Vasoactive Intestinal Peptide Messenger RNA Research Articles

Aging selectively suppresses vasoactive intestinal peptide messenger RNA expression in the suprachiasmatic nucleus of the Syrian hamster

Aging leads to many changes in the expression of circadian rhythms, including reduced amplitude, altered relationship to the environmental illumination cycle, and reduced sensitivity to phase resetting signals. Neuropeptide synthesizing neurons in the suprachiasmatic nucleus (SCN), the principal circadian pacemaker in mammals, play a role in regulating pacemaker function and in coupling the pacemaker to overt circadian rhythms. Aging may alter the activity of neuropeptide neurons in the SCN, which could be reflected in changes in mRNA expression. Therefore, this study investigated whether aging alters the level or rhythm of expression of neuropeptide mRNAs in the SCN of male Syrian hamsters, a well established model for the study of age-related changes in circadian rhythms. Three age groups of hamsters ( young [3–5 months old], middle-aged [12–15 months old] and old [19–22 months old] were sacrificed at five times of day. Their brains were dissected and sections through the suprachiasmatic nucleus were prepared and used for in situ hybridization for mRNAs for vasoactive intestinal peptide (VIP), arginine vasopressin (AVP) and somatostatin (SS). Aging selectively decreased the SCN expression of VIP mRNA without affecting AVP mRNA or SS mRNA. Also, only AVP mRNA expression exhibited a robust 24-h rhythm, in contrast to previous findings in other species that VIP mRNA and SS mRNA, as well as AVP mRNA, exhibit 24-h rhythms in the SCN. The present findings suggest that age-related reductions in VIP mRNA expression may contribute to the alterations in entrainment and attenuated sensitivity to phase resetting signals that are characteristic of aging. Furthermore, the results demonstrate that neuropeptide gene expression in the SCN is differentially regulated by aging and varies among species.

Maternal regulation of embryonic growth: the role of vasoactive intestinal peptide.

Vasoactive intestinal peptide (VIP) is an important growth regulator of the embryonic day (E)9-E11 mouse. In comparably aged rat embryos, VIP messenger RNA (mRNA) is not detectable; however, peak concentrations of VIP in maternal rat serum indicate a nonembryonic source. In the current study, mouse maternal and embryonic tissues were examined from E6-E12. Although RT-PCR revealed VIP mRNA in E6-E7 conceptuses, by E8 (when extraembryonic tissues could be separated from the embryo), VIP mRNA was detected only in the decidua/trophoblast. Decidual/trophoblastic VIP mRNA decreased until E10, after which it was not detectable. VIP mRNA was not apparent in the embryo until E11-E12. At E9, VIP immunoreactivity was localized to abundant, diffuse cells in the decidua basalis, which were also immunoreactive for T cell markers. VIP binding sites were dense in the decidua/trophoblast at E6, but gradually decreased until E10, after which they were not apparent. VIP binding sites were detected in embryonic neuroepithelium by E9. The transient presence of VIP binding sites and mRNA in the decidua/trophoblast correlate with the critical period of VIP growth regulation, when VIP mRNA is absent in the embryo. These findings suggest that maternal lymphocytes are the source of VIP's regulating early postimplantation embryonic growth.

Vasoactive intestinal peptide is an important endocrine regulatory factor of fetal rat testicular steroidogenesis.

This study elaborates our recent preliminary finding that vasoactive intestinal peptide (VIP) has a specific stimulatory effect on fetal rat Leydig cells. We examined the dose-response relationship for the effect of VIP on cAMP and testosterone production by dispersed fetal Leydig cells isolated from rat testes on embryonic day (E) 18.5. Further, we used RT-PCR to examine the expression of the VIP gene in fetal brain and testes and that of the VIP receptor genes in fetal testes and used RIA to measure VIP in testes and plasma during the fetal period. VIP stimulated fetal testicular cAMP production at a dose of 10(-9) mol/liter, whereas a dose as low as 10(-12) mol/liter stimulated testosterone production. This suggests that VIP at low doses may stimulate testosterone production using second messenger pathways other than cAMP. RT-PCR analysis could not reveal either VIP messenger RNA (mRNA) in fetal tissues or VIP1 receptor mRNA in the fetal or newborn testes, whereas VIP2 receptor mRNA was detected in fetal testes as early as E15.5. Northern hybridization analysis showed that the level of expression of VIP2 receptor mRNA is very low in fetal and neonatal testes and increases with age. The testicular VIP content was unmeasurable by our RIA method (i.e. <1 fmol/testis), whereas the circulating level of VIP was 82.9 +/- 1.1 pmol/liter on E17.5 and decreased with advancing fetal age. In conclusion, our results suggest that VIP from an extratesticular source, possibly from the maternal compartment, may regulate fetal testicular steroidogenesis through type 2 receptors as early as E15.5. These findings may be of physiological significance, because the onset of fetal testicular steroidogenesis occurs at an age (E15.5-19.5) before the onset of pituitary LH secretion.

Protection against developmental retardation in apolipoprotein E-deficient mice by a fatty neuropeptide: implications for early treatment of Alzheimer's disease.

Stearyl-Nle17-VIP (SNV) is a novel agonist of vasoactive intestinal peptide (VIP) exhibiting a 100-fold greater potency than the parent molecule and specificity for a receptor associated with neuronal survival. Here, mice deficient in apolipoprotein E (ApoE), a molecule associated with the etiology of Alzheimer's disease, served as a model to investigate the developmental and protective effects of SNV. In comparison to control animals, the deficient mice exhibited (a) reduced amounts of VIP messenger RNA; (b) decreased cholinergic activity (c) significant retardation in the acquisition of developmental milestones: forelimb placing behavior and cliff avoidance behavior; and (d) learning and memory impairments. Daily injections of SNV to ApoE-deficient newborn pups resulted in increased cholinergic activity and marked improvements in the time of acquisition of behavioral milestones, with peptide-treated animals developing as fast as control animals and exhibiting improved cognitive functions after cessation of peptide treatment. Specificity was demonstrated in that treatment with a related peptide (PACAP), pituitary adenylate cyclase-activating peptide, produced only limited amelioration. As certain genotypes of ApoE increase the probability of Alzheimer's disease, early counseling and preventive treatments may now offer an important route for therapeutics design.

Prolactin gene expression and secretion during pregnancy and lactation in the rat: role of dopamine and vasoactive intestinal peptide

It is known that dopamine (DA) is the major PRL-inhibiting factor, and vasoactive intestinal peptide (VIP) is one of the most potent and physiological PRL-releasing factors. We have investigated the implication of DA and VIP in PRL gene expression and peptide secretion regulation during the physiological hyperprolactinemic states of pregnancy and lactation. Pregnant rats were studied on days 8, 15, and 20 of pregnancy. Lactating rats suckled by eight pups were studied on days 3 and 8 of postpartum, and nonsuckling postpartum rats were used as controls. Plasma estradiol, progesterone, and PRL were measured by RIA, as well as pituitary immunoreactive (IR-) PRL, pituitary IR-VIP, and hypothalamic IR-VIP. DA was studied by measuring changes in gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. TH, PRL, and VIP messenger RNA (mRNA) were assessed by Northern blot hybridization. The results showed very high plasma PRL levels in early pregnancy and during lactation, whereas plasma PRL concentrations were normalized at the end of gestation and in nonsuckling control rats. The physiological hyperprolactinemia of both early pregnancy and lactation correlated with higher pituitary PRL mRNA levels and lower pituitary IR-PRL content. Moreover, hypothalamic TH mRNA levels were lower in early pregnancy and lactation than at the end of gestation and in nonsuckling rats, respectively. The hypothalamic IR-VIP content was lower on day 8 of pregnancy than on days 15 and 20. However, VIP gene expression in the hypothalamus did not change throughout pregnancy. During lactation, neither hypothalamic IR-VIP content nor VIP mRNA was significantly altered. In the pituitary, IR-VIP content did not significantly change, and VIP mRNA levels were higher on day 15 of pregnancy than on the other days. During lactation, the pituitary IR-VIP content was very low on day 8 compared with those on day 3 of lactation and in nonsuckling control rats. VIP mRNA 1.0-kilobase transcript levels were higher in the lactating rats than in the control animals. These data show that both early pregnancy and lactation are physiological hyperprolactinemic states in which increased PRL mRNA accumulation coincides with decreased IR-PRL content in the pituitary and higher plasma IR-PRL, indicating regulation at the gene expression level and of PRL secretion. Low TH gene expression also occurs during hyperprolactinemia, suggesting that the diminution of DA activity that occurs during early pregnancy and lactation might be the major regulator of PRL alterations. If hypothalamic VIP plays a role as a neuroendocrine PRL-releasing factor during pregnancy and lactation, this may occur at the secretory level, as suggested by the alterations in IR-VIP, with no modifications in VIP mRNA accumulation, in the hypothalamus. Pituitary VIP does not seem to be a major regulator of PRL secretion during pregnancy, whereas during lactation, it regulates PRL secretion in a paracrine and/or autocrine manner.

Prolactin gene expression and secretion during pregnancy and lactation in the rat: role of dopamine and vasoactive intestinal peptide.

It is known that dopamine (DA) is the major PRL-inhibiting factor, and vasoactive intestinal peptide (VIP) is one of the most potent and physiological PRL-releasing factors. We have investigated the implication of DA and VIP in PRL gene expression and peptide secretion regulation during the physiological hyperprolactinemic states of pregnancy and lactation. Pregnant rats were studied on days 8, 15, and 20 of pregnancy. Lactating rats suckled by eight pups were studied on days 3 and 8 of postpartum, and nonsuckling postpartum rats were used as controls. Plasma estradiol, progesterone, and PRL were measured by RIA, as well as pituitary immunoreactive (IR-) PRL, pituitary IR-VIP, and hypothalamic IR-VIP. DA was studied by measuring changes in gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. TH, PRL, and VIP messenger RNA (mRNA) were assessed by Northern blot hybridization. The results showed very high plasma PRL levels in early pregnancy and during lactation, whereas plasma PRL concentrations were normalized at the end of gestation and in nonsuckling control rats. The physiological hyperprolactinemia of both early pregnancy and lactation correlated with higher pituitary PRL mRNA levels and lower pituitary IR-PRL content. Moreover, hypothalamic TH mRNA levels were lower in early pregnancy and lactation than at the end of gestation and in nonsuckling rats, respectively. The hypothalamic IR-VIP content was lower on day 8 of pregnancy than on days 15 and 20. However, VIP gene expression in the hypothalamus did not change throughout pregnancy. During lactation, neither hypothalamic IR-VIP content nor VIP mRNA was significantly altered. In the pituitary, IR-VIP content did not significantly change, and VIP mRNA levels were higher on day 15 of pregnancy than on the other days. During lactation, the pituitary IR-VIP content was very low on day 8 compared with those on day 3 of lactation and in nonsuckling control rats. VIP mRNA 1.0-kilobase transcript levels were higher in the lactating rats than in the control animals. These data show that both early pregnancy and lactation are physiological hyperprolactinemic states in which increased PRL mRNA accumulation coincides with decreased IR-PRL content in the pituitary and higher plasma IR-PRL, indicating regulation at the gene expression level and of PRL secretion. Low TH gene expression also occurs during hyperprolactinemia, suggesting that the diminution of DA activity that occurs during early pregnancy and lactation might be the major regulator of PRL alterations. If hypothalamic VIP plays a role as a neuroendocrine PRL-releasing factor during pregnancy and lactation, this may occur at the secretory level, as suggested by the alterations in IR-VIP, with no modifications in VIP mRNA accumulation, in the hypothalamus. Pituitary VIP does not seem to be a major regulator of PRL secretion during pregnancy, whereas during lactation, it regulates PRL secretion in a paracrine and/or autocrine manner.

Autoparacrine action of vasoactive intestinal peptide on dopaminergic control of prolactin secretion.

We have previously reported that pituitary vasoactive intestinal peptide (VIP) mediates through autoparacrine mechanisms insulin-like growth factor-I and TRH-stimulated PRL release. In the present study, we have investigated whether VIP might also be implicated in the PRL increase that follows dopamine (DA) withdrawal. The experiments were carried out in defined medium supplemented or not with T3, as in a previous study we had found that PRL release increases under low T3 culture conditions due to mediation by concomittant stimulus of VIP. Anterior pituitary (AP) cells from adult male rats were incubated for 1 h in the presence of DA (10 microM), a VIP-receptor antagonist (VIP-At) (200 nM), or DA plus VIP-At. Then media were removed and the cells were washed with PBS and reincubated under the same conditions but without the addition of DA. In the first incubation, DA inhibited PRL release by 80%, and VIP release by 20% in both T3-free and T3 medium. In the second period of incubation, PRL and VIP release were increased in AP cells previously exposed to DA. These effects were greater when the cells were cultured in T3-free medium than in T3-medium (225% and 150%, respectively for PRL release; and 155 and 135%, respectively for VIP release). PRL response to DA withdrawal was inhibited by the simultaneous presence of VIP-At. This inhibition was again greater when the cells were incubated in medium supplemented with T2. Thus, the stimulus of DA withdrawal was inhibited by 88% in T2-free medium and by 37% in T3-medium. To investigate whether pituitary VIP messenger RNA (mRNA) expression is under dopaminergic control, AP cells were incubated in the presence or absence of bromocriptine (BC) (10 nM) for 2 and 24 h. After 24 h of incubation, BC decreased mRNA levels of PRL and of the two transcripts which VIP expresses in AP. As with DA, BC also inhibited PRL and VIP release both at 2 and 24 h. These data demonstrate that VIP, at least partially, mediates DA withdrawal-induced PRL release through an autoparacrine action. They also suggest that simultaneous inhibition of pituitary VIP mRNA expression and VIP release may be a necessary mechanism for the dopaminergic inhibition of PRL mRNA expression and PRL release.

Autocrine and/or paracrine action of vasoactive intestinal peptide on thyrotropin-releasing hormone induced prolactin release

Several in vitro studies have demonstrated that vasoactive intestinal peptide (VIP) modulates basal PRL release in normal and hypothyroid anterior pituitary (AP) cells through an autocrine or paracrine action. As thyroid hormone is an important factor in the regulation of pituitary VIP synthesis and secretion, we analyzed the influence of the absence of thyroid hormone on basal PRL release in vitro to study whether the release of PRL induced by TRH might be mediated by a local action of pituitary VIP. When normal AP cells were cultured in a medium supplemented with a near-physiological concentration of free T2 (0.5 nM), basal PRL and VIP release decreased and PRL secretion was not altered by the blockade of VIP action. This finding allowed us to establish the culture conditions in which basal PRL secretion is apparently not under VIP influence. Consequently, we were able to study whether pituitary VIP may be implicated in TRH-induced PRL release. TRH (100 nM) increased PRL and VIP release in a parallel manner and decreased PRL and VIP intracellular content in incubations from 15-180 min. When AP cells wee incubated simultaneously with TRH and a VIP receptor antagonist, TRH-induced PRL release decreased when incubation lasted more than 30 min, whereas the depletion of PRL intracellular content induced by TRH was unchanged. TRH also slightly increased VIP messenger RNA levels at 3 and 24 h, but PRL messenger RNA levels were not modified. These data demonstrate that pituitary VIP participates in in TRH-induced PRL release and that the effect of thyroid hormone on basal pituitary VIP secretion should be borne in mind when studies on its effect, through autocrine and/or paracrine mechanisms, on PRL release stimulated by PRL-releasing factors are conducted.

Autocrine and/or paracrine action of vasoactive intestinal peptide on thyrotropin-releasing hormone induced prolactin release.

Several in vitro studies have demonstrated that vasoactive intestinal peptide (VIP) modulates basal PRL release in normal and hypothyroid anterior pituitary (AP) cells through an autocrine or paracrine action. As thyroid hormone is an important factor in the regulation of pituitary VIP synthesis and secretion, we analyzed the influence of the absence of thyroid hormone on basal PRL release in vitro to study whether the release of PRL induced by TRH might be mediated by a local action of pituitary VIP. When normal AP cells were cultured in a medium supplemented with a near-physiological concentration of free T2 (0.5 nM), basal PRL and VIP release decreased and PRL secretion was not altered by the blockade of VIP action. This finding allowed us to establish the culture conditions in which basal PRL secretion is apparently not under VIP influence. Consequently, we were able to study whether pituitary VIP may be implicated in TRH-induced PRL release. TRH (100 nM) increased PRL and VIP release in a parallel manner and decreased PRL and VIP intracellular content in incubations from 15-180 min. When AP cells wee incubated simultaneously with TRH and a VIP receptor antagonist, TRH-induced PRL release decreased when incubation lasted more than 30 min, whereas the depletion of PRL intracellular content induced by TRH was unchanged. TRH also slightly increased VIP messenger RNA levels at 3 and 24 h, but PRL messenger RNA levels were not modified. These data demonstrate that pituitary VIP participates in in TRH-induced PRL release and that the effect of thyroid hormone on basal pituitary VIP secretion should be borne in mind when studies on its effect, through autocrine and/or paracrine mechanisms, on PRL release stimulated by PRL-releasing factors are conducted.

Distribution of arginine-vasopressin and vasoactive intestinal peptide messenger RNA in the suprachiasmatic nucleus of the sheep

The distribution of mRNAs for vasoactive intestinal peptide (VIP) and arginine-vasopressin (VP) was studied in the hypothalamus of the sheep by in situ hybridization. VIP mRNA was detected in the supraoptic nucleus (SON) and in the median part of the suprachiasmatic nucleus (SCN). VP mRNA was observed in the magnocellular system of the hypothalamus and in the dorso-lateral part of the SCN. These results confirm that the SCN is a site of synthesis of both peptides. Therefore, VIP and VP may be involved in diverse physiological functions including the functioning of the biological clock constituted by the SCN.

Induction of vasoactive intestinal peptide gene expression and prolactin secretion by insulin-like growth factor I in rat pituitary cells: evidence for an autoparacrine regulatory system.

The effects of recombinant human insulin-like growth factor I (IGF-I) on both vasoactive intestinal peptide (VIP) and PRL production and gene expression were studied using rat anterior pituitary cell cultures grown in serum-free defined medium. We also examined whether pituitary VIP could be involved in the PRL response to IGF-I and hence in a paracrine regulatory system. Exposure of cultured anterior pituitary cells to IGF-I (2.6 nM) for 3 h caused a significant decrease in both VIP content and media PRL. Treatment with IGF-I (from 0.65-5.2 nM) for 48 h increased VIP production and VIP messenger RNA (mRNA) accumulation, whereas only an increase in media and intracellular PRL content without changes in mRNA was observed. In all these experiments, IGF-I led to a decrease in both GH secretion and expression. Immunoglobulins G purified from VIP antiserum inhibited IGF-I-induced PRL release without affecting intracellular and mRNA levels. The inhibition of both GH secretion and gene expression induced by IGF-I was not blocked by VIP antiserum. In conclusion, these results indicate that IGF-I induces VIP gene expression, and its secretion and also increases PRL secretion. The effect of IGF-I on PRL release is specifically mediated by VIP through a paracrine or autocrine mechanism.

Induction of vasoactive intestinal peptide gene expression and prolactin secretion by insulin-like growth factor I in rat pituitary cells: evidence for an autoparacrine regulatory system

The effects of recombinant human insulin-like growth factor I (IGF-I) on both vasoactive intestinal peptide (VIP) and PRL production and gene expression were studied using rat anterior pituitary cell cultures grown in serum-free defined medium. We also examined whether pituitary VIP could be involved in the PRL response to IGF-I and hence in a paracrine regulatory system. Exposure of cultured anterior pituitary cells to IGF-I (2.6 nM) for 3 h caused a significant decrease in both VIP content and media PRL. Treatment with IGF-I (from 0.65-5.2 nM) for 48 h increased VIP production and VIP messenger RNA (mRNA) accumulation, whereas only an increase in media and intracellular PRL content without changes in mRNA was observed. In all these experiments, IGF-I led to a decrease in both GH secretion and expression. Immunoglobulins G purified from VIP antiserum inhibited IGF-I-induced PRL release without affecting intracellular and mRNA levels. The inhibition of both GH secretion and gene expression induced by IGF-I was not blocked by VIP antiserum. In conclusion, these results indicate that IGF-I induces VIP gene expression, and its secretion and also increases PRL secretion. The effect of IGF-I on PRL release is specifically mediated by VIP through a paracrine or autocrine mechanism.

Plasticity of tyrosine hydroxylase and vasoactive intestinal peptide messenger RNAs in visceral afferent neurons of the nodose ganglion upon axotomy-induced deafferentation

The nodose ganglion contains placode-derived visceral sensory neurons of the vagus nerve. Previous study showed that axotomy-induced deafferentation reduced the number of tyrosine hydroxylaseimmunoreactive and increased the number of vasoactive intestinal peptide-immunoreactive neurons in the ganglion. The present study was conducted to determine whether the changes in neuropeptide/neurotransmitter enzyme content are associated with changes in the expression of tyrosine hydroxylase and vasoactive intestinal peptide messenger RNAs in the nodose ganglion. We used in situ hybridization histochemistry with 35S-labeled oligonucleotide probes for tyrosine hydroxylase and vasoactive intestinal peptide precursor messenger RNAs. Peripheral axotomy of visceral afferent inputs reduced tyrosine hydroxylase messenger RNA and increased vasoactive intestinal peptide messenger RNA expression in neurons of the nodose ganglion of the rat. The number of tyrosine hydroxylase messenger RNA-containing neurons was significantly reduced at three, seven and 14 days after axotomy-induced deafferentation compared with intact and sham-operated controls. Labeling density of tyrosine hydroxylase messenger RNA-containing neurons was significantly reduced at three and seven days. Conversely, the number of vasoactive intestinal peptide messenger RNA-containing neurons increased significantly at three, seven and 14 days, while the labeling density of vasoactive intestinal peptide messenger RNA-containing neurons also increased at one, three, seven and 14 days. The results of the present study indicate that the axotomy-induced down-regulation of tyrosine hydroxylase and up-regulation of vasoactive intestinal peptide in the neurons of the nodose ganglion are associated with changes in their messenger RNAs in response to axotomy-induced deafferentation.

Organotypic slice culture of the rat suprachiasmatic nucleus: Sustenance of cellular architecture and circadian rhythm

The suprachiasmatic nucleus of the mammalian brain is thought to be the anatomical locus of circadian rhythms. To examine the functional organization of the suprachiasmatic nucleus in vitro with intact intercellular connections for a prolonged period, we have established an organotypic slice culture system using a roller-tube technique. Brain slices (400 μm in thickness) containing the bilateral suprachiasmatic nuclei, were obtained from newborn rats at four to seven days old and were maintained in vitro for more than three weeks. During this three-week period, the slices flattened to one to three cell layers and two tightly packed neuronal cell-masses (neuronal zones), with diameters of about 1 mm were formed, which were surrounded by a peripheral glial cell-dispersed zone. In the neuronal cell zones, peptides and their messenger RNAs were found cytochemically with characteristic patterns similar to the suprachiasmatic nucleus in the brain. In situ hybridization and immunocytochemistry showed that vasoactive intestinal peptide messenger RNA expressing and vasoactive intestinal peptide-immunoreactive neurons were detected predominantly in the ventrolateral part of the neuronal zones in the suprachiasmatic nucleus slice culture. Vasopressin messenger RNA-expressing and vasopressin-immunoreactive cells were localized in the dorsomedial neuronal zones near the ependymal cell zone. The distribution of cell bodies and fibers containing these neuropeptides and their messenger RNAs in the neuronal zones of suprachiasmatic nucleus organotypic slice culture were similar to that of the suprachiasmatic nucleus in vivo. This suggests that the suprachiasmatic nucleus in these organotypic slice cultures retains the biological characteristics of these cells in vivo as the cells did develop, form compact neuronal masses and did establish connections. To examine the possibility that suprachiasmatic nucleus neurons in slice cultures show a persistent rhythmic activity, we also measured the amount of vasopressin released into the culture medium. Sampling at 4-h intervals combined with enzyme immunoassay revealed that vasopressin concentration in the medium embracing suprachiasmatic nucleus slice cultures fluctuated with a period of approximately 24 h. The present findings suggest that the intranuclear neuronal networks of the suprachiasmatic nucleus are maintained in vitro for a long duration and that organotypic cultures of the suprachiasmatic nucleus produce and release bioactive substances in an oscillating manner. The suprachiasmatic nucleus in slice cultures may be useful for future analysis of circadian rhythms in vitro.

Bromocriptine Inhibits Galanin Gene Expression in the Rat Pituitary Gland

The chronic effects of diethylstilbestrol (DES) and bromocriptine on the expression of galanin in the rat anterior pituitary were examined and compared with the expression of prolactin and vasoactive intestinal peptide (VIP) after the same type of hormonal manipulation. Total pituitary RNA was subjected to Northern blot analysis 2, 7, and 28 days after rats were implanted sc with a 10-mg DES pellet with or without concurrent treatment with bromocriptine (5 mg/kg per day). Estrogen treatment resulted in a rise in galanin, prolactin, and VIP messenger RNA (mRNA) that was particularly evident for galanin. Coadministration of bromocriptine with DES suppressed the response to estrogen with galanin, prolactin, and VIP mRNA levels being in general lower than when treatment with estrogen alone was used. In the combined treatment group there was an increase in the levels for the three mRNA species over the control group. Pituitary content of galanin and prolactin measured by radioimmunoassay exhibited a temporal pattern similar to that of corresponding mRNA levels after administration of estrogen alone or in combination with bromocriptine. The rise in galanin content (up to 78-fold) was higher than the increase in prolactin immunoreactivity (up to 6.5-fold compared to control). Bromocriptine partially suppressed the estrogen-induced increase in the content of these peptides. The changes in galanin and prolactin peptide levels in the plasma paralleled those found in the pituitary. This study demonstrates that the neuropeptides galanin and VIP are coregulated with prolactin after estrogen and bromocriptine administration. Since galanin and VIP are known to stimulate prolactin secretion it is possible that they mediate, at least in part, the effects of estrogen and bromocriptine on pituitary size and/or prolactin release.

Para-chlorophenylalanine treatment inhibits the expression of vasoactive intestinal peptide messenger RNA in rat anterior pituitary

Adult male Sprague-Dawley rats were treated with para-chlorophenylalanine (pCPA) or α-methyl tyrosine (α-MT) to study the effect of serotonin or catecholamine depletion on the expression of vasoactive intestinal peptide (VIP) messenger RNA in the anterior pituitary. Single injections of pCPA (300 mg/kg) for two consecutive days resulted on the third day in a dramatic depletion of serotonin in the medial basal hypothalamus, and a significant reduction in the pituitary content of VIP mRNA (1.0 and 1.7 kb). The effect of pCPA on VIP mRNA appeared to be relatively specific for the anterior pituitary since VIP message levels in the cerebral cortex did not decrease. α-MT treatment, (150 mg/kg) for 2 consecutive days, reduced dopamine concentrations in the MBH but had no significant effect on pituitary VIP levels. In a time-course study, hypothalamic serotonin and pituitary VIP mRNA levels were significantly depressed 1–3 days after initiation of pCPA treatment; however, 12 days after pCPA treatment, serotonin concentrations in the hypothalamus approached control values and pituitary VIP mRNA content increased an average of 2-fold over control levels in an apparent rebound effect. pCPA-treated rats injected i.p. twice a day with 5-hydroxytryptophan (5-HTP; 50 mg/kg) experienced a partial reversal in the decline in the 1.7 kb VIP mRNA seen 24 h after the first pCPA injection. However, at 72 h, supplementation with 5-HTP did not prevent the pCPA-induced decreased of pituitary VIP mRNA. These data indicate that serotonergic pathways have a major role in the control of VIP mRNA expression in the rat anterior pituitary.

Increased expression of preprotachykinin, calcitonin gene-related peptide, but not vasoactive intestinal peptide messenger RNA in dorsal root ganglia during the development of adjuvant monoarthritis in the rat

Neuropeptides in dorsal root ganglia (DRG) have been implicated in the pathogenesis of pain and neurogenic inflammation in experimental and clinical arthritis. Recently we demonstrated increased levels of substance P (SP) and calcitonin gene-related peptide (CGRP) confined to innervating DRG in adjuvant-mediated monoarthritis. We have now investigated whether changes in peptide content are reflected in altered neuropeptide gene expression and the time course involved. Using in situ hybridization we found marked increases in expression of β-preprotachykinin (PPT; 81±24% rise) and α-CGRP (44±6% rise)mRNAs in innervating (ipsilateral L 5) DRG neurones only. These a increases occured at the onset of acute inflammation (8 h) and persisted until chronic arthritis developed after 14 days. There were no changes in the proportion of DRG neurones expressing PPT or CGRP mRNAs. Messenger RNA encoding vasoactive intestinal polypeptide (VIP) was not induced. These data suggest that increased synthesis of PPT and CGRP peptides in DRG may play a role in the pathogenesis both of adjuvant-mediated acute inflammation and chronic arthritis.

Tissue-specific regulation of vasoactive intestinal peptide messenger ribonucleic acid levels by estrogen in the rat.

The early and chronic effects of 17 beta-estradiol on vasoactive intestinal peptide (VIP) gene expression in rats were examined. Total RNA of four VIP-producing tissues were subjected to Northern blot analysis 15 and 30 min, and 1, 3, 6, and 24 h after a single injection of 17 beta-estradiol (100 micrograms/kg ip). Pituitary, hypothalamus, brain, and ileum VIP messenger RNA (mRNA) levels rose in a time-dependent manner after estrogen treatment. In the pituitary, the increase was maximal at 30-60 min, whereas in the hypothalamus, the increase reached significance only at 3 h but then persisted until at least 24 h. In the brain, a transient increase in VIP mRNA was observed at 30 min, whereas VIP mRNA levels in the ileum responded in a biphasic pattern; the initial early increase was followed by a second elevation occurring at 6 h. A smaller 1-kilobase VIP-related transcript particularly abundant in the pituitary was regulated in parallel with the 1.7-kilobase mature VIP mRNA species. Continuous estrogen stimulation for 7 weeks dramatically increased both mRNA species in the pituitary but did not affect VIP mRNA levels in the other tissues. These data suggest that the regulation of VIP gene expression by transient increases in estrogen levels is rapid and that the pattern of induction is tissue specific.