Effect of Tetanus Toxin on Oxytocin and Vasopressin Release from Nerve Endings of the Neurohypophysis

The effect of tetanus toxin on neuropeptide hormone release from isolated nerve endings of the neural lobe of rat pituitaries (neurosecretosomes) was measured in a perfusion system. Tetanus toxin inhibited depolarization-evoked release of oxytocin and vasopressin in a time- and dose-dependent manner. At 1 microgram/ml, tetanus toxin blocked stimulated release by 85%. Tetanus toxin that was preincubated with a neutralizing monoclonal antibody or heated to 100 degrees C had no effect on hormone release. The ionophores A23187 and ionomycin were potent stimulators of hormone release in control nerve endings, but were not able to overcome the effect of tetanus toxin in intoxicated nerve endings. 8-Bromo-cyclic GMP, which has been reported to reverse the action of tetanus toxin in PC12 cells, had no effect on the action of tetanus toxin in neurosecretosomes. Neurosecretosomes are the first system in which tetanus toxin has been shown to block release from peptidergic nerve terminals. They appear to be a valuable in vitro system for studying the biochemical mechanism of tetanus toxin action.     

Immunoreactive galanin-like material in magnocellular hypothalamoneurohypophysial neurones of the rat

Summary Immunoreactive galanin-like material was recently shown to co-exist with vasopressin in parvocellular and magnocellular perikarya of the paraventricular nucleus in the anterior hypothalamus of the rat (Melander et al. 1986). Since this distribution pattern differed from our observation of oxytocin-associated galanin-like immunoreactivity (LI) in the neurohypophysis, we compared in series of 0.5-m thick sections the localisation of galanin-LI with the localisation of oxytocin and vasopressin/dynorphin in the hypothalamus, the median eminence and the neurohypophysis. In the oxytocin system, galanin-LI was intense in oxytocin varicosities of the neurohypophysis. Oxytocin perikarya of the hypothalamic supraoptic and paraventricular nuclei exhibited galanin-LI only after intraventricular injection of colchicine and when sections were treated with trypsin prior to application of the antibody. In the vasopressin/dynorphin system galanin-LI was intense in hypothalamic perikarya after colchicine injection and in neurohypophysial varicosities after treatment of the sections with trypsin. In these neurones, galanin-LI was absent or weak in all elements when treatments with colchicine or trypsin were omitted. Galanin-LI in the neurohypophysis was not co-localised with the numerous fine endings showing GABA-LI. These observations indicate that galanin-like material coexists with vasopressin and oxytocin in the respective magnocellular neurones, although not always in an immunoreactive form.     

Effect of hypophysectomy on immunocytochemically demonstrated growth hormone releasing factor (GHRF) in the rat brain

The effect of removal of CH by long-term hypophysectomy (HPX) on the GHRF-immunoreactivity in the rat hypothalamus was studied using immunocytochemical methods. HPX resulted in a dramatic reduction in immunostaining in the median eminence (ME), indicating a significant reduction in storage of GHRF. Other regions of the hypothalamus, including the dorsomedial nucleus, the medial preoptic and suprachiasmatic regions, and the lateral hypothalamus showed the distribution and intensity of GHRF-immunoreactive structures as seen in intact animals; i.e., scattered fibers only. A few weakly stained cell bodies were seen in experimental and control animals. This could be due to a lack of storage, and/or this peptide may possibly not be present in its matured and fully immunoreactive form in the cell bodies.     

Vasopressin potentiates the stimulation of cyclic AMP accumulation by norepinephrine

Vasopressin, a peptide that appears to enhance the consolidation process of memory was studied for its ability to stimulate the accumulation of cyclic AMP in slices of the mouse hippocampus. While vasopressin alone exhibited no effects in this system, it substantially potentiated the effects of norepinephrine. Such actions are discussed in reference to an endogenous brain vasopressin system, and they suggest a possible neuromodulator role for vasopressin.     

Specific immunoelectronmicroscopic localization of vasopressin and oxytocin in the neurohypophysis of the rat

Summary An immunoelectronmicroscopic method for the specific localization of neurohypophyseal hormones was developed in neurohypophyses of Wistar and Brattleboro rats, the latter strain being homozygous for diabetes insipidus. If the proper precautions were omitted, a marked cross reactivity between antivasopressin and antioxytocin preparations was found. Cross reaction of an antivasopressin plasma with oxytocin, at a dilution of less than 11600, resulted in electron density of all granules within neurosecretory fibres of the Brattleboro and Wistar neurohypophyses. However, this cross reactivity could be eliminated either by sufficient dilution of the antiplasma, or by its purification. Purification of the antibodies was performed by absorption to agarose beads coated with the cross reacting component. Upon incubation with antivasopressin (diluted unpurified 11600 or purified 180) and unpurified antioxytocin (1400) plasma, sections of a Wistar neurohypophysis revealed two types of neurosecretory fibres, containing either electron dense or lucent granules. Oxytocin and vasopressin containing neurosecretory fibres were found as clusters in the neurohypophysis. The specificity of both unpurified antivasopressin (11600) and antioxytocin (1400) plasma was confirmed on serial sections of a Wistar neurohypophysis, alternately incubated with the solutions of the two antibodies.These data prove that the one-cell-one-hormone hypothesis holds true for the hypothalamic-neurohypophyseal system.The authors wish to thank Dr. L.A. Sternberger (Edgewood Arsenal, Md., U.S.A.) for the peroxidase-anti-peroxidase complex, Dr. J.G. Streefkerk (Free University, Amsterdam) and the members of our project group on neuroendocrinology for their suggestions and critical remarks, and Mrs. M. Mud, Mr. P. Wolters and Mrs. A. van der Velden for their skilful assistance     

Immunocytochemical localization of substance P in the neurohypophysis and hypothalamus of the mouse compared with the distribution of other neuropeptides

Summary Substance-P immunoreactivity has been located in semithin sections of mouse hypothalami and pituitaries and compared with the distribution of other hypothalamic peptides. In the mouse, nerve fibres and terminals reacting with antibodies against substance P (SP) were detected both in the external zone of the median eminence (ME) and in the neural lobe of the pituitary. Immunoreactive SP (ISP) axons of the ME did not react with antibodies against other peptides, i.e. arginine-vasopressin (AVP), oxytocin (OT), somatostatin and enkephalin, and were also negative with an antibody to serotonin. In the neural lobe, SP immunostaining occurred in AVP but not in OT axons. In the hypothalamus, ISP axons were widespread but conspicuously lacking in areas containing AVP neurones, i. e. in the suprachiasmatic nucleus and the clusters of AVP cells in the SO and PV nuclei. In contrast, multiple ISP endings were seen in contact with OT neurones. Immunoreactive cell bodies, only detected after colchicine treatment, belonged to two distinct classes of neurones: 1) single AVP neurones of the SO and PV nuclei; 2) specific (staining only for SP) neurones, scattered or grouped in different areas of the hypothalamus, not showing relationship with any circumscribed nucleus. These results reinforce the opinion that SP can be released as a neurohormone into the vascular portal system and can directly affect the pars distalis. The presence of immunoreactive SP in the neural lobe, which has not been reported in species other than the mouse, may have a different physiological significance.     

Coexpression of Glucagon-Like Peptide-1 (GLP-1) Receptor, Vasopressin, and Oxytocin mRNAs in Neurons of the Rat Hypothalamic Supraoptic and Paraventricular Nuclei

Abstract : This study was designed to gain better insight into the relationship between glucagon-like peptide-1 (GLP-1) (7-36) amide and vasopressin (AVP) and oxytocin (OX). In situ hybridization histochemistry revealed colocalization of the mRNAs for GLP-1 receptor, AVP, and OX in neurons of the hypothalamic supraoptic and paraventricular nuclei. To determine whether GLP-1(7-36)amide alters AVP and/or OX release, both in vivo and in vitro experimental study designs were used. In vivo, intravenous administration of 1 μg of GLP-1(7-36)amide into the jugular vein significantly decreased plasma AVP and OX concentrations. In vitro incubation of the neurohypophysis with either 0.1 or 1 μg of GLP-1(7-36)amide did not modify the release of AVP. However, addition of 1 μg of GLP-1(7-36)amide to the incubation medium increased slightly the secretion of OX. The coexpression of GLP-1 receptor and AVP mRNAs in hypothalamic supraoptic and paraventricular nuclei gives further support to the already reported central effects of GLP-1(7-36)amide on AVP. Our findings also suggest a dual secretory response of AVP and OX to the effect of GLP-1(7-36)amide, which most likely is related to the amount and/or the route of peptide administration.     

Substance P-immunoreactive neurons in the brainstem of the cat related to cardiovascular centers

Summary The distribution of substance P-immunoreactivity (SP-IR) in the brainstem and spinal cord of normal and colchicine-pretreated cats was analysed using the peroxidase-antiperoxidase (PAP) technique. Numerous SP-IR fibers are present in the nucleus solitarius, nucleus dorsalis nervi vagi and nucleus spinalis nervi trigemini, various parts of the formatio reticularis, substantia grisea centralis mesencephali, locus coeruleus and nucleus parabrachialis. SP-IR perikarya occur in the substantiae gelatinosa and intermedia of the spinal cord, the nucleus spinalis nervi trigemini-pars caudalis, the nucleus dorsalis nervi vagi, and the nucleus solitarius, as well as in the adjacent formatio reticularis and the medullary nuclei of the raphe. In addition, SP-IR cell bodies are located in the nuclei raphe magnus and incertus, ventral and dorsal to the nucleus tegmentalis dorsalis (Gudden), nucleus raphe dorsalis, substantia grisea centralis mensencephali, locus coeruleus, nucleus parabrachialis and colliculus superior.The results indicate that SP-IR neurons may be involved in the regulation of cardiovascular functions both at the central and peripheral level. A peripheral afferent portion seems to terminate in the nucleus solitarius and an efferent part is postulated to originate from the nucleus dorsalis nervi vagi and from the area of the nuclei retroambiguus, ambiguus and retrofacialis.     

Hypothalamic CRF-like immunoreactivity in the rat after hypophysectomy or adrenalectomy

Hypothalamic CRF-like immunoreactivity was measured in normal, hypophysectomized or adrenalectomized adult male rats. As expected, adrenalectomy resulted in decreased levels in plasma corticosterone and increased plasma levels of ACTH; hypophysectomy resulted in decreased levels in both corticosterone and ACTH. The hypothalamic content of CRF-like immunoreactivity in animals two weeks post-hypophysectomy or adrenalectomy was approximately seven times greater than that found in intact animals. At one week, post-surgery, small but statistically significant decreases in content of CRF-like immunoreactivity were observed. The results at one week are consistent with removal of feedback effects of ACTH and corticosterone causing increased release of CRF and decreased content. The increase in CRF-like immunoreactivity two weeks post-surgery is probably not related to direct feedback effects on release but may be due to increased synthesis secondary to long term removal of feedback inhibition.     

Ultrastructural immunohistochemical localization of vasopressin in the hypothalamic-neurohypophysial system of three murids

Vasopressin was immunohistochemically localized at the electron microscopic (EM) level in the hypothalamic-neurohypophysial system (HNS) of three murids. Antiserum to vasopressin was produced in rabbits injected with lysine vasopressin (LVP) conjugated to egg albumin (EA), anti-EA being precipitated prior to staining. Sternberger's unlabeled antibody peroxidase technique was employed, immunoreactivity being designated by peroxidase anti-peroxidase (PAP) molecules and electron opacity. Immunoreactive neurosecretory granules (NSG) were found in the perikarya of the supraoptic nucleus (SON) in all three murids investigated, although far more profusely in the two wild strains. Immunoreactive axonal NSG were observed in the inner and outer zones of the median eminence (ME), and within most of the axons and terminals in the neurohypophysis. The concentration of primary serum effective for staining the SON (110–150) was far higher than that required for the ME and the neurohypophysis (1:500–1:1,200). AntiLVP also induced electron opacity of granules in cells of the pars intermedia (PI). Discussion centers on the significance of immunoreactive NSG in the neurosecretory (NS) perikarya, on the possibility of an extragranular pool of hormone, and on speculation about the electron opacity of the PI granules.     

Measurement of nicotine and cotinine in human milk by high-performance liquid chromatography with ultraviolet absorbance detection

A high-performance liquid chromatographic (HPLC) assay for the determination of nicotine and cotinine in human milk was developed using an extraction by liquid-liquid partition combined with back extraction into acid, and followed by reverse-phase chromatography with UV detection of analytes. The assay was linear up to 500 microg/l for both nicotine and cotinine. Intra- and inter-day relative standard deviations (R.S.D.) were <10% (25-500 microg/l) for both nicotine and cotinine. Limits of quantitation (LOQ) were 10 and 12 microg/l for nicotine and cotinine, respectively, while the limits of detection (LOD) were 8 and 10 microg/l for nicotine and cotinine, respectively. The mean recoveries were 79-93% (range 25-500 microg/l) for nicotine and 78-89% (range 25-500 microg/l) for cotinine. The amount of fat in the milk did not affect the recovery. We found that this method was sensitive and reliable in measuring nicotine and cotinine concentrations in milk from a nursing mother who participated in a trial of the nicotine patch for smoking cessation.     

Intermittent nicotine administration modulates food intake in rats by acting on nicotine receptors localized to the brainstem

Previous studies have shown nicotine (NIC) administration leads to decreased food intake, while other investigations have reported that NIC stimulates c-Fos expression in the brainstem. Whether there is a causal relationship between NIC effects on ingestion and its effect on brainstem neurons is uncertain, however we hypothesized that blocking NIC action in the brainstem would prevent, to some extent, the hypophagic effects of NIC. In the present study, cannulas were placed in the fourth ventricle of rats. A dose of NIC or saline was injected i.p. in four equal injections during the dark phase for four days. At the start of the second day of injections the NIC receptor antagonist mecamylamine (MEC) or artificial cerebrospinal fluid (a-CSF) was infused intracerebroventricularly (i.c.v.). Thus, four experimental groups were examined: a-CSF + SAL; a-CSF + NIC; MEC + SAL; MEC + NIC. Meal patterns were recorded using a computerized system and water intake and body weight were measured daily. Peripheral NIC injections suppressed food intake by decreasing meal size, whereas infusion of the NIC receptor antagonist MEC (4 microg) into the fourth ventricle blocked the NIC suppression of food intake. Moreover, the MEC effect was due primarily to an increase in dark phase meal size, which suggests neurons localized to the brainstem transmit NIC signals that regulate feeding behavior by affecting meal size.     

Ultrastructure of the rat posterior pituitary gland and evidence of hormone release by exocytosis as revealed by freeze-fracturing

Summary Posterior pituitary glands from normal rats, and rats which had been deprived of water for varying periods, were examined by the freeze-fracture method. This technique reveals large areas of the nerve cell membrane. Images consistent with exocytosis as the mechanism of release of the neurohypophysial hormones were observed. These modifications were most numerous after the rat had been starved of water for 2 days.In normal rats, the large number of neurosecretory granules within the nerve fibres caused a bulging of the nerve cell membrane. The bulges disappeared 2 days after removal of drinking water. Regions of the membrane displaying bulges were characterised by the absence of the typical membrane-associated particles.It is postulated that the close proximity of the neurosecretory granules to the nerve cell membrane may result in rapid fusion of the neurosecretory granules on stimulation of the gland. The change in properties of the nerve cell membrane overlying the neurosecretory granules, as suggested by the loss of membrane-associated particles, may represent a change in the structure of the membrane to a form which is more favourable for fusion.This work was supported in part by a grant from the New Zealand Medical Research Council.     

Ultrastructural characteristics of vasopressin-containing neurons in the paraventricular nucleus of the hypothalamus

Summary Vasopressin-containing neurons, identified by immunocytochemistry, are located predominantly in the posterior magnocellular division of the paraventricular nucleus of the rat hypothalamus. By electron microscopy, the immunoreaction product is seen within the cell bodies and neuronal processes. In the perikarya and dendritic processes, the immunoreactive material is associated primarily with neurosecretory granules. Axonal processes, identified by their content of microtubules and accumulation of neurosecretory granules, show the immunoreaction product in association with both of these organelles. Afferent axo-dendritic, axo-somatic and putative axo-axonic synapses with immunostained vasopressinergic neurons can be identified. The presynaptic profiles do not contain immunoreactive material. This study contributes to the ultrastructural characterization of vasopressinergic neurons in the paraventricular nucleus and of their afferent synaptic input.Supported by NIH Grants HD-12956 and 2SO7RR05403     

Rapid method for the simultaneous measurement of nicotine and cotinine in urine and serum by gas chromatography–mass spectrometry

A simple, sensitive, and rapid gas chromatographic–mass spectrometric method is described for the simultaneous detection and quantitation of nicotine and its metabolite, cotinine, in urine and serum. The analytes and their respective deuterated internal standards were extracted by liquid–liquid extraction coupled to centrifugation and evaporation. The detection limit of the assay was 0.16 ng/ml for both nicotine and cotinine. The limit of quantitation for each analyte was 1.25 ng/ml.     

Presence of vasopressin, oxytocin and neurophysin in the retina of mammals, effect of light and darkness, comparison with the neuropeptide content of the neurohypophysis and the pineal gland

Arginine vasopressin (AVP) and oxytocin (OT) as well as their CNS carrier neurophysins (Np) have been found in the pineal gland. In view of the analogy between the pineal gland and the retina, the contents of these neuropeptides in rat, human and bovine retinae were determined. AVP, OT and Np were detected by specific radioimmunoassay (RIA) and their presence confirmed by RIA measurements (1) in rat and human retinae on HPLC fractions and (2) by the detection of the C-terminal portion of the precursor to AVP and its associated Np = propressophysin (CPP). The AVP and OT content in the retina of the rat was modified by light: AVP and OT content was smaller at 2 a.m. than at 2 p.m., but was increased by a 7 day constant exposure to darkness. In contrast, pituitary content was decreased after 7 days of constant darkness. If one optic nerve was cut we observed a decrease in retinal AVP content compared to the contralateral side and a decrease in pituitary AVP content. Our data clearly demonstrated the presence of AVP, OT and Np in the retina and their variation induced by light. It is probable that these peptides are of central origin.     

Intra- and extrahypothalamic vasopressin and oxytocin pathways in the rat

Summary Perfusion of rat brain followed by immersion fixation with 2.5% glutaraldehyde-1% paraformaldehyde, purification of the first antisera and application of the unlabelled antibody enzyme method were used to specifically identify vasopressin and oxytocin containing cells and fibres. The conventional sites of production of these hormones were confirmed as follows: supraoptic and paraventricular nuclei, suprachiasmatic nucleus (only vasopressin), and other cells and cell groups of the hypothalamus. Fibres from the suprachiasmatic nucleus spread out in various directions, and probably project to the nucleus praeopticus periventricularis, organum vasculosum laminae terminalis and in the direction of the supraoptic nucleus. Oxytocin and vasopressin containing pathways could be traced from the paraventricular nucleus to the lateral ventricle, the stria terminalis and the stria medullaris. Some of the oxytocin and vasopressin containing tracts appear to continue onto the septum. The possible importance of these morphological findings for the behavioural effects of vasopressin and oxytocin is discussed.The authors wish to thank Dr. L. Sternberger for his generous gift of peroxidase-antiperoxidase complex, and Miss M.M. Smidt, Mr. A. Potjer and Mr. P. Wolters for their assistance. This work was supported in part by the Foundation for Medical Research FUNGO     

Determination of nicotine and cotinine in human plasma by liquid chromatography-tandem mass spectrometry with atmospheric-pressure chemical ionization interface

Here we report a sensitive liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method capable of quantifying nicotine down to 1 ng/ml and cotinine to 10 ng/ml from 1.0 ml of human plasma. The method was validated over linear ranges of 1.0–50.0 ng/ml for nicotine and 10.0–500.0 ng/ml for cotinine, using deuterated internal standards. Compounds were simply extracted from alkalinized human heparinized plasma with methylene chloride, reconstituted into a solution of acetonitrile, methanol and 10 mM ammonium acetate (53:32:15, v/v) after the organic phase was dried down, and analyzed on the LC-MS-MS, which is a PE Sciex API III system equipped with a Keystone BDS Hypersil C₁₈ column and atmospheric pressure chemical ionization (APCI) interface. The between-run precision and accuracy of the calibration standards were ≤6.42% relative standard deviation (R.S.D.) and ≤11.8%n relative error (R.E.) for both nicotine and cotinine. The between-run and within-run precision and accuracy of quality controls. (2.5, 15.0, 37.5 ng/ml for nicotine and 25.0, 150.0, 375.0 ng/ml for cotinine), were ≤6.34% R.S.D. and ≤7.62% R.E. for both analytes. Sample stabilities in chromatography, in processing and in biological matrix were also investigated. This method has been applied to pharmacokinetic analysis of nicotine and cotinine in human plasma.     

The location of LH-RH neurons in the rat hypothalamus and their pathways to the median eminence

Summary The location of the perikarya of LH-RH neurons in the rat hypothalamus and their pathways to the median eminence were studied by immunohistochemistry and radioimmunoassay after placing stereotaxic electrolytic lesions in several parts of the hypothalamus. The principal location of the cell somata was found to be in the ventral part of the medial preoptic area; their pathways were classified into a main baso-lateral pathway and an accessory descending pathway branching off from the former. The main pathway was found to cross in the vicinity of the corresponding neuronal perikarya. The central median eminence and the dorsal and ventral walls of the tubero-infundibular sulcus of the caudal part of the median eminence are innervated mainly by the baso-lateral pathway. On the other hand, the rostral and most caudal portions of the median eminence are innervated principally by the descending pathway and have a subsidiary dual innervation. The projection of LH-RH neurons to the OVLT is believed to originate from perikarya adjacent to this circumventricular organ.This work was supported in part by a grant (No. 248093, 321426) from the Ministry of Education, Science and Culture, Japan     

Sensitive and simple method for the determination of nicotine and cotinine in human urine, plasma and saliva by gas chromatography-mass spectrometry

A method is proposed for the determination of nicotine and cotinine in human urine, plasma and saliva. Nicotine and cotinine were extracted from alkalinized sample with ethyl ether and concentrated to minimum volume with nitrogen stream. The volatility of nicotine was prevented by the addition of acetic acid to the organic solvent during evaporation. Peak shapes and quantitation of nicotine and cotinine are excellent, with linear calibration curves over a wide range of 1-10,000 ng/ml. The detection limits of nicotine and cotinine are 0.2 ng/ml in urine and 1.0 ng/ml in plasma and saliva. The intra-day precision of nicotine and cotinine in all samples was <5% relative standard deviation (RSD). Urine, plasma and saliva samples of 303 non-smoking and 41 smoking volunteers from a girl's high school in Korea were quantified by the described procedure. As a result, the concentrations of nicotine and cotinine in plasma ranged from 6 to 498 ng/ml and 4 to 96 ng/ml. Otherwise, those of nicotine and cotinine in saliva ranged from 0 to 207 ng/ml and 0 to 42 ng/ml, and those of nicotine and cotinine in urine ranged from 0 to 1,590 ng/ml and 0 to 2,986 ng/ml, respectively. We found that the concentration of cotinine in plasma was successfully predicted from the salivary cotinine concentration by the equation y=2.31x+4.76 (x=the concentration of cotinine in saliva, y=the concentration of cotinine in plasma). The results show that through the accurate determination of cotinine in saliva, the risk of ETS-exposed human can be predicted.