Protein kinase D2 regulates chromogranin A secretion in human BON neuroendocrine tumour cells

Chromogranin A is a member of the granin family of acidic secretory glycoproteins that is found in secretory granules of many endocrine cells including neuroendocrine tumour cells. This hormone serves as a model system for autonomous hormone secretion by the so called functional neuroendocrine tumours of the gastrointestinal tract. The precise regulation of chromogranin secretion at the level of the Golgi apparatus is a subject of intense research. The protein kinase D (PKD) family of serine threonine kinases has so far been implicated in the regulation of constitutive secretion in epithelial cells. Here we examined whether PKD2 expression and activity could also play a role in the release of secretory granules from the trans Golgi network (TGN) in neuroendocrine tumour cells and hence be a target to block autonomous secretion by these tumours. Our data show that expression and catalytic activity of PKD2 are required for the release of chromogranin A containing secretory vesicles. Inhibition of PKD2 activity or siRNA knockdown of PKD2 resulted in a marked perinuclear retention of chromogranin A immunofluorescence in the trans Golgi network and led to a marked reduction in basal as well as phorbol ester stimulated secretion of chromogranin A into the supernatant of cells. Thus, PKD2 controls the release of secretory granules in neuroendocrine tumour cells at the level of the Golgi apparatus and could hence serve as a novel target to block hormone secretion in functional neuroendocrine tumours.     

Neonatal atria and ventricles secrete atrial natriuretic factor via tissue-specific secretory pathways

The cellular mechanisms regulating secretion of the peptide hormone atrial natriuretic factor (ANF) differ in neonatal atrial and ventricular cardiocytes. We demonstrate that although both cell types synthesize and secrete ANF, only atrial cells store peptide in abundant secretory granules. Neonatal ventricular cells secrete ANF rapidly after synthesis and lack secretory granules. We propose that ventricular ANF is released by a constitutive secretory pathway whereas atrial ANF is stored and released by a regulated pathway. Furthermore, ventricular ANF mRNA and hormone concentrations decrease during the first week of life. Developmental variation in the use of ANF secretory pathways may reflect changing requirements for maintenance of intravascular volume and pressure. Tissue-specific modulation of hormone secretory pathways appears to be a novel response to developmentally induced changes in the requirements for a peptide hormone.     

Secretory cargo composition affects polarized secretion in MDCK epithelial cells

Polarized epithelial cells secrete proteins at either the apical or basolateral cell surface. A number of non-epithelial secretory proteins also exhibit polarized secretion when they are expressed in polarized epithelial cells but it is difficult to predict where foreign proteins will be secreted in epithelial cells. The question is of interest since secretory epithelia are considered as target tissues for gene therapy protocols that aim to express therapeutic secretory proteins. In the parathyroid gland, parathyroid hormone is processed by furin and co-stored with chromogranin A in secretory granules. To test the secretion of these proteins in epithelial cells, they were expressed in MDCK cells. Chromogranin A and a secreted form of furin were secreted apically while parathyroid hormone was secreted 60% basolaterally. However, in the presence of chromogranin A, the secretion of parathyroid hormone was 65% apical, suggesting that chromogranin can act as a “sorting escort” (sorting chaperone) for parathyroid hormone. Conversely, apically secreted furin did not affect the sorting of parathyroid hormone. The apical secretion of chromogranin A was dependent on cholesterol, suggesting that this protein uses an established cellular sorting mechanism for apical secretion. However, this sorting does not involve the N-terminal membrane-binding domain of chromogranin A. These results suggest that foreign secretory proteins can be used as “sorting escorts” to direct secretory proteins to the apical secretory pathway without altering the primary structure of the secreted protein. Such a system may be of use in the targeted expression of secretory proteins from epithelial cells. David V. Cohn—Deceased.     

The spermatheca of Melanoplus sanguinipes (Fabr.). I. Morphology,histology, and histochemistry

The spermatheca of Melanoplus sanguinipes consists of a preapical and an apical diverticulum, and a long, thin ductus seminalis. Histologically, the three components are identical. The wall of the spermatheca includes a basement membrane, secretory and epithelial cells, and a cuticular intima. Small, discrete bundles of muscle occur outside the basement membrane. In each secretory cell is a large central cavity which connects with a cuticular channel (efferent ductule) running through the epithelial cell to the spermathecal lumen. During sexual maturation, light- and dark-staining vesicles accumulate in the secretory cells and discharge their contents into the central cavity. Simultaneously, glycogen accumulates in the epithelial cells. Allatectomy of newly emerged females renders the secretory cells unable to produce material, an effect which can be reversed by topical application of synthetic juvenile hormone. The secretion contains protein and acidic mucopolysaccharide. After insemination the quantities of secretion in the lumen and of glycogen in the epithelial cells diminish in the preapical diverticulum where almost all sperm are stored. As the number of sperm declines, the secretion and glycogen are replenished.     

Molecular machinery and mechanism of cell secretion

Secretion occurs in all living cells and involves the delivery of intracellular products to the cell exterior. Secretory products are packaged and stored in membranous sacs or vesicles within the cell. When the cell needs to secrete these products, the secretory vesicles containing them dock and fuse at plasma membrane-associated supramolecular structures, called porosomes, to release their contents. Specialized cells for neurotransmission, enzyme secretion, or hormone release use a highly regulated secretory process. Similar to other fundamental cellular processes, cell secretion is precisely regulated. During secretion, swelling of secretory vesicles results in a build-up of intravesicular pressure, allowing expulsion of vesicular contents. The extent of vesicle swelling dictates the amount of vesicular contents expelled. The discovery of the porosome as the universal secretory machinery, its isolation, its structure and dynamics at nanometer resolution and in real time, and its biochemical composition and functional reconstitution into artificial lipid membrane have been determined. The molecular mechanism of secretory vesicle swelling and the fusion of opposing bilayers, that is, the fusion of secretory vesicle membrane at the base of the porosome membrane, have also been resolved. These findings reveal, for the first time, the universal molecular machinery and mechanism of secretion in cells.     

Thyroid stimulating hormone upregulates secretion of cathepsin B from thyroid epithelial cells

Constant levels of thyroid hormones in the blood are principal requirements for normal vertebrate development. Their release depends on the regulated proteolysis of thyroglobulin which is extracellularly stored in the follicle lumen under resting conditions. Thyroglobulin is proteolytically degraded to a major part in lysosomes, but in part also extracellularly leading to the release of thyroxine. Extracellularly occurring lysosomal enzymes are most probably involved in the proteolytic release of thyroxine. In this study we have analyzed the secretion of cathepsin B by thyroid follicle cells (primary cells as well as FRTL-5 cells) and its regulation by thyroid stimulating hormone, which stimulated the secretory release of the proenzyme as well as of mature cathepsin B. Within one to two hours of stimulation with thyroid stimulating hormone, the cathepsin B activity associated with the plasma membrane increased significantly. This increase correlated closely with the localization of lysosomes in close proximity to the plasma membrane of cultured thyrocytes as well as with the thyroxine liberating activity of thyrocyte secretion media. These observations indicate that thyroid stimulating hormone induces the secretion of cathepsin B, which contributes to the extracellular release of thyroxine by thyrocytes.     

The trafficking of alpha 1-antitrypsin,a post-Golgi secretory pathway marker,in INS-1 pancreatic beta cells

A sulfated alpha1-antitrypsin (AAT), thought to be a default secretory pathway marker, is not stored in secretory granules when expressed in neuroendocrine PC12 cells. In search of a constitutive secretory pathway marker for pancreatic beta cells, we produced INS-1 cells stably expressing wild-type AAT. Because newly synthesized AAT arrives very rapidly in the Golgi complex, kinetics alone cannot resolve AAT release via distinct secretory pathways, although most AAT is secreted within a few hours and virtually none is stored in mature granules. Nevertheless, from pulse-chase analyses, a major fraction of newly synthesized AAT transiently exhibits secretogogue-stimulated exocytosis and localizes within immature secretory granules (ISGs). This trafficking occurs without detectable AAT polymerization or binding to lipid rafts. Remarkably, in a manner not requiring its glycans, all of the newly synthesized AAT is then removed from granules during their maturation, leading mostly to constitutive-like AAT secretion, whereas a smaller fraction (approximately 10%) goes on to lysosomes. Secretogogue-stimulated ISG exocytosis reroutes newly synthesized AAT directly into the medium and prevents its arrival in lysosomes. These data are most consistent with the idea that soluble AAT abundantly enters ISGs and then is efficiently relocated to the endosomal system, from which many molecules undergo constitutive-like secretion while a smaller fraction advances to lysosomes.     

Differential sorting of lutropin and the free alpha-subunit in cultured bovine pituitary cells.

The glycoprotein hormones lutropin (LH) and follitropin (FSH) are both synthesized by gonadotrophs in the anterior pituitary but are stored in separate secretory granules prior to secretion. Despite having highly homologous beta-subunits and alpha-subunits with the identical amino acid sequence, the Asn-linked oligosaccharides on LH terminate with SO4-GalNAc while those on FSH terminate with sialic acid-Gal. In addition to LH and FSH, gonadotrophs secrete uncombined (free) alpha-subunit which bears the same sulfated oligosaccharides as LH. We have examined the synthesis and secretion of LH and free alpha-subunit in primary cultures of bovine pituitary cells in order to determine if the sulfated oligosaccharides have any impact on sorting. Our results show that newly synthesized free alpha-subunit is secreted exclusively via the constitutive pathway with a t1/2 of 1.8 h and is never found in dense-core secretory granules. In contrast, LH dimer is secreted by both the constitutive and the regulated pathways. Constitutive secretion and arrival in a dense secretory granule both occur with t1/2 values of 1-1.5 h for newly synthesized LH. Sulfation occurs immediately prior to arrival of LH in the secretory granule and is followed by a period of 1-1.5 h before the LH-containing granules become sensitive to release by gonadotropin releasing hormone. As a result the t1/2 for LH secretion in the presence of gonadotropin releasing hormone is 3.5 h. Sulfation of the free alpha-subunit oligosaccharides is not, therefore, sufficient to direct the alpha-subunit to secretory granules, and the information required for directing LH to granules must reside either in the beta-subunit or the alpha beta-complex.     

Orexin-B-like immunoreactivity localizes in both luteinizing-hormone-containing cells and melanin-concentrating hormone-containing fibers in the red-bellied piranha (Pygocentrus nattereri) pituitary

We examined orexin-like immunoreactivity in the pituitary of the red-bellied piranha (Pygocentrus nattereri). Orexin-B-immunoreactive (IR) cells corresponded to luteinizing hormone (LH)-containing cells in the pars distalis, and orexin-B-IR fibers corresponded to melanin-concentrating hormone (MCH)-containing fibers in the pars nervosa. In the pars distalis, orexin-B-IR puncta that were also immunoreactive for MCH were observed around the orexin-B-IR cells. In the ventral hypothalamus, orexin-B-IR and MCH-IR neurons were found in the nucleus lateralis tuberis. Immunoelectron-microscopic analysis revealed that the orexin-B-like substance co-localized with LH in secretory granules and with MCH in MCH-containing neurons. Some of the MCH secreted in the pituitary might participate in the modulation of LH secretion from the gonadotrophs, together with orexin-B, leading to food intake by the stimulation of growth hormone secretion from the somatotrophs.     

Secretory lysosomes

Regulated secretion of stored secretory products is important in many cell types. In contrast to professional secretory cells, which store their secretory products in specialized secretory granules, some secretory cells store their secretory proteins in a dual-function organelle, called a secretory lysosome. Functionally, secretory lysosomes are unusual in that they serve both as a degradative and as a secretory compartment. Recent work shows that cells with secretory lysosomes use new sorting and secretory pathways. The importance of these organelles is highlighted by several genetic diseases, in which immune function and pigmentation--two processes that normally involve secretory lysosomes--are impaired.     

Secretion and degradation of parathormone as a function of intracellular maturation of hormone pools

The biosynthesis, processing, and secretion of parthormone and the effect of calcium on these processes were measured in dispersed porcine parthyroid cells incubated with [(35)S]methionine. Proparathormone was detected at 10 min, the earliest time measured, and was rapidly and apparently quantitatively converted to parathormone. The half-life of the prohomormone pool was 15 min. Secretion of parathormone was detected by 20 min. In pulse-chase experiments there was a period between 20 and 40 min during which the wave of newly-synthesized parathormone was secreted. After 40 min during little additional radioactive hormone was secreted, but dibutyryl cyclic AMP, an agent that can mobilize stored parathormone, when added to the incubation mixtures enhanced radioactive parathormone secretion but only after 60 min, although it increased net hormone secretion as determined by radioimmunoassay to the same extent at all times studied. When the ionized calcium concentration of the medium was lowered, more radioactive hormone was secreted at all times but the effect was greatest on that hormone that was synthesized less than 60 min previously ; however, net hormone secretion in contrast to radioactive hormone was enhanced equally at all intervals. These data could mean that the refractoriness to secretion of parathormone 40-60 min of age was related to maturation of secretory container preparatory to storage. Low calcium (0.5 mM) stimulated hormone secretion up to fivefold compared to high calcium (3.0 mM) but did not affect synthesis of parathormone or proparathormne or conversion of the latter to hormone. During processing at least 70 percent of the intracellular parathormone was lost, presumably through proteolysis and this degradation was greater at high calcium. These data have been interpreted in light of the concept that two secretable pools of parathormone exist within the parathyroid.     

Islet amyloid polypeptide (IAPP) does not inhibit glucose-stimulated insulin secretion from isolated perfused rat pancreas

Islet amyloid polypeptide (IAPP) is a recently discovered pancreatic islet hormone which is stored with insulin in the secretory vesicles of beta cells. Several lines of evidence suggested that IAPP might affect glucose-stimulated insulin secretion and, therefore, might play a role in the development of impaired insulin secretion which is typical of type 2 diabetes. In this study, the effects of human IAPP (amide) on glucose-stimulated insulin secretion was evaluated in the isolated perfused rat pancreas. IAPP in concentrations from 5 x 10(-12) to 10(-7) M had no significant effects on insulin secretion. IAPP, therefore, does not appear to be a significant modulator of glucose-stimulated insulin secretion at concentrations that are physiologically relevant.     

Mitochondrial deformation and apocrine secretory mechanism in the rabbit submandibular organ as revealed by electron microscopy

Summary The submandibular organ (a sort of apocrine sweat glands) of the rabbit was observed with the electron microscope. The cell structure of glandular tubules varies depending upon the secretory activity; there are three functional stages. The secretory cells at the resting stage are characterized by low height, absence of secretory substance, and presence of small and slender mitochondria.In the synthesizing stage, enlargement and peculiar deformation of mitochondria are observed. Secretory substance always occurs near the deformed mitochondria. The part of a mitochondrion closely abutting on the secretion mass is extremely thin, and contains longitudinally oriented cristae. Sometimes a direct continuity is observed between the thinned portion of the deformed mitochondria and the mass of secretory substance. It is presumed that the secretion is initially produced in the mitochondria and then discharged from them. The Golgi apparatus and the rough surfaced endoplasmic reticulum may be involved indirectly. Smooth surfaced vesicles, probably related to the transport of raw material, are extremely abundant in the cells of this stage.The development of a generally homogeneous projection into the gland lumen is characteristic of the stage of secretion discharge. The mitochondria are again small and slender, and the secretion is liquefied. At the base of the full-grown projection, cytoplasm is condensed to form a demarcation zone from which the projection may become detached. This mechanism of release of secretory product is quite the same as the so-called apocrine secretory process long postulated by light microscopists.     

A model for intracellular calcium signaling and the coordinate regulation of hormone biosynthesis, receptors and secretion.

A two-state model for the stimulus-induced nongraded response of a single cell is formulated. Individual metestrus gonadotropes stimulated with LHRH operate as a simple switch: either on or off. At a given concentration of stimulus some gonadotropes switch on, while others do not switch on, secretion. The probability of a gonadotrope being in the secretory state is enhanced with each increment of LHRH concentration. Individual gonadotropes in a secretory state are envisioned to decrease their number of LHRH receptors and to switch off LH biosynthesis. On the other hand, individual gonadotropes that are not in a secretory state are thought to increase their number of LHRH receptors and to switch on LH biosynthesis. The group of individuals in the population that have thresholds falling in the range of a given stimulus initiate secretion. And, the group of individuals in the population that have thresholds that fall above the range of a given stimulus do not initiate secretion. More remarkable is evidence that the cells that are protected from hormone secretion nevertheless respond with a set of intracellular signals and this provides a new perspective of how they switch on hormone biosynthesis and up-regulate the LHRH receptors. These changes are envisioned to reduce the threshold of an individual cell and accordingly to enhance the probability that the cell responds in the secretory state with the next stimulus. This scheme would appear to lead to automatic cycles of secretion and biosynthesis since an individual cell can occupy only one of two states at any time and occupancy of either state promotes change to the other. This may provide a solution to the problem of how an endocrine gland might reconcile differences in the time-course of hormone secretion which occurs rapidly and hormone biosynthesis that requires a longer period of time. Parenthetically, the model may also be adapted to the case where the vast majority of individuals in the population are generally subthreshold in relation to the physiological stimulus: such an adaption leads to interesting ways of viewing the mammalian reproductive cycle and the regulation of the preovulatory LH surge. A two-state model of the internal Ca2+ store is outlined here to stimulate thought on how the intracellular signals of each binary state may switch a variety of cellular responses either on or off. The model provides a new perspective on the coordinate regulation of hormone biosynthesis, receptors, and secretion that may be useful in the final reconciliation of population studies with insights about individual cells.     

Somatostatin is targeted to the regulated secretory pathway of gonadotrophs in transgenic mice expressing a metallothionein-somatostatin gene

The pituitaries of transgenic mice that express a metallothionein-somatostatin fusion gene contain high concentrations of somatostatin-14 exclusively in the gonadotrophic cells. The purpose of this study was to determine whether somatostatin expressed from the foreign fusion gene enters the normal secretory pathway within these cells. Immuno-gold labeling of serial thin sections localized somatostatin to the secretory granules of gonadotropin-producing cells. The gonadotroph-specific hypophysiotropic factor, luteinizing hormone-releasing hormone caused a dose-dependent secretion of somatostatin when applied to primary pituitary cultures from these mice. Growth hormone-releasing hormone, thyrotropin-releasing hormone, corticotropin releasing factor, and dopamine did not affect somatostatin secretion. These experiments demonstrate that a neurosecretory peptide encoded by a foreign gene can enter the regulated secretory pathway of pituitary cells from transgenic mice.     

Correlation between electrical activity and ACTH/beta-endorphin secretion in mouse pituitary tumor cells

The electrical and secretory activities of mouse pituitary tumor cells (AtT-20/D-16v), which contain and release the ACTH/beta-endorphin family of peptides, were studied by means of intracellular recordings and radioimmunoassays. Injection of depolarizing current pulses evoked action potentials in all cells and the majority (82%) displayed spontaneous action potential activity. Action potentials were found to be calcium-dependent. Barium increased membrane resistance, action potential amplitude and duration, and release of ACTH and beta- endorphin immunoactivity. Isoproterenol increased both action potential frequency and hormone secretion. Raising the external calcium concentration increased the frequency and amplitude of the action potentials and stimulated secretion of ACTH and beta-endorphin immunoactivity. Thus, stimulation of secretory activity in AtT-20 cells was closely correlated with increased electrical activity. However, a complete blockade of action potential activity had no effect on basal hormone secretion in these cells. These results suggest that the mechanisms underlying stimulated hormone secretion are different from those responsible for basal secretory activity. It is proposed that the increased influx of calcium due to the increased action potential frequency initiates the stimulated release of hormone from these cells.     

Snythesis of mucoprotein secretion in the salivary glands of Drosophila melanogaster IIIrd instar larvae]

The process of mucoprotein (glue) accumulation is described. This substance is secreted by salivary glands of the III instar larvae of Drosophila melanogaster. Granules of the secretory substance appear in the glandular cells 90 hrs after egg laying. Its secretion takes place within 120 hrs. The template RNA of glue proteins appears to be synthesized within the range of 72-85 hrs since actinomycin treatment of larvae at this time suppresses the formation of secretory substance. The portion of secretory proteins amounts to 23-32% of the total gland protein. In a mutant, 13tl, deficient by the absence of pupation, no PAS-positive, staining of glands or secretory granules identified under the phase-contrast microscope was found. A possible participation of fat body in the formation of mucoprotein secretory substance is discussed.     

Inhibitory effect of colchicines on amylase secretion by rat parotid glands: possible localization in the golgi area

Colchicine inhibited amylase secretion by isolated rat parotid glands only 6 h after administration of the drug in vivo. This delayed effect was not the result of the inability of the drug to reach its reaction site. When parotid glands were emptied of their secretory granules by isoproterenol treatment, the subsequent replenishment of cells with granules was inhibited by colchicines. Colchicine concomitantly produced alterations of the Golgi complexes, the cisternae of which were reduced in size and surrounded by clusters of microvesicles. Incubation of parotid glands with colchicines for prolonged durations failed to alter stored amylase secretion as stimulated by isoproterenol, but it inhibited the release of de novo synthesized enzyme. Another colchicines-binding activity, firmly bound to the particular fraction of homogenates, was found, of which a part may represent membrane located microtubular protein. An assembly-disassembly cycle of microtubules appears to exist in the parotid gland, as in the liver. However, only 14 percent of tubulin was found to be polymerized as microtubules in parotid glands as opposed to 40 percent in the liver. The present data suggest that colchicine primarily inhibits the transfer of secretory material towards or away from the Golgi complexes but not the hormone-stimulated secretion of stored amylase.     

Regulated and constitutive secretion. Differential effects of protein synthesis arrest on transport of glycosaminoglycan chains to the two secretory pathways.

Many neural and endocrine cells possess two pathways of secretion: a regulated pathway and a constitutive pathway. Peptide hormones are stored in granules which undergo regulated release whereas other surface-bound proteins are externalized constitutively via a distinct set of vesicles. An important issue is whether proper function of these pathways requires continuous protein synthesis. Wieland et al. (Wieland, F.T., Gleason, M.L., Serafini, T.A., and Rothman, J.E. (1987) Cell 50, 289-300) have shown that a tripeptide containing the sequence Asn-Tyr-Thr can be glycosylated in intracellular compartments and secreted efficiently from Chinese hamster ovary and HepG2 cells, presumably via the constitutive secretory pathway. Secretion is not affected by cycloheximide, suggesting that operation of this pathway does not require components supplied by new protein synthesis. In this report we determined the effects of protein synthesis inhibitor on membrane traffic to the regulated secretory pathway in the mouse pituitary AtT-20 cells. We examined transport of glycosaminoglycan chains since previous studies have shown that these chains enter the regulated secretory pathways and are packaged along with the hormone adrenocorticotropin (ACTH). We found that cycloheximide treatment severely impairs the cell's ability to store and secrete glycosaminoglycan chains by the regulated secretory pathway. In marked contrast, constitutive secretion of glycosaminoglycan chains remains unhindered in the absence of protein synthesis. The differential requirements for protein synthesis indicate differences in the mechanisms for sorting and/or transport of molecules through the constitutive and the regulated secretory pathways. We discuss the possible mechanisms by which protein synthesis may influence trafficking of glycosaminoglycan chains to the regulated secretory pathway.     

Cell Volume-Induced Hormone Secretion: Signal Transduction and Specificity

Maintenance of the cell volume within physiological limits under anisosmotic conditions is an important prerequisite for survival and functioning of the cell. Cell volume alterations are also involved in numerous cellular events and are recently considered to be integrated into a physiological signal transduction network. Cell swelling induced by anisosmotic environment, hormones, oxidative stress, or substrate uptake evokes an immediate secretory burst of the material (peptide hormones, enzymes) stored in secretory vesicles from various types of cells (endocrine cells, neurons, leukocytes, exocrine pancreatic cells). The dynamics of this secretion are indistinguishable from those induced by specific secretagogues. This regulated secretion does not require a rise in the intracellular Ca²⁺. Using various tissues (pituitary, pancreatic islets, brain structures), hormones (prolactin, insulin, thyrotropin - releasing hormone - TRH, oxytocin), and inhibitors, we found that hormone secretion induced by cell swelling is not depressed by inhibition of stretch-activated channels (GdCl₃), mercury-sensitive aquaporins, protein kinase C (bisindolylmaleimide), microtubules and microfilaments (colchicine, cytochalasin)and does not involve arachidonic acid metabolites, prostaglandins and leukotrienes (indomethacin, NDGA). The blockade of Na⁺-K⁺-dependent ATPase, that of Na⁺ channels, or that of K⁺ channels exerted no effect on hyposmolarity-induced hormone secretion in pituitary cells. Norepinephrine, a physiological inhibitor of secretion of insulin, did not inhibit hypotonicity-induced secretion from pancreatic islets. The participation of such a general biophysical phenomenon in physiological reactions raises a question of its specificity. Cell swelling induced by an isosmotic ethanol-containing medium evoked release of TRH from hypothalamic paraventricular nucleus and posterior pituitary, while oxytocin (known to be engaged in the water and salt regulation) release was not stimulated. Neirofiziologiya/Neurophysiology, Vol. 37, No. 2, pp. 177–180, March–April, 2005.