Celia D. Sladek

Neurotransmitter/neuropeptide interactions in the regulation of neurohypophyseal hormone release

Regulation of neurohypophyseal hormone release reflects the convergence of a large number of afferent pathways on the vasopressin (VP)- and oxytocin-producing neurons. These pathways utilize a broad range of neurotransmitters and neuropeptides. In this review, the mechanisms by which this information is coordinated into appropriate physiological responses is discussed with a focus on the responses to agents that are coreleased from A1 catecholamine nerve terminals in the supraoptic nucleus. The A1 pathway transmits hemodynamic information to the vasopressin neurons by releasing several neuroactive agents including ATP, norepinephrine, neuropeptide Y, and substance P. These substances stimulate VP release from explants of the hypothalamo-neurohypophyseal system and certain combinations of these agents elicit potent but selective synergism. Evaluation of the signal cascades elicited by these agents provides insights into mechanisms underlying these synergistic interactions and suggests mechanisms responsible for coordinated responses of the VP neurons to activation of a range of ion-gated ion channel and G-protein-coupled receptors.

N‐Methyl‐d‐Aspartic Acid Stimulation of Vasopressin Release: Role in Osmotic Regulation and Modulation by Gonadal Steriods

Previous experiments demonstrated that excitatory amino acids participate in the osmotic regulation of vasopressin secretion, but the specific involvement of N‐methyl‐ d‐aspartic acid (NMDA) receptors was not evaluated. This was demonstrated in the present studies. NMDA stimulated vasopressin release from perifused explants of the hypothalamo‐neurohypophyseal system (HNS), and osmotic stimulation of vasopressin release was inhibited by MK‐801 (10u2003μM) and AP5 (100u2003μM) NMDA receptor antagonists. The effective concentration of NMDA was dependent upon the Mg2+ concentration of the perifusate with stimulation observed at 1u2003μM NMDA in Mg2+‐replete compared with 5u2003μM in low‐Mg2+ medium. Previous experiments also demonstrated that estradiol and dihydrotestosterone (DHT) inhibited osmotically stimulated vasopressin secretion, and a nongenomic mechanism of action was suggested by the ability of steroids conjugated to bovine serum albumin to replicate the effect. Experiments were performed to explore the potential role of NMDA receptors in this mechanism. Estradiol (50u2003pg/ml) and DHT (3u2003ng/ml) inhibited NMDA stimulated vasopressin release in perifused HNS explants. These results suggest a role of NMDA receptors in the mediation of vasopressin secretion in osmotically stimulated release. Furthermore, estradiol and DHT may exert their inhibitory effect on osmotically stimulated vasopressin release via the NMDA receptor.

Prolactin modulates oxytocin mRNA during lactation by its action on the hypothalamo-neurohypophyseal axis

Since prolactin (PRL) can increase electrically stimulated oxytocin (OT) release by a direct action on the neurohypophysis, experiments were done to test the effect of PRL on OT mRNA content of explants of the hypothalamo-neurohypophyseal system (HNS) obtained from Day 10 lactating Sprague-Dawley rats, to determine if PRL could alter OT mRNA by a direct effect on the HNS. The effect of PRL was evaluated alone and in conjunction with bicuculline, an antagonist at gamma amino butyric acid receptors, in order to provide a concomitant stimulus for OT release. Neither PRL nor bicuculline alone altered OT or VP release. However, the simultaneous administration of bicuculline and PRL caused a statistically significant increase in the release of OT and in OT mRNA content of the explants (P < 0.05). On the other hand, PRL did not cause vasopressin (VP) release nor an increase in VP mRNA in these explants even in the presence of bicuculline, thus ruling out a direct effect of PRL on the HNS VP neurons. In conclusion, during lactation, PRL, in combination with other stimuli can increase OT mRNA content in the hypothalamus by a direct action on the hypothalamo-neurohypophyseal system. This effect may be secondary to the release of OT. This effect is specific to OT, because PRL did not alter either the release of VP or VP mRNA content.

The role of steroid hormones in the regulation of vasopressin and oxytocin release and mRNA expression in hypothalamo neurohypophysial explants from the rat

Vasopressin and oxytocin release from the neural lobe, and the vasopressin and oxytocin mRNA contents of the supraoptic and paraventricular nuclei are increased by hypertonicity of the extracellular fluid. The factors regulating these parameters can be conveniently studied in perifused explants of the hypothalamo‐neurohypophysial system that include the supraoptic nucleus (but not the paraventricular nucleus) with its axonal projections to the neural lobe. Vasopressin and oxytocin release and the mRNA content of these explants respond appropriately to increases in the osmolality of the perifusate. This requires synaptic input from the region of the organum vasculosum of the lamina terminalis. Glutamate is a likely candidate for transmitting osmotic information from the organum vasculosum of the lamina terminalis to the magnocellular neurones, because agonists for excitatory amino acid receptors stimulate vasopressin and oxytocin release, and because increased vasopressin release and mRNA content induced in hypothalamo‐neurohypophysial explants by a ramp increase in osmolality are blocked by antagonists of both NMDA (N‐methyl‐D‐aspartate) and non‐NMDA glutamate receptors. Osmotically stimulated vasopressin release is also blocked by testosterone, dihydrotestosterone, oestradiol and corticosterone. Both oestrogen and dihydrotestosterone block NMDA stimulation of vasopressin release, and in preliminary studies oestradiol blocked AMPA stimulation of vasopressin release. Thus, steroid inhibition of osmotically stimulated vasopressin secretion may reflect inhibition of mechanisms mediated by excitatory amino acids. Recent studies have demonstrated numerous mechanisms by which steroid hormones may impact upon neuronal function. Therefore, additional work is warranted to understand these effects of the steroid hormones on vasopressin and oxytocin secretion and to elucidate the potential contribution of these mechanisms to regulation of hormone release in vivo.

Expression of Floor Plate in Dispersed Mesencephalic Cultures: Role in Differentiation of Tyrosine Hydroxylase Neurons

The availability of sufficient numbers of dopaminergic neurons for transplantation has been an important issue. Recently, it has been shown that the ventral floor plate (FP4-positive) cells and the transcription factor HNF-3beta are important in the signals that terminate proliferation and produce differentiation of the dopaminergic phenotype. In this study, dispersed mesencephalon from embryonic rats at Day 11 postcoitus (E-11), 1 day prior to the birth of TH cells, were cultured for 48 h and 1 week to evaluate TH neuronal differentiation and/or proliferation in vitro. The number of TH cells increased 14x between 48 h and 1 week in culture. In dispersed E-14 cultures, the presence of FP4 and HNF-3beta markers was demonstrated using immunohistochemistry. The majority of FP4-positive cell clusters were associated with TH neurons, suggesting that floor plate cells may have participated in TH neuron differentiation in culture. Antisense oligonucleotide probe for HNF-3beta mRNA added daily to cultured E-14 cells blocked the HNF-3beta expression, but had no effect on the FP4 or TH expression. These studies suggest a potentially important role for floor plate cells in the differentiation of TH cells, and differentiation and/or proliferation of TH cells in dispersed cultures of E-11 is demonstrated.

Regulation of Vasopressin Release by Ionotropic Glutamate Receptor Agonists

Immunocytochemistry, in situ hybridization, and electrophysiological techniques have demonstrated the presence of excitatory amino acid (EAA) receptors in the supraoptic nuclei(1–4). A role for EAA regulation of vasopressin (VP) release is suggested by the ability of the non-specific EAA antagonist, kynureic acid, to inhibit osmotically stimulated VP release from explants of the hypothalamo-neurohypophyseal system (HNS)(5). In addition, both N-methyl-D-aspartate (NMDA) receptor antagonists (AP5 and MK801) and the non-NMDA receptor antagonist, DNQX, applied independently inhibit osmotically stimulated VP release implicating the importance of both classes of glutamate receptors in the regulation of VP release.

cAMP Stimulation of Vasopressin and Oxytocin Release and Regulation of Vasopressin mRNA Stability: Role of Auto‐Facilitation

The effects of cycloheximide and actinomycin on 8‐bromo‐cAMP (8‐Br‐cAMP) stimulated vasopressin and oxytocin release from the posterior pituitary and vasopressin mRNA content of the supraoptic nucleus were studied with perifused explants of the hypothalamo‐neurohypophyseal system. 8‐Br‐cAMP stimulated vasopressin and oxytocin release from the explant for up to 6u2003h. Inhibition of protein synthesis by cycloheximide completely suppressed the response to 8‐Br‐cAMP. When gene transcription was inhibited by actinomycin, vasopressin release was stimulated by 8‐Br‐cAMP for approximately 2u2003h, but the response was not sustained. Vasopressin mRNA content was not changed by 8‐Br‐cAMP in the absence or presence of cycloheximide, but it was significantly decreased by simultaneous exposure to 8‐Br‐cAMP and actinomycin. Actinomycin alone did not change vasopressin mRNA content. Since other studies have demonstrated that cAMP stimulates vasopressin gene transcription, and since vasopressin mRNA content reflects the balance between gene transcription and mRNA degradation, the effect of actinomycin and 8‐Br‐cAMP on vasopressin mRNA content suggests that 8‐Br‐cAMP also decreased vasopressin mRNA stability and thereby induced a rapid turnover of vasopressin mRNA. The effects of cycloheximide and actinomycin on vasopressin and oxytocin release suggest that ongoing protein synthesis is required for stimulation of hormone release. Since the posterior pituitary hormone stores are not depleted with a stimulus for release that is even more potent than cAMP, it is possible that cycloheximide and actinomycin depleted smaller pools of the peptides such as those responsible for intranuclear vasopressin and oxytocin release. Further evidence that intranuclear release of vasopressin and oxytocin is a prerequisite for cAMP stimulation of vasopressin and oxytocin release was obtained by demonstrating that d(CH2)5‐D‐Tyr(Me)VAVP, a potent combined V1a/V2/oxytocin receptor antagonist blocked stimulation of vasopressin and oxytocin release by 8‐Br‐cAMP.

Effect of Age and Testosterone on the Vasopressin Response to Dehydration in F344BNF1 Male Rats

Testosterone has been implicated in the dehydration-induced increase in vasopressin (VP) mRNA observed in young rats (1). Since testosterone is diminished in aged rats, and since prior studies have demonstrated a deficit in the dehydration-induced increase in VPmRNA in aged male Fischer 344 rats (2), the hypothesis that aging-associated deficits in testosterone result in a diminished VP response to dehydration was evaluated.

Increased vasopressin secretion from hypothalamic cultures following administration of exogenous vasopressin mRNA.

In Brattleboro rats, exogenous vasopressin (VP) mRNA can be accumulated, transported, and translated by magnocellular neurons. To determine whether this phenomenon may also occur in magnocellular neurons of normal rats, dispersed hypothalamic neuronal cultures of fetal Sprague-Dawley rats were exposed to VP mRNA. The cultures were maintained in either control medium or medium containing the cAMP elevating drugs, IBMX (3-isobutyl-1-methylxanthine), and forskolin for 23 or 30 days of culture to induce VP synthesis and secretion. Following removal of the IBMX and forskolin at Day 23, VP secretion into the medium declined to baseline by 30 days in vitro, but administration of VP mRNA to these cultures on Day 30 resulted in a 5-fold increase in VP content of the medium (P = 0.005) after 6 h and a 2.5-fold increase after 24 h (P = 0.002). Administration of VP mRNA to the cultures treated continuously with IBMX and forskolin also resulted in a small increase in VP secretion which did not reach significance after either 6 or 24 h. When cultures prepared with continuous I/F were exposed to antisense VP mRNA, VP secretion into the media was decreased by 58%, and VP immunoreactive perikayra were difficult to observe. This demonstrates that the increase in VP release observed after the addition of sense VPmRNA did not reflect a nonspecific effect of the addition of mRNA to the culture medium. Autoradiography of cultures administered 3H- or 32P-VP mRNA for 24 h revealed silver grains associated with varicosities, perikarya, and neuritic processes of neurophysin (NP)-positive, but not NP-negative neurons. These results suggest that exogenously administered mRNA has access to cell translation systems in cultured hypothalamic neurons as well as magnocellular neurons of Brattleboro rats.

Effect of forskolin and exogenously administered oxytocin mRNA on oxytocin release by dispersed hypothalamic cultures.

Differential vasopressin (VP) gene expression and oxytocin (OT) gene expression were observed in hypothalamic cultures derived from 14-day-old rat fetuses, with VP but not OT being induced by treatment with forskolin and 3-isobutyl-1-methylxanthine. These cultures were used to demonstrate that exogenous VP mRNA could be taken up and translated into releasable VP. In the current studies a similar culture preparation was used to test the hypothesis that, due to the similarity in the mRNA and prohormone structures of VP and OT, the VP-expressing neurons in the cultures would be capable of utilizing exogenous OT mRNA for synthesis of releasable OT. Although OT release was increased by the administration of exogenous OT mRNA, endogenous OT gene expression was also observed. To determine what had induced OT gene expression in the current cultures, the undefined components of the culture preparation, e.g., the glial feeder layer and the serum component of the culture medium, were evaluated. Restraining growth of the glial carpet with cytosine-arabinoside did not alter OT gene expression. Use of a defined medium supplemented with B-27 induced optimal OT gene expression. From this, it is possible to conclude that the components included in B-27 are sufficient for OT gene expression.Factors included in earlier lots of sera may have been responsible for suppression of OT gene expression. Cultures maintained in serum-free, B-27-supplemented medium may provide a useful model system for studying OT gene regulation.