Radiolabeled gastrin/CCK analogs in tumor diagnosis: towards higher stability and improved tumor targeting.

Cholecystokinin subtype 2 receptors (CCK2R) are overexpressed in several human cancers, including medullary thyroid carcinoma. Gastrin and cholecystokinin (CCK) peptides that bind with high affinity and specificity to CCK2R can be used as carriers of radioactivity to CCK2R-expressing tumor sites. Several gastrin and CCK related peptides have been proposed for diagnostic imaging and radionuclide therapy of primary and metastatic CCK2R-positive human tumors. Their clinical application has been restricted to a great extent by their fast in vivo degradation that eventually compromises tumor uptake. This problem has been addressed by structural modifications of gastrin and CCK motifs, which, however, often lead to suboptimal pharmacokinetic profiles. A major enzyme implicated in the catabolism of gastrin and CCK based peptides is neutral endopeptidase (NEP), which is widely distributed in the body. Coinjection of the NEP inhibitor phosphoramidon (PA) with radiolabeled gastrin and other peptide analogs has been recently proposed as a new promising strategy to increase bioavailability and tumor-localization of radiopeptides in tumor sites. Specifically, co-administration of PA with the truncated gastrin analog [(111)In-DOTA]MG11 ([((111)In-DOTA)DGlu(10)]gastrin(10-17)) impressively enhanced the levels of intact radiopeptide in mouse circulation and has led to an 8-fold increase of CCK2R-positive tumor uptake in SCID mice. This increased tumor uptake, visualized also by SPECT/CT imaging, is expected to eventually translate into higher diagnostic sensitivity and improved therapeutic efficacy of radiolabeled gastrin analogs in CCK2R-expressing cancer patients.

Optimised labeling, preclinical and initial clinical aspects of CCK-2 receptor-targeting with 3 radiolabeled peptides.

Medullary thyroid carcinoma (MTC) expresses CCK-2 receptors. (111)In-labeled DOTA-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH(2) (DOTA-MG11), DOTA-DAsp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH(2) (DOTA-CCK), and (99m)Tc-labeled N(4)-Gly-DGlu-(Glu)(5)-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH(2) ((99m)Tc-Demogastrin 2) are analogs developed for CCK-2 receptor-targeted scintigraphy. All 3 radiolabeled analogs were selected on the basis of their high CCK-2 receptor affinity and their good in vitro serum stability, with in vitro serum t(1/2) values of several hours. Radiolabeling of DOTA-peptides with (111)In requires a heating procedure, typically in the range of 80 degrees -100 degrees C up to 30 min. Following this procedure with DOTA-MG11 resulted in a >98 % incorporation of (111)In, however, with a radiochemical purity (RCP) of <50 %. The decrease in RCP was found to be due to oxidation of the methionine residue in the molecule. Moreover, this oxidized compound lost its CCK-2 receptor affinity. Therefore, conditions during radiolabeling were optimised: labeling of DOTA-MG11 and DOTA-CCK with (111)In involved 5 min heating at 80 degrees C and led to an incorporation of (111)In of >98 %. In addition, all analogs were radiolabeled in the presence of quenchers to prevent radiolysis and oxidation resulting in a RCP of >90 %. All 3 radiolabeled analogs were i.v. administered to 6 MTC patients: radioactivity cleared rapidly by the kidneys, with no significant differences in the excretion pattern of the 3 radiotracers. All 3 radiolabeled analogs exhibited a low in vivo stability in patients, as revealed during analysis of blood samples, with the respective t(1/2) found in the order of minutes. In patient blood, the rank of radiopeptide in vivo stability was: (99m)Tc-Demogastrin 2 (t(1/2) 10-15 min)>(111)In-DOTA-CCK (t(1/2) approximately 5-10 min)>(111)In-DOTA-MG11 (t(1/2)<5 min).

3D solution structure of [Tyr3]octreotate derivatives in DMSO: structure differentiation of peptide core due to chelate group attachment and biologically active conformation.

The solution models of [Tyr3]octreotate (DPhe1-Cys2-Tyr3-DTrp4-Lys5-Thr6-Cys7-Thr8-COOH, disulfide bridged) (I), its analogs functionalized with an open chain tetraamine chelator, N4-[Tyr3]octreotate (II), and the N4-(Asp)2-[Tyr3]octreotate (III) peptide have been determined through 2D 1H NMR spectroscopy in DMSO. Chemical shift analysis has been performed in an attempt to elucidate structural changes occurring during attachment of the tetraamine to the peptide backbone. NMR-derived geometrical constraints have been used in order to calculate high resolution conformers of the above peptides. Conformational analysis of the three synthetic analogues, have shown that these somatostatin analoges adopt a predominant antiparallel beta-sheet conformation characterized by a beta-like turn spanning residues DTrp4 and Lys5 which is supported in the case of N4-(Asp)2-[Tyr3]octreotate and N4-[Tyr3]octreotate by medium range NOEs. These data indicate that the above-mentioned molecules adopt a rather constrained structure in the 4-residue loop Tyr3-Thr6. Additionally, the C-terminal of [Tyr3]octreotate, comprising Cys7 and Thr8, appears to form a turn-like structure manifested by characteristic side-chain NOEs between Lys5 and Thr8, which have not been detected for the other two compounds. These data are discussed in the light of previous structural data of Sandostatin (octreotide) and suggest that attachment of the N4-chelator and two Asp residues at the N-end of [Tyr3]octreotate impose considerable structural changes and affect the binding properties of these peptides. Indeed, the IC50 values determined during competition binding assays against the sst2 (somatostatin subtype 2 receptor) suggest that the presence of the N4 group enhances receptor affinity, while extension of peptide chain by two negatively-charged Asp residues impairs receptor affinity at approximately one order of magnitude.

Neurotransmitter modulation of steroid action in target cells that mediate reproduction and reproductive behavior.

Two major functional interactions between steroid hormones and neurotransmitters are generally recognized. First, steroids affect neurotransmission, and second, through effects on hypothalamic peptides that regulate anterior pituitary function neurotransmitters affect steroid secretion. In recent years, evidence has accumulated which indicates that neurotransmitters can also affect steroid action within postsynaptic steroid target cells. We review evidence for this relationship in pineal, uterus and hypothalamus and propose that the modulation of target cell responsiveness to steroids is an important mechanism by which neurotransmitters affect steroid-dependent processes. The operation of such a mechanism provides a means for environmental, behavioral and emotional events to rapidly and selectively alter steroid effects on behavior and physiology.

In vivo evidence for a direct effect of naloxone on testicular steroidogenesis in the male rat.

It has been suggested that endogenous opioid peptides (EOP) exert paracrine or autocrine effects in the testes. To assess this hypothesis, we examined whether naloxone, by blocking the effects of EOP, influenced serum testosterone levels, apart from its effects on LHRH/LH, in the intact male rat. We found that naloxone increased serum LH and testosterone levels over essentially the same time course and produced dose-dependent increases in serum testosterone levels even though LH levels were maximally elevated at all doses. These data are not consistent with the view that naloxone exerts its effects on testosterone exclusively by altering LHRH/LH release. Additional, perhaps more definitive, evidence of a direct effect of naloxone on testosterone's biosynthesis was provided by our observations that 1) naloxone generated increases in serum testosterone levels in male rats in which the naloxone-induced surge in LH was blocked by nembutal; and 2) intratesticular injections of naloxone increased serum testosterone levels without increasing LH. Although these data suggest that naloxone influences steroidogenesis independently of its effects on LH, we found that the antagonist failed to increase serum testosterone levels in hypophysectomized animals or when serum LH levels were allowed to reach undetectable levels in nembutal-blocked animals. Consequently, our data are consistent with the hypothesis that naloxone facilitates the effects of LH on testosterone's biosynthesis rather than exerting an independent effect of its own. Whether the effects observed in these studies represent a negative autocrine effect of EOP on Leydig cells or a paracrine effect on Sertoli cells remains to be determined. Nevertheless, our results provide, to our knowledge, the first in vivo evidence that EOP modulate testicular steroidogenesis in the intact animal.

Muscarinic cholinergic receptors in the songbird and quail brain: a quantitative autoradiographic study.

In order to clarify the neuroanatomical basis for postulated muscarinic cholinergic control of a wide array of physiological processes in birds, the distribution of muscarinic cholinergic receptors in the brain of three avian species was investigated by quantitative autoradiography. The species consisted of two passerines (songbirds), the European starling (Sturnus vulgaris) and the song sparrow (Melospiza melodia), and one galliform, the Japanese quail (Coturnix coturnix japonica). [3H]N-methyl scopolamine (NMS), a muscarinic cholinergic antagonist was used as the ligand to label the receptors. Initial experiments demonstrated that the binding of this ligand in the three species is saturable in the nanomolar range and has a high affinity (Kd = +/- 0.6 nM). Displacement experiments revealed that three muscarinic ligands competed in an order of potency characteristic of the mammalian muscarinic receptor (i.e., atropine greater than oxotremorine greater than carbachol) for NMS binding in the avian brain. In all three species, portions of the basal ganglia, such as the parolfactory lobe and the paleostriatum augmentatum, exhibited the highest density of binding. On the other hand, the paleostriatum primitivum, the avian homologue of the mammalian globus pallidus, contained very few binding sites. Other telencephalic sites, such as the ventral and dorsal hyperstriatum, also revealed relatively high receptor density. However, the neostriatum and especially the ectostriatum showed much lower levels. In the hypothalamus, in all three species, specific binding could be observed in the ventromedial nucleus and adjacent areas. The paraventricular nucleus also showed moderate levels of binding density, especially in the two songbird taxa. At a more rostral level, the preoptic area showed low levels of binding. In the quail, the sexually dimorphic nucleus of the preoptic area was clearly outlined in the autoradiograms by the low level of binding sites compared to the surrounding areas. In the two passerine species, nuclei of the song system were identified by either high or low levels of NMS binding. High binding defined area X and the mesencephalic nucleus, intercollicularis (ICo). In contrast, the robust nucleus of the archistriatum and the magnocellular nucleus of the anterior neostriatum showed low levels of binding in comparison with the surrounding tissue. None of these nuclei were visible in the quail autoradiograms except for ICo, which appeared as in the passerines as a heavily labelled area surrounding the lightly labelled nucleus mesencephalicus lateralis pars dorsalis. In all three species, the hippocampal complex was devoid of NMS binding except for two lateral dark bands that were present along the entire rostral to caudal extent of the hippocampus.(ABSTRACT TRUNCATED AT 400 WORDS)

Glutathione-mediated metabolism of technetium-99m SNS/S mixed ligand complexes: a proposed mechanism of brain retention.

Two series of [99mTc](SNS/S) mixed ligand complexes each carrying the N-diethylaminoethyl or the N-ethyl-substituted bis(2-mercaptoethyl)amine ligand (SNS) are produced at tracer level using tin chloride as reductant and glucoheptonate as transfer ligand. The identity of [99mTc](SNS/S) complexes is established by high-performance liquid chromatographic (HPLC) comparison with authentic rhenium samples. The para substituent R on the phenylthiolate coligand (S) ranges from electron-donating (-NH2) to electron-withdrawing (-NO2) groups, to study complex stability against nucleophiles as a result of N- and R-substitution. The relative resistance of [99mTc](SNS/S) complexes against nucleophilic attack of glutathione (GSH), a native nucleophilic thiol of 2 mM intracerebral concentration, is investigated in vitro by HPLC. The reaction of [99mTc](SNS/S) complexes with GSH is reversible and advances via substitution of the monothiolate ligand by GS- and concomitant formation of the hydrophilic [99mTc](SNS/GS) daughter compound. The N-diethylaminoethyl complexes are found to be more reactive against GSH as compared to the N-ethyl ones. Complex reactivity as a result of R-substitution follows the sequence -NO2 >> -H > -NH2. These in vitro findings correlate well with in vivo distribution data in mice. Thus, brain retention parallels complex susceptibility to GSH attack. Furthermore, isolation of the hydrophilic [99mTc](SNS/GS) metabolite from biological fluids and brain homogenates provides additional evidence that the brain retention mechanism of [99mTc](SNS/S) complexes is GSH-mediated.

Behavioural responses to sex steroids of gonadectomized and sexually regressed quail.

The purpose of these experiments was to compare the behavioural and morphological effects of exogenous sex hormones in gonadectomized quail (Coturnix coturnix japonica) with those in quail having regressed gonads as a result of exposure to short days. In Expt 1, male quail were assigned to one of three treatment groups: (1) intact, exposed to 16 h light: 8 h darkness and injected with oil (group 16L); (2) gonadectomized, exposed to 16 h light: 8 h darkness and injected with 2-5 mg testosterone propionate (TP)/day (group 16L-castrated); and (3) intact, exposed to 8 h light: 16 h darkness, and injected with 2-5 mg TP/day (group 8L). Groups 16L-castrated and 8L responded similarly to testosterone, copulating with equal frequency and rapidity after the same number of days of treatment, and also developing proctodeal (foam) glands of a similar size. Only on day 7 of testosterone treatment did the results for these two groups differ. By day 14, the behaviour of both groups resembled that of the 16L birds. In Expt 2 female quail were assigned to the same three treatment groups, except that the hormone treatment was 25 mug oestradiol benzoate/day. Group 8L became sexually receptive sooner than the 16L-ovariectomized quail, but by day 13 both groups had oviducts of similar size, were equally receptive, and were as receptive as the 16L females. The results suggest that the effects of photoperiod on sexual behaviour in this species are mediated largley, if not wholly , by the gonads. They also suggest exposure to short days and surgical gonadectomy are rather similar experimental procedures in the quail.

Extracti-Gel D chromatography is a simple, efficient method for removing digitonin during receptor purification: application to the kappa 1 opioid receptor.

Digitonin is widely used for extracting active neurotransmitter receptors from membranes. However, its low critical micellar concentration has made its removal from samples problematic. Here we report that digitonin can be efficiently removed (> 90%) from solution using Extracti-Gel D, a detergent-absorbing matrix. Active kappa 1 opioid receptors solubilized from brain survive Extracti-Gel D chromatography with a recovery of 50-55% and 25% dilution by added volume. The loss of receptor and the dilution, however, are compensated for to a large extent by the disinhibition of binding that results from the removal of digitonin. Extracti-Gel D chromatography had little or no effect on the apparent equilibrium dissociation constant for [3H]U-69,593 binding to the kappa 1 receptor. We conclude that Extracti-Gel D column chromatography is a simple, highly efficient and practical method for markedly reducing the concentration of digitonin in biological samples. Application of the procedure should allow characterization of digitonin-solubilized receptors with minimal complications from bound digitonin and extend the usefulness of digitonin to studies going beyond the initial stages of receptor purification.

A simple, highly sensitive assay for measurement of digitonin during receptor solubilization.

A simple and highly sensitive assay for measuring total digitonin in biological samples is described. The assay is based on the ability of digitonin to hemolyze red blood cells. The precision and reproducibility of the assay was excellent with intra- and inter-assay variabilities of less than 1% and 6%, respectively. The assay was used to evaluate several potential methods for removing digitonin from biological samples (digitonin extracts from guinea pig brain membranes). Dialysis and G-25 Sephadex chromatography were ineffective. However, protein and digitonin can be effectively separated by ammonium sulfate precipitation followed by dialysis. The kappa 1 opioid receptor survived these procedures with no change in affinity for [3H]U-69,593. In conclusion, the hemolytic assay for digitonin appears to provide a practical means for determining detergent concentrations during receptor purification and characterization and for evaluating potential methods for detergent removal. Although an in depth analysis of the assay was carried out only for digitonin, CHAPS and deoxycholate also caused 50% hemolysis at concentrations well below those commonly used for receptor solubilization and, therefore, the general assay procedures might have applicability for measurement of these and perhaps other detergents used in receptor solubilization as well.

Noradrenergic transmission and female sexual behavior of guinea pigs.

Treatment with the dopamine beta-hydroxylase (DBH) inhibitor U-14,624 (50, 100, or 150 mg/kg) blocked the induction of lordosis behavior be estradiol benzoate (EB) and progesterone (P) in ovariectomized guinea pigs. After treatment with U-14,624 (100 mg/kg), norepinephrine (NE) content of medial basal hypothalamus, preoptic area and cortex was reduced (by 55%) and dopamine (DA) content of medial basal hypothalamus was increased (by 155%) during the period when females treated with EB and P normally display lordosis. Treatment with the NE receptor stimulator clonidine (1.0 mg/kg) restored lordosis behavior in females treated with EB, P, and U-14,624 (100 mg/kg), but the putative DA and serotonin (5-HT) receptor blockers pimozide (1.0 mg/kg) and methysergide (20.0 mg/kg) were ineffective in this respect. Thus, inhibition of lordosis after treatment with U-14,624 appeared to be attributable primarily to a reduction in NE neurotransmission, rather than to increase in DA or 5-HT activity. Because clonidine induced lordosis in females treated with EB, P, and U-14,624, it seemed unlikely that the facilitatory effects of clonidine on lordosis were mediated by activation of presynaptic alpha-adrenergic receptors (i.e. inhibitory NE autoreceptors) rather than by postsynaptic alpha-receptors. In addition, pretreatment with the postsynaptic alpha-adrenergic antagonist phenoxybenzamine (20.0 mg/kg) blocked the facilitation of lordosis by clonidine (1.0 mg/kg) in females primed with EB alone and with EB plus P. Thus, the facilitatory effects of clonidine on lordosis appear to be mediated by activation of postsynaptic alpha-adrenergic (i.e. NE) receptors. The results of this study provide further evidence that NE neurotransmission facilitates the expression of female sexual behavior in guinea pigs.

Characteristics of [3H]prazosin binding to guinea pig brain alpha-adrenergic receptors: effects of estradiol and progesterone.

[3H]Prazosin was found to bind to sites on guinea pig brain membranes with alpha 1-adrenergic receptor characteristics. Treatment of ovariectomized guinea pigs with estradiol benzoate (EB) or EB followed by progesterone (P) did not affect [3H]prazosin binding to membranes from hypothalamus, preoptic area, amygdala or cerebral cortex. When added to the incubation mixture of the assay, estradiol, P, and other steroids decreased [3H]prazosin binding but only at high concentrations. These results do not support the idea that estrogen and progestin influence reproductive physiology through effects on brain alpha 1-receptors, although limitations of the methodology employed do not completely rule out this possibility.

Alpha 1-noradrenergic regulation of hypothalamic progestin receptors and guinea pig lordosis behavior.

Experiments were conducted to determine whether alpha 1- or alpha 2-receptors mediate noradrenergic (NA) regulation of guinea pig lordosis behavior and hypothalamic progestin receptors. When infused into a lateral cerebroventricle at a dose that inhibits lordosis and that decreases the concentration of estradiol-inducible hypothalamic progestin receptors, phenoxybenzamine decreased binding of the alpha 1-ligand [3H]WB4101 but not the alpha 2-ligand [3H]clonidine to brain membranes. Thus, under the conditions used, phenoxybenzamine appears to block alpha 1-receptors with little or no effect on alpha 2-receptors. Experiments with the selective alpha 1-antagonist prazosin also indicated alpha 1-receptor regulation of lordosis and hypothalamic progestin receptors. Prazosin inhibited lordosis induced by estradiol benzoate (EB) plus progesterone and by EB + clonidine and decreased the concentration of cytoplasmic progestin receptors in hypothalamus (but not in preoptic area or frontal cortex) of EB-primed females. The inhibition of lordosis is apparently not due to some unknown side effect of prazosin because pretreatment with a high dose of clonidine attenuated the inhibition. The possibility that a causal relationship exists between effects of alpha 1-NA transmission on hypothalamic progestin receptors and lordosis was discussed. Also, because effects of NA transmission on hypothalamic progestin receptors are dependent on prior treatment with EB, it was suggested that NA transmission might influence estradiol action in addition to progestin action in hypothalamic cells.

Effects of 5,7-dihydroxytryptamine on serotonin1 and serotonin2 receptors throughout the rat central nervous system using quantitative autoradiography.

The effects of the serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), on serotonin1 (5-HT1) and 5-HT2 receptors were investigated using the high degree of resolution provided by quantitative autoradiography in an effort to determine the synaptic location of these receptors. 5,7-DHT treatment resulted in a decrease in 5-HT1 binding in the dentate gyrus and CA3c/4 of the anterior hippocampus and in the dorsal raphe nucleus, whereas no changes were observed in the posterior hippocampus nor in many other brain structures. 5-HT2 receptors exhibited no changes in any brain area examined in response to 5,7-DHT treatment, despite over 90% serotonin depletion in most of the forebrain nuclei examined. The results indicate that at least some of the 5-HT1 sites labelled by [3H]5-HT in the hippocampus and dorsal raphe nucleus are presynaptic, whereas 5-HT2 receptors are probably postsynaptic. In addition, the distribution profiles of 5-HT1 and 5-HT2 binding sites were compared in the rat central nervous system at various anatomical levels. 5-HT1 binding sites were identified using [3H]5-HT, while 5-HT2 binding sites were labelled with [3H]ketanserin. Both receptor subtypes displayed distinctly different localization patterns, which, in most cases was the inverse of the other pattern. In the brainstem it is significant that 5-HT2 receptors are concentrated in the facial nucleus and the motor nucleus of the trigeminal nerve, areas known to influence head and facial movement. The serotonin-mediated head-shake response occurs when 5-HT2 receptors are activated. In contrast, 5-HT1 receptors are distributed throughout the brainstem and in specific portions of the spinal cord. These areas are thought to control the serotonin behavioral syndrome and this behavior is 5-HT1A-mediated. All raphe nuclei were devoid of 5-HT2 receptors; only 5-HT1 receptor were found in these nuclei. Correlations with serotonin terminal distribution patterns are discussed. The pattern of 5-HT2 receptor distribution was also compared with the pattern of alpha 1 receptors, using [3H]prazosin in order to determine whether [3H]ketanserin significantly labels alpha 1 receptors. Although some similarities exist, overlap of binding did not occur in other nuclei, indicating that alpha 1 contamination of this system is probably negligible.

Photoperiodic changes in opiate binding and their functional implications in golden hamsters.

Daylength modulates gonadotropin secretion, gonadal steroid hormone feedback, sexual behavior and body weight in male golden hamsters. Endogenous opiates regulate each of these phenomena, and the ability of opiate receptor blockade to elevate serum LH secretion is photoperiod-dependent. We used in vitro autoradiography to localize and quantify effects of daylength in golden hamsters. Hamsters were exposed to stimulatory (14 h light: 10 h dark) or inhibitory (10 h light: 14 h dark) photoperiods for 10 weeks before specific [3H]naloxone binding was assessed. Short days significantly decreased binding in medial amygdala and the intercalated amygdaloid nucleus. This effect was reversed by superior cervical ganglionectomy. No significant effects of daylength were observed in other amygdaloid, hypothalamic or preoptic areas. Lesions of the medial amygdala decreased copulatory behavior, short day-induced weight loss, and anogenital chemoinvestigation but did not affect gonadal regression or other forms of chemoinvestigation. These lesions facilitated testosterone's negative feedback on luteinizing hormone in long days but did not interfere with the potentiation of negative feedback by short days.

Alpha 1- and alpha 2-noradrenergic receptors in steroid-sensitive brain areas: development and response to estradiol-17 beta benzoate in neonatal guinea pigs.

Immature female guinea pigs are relatively insensitive to the effects of ovarian steroids on certain reproductive processes including the facilitation of sexual receptivity and the induction of hypothalamic progestin receptors by estrogen. In adult guinea pigs, alpha-receptor-mediated noradrenergic transmission is known to affect both of these processes. In the experiments reported here, we investigated the possibility that age-related differences in alpha 1- and alpha 2-noradrenergic receptors might underlie the insensitivity of neonates to ovarian steroids. With tritium-sensitive film autoradiography, the distribution of alpha 1- and alpha 2-receptors in neonatal and adult female guinea pigs was compared and the effects of exogenous estradiol-17 beta on alpha-receptor binding was examined in neonatal guinea pigs. The results of these experiments showed that in animals not treated with estrogen the binding of both alpha-receptor subtypes differed between adults and neonates in several brain regions including areas known to be involved in the regulation of reproduction. In all regions where differences occurred, alpha-receptor levels were higher in neonatal females than in adult females. In addition, in contrast to previously reported results in adults, estrogen did not affect alpha-receptor binding in any region of neonatal guinea pig brain.

Noradrenergic modulation of hypothalamic progestin receptors in female guinea pigs is specific to the ventromedial nucleus.

The concentration of estrogen-induced cytosolic progestin receptors (CPRs) in the hypothalamus of alpha 1-noradrenergic antagonist-treated female guinea pigs is reduced relative to non-drug-treated controls. The present study determined where within the hypothalamus this reduction occurs. Ovariectomized, estradiol-treated guinea pigs were given either the alpha 1-noradrenergic antagonist prazosin or vehicle. Microdissected brain regions were assayed for CPR levels. Prazosin caused a selective relative decrease in CPR levels of the ventromedial nucleus of the hypothalamus. No significant effects of prazosin were seen in other hypothalamic or preoptic area nuclei.

Alpha 1- and alpha 2-noradrenergic receptor binding in guinea pig brain: sex differences and effects of ovarian steroids.

Noradrenergic transmission mediated by alpha 1-receptors is involved in the regulation of ovarian steroid-dependent reproductive processes in female guinea pigs. In addition, estradiol treatment is known to affect alpha 2-receptor binding in some steroid-sensitive regions of guinea pig brain. In these experiments, we further assessed the effects of ovarian hormones on alpha-noradrenergic receptors with quantitative autoradiographic methods. Binding of alpha 1- and alpha 2-noradrenergic receptors was measured in male and female guinea pigs treated with either estradiol or oil to determine if sex differences in steroid sensitivity might be related to differences in the distribution or modulation of alpha-receptors. In a second experiment, the possibility was tested that progesterone might alter the sensitivity to norepinephrine by modulating alpha 1- or alpha 2-receptor binding in estradiol-primed female guinea pigs. Results of these studies showed that estradiol treatment reduced alpha 1- and alpha 2-receptor binding of the ventromedial hypothalamic nucleus only in females and increased alpha 2-receptor binding in several preoptic nuclei of both sexes. Progesterone administration, however, had no effect on either alpha-receptor subtype. These studies indicate that estradiol-induced changes in alpha-receptor binding might be involved in steroid-dependent reproductive processes and in the differential responsiveness of males and females to ovarian hormones. Changes in these receptors do not appear to be involved in the stimulatory effects of progesterone on neuroendocrine processes.

Distribution of alpha 2-adrenergic receptors in the brain of the Japanese quail as determined by quantitative autoradiography: implications for the control of sexually dimorphic reproductive processes.

With the use of [3H]p-aminoclonidine (PAC), alpha 2-adrenergic binding sites were mapped in the brain of the Japanese quail (Coturnix coturnix japonica). The sites were labeled with the use of in vitro quantitative autoradiography. Special attention was given to areas implicated in the control of sexually dimorphic reproductive processes including sexual behavior. Preliminary competition experiments found that [3H]PAC binding on tissue sections exhibited a pharmacology appropriate to the alpha 2 receptor. Binding sites were found to be heterogeneously distributed throughout the brain. Some of the highest levels of specific binding were found in several areas regulating reproductive function such as the preoptic area, the supraoptic nucleus, the infundibulum, and the medial mammillary nucleus of the infundibulum. [3H]PAC labeled precisely the morphologically dimorphic preoptic medial nucleus but no sexual dimorphism in density of receptor binding was identified. However, dimorphism in density of receptor binding was identified in two areas: the medial mammillary nucleus and the mesencephalic intercollicular nucleus. The former area appears to be involved in the regulation of gonadotrophin secretion and the latter area has been implicated in the control of vocal behavior. These neurochemical dimorphisms may contribute to the regulation of two sexually dimorphic reproductive processes, gonadotropin secretion and courtship vocalizations.

Sex differences in cytosolic progestin receptors in microdissected regions of the hypothalamus/preoptic area of guinea pigs.

Cytosolic progestin receptors (CPRs) were measured in microdissected nuclei of the hypothalamus and preoptic area of male and female guinea pigs. Adult gonadectomized animals were given 3 daily injections of 20 micrograms/day estradiol benzoate (EB) or oil vehicle. 24 h later, animals were sacrificed and cytosolic progestin receptors were measured using the synthetic progestin 3H-R5020. CPR levels did not differ significantly between oil treated males and oil treated females in any brain areas examined. With EB treatment, males showed significant increases in CPRs in most of the brain areas in which females showed increases, i.e. in the medial preoptic area, the periventricular part of the preoptic area, the periventricular part of the anterior hypothalamus, the ventromedial nucleus of the hypothalamus, the periventricular part of the medial hypothalamus and the arcuate-median eminence. However, EB treated males showed significantly lower CPR levels than EB treated females in both the periventricular part of the preoptic area and the periventricular part of the medial hypothalamus.