Immunocytochemical localization of peptidergic cells in the neuro-endocrine system of the Colorado potato beetle, Leptinotarsa decemlineata, with antisera against vasopressin, vasotocin and oxytocin

Antisera against vasopressin, vasotocin, oxytocin, neurophysin-1 and neurophysin-2 were used to investigate immunocytochemically the presence of neurons containing substances antigenically related to these peptides in the nervous system of the Colorado potato beetle. Ten different antisera were used, four against vasopressin, three against oxytocin and one against vasotocin, neurophysin-1, and neurophysin-2. Immunoreactivity was shown by all antisera except those against the neurophysins. The vasopressin antisera all gave different results. One antiserum revealed only a single neuron pair, whereas others revealed in addition one or two other different cell groups. The oxytocin antisera likewise revealed different neurons. The fixation procedure influenced the outcome of the immunocytochemical reaction. Immunoreactivity as revealed by vasopressin, vasotocin and oxytocin antisera is often co-localized in the same neurons; solid phase adsorptions showed that this is due to cross-reactivity of the antisera. Some of the immunoreactive neurons are identical to those recently described to contain a bovine pancreatic polypeptide/FMRFamide-like peptide. This co-localization is probably not due to a cross-reaction. These findings indicate the presence of several vasopressin-like and oxytocin-like substances which in the Colorado potato beetle all have a different degree of immunocytochemical resemblance to vasopressin and oxytocin.     

Immunocytochemical localization of peptidergic neurons and neurosecretory cells in the neuro-endocrine system of the Colorado potato beetle with antisera to vertebrate regulatory peptides

A large number of antisera to regulatory vertebrate peptides was tested immunocytochemically on the nervous system of the Colorado potato beetle to further characterize the peptidergic cells of the neuro-endocrine system and to reveal cells participating in endocrine control mechanisms. Neurons, neurosecretory cells, axons and axon terminals were revealed by antisera to ACTH, gastrin, CCK, alpha-endorphin, beta-endorphin, gamma 1-MSH, insulin, motilin, human calcitonin, growth hormone, somatostatin, CRF, ovine prolactin and rat prolactin. Together with previously described results these findings demonstrate that at least 19 different peptidergic cell types are present in the Colorado potato beetle. Several of these cell types are identical with the known neurosecretory cells, while others have not been identified before. The functions of the immunoreactive neurons are as yet unclear, although in two cases the localization of these cells gives some clues. Thus the lateral neurosecretory cells, which are immunoreactive with antisera to beta-endorphin and ovine prolactin, may regulate corpus allatum activity, whereas a CRF immunoreactive substance seems to be used as neurotransmitter by antennal receptors. These immunocytochemical findings do not imply that the immunoreactive substances are evolutionarily related to the vertebrate peptides to which the antisera were raised. It is postulated that if the part of the substance recognized by a certain antiserum is functionally important for the insect, which should be so if the insect peptide is evolutionarily related to its vertebrate homologue, the antiserum should reveal homologous cells in different insect species. The consequence of this hypothesis is, that if an antiserum does not reveal homologous neurons in different insect species, the immunologically demonstrated substance is probably of little physiological importance, and will not be related evolutionarily to the vertebrate analogue. The positive immunocytochemical results in the Colorado potato beetle are discussed in relation to these considerations.     

Secretory glycoproteins of the subcommissural organ of the dogfish (Scyliorhinus canicula): evidence for the existence of precursor and processed forms

The probable presence of oxytocin in the hypothalamo-hypophysial system of two reptilian species, the snake Natrix maura and the turtle Mauremys caspica, was re-investigated. A high-pressure liquid chromatographic analysis of the turtle neural lobe revealed the existence of vasotocin, mesotocin, and a third compound co-eluting with oxytocin. Brains from both species were fixed by vascular perfusion with Bouin's fluid. Adjacent paraffin sections were immunostained using antisera against the following substances: (1) bovine oxytocin-neurophysin; (2) a mixture of bovine oxytocin-neurophysin and vasopressin-neurophysin; (3) dogfish neurophysins; (4) oxytocin; (5) arginine-vasotocin; (6) mesotocin; (7) somatostatin. Immunoreactivity against oxytocin was found in parvocellular neurons of the snake suprachiasmatic nucleus and cerebrospinal-fluid contacting neurons of the medial nucleus of the infundibular recess of both species, the latter immunoreactivity being much more conspicuous in the turtle. Numerous fibers containing immunoreactive oxytocin extended between the medial nucleus of the infundibular recess, and the internal region of the medium eminence and the neural lobe. The oxytocin-immunoreactivity in all locations was completely abolished by preabsorption of the anti-oxytocin serum with three different oxytocin preparations. None of the neurons of the suprachiasmatic and medial nucleus of the infundibular recess, including the oxytocin-immunoreactive elements, reacted with either the antineurophysin sera used, or the anti-vasotocin or anti-mesotocin antibodies. The possible existence of a reptilian oxytocin-neurophysin is discussed. The alternative that, in the reptilian hypothalamus, neurons synthesize a compound closely related to, but different from oxytocin is also considered.     

A proctolin-like peptide and its immunocytochemical localization in the Colorado potato beetle,Leptinotarsa decemlineata

Summary Evidence is presented that neurons in the adult Colorado potato beetle contain a proctolin-like substance. By use of immunocytochemical methods the location of immunoreactive neurons in the central and stomatogastric nervous systems is described. No such neurons were found in the proto- and deutocerebrum or optic lobe. Few immunoreactive neurons are present in the tritocerebrum and numerous proctolin-immunoreactive neurons occur in all ventral ganglia and in the frontal ganglion. Two groups of neurosecretory cells in the suboesophageal ganglion contain a proctolin-immunoreactive substance. In these cells this material is co-localized with a bovine pancreatic polypeptide/FMRF amide-like substance and with a vasopressin/vasotocin/oxytocin-like substance. Proctolin-immunoreactive axon terminals were found on the musculature of the fore- and hindgut and of the vas deferens, and on some segmental muscles. Furthermore, proctolin-immunoreactive neurosecretory axon terminals were found in the corpus cardiacum. The proctolin-like substance may therefore function both as a neurotransmitter/neuromodulator and as a neurohormone. The presence of a proctolin-like substance was also demonstrated with a sensitive bioassay. On fractionation of extracts of the nervous systems of Leptinotarsa decemlineata with high performance liquid chromatography most of the proctolin-like bioactive material comigrated with authentic proctolin. This shows that a proctolin-like substance in this insect is very similar to, if not identical with, the known pentapeptide proctolin.     

Immunocytochemical localization of peptidergic neurons and neurosecretory cells in the neuro-endocrine system of the Colorado potato beetle with antisera to vertebrate regulatory peptides

Summary A large number of antisera to regulatory vertebrate peptides was tested immunocytochemically on the nervous system of the Colorado potato beetle to further characterize the peptidergic cells of the neuro-endocrine system and to reveal cells participating in endocrine control mechanisms. Neurons, neurosecretory cells, axons and axon terminals were revealed by antisera to ACTH, gastrin, CCK, -endorphin, -endorphin, 1-MSH, insulin, motilin, human calcitonin, growth hormone, somatostatin, CRF, ovine prolactin and rat prolactin. Together with previously described results these findings demonstrate that at least 19 different peptidergic cell types are present in the Colorado potato beetle. Several of these cell types are identical with the known neurosecretory cells, while others have not been identified before.The functions of the immunoreactive neurons are as yet unclear, although in two cases the localization of these cells gives some clues. Thus the lateral neurosecretory cells, which are immunoreactive with antisera to -endorphin and ovine prolactin, may regulate corpus allatum activity, whereas a CRF immunoreactive substance seems to be used as neurotransmitter by antennal receptors. These immunocytochemical findings do not imply that the immunoreactive substances are evolutionarily related to the vertebrate peptides to which the antisera were raised. It is postulated that if the part of the substance recognized by a certain antiserum is functionally important for the insect, which should be so if the insect peptide is evolutionarily related to its vertebrate homologue, the antiserum should reveal homologous cells in different insect species. The consequence of this hypothesis is, that if an antiserum does not reveal homologous neurons in different insect species, the immunologically demonstrated substance is probably of little physiological importance, and will not be related evolutionarily to the vertebrate analogue. The positive immunocytochemical results in the Colorado potato beetle are discussed in relation to these considerations.     

Immunocytochemical localization of neurons in the nervous system of the Colorado potato beetle with antisera against FMRFamide and bovine pancreatic polypeptide

Summary Particular neurons in the nervous system of the Colorado potato beetle, Leptinotarsa decemlineata, are recognized by antisera against bovine pancreatic polypeptide and FMRFamide. Both antisera react with the same neurons. Solid phase absorptions showed that antiserum against bovine pancreatic polypeptide cross-reacts with FMRFamide, whereas antiserum against FMRFamide cross-reacts with bovine pancreatic polypeptide. Some of the immunoreactive neurons have axons branching extensively within the neuropile, which suggests that the peptide is used as transmitter. In the corpus cardiacum, a neurohaemal organ in insects, numerous immunoreactive axon terminals are present. Here, the peptide material is presumably released as a hormone.     

The distribution of vasopressin-, oxytocin-, and neurophysin-producing neurons in the guinea pig brain

Summary The location, cytology and projections of vasopressin-, oxytocin-, and neurophysin-producing neurons in the guinea pig were investigated using specific antisera against vasopressin, oxytocin or neurophysin in the unlabeled antibody enzyme immunoperoxidase method. Light microscopic examination of the neurons of the supraoptic and paraventricular nuclei shows that hormone is transported not only in axons, but also in processes having the characteristics of dendrites. Neurons were found to contain only vasopressin or oxytocin; all neurons containing neurophysin appear to contain either vasopressin or oxytocin. In the neural lobe, vasopressin and oxytocin terminals are intermingled. In the median eminence, vasopressin and oxytocin fibers are intermingled in the internal zone. In a caudal portion of the median eminence, a number of vasopressin and neurophysin (but few oxytocin) axons enter the external zone from the internal zone, and surround portal capillaries. In the supraoptic nucleus, vasopressin neurons outnumber oxytocin neurons with a ratio of at least 5:1. The paraventricular nucleus is separated into two distinct groups of neurons, a lateral group consisting of only vasopressin neurons, and a medial group consisting of only oxytocin neurons. In addition to axons passing to the neurohypophysis, a number of axons appear to interconnect the supraoptic and paraventricular nuclei.Supported by the Deutsche Forschungsgemeinschaft (SFB 51, C/21 and C/27), (We 608/3)Acknowledgements. The authors are greatly indebted to Mmes. R. Köpp-Eckmann, B. Reijerman, A. Scheiber, I. Wild and Mr. U. Schrell for technical assistance, to Mmes. P. Campbell and U. Wolf for editorial assistance, and to Dr. R.R. Dries and Ferring Pharmaceuticals, Kiel, for the generous provision of high quality peptides     

A monoclonal antibody to vasopressin: preparation, characterization, and application in immunocytochemistry

The hypothalamo-neurohypophysial system, containing the hormones oxytocin (OT) and vasopressin (VP) and their associated carrier proteins, the neurophysins (NPS), has been the subject of extensive investigation for more than 40 years. This system has been reinvestigated during the last decade by application of immunocytochemical methods employing the rabbit antisera to the hormones and NPS. In this study we describe the preparation and characterization of a monoclonal antibody to VP and its application in immunohistochemistry. The antibody did not cross-react with OT or arginine vasotocin (AVT). Its antigenic determinants as characterized by absorption with various VP analogs included two aromatic amino acids: Phe in position 3, and to a lesser extent Tyr in 2. Tissue fixation with formaldehyde resulted in inadequate immunostaining as compared to glutaraldehyde, most likely due to interference with the aromatic amino acid determinants by the former fixative.     

Co-localization of putative vasopressin receptors and vasopressinergic neurons in rat hypothalamus

Summary Vasopressin and oxytocin are synthesized by neurons in the paraventricular and supraoptic nuclei of hypothalamus. Dense concentrations of vasopressin binding sites have also been localized in these nuclei. Using a vasopressin anti-idiotypic antiserum, a dual immunocytochemical labeling procedure has been employed to elucidate the distribution of putative vasopressin receptors in anatomical relation to vasopressin and oxytocin immunoreactive cells in rat brain. Putative vasopressin receptors are observed in relation to magnocellular neurons in hypothalamus that are vasopressin immunoreactive. They do not appear to be associated with parvocellular vasopressinergic cells or oxytocin immunoreactive neurons. The presence of these presumed autoreceptors would support evidence that vasopressin may autoregulate the activity of magnocellular vasopressinergic neurons in hypothalamus.     

Co-localization of putative vasopressin receptors and vasopressinergic neurons in rat hypothalamus

Vasopressin and oxytocin are synthesized by neurons in the paraventricular and supraoptic nuclei of hypothalamus. Dense concentrations of vasopressin binding sites have also been localized in these nuclei. Using a vasopressin anti-idiotypic antiserum, a dual immunocytochemical labeling procedure has been employed to elucidate the distribution of putative vasopressin receptors in anatomical relation to vasopressin and oxytocin immunoreactive cells in rat brain. Putative vasopressin receptors are observed in relation to magnocellular neurons in hypothalamus that are vasopressin immunoreactive. They do not appear to be associated with parvocellular vasopressinergic cells or oxytocin immunoreactive neurons. The presence of these presumed autoreceptors would support evidence that vasopressin may autoregulate the activity of magnocellular vasopressinergic neurons in hypothalamus.     

Binding of GRP(14-27) but not bombesin or GRP(1-27) to hypothalamic magnocellular elements: an immunohistochemical study

Bombesin, gastrin-releasing peptide (1-27), and gastrin-releasing peptide (14-27) abolished the specific immunocytochemical staining revealed by antiserum directed to the C-terminus of gastrin releasing peptide (GRP) and bombesin (BN) in rat hypothalamus. When the antiserum was preabsorbed with GRP(14-27), a strong reaction appeared in hypothalamic magnocellular neurons. This staining of magnocellular elements was produced by lower concentrations of GRP(14-27) than were needed to block immunocytochemical staining revealed by the antiserum in other hypothalamic locations. The distribution of GRP(14-27)-induced immunostaining was similar to that of neurophysin. Since only GRP(14-27) but not GRP(1-27) or bombesin was found to bind to magnocellular cells, it was concluded that binding was due to the N-terminus of GRP(14-27), which resembles the structure of oxytocin and vasopressin. In agreement with this, oxytocin and vasopressin were found to prevent the binding of GRP(14-27) to magnocellular cells. The similarity in localization and the effect of oxytocin and vasopressin suggest that GRP(14-27) may bind to neurophysin at low concentrations. The results suggest that enhancement of staining after preabsorption of antisera with antigens must be interpreted with care. Enhancement can occur at antigen concentrations lower than those required to block the immunostaining. These results fail to support the premise that antigen-induced enhancement of staining is due to antigen binding to specific receptors and subsequent detection of the receptor-bound antigen with the antiserum.     

The topography of mesotocin and vasotocin systems in the brain of the domestic mallard and japanese quail: Immunocytochemical identification

Summary The neurosecretory systems producing mesotocin (MT) and vasotocin (VT) (the avian homologues of oxytocin and vasopressin, respectively) were characterized in the brains of the domestic mallard and Japanese quail by means of indirect immunofluorescence techniques using specific antisera. In the anterior preoptic region, including the organum vasculosum of the lamina terminalis, and at different levels of the supraoptic and paraventricular nuclei, separate mesotocin- and vasotocin-producing neurons were identified. Mesotocinergic and vasotocinergic neurons were also located in the tuberomammillary area, among the ectomammillary tract fibers. The supraoptico-neurohypophysial tract, formed by vasotocin- and mesotocincontaining axons, enters the internal zone of the median eminence and ends in the posterior lobe of the pituitary. The external zone of the rostral median eminence appears to contain vasotocin and mesotocin fibers, which terminate in close contact with the capillaries of the hypophysial portal system.With contributions by Dr. B. Kerdelhué, Laboratoire des Hormones Polypeptidiques du CNRS, 91190 Gif-sur-Yvette, France     

Colloid droplets in the magnocellular secretory neurons of the reptilian hypothalamus: An immunocytochemical and lectin-histochemical study

Summary The immunocytochemical and lectin-binding properties of the magnocellular neurosecretory neurons in the hypothalamus of 2 reptilian species, the snake Natrix maura and the lizard Liolaemus cyanogaster, were investigated. Particular attention was paid to the secretory droplets present in these neurons. Antisera against bovine neurophysins I+II, arginine-vasotocin, and mesotocin were used. The following lectins were applied: concanavalin A (Con A), wheat-germ agglutinin (WGA), and Limax flavus agglutinin (LFA). Adjacent 1-m-thick methacrylate sections were used to investigate the same secretory neuron and the same colloid droplets with all three antisera and all three lectins. Several sections were treated with trypsin and urea before immunostaining or lectin binding. Con A bound to both vasotocin- and mesotocin-immunoreactive neurons, WGA exclusively to vasotocin neurons; neither of these neurons reacted with LFA. The colloid droplets were present in vasotocin neurons but absent in the mesotocin neurons. These secretory droplets showed an affinity for Con A but not for WGA, and reacted with antisera against neurophysins and vasotocin. In Natrix maura, the colloid droplets became reactive with Con A and the antisera used only after pretreatment of the sections with trypsin and urea. Within the hypothalamo-neurohypophyseal system, antiserum against vasotocin and WGA revealed the same fiber bundles. It is concluded (i) that in reptiles the vasotocin-neurophysin precursor is glycosylated, (ii) that vasotocin neurons have the exclusive capacity to form colloid droplets, and (iii) that these droplets are an intracisternal (RER) storage form of the vasotocin-neurophysin precursor.This work was partially supported by Grants BOJA 27/9/88 from the Dirección General de Universidades e Investigación de Junta de Andalucía and DGICYT PB87-0710 from the Comisión Interministerial de Ciencia y Tecnología, Madrid, to P.F.-LL.; and Grant 89-01 from the Dirección de Investigaciones, Universidad Austral de Chile, to E.M.R.     

Pymetrozine causes a nontarget pest, the Colorado potato beetle (Coleoptera: Chrysomelidae), to leave potato plants

Pymetrozine is a selective insecticide that targets aphids. Published assessments of the effects of pymetrozine on nontarget organisms focus mainly on predatory insects, and they rarely indicate toxicity. In a laboratory bioassay, survival of Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), larvae was not affected by pymetrozine exposure. We subsequently used pymetrozine to implement low-aphid-density treatments in a field experiment that involved separate manipulations of Colorado potato beetle density. Unexpectedly, the addition of Colorado potato beetle adults and eggs did not increase the densities of Colorado potato beetle larvae in plots that were sprayed with pymetrozine (applied with water and an adjuvant). In control plots sprayed with water and adjuvant (without pymetrozine), addition of Colorado potato beetles increased densities of their larvae. Data collected on a smaller scale suggest that a behavioral mechanism underlies the population-level pattern: Colorado potato beetle larvae become more active and are less likely to remain on a host plant after exposure to pymetrozine. Thus, potato, Solanum tuberosum L., growers who use pymetrozine against aphids also might benefit in terms of Colorado potato beetle control.     

Pharmacological characterization of two specific binding sites for neurohypophyseal hormones in hippocampal synaptic plasma membranes of the rat

Synaptic plasma membranes containing binding sites for tritiated oxytocin and arginine vasopressin were isolated from rat hippocampus. The binding parameters for oxytocin and vasopressin sites were determined and statistically analysed. The fitted curve for oxytocin binding was compatible with a model where the ligand interacts with two classes of receptors with different capacities and affinities. The sites with low binding capacity had an apparent dissociation constant at equilibrium of 1.8 nM and a maximal binding capacity of 17 fmol/mg protein. By contrast, the Scatchard plot failed to reveal a marked heterogeneity in the population of sites labelled with [3H]vasopressin with an affinity of 1.5 nM and a maximal binding capacity of 39 fmol/mg protein. The specificity of these binding sites, tested in competition experiments, revealed that these neurohypophyseal hormones labelled two distinct populations of sites. One population with a high affinity for vasopressin, oxytocin and vasotocin, the other population with a high affinity for vasopressin and vasotocin and a low affinity for oxytocin. Adenylate cyclase activity was not affected by arginine-vasopressin or oxytocin. These receptors are compared with previously characterized peripheral receptors.     

Vasopressin and oxytocin excite MCH neurons, but not other lateral hypothalamic GABA neurons

Neurons that synthesize melanin-concentrating hormone (MCH) colocalize GABA, regulate energy homeostasis, modulate water intake, and influence anxiety, stress, and social interaction. Similarly, vasopressin and oxytocin can influence the same behaviors and states, suggesting that these neuropeptides may exert part of their effect by modulating MCH neurons. Using whole cell recording in MCH-green fluorescent protein (GFP) transgenic mouse hypothalamic brain slices, we found that both vasopressin and oxytocin evoked a substantial excitatory effect. Both peptides reversibly increased spike frequency and depolarized the membrane potential in a concentration-dependent and tetrodotoxin-resistant manner, indicating a direct effect. Substitution of lithium for extracellular sodium, Na(+)/Ca(2+) exchanger blockers KB-R7943 and SN-6, and intracellular calcium chelator BAPTA, all substantially reduced the vasopressin-mediated depolarization, suggesting activation of the Na(+)/Ca(2+) exchanger. Vasopressin reduced input resistance, and the vasopressin-mediated depolarization was attenuated by SKF-96265, suggesting a second mechanism based on opening nonselective cation channels. Neither vasopressin nor oxytocin showed substantial excitatory actions on lateral hypothalamic inhibitory neurons identified in a glutamate decarboxylase 67 (GAD67)-GFP mouse. The primary vasopressin receptor was vasopressin receptor 1a (V1aR), as suggested by the excitation by V1aR agonist [Arg(8)]vasotocin, the selective V1aR agonist [Phe(2)]OVT and by the presence of V1aR mRNA in MCH cells, but not in other nearby GABA cells, as detected with single-cell RT-PCR. Oxytocin receptor mRNA was also detected in MCH neurons. Together, these data suggest that vasopressin or oxytocin exert a minimal effect on most GABA neurons in the lateral hypothalamus but exert a robust excitatory effect on presumptive GABA cells that contain MCH. Thus, some of the central actions of vasopressin and oxytocin may be mediated through MCH cells.     

Coexistence of NADPH-diaphorase with vasopressin and oxytocin in the hypothalamic magnocellular neurosecretory nuclei of the rat

Coexistence of NADPH-diaphorase with vasopressin and oxytocin was studied in the magnocellular neurosecretory nuclei of the rat hypothalamus by use of sequential histochemical and immunocytochemical techniques in the same sections. Coexistence was found in all the nuclei examined (supraoptic, paraventricular, circular, fornical, and in some isolated neurons located in the hypothalamic area between the paraventricular and supraoptic nuclei). The ratios of neurons expressing both markers (NADPH-diaphorase and vasopressin, NADPH-diaphorase and oxytocin) in each of the nuclei were very similar. Although further studies must be carried out, the partial coexistence found in all nuclei suggests that NADPH-diaphorase is probably not related to general mechanisms involving vasopressin and oxytocin, but rather in specific functions shared by certain hypothalamic neuronal cell populations.     

Effect of crop rotation distance on populations of Colorado potato beetle (Coleoptera: Chrysomelidae): development of areawide Colorado potato beetle pest management strategies

The Colorado potato beetle, Leptinotarsa decemlineata (Say), overwinters adjacent to field edges and infests nearby fields in the spring, primarily by walking. Crop rotation is known to be an effective cultural control against Colorado potato beetle populations limiting spring infestations. Spatial separation is an important consideration in optimizing the rotational effect because long-distance rotations have been shown to reduce Colorado potato beetle infestations. To determine the effect of long-distance rotations on Colorado potato beetle populations in commercial Wisconsin potato production, a geographic area of >18,200 ha (>45,000) acres in southern Portage County, Wisconsin, was selected as an experimental area for areawide pest management of the Colorado potato beetle. From 1997 to 1999, beetle populations at edges within each potato field in this region were determined by spring and fall field sampling. The rotational distance between current and previous potato fields was measured and analysis was run between the distance and Colorado potato beetle populations. Long-distance rotations of >400 m were an effective cultural control management strategy to limit adult beetle infestations in the spring. This strategy can be optimized when collaborating growers are able to maximize their rotational distances by coordinating their rotational schemes within large areawide, geographic locations. Deploying long-distance rotations within a geographic area over many years would limit Colorado potato beetle populations and could result in a significantly reduced Colorado potato beetle populations entering fields in the spring.     

Circular-dichroic spectra of vasopressin analogues and their cyclic fragments.

The circular dichroic spectra of [Arg8]vasopressin, [Mpr1, Arg8]vasopressin, [Mpr1, D-Arg8]-vasopressin, pressinamide, deaminopressinamide, tocinamide, deaminotocinamide, [Leu4, D-Arg8]-vasotocin, [Mpr1, Leu4, D-Arg8]vasotocin and [Phe2, Lys8]vasopressin have been studied. All these substances showed a characteristic positive dichroic band at about 225 nm due to the presence of tyrosine in sequence position 2. The intensity of this band was affected by interactions between the tyrosine side-chain and other structural elements in the molecule, such as the Na-amino group, the side-chain of phenylalanine in position 3 and the linear C-terminal peptide. Analysis of the response of this band to structural modifications of the molecule and change in the solvent (particularly comparing neutral aqueous solutions with hexafluoroacetone solutions) allowed some conformational conclusions. The linear C-terminal tripeptide is probably situated over the cyclic portion of the molecule both in vasopressin and oxytocin substances. Its steric interaction with the tyrosine side-chain seems to be particularly efficient in molecules containing D-arginine in position 8. In the vasopressin series the stacking interaction of neighbouring aromatic amino acid residues furthermore limits the conformational freedom of the tyrosine side-chain and also probably distorts the dihedral angles of residues 1-3 in comparison with oxytocin. The interactions of phenylalanine and arginine with tyrosine relatively decrease the conformational effects of the primary amino group. Consequently the local conformation of vasopressin in the region of the tyrosine residue is more rigid and less sensitive to changes in medium than that of oxytocin. The circular dichroic spectra did not show any basic conformational differences in the backbone peptide chain of oxytocin and vasopressin substances. A weak negative disulphide band at about 290 nm could be observed in the spectra of both series of substances.     

Central cardiovascular effects of mammalian neurohypophyseal peptides in conscious rats

To confirm and extend the results of previous studies which demonstrated central cardiovascular effects of vasopressin in anesthetized rats, we determined blood pressure and heart rate changes for 30 minutes after intracerebroventricular injections of arginine vasopressin, arginine vasotocin and oxytocin in conscious rats. As compared to sham injections, significantly greater increases in either systolic or diastolic blood pressure were noted over the 30 minutes which followed the injection of 0.15, 1.0 or 10.0 nM of either vasopressin or vasotocin. In animals given vasopressin, plasma levels of the peptide were determined. There was a substantial increase in plasma vasopressin only after the highest dose. Overall blood pressure responses to doses of oxytocin as high as 100 nM were not significantly different than sham injections. Heart rate following both vasopressin and vasotocin was increased at 0.15 nM, was initially decreased then increased at 1.0 nM and was substantially decreased after the 10.0 nM dose. There was a significant increase in heart rate at the 10.0 nM and 100 nM doses of oxytocin. Dose response curves for systolic blood pressure and heart rate 20 minutes after injection were similar for vasopressin and vasotocin. We conclude that arginine vasopressin has significant central pressor and tachycardic effects in conscious rats, and it is related, at least in part, to the tail structure of the peptide, which is shared with arginine vasotocin.