Distribution of prolactin-releasing peptide-immunoreactive neurons in the rat hypothalamus.

A newly identified hypothalamic peptide whose specific receptors are present in the anterior pituitary gland is a selective and potent stimulator of prolactin secretion and is therefore termed prolactin-releasing peptide (PRP). We investigated the distribution of PRP-containing neurons in the hypothalamus of female rats by immunocytochemical techniques. Immunocytochemistry using a specific antibody raised to PRP revealed that PRP-immunoreactive perikarya were located in the posteroventral part of the dorsomedial nucleus of the hypothalamus. PRP-immunoreactive nerve terminals were present in high concentrations in a region ventral to the bed nucleus of the stria terminalis, but scarcely observed in the external layer of the median eminence in which well known hypothalamic hormones such as growth hormone-releasing hormone and somatostatin were abundantly detected. This specific distribution in the hypothalamus suggests a novel route of the hypophysiotropic action of PRP.

Antiarrhythmic effects of an aconitine-like compound, TJN-505, on canine arrhythmia models.

We examined the effects of an aconitine-like compound, TJN-505 (1alpha-16beta-dimethoxy-20-ethyl-14alpha-(4-methox ybenzoyloxy)-aconitan-8,13-diol hydrochloride), on canine arrhythmias provoked by digitalis, two-stage coronary ligation, adrenaline, programmed electrical stimulation, or aconitine. TJN-505 (2-2.5 mg/kg i.v.) suppressed digitalis-, two-stage coronary ligation- and adrenaline-induced ventricular arrhythmias. The antiarrhythmic plasma concentrations (IC50) of TJN-505 for these arrhythmia models were 1.26, 0.94 and 1.31 microg/ml, respectively. TJN-505 (2 mg/kg i.v. followed by the infusion of 0.1 mg/kg per min) prolonged PR, QRS, QTc and JTc intervals and the ventricular effective refractory period and reduced the incidence of programmed electrical stimulation-induced arrhythmias in dogs with 7-day-old myocardial infarction (P < 0.05). TJN-505 (2 mg/kg i.v.) also suppressed the aconitine-induced atrial arrhythmias. In conclusion, TJN-505 suppressed various canine ventricular and atrial arrhythmias and seems to act as a blocker of multiple channels.

Estradiol-induced diurnal changes in lactotroph proliferation and their hypothalamic regulation in ovariectomized rats.

Pentobarbital anesthesia during the proestrous afternoon delays proliferation of lactotrophs of the anterior pituitary from estrus to diestrus 1 in cycling female rats. We determined whether estradiol treatment induced diurnal changes in rates of lactotroph proliferation in ovariectomized rats, and if so, examined whether hypothalamic neural activity was involved in the occurrence of the estradiol-induced diurnal changes. Dispersed anterior pituitary cells were obtained from ovariectomized rats bearing estradiol implants that had been treated with 5-bromo-2'-deoxyuridine (BrdU) 3 h before decapitation. BrdU-labeling indices representative of the proliferation rate of lactotrophs were determined by double immunofluorescence staining for BrdU and PRL. After treatment of ovariectomized rats with estradiol on day 0, BrdU-labeling indices of lactotrophs as determined by injecting BrdU at 1000 h increased markedly with time peaking on days 4-7. Levels of BrdU-labeling indices at 1000 h on day 4 were 2.8-fold higher than those at 2200 h on day 3 or 4 after estradiol treatment. However, levels of BrdU-labeling indices at 1000 h on day 14 were 35% lower than those at the same time on day 4 and did not differ from those at 2200 h on day 13 or 14. In addition, a difference in BrdU-labeling indices as observed between 1000 and 2200 h on day 4 in the ovariectomized rats was not detected in estradiol-treated orchidectomized male rats. Serial determinations of BrdU-labeling indices throughout day revealed that the difference in BrdU-labeling indices between 1000 and 2200 h on day 4 in the ovariectomized rats reflected estradiol-induced diurnal changes that were characterized by a peak between 0700-0900 h and a nadir between 1900-2200 h. Pentobarbital injected at 0900 or 2100 h on day 3 decreased slightly high levels of BrdU-labeling indices at 0800 h on day 4. However, pentobarbital injection at 1345 h on day 3, which was effective in blocking estradiolinduced surges of LH and PRL secretion, suppressed markedly the high levels at 0800 h on day 4. In these pentobarbital-blocked rats, the diurnal changes in BrdU-labeling indices whose peak would normally have occurred at 0700-0900 h on day 4 were delayed not by the time corresponding to the duration of pentobarbital anesthesia but exactly by 24 h. These results suggest that 1) hypothalamic and sexually dependent diurnal changes in lactotroph proliferation can be induced by short-term estradiol treatment in ovariectomized rats as well as in cycling rats, and 2) estradiol treatment for 14 days rather prevents the diurnal changes in lactotroph proliferation.

Pentobarbital anesthesia during the proestrous afternoon blocks lactotroph proliferation occurring on estrus in female rats.

Estradiol stimulates the synthesis and secretion of PRL and lactotroph proliferation, and its long-term administration induces PRL-secreting pituitary tumors. We examined changes in the number of proliferating lactotrophs throughout the estrous cycle in female rats and the involvement of the brain in the regulation of the lactotroph proliferation by anesthetizing with pentobarbital. Female rats revealing regular 4-day estrous cycles were injected ip with the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) 3 h before decapitation to label DNA-replicating cells. Dispersed anterior pituitary cells obtained from these rats were stained for PRL and BrdU with a double labeling immunofluorescence technique. The rate of lactotroph proliferation was represented by the BrdU-labeling index (BrdU-labeled lactotrophs as a percentage of total lactotrophs counted). Lactotrophs from rats injected with BrdU at 1000 h showed a high BrdU-labeling index of 2.6% on estrus, whereas they showed almost undetectable levels of the BrdU-labeling index during the other stages of the estrous cycle. The anterior pituitary cells other than lactotrophs were scarcely labeled during any stages. The BrdU-labeling index began to rise by the midnight between proestrus and estrus, peaked between 0800 and 1200 h, and returned to undetectable levels by the midnight between estrus and diestrus I. Pentobarbital (35 mg/kg, i.p.) injected at 1345 h on proestrus, which was effective in blocking ovulation on estrus, eliminated completely the increase in the BrdU-labeling index as determined by BrdU injection at 1000 h on estrus. In contrast, the high BrdU-labeling index on estrus was partly suppressed to a level of 1.4% by pentobarbital injection at 0900 or 2100 h on proestrus. The rats injected with pentobarbital at 1345 h on proestrus did not show any increases in the BrdU-labeling index even after BrdU injection was delayed from 1000 to 1400 h on estrus. However, a high BrdU-labeling index of 3.7% was obtained in these animals when BrdU was injected at 1000 h on diestrus I. We conclude that 1) lactotrophs of cycling female rats proliferate selectively on the day of estrus; and 2) the proliferation of lactotrophs on estrus is not due to a direct action on the anterior pituitary of estradiol secreted from the ovaries but triggered by neural events occurring during the proestrous afternoon, which are closely related with the regulation of preovulatory surges of gonadotropin and PRL secretion.

The activity of catecholamine synthesis in the hypothalamus of female normotensive Wistar Kyoto and spontaneously hypertensive rats.

The activity of catecholamine synthesis in the hypothalamus, as determined by the rate of 3,4-dihydroxyphenylalanine (DOPA) accumulation after the administration of a DOPA decarboxylase inhibitor, was compared among Wistar, spontaneously hypertensive (SH), and genetically matched normotensive Wistar Kyoto (WKY) rats. DOPA accumulation in the median eminence, an index of the activity of tuberoinfundibular dopaminergic neurons, was greater in SH rats than Wistar and WKY rats while DOPA accumulation in the medial preoptic area was smaller in Wistar rats than SH and WKY rats. No strain difference was found in DOPA accumulation in the corpus striatum, which represents the activity of nigrostriatal dopaminergic neurons. These results suggest that there are differences in catecholamine synthesis in the hypothalamus not only between SH and WKY rats but also between WKY and Wistar rats.

Two flavonol glycosides from seeds of Camellia sinensis.

Two novel flavonol triglycosides, camelliaside A and B, have been isolated from seeds of Camellia sinensis. The structures were determined to be kaempferol 3-O-[2-O-beta-D- galactopyranosyl-6-O-alpha-L-rhamnopyranosyl]-beta-D-glucopyranoside and kaempferol 3-O-[2-O-beta- D-xylopyranosyl-6-O-alpha-L-rhamnopyranosyl]-beta-D-glucopyranoside on the basis of spectroscopic, chemical and enzymatic studies. These types of interglycosidic linkages, Gal(1----2)[Rha(1----6)]Glc and Xyl(1----2)[Rha(1----6)]Glc, have not been reported previously in flavone and flavonol glycosides.

Enkephalin inhibits dopamine synthesis in vitro in the median eminence portion of rat hypothalamic slices.

The effect of enkephalin on dopamine synthesis in vitro in tuberoinfundibular dopaminergic (TIDA) neurons was investigated in rat hypothalamic slices. Dopamine synthesis in vitro in TIDA neurons was estimated by 3,4-dihydroxyphenylalanine (DOPA) accumulation in the median eminence after incubation of slices with a DOPA decarboxylase inhibitor. The enkephalin agonist [D-Ala2]Met-enkephalinamide (ENKamide) decreased the rate of basal DOPA accumulation in the median eminence portion of hypothalamic slices from ovariectomized rats at concentrations over 2 microM. The inhibitory action of ENKamide was more pronounced in hypothalamic slices from haloperidol-treated rats in which basal DOPA accumulation in the median eminence was stimulated by increased PRL secretion. In contrast, ENKamide decreased neither the rate of depolarization- induced CA2+-dependent DOPA accumulation nor the rate of (Bu)2cAMP- or forskolin-induced DOPA accumulation in the median eminence of normal or haloperidol-treated rats. The rank order of the potencies of enkephalins and their analogs for inhibition of DOPA accumulation in the median eminence was similar to that of their binding capacities for opioid receptors. ENKamide inhibited basal DOPA accumulation even when hypothalamic slices were incubated in Ca2+-free medium to which tetrodotoxin was added or when the median eminence was incubated alone without the remainder of the hypothalamic slice. These results suggest that enkephalin, by acting directly on axon terminals of TIDA neurons in the median eminence, inhibits basal dopamine synthesis.

Direct inhibitory effect of long term estradiol treatment on dopamine synthesis in tuberoinfundibular dopaminergic neurons: in vitro studies using hypothalamic slices.

The mechanism of the inhibitory effect of long term treatment with estradiol on dopamine synthesis in tuberoinfundibular dopaminergic (TIDA) neurons was studied by using hypothalamic slices from ovariectomized rats. Treatment with 2 mg estradiol valerate (EV) at a 3-week interval increased the weight of the anterior pituitary gland and the concentration of serum PRL. In vivo and in vitro dopamine synthesis in TIDA neurons were estimated in EV-treated animals by 3,4-dihydroxyphenylalanine (DOPA) accumulation in the median eminence after injections of 3-hydroxybenzylhydrazine (NSD 1015), a DOPA decarboxylase inhibitor, and after incubation of hypothalamic slices with NSD 1015, respectively. In vivo DOPA accumulation in the median eminence was less in EV-treated rats than in control rats. The basal rate of in vitro DOPA accumulation in the median eminence of hypothalamic slices from EV-treated rats was lower than that in control rats. Ca2+-dependent DOPA accumulation in the median eminence, determined by incubation in medium containing depolarization agents such as 50 mM K+ and veratridine, was decreased in EV-treated rats. Furthermore, cAMP-dependent DOPA accumulation, determined by incubation with Bu2cAMP or forskolin, was also suppressed in EV-treated rats. The decreased depolarization-induced DOPA accumulation in the median eminence recovered after cessation of EV treatment. Hyperprolactinemia lasting for 6 weeks, achieved by transplantation of anterior pituitaries under the kidney capsule, increased the rate of depolarization-induced DOPA accumulation in the median eminence. On the other hand, EV treatment was effective in inhibiting depolarization-induced DOPA accumulation in hypophysectomized rats regardless of the presence of anterior pituitary transplants. These results suggest that chronically administered estradiol inhibits dopamine synthesis in TIDA neurons via a direct action on the hypothalamus and overcomes the facilitatory action of PRL on dopamine synthesis; and estradiol inhibits all three distinct systems that regulate basal, Ca2+-dependent, and cAMP-dependent dopamine synthesis in TIDA neurons.

Estimation of in vitro activity of tuberoinfundibular dopaminergic neurons by measurement of DOPA synthesis in the median eminence of hypothalamic slices.

A new method for estimation of in vitro neurosecretory activity of tuberoinfundibular dopaminergic (TIDA) neurons was developed by measuring the rate of synthesis of dihydroxyphenylalanine (DOPA) in the median eminence of hypothalamic slices. Sagittal hypothalamic slices of ovariectomized rats were incubated in a medium containing 3-hydroxybenzylhydrazine (NSD 1015), an inhibitor of DOPA decarboxylase. DOPA accumulated in the median eminence following incubation with NSD 1015 was determined by high-performance liquid chromatography with electro-chemical detection. The amount of DOPA accumulated in vitro in the median eminence was maximal in a medium containing 10 mM NSD 1015 and linear up to 120 min at 37 degrees C. Increasing the concentration of tyrosine in medium stimulated the synthesis of DOPA in the median eminence. The synthesis of DOPA was blocked by 1 mM alpha-methyltyrosine, an inhibitor of tyrosine hydroxylase. The rate of in vitro synthesis of DOPA in the median eminence was 33% of that of in vivo synthesis. Incubation in a medium containing 50 mM K+ to depolarize neurons caused a 2.4-fold increase in DOPA synthesis in the median eminence. The high K+-induced increase in DOPA synthesis was blocked by omission of Ca2+ and addition of 1 mM EGTA into the medium, suggesting Ca2+ dependency of depolarization-activated DOPA synthesis. These results indicate that this in vitro assay is a useful means to study the regulatory mechanisms of TIDA neurons.

Dopaminergic neurons in the mediobasal hypothalamus of old rats: evidence for decreased affinity of tyrosine hydroxylase for substrate and cofactor.

The effect of aging on the activity of tyrosine hydroxylase (TH) and on the number of TH-positive perikarya in the hypothalamus was studied in old and young female rats. The activity of TH in the mediobasal hypothalamus (MBH) of old rats was significantly (P less than 0.025) less than that in young rats. In old rats, the Km of TH for tyrosine as well as cofactor, 6-methyl-5,6,7,8-tetrahydropterine (6MPH4), was markedly greater than the Km in young rats. The maximal velocity was only slightly reduced in old animals. Contiguous coronal sections of the brain of an old and a young female rat were immunocytochemically stained for TH, and the TH-positive perikarya in the hypothalamus were counted. In the circumventricular region, 6793 TH-positive perikarya were present in the young brain and 6632 in the old brain. In the arcuate region, 2868 and 2760 TH-positive perikarya were counted in the young and old brain, respectively. It is concluded that the reduced TH activity in the MBH of old rats is not a consequence of a reduction in the number of TH-positive perikarya in the arcuate or circumventricular regions of the hypothalamus but is due to a reduction in the affinity of TH for its substrate and cofactor.

Role of the medial preoptic area in the neural control of the nocturnal prolactin surge in the rat.

In the present study, the role of the medial preoptic area (MPO) in the neural control of the nocturnal prolactin (PRL) surge was investigated in ovariectomized rats. Cervical stimulation (CS) or bilateral MPO lesions caused a marked nocturnal PRL surge at 0400 h on the fourth day after CS or the lesions in ovariectomized rats in which blood samples were obtained by decapitation. However, operation for indwelling a catheter and serial blood collection completely eliminated the MPO lesion induced nocturnal surge while they did not affect the CS-induced surge. On the other hand, MPO lesions could not induce the nocturnal PRL surge in neonatally androgenized female rats. These results suggest that the MPO not only tonically inhibits the initiation of the nocturnal PRL surge but also has a buffer action on the PRL surge-suppressing action of stress. Furthermore, it may be possible that the failure of CS to initiate and maintain the nocturnal PRL surge in neonatally androgenized rats is not due to the inability of CS to disinhibit the inhibitory action of the MPO, but rather due to the extinction of the circadian rhythm itself of the nocturnal PRL surge in these rats.

Circadian rhythm of prolactin surges induced by stimulation of the uterine cervix in the ovariectomized rat: its sexual differentiation and preoptic regulation.

Stimulation of the uterine cervix (CS) induced a nocturnal surge of prolactin at 04.00 h and a diurnal surge at 17.00h in normal ovariectomized rats. However, the CS-induced prolactin propionate during the neonatal period. Chronic bilateral lesions of the suprachiasmatic nucleus (SCN) completely abolished the CS-induced nocturnal and diurnal surges of prolactin release which were observed in sham-lesioned, ovariectomized rats. Furthermore, bilateral lesions of the medial basal part of the suprachiasmatic area (MBSC), lying rostral to the SCN, were also effective in blocking the CS-induced nocturnal and diurnal surges. Lesions which destroyed mainly the optic chiasma and extended partially into the MBSC and SCN did not block the CS-induced prolactin surges. These results suggest that one reason for the failure of ovary-grafted male rats and neonatally androgenized female rats to maintain pseudopregnancy is the extinction of the circadian rhythm of the two daily prolactin surges, and that the MBSC, in addition to the SCN which is known to be a generator of other circadian rhythms, is involved in generation of the rhythm of prolactin surges.

Effects of lesions in the medial basal part of the suprachiasmatic area on prolactin and gonadotropin surges induced by estrogen and progesterone treatment in ovariectomized rats.

Bilateral lesions in the medial basal part of the suprachiasmatic area (MBSC) lying immediately rostral to the suprachiasmatic nuclei caused persistent vaginal cornification. Estradiol benzoate (EB) injections induced a surge in serum prolactin, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels between 15.00 and 18.00 h in ovariectomized rats bearing sham lesions in the MBSC. The EB-induced surge of LH and FSH release was eliminated in ovariectomized animals with lesions in the MBSC. Serum prolactin levels showed consistently low levels during 24 h 3 out of the 7 lesioned animals and considerable fluctuations but no surge in the remaining 4 animals. Progesterone injections into the EB-treated rats with MBSC lesions did not elicit the marked surge in serum LH and FSH which was observed in sham-lesioned animals. In contrast, the EB-treated rats with MBSC lesions showed a significant rise of serum prolactin levels after progesterone injections, similar to that of sham lesioned animals. These results suggest that MBSC plays an essential role in the neural control of the steroid-induced surges of prolactin and gonadotropin release.

Negative feedback effects of chlormadinone acetate and ethynylestradiol on gonadotropin secretion in patients with prostatic cancer and male rats.

Serum LH and FSH levels were determined before and after LH-RH injection (100 micrograms, i.m.) in patients with prostatic cancer who were chronically treated with either chlormadinone acetate (CMA, 100 mg/day) or ethynylestradiol (EE, 1 mg/day). In patients treated with EE, the levels of serum LH and FSH before and after injection of LH-RH were significantly lower than those in controls. On the other hand in patients treated with CMA, the basal levels of serum gonadotropins did not differ from those in controls, and the increase in gonadotropin after LH-RH injection was comparable to that in controls. To examine the effects of these steroids on the hypothalamo-hypophysial axis in the regulation of gonadotropin secretion, CMA or EE was implanted in castrated male rats. CMA, EE or cholesterol (control) was implanted in the hypothalamic median eminence-arcuate nucleus region through a stainless doublecannula. EE implantation resulted in a 75% decrease in serum LH (p < 0.001) and a 38% decrease in serum FSH (p < 0.05) from the control levels on day 5 of implantation. On the other hand, CMA implantation induced a 33% decrease in serum LH (p < 0.05) from the control level on day 3 of implantation, but no significant change in serum FSH levels. The injection of 2 micrograms/kg of LH-RH on day 7 of implantation induced significant lowering of LH and FSH levels. There was no significant difference between serum levels of the hormones 20 min after LH-RH injection for these two groups and those for the control group. These studies suggest that EE has a potent negative feedback effect on both LH and FSH secretion, and that CMA has a mild negative feedback effect on LH secretion.

Data on the sites of stimulatory feedback action of gonadal steroids indispensable for luteinizing hormone release in the rat.

The sites of the stimulatory feedback action of gonadal steroids on LH release were investigated in estrogen-primed ovariectomized rats. Either estradiol benzoate (E2) or estrone (E1) injections 72 h after E2 priming induced a significant increase in serum LH 30 h later, whereas injections of progesterone (P) did so 6 h later. Horizontal sections placed above the medial preoptic area prevented the increase after E2 injections but not after E1 and P injections. Retrochiasmatic sections or bilateral lesions placed in the medial-basal part of the suprachiasmatic area prevented any increases in serum LH induced by steroid injections. Intracerebral implantations of E2 and P in E2-primed ovariectomized rats induced a similar significant increase in serum LH when E2 was implanted into the bed nucleus of stria terminalis (BST), the lateral septum (l-SEPT) and the preoptic suprachiasmatic area (POSC), and when P was implanted in to the medial amygdala, the diagonal band of Broca (DBB), the POSC, the l-SEPT and the anterior hypothalamic area (AHA). These results suggest that the stimulatory feedback effect of E2 is exerted on the limbic structures, especially the BST and the l-SEPT, while the main sites of the stimulatory feedback action of P are located in the DBB, the POSC, and the AHA.