Expression of pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R) in the ewe hypothalamus: distribution and colocalization with tyrosine hydroxylase-immunoreactive neurones.

We have examined the distribution of the pituitary adenylate cyclase activating polypeptide type I receptor (PAC1R) in the ewe hypothalamus by reverse transcription-polymerase chain reaction, in situ hybridization and immunohistochemistry. PAC1R mRNA was highly expressed in the mediobasal hypothalamus of the ewe, particularly in the arcuate nucleus and ventromedial hypothalamus, compared to other hypothalamic regions. Similar results were obtained from immunohistochemistry using a specific PAC1R antibody. Intense immunolabelling was observed in the arcuate nucleus, external zone of the median eminence and ventromedial hypothalamus. Only relatively weak immunolabelling was observed in other hypothalamic regions, including the paraventricular nucleus and supraoptic nucleus. In the ewe, PACAP acts via the arcuate nucleus to suppress prolactin secretion. Therefore we examined whether PAC1R was present on the tuberoinfundibular dopamine (TIDA) neurones in this nucleus. Dual immunofluorescence labelling for PAC1R and tyrosine hydroxylase revealed that 21.2 +/- 1.7% of dopaminergic neurones in the arcuate nucleus (A12 cell group) also stained for PAC1R. By contrast, other hypothalamic dopaminergic cell groups (A11, A13, A14 and A15) exhibited little (< 3%) or no colocalization. Overall, our results indicate that, in the ewe hypothalamus, PAC1R is most concentrated in the arcuate nucleus, where it is localized on a substantial proportion of dopaminergic neurones. These observations, together with previous in vivo studies, suggest that PACAP could act directly on TIDA neurones via PAC1R to increase dopamine release and consequently inhibit prolactin secretion in the sheep.

Prolactin-releasing peptide in the ewe: cDNA cloning, mRNA distribution and effects on prolactin secretion in vitro and in vivo.

RT-PCR followed by 5'- and 3'- rapid amplification of cDNA ends was used to clone and sequence ovine prolactin-releasing peptide (PrRP). The cDNA was characterised by short 5'- and 3'-untranslated regions and a GC-rich (71%) coding region. The nucleotide and deduced amino acid sequences for the coding region showed 95.6 and 94.9% identity with bovine PrRP but the amino acid sequence of PrRP31 was conserved between these species. Northern blot analysis and RT-PCR showed that, as in the rat, the peptide was more abundantly expressed in the brainstem than the hypothalamus. However, in the ovine hypothalamus, PrRP mRNA expression was more widespread than in the rat, with expression detected in both rostral and caudal parts of the mediobasal hypothalamus. The effects of synthetic ovine PrRP on prolactin secretion both in vitro and in vivo were also examined. In primary cultures of sheep pituitary cells, PrRP significantly (P<0.01) increased prolactin concentrations in the culture medium but the response was not observed in every experiment and was only seen when pituitary glands were dispersed with collagenase rather than trypsin. PrRP was much less potent than TRH which caused a significant (P<0.01) two- to threefold increase in prolactin concentrations in every experiment. Intravenous (10 and 50 nmol) or intracerebroventricular (10 and 50 nmol) injection of PrRP had no significant effect on either plasma prolactin concentration or pulsatile LH secretion whereas intravenous injection of TRH (10 nmol) produced a highly significant (P<0.01) and more than sevenfold stimulation of plasma prolactin concentrations. In conclusion, these results suggest that PrRP is unlikely to be an important prolactin-releasing factor in this species.

Hypothalamic dopamine D1 receptors are involved in the stimulation of prolactin secretion by high environmental temperature in the female sheep.

Recent evidence suggests that dopamine, acting via its D1 receptors, may function as a neurotransmitter in intrahypothalamic pathways involved in the stimulation of prolactin secretion. Functional dopamine D1 receptors are present in the ventromedial hypothalamic nucleus (VMH) and we hypothesized that they might be part of a prolactin-stimulatory pathway activated by stress. We tested this hypothesis in a series of experiments on sheep involving two different forms of stressors, audiovisual (barking dog) and high environmental temperature. We attempted to block the stimulation of prolactin secretion by infusion into the VMH of an antagonist specific for the D1 receptor. Ovariectomised, oestradiol-implanted merino ewes were surgically implanted with bilateral guide tubes directed at the VMH. After a 180 min pretreatment period, the ewes either were or were not exposed to a stressor (30 min of barking dog or 120 min at 35 degrees C, 65% relative humidity). D1 receptor antagonist, SCH23390 or vehicle (0.9% saline) was infused into the VMH (1.7 microliters/h, 120 nmol/h) for 60 min prior to and during the stressor period. Blood was sampled every 15 min via jugular cannulae and the plasma was assayed for prolactin, cortisol and growth hormone (GH). Both stressors significantly increased prolactin concentrations over control levels. SCH23390 infusion significantly attenuated the prolactin response to high environmental temperature, but had no effect on the prolactin response to audiovisual stress. Cortisol concentrations were significantly increased by audiovisual stress only and were not affected by SCH23390. GH concentrations were not changed by either stressor or infusion. Drug infusion alone did not affect the concentration of the hormones. The data suggest that the VMH D1 receptors are involved in a prolactin stimulatory pathway in response to high environmental temperature. The inability of the D1 antagonist to affect the response to the barking dog indicates that this pathway is stress-specific, implying that there is more than one mechanism or pathway involved in the prolactin response to different stressors.

Localization and characterization of dopamine D1 receptors in sheep hypothalamus and striatum.

Dopamine receptors are pharmacologically grouped as D1 and D2 receptors. Previous research in the ewe has shown that central D1 receptors may have a role in facilitating prolactin release. The aims of this study were therefore to localize and characterize D1 binding sites in the hypothalamus of sheep. For comparison, a known D1 receptor-rich tissue (striatum) was also studied. The bioactivities of several D1 analogues were also assessed for their efficacy in sheep tissue. In vitro autoradiography with [125l]-SCH23982 was used to localize D1 binding sites. The ventromedial hypothalamic nucleus (VMH) displayed moderate levels of specific binding, localized to the medial portion of the nucleus. Low levels of specific binding were seen in the preoptic area, supraoptic nucleus and anterior hypothalamic area. The suprachiasmatic nucleus, median eminence and arcuate nucleus did not show specific binding. As expected the striatum displayed high levels of specific binding. The VMH, preoptic area, median eminence, striatum and anterior pituitary were examined with radioligand binding studies to quantify and characterize D1 binding sites. Scatchard analysis gave KD 1.04 nM and Bmax 127.4 fmol/mg protein for VMH and KD 1.99 nM and Bmax 454.6 fmol/mg protein for striatum. While specific binding occurred in the preoptic area and median eminence this binding did not show saturation characteristics. Specific binding was not observed in the anterior pituitary. Affinities determined by competitive binding studies showed that the binding sites in both VMH and striatum have the characteristics of a D1 receptor, that is, high affinity for the D1 agonists and antagonists, low affinity for dopamine and the serotonergic antagonist ketanserin and extremely low affinity for the D2 agonists and noradrenaline. Adenylate cyclase studies showed that in the striatum dopamine and the D1 agonists, fenoldopam and SKF38393, were able to cause significant dose-dependent increases in adenylate cyclase activity. In contrast the D1 agonist, SKF82958, was inactive in this system. The D1 antagonists SCH23390 and SCH39166, but not SKF83566, abolished the adenylate cyclase response to 50 microM dopamine. In the VMH the D1 agonist SKF38393, but not dopamine, stimulated adenylate cyclase activity. In conclusion, these results demonstrate that D1 binding sites exist within the hypothalamus in the VMH and that these binding sites have the characteristics of D1 receptors. These receptors are a potential site of action for dopamine in facilitating prolactin release. In addition, the results show that at least for some dopamine analogues, receptor binding affinity does not always correlate with biological activity.

Effect of hypothalamic infusion of a dopamine D1 receptor antagonist on prolactin secretion in the ewe.

In this study we investigated whether dopamine D1 receptors in the hypothalamus are involved in the control of prolactin secretion in ovariectomised, oestradiol implanted ewes. The D1 antagonist SCH23390 or vehicle was infused into either the preoptic area (POA) or the ventromedial hypothalamus (VMH). During infusion into the VMH, prolactin concentrations declined significantly and did not return to control values until more than 60 min after the infusions had stopped. In contrast, infusion into the POA had no significant effect. These results are in accord with the hypothesis that dopaminergic pathways within the hypothalamus stimulate prolactin secretion via dopamine D1 receptors in the VMH.

Effects of chronic long-acting bromocriptine treatment on liveweight, voluntary food intake, coat growth and breeding season in non-pregnant red deer hinds.

Seventeen red deer hinds were housed in individual pens and from 28 February until 11 November were injected each week with vehicle (group A; n = 6) or 5 (group B; n = 6) or 12.5 mg (group C; n = 5) of a long-acting formulation of bromocriptine. Liveweight and voluntary food intake (VFI) were recorded for each hind, and blood was collected for determination of progesterone, prolactin, tri-iodothyronine (T3) and cortisol concentrations by radioimmunoassay. Treatment with the high dose of bromocriptine was associated with a significant (P less than 0.05) reduction in VFI, with the effect being greatest between March and July. There was no treatment effect on liveweight, but there was a significant (P less than 0.01) interaction between time and treatment due to the faster rate of weight gain in control animals at the beginning of the experiment. Changes in liveweight could be explained by changes in VFI rather than by changes in the efficiency of utilization of intake. Termination of the breeding season was significantly (P less than 0.01) delayed by 54 days in group C hinds. Growth of the summer coat and subsequent winter coats was delayed by 1 and 3 months respectively in group C hinds, and in groups B and C the duration that animals were in summer coat was increased by about 1 month. The seasonal increase in prolactin concentrations was seen in all groups, but levels were significantly (P less than 0.05) lower in group C hinds. Concentrations of T3 and cortisol were not affected by bromocriptine.