pituitrin and preproenkephalin

Peptides derived from prodynorphin and provasopressin precursors coexist within neurosecretory vesicles of magnocellular neurons of the rat hypothalamus projecting to the posterior pituitary. The secretory activity of these neurons can be stimulated with physiological manipulations that elevate plasma levels of vasopressin (VP), such as dehydration and salt-loading. Evidence indicates that both VP- and prodynorphin-derived peptides are secreted under such conditions. With chronic osmotic challenge, the mRNAs for both prodynorphin and provasopressin increase in parallel in the supraoptic and paraventricular nuclei of the hypothalamus, and not within nonmagnocellular cell groups projecting elsewhere in the brain. The results indicate an example of coordinate regulation of mRNA expression for coexisting peptides within the brain. These results from microdissected tissues have been coupled with the more anatomically precise method of in situ hybridization histochemistry. Using 35S-radiolabeled synthetic oligonucleotides complementary to VP and dynorphin mRNAs, these mRNAs have been autoradiographically localized to magnocellular parikarya in the rat hypothalamus. Results also indicate that this technology can be used for regulatory studies, as evidenced by the increased hybridization of VP oligonucleotide to hypothalamic nuclei from salt-loaded rats.

Topics: Animals; Dynorphins; Enkephalins; Histocytochemistry; Hypothalamus; Nucleic Acid Hybridization; Protein Precursors; Rats; Rats, Brattleboro; RNA, Messenger; Vasopressins

The biosynthesis of neuro and hormonal peptides is in a state of rapid progress. The development of protein microsequencing is making it possible to characterize more and more new important molecules with nanomolar quantities, while the DNA structural studies favor the sequencing of the precursors of all these new peptides. These precursors are often polyproteins containing more than one active end-product. Their maturation processes are following a highly similar pattern with cleavage of the precursors at sites characterized by the presence of pairs of basic amino acid as noted in 1967-68 for the LPH and the pro-insulin models. This now constitutes a general concept which has good chances to be applicable to all the exciting neuro and hormonal peptides recently identified or yet to be discovered.

Topics: Amino Acid Sequence; Animals; Arginine Vasopressin; Calcitonin; Corticotropin-Releasing Hormone; DNA; Endorphins; Enkephalins; Gastrins; Glucagon; Humans; Models, Biological; Neurophysins; Oxytocin; Parathyroid Hormone; Pituitary Gland, Anterior; Pituitary Hormones, Anterior; Pro-Opiomelanocortin; Proglucagon; Proinsulin; Protein Precursors; Somatostatin; Vasopressins

Dehydration increases vasopressin (antidiuretic hormone) secretion from the posterior pituitary gland to reduce water loss in the urine. Vasopressin secretion is determined by action potential firing in vasopressin neurones, which can exhibit continuous, phasic (alternating periods of activity and silence), or irregular activity. Autocrine kappa-opioid inhibition contributes to the generation of activity patterning of vasopressin neurones under basal conditions and so we used in vivo extracellular single unit recording to test the hypothesis that changes in autocrine kappa-opioid inhibition drive changes in activity patterning of vasopressin neurones during dehydration. Dehydration increased the firing rate of rat vasopressin neurones displaying continuous activity (from 7.1 +/- 0.5 to 9.0 +/- 0.6 spikes s(1)) and phasic activity (from 4.2 +/- 0.7 to 7.8 +/- 0.9 spikes s(1)), but not those displaying irregular activity. The dehydration-induced increase in phasic activity was via an increase in intraburst firing rate. The selective -opioid receptor antagonist nor-binaltorphimine increased the firing rate of phasic neurones in non-dehydrated rats (from 3.4 +/- 0.8 to 5.3 +/- 0.6 spikes s(1)) and dehydrated rats (from 6.4 +/- 0.5 to 9.1 +/- 1.2 spikes s(1)), indicating that kappa-opioid feedback inhibition of phasic bursts is maintained during dehydration. In a separate series of experiments, prodynorphin mRNA expression was increased in vasopressin neurones of hyperosmotic rats, compared to hypo-osmotic rats. Hence, it appears that dynorphin expression in vasopressin neurones undergoes dynamic changes in proportion to the required secretion of vasopressin so that, even under stimulated conditions, autocrine feedback inhibition of vasopressin neurones prevents over-excitation.

Topics: Action Potentials; Animals; Cholecystokinin; Dehydration; Electrophysiology; Enkephalins; Female; Hypernatremia; Hyponatremia; Immunohistochemistry; In Situ Hybridization; Naltrexone; Narcotic Antagonists; Neurons; Oxytocin; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; RNA, Messenger; Vasopressins

Oxytocin receptors (OTR) and vasopressin V1a receptors (V1aR) in the ventral forebrain play critical roles in the formation of pair bonds in the monogamous prairie vole. Previous reports have been inconsistent in the identification of the specific brain regions in the ventral forebrain that express these receptors. To delineate more clearly the neuroanatomical boundaries of the OTR and V1aR fields in this species, we compared OTR and V1aR binding in adjacent brain sections and also with markers that delineate neuroanatomical boundaries in the ventral forebrain. OTR binding displayed an overlapping distribution with substance P mRNA and preproenkephalin mRNA, both markers for the shell and core of the nucleus accumbens. V1aR binding was nonoverlapping with each of these markers but colocalized with iron accumulation as shown by Perls' iron stain as well as leucine-enkephalin immunoreactivity, both markers for the ventral pallidum. OTR and V1aR mRNA were also restricted within the nucleus accumbens and ventral pallidum, respectively. Furthermore, destruction of ventral striatal dopaminergic terminals with 6-hydroxydopamine infusions into the nucleus accumbens did not alter OTR binding. Immunocytochemical analysis of oxytocin and vasopressin in the ventral forebrain demonstrated the presence of oxytocin-immunoreactive fibers in the nucleus accumbens and vasopressin-immunoreactive fibers in the ventral pallidum, with males showing a greater density of vasopressin fibers than females, but there was no such sex difference in the oxytocin system. Based on these results, we discuss potential neural mechanisms by which receptors in these brain regions mediate pair bond formation in this monogamous species. J. Comp. Neurol. 468:555-570, 2004.

Topics: Animals; Arvicolinae; Basal Ganglia; Binding Sites; Dopamine; Enkephalins; Female; Globus Pallidus; Immunohistochemistry; Male; Mice; Neural Pathways; Nucleus Accumbens; Oxidopamine; Oxytocin; Presynaptic Terminals; Protein Precursors; Receptors, Oxytocin; Receptors, Vasopressin; RNA, Messenger; Sex Characteristics; Sexual Behavior, Animal; Substance P; Vasopressins

Vasopressin released in the central nervous system has been shown to be involved both in homeostatic mechanisms (e.g., water balance, thermoregulation, cardiovascular regulation, metabolism, and antinociception) and in higher brain functions (e.g., social recognition and communication, and learning and memory). Many nuclear groups have been proposed to synthesize vasopressin, but available data are conflicting. We have used a sensitive in situ hybridization technique to identify the distribution of the neurons that may be the origin of the vasopressin in the central nervous system of the male Sprague-Dawley rat. Vasopressin mRNA-expressing neurons were most abundant in the hypothalamus (e.g., the paraventricular, supraoptic, and suprachiasmatic nuclei) but were also seen in the medial amygdaloid nucleus, the bed nucleus of stria terminalis, and the nucleus of the horizontal diagonal band. Previously unreported vasopressinergic neurons were seen in the entorhinal and piriform cortices, the ventral lateral portion of the parabrachial nucleus, the pedunculopontine nucleus, and the rostral part of the ventral periaqueductal gray matter and the adjacent portion of the mesencephalic reticular nucleus. Vasopressin mRNA expression suggestive of neuronal labeling was seen in the pyramidal layer of the CA1-3 fields and the dentate gyrus of the hippocampus. In addition, vasopressin mRNA expression, probably representing axonal mRNA, was detected over the hypothalamopituitary tract. No or insignificant preprovasopressin mRNA expression was present in the cerebellum, locus coeruleus, subcoeruleus, or the spinal cord. These findings provide novel information on the distribution of vasopressin neurons that are important for our understanding of how vasopressin acts in the brain.

Topics: Animals; Brain; Brain Stem; Cerebellum; Enkephalins; Hypothalamus; In Situ Hybridization; Male; Neurons; Organ Specificity; Oxytocin; Prosencephalon; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord; Transcription, Genetic; Vasopressins

This study, the first using the pig, examined expression of mRNAs for vasopressin (VP), oxytocin (OT), preproenkephalin (PENK) and pro-opiomelanocortin (POMC) in the forebrain, and of POMC and prolactin in the pituitary. High basal expression of VP and OT mRNAs was present in the paraventricular (PVN) and supraoptic (SON) nuclei. In the PVN, VP was found in magnocellular regions whereas OT was also seen in the parvocellular portion; the distribution of VP and OT mRNAs in the SON was as reported in other species. The suprachiasmatic nucleus contained VP mRNA but only OT message was present in the dorsomedial SON, a structure peculiar to swine. Gene expression for PENK occurred in the caudate putamen (CPu), for POMC in the mediobasal hypothalamus (MBH) and for prolactin and POMC in the hypophysis. Following restraint, VP message increased in the magnocellular PVN, as did PENK in the CPu and POMC in the MBH.

Topics: Acute Disease; Animals; Basal Metabolism; Enkephalins; Gene Expression; Hypothalamus; Male; Oxytocin; Pituitary Gland; Pro-Opiomelanocortin; Prolactin; Prosencephalon; Protein Precursors; Restraint, Physical; Stress, Physiological; Swine; Vasopressins

We have investigated the influence of endogenous opioids on oxytocin secretion during pregnancy. In blood-sampled conscious rats on days 18 and 21 of pregnancy plasma oxytocin concentration, measured by radioimmunoassay, was significantly increased compared to non-pregnant or post-partum rats. On days 15, 18 and 21 of pregnancy but not in non-pregnant, early pregnant or post-partum rats, the opioid antagonist naloxone caused a significant increase in plasma oxytocin compared to vehicle injection, indicating activation of an endogenous opioid restraint over oxytocin secretion. Electrically stimulated neural lobes isolated from 16- and 21-day pregnant rats released more oxytocin than those from non-pregnant rats. However, naloxone (10(-5) M) was less effective at potentiating, and the kappa-opioid agonist U50,488 (10(-5)M) was less effective at inhibiting, stimulated release at the end of pregnancy than in non-pregnant rats suggesting desensitization of oxytocin nerve terminals to actions of endogenous opioids. Neural lobes from male rats drinking 2% saline for 4 days also showed desensitization of oxytocin nerve endings to naloxone. Neither neural lobe content of dynorphin A(1-8), an endogenous kappa-opioid, nor prodynorphin mRNA expression, measured by in situ hybridization histochemistry in the supraoptic nucleus altered during pregnancy. However, neural lobe content of Met5-enkephalin significantly decreased by day 21 of gestation suggesting enhanced release.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Dynorphins; Electric Stimulation; Endorphins; Enkephalin, Methionine; Enkephalins; Female; Male; Naloxone; Oxytocin; Peptide Fragments; Pituitary Gland, Posterior; Pregnancy; Pregnancy, Animal; Protein Precursors; Pyrrolidines; Rats; Rats, Wistar; RNA, Messenger; Vasopressins

Following our recent demonstration of metrazole-induced immediate-early gene expression in the hypothalamic supraoptic nucleus (SON), we have now performed a mRNA and transcription analysis to determine the consequences of metrazole treatment for neurohypophyseal peptide gene expression in male rats. Levels of hypothalamic vasopressin (VP) and oxytocin (OT) mRNA were significantly reduced at 2 and 4 h after metrazole (50 mg/kg, i.p.), whereas pro-dynorphin mRNA was significantly elevated at 2 h. No changes in mRNA levels were found at 8, 24 or 48 h after treatment. Another convulsant (kainic acid, 8 mg/kg, i.p.) elicited similar effects on VP and OT mRNAs at 2 h. Specific analysis of the SON, following metrazole, revealed an equivalent effect on VP and OT mRNA levels but a nuclear run-on assay did not detect any change in SON VP gene transcription at 0.5, 1 and 2 h after treatment. The results provide evidence of a novel mechanism which may provide an additional level of control in the regulation of neuropeptide gene expression.

Topics: Animals; Blotting, Northern; Cell Nucleus; Enkephalins; Gene Expression; Hypothalamus; Male; Oxytocin; Pentylenetetrazole; Pituitary Gland, Posterior; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Seizures; Supraoptic Nucleus; Transcription, Genetic; Vasopressins

Opioid peptides derived from the precursor, prodynorphin, are co-localized with vasopressin in the hypothalamus and posterior pituitary, and vasopressin and prodynorphin synthesis are coordinately regulated during salt-loading. We had previously found that chronic ethanol ingestion resulted in decreased levels of hypothalamic and extrahypothalamic vasopressin mRNA, and the current study investigated the effect of ethanol ingestion on prodynorphin mRNA levels. A cRNA probe was constructed from a PCR product amplified from mouse genomic DNA. Cloning and sequencing of the PCR product revealed that the sequence of the mouse prodynorphin gene used to synthesize the probe is highly conserved, with high sequence similarity to corresponding regions of the gene in other mammalian species. In situ hybridization using the cRNA probe showed a widespread distribution of prodynorphin mRNA in mouse brain. In dehydrated mice, prodynorphin mRNA was significantly increased in the hypothalamus and nearly all other brain areas examined. In ethanol-fed mice, prodynorphin mRNA was also significantly increased in hypothalamus (50-60%) and in most brain areas. In the same mice, measurement of hypothalamic vasopressin mRNA confirmed a significant (approximately 60%) decrease. These results indicate that hypothalamic vasopressin and prodynorphin mRNA can be differentially regulated in certain situations.

Topics: Alcoholism; Amino Acid Sequence; Animals; Base Sequence; Brain; Enkephalins; Ethanol; Gene Expression Regulation; Hypothalamus; In Situ Hybridization; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Polymerase Chain Reaction; Protein Precursors; RNA, Messenger; Vasopressins

To clarify the role of endogenous opioid peptides in the control of vasopressin (AVP) secretion, the effects of an endogenous kappa-agonist, leumorphin, derived from proenkephalin B and an opioid antagonist, naloxone, on AVP secretion were examined in conscious and freely moving rats. Intraperitoneal injection of nicotine markedly increased AVP secretion in rats. The nicotine-induced AVP secretion was significantly suppressed by intracerebroventricular (i.c.v.) pretreatment with leumorphin. Intravenous injection of naloxone significantly increased the basal AVP level and carbachol-induced AVP secretion. These results indicate that endogenous opioid peptides have an inhibitory effect on AVP secretion in rats.

Topics: Animals; Blood Pressure; Carbachol; Endorphins; Enkephalins; Heart Rate; Male; Naloxone; Nicotine; Protein Precursors; Rats; Rats, Inbred Strains; Vasopressins

Peptides derived from pro-dynorphin and pro-vasopressin precursors coexist within neurosecretory vesicles of magnocellular neurons in the rat hypothalamus projecting to the posterior pituitary. The secretory activity of these neurons can be stimulated using physiological manipulations known to increase plasma vasopressin levels, such as dehydration and salt-loading. With chronic osmotic challenge, the mRNAs for both pro-dynorphin and pro-vasopressin increase in parallel in the supraoptic and paraventricular nuclei of the hypothalamus, but not within the nonmagnocellular suprachiasmatic nucleus cell groups projecting elsewhere than the neural lobe. The results indicate an example of coordinate regulation of mRNA expression for coexisting peptides within the brain.

Topics: Animals; Arginine Vasopressin; Autoradiography; Electrophoresis, Agar Gel; Enkephalins; Gene Expression Regulation; Hypothalamus; Male; Neurophysins; Nucleic Acid Hybridization; Osmolar Concentration; Oxytocin; Poly A; Protein Precursors; Rats; Rats, Inbred Strains; RNA; RNA, Messenger; Vasopressins

The postnatal development of several pro-enkephalin-B-derived opioid peptides - dynorphin 1-17, dynorphin 1-8, dynorphin B, alpha-neo-endorphin and beta-neo-endorphin - was examined in rat pituitary lobes. The concentrations of pro-enkephalin-B-derived peptides from the anterior pituitary were between 4- and 12-fold and those from the neurointermediate pituitary between 17- and 122-fold lower in newborn as compared to adult rats. Similarly, the concentrations of vasopressin in the neurointermediate pituitary increased 50-fold between birth and adulthood; those of oxytocin, however, increased more than 540-fold over this period. The molecular weight pattern of dynorphin 1-17, dynorphin 1-8, dynorphin B, alpha- and beta-neo-endorphin-immunoreactive peptides in the anterior and neurointermediate pituitary did not differ between 3-day-old pups and adult rats. In the neurointermediate pituitary, the major immunoreactive components had the same chromatographic properties as synthetic dynorphin 1-17, dynorphin 1-8, dynorphin B, alpha- and beta-neo-endorphin, respectively, on gel filtration and high-performance liquid chromatography (HPLC). This indicates that neonatal rats were already capable of processing the precursor pro-enkephalin B into these various opioid peptides. In newborn rats, however, the amount of dynorphin 1-8 in the neurointermediate pituitary was three times lower than that of its putative intermediate precursor peptide dynorphin 1-17. Similarly, the amount of beta-neo-endorphin was almost four times lower than that of its putative precursor alpha-neo-endorphin. In contrast, in the neurointermediate pituitary of adult rats, dynorphin 1-17 and dynorphin 1-8, in addition to a alpha- and beta-neo-endorphin, occurred in equimolar amounts.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Animals, Newborn; Cell Differentiation; Chromatography, High Pressure Liquid; Dynorphins; Endorphins; Enkephalin, Leucine; Enkephalins; Female; Male; Molecular Weight; Oxytocin; Peptide Fragments; Peptides; Pituitary Gland, Anterior; Pituitary Gland, Posterior; Pregnancy; Protein Precursors; Rats; Rats, Inbred Strains; Vasopressins

[he concentrations of immunoreactive (ir-) peptides derived from the opioid peptide precursors proenkephalin A (Met-enkephalin), proenkephalin B [dynorphin (DYN)-(1-17), dynorphin-(1-8), dynorphin B, alpha-neoendorphin (alpha-NEO-E), beta-NEO-E] and proopiomelanocortin [beta-endorphin (beta-END)], and of the neurosecretory hormones vasopressin and oxytocin increased between approximately 10-fold and 50-fold from birth to adulthood in the rate hypothalamus. Gel filtration and HPLC analysis of proenkephalin B-derived opioid peptides revealed that in 3-day-old rats the predominant portion of ir-dynorphin-(1-17) and a substantial part of ir-dynorphin B consisted of a high (6000) mol wt species, a common precursor peptide for DYN-(1-17) and DYN B. In adults rats, however, authentic DYN-(1-17) and DYN B were found to be the major ir-forms. The mol wt patterns of ir-DYN-(1-8), ir-alpha-NEO-E and ir-beta-NEO-E did not differ between 3-day-old and adult rats and reflected predominantly the respective authentic opioid peptides. Taking into consideration the developmental changes in the mol wt pattern of ir-DYN-(1-17), authentic DYN-(1-17) was 5 times lower in concentration than DYN-(1-8) in 3-day-old rats, whereas in adults these opioid peptides occurred in equimolar concentrations. These findings suggest that the posttranslational processing of the precursor proenkephalin B changes in the course of postnatal development. Ir-beta-END in the hypothalamus of newborn and adult rats consisted exclusively of beta-END-sized peptides which were not (unlike those in the intermediate pituitary lobe) alpha-N-acetylated. Thus, in the hypothalamus, the enzymatic processing of the opioid peptide precursor proopiomelanocortin to beta-END seems to be fully active at birth, in contrast to that of proenkephalin B.

Topics: Age Factors; Animals; Animals, Newborn; Chromatography, Gel; Chromatography, High Pressure Liquid; Enkephalins; Female; Hypothalamus; Male; Molecular Weight; Oxytocin; Pituitary Hormones, Anterior; Pregnancy; Pro-Opiomelanocortin; Protein Precursors; Rats; Rats, Inbred Strains; Sex Factors; Vasopressins