Release of amines from acidified stores following accumulation by Transport-P.

1. Transport-P is an uptake process for amines in peptidergic neurones of the hypothalamus. It differs from other uptake processes by its anatomical location in post-synaptic neurones, its functional properties and by the structure of its ligands. Transport-P accumulates amines in intracellular vesicles, derives its energy from the electrochemical proton gradient and is linked to vacuolar-type ATPase (V-ATPase). Transport-P is blocked by antidepressants. We have now studied the release of amines following uptake by Transport-P in a cell line of hypothalamic peptidergic neurones. 2. Release of prazosin was not inhibited by the antidepressant desipramine; as Transport-P is blocked by desipramine, this indicated that amines are released by a mechanism which is independent of Transport-P. 3. Release of prazosin was sensitive to temperature and conformed to the Arrhenius equation. Release was minimal in the range 0-25 degrees C but accelerated exponentially at higher temperatures up to 33 degrees C. The activation energy for the release of prazosin is 83.1 kJ x mol(-1), corresponding to a temperature quotient (Q10) value of 3. 4. Release was accelerated by the organic base chloroquine, the ionophore monensin, bafilomycinA1 which inhibits V-ATPase and by increasing extracellular pH. Thus, retention of prazosin requires an intracellular proton gradient which is generated by V-ATPase. 5. Fluorescence microscopy demonstrated that release of BODIPY FL prazosin was temperature dependent and was accelerated by chloroquine and monensin. 6. Thus, following uptake by Transport-P, amines are accumulated in acidified intracellular stores. Their retention in peptidergic neurones requires intracellular acidity. The amines are released by a temperature-dependent process which is resistant to antidepressants.

alpha(1B) adrenergic receptors in gonadotrophin-releasing hormone neurones: relation to Transport-P.

1. Peptidergic neurones accumulate amines via an unusual uptake process, designated Transport-P. [(3)H]-prazosin binds to alpha(1) adrenoceptors on these cells and is displaceable by unlabelled prazosin in concentrations up to 10(-7) M. However, at greater concentrations of prazosin, there is a paradoxical accumulation of [(3)H]-prazosin which we have attributed to Transport-P. Uptake of prazosin via Transport-P is detectable at 10(-10) M prazosin concentration, is linear up to 10(-7) M and at greater concentrations becomes non-linear. In contrast, in noradrenergic neurones, noradrenaline uptake is linear and saturates above 10(-7) M. In noradrenergic neurones and in non-neuronal cells, there is no uptake of prazosin in concentrations up to 10(-6) M, suggesting that Transport-P is a specialised function of peptidergic neurones. 2. Using a mouse peptidergic (gonadotrophin-releasing hormone, GnRH) neuronal cell line which possesses Transport-P, we have studied the interaction of alpha(1) adrenoceptors with Transport-P. Polymerase chain reactions and DNA sequencing of the products demonstrated that only the alpha(1B) sub-type of adrenoceptors is present in GnRH cells. 3. In COS cells transfected with alpha(1b) adrenoceptor cDNA and in DDT(1) MF-2 cells which express native alpha(1B) adrenoceptors, [(3)H]-prazosin was displaced by unlabelled prazosin in a normal equilibrium process, with no prazosin paradox in concentrations up to 10(-6) M. In DDT(1) MF-2 cells, [(3)H]-prazosin was displaced likewise by a series of alpha(1) adrenergic agonists, none of which increased the binding of [(3)H]-prazosin. Hence, the prazosin paradox is not due to some function of alpha(1) adrenoceptors, such as internalization of ligand-receptor complexes. 4. In neurones which possess Transport-P, transfection with alpha(1b) adrenoceptor cDNA resulted in over-expression of alpha(1B) adrenoceptors, but the prazosin paradox was unaltered. Thus, alpha(1) adrenoceptors and Transport-P mediate distinct functions in peptidergic neurones.

Structural properties of phenylethylamine derivatives which inhibit transport-P in peptidergic neurones.

1. Transport-P is an antidepressant-sensitive, proton-dependent, V-ATPase-linked uptake process for amines in peptidergic neurones of the hypothalamus. It is unusual in its anatomical location in postsynaptic neurones and in that it is activated by its substrate (prazosin). This study examined the structural properties of phenylethylamine derivatives which are substrates for transport-P, as judged by competitive inhibition of the uptake of prazosin 10(-6) M in immortalized hypothalamic peptidergic neurones. 2. A basic amine was essential for activity; absence of the amine or neutralization with a carboxyl group abolished activity. Primary, secondary and tertiary amines were active but quaternary and guanyl amines were inactive. 3. A phenyl group was essential for activity at transport-P. Potency at transport-P was reduced by phenolic hydroxyl groups and enhanced by phenolic halogens. Thus, for maximal potency, the phenyl group should be hydrophobic. Phenolic methoxyl groups had no effect on potency at transport-P. 4. A side chain was necessary for activity at transport-P. Potency at transport-P was reduced by beta-hydroxyl and enhanced by alpha-methyl groups. 5. These findings further distinguish transport-P from other amine uptake processes in the brain.

Visual detection of transport-P in peptidergic neurones.

1. Hypothalamic peptidergic neurones possess an uptake process for amines (transport-P), for which prazosin is a substrate. It is characterized by a paradoxical increase in the accumulation of [3H]-prazosin when the concentration of unlabelled prazosin is increased above 10(-7) M. This increase is due to activation of a proton-dependent, vacuolar-type ATPase-linked pump that is blocked by tricyclic antidepressants. This study utilized a fluorescence method to detect amine uptake in individual cells. 2. Prazosin is fluorescent but most of its emission spectrum is in the ultraviolet range. We therefore used an analogue of prazosin in which the furan ring had been substituted with a fluorescent group, BODIPY FL. This compound's emission maximum is in the green part of the visible spectrum. 3. BODIPY FL prazosin accumulated in immortalised peptidergic neurones and the characteristic emission spectrum of the compound was evident in these cells. Accumulation of BODIPY FL prazosin was saturable and was inhibited by the tricyclic antidepressant desipramine and by unlabelled prazosin. As previously described for prazosin, uptake of BODIPY FL prazosin was blocked by cold temperature and by the organic base chloroquine. Thus, prazosin and BODIPY FL prazosin were accumulated by the same uptake process. 4. BODIPY FL prazosin accumulated in a granular distribution, which is compatible with storage in intracellular vesicles. 5. Hypothalamic cells from foetal rats in primary culture also accumulated BODIPY FL prazosin by a desipramine-sensitive process. Uptake was predominantly in neurones and glial cells did not accumulate the amine. 6. Fluorescent detection provides visual evidence for amine uptake in peptidergic neurones and should enable detailed study of the distribution of this process in the brain.

Binding and competitive inhibition of amine uptake at postsynaptic neurones (transport-P) by tricyclic antidepressants.

1. We have provided evidence for a novel amine uptake process for which prazosin is a substrate in postsynaptic neurones, characterized by a paradoxical increase in accumulation of the radioligand when the concentration of the unlabelled drug is increased above 10(-7) M. This increase is due to activation of a proton-dependent, vacuolar type-ATPase-linked uptake process which is blocked by desipramine but is resistant to reserpine. We have now examined the effects of tricyclic antidepressants on this uptake system in a cell line derived from hypothalamic peptidergic neurones, known to be innervated by noradrenergic nerve terminals in vivo. 2. [3H]-imipramine bound to the cells and was displaced by unlabelled imipramine, desipramine, amitriptyline and nortriptyline. The data fitted a single binding site model. This is the first demonstration of antidepressant binding sites in postsynaptic neurones. 3. There was no increase in the binding of [3H]-imipramine at high concentrations of unlabelled imipramine, suggesting that antidepressants inhibit uptake but are not themselves accumulated by peptidergic gonadotrophin releasing hormone neurones. 4. Accumulation of prazosin was competitively inhibited by antidepressants. Tertiary amines were slightly more potent than secondary amines and the presence of a nitrogen atom in the heterocyclic ring enhanced blocking activity. 5. The affinities of the antidepressants for the uptake process are within the range of plasma concentrations that are observed during therapeutic use of these compounds. Since it is likely that this uptake process has a physiological function, its inhibition by antidepressants may provide a new avenue for investigating the mechanism of action of these compounds.

Functional properties of the uptake of amines in immortalised peptidergic neurones (transport-P).

1. Most neurotransmitters are inactivated by uptake into presynaptic nerve terminals and into glial cells. We recently provided evidence for uptake of amines in postsynaptic neurones. Uptake was evident at nanomolar concentrations of prazosin, but at concentrations of unlabelled prazosin greater than 10(-7) M, there was a further activation of uptake, manifested by a paradoxical increase in accumulation of the radioligand. We have now studied further characteristics of amine uptake in immortalised gonadotrophin-releasing hormone (GnRH) neurones. Control cells included SK-N-SH neuroblastoma cells (which possess presynaptic type amine transporters) and non-neuronal (COS-7) cells. 2. [3H]-prazosin bound to intact GnRH cells and was displaced by unlabelled prazosin in concentrations of 10(-9) to 10(-7) M. However, at higher concentrations of unlabelled prazosin, there was an increase in apparent [3H]-prazosin binding, as we had previously described. This paradoxical increase in accumulation of the radioligand was abolished by desipramine. 3. Desipramine had no effect on the association of prazosin with COS-7 cells. There was no paradoxical increase in accumulation of [3H]-prazosin in COS-7 cells, indicating that this effect requires the presence of a desipramine-blockable uptake process. 4. The increase in binding of the radioligand that was observed in the GnRH cells is not a general property of neuronal transporters; in SK-N-SH cells, there was no increase in accumulation of (-)-[3H]-noradrenaline in the presence of concentrations of unlabelled (-)-noradrenaline greater than 10(-7) M. 5. The uptake of prazosin and the increase in accumulation of [3H]-prazosin were abolished in the cold, indicating that this is an active, energy-requiring process. 6. Desipramine-sensitive uptake of prazosin was demonstrable in the GnRH cells in the absence of sodium. Further, the Na+/K(+)-ATPase inhibitor, vanadate, abolished noradrenaline uptake in SK-N-SH cells but had no effect on prazosin uptake in GnRH cells. Thus, the uptake of prazosin does not derive its energy from the sodium pump. 7. Prazosin uptake was inhibited by the V-ATPase inhibitor bafilomycin A1, the H+/Na+ ionophore, monensin and the organic base, chloroquine, indicating that uptake derives its energy from a proton pump. In contrast to other proton-dependent amine transporters, the uptake of prazosin was unaffected by reserpine. 8. Increasing extracellular pH did not increase the uptake of prazosin into GnRH cells, indicating that it is unlikely to be due to non-specific diffusion and concentration of a lysosomotropic drug into intracellular acidic particles. 9. The uptake of prazosin was unaffected by steroid hormones. 10. In COS-7 cells transfected with alpha 1-adrenoceptor cDNA, [3H]-prazosin was displaced by unlabelled prazosin without causing an increase in binding of the radioligand. This indicated that the increase in accumulation of the radioligand is unlikely to be due simply to some function of alpha 1-adrenoceptors. 11. Thus, peptidergic neurones possess an uptake process with properties that are distinguishable from known amine transporters.

High-affinity uptake of noradrenaline in postsynaptic neurones.

1. Neurotransmitters released from nerve endings are inactivated by re-uptake into the presynaptic nerve terminals and possibly into neighbouring glial cells. While analysing the functional properties of alpha 1-adrenoceptors in the hypothalamus, we observed a high-affinity uptake process for noradrenaline in postsynaptic peptidergic neurones. 2. In primary hypothalamic cell cultures and in a hypothalamic neuronal cell line, [3H]-prazosin bound with high affinity and was displaced by unlabelled prazosin in concentrations of 10(-10) to 10(-7) M. However, at concentrations of unlabelled prazosin above 10(-7) M, there was a paradoxical increase in apparent [3H]-prazosin binding. 3. Methoxamine, an alpha 1-adrenoceptor ligand that is not subject to significant neuronal uptake, displaced [3H]-prazosin but did not cause the paradoxical increase in the apparent binding of [3H]-prazosin. Cooling the cells to 4 degrees C reduced the total amount of prazosin associated with the cells; under these conditions, methoxamine almost completely inhibited [3H]-prazosin binding to the cells. 4. In the presence of desipramine (DMI), unlabelled prazosin displaced [3H]-prazosin as before, but no paradoxical increase in apparent binding was seen above 10(-7) M. 5. The paradoxical increase of [3H]-prazosin binding was not observed in membrane preparations of hypothalamic neurones. These findings indicated that the paradoxical increase in apparent [3H]-prazosin binding was due to a cellular uptake process that becomes evident at high concentrations of the ligand. 6. DMI (10(-5) M) had no effect on the specific binding of [3H]-prazosin. The presence of alpha1-adrenoceptors was confirmed by binding of [125]-HEAT, but [3H]-idazoxan (an alpha2- ligand) did not bind to the cells.7. The uptake of prazosin obeyed the Michaelis-Menten model, with similar Km and Vmax values in both types of cultures.8. Noradrenaline was taken up with high affinity by both types of cultures. (+/-)-[3H]-noradrenaline uptake was reduced by DMI and by excluding sodium from the medium, indicating that this process has some of the properties of uptake 1. (+/-)-[3H]-noradrenaline uptake in the cell line was unaffected by testosterone.9. The measured uptake of (-)-noradrenaline in the cell line was considerably increased by blockade of catechol-omicron-methyl-transferase and monoamine oxidase, suggesting that (-)-noradrenaline is metabolized to lipophilic products that escape across the plasma membrane.10. Studies in rats, in which the noradrenaline isomer 6-hydroxydopamine was used, suggested that the post synaptic uptake process is operative in hypothalamic CRH and vasopressin neurones in vivo.11. The Km for (-)-noradrenaline was within the range for the high affinity uptake, process in noradrenergic neurones. Uptake takes place in concentrations at which noradrenaline activates alpha1-adrenoceptors.Removal of noradrenaline from the vicinity of the receptors may prevent desensitization,thus maintaining the responsiveness of postsynaptic neurones to the actions of the neurotransmitter.

Adrenergic control of the secretion of anterior pituitary hormones.

The hypothalamic hypophysiotrophic neurones are densely innervated by adrenergic and noradrenergic nerve terminals. Activation of alpha 1-adrenoceptors located in the brain stimulates the secretion of ACTH, prolactin and TSH. The effects of the alpha 1-adrenoceptors seem to be exerted on hypothalamic neurones that secrete vasopressin, CRH-41 and TRH. These mechanisms are important in the physiological control of the secretion of ACTH and TSH in humans. alpha 2-Adrenoceptors are not involved in the control of secretion of these hormones under basal conditions in humans. However, alpha 2-adrenoceptors exert an inhibitory effect that acts as a negative feedback mechanism, limiting excessive secretion of these hormones. There is no convincing evidence for the involvement of beta-adrenoceptors in the control of the secretion of these three hormones in humans. Studies on cultured anterior pituitary cells suggested that adrenaline and noradrenaline may influence the secretion of ACTH, prolactin and TSH directly at the level of the pituitary. However, these effects are not demonstrable in humans, and are likely to be due to alterations in the pituitary adrenoceptors during culture. In the case of growth hormone, activation of alpha 2-adrenoceptors located in the brain stimulates secretion of this hormone both by increasing the secretion of GHRH and by inhibiting the secretion of somatostatin. Activation of beta-adrenoceptors inhibits the secretion of growth hormone via an increase in the secretion of somatostatin. The effects of the central alpha 2- and beta-adrenoceptors are important in the physiological control of growth hormone secretion in humans. A considerable amount of evidence implicates brain alpha 1-adrenoceptors in the control of secretion of the gonadotrophins in experimental animals, but, despite intensive study, no convincing evidence has been found in humans of reproductive age.

Activation of central alpha 1-adrenoceptors in humans stimulates secretion of prolactin and TSH, as well as ACTH.

In normal male volunteers, intravenous infusions of the alpha 1-adrenergic agonist methoxamine stimulated the secretion of prolactin, thyroid-stimulating hormone (TSH), and adrenocorticotropic hormone (ACTH), and the effects were abolished by pretreatment with the alpha 1-antagonist prazosin. To investigate the site of action of methoxamine, its effects were compared with those of equipotent doses of norepinephrine, an alpha 1-agonist that reaches the pituitary gland and the median eminence after an intravenous infusion but, unlike methoxamine, does not cross the blood-brain barrier. Norepinephrine did not stimulate secretion of prolactin, TSH, or ACTH, suggesting that the stimulant alpha 1-adrenoceptors are located in the central nervous system and not directly on the pituitary gland or in the periphery. The alpha 2- and beta-adrenoceptor agonist properties of norepinephrine could not account for the differences from methoxamine, as pretreatment with prazosin did not modify hormone concentrations after norepinephrine. Methoxamine had no behavioral stimulant effects, as judged by visual analog scales that were sensitive to physiological changes in behavioral arousal. In four patients with hypothalamic dysfunction but responsive pituitary corticotrophs, methoxamine had no stimulant effect on the secretion of ACTH, confirming that the alpha 1-adrenoceptors that stimulate ACTH secretion are not located directly on the pituitary. None of the drugs had an effect on the secretion of growth hormone or the gonadotrophins.

Central noradrenergic lesion impairs the adrenocorticotrophin response to release of endogenous catecholamines.

Activation of hypothalamic α(1) -adrenoceptors stimulates the secretion of corticotrophin-releasing factors which in turn stimulate pituitary adrenocorticotrophin (ACTH). This mechanism is important in the physiological control of ACTH secretion. This study assesses the feasibility of using the ACTH response to release of endogenous catecholamines as a means of detecting a hypothalamic noradrenergic lesion in vivo. Intracerebroventricular infusion of the catecholamine neurotoxin, 6-hydroxydopamine, was used to destroy noradrenergic nerve endings in rats, with the purpose of producing a model that could be used to study alterations in ACTH responses that may result from a lesion involving central noradrenergic neurons. 6-Hydroxydopamine (250 µg icv) significantly reduced hypothalamic noradrenaline content, indicating damage to noradrenergic nerve endings, without affecting postsynaptic receptor function, as judged by preservation of the effect of a selective α(1) -adrenergic agonist. Pharmacological release of endogenous catecholamines, effected by combined administration of a catecholamine precursor and an α(2) -adrenergic antagonist, stimulated the secretion of ACTH in control, but not in 6-hydroxydopamine-treated rats. Degeneration of hypothalamic noradrenergic nerve endings is not followed by denervation hypersensitivity, and is therefore accompanied by impairment of the ACTH response to release of endogenous catecholamines.

Receptors and neurosecretory actions of endothelin in hypothalamic neurons.

Primary cultures of rat hypothalamic neurons were found to secrete the potent calcium-mobilizing and mitogenic peptide endothelin (ET) and to contain specific ET binding sites with higher affinity for ET-1 and ET-2 than ET-3. ET receptors of similar specificity were also identified in two gonadotropin-releasing hormone (GnRH) neuronal cell lines (GT1-1 and GT1-7). In both primary cultures and GnRH neurons, receptor binding of ETs led to marked and dose-dependent increases of inositol phosphates; inositol bis-, tris-, and tetrakisphosphates increased promptly, reached a peak within 2 min, and returned toward the steady-state levels during the next 10 min. ET-1 was more potent than ET-3 in mobilizing inositol phosphates, consistent with its greater affinity for the ET receptors in these cells. ET also stimulated GnRH secretion from perifused hypothalamic cultures and GnRH cell lines, with a sharp increase followed by a prompt decline to the basal level. These data show that ET is produced in the hypothalamus and acts through calcium-mobilizing ET receptors in normal and transformed secretory neurons to stimulate GnRH release. These actions of locally produced ETs upon GnRH-secreting neurons indicate that the vasoconstrictor peptides have the capacity to regulate neurosecretion and could participate in the hypothalamic control of anterior pituitary function and gonadotropin secretion.

FMRF-amide and L-Arg-L-Phe increase blood pressure and heart rate in the anaesthetised rat by central stimulation of the sympathetic nervous system.

The neuropeptide FMRFamide (L-Phe-L-Met-L-Arg-L-Phe-NH2) increases mean arterial blood pressure (MABP) and heart rate (HR) in the anaesthetised rat at concentrations ranging from 10-1000 micrograms/kg. Here, we demonstrate that peptides containing L-arginyl-L-phenylalanine (L-Arg-L-Phe), the C-terminal sequence of FMRFamide, mimic its haemodynamic effects. L-Arg-L-Phe was approximately 4 fold more potent in increasing MABP and HR than FMRFamide. In 40 different peptides investigated, the following order of potency of the effective compounds was established: L-Arg-L-Phe-L-Ala = L-Arg-L-Phe greater than FMRFamide greater than L-Met-L-Arg-L-Phe = L-Arg-L-Trp greater than L-Arg-L-Tyr greater than D-Arg-L-Phe = L-Arg-L-Phe-OMe greater than L-Arg-L-Leu = L-Arg-L-Ile greater than L-Lys-L-Phe greater than L-Arg-L-Met. L-Arg-L-Phe or FMRFamide did not cause any pressor response in pithed rats, indicating a central mechanism of action. In anaesthetised rats, intravenous injections of FMRFamide or L-Arg-L-Phe (100 micrograms/kg) were associated with a 2-3 fold increase in plasma noradrenaline levels, whereas plasma adrenaline levels remained unchanged. Thus, L-Arg-L-Phe may represent the active principle of FMRFamide acting by a central mechanism involving the release of noradrenaline from sympathetic nerve terminals.

Brattleboro rats have deficient adrenocorticotropin responses to activation of central alpha 1-adrenoceptors.

This experiment was designed to test further the hypothesis that vasopressin is the major mediator of the ACTH response to activation of central alpha 1-adrenoceptors in the rat. The alpha 1-adrenergic agonist methoxamine was given intracerebro-ventricularly to conscious vasopressin-deficient (homozygous Brattleboro) and normal rats bearing venous and intracerebro-ventricular cannulae. Methoxamine stimulated the secretion of ACTH in the normal, but not in the vasopressin-deficient, rats. The data confirm that vasopressin, rather than CRH-41 or oxytocin, is the major hypothalamic peptide that mediates the effects of central alpha 1-adrenoceptors on the pituitary corticotrophs.

Vasopressin mediates alpha 1-adrenergic stimulation of adrenocorticotropin secretion.

In conscious rats bearing venous and cerebroventricular cannulae, central administration of the alpha 1-adrenergic agonist methoxamine stimulated the secretion of ACTH, and the effect was reduced by the alpha 1-antagonist prazosin. Methoxamine was more potent in stimulating ACTH secretion when injected icv than peripherally, suggesting that the stimulant alpha 1-adrenoceptors are located in the brain rather than in the periphery. In order to investigate the relative roles of hypothalamic CRF-41 and vasopressin as mediators of the stimulant effects of alpha 1-adrenoceptors on ACTH secretion, we examined the effects of equipotent doses of antagonists to CRF-41 and vasopressin on the ACTH responses to methoxamine. The effect of methoxamine was reduced by the vasopressin antagonist dPTyr(Me) arginine vasopressin but not by the CRF-41 antagonist alpha-helical CRF-9-41, suggesting that vasopressin is more important than CRF-41 in mediating the effects of alpha 1-adrenoceptors on ACTH secretion. However, the combination of the two antagonists caused a reduction in the ACTH response to methoxamine that was greater than that of the vasopressin antagonist alone. This suggested that CRF-41 plays some role in this response, possibly by enhancing the activity of vasopressin in a synergistic manner. These two hypothalamic peptides seem to account for most of the ACTH releasing activity of alpha 1 adrenoceptor activation.

The role of alpha-2-adrenoceptors in the control of ACTH secretion; interaction with the opioid system.

This study examined the effects of an alpha-2-adrenoceptor antagonist on the secretion of ACTH basally and in response to the opioid antagonist naloxone, which is known to stimulate ACTH secretion by an adrenergic mechanism. Eight normal men were given, in double-blind, random order, intravenous infusions of normal saline (placebo), idazoxan (alpha-2-adrenoceptor antagonist), naloxone and the combination of idazoxan and naloxone. Naloxone increased plasma ACTH and cortisol concentrations in comparison to placebo. Idazoxan significantly enhanced the ACTH and cortisol responses to naloxone but had no effect on plasma ACTH or cortisol concentrations when given alone. These findings suggest that during some conditions of increased ACTH secretion, inhibitory alpha-2-adrenoceptors are activated and that these receptors limit the ACTH response. This provides an explanation for some of the apparent contradictions in interpreting the data from previous studies on the effects of catecholamines on the secretion of ACTH.

Modulation of the actions of tyrosine by alpha 2-adrenoceptor blockade.

1. Eight normal subjects were given, in double-blind, random order L-tyrosine 50, 250 and 500 mg kg-1 and placebo orally. Plasma tyrosine concentrations rose in a dose-dependent manner, without affecting the concentrations of the other large neutral amino acids. Tyrosine stimulated the secretion of prolactin and thyrotrophin (TSH) but had no effect on the plasma concentrations of adrenocorticotrophic hormone (ACTH), cortisol, growth hormone or the gonadotrophins. 2. The lack of a stimulant effect of tyrosine on ACTH secretion was presumed to be due to activation of one of the negative feedback mechanisms that control the rate of synthesis and release of the catecholamines, and this hypothesis was tested by examining the effects of the alpha 2-adrenoceptor antagonist idazoxan on the actions of tyrosine. 3. Seven normal males were given on 6 separate occasions tyrosine 250 and 500 mg kg-1 and placebo orally following pretreatment with saline and idazoxan (0.1 mg kg-1 i.v.). Following pretreatment with idazoxan, tyrosine stimulated the secretion of ACTH and noradrenaline in a dose-dependent manner, although neither tyrosine nor idazoxan on their own had any effect on the secretion of either substance. 4. The lack of effect of tyrosine when given on its own appears to be due, to partly, to activation of alpha 2-adrenoceptors, which inhibit the release of noradrenaline. Idazoxan caused a small increase in systolic blood pressure, both when given on its own and in combination with tyrosine. Neither tyrosine nor idazoxan had any significant effect on the state of behavioural arousal, as measured by visual analogue scales, or on the secretion of growth hormone or the gonadotrophins.

Adrenergic mechanisms in the control of corticotrophin secretion.

Although the data on the effects of adrenergic mechanisms on the secretion of ACTH had seemed confusing, most of the discrepancies are probably explicable on the basis of methodological differences. In the present state of knowledge, the following conclusions seem reasonable. (1) In both man and rat, activation of central alpha-1 adrenoceptors is followed by increased ACTH secretion and this mechanism is important in the control of secretion of this hormone under some physiological circumstances. (2) In man, peripheral circulating catecholamines do not stimulate ACTH secretion under physiological conditions. This conclusion probably also applies to the anterior pituitary corticotrophs of the rat. (3) In the rat, beta-2 adrenoceptor agonists stimulate the intermediate lobe by a direct action that is physiologically relevant. (4) The role of central beta and alpha-2 adrenoceptors requires further investigation.

Effects of catecholamines on secretion of adrenocorticotrophic hormone (ACTH) in man.

The hypothalamus receives a rich supply of adrenergic and noradrenergic nerve fibres from the brain stem, terminating in many hypothalamic regions, including the paraventricular nucleus, which is the site of the cell bodies of corticotrophin releasing factor (CRF) neurones in man. Experimental evidence has shown that an alpha 1 adrenoceptor mechanism stimulates adrenocorticotrophic hormone (ACTH) secretion in man. The site of action of this mechanism seems to be within the blood brain barrier, presumably modulating the secretion of the CRF complex. This mechanism is important in the control of ACTH secretion in some physiological conditions in healthy subjects.

Thymoxamine: lack of antihistaminic effects in clinical doses in man.

The purpose of this study was to investigate whether the alpha 1-adrenoceptor antagonist thymoxamine possesses antihistaminic activity in clinical doses in man, as has been reported on the guinea pig ileum in vitro. Five normal subjects were given on three separate occasions intravenous infusions of thymoxamine (0.15 mg kg-1 loading dose followed by 0.15 mg kg-1 h-1), chlorpheniramine (1.5 mg loading dose followed by 1.5 mg h-1) and normal saline (placebo). Intravenous bolus doses of histamine (1 and 2 micrograms kg-1) were given after pretreatment with propranolol 10 mg to block the beta-adrenoceptor agonist effects of the catecholamines released by the histamine injections. Histamine caused a dose-dependent reduction of FEV1 and FVC that was antagonised by chlorpheniramine but not by thymoxamine, suggesting that thymoxamine has no antihistaminic activity in the doses used in man. Thymoxamine caused a small enhancement of the bronchoconstrictor effect of the lower dose of histamine. The relatively selective action of thymoxamine makes it a suitable agent for the investigation of alpha 1-adrenoceptors.