into the neurosecretory nerve terminals from the extracellular space both in vivo and in vitro 2,3,6,7. Moreover, experimental data suggested that the significant increase in intra-axonal free Ca 2+ concentration is essential for the liberation of vasopressin 9. In the present study, using light and electron microscopic and X-ray microanalytical methods, the histochemical localization of Ca 2+ has been attempted in the rat neural lobe following intravenous injection of hypertonic saline, known to evoke vasopressin secretion 1,1°. Adult male C F Y rats were used. Animals were given an intravenous injection of either 2 M NaCI or physiological saline in a dose of 6 ml/kg, and were sacrificed 15, 30 or 60 min after the injection. For light microscopy, ionic Ca 2+ was histochemically localized by using the method of Kashiwa and Atkinson 4 or that of Shvay and Csillik s, with slight modifications. The procedure of Oschman and Wall '5 was applied to detect the Ca z+ binding sites at the ultrastructural level. Tissue samples were postfixed with OsO4, dehydrated in graded alcohol and embedded in Araldite (Durcupan ACM, Fluka). Ultrathin sections were double-stained with uranyl acetate and lead citrate. For X-ray microanalysis, sections with a gold to purple interference color were also used without staining. The analysis was performed in a JEM 100 C electron microscope equipped with an A S I D 4D scanning attachment and an O R T E C energydispersive X-ray analyzer (tilt angle of the preparation 35 °, accelerating voltage 80 kV, 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
Fig. 1. Light microscopic histochemical localization of ionic Ca 2+ in rat neurohypophysis 60 min after injection of hypertonic saline. Method of S~tvay and Csillik. x 350. Figs. 2 and 3. Fine-structural localization of electron-dense deposits in mitochondria of neurosecretory axon terminals 15 min after injection of hypertonic NaCI. Note absence of dense deposits in mitochondria of adjacent pituicyte (arrow). Fig. 2 × 37,000; Fig. 3 x 47,000. Fig. 4. Characteristic appearance of intramitochondrial granules 60 min after hypertonic saline, x 47,000. Figs. 5 and 6. Mitochondria containing electron-dense deposits before (Fig. 5) and after (Fig. 6) X-ray microanalysis. No contrast staining. Arrowhead, spot of contamination accentuated by the photographic procedure around the two granules analyzed. Both x 27,000. Fig. 7. X-ray energy spectrum from the two granules shown in Fig. 5 (arrow) containing a marked Ca Ka peak. The P Ka+a and Os Ma+a peaks overlap. The Cu Ka and Ks as well as the Mo Ka (Compton) peaks are instrumental artefacts.
280 area scan mode with the area slightly exceeding that of the analyzed intramitochondrial particles). By light microscopy ionic Ca 2+ could be demonstrated in the form of small, darkly stained granules as early as 15 min after the injection of hypertonic NaCI, but not after physiological saline in the rat neural lobe. The histochemical picture suggested the accumulation of Ca 2+ in secretory nerve endings (Fig. l). The adenohypophysis and the intermediate lobe were completely devoid of reaction product. Electron microscopy revealed that 15 min after the administration of hypertonic saline the mitochondria of certain secretory axon terminals in the neural lobe appeared swollen and contained electron-dense deposits in their matrix. These terminals showed typical signs of secretory activity: a paucity of large neurosecretory vesicles and an abundance of microvesicles (Figs. 2 and 3). At 60 rain the electron-dense deposits in the mitochondria were increased both in number and in size (Fig. 4), but such deposits were never seen in other intraterminal organelles. These axon terminals were always found to be in contact with the outer lamina of the basement membrane of the capillaries or were situated in the perivascular space. Neurosecretory axonal profiles distant from the capillaries and the mitochondria of pituicytes (Fig. 3) were devoid of dense deposits. Similarly, no granules were observed in any structure of the neural lobe of control animals after perfusion with the Ca 2 ~--containing fixative. X-ray microanalysis revealed that the electron-dense granules within the mitochondria of neurosecretory nerve terminals contained appreciable amounts of calcium (Figs. 5-7). Under the same analysis conditions, however, significant quantities of calcium could not be detected in the mitochondria of either the normally appearing nerve terminals or the pituicytes. It is worthy of mention that only traces of calcium were detectable by X-ray microanalysis in the dense intramitochondrial granules in contrast-stained sections; a high amount of lead and a small amount of uranium were found instead. It is generally accepted that entry of Ca 2+ into the nerve terminal, by triggering stimulus-secretion coupling, plays an important role in the mechanism of vasopressin release from the neurohypophysis z,3,6,9. Accordingly, stimulation of secretory nerve endings depolarizes the axonal membrane and thereby causes the influx of Ca 2+ from the extracellular space. The resulting rise in the intraterminal free Ca 2+ concentration triggers the release of vasopressin. Restoration of the normal intra-axonal Ca 2+ level following secretion is of prime importance for the neurosecretory nerve endings to remain functional. Sequestration of Ca 2 ~ in mitochondria is one of the essential mechanisms whereby the intra-axonal level of Ca z~ is regulated in the short term 7,9. The present light microscopic histochemical results are in correlation with previous biochemical findings showing a significant increase in the ionic Ca 2+ level of stimulated neurosecretory nerve endings. The electron histochemical and X-ray microanalytical observations provide a firm morphological basis for the view that axon terminal mitochondria play a decisive role in the temporary storage of Ca 2 ~ and, consequently, in the regulation of the free Ca z+ concentration in neurosecretory nerve terminals. Although morphological signs of Ca 2+ accumulation were not observed in other intraaxonal organelles, their possible role in the short-term regulation cannot be
281 excluded. I n addition, the present experimental a p p r o a c h offers a new possibility for the direct morphological identification of stimulated neurosecretory nerve endings which release vasopressin, as well as for the study of the cellular mechanisms involved in h o r m o n e secretion.
1 De Wied, D. and L~iszi6,F. A., Effect of autonomic blocking agents on ADH-release induced by hyperosmoticity, J. Endocr., 37 (1967) 16. 2 Douglas, W. W., A possible mechanism of neurosecretion: release of vasopressin by depolarization and its dependence on calcium, Nature (Lond.), 197 (1963) 81-82. 3 Douglas, W. W. and Poisner, A. M., Stimulus-secretion coupling in a neurosecretory organ: the role of calcium in the release of vasopressin from the neurohypophysis, J. Physiol. (Lond.), 172 (1964) 1-18. 4 Kashiwa, H. K. and Atkinson, W. B., The applicability of a new Schiff base, glyoxal bis(2-hydroxyanil), for the cytochemical localization of ionic calcium, J. Histochem. Cytochem., 11 (1963) 258-264. 50schman, J. L. and Wall, B. J., Calcium-bindingto intestinal membranes, J. Cell Biol., 55 (1972) 58-73. 6 Russel, J. T. and Thorn, N. A., Calcium and stimulus-secretion coupling in the neurohypophysis. I. 45-Calcium transport and vasopressin release in slices from ox neurohypophyses stimulated electrically or by a high potassium concentration, Acta endocr., 76 (1974) 449470. 7 Russel, J. T. and Thorn, N. A., Adenosine triphosphate dependent calcium uptake by subcellular fractions from bovine neurohypophyses, Acta physiol, scand., 93 (1975) 364--377. 8 S~ivay, G. and Csillik, B., Acetylcholine-induced calcium release in the post-junctional sarcoplasm, Syrup. BioL Hung., 5 (1965) 149-157. 9 Thorn, N. A., Torp-Pedersen, C., Treiman, M., Dartt, D. A. and Worm-Petersen, S., Biochemical mechanism of release of vasopressin. In F. A. L~.szl6 (Ed.), Recent Results in Peptide Hormone and Androgenic Steroid Research, Akad6miai Kiad6, Budapest, 1979, pp. 111-119. 10 Verney, E. B., Croonian Lecture. The antidiuretic hormone and the factors which determine its release, Proc. roy. Soc. B, 135 (1947/48) 25-106.
Brain Research, 238 (1982) 278-281 Elsevier Biomedical Press
Morphological evidence for the involvement of calcium in neurohypophysial hormone release
SAROLTA KARCSO, FERENC A. LASZLO, G/~BOR JANCS0, LAJOS TOTH and ERNO B~,CSY Endocrine Unit, First Department of Medicine; (G.J.) Department of Physiology and (L.T.) Department of Anatomy, University Medical School, Szeged and (E.B.) Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest (Hungary)
(Accepted December 24th, 1981) Key words: neurohypophysis - - hyperosmoticity - - calcium - - microscopy histochemistry - - X-ray microanalysis
An elevated intracellular Ca 2+ concentration in the secretory nerve endings of the rat neurohypophysis was detected histochemically by means of light microscopy concomitant with the vasopressin secretion evoked by hypertonic saline. The electron microscopic and X-ray microanalytical results furnish morphological evidence for the function-dependent Ca 2+ storage capacity of the mitochondria, and suggest their role in the regulation of the free Ca2+ level in the neurosecretory axon terminal. A large body of biochemical evidence indicates that the release of neurohypophysial hormones is accompanied by the uptake of Ca 2+ into the neurosecretory nerve terminals from the extracellular space both in vivo and in vitro 2,3,6,7. Moreover, experimental data suggested that the significant increase in intra-axonal free Ca 2+ concentration is essential for the liberation of vasopressin 9. In the present study, using light and electron microscopic and X-ray microanalytical methods, the histochemical localization of Ca 2+ has been attempted in the rat neural lobe following intravenous injection of hypertonic saline, known to evoke vasopressin secretion 1,1°. Adult male C F Y rats were used. Animals were given an intravenous injection of either 2 M NaCI or physiological saline in a dose of 6 ml/kg, and were sacrificed 15, 30 or 60 min after the injection. For light microscopy, ionic Ca 2+ was histochemically localized by using the method of Kashiwa and Atkinson 4 or that of Shvay and Csillik s, with slight modifications. The procedure of Oschman and Wall '5 was applied to detect the Ca z+ binding sites at the ultrastructural level. Tissue samples were postfixed with OsO4, dehydrated in graded alcohol and embedded in Araldite (Durcupan ACM, Fluka). Ultrathin sections were double-stained with uranyl acetate and lead citrate. For X-ray microanalysis, sections with a gold to purple interference color were also used without staining. The analysis was performed in a JEM 100 C electron microscope equipped with an A S I D 4D scanning attachment and an O R T E C energydispersive X-ray analyzer (tilt angle of the preparation 35 °, accelerating voltage 80 kV, 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
Fig. 1. Light microscopic histochemical localization of ionic Ca 2+ in rat neurohypophysis 60 min after injection of hypertonic saline. Method of S~tvay and Csillik. x 350. Figs. 2 and 3. Fine-structural localization of electron-dense deposits in mitochondria of neurosecretory axon terminals 15 min after injection of hypertonic NaCI. Note absence of dense deposits in mitochondria of adjacent pituicyte (arrow). Fig. 2 × 37,000; Fig. 3 x 47,000. Fig. 4. Characteristic appearance of intramitochondrial granules 60 min after hypertonic saline, x 47,000. Figs. 5 and 6. Mitochondria containing electron-dense deposits before (Fig. 5) and after (Fig. 6) X-ray microanalysis. No contrast staining. Arrowhead, spot of contamination accentuated by the photographic procedure around the two granules analyzed. Both x 27,000. Fig. 7. X-ray energy spectrum from the two granules shown in Fig. 5 (arrow) containing a marked Ca Ka peak. The P Ka+a and Os Ma+a peaks overlap. The Cu Ka and Ks as well as the Mo Ka (Compton) peaks are instrumental artefacts.
280 area scan mode with the area slightly exceeding that of the analyzed intramitochondrial particles). By light microscopy ionic Ca 2+ could be demonstrated in the form of small, darkly stained granules as early as 15 min after the injection of hypertonic NaCI, but not after physiological saline in the rat neural lobe. The histochemical picture suggested the accumulation of Ca 2+ in secretory nerve endings (Fig. l). The adenohypophysis and the intermediate lobe were completely devoid of reaction product. Electron microscopy revealed that 15 min after the administration of hypertonic saline the mitochondria of certain secretory axon terminals in the neural lobe appeared swollen and contained electron-dense deposits in their matrix. These terminals showed typical signs of secretory activity: a paucity of large neurosecretory vesicles and an abundance of microvesicles (Figs. 2 and 3). At 60 rain the electron-dense deposits in the mitochondria were increased both in number and in size (Fig. 4), but such deposits were never seen in other intraterminal organelles. These axon terminals were always found to be in contact with the outer lamina of the basement membrane of the capillaries or were situated in the perivascular space. Neurosecretory axonal profiles distant from the capillaries and the mitochondria of pituicytes (Fig. 3) were devoid of dense deposits. Similarly, no granules were observed in any structure of the neural lobe of control animals after perfusion with the Ca 2 ~--containing fixative. X-ray microanalysis revealed that the electron-dense granules within the mitochondria of neurosecretory nerve terminals contained appreciable amounts of calcium (Figs. 5-7). Under the same analysis conditions, however, significant quantities of calcium could not be detected in the mitochondria of either the normally appearing nerve terminals or the pituicytes. It is worthy of mention that only traces of calcium were detectable by X-ray microanalysis in the dense intramitochondrial granules in contrast-stained sections; a high amount of lead and a small amount of uranium were found instead. It is generally accepted that entry of Ca 2+ into the nerve terminal, by triggering stimulus-secretion coupling, plays an important role in the mechanism of vasopressin release from the neurohypophysis z,3,6,9. Accordingly, stimulation of secretory nerve endings depolarizes the axonal membrane and thereby causes the influx of Ca 2+ from the extracellular space. The resulting rise in the intraterminal free Ca 2+ concentration triggers the release of vasopressin. Restoration of the normal intra-axonal Ca 2+ level following secretion is of prime importance for the neurosecretory nerve endings to remain functional. Sequestration of Ca 2 ~ in mitochondria is one of the essential mechanisms whereby the intra-axonal level of Ca z~ is regulated in the short term 7,9. The present light microscopic histochemical results are in correlation with previous biochemical findings showing a significant increase in the ionic Ca 2+ level of stimulated neurosecretory nerve endings. The electron histochemical and X-ray microanalytical observations provide a firm morphological basis for the view that axon terminal mitochondria play a decisive role in the temporary storage of Ca 2 ~ and, consequently, in the regulation of the free Ca z+ concentration in neurosecretory nerve terminals. Although morphological signs of Ca 2+ accumulation were not observed in other intraaxonal organelles, their possible role in the short-term regulation cannot be
281 excluded. I n addition, the present experimental a p p r o a c h offers a new possibility for the direct morphological identification of stimulated neurosecretory nerve endings which release vasopressin, as well as for the study of the cellular mechanisms involved in h o r m o n e secretion.
1 De Wied, D. and L~iszi6,F. A., Effect of autonomic blocking agents on ADH-release induced by hyperosmoticity, J. Endocr., 37 (1967) 16. 2 Douglas, W. W., A possible mechanism of neurosecretion: release of vasopressin by depolarization and its dependence on calcium, Nature (Lond.), 197 (1963) 81-82. 3 Douglas, W. W. and Poisner, A. M., Stimulus-secretion coupling in a neurosecretory organ: the role of calcium in the release of vasopressin from the neurohypophysis, J. Physiol. (Lond.), 172 (1964) 1-18. 4 Kashiwa, H. K. and Atkinson, W. B., The applicability of a new Schiff base, glyoxal bis(2-hydroxyanil), for the cytochemical localization of ionic calcium, J. Histochem. Cytochem., 11 (1963) 258-264. 50schman, J. L. and Wall, B. J., Calcium-bindingto intestinal membranes, J. Cell Biol., 55 (1972) 58-73. 6 Russel, J. T. and Thorn, N. A., Calcium and stimulus-secretion coupling in the neurohypophysis. I. 45-Calcium transport and vasopressin release in slices from ox neurohypophyses stimulated electrically or by a high potassium concentration, Acta endocr., 76 (1974) 449470. 7 Russel, J. T. and Thorn, N. A., Adenosine triphosphate dependent calcium uptake by subcellular fractions from bovine neurohypophyses, Acta physiol, scand., 93 (1975) 364--377. 8 S~ivay, G. and Csillik, B., Acetylcholine-induced calcium release in the post-junctional sarcoplasm, Syrup. BioL Hung., 5 (1965) 149-157. 9 Thorn, N. A., Torp-Pedersen, C., Treiman, M., Dartt, D. A. and Worm-Petersen, S., Biochemical mechanism of release of vasopressin. In F. A. L~.szl6 (Ed.), Recent Results in Peptide Hormone and Androgenic Steroid Research, Akad6miai Kiad6, Budapest, 1979, pp. 111-119. 10 Verney, E. B., Croonian Lecture. The antidiuretic hormone and the factors which determine its release, Proc. roy. Soc. B, 135 (1947/48) 25-106.