Mechanism of the post-haemorrhagic vasopressin release from the posterior pituitary lobe

The mechanism of post-haemorrhagic vasopressin release from the neurohypophysis was studied in rats anaesthetized with urethane. Neurohypophysial vasopressin content was determined according to Dekański and plasma renin activity by radioimmunoassay. In animals bled (1.5% body weight) 60 min after induction of anaesthesia and 30 min after bilateral nephrectomy vasopressin content of the posterior pituitary was significantly higher than in sham-nephrectomized rats. However, when haemorrhage was produced 240 min after induction of anaesthesia and 210 min after nephrectomy, the neurohypophysial vasopressin content was low and similar as that in non-nephrectomized animals. It is concluded that in the phase directly following haemorrhage vasopressin release depends on acute activation of the renin-angiotensin system. Other mechanisms, possibly circulatory reflexes, are involved in the late phase, during prolonged anaesthesia.     

Pituitary astrocytes from the neural lobe of rats

Summary Tissue culture preparations of adult and neonatal rat pituitary neural lobes were examined by use of cell-type specific immunohistochemical markers. Cultures obtained from explanted or dissociated adult tissue or explanted neonatal tissue produced cells immunoreactive for endothelial and fibroblast markers. In contrast, dissociated neonatal tissue produced, in addition, two distinct forms of astrocytic glial cells immunoreactive for glial fibrillary acidic protein, one of which was also immunoreactive for the ganglioside GD3.     

Morphometric classification of the neurosecretory granules in the rat pars nervosa

Summary The distribution of the diameters of the neurosecretory granules in the rat pars nervosa (measured from electron micrographs taken at 40 000 × ) was compared among axons by nonparametric statistical methods and the axons were classified into five groups with median granule diameters of 143, 155, 167, 180 and 193 nm. We suggested that these five axon types carried different secretory substances contained in the pars nervosa. This investigation is supported by a grant from the Population Council, New York and grant from the Ministry of Education. Authors are grateful to Japan Electron Optics Laboratory Company for their technical assistance with the electron microscopy and to Miss Kazue Yamamoto for her help in preparing the figures.     

Electron-microscopic cytochemistry of the catecholaminergic innervation of TRH neurons in the rat hypothalamus

Summary The catecholaminergic innervation of thyrotropin-releasing hormone (TRH) neurons was examined by use of a combined method of 5-hydroxydopamine (5-OHDA) uptake or autoradiography after intraventricular injection of ³H-noradrenaline (³H-NA) and immunocytochemistry for TRH in the same tissue sections at the electron-microscopic level.TRH-like immunoreactive nerve cell bodies were distributed abundantly in the parvocellular part of the paraventricular nucleus (PVN), in the suprachiasmatic preoptic nucleus and in the dorsomedial nucleus of the rat hypothalamus. In the PVN, a large number of immunonegative axon terminals were found to make synaptic contact with TRH-like immunoreactive cell bodies and fibers. In the combined autoradiography or 5-OHDA labeling with immunocytochemistry, axon terminals labeled with ³H-NA or 5-OHDA were found to form synaptic contacts with the TRH immunoreactive nerve cell bodies and fibers. These findings suggest that catecholamine-containing neurons, probably noradrenergic, may innervate TRH neurons to regulate TRH secretion via synapses with other unknown neurons in the rat PVN.This study was supported by grants from the Ministry of Education, Science and Culture, Japan     

Mercuric chloride inhibition of vasopressin release from the isolated neurointermediate lobe of the rat pituitary

The effects of HgCl2 and ouabain on vasopressin release and Ca2+ uptake and distribution was examined in the neurointermediate lobe of the rat pituitary. HgCl2 (0.5 mM) inhibited vasopressin release by approx. 90% in both basal and potassium depolarized states. With 0.1 mM HgCl2 vasopressin release was inhibited by 50% in the depolarized state, but release was not effected in basal state. On the other hand, ouabain (0.5 mM) caused a 3-fold stimulation of vasopressin release in the depolarized state. Both HgCl2 (0.5 mM) and ouabain (0.5 mM) increased net 45Ca+2 uptake by about 80% in groups of neurointermediate lobes. Following 45Ca+2 uptake, HgCl2 (0.5 mM), which is absorbed by the neurointermediate lobe, produced an increase in cytosolic 45Ca+2 content and a decrease in mitochondrial 45Ca+2 content compared to control. In comparison, ouabain (0.5 mM), which does not penetrate the neurointermediate lobe, gave no change in cytosolic 45Ca+2, but an increase in mitochondrial 45Ca+2. These results suggest that HgCl2 inhibits vasopressin release from the neurointermediate lobe of the rat pituitary at a point distal to Ca+2 uptake by the gland.     

Centrally administered galanin modifies vasopressin and oxytocin release from the hypothalamo-neurohypophysial system of euhydrated and dehydrated rats.

Galanin (Gal) as a neuropeptide with widespread distribution in the central nervous system may be involved in the mechanisms of vasopressin (AVP) and oxytocin (OT) release from the hypothalamo-neurohypophysial system. Vasopressin and oxytocin content in the hypothalamus and neurohypophysis as well as plasma level of both neurohormones were studied after galanin treatment in euhydrated and dehydrated rats. In not dehydrated rats intracerebroventricular (i.c.v.) injections of Gal did not affect the hypothalamic and neurohypophysial OT content, however, distinctly increased plasma OT concentration. In the same animals Gal diminished the hypothalamic AVP content but was without the effect on neurohypophysial AVP storage; plasma AVP level then raised. Galanin, administered i.c.v. to rats deprived of water, distinctly inhibited AVP and OT release from the hypothalamo-neurohypophysial system. Simultaneously, plasma AVP and OT level was significantly diminished after Gal treatment in dehydrated rats. These results suggest that modulatory effect of galanin on vasopressin and oxytocin release depends on the actual state of water metabolism. Gal acts as an inhibitory neuromodulator of AVP and OT secretion under conditions of the dehydration but stimulates this process in the state of equilibrated water metabolism.     

On the fine structure of the perivascular cells in the neural lobe of rats

Summary The purpose of this study is to investigate the ultrastructural features of perivascular cells as found in the neural lobe of the hypophysis. In particular, an inquiry was made into the nature of the relationship of such cells to neurosecretory fibers and endings. The latter, in fact, are often invaginated within the perivascular cells and enveloped by their processes; furthermore, they often reveal a certain number of empty granules as well as characteristics of degenerative nature. In the course of this study the localization of the perivascular cells has been investigated as well as that of their processes within the extensive interlobular network typical of the hypophysial neural lobe of rodents. Based on the data gathered, the hypothesis is put forward that the perivascular cells play an important role in the turnover of neurosecretory endings, both under physiological and experimental conditions, contributing thereby to the release of post-hypophysial hormones.With the technical assistance of Vincenzo Panetta.     

Biosynthesis, processing and release of pro-opiomelanocortin related peptides in the intermediate lobe of the pituitary gland of the frog (Rana ridibunda)

The biosynthesis of pro-opiomelanocortin (POMC) and related peptides by the intermediate lobe of the pituitary gland was studied in the frog Rana ridibunda using the pulse-chase technique. Analysis of radioactive proteins by dodecyl sulfate polyacrylamide gel electrophoresis showed that during pulse incubations a 36,000 dalton (36K) glycosylated prohormone was synthesized. It disappeared slowly during chase incubations, giving rise to another glycosylated protein (Mr 18K), identified as the N-terminal fragment of POMC. This latter protein was secreted to the incubation medium. High performance liquid chromatography analysis of peptides synthesized during chase incubations revealed the biosynthesis of two peptides related to gamma-MSH, three peptides related to alpha-MSH, one endorphin-related and one CLIP-related peptides. These newly synthesized peptides were slowly secreted to the incubation medium. Among the alpha-MSH related peptides, only the des-N alpha-acetyl alpha-MSH form of the peptide was found to be present within the cells, in contrast to the incubation medium where the presence of des-N alpha-acetyl alpha-MSH and a modified alpha-MSH was demonstrated.     

Effect of salt loading and salt deprivation on the vasopressin and oxytocin content of the median eminence and the neural lobe in adrenalectomized rats

Summary In adrenalectomized rats the influence of salt loading or salt deprivation on the vasopressin and oxytocin content of the median eminence (ME) and the neural lobe (NL) was studied by means of various methods: (1) morphometric and microphotometric analysis of aldehyde fuchsin-stained sections of ME and NL; (2) immunohistochemical demonstration of neurophysin, oxytocin, and vasopressin in the ME and in the NL; (3) radioimmunological measurement of oxytocin and vasopressin in the ME and in the NL. Adrenalectomy in salt-substituted rats raised the vasopressin content of the outer layer of the ME (OLME) but had no influence on the amount of vasopressin in the inner layer of the ME and in the NL. Osmotic stimulation of adrenalectomized rats by hypertonic saline markedly diminished vasopressin and oxytocin in the inner layer of the ME and in the NL but did not, or only slightly reduced vasopressin in the OLME. Withdrawal of salt supplementation in adrenalectomized rats resulted in a decrease of plasma sodium and plasma volume. It did not change the vasopressin or oxytocin content of the inner layer of the ME and of the NL, but it was correlated with a decrease of vasopressin in the OLME. The present findings may suggest that vasopressin in the OLME is involved in salt and/or volume regulation by influencing the hypophysial-adrenal axis.The study was supported by the Deutsche Forschungsgemeinschaft (Bo 392/6-5¹ and SFB 90, Cardiovasculäres System, A5²). The morphometric measurements with the TAS plus were carried out at the Max-Planck-Institut für Psychiatrie, Munich, FRG. We are particularly indebted to Prof. G. W. Kreutzberg and Prof. P. Schubert for their help     

Ultrastructure of the rat neural lobe during recovery from hypertonic saline treatment

Summary Neural lobes from rats which had been allowed to drink hypertonic saline for five days were examined electron microscopically and by bioassay of oxytocin levels. The profound changes in the ultrastructure were examined and the reversal of these changes in morphology was followed until the hormone levels returned to normal. The recovery of the gland as measured by the two parameters of structure and hormone content indicated that the morphological recovery apparently preceded the hormone level recovery, a factor which might be explained by continued release of hormone during the period of recovery.     

Catecholaminergic innervation of GRF-containing neurons in the rat hypothalamus revealed by electron-microscopic cytochemistry

Summary The Catecholaminergic innervation of neurons containing growth hormone-releasing factor (GRF) was examined by use of a method which combined either 5-hydroxydopamine (5-OHDA) uptake or autoradiography after intraventricular injection of ³H-noradrenaline with immunocytochemistry for GRF in the same tissue sections at the electron-microscopic level. In the ventrolateral part of the arcuate nucleus of the rat hypothalamus a large number of immunonegative axon terminals were found to make synaptic contact with GRF-like immunoreactive (GRF-LI) cell bodies and processes. ³H-noradrenaline autoradiography or 5-OHDA-labeling combined with GRF immunocytochemistry revealed that axon terminals labeled with ³H-noradrenaline or 5-OHDA make synaptic contact with the GRF-LI nerve cell bodies and processes. These findings indicate that catecholamine-containing neurons innervate GRF neurons to regulate GRF secretion via synapses in the rat arcuate nucleus.     

An investigation of N-terminal pro-opiocortin peptides in the rat pituitary

Extracts of neurointermediate lobe (NIL) and anterior lobe (AL) of the rat pituitary, and material released from perfused rat pars distalis (PD) and pars intermedia (PI) cells were gel chromatographed and monitored using three antisera, each recognizing different regions of the non-corticotropin (ACTH)-lipotropin (LPH) portion of pro-opiocortin (POC). Two peaks (termed N-POC I) which emerged close to the elution position of rat beta-LPH were detected. The first peak was reduced significantly in the PI. Two smaller N-POC fragments which eluted near beta-endorphin were detected only in extracts and secretions of intermediate lobe tissue. One peak cross-reacted in the gamma 3-melanotropin (MSH) assay (N-POC III) whereas the other peak possessed amino (N)-terminal N-POC immunoreactivity (N-POC II). The results demonstrated differences in the distribution and nature of N-POC peptides released and extracted from the PD and PI of the rat pituitary, and suggest that the enzymic processing of N-POC is different in the two pituitary lobes.     

Evidence for serotonergic stimulation of pituitary beta-endorphin release: preferential release from the anterior lobe in vivo

Using gel filtration chromatography (Sephadex G-50) and radio-immunoassay for beta-endorphin (beta-END) and beta-lipotropin (beta-LPH) we investigated the site [anterior lobe (AL) vs. intermediate lobe (IL)] for serotonergic control of pituitary beta-END-like immunoreactivity (beta-END-LI) in the rat. Since the secretion of beta-LPH in vitro clearly distinguishes beta-END-LI release by the AL as compared to the IL, we interpreted changes in plasma levels of immunoreactivity resembling beta-LPH to reflect beta-END-LI release from the AL. Following the administration of L-tryptophan (200 mg/kg, 30 min, ip), a serotonin precursor, nearly all of the rise in total plasma beta-END-LI was due to the form of immunoreactivity resembling beta-LPH in molecular size. Similarly, 5-hydroxytryptophan (30 mg/kg, 30 min, ip), a serotonin precursor, and fluoxetine (10 mg/kg, 15 min, ip), a serotonin reuptake blocker, predominantly increased circulating levels of beta-LPH-sized immunoreactivity with little effect on beta-END-sized immunoreactivity. Quipazine (2.5 and 5.0 mg/kg, 30 min, ip), a serotonin receptor agonist, elevated plasma levels of both forms of beta-END-LI; however, the immunoreactive peak coeluting with beta-LPH was primarily affected, being increased 9.5-fold while that resembling beta-END was increased less than 1-fold. Immobilization stress (30 min) dramatically elevated plasma levels of both forms of immunoreactivity, however, a greater relative rise in beta-LPH than beta-END was observed. Intraventricular administration of 5,7-dihydroxytryptamine (75 micrograms, free base, 10 d), a serotonin neurotoxin, lowered plasma levels of both forms of immunoreactivity about equally in stressed animals. Further, dexamethasone, a synthetic glucocorticoid which selectively inhibits AL corticotroph secretion in vitro, attenuated the beta-LPH response to serotonergic activation in vivo. Together, these findings indicate that serotonergic drugs predominantly influence the release of beta-END-LI resembling beta-LPH and further suggest that serotonin neurons preferentially regulate the release of beta-END-LI from AL corticotrophs in vivo.     

Intercellular communications within the rat anterior pituitary. XVI: Postnatal changes of distribution of S-100 protein positive cells,connexin 43 and LH-RH positive sites in the pars tuberalis of the rat pituitary gland. An immunohistochemical and electron microscopic study

The architecture of luteinizing hormone-releasing hormone (LH-RH) nerve ends and the S-100 protein containing folliculo-stellate cells forming gap junctions in the pars tuberalis is basically important in understanding the regulation of the hormone producing mechanism of anterior pituitary glands. In this study, intact male rats 5–60 days old were prepared for immunohistochemistry and electron microscopy. From immunostained sections, the S-100 containing cells in pars tuberalis were first detected on day 30 and increased in number to day 60; this was parallel to the immunohistochemical staining of gap junction protein, connexin 43. LH-RH positive sites were clearly observed on just behind the optic chiasm and on the root of pituitary stalk on day 30. On day 60, the width of layer increased, while follicles and gap junctions were frequently observed between agranular cells in 10 or more layers of pars tuberalis.     

Stress-induced ACTH release after removal of the hypothalamus in rats with atrophied neural lobe.

Twenty-four hours after isolation of the pituitary by surgical removal of the medial hypothalamus, i.e. in rats with pituitary island, E. coli endotoxin significantly increased the plasma corticosterone level. Atrophy of the neural lobe, due to pituitary stalk section performed one month prior to removal of the medial hypothalamus, did not prevent the increase of ACTH release by E. coli endotoxin. E. coli endotoxin-induced ACTH release in MBH-deprived animals does not appear to be a function of mechanisms operating only in the innervated lobe.     

Organization and ultrastructural identification of the catecholamine nerve terminals in the neural lobe and pars intermedia of the rat pituitary

Summary The central catecholamine innervation of the pituitary neural lobe and pars intermedia of the rat have been identified ultrastructurally and their organization has been investigated in a combined fluorescence histochemical and electron microscopical study. The dopamine analogues, 5-hydroxydopamine and 6-hydroxydopamine, were used to label the catecholamine terminals, and to enable the direct correlation between the fluorescence microscopical and the electron microscopical pictures.The fibre type that was identified as catecholamine-containing was ultrastructurally chiefly characterized by dense-cored vesicles, 500–1200 Å in diameter, intermingled with varying numbers of small empty vesicles. 5-hydroxydopamine was selectively accumulated in these fibres and caused an increased electron density of the granular vesicles as well as of some small normally agranular vesicles, and systemically administered 6-hydroxydopamine caused a selective degeneration of these fibres, most prominently within the neural lobe. The dopaminergic terminals of the neural lobe showed frequent close contacts (80–120 Å), without real membrane thickenings, to neurosecretory axons and to pituicyte processes. It is suggested that these close contacts might signify a direct dopaminergic influence on the neurosecretory axons and/or on the pituicyte processes. The identified central catecholamine fibres were also found to make common synapse-like contacts on the pars intermedia cells, whereas the innervation by neurosecretory fibres was very rare. This suggests that the direct central nervous control of the rat pars intermedia is exerted by the catecholamine neurons. A very special feature of the catecholamine fibres in the pituitary is the occurrence of peculiar, large dopamine-filled droplet-like swellings. Electron microscopically, such large axonal swellings (more than 2 in diameter) were found to contain, in addition to the characteristic vesicles and organelles, strongly osmiophilic lamellated membrane complexes resembling myelin bodies and multivesicular bodies encircling disintegrated vesicles, suggesting that these droplet fibres represent dilated stumps of spontaneously degenerating dopaminergic axons. It is suggested that the dopaminergic neural lobe fibres are undergoing continuous reorganization through degeneration—regeneration cycles, a phenomenon previously suggested for the neurosecretory axons of the neural lobe.Supported by the Deutsche Forschungsgemeinschaft.Supported by Svenska Livförsäkringsbolags Nämnd för Medicinsk Forskning, by The Medical Faculty, University of Lund and by the Ford Foundation.     

Quantitative assessment of GABA-uptake sites in the neural lobe by electron-microscopic autoradiography

Summary Distribution of (³H)GABA in the rat neural lobe was investigated 5 min after intracarotid administration using quantitative electron-microscopic autoradiography. Specificity of (³H)GABA-uptake was tested by pretreatment of control animals with nipecotic acid. It was concluded that, apart from a small fraction in the perivascular spaces, radioactivity was present exclusively in pituicytes. The results confirm and quantify earlier in-vitro observations; they are compared with recent immunocytochemical findings that reveal the presence of glutamate-decarboxylase-containing axons in the neural lobe. It is concluded that there may be GABAergic terminals that lack an uptake mechanism for exogenous transmitter. Nevertheless, (³H)GABA autoradiography is useful in demonstrating other functional components of GABAergic systems, i.e., glial cells.     

Subcellular Distribution of Tyrosine Hydroxylase in Some Catecholaminergic Rat Brain Areas Determined by a Quantitative Immunoblot Assay

The subcellular distribution of the protein tyrosine hydroxylase (TH) after fractionation of rat brain tissue was studied by a sensitive technique of immunoblot quantification in the dopaminergic nigrostriatal and the dorsal noradrenergic pathways and in the ventrolateral medulla. This repartition indicates that in all catecholaminergic regions of the cell bodies studied, the contribution of the nerve endings to the total TH amount is very low (less than 7%), in contrast to that observed in the terminal fields. The correlative subcellular determination of the TH amount and activity in the same tissue could be a useful approach for studying experimentally induced mechanisms of catecholamine synthesis modulation in different brain catecholaminergic pathways.     

Intercellular communication within the rat anterior pituitary: relationship between LH-RH neurons and folliculo-stellate cells in the pars tuberalis

The distribution of LH-RH-positive nerve fibers in the median eminence was demonstrated in the 1970s and 1980s. A few LH-RH fibers have been reported to be present in the adjacent pars tuberalis of the pituitary, but their functional significance has not been clarified and still remains enigmatic. Adult male Wistar-Imamichi rats were separated into two groups: one for immunohistochemistry of LH-RH and S-100 protein (for the identification of folliculo-stellate cells) and the other for electron microscopy. For both immunohistochemistry and electron microscopy, the specimens obtained contained the pituitary gland connected with the hypothalamus. Numerous LH-RH-positive fibers were observed as tiny lines with several varicosities both on the primary vascular plexus and in the hypothalamus corresponding to the posterior half of the portal vein area. LH-RH-positive fibers were also noted around S-100-positive cells in the pars tuberalis. Weakly reactive S-100 cells were scattered in the pars tuberalis in the midsagittal plane, while clusters of strong reactive elements occurred 100–300 m from the center. Similar observations were made using fluorescence immunohistochemistry for LH-RH and S-100, and at the electron-microscopic level. At the posterior portion of the portal vein system, bundles of the LH-RH-immunoreactive fibers invaded the pars tuberalis and terminated on agranular cells. Gap junctions were clearly seen among agranular cells corresponding to folliculo-stellate cells. It is postulated that the LH-RH message might be transmitted not only by the established hypophyseal portal vein system but also via the folliculo-stellate cells in the pars tuberalis to aid in the modulation of LH release.     

Effects of experimental hypo-or hypernatremia on the fine structure of the pars intermedia of the murine pituitary

Summary Quantified ultrastructural observations of the pars intermedia (PI) of the murine hypophysis enable evaluation and kinetic study of relatively fine secretory changes in the gland. Changes in volume of rER and newly formed dense secretory granules (Golgi granules) appear to best translate functional variations in the PI, as shown by the morphological effects of drugs affecting the dopaminergic control of the gland. Our morphometric results show that the PI is stimulated, but only briefly (no longer than 8–12 days), by both salt-loading and Na deprivation. However, the PI displays different secretory patterns in salt-loaded and Na-deprived mice; moreover, bromocriptine, which abolishes PI stimulation in Na-deprived mice, has only a slight inhibitory effect in salt-treated animals. Thus, it appears that the stimulation of the PI under both experimental conditions is triggered by different mechanisms. These results underline the plurifactorial control of the PI and show that the gland may have complex effects on hydromineral regulation.