The effect of tubero-infundibular dopaminergic neurons on prolactin and alpha-melanocyte stimulating hormone release.

The effect of tubero-infundibular dopaminergic neurons (TIDA) on the release of prolactin (PRL) and alpha-melanocyte stimulating hormone (alpha-MSH) was studied in median eminence-lesioned (MEL) male rats (N = 6-28). Plasma PRL and alpha-MSH levels were significantly elevated 2 (86.1 +/- 19.8 and 505.1 +/- 19.1 ng/ml), 4 (278.7 +/- 15.5 and 487.4 +/- 125.1 ng/ml), 7 (116.2 +/- 16.2 and 495.8 +/- 62.6 ng/ml) and 14 (247.3 +/- 26.1 and 448.4 +/- 63.8 ng/ml) days after MEL when compared to sham-operated control animals (55.5 +/- 13.4 and 56.2 +/- 6.1 ng/ml, respectively). MEL altered plasma PRL and alpha-MSH levels in a differential manner, with a 1.5- to 5.0-fold increase in PRL and an 8.0- to 9.0-fold increase in alpha-MSH. The increase of alpha-MSH levels occurred abruptly and remained constant from days 2 to 14. These observations indicate that TIDA plays an important role in the pituitary release of PRL and alpha-MSH and provide evidence that the release of the two hormones occurs in a differential manner.

The effect of tubero-infundibular dopaminergic neurons on prolactin and alpha-melanocyte stimulating hormone release

The effect of tubero-infundibular dopaminergic neurons (TIDA) on the release of prolactin (PRL) and alpha-melanocyte stimulating hormone (alpha-MSH) was studied in median eminence-lesioned (MEL) male rats (N = 6-28). Plasma PRL and alpha-MSH levels were significantly elevated 2(86.1 ñ 19.8 and 505.1 ñ 19.1 ng/ml), 4(278.7 ñ 15.5 and 487.4 ñ 125.1 ng/ml), 7 (116.2 ñ 16.2 and 495.8 ñ 62.6 ng/ml) and 14 (247.3 ñ 26.1 and 448.4 ñ 63.8 ng/ml) days after MEL when compared to sham-operated control animals (55.5 ñ 13.4 and 56.2 ñ 6.1 ng/ml, repectively). MEL altered plasma PRL and alpha-MSH levels in a diffential manner, with 1.5-to5.0-fold increase in PRL and an 8.0- to 9.0-fold increase in alpha-MSH. The increase of alpha-MSH levels occured abruptly and remained constant from days 2 to 14. These observations indicate that TIDA plays an important role in the pituitary release of PRL and alpha-MSH and provide evidence that the release of the two hormones occurs in a differential manner

Involvement of beta-adrenergic receptors in the differential release of gonadotropins in acutely orchidectomized rats.

To determine the differential release of gonadotropins in acutely orchidectomized rats, a single injection of the beta-adrenergic blocker Bornaprolol (FM 24) was administered to the animals and plasma FSH and LH levels were determined. FM 24 produced low plasma FSH levels only when injected 16 h before starting the blood collection and had no effect on FSH levels at 0, 30 and 46 h after its administration. However, it produced low LH plasma levels at 0, 16 and 30 h after administration. These findings confirm that pituitary beta-adrenergic receptors are involved in plasma LH release and suggest that they could also be involved in the differential release of FSH/LH.

Involvement of beta-adrenergic receptors in the differential release of gonadotropins in acutely orchidectomized rats

To determine the diferential release fo gonadotropins in acutely orchidectomized rats, a single injection of the beta-adrenergic blocker Bornaprolol (FM 24) was administered to the animals and plasma FSH levels weredetermined. FM 24 produced low plasma FSH levels only when injected 16 h before starting the blood collection and had no effect on FSH levels at 0,30 and 46 h after its administration. However, it produced low LH plasma levels at 0,16 and 30 h after administration. These findings confirm that pituitary beta-adrenergic receptors are involved in plasma LH release and suggest that they could also be involved in the differential release of FSH/LH

Role of opioid peptides in pulsatile release of gonadotropins and prolactin in the rat.

To determine the role of endogenous opioid peptides in the pulsatile release of gonadotropins and prolactin in the ovariectomized rat, the opiate receptor blocker, naloxone, was administered intravenously, and its effect on plasma FSH, LH and prolactin was determined by multiple sampling prior to and after injection. Naloxone produced a dose-related increase in plasma LH and to a lesser extent FSH and decreased prolactin levels in the experiment in which they were examined. Higher doses of naloxone produced a significant increase in plasma LH pulse amplitude and lengthened the interpulse interval with a consequent decrease in pulse frequency. Minimum values between pulses were also increased. There was no clear effect on FSH pulsations but pulses of prolactin were blocked. Intraventricular (third ventricle) injection of a specific anti beta endorphin antiserum (3 microliter) produced an initial decline followed by an elevation of LH but had no effect on plasma FSH. The normal rabbit serum control injections were without effect. It is hypothesized that initiation of LH pulses in the castrated rat may be related to a periodic removal of tonic beta endorphinergic tone.

Beta-adrenergic blockers decrease levels of luteinizing hormone in plasma of orchidectomized rats.

The beta-adrenergic antagonists, propranolol and bornaprolol (FM-24), at greater than 2 mg/kg (as [-] form) significantly depressed plasma levels of luteinizing hormone (LH) in orchidectomized rats. This occurred in the absence of consistently significant changes in interpulse intervals or amplitudes of pulsatile LH release. Nadirs of plasma LH decreased significantly even at low blocker doses, with a clear dose dependence for both drugs. The highly significant decrease of plasma LH induced by blocker dosages causing greater than 93% inhibition of beta-adrenergic binding in the anterior pituitary gland was shown to occur without significant changes in binding of specific ligands at pituitary dopamine receptors and hypothalamic alpha 1-adrenergic receptors. The above evidence indicates that beta-blockers may lower LH release in vivo at the level of pituitary beta-adrenergic receptors.

Effects of unilateral decortication on beta-adrenergic receptors in the remaining cortex and in the hypothalamus of female rats.

Many experiments have been performed to evaluate the physiological role of catecholaminergic mechanisms in gonadotropin release. The purpose of the present study was to determine the concentration of beta-adrenoreceptors in the remaining (right) cerebral cortex and in right and left hypothalamic halves of hemi-decorticated female rats which exhibited elevated plasma gonadotropin levels as observed previously. The density of beta-receptors was measured using a high-affinity beta-adrenergic ligand, iodocyanopindolol (ICYP). Scatchard estimates were obtained for maximum binding (Bmax fmol/mg of tissues) from pooled cerebral cortical and hypothalamic tissue of animals under several experimental conditions after hemi-decortication and sham operation. There was an increase in beta-adrenoreceptor density in the remaining (right) cerebral cortex at all times examined in hemi-decorticate in comparison with the sham-operated animals (7 days, +10.9%; 21 days, +8.4%; 90 days, +22%; and 90 days plus ovariectomy, +34.8%). The number of beta-adrenoreceptors in the right hypothalamic half in hemi-decorticates decreased at 21 days (-42.20%) and then increased at 90 days (+76.63%) and 90 days plus ovariectomy (+51.75%) when compared with the left hypothalamic half. At the same time there were no significant changes in the sham-operated animals when comparing the receptor density in the right and left hypothalamic halves, respectively. Thus, our results suggest a direct or indirect adrenergic pathway by which the left cortex can influence the right cortex and a crossed pathway to the contralateral hypothalamus changing adrenergic activity which can alter the beta-adrenergic receptor binding capacity in the hypothalamus.

Beta-adrenergic agonists increase amplitude of LH release in orchidectomized rats.

The role of intravenously (iv) injected adrenergic agonists in the pulsatile secretion of luteinizing hormone (LH) was examined in unanesthesized, freely behaving, castrated male rats. The alpha 2-adrenergic receptor agonist, clonidine (25 micrograms/kg), and the alpha 1-adrenergic agonist, (-)-phenylephrine (12.5 micrograms/kg), did not significantly alter pulsatile release of LH. The physiological beta 2-adrenergic receptor agonist, (-)-epinephrine (2.5 micrograms/kg), significantly increased the mean plasma concentrations of plasma LH and the amplitude of the LH pulses over a period of 70 min. The specific beta 2-receptor agonist, salmefamol, significantly increased the mean plasma concentrations of LH and especially the average amplitude of LH pulses over 70-80 min in a dose-related fashion following the injection of doses from 25 to 125 micrograms/kg. The frequency of LH pulses was not significantly increased by either agonist at any of the doses employed. Salmefamol-induced increases in plasma LH could be prevented by the beta-adrenergic blocker, bornaprolol (FM-24), in a dose-related manner. When injected together with synthetic LH-releasing hormone (400 ng/kg), salmefamol (125 micrograms/kg) significantly increased the mean plasma concentrations of LH over 70 min compared with values in controls receiving LH-releasing hormone only. The data support the concept that beta-agonists act on their receptors in the pituitary to facilitate LH-releasing hormone-induced discharge of LH.

The effect of unilateral decortication on the patterns of pulsatile follicle stimulating hormone (FSH) and luteinizing hormone (LH) release and on mean plasma prolactin (Prl) levels in the ovariectomized rat.

Previous experiments have shown that unilateral decortication is followed by an elevation in FSH and LH secretion, but there has been no study of the rhythms of release of gonadotropins in these animals. Consequently, this study was undertaken to determine if there are alterations in the pulsations of gonadotropins following hemi-decortication and also to measure plasma Prl in these animals to determine if it might also be under the influence of extrahypothalamic pathways. Our results show an increased mean plasma LH in unilateral decorticate animals that occurred because of an increase in pulse frequency without a significant increase in pulse amplitude. Mean plasma FSH was also elevated significantly in unilaterally decorticate animals, but there was no significant change in pulse frequency or amplitude. The length of the interpulse interval for LH release secretion decreased in unilateral decorticate animals, whereas the length of the cycle of FSH secretion increased in this circumstance. The mean levels of Prl were reduced in unilateral decorticate animals. These changes indicate that unilateral removal of cortico-rhinencephalic structures has an excitatory influence on gonadotropin secretion mediated by an increased frequency of LH and decreased frequency of FSH pulsations in the castrate, while at the same time there is a lowering of plasma Prl, probably due to removal of excitatory cortico-rhinencephalic pathways.

Regulation of anterior pituitary and brain beta-adrenergic receptors by ovarian steroids.

Ovariectomy of adult female rats (200-230g) resulted in an increase in beta-adrenergic receptors in the cerebral cortex, hypothalamus and anterior pituitary. The anterior pituitary had the largest overall increase as well as the most rapid increase in beta-adrenergic receptor density of the tissues examined. The increase in hypothalamic or cerebral cortical beta-adrenergic receptors became apparent only long after ovariectomy (7-14 days). Fourteen days after ovariectomy, the density of beta-adrenergic receptors was 79%, 40%, and 24% in excess of control values in crude membranes prepared from anterior pituitary, hypothalamus and cerebral cortex, respectively. Over the same interval, the plasma concentration of luteinizing hormone (LH) increased 28-fold, while the concentration of follicle-stimulating hormone (FSH) rose 5-fold compared to control levels. Estradiol replacement (20 micrograms/kg/day) in these animals for four days before sacrifice concomitantly reduced plasma levels of the gonadotropins as well as the density of beta-adrenergic receptors in both the anterior pituitary and the hypothalamus. Long-term steroid replacement during the fifth and sixth week after ovariectomy, with implants of estradiol and progesterone which released the steroids in approximately physiological concentrations, significantly reduced beta-adrenergic density in anterior pituitary, but not in the hypothalamic membranes. This treatment significantly reduced plasma LH, but not FSH. Beta-adrenergic receptor density was also found to fluctuate significantly during the 4-day estrous cycle. The highest values were found on proestrus, and the lowest on diestrus 1. These studies indicate that changes in plasma concentrations of gonadal steroids (e.g. during the estrous cycle) influence the density of beta-adrenergic receptors in tissues involved in the control and release of anterior pituitary gonadotropins.

Testosterone control of brain and anterior pituitary beta-adrenergic receptors.

Orchidectomy of adult albino rats resulted in a quick, (approximately 70%) increase in the density of beta-adrenergic receptors in the anterior pituitary gland within the first day. There was a concurrent rapid increase in plasma levels of pituitary gonadotropins. The beta-receptor density continued to increase slowly for at least 16 days after castration, but it could be lowered significantly to the levels of sham-operated animals by treatment with testosterone (3 mg/kg/day) beginning on the fourth day after castration and continuing for 4 days. This treatment also completely reversed the elevation in plasma levels of luteinizing hormone (LH), and significantly reduced the circulating follicle-stimulating hormone (FSH) levels. Prolactin levels were not significantly altered by the treatments used in these studies. Most of the beta-adrenergic receptors induced by orchidectomy in the anterior pituitary were shown, using a beta 1-selective antagonist, practolol, or a beta 2-selective antagonist, IPS-339, to be of the beta 2-subtype. The density of the beta-adrenergic receptors in the cerebral cortex also increased significantly (10-24%) after castration, and returned to the levels of sham-operated animals following treatment with testosterone. No significant change in the density of the beta-adrenergic receptors in the hypothalamus resulted from either castration or testosterone replacement.

Characterization of beta-adrenergic receptor subtypes in androgen-induced mouse kidney hypertrophy using a new high-affinity ligand, [125I]iodocyanopindolol.

In fully developed androgen-induced hypertrophy of female mouse kidney, beta-adrenergic receptors per unit membrane protein were increased approx. 2.5-fold, as measured by the binding of [125I]iodocyanopindolol, with no change in apparent dissociation constants (Kd range 20-25 pM). Membrane protein relative to total kidney protein, Na+/K+-dependent ATPase (EC 3.6.1.3) and 5'-nucleotidase (EC 3.1.3.5) activities and cholesterol content per unit membrane protein did not differ significantly in preparations from control and treated animals. The binding of iodocyanopindolol to kidney membranes was characterized with respect to association and dissociation kinetics, and also in regard to the less-specific contributions of other major catecholamine or indolamine receptors, using mixtures of the corresponding specific competitors. beta 1-selective drugs, practolol and metoprolol, and beta 2-selective agents, IPS-339 and zinterol, were competed with iodocyanopindolol to assess the receptor type specificity, and the ensuing binding profiles were dissected by a nonlinear regression analysis as described by Munson, P.J. and Rodbard, D. (Anal. Biochem. (1982) 107, 220-239). Most of the androgen-induced beta-adrenergic receptors had the binding properties corresponding to beta 2-subtype. No consistent increase in the density of beta 1-adrenergic receptors could be shown.

Characterization of beta-adrenergic receptors in rat brain and pituitary using a new high-affinity ligand, [125I]iodocyanopindolol.

The binding of [125I]iodocyanopindolol (ICYP) to membrane preparations from rat cerebral cortex, hypothalamus and anterior pituitary gland was characterized in regard to specificity, density, and the proportion of beta-adrenergic receptor subtypes. By employing a mixture of ligands specific for alpha-adrenergic, serotoninergic and dopaminergic receptors, it was possible to eliminate most of the less-specific contributions to ICYP binding profiles, which resulted in narrowing the range of measured dissociation constants to 35-50 pM for all neural tissues studied. These values corresponded well with constant for the 'slow' component discernible in ICYP association with cerebral cortical membranes at 37 degrees C. The maximum binding values were 63, 29 and 5.6 fM/mg membrane protein in cortical, hypothalamic and anterior pituitary membrane fractions, respectively. Evaluation of the beta-adrenergic receptor subtypes using 4 selective competitors indicated an average 19% content of the beta 2-subtype in cortical membranes, while in hypothalamic membranes 47% of the receptors could be assigned to that subtype. In the anterior pituitary as well as in the cerebellum, the receptors were predominantly of beta 2-subtype. These findings are discussed in terms of possible physiological functions of beta-receptors in these tissues, including the regulation of the release of pituitary hormones.

The development of beta-adrenergic receptors in the visual cortex of the rat.

The development of beta-adrenergic receptors in the rat visual cortex was examined and the density of beta-receptors and associated subtypes (beta 1 and beta 2) was compared between visual and non-visual or whole cortical tissues using radioreceptor assays employing [125I]iodohydroxybenzylpindolol and [125I]iodocyanopindolol as ligands. Saturation assays revealed not only similar affinities of beta-receptors for [125I]iodohydroxybenzylpindolol in visual cortical samples at 10, 24 and 160 days after birth but also practically identical saturation curves for visual and non-visual cortical samples at 160 days of age. Displacement of [125I]iodohydroxybenzylpindolol with propranolol in visual cortical membranes at various postnatal ages showed a gradual increase in receptor density from day 4 to day 24 with no change thereafter. No significant differences were observed in the overall density of beta-receptors or in the distribution and density of beta 1 and beta 2-receptors between visual and non-visual or whole cortical samples; however, there was a definite decline in the density of beta-receptors in these samples between 40 and 160 days of age. The results indicate that the developmental pattern of beta-receptor density and the distribution of beta 1 and beta 2-receptors are similar between visual and whole cortical tissues. In addition, the results emphasize the importance of maintaining the dissociation constant at a fixed value when comparing receptor densities between experiments, and also show the utility of employing the high-affinity ligand, [125I]iodocyanopindolol, with a combination of serotoninergic, dopaminergic and alpha-adrenergic antagonists to examine beta-adrenergic receptors in a specific region of the brain. Study of beta-receptors in the visual cortex may be beneficial in elucidating the role of norepinephrine in this region.

Increased beta-adrenergic receptor complement in androgen-induced mouse kidney hypertrophy.

Treatment of female mice with testosterone propionate led to a pronounced, but gradual increase in kidney beta-adrenergic receptor complement. The specific binding of [125I]iodohydroxybenzylpindolol rose 2-3-fold above the control levels after 8-12 days of the treatment. No significant changes were detected prior to the fourth day of androgen administration. No gross changes in either the binding strength or cooperativity of the binding were apparent in membrane preparations from treated animals. Averages of the high-affinity binding constant estimates were 1.3 +/- 0.3 nmol in controls, vs. 1.6 +/- 0.5 nmol in treated animals (15 groups each) in competition with pindolol, with the Hill slope factors of 0.98 +/- 0.08 for controls, and 0.91 +/- 0.07 for the treated animal membrane preparations. Scatchard estimates of the binding constants in self-competed [125I]iodohydroxybenzylpindolol binding were about 160 pmol in both control and treated animals. Competition experiments using isoproterenol also indicated similar dissociation constants (151 +/- 16 nmol) for control and treated groups. Na+/K+-activated ATPase (EC 3.6.1.3) was also found to be increased at the 12th day of the androgen treatment (to 74% above control levels).

Binding of Lens culinaris lectin to sea urchin embryo chromatin.

Lentin binds specifically to sea urchin embryo chromatin. This binding is saturable and inhibited by alpha-methyl-mannose. Scatchard plot analysis of the binding reaction suggests a single binding site.