Neurotrophic ACTH analogue promotes plasticity of type I corticosteroid receptor in brain of senescent male rats.

Age-related changes were studied in the concentration of type-I and type-II corticosteroid receptors in the hippocampus of young adult (3 months) and aged (28.5 to 30.5 months) male rats. Using 3H-labelled ligands, in vitro binding of type-I and type-II corticosteroid receptors in the soluble cell fraction (cytosol) revealed an age-related decrease in concentration of both receptor types of 52% and 28%, respectively. Infusion of young and aged male rats for 2 weeks with the ACTH4-9 [adrenocorticotropin4-9] peptide analogue ORG 2766 (0.5 micrograms/0.5 microliter/hr) resulted in only a minor increase (+8%) in the number of type-I receptors in young rats. In the aged animals, however, the type-I receptor concentration was 68% higher than in the vehicle-treated aged animals. In contrast, no effect of the peptide treatment was noted on the concentration of type-II receptors in either young or aged rats. Furthermore, no effect was found for either age or treatment with peptide on the affinity of type-I and type-II receptors for their respective ligands. Binding of 3H-labelled ligands to brain sections of young and aged rats was performed using in vitro autoradiography. Quantitative image analysis of the film showed that in senescence there is a marked reduction in both type-I (62-75%) and type-II (29-56%) receptor concentrations in the hippocampal subregions (CA1, CA2, CA3 and dentate gyrus) as well as in the lateral septum. Treatment of aged rats with ORG 2766 selectively reversed the age-associated reduction in type-I receptors, while the peptide did not affect the type-II receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging of the serotonergic system in the rat forebrain: an immunocytochemical and neurochemical study.

Age-related changes in both morphological and neurochemical parameters of indol- and catecholaminergic system in the rat brain were examined. A qualitative histochemical survey of the occurrence of aberrant serotonergic fibers in the aged rat brain suggests region-specificity in the process of degeneration. Forebrain areas, such as the caudate-putamen complex, globus pallidus, prefrontal and frontoparietal cortices were consistently affected, whereas serotonergic fibers were only infrequently affected in other areas like septal and amygdaloid nuclei. Neurochemical data similarly revealed regional differences. 5-Hydroxytryptamine levels were increased in the frontoparietal cortex, hippocampus, hypothalamus and the mesencephalic raphe region but remained unchanged in the caudate-putamen complex. 5-Hydroxyindolacetic acid levels were also enhanced in all these areas. Examination of brains of 12-, 18- and 24-month-old rats revealed that aberrant serotonergic fibers were already present at the age of 12 months and their incidence increase with age. There was no difference in the number of serotonergic cells in the dorsal raphe nucleus of young and aged rats. Aberrant tyrosine hydroxylase-immunoreactive fibers were observed only infrequently. Their occurrence showed no overlap with the areas containing aberrant serotonergic fibers. Neurochemical estimates of the levels of catecholamines in young versus aged rat brain areas similarly revealed regional and neurotransmitter specific differences to occur during the process of aging.

Morphological, neurochemical, and behavioral studies on serotonergic denervation and graft-induced reinnervation of the rat hippocampus.

A procedure was developed to conduct simultaneously immunocytochemical and neurochemical studies on the serotonergic system in adjacent 300-micron-thick slices of rat hippocampus. This procedure was applied to correlate morphological (innervation pattern and density), neurochemical (5-hydroxytryptamine and 5-hydroxyindolacetic acid levels and [3H]5-hydroxytryptamine uptake and release) and behavioral (spatial learning) effects of neurotoxin-induced denervation and reinnervation by grafting fetal mesencephalic raphe cells. Intracerebroventricular injections of a low dose of 5,7-dihydroxytryptamine caused a discrete serotonergic denervation of the hippocampus. Eleven months after lesioning, 5-hydroxytryptamine and 5-hydroxyindolacetic acid levels and [3H]5-hydroxytryptamine uptake capacity were decreased by 50-60%. By this time, the residual fibers displayed an enhanced vulnerability towards K(+)-induced depolarization. Grafting of a fetal raphe cell suspension resulted in a reinnervation of the host hippocampus. The pattern of reinnervation was comparable to control innervation and the density was supranormal at the level of the graft. As observed semiquantitatively, the innervation density decreased with distance from the core of the graft. Neurochemical studies showed that the fibers were capable of synthesizing, metabolizing and releasing 5-hydroxytryptamine. The turnover of 5-hydroxytryptamine in both the denervated and the reinnervated hippocampus was comparable to that in control tissue. Previous behavioral testing of the denervated and of the denervated and implanted animals did not reveal any effect on spatial learning, either in an individual or in a social test paradigm. The latter data substantiate the notion that interference with the hippocampal serotonergic innervation does not hamper adequate spatial learning.

Ovariectomy and subchronic estradiol-17 beta administration decrease dopamine D1 and D2 receptors in rat striatum.

Ovariectomy and subchronic estradiol-17 beta cause a down-regulation of dopamine D1 and, to a lesser extent, D2 receptors in rat striatum. An intracellular mechanism mediates the DA receptor down-regulation, as various estrogens do not interact with membrane-bound DA receptors in vitro. A common denominator, e.g. enhanced DA turnover, is suggested to mediate the estradiol-induced DA receptor down-regulation. Ovarian factors other than estradiol are additionally proposed to be involved in the regulation of striatal DA receptors.

Cholinergic innervation and topographical organization of muscarinic binding sites in rat brain: a comparative autoradiographic study.

Employing [3H]hemicholinium-3 ([3H]HC), [3H]pirenzepine([3H]PZ) and [3H]quinuclidinyl benzilate ([3H]QNB), autoradiographic binding studies were performed to identify and quantitate the localization of high-affinity choline carriers, M1-subtype of muscarinic binding sites and a mixed population of M1- and M2-subtypes of muscarinic binding sites, respectively, in 38 anatomically defined areas of rat brain. Labelling of adjacent brain sections with [3H]HC, [3H]PZ and [3H]QNB revealed different topographical binding patterns. [3H]HC binding, which is supposed to reflect cholinergic innervation, was dense in the nucleus accumbens, olfactory tubercle, caudate putamen, basolateral amygdaloid nucleus and the interpeduncular nucleus. Moderate but heterogeneous binding was found in thalamic, hypothalamic, hippocampal and cortical areas. Maximal [3H]PZ binding was observed in the nucleus accumbens, olfactory tubercle and in discrete substructures of the hippocampus, e.g. CA1 and dentate gyrus. Binding to other hippocampal and cortical areas was intermediate, whilst minor binding was found in thalamic, hypothalamic and brain stem areas. The binding of [3H]QNB was more evenly distributed over the brain as compared to that of [3H]PZ. [3H]QNB clearly exceeded the binding of [3H]PZ in the thalamus, hypothalamus and brain stem. A relationship was found between the topography patterns of the [3H]PZ and [3H]QNB binding sites. However, some brain areas showed preference for one of the two ligands, pointing to a distinct localization of M1- and M2-subtypes of muscarinic binding sites. Although M1 sites appeared to predominate in the basal ganglia, hippocampus and cortex, some heterogeneity was observed indicative of the minor occurrence of M2 sites within these structures. There was no relationship between the density of the presumed cholinergic innervation and the binding capacity of either of the muscarinic sites in the various brain areas. However, a relationship was found between M2-selectivity and [3H]HC binding, pointing to a possible presynaptic localization of the M2-sites. In addition, it is suggested that distinct cholinergic cell groups might project their fibres to brain areas containing particular subsets of postsynaptic muscarinic binding sites.

Serotonergic fibres degenerating in the aging rat brain or sprouting from grafted fetal neurons are not affected by the neurotrophic ACTH analogue Org 2766.

The effects of chronic treatment with the purported neurotrophic factor ACTH(4-9) analogue Org 2766 were studied on age-related degeneration of serotonergic fibres and on gliosis in the rat hippocampus and caudate putamen complex. In addition, the potential growth-promoting effects of Org 2766 were investigated on fetal serotonergic cells implanted in a previously denervated hippocampus of young adult rats. Chronic treatment of rats from the age of 11 months to 17-18 months did not affect the incidence of aberrant serotonergic fibres in the caudate-putamen complex or the fibres densities in the hippocampus or the caudate-putamen complex. Gliosis was unaffected by Org 2766 treatment as indicated by increased number and staining intensity of glial fibrillary acidic protein-immunoreactive cell bodies in both brain areas. Grafting of fetal raphe cells in young adult rats caused a time-dependent reinnervation of the previously denervated hippocampus. The reinnervation was not affected by treatment of the rats with Org 2766 for 4 weeks following implantation.

Aging and regenerative capacity of the rat serotonergic system. A morphological, neurochemical and behavioral analysis after transplantation of fetal raphe cells.

Morphological dissimilarities between the brains of young (3 months) and aged (28 months and older) rats were demonstrated using serotonin-immunocytochemistry. A degeneration of the serotonergic system, noted as a decreased innervation and the appearance of enlarged or swollen varicosities, was observed particularly in the frontoparietal cortex, and the neostriatum of the aged rat brain. No direct relationship between this aberrant morphology and decrease in density of serotonin-innervation was found as we demonstrated a decline in fiber density without the appearance of aberrant serotonergic fibers in the hippocampus. HPLC analysis revealed that serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) levels in the frontoparietal cortex, hippocampus and raphe area were increased in the aged rat, while the 5-HT level in the caudate-putamen complex was not different from the young adult rat. The ratio 5-HIAA/5-HT, indicative of 5-HT turnover, appeared increased in the frontoparietal cortex, sensoric part, the caudate-putamen and the raphe area, while this ration in the frontoparietal cortex, motoric part and the hippocampus was not altered in the aged rat. Behavioral screening revealed a decrease spatial performance of aged males in a Morris Water-Maze task. To investigate whether the age of the host recipient was of influence on the regenerative capacity, a fetal raphe cell suspension of embryonic day E 15 was implanted in the caudate-putamen of young adult as well as aged rats. Neither differences in survival of the serotonergic cells nor in fiber outgrowth between both groups appeared five weeks after transplantation. Subsequently, transplantation of raphe cells in the hippocampus of young adult rats, after lesioning the hippocampal serotonergic innervation with 5,7-DHT, was performed to compare behavioral, morphological and neurochemical effects of the implants. It appeared that 11 months after transplantation the serotonergic innervation of the previously denervated hippocampus was greatly restored. There was a striking resemblance between the immunohistochemical and neurochemical data with respect to the increase in the amount of newly formed serotonergic fibers, the increase in uptake of [3H]-5-HT and in 5-HT and 5-HIAA levels. Also the behavior of lesioned and lesioned + transplanted males was rather similar to controls. In the behavioral tests we were mainly interested in hippocampal functioning, therefore orientation was of our prime interest. The other behavioral tests were only to confirm that the possible changes were linked to hypothalamic or extra-hypothalamic functions.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional activity of raphe neurons transplanted to the hippocampus and caudate-putamen. An immunohistochemical and neurochemical analysis in adult and aged rats.

Adult (3-month-old) and aged (28-month-old) rats that had been pretreated with 5,7-DHT in both lateral ventricles received grafts of cell suspensions taken from the RR or MR regions taken from the embryonic stages E12-21. These cell suspensions were implanted unilaterally into the rostral part of the hippocampus or the caudate-putamen for immunohistochemical and neurochemical studies. MR and RR cell suspensions have the potency to regenerate serotonergic fibers in the previously denervated adult and aged hippocampus and caudate-putamen. The RR cell suspension, however, also showed outgrowth of other transmitter-specific neuronal systems, specifically noradrenaline and substance P. To evaluate the functional activity of the serotonergic reinnervation, we have combined immunohistochemistry and neurotransmitter release studies on adjacent hippocampus slices of adult rats. Results showed that after a survival time of 10 weeks, the serotonergic innervation of the hippocampus was greatly restored and, moreover, that the K+-induced Ca2+-dependent release of 5-HT amounted to about 80% of normal values. There appeared to be a striking similarity between the immunohistochemical and neurochemical data regarding the increase in the number of newly formed serotonergic fibers, the increase of the release of radiolabeled 5-HT, and the extent of the outgrowth in the hippocampus.

In vitro interaction of ACTH with rat brain muscarinic receptors.

ACTH-(1-24) inhibits the in vitro binding of the muscarinic antagonist [3H]QNB to membranes from rat brain. The magnitude of inhibition is dependent on the concentration of ACTH-(1-24). Kinetic analysis indicates a pure competitive inhibition which is suggestive of a reversible interaction of ACTH with muscarinic receptors. A mechanism involving an interaction of ACTH-(1-24) with the phospholipid core of the receptors is suggested. Structure activity studies point to a relation with reported effects of intracerebroventricularly administered ACTH on the turnover rate of acetylcholine and the ACTH-induced stretching and yawning syndrome.

Melanocortin analogue Org2766 binds to rat Schwann cells, upregulates NGF low-affinity receptor p75, and releases neurotrophic activity.

Binding of the stable melanocortin(4-9) analogue, Org2766 [Met(O2)-Glu-His-Phe-D-Lys-Phe] to cultured rat sciatic nerve Schwann cells was demonstrated using a biotinylated derivative in semiquantitative histochemical and CELISA assays. Org2766 bound to Schwann cells, but not to fibroblasts, and was displaced maximally by unlabeled Org2766, alpha-MSH and ACTH(1-24). Displacement of Org2766 from the binding sites was considerably reduced by N- and C-truncation of the peptide. Specific binding of Org2766 was also demonstrated in the immortal rat Schwann cell line SCL4.1/F7 and was more pronounced in cells displaying a differentiated morphology. Org2766 and alpha-MSH increased cyclic AMP content of Schwann cells but neither stimulated DNA synthesis when applied alone. However, in the presence of a priming (subthreshold) concentration of the mitogen, cholera toxin, Org2766 and alpha-MSH caused a delayed increase in DNA synthesis. Org2766 did not modulate the expression of several differentiation-related Schwann cell markers. However, Org2766 increased immunoreactivity for p75 low-affinity NGF receptor on Schwann cells and evoked the release of neurotrophic factor(s) that synergized with NGF in stimulating neurite outgrowth in rat DRG neurons. The results indicate that Schwann cells are a primary target for the action of Org2766 and provide evidence for an indirect mechanism by which melanocortins might stimulate neurite sprouting in regenerating peripheral nerve axons.

Binding of a biotinylated neurotrophic ACTH(4-9) analogue, Org 2766, to neurofilament-positive cells in primary or cell line cultures.

To study the putative binding sites of the neurotrophic peptide Org 2766, an analogue of ACTH(4-9) [H-Met(O2)-Glu-His-Phe-D-Lys-Phe-OH], biotinylated forms of the peptide were used. After fixation, cultures of rat spinal cord and dorsal root ganglia were incubated with 4-10 microM of biotinyl-Org 2766 (b-Org 2766). Binding of both N- and C-terminally biotinylated Org 2766 was seen to phase-bright, round cells with thin processes, but not to flat, orthogonal-shaped cells with tapering processes. The b-Org 2766 binding was displaceable by an excess of nonbiotinylated Org 2766. Light and electron microscopy showed that the biotinylated peptide binds to a cytoplasmatic component as well as to the cell membrane. Double-labeling experiments with b-Org 2766 and an antibody (RT-97) to a high molecular weight neurofilament protein in dorsal root ganglion cultures showed, using fluorescence and confocal scanning laser microscopy, that all b-Org 2766 binding cells were neurofilament positive. Biotinylated Org 2766 did also bind to the neuronally differentiated cells in cultures of the human neuroblastoma cell line SK-N-SH, but not to those differentiated into epithelial cells. The present data suggest that the neurotrophic peptide Org 2766 binds specifically to cell types with neuronal characteristics.

Local cerebral glucose uptake in anatomically defined structures of freely moving rats.

Two limitations of the classical [14C]2-deoxyglucose (DG) method are the severe stress to which the restrained animals are exposed, and the difficulties with the anatomical analysis of the autoradiograms. The present study describes modifications which circumvent these limitations. Firstly, rats are provided with two chronic indwelling cannulas to allow blood sampling under unrestrained conditions. Absence of stress is demonstrated by low plasma corticosterone levels in the cannulated rats at the start of the experiment. The second modification concerns the image analysis system. The image of the autoradiogram is superimposed on the image of the identical histologically stained section in order to improve the accuracy of the structure identification. This approach enables the operator to delineate the anatomical brain structure in the histologically stained section and quantify the glucose uptake in the autoradiogram generated from this section. The reproducibility of the present quantitative measuring system is illustrated by glucose uptake measurements in different laminar zones of the various fields in the dorsal hippocampal formation. It is concluded that the present technical improvements of the classically applied [14C]2-deoxyglucose technique provide favourable conditions for the quantitative study on cerebral glucose uptake in normally behaving animals.

Selective fimbria lesions impair acquisition of working and reference memory of rats in a complex spatial discrimination task.

It has been reported that transections of the fimbria-fornix or lesions of the hippocampus selectively impair spatial working memory. Disruptive effects of these lesions on reference memory performance, however, have also been reported. We studied the effects of selective fimbria lesions on the acquisition of a complex spatial discrimination in the cone field. The cone field task is a place learning task that permits the simultaneous assessment of working and reference memory performance. Reproducible bilateral stereotaxic lesions were made by knife-cuts parallel to the midline. Sham lesions consisted of similar knife-cuts that were restricted to the overlying tissue. The rats were randomly started from 1 of 4 positions in order to prevent the development of a fixed food search pattern. On both memory components, fimbria-lesioned rats made about twice as many errors as the sham-lesioned and intact subjects, even after extensive training. Transection of the fimbria caused pronounced cholinergic denervation, predominantly at the more ventral part of the hippocampus, as indicated by reduced acetylcholinesterase histochemistry. Our results suggest a major role of the cholinergic innervation of the ventral hippocampus in spatial discrimination.

Binding of the neurotrophic peptide Org 2766 to rat spinal cord sections is affected by a sciatic nerve crush.

The binding of the neurotrophic peptide, [3H]Org 2766 (55 nM), to rat spinal cord sections was studied, employing quantitative autoradiography. The binding was unevenly distributed over spinal cord structures and was displaceable by non-labelled Org 2766 to a limited extent (35%). Binding could not be displaced by the opiate antagonist, naloxone, indicating that [3H]Org 2766 binding sites are distinct from opiate receptors. However, the exact nature of the binding sites remains to be elucidated. A marked left-right difference in [3H]Org 2766 binding in the dorsal horns of the spinal cord at level L2 was observed, 6 days after unilateral crush lesioning of the sciatic nerve. No such effect was found at level T10. After 28 days, when sensorimotor functioning had completely recovered, the [3H]Org 2766 binding pattern was comparable to that in sham-operated rats again. It is suggested that Org 2766 binds to axonal sprouts or glia in the dorsal horn of the spinal cord.

Angiotensin generation in the brain and drinking: indications for the involvement of endopeptidase activity distinct from cathepsin D.

The dipsogenic activity of two artificial renin substrates, tetradecapeptide and tridecapeptide, was studied. The dose-response curves obtained with these peptides, following intracerebroventricular administration, were similar to that of angiotensin I. The angiotensin II antagonist, Sar1, Ala8-angiotensin II, inhibited the dipsogenic effect of tetradecapeptide, indicating the conversion of the latter peptide into angiotensin II. The lower dipsogenic activity of tridecapeptide points to a conversion of this renin substrate into angiotensin III. Specific inhibition of tetradecapeptide induced drinking by the endopeptidase inhibitor N-acetyl-pepstatin suggests the involvement of an endopeptidase in the conversion of the renin substrates in the brain. Two endopeptidases present in the brain (cathepsin D and renin), were compared with respect to their capacity to generate angiotensin I from artificial renin substrate in vitro. Cathepsin D was active under only acidic pH conditions, whereas renin showed a wider pH range with maximal activity in the non-acidic region. Moreover, cathepsin D did not generate angiotensin I from natural, cerebrospinal fluid-angiotensinogen in vitro, and lacked dipsogenic activity following central administration. Small amounts of renin, however, were able to release angiotensin I from cerebrospinal fluid in vitro. In addition, this enzyme induced high dipsogenic activity upon intracerebroventricular injection. These results support the existence of a functionally active central renin-angiotensin system and provide an argument against the involvement of cathepsin D in the formation of angiotensin I in the brain.

Facilitation of amphetamine-induced rotation by muscarinic antagonists is correlated with M2 receptor affinity.

This study examined the relationship between the affinity of cholinergic drugs for muscarinic receptor subtypes and their potency in potentiating or inhibiting amphetamine-induced rotation. The ascending nigrostriatal dopaminergic pathway was unilaterally lesioned in male Wistar rats using 6-hydroxydopamine. In these rats, ipsiversive rotation induced by amphetamine sulphate (1 mg/kg, s.c.) was dose-dependently inhibited by the cholinergic agonists oxotremorine, RS86 and pilocarpine and by the acetylcholinesterase inhibitor physostigmine. In contrast the cholinergic antagonists scopolamine, secoverine and dicyclomine facilitated amphetamine-induced rotation. Agonist and antagonist potencies were then compared with M1 and M2 binding site affinities estimated by displacing [3H]pirenzepine from forebrain and [3H]QNB from brainstem homogenates. The data suggest a relationship between antagonist potency and M2 binding site affinity.

Immunohistochemical evidence for degeneration of cholinergic neurons in the forebrain of the rat following injection of AF64A-picrylsulfonate into the dorsal hippocampus.

The cholinergic neurotoxin, AF64A-picrylsulfonate, was unilaterally infused into the dorsal hippocampus of Wistar rats (2 nmol/2 microliters/4 min; A 6.2, Ls 1.5, H 6.5, Paxinos and Watson). After 19 days the for immunohistochemical staining of choline acetyltransferase (ChAT). Morphometry and counting of ChAT-immunoreactive profiles revealed shrinkage and disappearance of cholinergic neurons in the medial septum and diagonal band of Broca at the lesioned brain side. These data indicate a retrograde degeneration of cholinergic neurons following injection of AF64A-picrylsulfonate into the dorsal hippocampus of the rat.

Central effects of angiotensins on drinking and blood pressure: structure-activity relationships.

The dipsogenic and the pressor effect following intracerebroventricular injection of angiotensins and several C-terminal fragments were studied. Angiotensin I (ANG I), ANG II, ANG III and C-terminal hexa-, penta, tetra- and tripeptide stimulated water intake in water-replete rats and induced significant pressor responses. In both paradigms the most active peptides (in the pmol range) were ANG II, ANG I and ANG III, in that order. Shorter C-terminal peptides appeared to be active, but had to be injected in the nmol range. Latencies to the onset of drinking were less than 45 s for all peptides tested. The C-terminal dipeptide and other dipeptide fragments did not possess detectable dipsogenic activity. The dipsogenic effect of ANG I was inhibited by pretreatment of animals with the converting enzyme inhibitor SQ 14,225. Drinking induced by both ANG I and ANG (4-8) was antagonized by the ANG II-receptor blocking agent Sar1-Ala8-ANG II. It is concluded that conversion of ANG I into ANG II is a prerequisite for the expression of the observed biological activity in the brain. Short C-terminal fragments are capable of stimulating the ANG II receptors, but a peptide chain of 7 amino acids appears necessary for maximal agonistic activity.

Topography and characteristics of specific binding sites for non-opioid gamma-type endorphins in the rat brain as studied by autoradiography with [35S]Met-desenkephalin-gamma-endorphin.

An in vitro autoradiographic study was performed to characterize specific rat brain binding sites for non-opioid neuroleptic-like gamma-type endorphins, using [35S]Met-des-enkephalin-gamma-endorphin ([35S]Met-DE gamma E; [35]S-beta-endorphins(5-17)) with high specific activity as radioligand. The binding sites appeared to be confined to rat forebrain regions, e.g., orbital cortex, frontal cortex, cingulate cortex, piriform cortex, nucleus accumbens, amygdala, mediodorsal nucleus of the thalamus and arcuate and periventricular nuclei of the hypothalamus. These regions are part of the mesocorticolimbic feedback circuit. Densitometric analysis of the autoradiographs revealed that the density of the binding sites was highest in the mediodorsal nucleus of the thalamus and the amygdala. Concentration-dependent displacement of [35S]Met-DE gamma E (500 pM) with DE gamma E yielded an IC50 of 0.6 nM whereas DE alpha E (beta-endorphin(6-16)) had an IC50 of 210 nM. Various endorphins, sharing the gamma-endorphin C terminus, displaced [35S]Met-DE gamma E to the same extent as non-labelled DE gamma E (at 10(-6) M) whereas non-endorphin peptides did not show displacing capacity. Possible relationships of the binding sites with opioid receptors were investigated. DAMGO (mu) and DPDPE (delta) displaced [35S]Met-DE gamma E to some extent at 10(-6) M whereas U69,593 (kappa) was inactive, suggesting that the binding sites for gamma-type endorphins may resemble mu- and delta-opioid receptors in some aspects. Similarly, relationships with dopamine receptors were investigated. Haloperidol partially displaced [35S]Met-DE gamma E whereas sulpiride, SKF38,393 and 3-PPP at 10(-6) M did not induce significant displacement. Thus, binding sites are distinct from dopamine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

The endogenous muscarinic acetylcholine receptor in Xenopus oocytes is of the M3 subtype.

The muscarinic acetylcholine receptor of the Xenopus oocyte was characterized electrophysiologically. Iontophoretic responses to acetylcholine were inhibited by the muscarinic antagonists 4-DAMP, pirenzepine and AF-DX 116 at respective IC50 values of 7 nM, 15 microM and 2 microM. This antagonist sensitivity order indicates that the receptor is of the M3 muscarinic subtype.