Effects of naloxone on basal and stress-induced ACTH and corticosterone secretion in the male rat - site and mechanism of action

The acute effects of naloxone upon basal and stress-induced secretion of ACTH and corticosterone (CS) in the adult male rat were investigated. Forty-five minutes subsequent to naloxone injection (5 mg/kg body wt, i.p.), basal serum levels of ACTH (by radioimmunoassay) and of CS (by corticosterone-binding globulin) were more than doubled, as compared to vehicle-treated animals. Upon exposure to either photic or audiogenic stress, the ACTH and CS secretory responses were greater in the naloxone-injected groups. In animals with complete hypothalamic deafferentation basal serum ACTH concentrations were significantly greater than in intact controls (2-fold), and naloxone elicited a further doubling of this parameter. In dexamethasone-pretreated rats (50 μg/animal,4 h prior to naloxone), naloxone had no effect upon ACTH and CS secretion. This study demonstrates: (1) that acute naloxone administration leads to hypersecretion of ACTH, as well as of CS, in the adult male rat; and (2) that its effect is due to an action within the hypothalamo-hypophyseal unit. The data also suggest that these naloxone effects are not mediated by glucocorticoid hormones.     

Oxaprotiline enantiomers stimulate ACTH and corticosterone secretion in the rat

Summary The effect of oxaprotiline (OXA) enantiomers — of which (+)-OXA inhibits noradrenaline (NA) uptake, whereas (–)-OXA does not — on the secretion of adrenocorticotropin hormone (ACTH) and corticosterone was studied in rats. Both enantiomers dose-dependently and with a similar potency increased the plasma level of ACTH and corticosterone, the effect of (–)-OXA on corticosterone being of a longer duration. The stimulation of ACTH secretion and the inability of (+)- and (–)-OXA to increase the plasma corticosterone concentration in animals pretreated with dexamethasone indicate that secretion of the latter hormone results from the action of the enantiomers at a level superior to the adrenal cortex, i.e. the hypothalamus/pituitary.The corticosterone response to (+)- or (–)-OXA was not modified in rats with a selective lesion of NA nerve endings induced by the neurotoxin DSP-4, nor was it affected by the selective ₁-antagonist prazosin, the selective ₂-antagonist yohimbine, the mixed ₁/₂-antagonist phentolamine, the selective dopamine (D₂) receptor antagonist sulpiride and the non-selective 5-hydroxytryptamine (5-HT) receptor antagonist metergoline. These results indicate that neither the NA system nor D₂ and 5-HT receptors are involved in the hormonal response to the OXA enantiomers.Although the (+)- and (–)-OXA-induced stimulation of corticosterone secretion was not antagonized by diazepam, ipsapirone, naloxone, or propranolol, it cannot be excluded that both these enantiomers act as non-specific Stressors.     

Concanavalin A inhibits nicotinic acetylcholine receptor function in cultured chick ciliary ganglion neurons

The effects of various lectins and toxins on neuronal nicotinic acetylcholine receptor function have been studied in primary cultures of chick ciliary ganglion neurons. Neuronal response to acetylcholine receptor activation was measured by a cation flux method at 4 degrees C in a high potassium-low sodium medium designed to stabilize membrane potential near zero, with acetylcholine as the agonist and cesium-137 as the tracer ion. Exposure to 1 mM acetylcholine for 30 s produced a 5-10-fold stimulation of cesium-137 influx. Acetylcholine-stimulated influx was inhibited more than 95% by 10 microM D-tubocurarine, but was insensitive to both 1 microM tetrodotoxin and 1 microM alpha-bungarotoxin. Concanavalin A (50 micrograms/ml) inhibited agonist-induced ion flux by 80% at 4 degrees C. Succinyl-concanavalin A was ineffective at concentrations up to 250 micrograms/ml, and could not protect against the concanavalin A inhibition. However, inhibition by concanavalin A was eliminated by prior incubation of the lectin with 0.2 M alpha-methyl-D-mannoside and subsequent co-incubation with the sugar. Wheat germ agglutinin, lentil lectin, cholera toxin and tetanus toxin were without effect at either 4 degrees C or 37 degrees C. These results suggest a specific interaction between concanavalin A and neuronal nicotinic acetylcholine receptors.     

Calcium permeability of neuronal nicotinic acetylcholine receptor channels in PC12 cells

The conductance properties of neuronal nicotinic acetylcholine receptors (neuronal nAChR channels) in PC12 cells were studied using single channel and whole cell gigaohm seal voltage clamp techniques. Acetylcholine receptor agonists were applied using external pipettes. Neuronal nAChR channels were selective for monovalent cations, and were impermeable to anions. Based on measurements of shifts in reversal potential with changes in external Na and K ion concentrations and evaluation of the GHK constant field relation,P_K/P_Na was 1.45 (ignoring Ca permeability).P_Ca/P_K was 1.75 based on the shift in reversal potential seen when raising the external Ca concentration from 2 to 50 mM (holding the external Na ion concentration constant), and determining the best fit to the extended GHK constant field relation using ionic activities. This value is larger than that determined for the muscle-type nAChR channel. Evaluation of the extended GHK current equations using these permeability coefficients indicated that at subthreshold voltages approximately 5% of total current through neuronal nAChR channels will be carried by Ca ions. Thus, these channels may be important mediators of activity-dependent Ca influx in the nervous system.     

Reserpine inhibits rat anterior pituitary hormone secretion in vitro: Effects of prolactin and ACTH and ultrastructural observations

We measured [³H]prolactin ([³H]Prl) synthesis and secretion in female rat anterior hemipituitary glands incubated in vitro, and immunoassayable Prl secretion from dispersed anterior pituitary cells in a perfused column. Anterior pituitary glands which were incubated in 9 μM reserpine showed a marked inhibition of [³H]Prl secretion but no change in hormone synthesis, thus causing [³H]Prl accumulation within the gland. The same concentration of reserpine produced a similar effect in pituitary glands taken from rats depleted of dopamine with a-methyl-p-tyrosine. Reserpine inhibited Prl secretion from dispersed anterior pituitary cells with a gradual onset and prolonged duration. Thyrotropin-releasing hormone (TRH), but not dibutyryl cyclic AMP (dbcAMP), the calcium ionophore A23187 or excess Ca²⁺, stimulated both [³H]Prl and Prl secretion in the presence of reserpine. In contrast, neither basal nor vasopressin-stimulated ACTH (bio- and immunoassayable) secretion was inhibited by 9 μM reserpine. Ultrastructurally, pituitary glands incubated in reserpine had an increased content of Prl secretory granules. Reserpine thus selectively inhibited Prl secretion, secondarily causing accumulation of both measurable hormone and Prl secretory granules within the pituitary gland. We hypothesize that reserpine interrupted calcium-dependent mechanisms in the stimulus-secretion coupling process to inhibit Prl release.     

Differential effects of basolateral amygdala lesions on behavior, corticosterone, and prolactin responses

The present experiment examines hormone-behavior relationships following manipulation of the amygdala. Affective behavior and levels of corticosterone and prolactin were compared in rats with lesions of the basolateral amygdala and in nonlesioned and sham-operated controls. Animals with lesions of the basolateral amygdala were found to be hyperreactive and to have normal resting levels of corticosterone and prolactin but potentiated corticosterone responses to stress. Normal prolactin stress responses were unaltered by the lesion. The results are discussed in relation to behavioral and endocrine changes seen following other limbic system lesions.     

Measurement of ACTH and prolactin in the dexamethasone suppression test

Plasma concentrations of ACTH and prolactin were measured in psychiatric inpatients at 8 a.m. and 4 p.m. before and after the standard 1 mg overnight Dexamethasone Suppression Test (DST). Plasma concentrations of cortisol were measured at 8 a.m. and 4 p.m., and 11 p.m. before and after 1 mg dexamethasone. Dexamethasone suppressed plasma concentrations of ACTH, prolactin and cortisol in the subject group as a whole. "Cut Points" obtained using Fisher's Exact Test identified plasma ACTH values at 8 a.m. baseline, 4 p.m. baseline and 8 a.m. post-dexamethasone and plasma prolactin values at all four times that significantly differentiated patients with bipolar depressive disorder and major depressive disorder from other psychiatric patients. There were no cut points found at any of the six times for plasma levels of cortisol that significantly differentiated between these two diagnostic groups. Of interest in this subject population, basal (pre-dexamethasone) plasma concentrations were of more diagnostic information than post-dexamethasone values. These pilot findings suggest that monitoring plasma prolactin and ACTH concentrations before and after dexamethasone might increase the sensitivity and specificity of this laboratory test for depression.     

Transcranial magnetic stimulation (TMS) effects on testosterone, prolactin, and corticosterone in adult male rats.

BACKGROUND: Transcranial magnetic stimulation is a relatively new technique for inducing small, localized, and reversible changes in living brain tissue. Although transcranial magnetic stimulation generally results in no immediate changes in plasma corticosterone, prolactin, and testosterone, it normalizes the dexamethasone suppression test in some depressed subjects and has been shown to attenuate stress-induced increases in adrenocorticotropic hormone in rats. METHODS: In this study, serum corticosterone and testosterone concentrations were assayed in male rats immediately and 3, 6, 9, 12, 24, and 48 hours following a single transcranial magnetic stimulation or sham application. Serum prolactin concentrations were determined immediately and 2 hours following a one-time application of either transcranial magnetic stimulation or sham. RESULTS: Transcranial magnetic stimulation animals displayed significantly lower corticosterone concentrations at 6 and 24 hours following a single application compared with sham-control values. Transcranial magnetic stimulation also resulted in lower corticosterone concentrations numerically but not statistically in transcranial magnetic stimulation animals immediately after application (p =.089). No significant differences were found between groups for serum prolactin or testosterone levels at any given collection time point. CONCLUSIONS: These findings 1) suggest that transcranial magnetic stimulation alters the hypothalamic-pituitary-adrenal stress axis and 2) provide time-course data for the implications of the hormonal mechanism that may be involved in the actions of transcranial magnetic stimulation.     

The influence of ACTH and corticosterone on [H]GABA receptor binding in rat brain

Bilateral adrenalectomy (ADX) induces a significant, regionally selective, increase in GABA, but not cholinergic muscarinic orα₁-adrenergic, receptor binding in rat brain. The increase in GABA receptor binding in the midbrain occurs within 72 h of surgery, whereas that found in the corpus striatum becomes evident between 1 and 2 weeks later. These ADX-induced receptor changes are counteracted by the administration of corticosterone, a reversal which can occur within 24 h following a single administration of the steroid. Unlike ADX, hypophysectomy causes a significant reduction in [³H]GABA receptor binding in these two brain areas, an action that is not reversed by corticosterone treatment. Furthermore, systemic administration of either ACTH_1–39 or ACTH_4–10 in unoperated animals causes an increase in midbrain and striatal GABA receptor binding similar to that observed in ADX animals. The increase in [³H]GABA binding observed after ACTH administration appears to be due to the apearance of low affinity, high capacity binding sites not observed in untreated animals. ADX had no effect on high affinity GABA uptake, glutamic acid decar☐ylase or GABA content in the brain regions where receptor modifications were noted. These findings indicate that GABA receptor binding in rat brain can be modified by changes in the circulating levels of ACTH.     

Pharmacological characterization of dopamine, norepinephrine and serotonin release in the rat prefrontal cortex by neuronal nicotinic acetylcholine receptor agonists

Neuronal nicotinic acetylcholine receptors (nAChRs) modulate synaptic transmission by regulating neurotransmitter release, an action that involves multiple nAChRs. The effects of four nAChR agonists, nicotine (NIC), 1,1-dimethyl-4-phenylpiperzinium iodide (DMPP), cytisine (CYT) and epibatidine (EPI) were investigated on [3H]-norepinephrine (NE), [3H]-dopamine (DA) and [3H]-serotonin (5-HT) release from rat prefrontal cortical (PFC) slices. All four agonists evoked [3H]-DA release to a similar magnitude but with a differing rank order of potency of EPI>DMPP approximately NIC approximately CYT. Similarly, all four agonists also increased [3H]-NE release, but with a differing rank order of potency of EPI>CYT approximately DMPP>NIC. NIC-induced [3H]-NE and [3H]-DA release responses were both calcium-dependent and attenuated by the sodium channel antagonist, tetrodotoxin (TTX) and by the nAChR antagonists mecamylamine (MEC) and dihydro-beta-erythroidine (DHbetaE), but not by D-tubocurare (D-TC). The modulation of [3H]-5-HT release by nAChR agonists was distinct from that seen for catecholamines. DMPP produced robust increases with minimal release observed with other agonists. DMPP-induced [3H]-5-HT release was neither sensitive to known nAChR antagonists nor dependent on external calcium. The differences between nicotinic agonist induced catecholamine and serotonin release suggest involvement of distinct nAChRs.     

Modulation of acetylcholine release by nicotinic receptors in the rat brain

Since physostigmine (Phy) is presently used in the experimental treatment of Alzheimer disease (AD) patients by means of intracerebral ventricular (i.c.v.) administration, we designed a study to determine the effect of the drug administered by the same route on the cholinergic system of the rat brain. Particularly, we studied the involvement of nicotinic cholinergic function. The specific conditions required in this experiment were achieved by a series of short-lasting periods of acetylcholinesterase (AChE) inhibition leading to short-lasting increases of acetylcholine (ACh). These were produced by periodic i.c.v. injections of Phy. At 7 days of Phy administration, a small effect on 3H-nicotine binding was seen only in the striatum of the injected side. In rats treated for 13 days, we observed a 120% increase in the stimulated release of 3H-ACh in hippocampal slices of the injected side of the brain. There also was a significant 88% increase in 3H-nicotinic binding in the hippocampus of the same side while muscarinic binding was unchanged. These results suggest a process of upregulation of presynaptic nicotinic autoreceptors in the hippocampus modulating ACh release but no effect on the muscarinic receptors. Our results also suggest that pulses of ACh in analogy to nicotinic stimulation can cause protracted desensitization and eventually inactivation of the receptor leading to its up-regulation. These results are consistent with findings on the release of ACh from cortical biopsies and of a sustained ACh release in the CSF of AD patients following the same treatment.     

Characterization of a putative nicotinic acetylcholine receptor in mammalian brain

The preponderance of evidence suggests that neurons respond electrophysio-logically to ACh and ACh antagonists in a manner consistent with the existence of AChRs, and at least some of these AChRs are nicotinic in nature. The binding of neurotoxins to brain tissue generally occurs in areas where other cholinergic markers exist. The biochemical properties of binding sites are remarkably similar to muscle tissues in size, the presence of a sulf hydryl group that alters ligand affinities, pharmacology, and glycoprotein nature. The dissimilarities, however, are also unmistakable: the association and dissociation rates of toxin binding are different, the inability of Butx to block electrophysiological activity in some tissues, the ratio of toxin sites blocked by MBTA and the apparent lack of antigenic similarity between peripheral nAChRs and brain. In at least one area the relationship to other cholinergic markers has been questioned. It is not likely that brain possesses non-functional sites with such similarity to nAChRs, but it is possible that some sites are non-synaptic and possibly non-specific (see ref. 74 for a discussion of some of these topics). We believe it is possible, however, that the dissimilarities between toxin binding sites in brain and muscle tissue reflect a structural difference between nAChRs. It is likely that toxins bind to nAChRs but do not prevent ACh binding to all sites.Since ACh has apparently not undergone evolutionary change, it is likely that evolutionary changes in cholinergic functionring are reflected in receptor specialization ³⁶. The neuromuscular synapse evolved for a highly specialized function requiring fast, excitatory post-synaptic depolarization. The muscle nAChR probably evolved from a primitive, possibly less specific, form of a molecule sensitive to acetylcholine. Although little is known regarding phylogenetic changes in receptor sites, evidence has recently been presented that the four Torpedo subunits are not immunologically distinct and may have evolved from a common precursor¹¹⁶. While some CNS neurons may have evolved with similar needs, other CNS neuronal groups might necessitate both the possibility of excitation and inhibition. Other synapses may have retained simpler evolutionary traits of mixed nicotinic-muscarinic responsiveness. There is, also, evidence for the interaction of ACh with neuromodulators and other putative neurotransmitters. It is presently not clear whether the characteristics of a receptor are similar when it is pre-synaptic, non-synaptic or inhibitory, in contrast to the peripheral excitatory post-synaptic nAChR.Future studies must determine if currently used neurotoxins bind to a functional CNS nAChR site in any of these cases. We must also find a ligand that specifically binds to and blocks nAChRs that are not affected by Butx. Only then will we be able to address the central questions. Specifically, do Butx and similar neurotoxins bind to an nAChR?, and are there nAChRs which are not bound by currently known neurotoxins?     

Inhibition in corticotrophin and corticosterone secretion following photic stimulation in rats with 6-hydroxydopamine injection into the medial forebrain bundle

Changes in corticotrophin (ACTH) and corticosterone (CS) serum levels, were examined in rats in which either 6-hydroxydopamine or vehicle were injected into the medial forebrain bundle (MFB) and which were sacrificed by decapitation 14 days later. The injection of the neurotoxin, which depleted very significantly the norepinephrine (NE) content of the mediobasal hypothalamus, elevated significantly the basal levels of serum ACTH and CS, but inhibited the rise in the levels of these hormones following photic stimulation. These results would suggest that the hypothalamic NE system has normally an inhibitory effect on basal ACTH and CS secretion and that the hormonal response to photic stimulation is mediated by that system and possibly by NE MFB fibers.     

Hidden function of neuronal nicotinic acetylcholine receptor beta2 subunits in ganglionic transmission: comparison to alpha5 and beta4 subunits

Neuronal nicotinic acetylcholine receptors (nAChR), which modulate fast excitatory postsynaptic potentials (f-EPSP), are located on both pre- and postganglionic sites in the autonomic nervous system (ANS). The receptor subunits alpha3, alpha5, alpha7, beta2 and beta4 are present in autonomic ganglia in various combinations and modulate acetylcholine (ACh) transmission. In the present study, autonomic functions were systemically examined in mice lacking beta2 subunits (beta2-/-) to further understand the functional role of beta2 subunits in modulating ganglionic transmission. The results show normal autonomic functions, both under physiological conditions and in perturbed conditions, on thermoregulation, pupillary size, heart rate responses and ileal contractile reactions. This suggests that the function of beta2-containing receptors in ganglionic transmission is hidden by the predominant beta4 containing receptors and confirms previous studies which suggest that alpha3alpha5beta4 nAChRs are sufficient for autonomic transmission. On the other hand, beta2-containing receptors have only a minor function on postsynaptic responses to ACh, but may modulate ACh release presynaptically, although there is no evidence for this.     

The concept of isoreceptors: Application to the nicotinic acetylcholine receptor and the gamma-aminobutyric acidA/benzodiazepine receptor complex

Summary The concept of isoreceptors offers a possible clue to account for pharmacological and biochemical heterogeneity in specific receptor systems. The existence of isozymes has set the foundation for the definition of isoreceptors. The resulting criteria are applied to two central receptor complexes. Accordingly, the nicotinic acetylcholine receptor is defined as an isoreceptor, and research results on the GABA/benzodiazepine receptor are interpreted under the consideration of the possible existence of isoreceptors.     

Differential adrenocortical responses to neural stimuli following intracerebroventricular injection of nicotinic acetylcholine receptor antibodies in rats

The aim of the present study was to investigate the involvement of central nervous system nicotinic receptors for acetylcholine in the adrenocortical responses to neural stimuli. Adult male rats received a daily intraventricular injection of partially purified nicotinic acetylcholine receptor antibodies (anti-AchR), obtained from a patient with myasthenia gravis, for 5 consecutive days. Control animals were treated similarly with immunoglobulins obtained from normal human serum. Blood samples were obtained under basal conditions or following photic, sciatic nerve stimulation, and acoustic stress. Treatment with anti-AchR increased basal ACTH and corticosterone levels (by approximately twofold) but inhibited the response to acoustic stress. The response to photic and sciatic nerve stimulation stress was not affected. This study demonstrated that nicotinic acetylcholine receptors may be differentially involved in the mediation of adrenocortical responses to neural stimuli.     

Nicotine-induced switch in the nicotinic cholinergic mechanisms of facilitation of long-term potentiation induction

Nicotine facilitates the induction of long-term potentiation (LTP) in the hippocampal CA1 region. The present study reveals the potential mechanisms underlying this effect of nicotine. Timed ACh-mediated activation of alpha7 nicotinic acetylcholine receptors (nAChRs) on pyramidal cells is known to promote LTP induction. Nicotine could suppress this timing-dependent mechanism by desensitizing nAChRs. Timed ACh-mediated activation of alpha7 nAChRs on feedforward interneurons can prevent LTP induction by inhibiting pyramidal cells. Nicotine diminished this ACh-mediated inhibition by desensitizing alpha7 nAChRs, thereby reducing the inhibitory influence on pyramidal cells. In addition to these desensitizing effects, nicotine activated presynaptic non-alpha7 nAChRs on feedforward interneurons to decrease the evoked release of gamma-aminobutyric acid (GABA) onto pyramidal cells. Furthermore, nicotine increased the frequency of spontaneous inhibitory postsynaptic currents (IPSCs) in pyramidal cells, and concomitantly caused a reduction in the size of responses to focal GABA application onto the dendrites of pyramidal cells, suggesting that the nicotine-induced increase in interneuronal activity leads ultimately to a use-dependent depression of evoked IPSCs in pyramidal cells. These nicotine-induced suppressions of inhibition of pyramidal cells were accompanied by enhanced N-methyl-D-aspartate (NMDA) responses in pyramidal cells. Thus, our results suggest that nicotine promotes the induction of LTP by diminishing inhibitory influences on NMDA responses while suppressing the ACh-mediated mechanisms. These ACh-independent mechanisms probably contribute to the nicotine-induced cognitive enhancement observed in the presence of cholinergic deficits, such as those in Alzheimer's disease patients.