David A. Carter

Oxytocin Responses to Stress in Lactating and Hyperprolactinaemic Rats

The plasma oxytocin (OT) response to acute stress was compared between virgin, lactating, and hyperprolactinaemic female rats. In virgin rats, brief immobilization was associated with a significant elevation of plasma OT to 24.7 +/- 3.7 pmol/l compared with 7.7 +/- 1.1 pmol/l in controls. In contrast, the stress response was absent in lactating (6 days post-partum) animals: control OT 9.4 +/- 2.2, immobilized OT 9.0 +/- 1.1 pmol/l. Hyperprolactinaemia produced by treatment with either dopamine antagonists (domperidone or haloperidol) or ovine prolactin was also associated with an impairment of the OT stress response in intact females, whereas domperidone treatment failed to modify the response in ovariectomized (OVX) rats. Following ovarian steroid replacement with oestradiol and progesterone, the inhibitory effect of domperidone was observed in OVX rats: control OT 11.1 +/- 2.5, immobilized OT 16.0 +/- 3.7 pmol/l. Treatment of OVX rats with oestradiol and progesterone, either separately or combined, did not modify the OT stress response. Plasma levels of vasopressin were not significantly modified in either control or immobilized rats of any experimental groups. The results indicate that hyperprolactinaemia may be a causative factor in the impairment of OT stress responses observed in lactating rats.

Characterization of a postjunctional 5-HT receptor mediating relaxation of guinea-pig isolated ileum

The 5-HT receptor mediating postjunctional relaxation of precontracted guinea-pig ileum has been characterized using several agonists and antagonists. Substance P precontracted tissues were potently relaxed by 5-HT (5-hydroxytryptamine, serotonin), 5-CT (5-carboxamidotryptamine) and several other indoles. The rank order of potency, with pEC50 values in parentheses, was 5-CT (7.6) > 5-methoxytryptamine (5.7) > 5-HT (5.5) > alpha-methyl-5-HT (4.7) > 2-methyl-5-HT (< 4.0) = tryptamine (< 4.0) = N,N-dimethyl-tryptamine (< 4.0) = N,N-dimethyl-5-HT (< 4.0) = dipropyl-5-CT (< 4.0) = sumatriptan (< 4.0). 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)-tetralin) acted as a potent (6.3), but partial, agonist with respect to 5-HT. The responses to 5-CT were antagonized by several compounds with the following rank order of affinity, with pKB values in parentheses: LSD (lysergic acid diethylamide; 8.1) = mesulergine (7.8) > methysergide (7.6) = spiperone (7.6) > clozapine (7.3) >> (-)-pindolol (< 6.0) > ketanserin (< 6.0) = ondansetron (< 6.0) = GR 113808 ([1-(2-methane-sulphonamido-ethyl)-piperidin-4-yl]-methyl-in dole-3- carboxylate maleate; < 6.0). The relaxant responses to 5-HT were also resistant to tetrodotoxin. These data are consistent with a functional 5-HT receptor, mediating relaxation of guinea-pig ileum, which exhibits an operational profile similar to that of the cloned guinea-pig 5-ht7 receptor. This study, therefore, provides evidence for a functional correlate of the 5-ht7 gene product.

Haemodynamic effects of arginine-vasopressin microinjections into the nucleus tractus solitarius: A comparative study of vasopressin, a selective vasopressin receptor agonist and antagonist, and oxytocin

Arginine-vasopressin (AVP) and related peptides were administered by microinjection into the nucleus tractus solitarius of the rat. AVP produced a rise both in mean arterial pressure and in heart rate. This effect was abolished by pretreatment with a specific antagonist of V1 receptors and was not seen either after injections of oxytocin or of the V2 agonist deamino-D-arginine-vasopressin. This study provides evidence for a specific action of vasopressin on the cardiovascular system in the nucleus of the tractus solitarius, which is mediated neither by V2 nor oxytocin receptors.

Regulation of c-fos and c-jun expression in the rat supraoptic nucleus

Summary1.We have investigated induction of the nuclear proto-oncogenes c-fos and c-jun in the rat supraoptic nucleus (SON) during physiological stimulation.2.Dehydration (0-24 hr) was asssociated with modest, but significant increases in both c-fos and c-jun mRNA at 8 hr and 16 hr as determined by Northern analysis of total RNA extracted from microdissected SON. Prior to 8 hr, and beyond 24 hr, no consistent changes in c-fos and c-jun mRNA were found. Levels of c-fos and c-jun mRNA in the hippocampus were not altered over 24 hr of dehydration.3.Acute stimulation with hypertonic saline (1.5M, i.p.) resulted in a marked increase in SON c-fos mRNA at 1 hr (6-fold) and 2 hr (3.5-fold). Small increases in SON c-jun mRNA were observed at these time points. Treatment with a similar volume of 0.9% saline did not elevate SON c-fos and c-jun mRNA levels.4.Analysis of transcriptional activity with a nuclear run-on assay showed that activation of transcription appears to mediate the induction of c-fos and c-jun mRNA following acute hypertonic saline treatment. During dehydration transcriptional activation is apparent for c-jun but is not well defined for c-fos.5.The results are discussed with reference to the hypothesis that products of c-fos and c-jun may mediate adaptive changes in hypothalamic gene expression.

Neuropeptide Y alters monoamine turnover in the rat brain

The effects of intracerebroventricular administration of neuropeptide Y (NPY) on central monoamine turnover were studied in rats treated with alpha-methyl-p-tyrosine (alpha-MPT). NPY decreased noradrenaline turnover in the brainstem, hypothalamus, midbrain and hippocampus. Dopamine turnover was decreased in the brainstem and striatum of NPY-treated rats. Although alpha-MPT did not seem to affect the turnover of serotonin, the concentration of this amine after the administration of NPY was higher in the brainstem, hypothalamus and striatum. Our results suggest that NPY may play a role in the modulation of monoamine turnover in the central nervous system.

Cardio-respiratory actions of substance P, TRH and 5-HT in the nucleus tractus solitarius of rats: Evidence for functional interactions of neuropeptides and amine neurotransmitters

The cardiovascular and respiratory effects of Substance P (SP) and Thyrotrophin-Releasing Hormone (TRH) microinjections into the nucleus tractus solitarius (NTS) of urethane anaesthetized rats have been investigated. Dual injections of the peptides with serotonin (5-HT) were given to investigate possible functional interactions. In addition, SP and TRH were injected into rats in which 5-HT in the NTS area had been depleted by prior treatment with 5,7-Dihydroxytryptamine (5,7-DHT). SP (65pmol) did not elicit significant effects on blood pressure (BP) or heart rate (HR), but produced a marked, acute reduction in respiration rate (RR). TRH (110pmol) elicited a small but significant reduction in mean arterial pressure (MAP), whereas 5-HT (15nmol) caused a rise in MAP. Neither TRH nor 5-HT modified RR when given alone. A dual injection of SP (6.5pmol, ineffective alone) and 5-HT (15nmol) resulted in a rise in MAP which was insignificantly different from the effect of 5-HT alone. However, a prolonged fall in RR, unlike the effect of SP alone was also observed. A dual injection of TRH (11pmol, ineffective alone) and 5-HT (15nmol) resulted in a profound fall of RR but no significant changes in MAP or HR. SP elicited similar effects in 5,7-DHT lesioned animals as in sham operated controls. In contrast, TRH microinjections in lesioned rats were associated with a profound fall in RR, and a blood pressure response significantly different to that observed in the corresponding sham group. The results are discussed in relation to other evidence suggesting functional interactions between neuropeptides and amine neurotransmitters in the mammalian brainstem.

Nuclear mechanisms mediate rhythmic changes in vasopressin mRNA expression in the rat suprachiasmatic nucleus

Vasopressin (VP) gene expression in the rat suprachiasmatic nucleus (SCN) is subject to a cyclical mode of regulation which is indicative of a close association with the circadian clock intrinsic to this area of the hypothalamus. Previous studies show that both the amount and size (due to differential polyadenylation) of VP mRNA are reduced during the dark phase of the daily cycle. We have now identified the cellular site wherein these changes are mediated. By transcriptional run-on analysis of nuclei isolated at different time points from the SCN we have shown that an attenuation of transcriptional activity can account for the dark-phase reduction in VP mRNA levels; by comparison with other genes expressed in this tissue, a significant, VP gene-specific reduction was observed which resulted in dark-phase transcriptional activity at 30% of light-phase activity (P less than 0.005). A similar diurnal variation was not found in the supraoptic nucleus. In addition, by Northern analysis of sub-cellular RNA pools, we have demonstrated that the smaller, dark-phase-specific VP RNA species is located, in abundance, within the nuclear fraction. These results provide clear evidence that the cyclical changes in SCN VP mRNA expression are primarily regulated within the nucleus, indicating that any potential regulation in the cytoplasm is of secondary importance. Further analysis of the molecular components which mediate the cyclical changes in transcriptional activity of the VP gene may identify fundamental aspects of neuronal timing mechanisms.

Posttranscriptional regulation of rat growth hormone gene expression: increased message stability and nuclear polyadenylation accompany thyroid hormone depletion.

In thyroid hormone-depleted rats, the rate of transcription of the growth hormone (GH) gene in the anterior pituitary gland is lower than the rate in euthyroid controls, and there is a corresponding reduction in the abundance of the GH mRNA. Concomitantly, the poly(A) tail of the GH mRNA increases in length. Examination of nuclear RNA from anterior pituitary glands of control and thyroid hormone-depleted rats revealed no difference in the length of pre-mRNAs containing the first and last introns of the GH gene. However, mature nuclear GH RNA is differentially polyadenylated in euthyroid and hypothyroid animals. We suggest that the extent of polyadenylation of the GH transcript is regulated in the cell nucleus concomitant with or subsequent to the splicing of the pre-mRNA. Experiments with anterior pituitary gland explant cultures demonstrated that the GH mRNA from thyroid hormone-depleted rats is more stable than its euthyroid counterpart and that the poly(A) tail may contribute to the differential stability of free GH ribonucleoproteins.

Diurnal rhythm of vasopressin mRNA species in the rat suprachiasmatic nucleus: independence of neuroendocrine modulation and maintenance in explant culture

Vasopressin mRNA in the suprachiasmatic nucleus (SCN) of the rat brain exhibits diurnal variation in poly(A) tail length; a single species of mRNA identical in size to that in other hypothalamic nuclei is expressed in the light phase of the daily cycle whereas a second, smaller species is expressed in the dark phase. We have investigated the neuroendocrine factors which may regulate this rhythm by comparing vasopressin mRNA size with Northern analysis of RNA extracted from SCN tissue taken at 09.00 h (light phase) and 21.00 h (dark phase). The consistent rhythmic variation observed in normal male rats was not modified in either adrenalectomized or castrated animals or in ovariectomized female rats. The rhythm was also not disrupted following treatment with the serotonin-depleting agent parachlorophenylalanine, or following treatment with either melatonin or the benzodiazepine, triazolam. We next investigated whether the daily pattern of expression was maintained in isolated SCN. Microdissected blocks containing the paired SCN were explanted into culture at various times of the day for a period of 4 h. Vasopressin mRNA extracted from light phase cultures (11.00-15.00 h) exhibited no size change from control SCN mRNA taken at either 11.00 h or 15.00 h. In contrast, mRNA from cultures maintained over the period 16.00-20.00 h (lights off: 18.00 h) exhibited a marked size difference from 16.00 h controls, being similar to the smaller species observed in 20.00 h controls. Similarly, vasopressin mRNA from cultures maintained over the period 05.00-09.00 h (lights on 06.00 h) was similar in size to 09.00 h controls and contrasted to the pattern of expression observed in 05.00 h controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurotransmitter-stimulated immediate-early gene responses are organized through differential post-synaptic receptor mechanisms

The products of the cellular immediate-early genes (IEGs) are thought to act as messengers in the coupling of trans-synaptic stimuli with altered neuronal gene expression. However, the manner in which neurotransmission specifies particular responses through the IEGs is undefined. In this report, mRNA and transcription analysis of a precisely-timed, physiological IEG response illustrates how an IEG signal may be organized through differential neurotransmitter receptor activation. The nocturnal pattern of IEG expression in the rat pineal gland has been shown to be differentially regulated through post-synaptic adrenergic receptors. Induction of the c-fos gene is primarily mediated through alpha 1-receptors, whereas the coordinately regulated jun-B gene exhibits dual regulation through alpha 1- and beta-receptors. A simultaneous repression of c-jun expression is partly mediated through a beta-receptor mechanism. In vitro analysis of IEGs in cultured pineal glands has confirmed the receptor-specific link between adrenergic neurotransmission and IEG induction. The pineal is a unique neuroendocrine model in which the characteristics and function of the IEG third messenger system may be defined.