Basal Cyclic AMP Production Research Articles

Neurotoxic effects of dietary aluminium.

Neurochemical responses to chronic oral aluminium administration have been studied in rats. Aluminium (0.3%) was added to drinking water of adult rats for four weeks or longer and weanling rats were given aluminium for eight weeks. Selective cognitive impairment was demonstrated in the adult rats. Aluminium inhibited calcium flux and phosphoinositide metabolism, one product of which (inositol 1,4,5-trisphosphate) modulates intracellular calcium levels. In weanling rats aluminium decreased the in vivo concentration of inositol 1,4,5-trisphosphate in the hippocampus. An increase in cyclic AMP concentrations by 30-70% in various brain regions in adult and weanling rats was found. Aluminium enhanced agonist-stimulated but not basal cyclic AMP production in vitro. It was postulated that aluminium inhibits the GTPase activity of the stimulatory G protein, Gs, leading to prolonged activation of Gs after receptor stimulation and increased cyclic AMP production. Aluminium treatment also increased the phosphorylation of microtubule-associated protein 2 (MAP-2) and the 200 kDa neurofilament protein (NF-H) but several other phosphoproteins were unaffected. Concentrations of seven structural proteins--MAP-2, tau, NF-H, NF-M (150 kDa), NF-L (68 kDa), tubulin and spectrin--were measured in rat brain regions by immunoblot methods. MAP-2 was most consistently decreased. These studies show that chronic oral aluminium administration to rats has significant neurochemical consequences. Three sites of action are implicated: altered calcium homeostasis, enhanced cyclic AMP production, and changes in cytoskeletal protein phosphorylation states and concentrations.

In resting COS1 cells a dominant negative approach shows that specific, anchored PDE4 cAMP phosphodiesterase isoforms gate the activation, by basal cyclic AMP production, of AKAP-tethered protein kinase A type II located in the centrosomal region

We employ a novel, dominant negative approach to identify a key role for certain tethered cyclic AMP specific phosphodiesterase-4 (PDE4) isoforms in regulating cyclic AMP dependent protein kinase A (PKA) sub-populations in resting COS1 cells. A fraction of PKA is clearly active in resting COS1 cells and this activity increases when cells are treated with the selective PDE4 inhibitor, rolipram. Point mutation of a critical, conserved aspartate residue in the catalytic site of long PDE4A4, PDE4B1, PDE4C2 and PDE4D3 isoforms renders them catalytically inactive. Overexpressed in resting COS1 cells, catalytically inactive forms of PDE4C2 and PDE4D3, but not PDE4A4 and PDE4B1, are constitutively PKA phosphorylated while overexpressed active versions of all these isoforms are not. Inactive and active versions of all these isoforms are PKA phosphorylated in cells where protein kinase A is maximally activated with forskolin and IBMX. By contrast, rolipram challenge of COS1 cells selectively triggers the PKA phosphorylation of recombinant, active PDE4D3 and PDE4C2 but not recombinant, active PDE4A4 and PDE4B1. Purified, recombinant PDE4D3 and PDE4A4 show a similar dose-dependency for in vitro phosphorylation by PKA. Disruption of the tethering of PKA type-II to PKA anchor proteins (AKAPs), achieved using the peptide Ht31, prevents inactive forms of PDE4C2 and PDE4D3 being constitutively PKA phosphorylated in resting cells as does siRNA-mediated knockdown of PKA-RII, but not PKA-RI. PDE4C2 and PDE4D3 co-immunoprecipitate from COS1 cell lysates with 250 kDa and 450 kDa AKAPs that tether PKA type-II and not PKA type-I. PKA type-II co-localises with AKAP450 in the centrosomal region of COS1 cells. The perinuclear distribution of recombinant, inactive PDE4D3, but not inactive PDE4A4, overlaps with AKAP450 and PKA type-II. The distribution of PKA phosphorylated inactive PDE4D3 also overlaps with that of AKAP450 in the centrosomal region of COS1 cells. We propose that a novel role for PDE4D3 and PDE4C2 is to gate the activation of AKAP450-tethered PKA type-II localised in the perinuclear region under conditions of basal cAMP generation in resting cells.

Impaired cyclic AMP production in the hippocampus of a Down syndrome murine model

Behavioral and learning disturbances have been found in mice with partial trisomy 16, a new model for Down syndrome. Basal production of cyclic AMP in the hippocampus of trisomic mice was shown to be impaired. In addition, the responses of adenylyl cyclase to the stimulation of β-adrenoceptors with isoprenaline and of the catalytic subunit with forskolin were both severely depressed.

Neuropeptide Y inhibits Ca2+ oscillations, cyclic AMP, and secretion in melanotrope cells of Xenopus laevis via a Y1 receptor

The melanotrope cells in the pituitary gland of Xenopus laevis are innervated by neurons containing neuropeptide Y (NPY). In the present study, the mechanism of action of NPY on the melanotropes has been investigated. NPY inhibited in vitro secretion from melanotropes in intact neurointermediate lobes as well as from isolated, single melanotropes. Inhibition of secretion from neurointermediate lobes was mimicked by the NPY analogues PYY and [Leu31,Pro34]NPY, whereas NPY(13-36) was inactive. Secretion from isolated melanotropes was inhibited by [Leu31,Pro34]NPY and NPY(13-36), but NPY(13-36) was 10-fold less potent than [Leu31,Pro34]NPY. Studies on isolated cells revealed that NPY and its analogues inhibited the occurrence of intracellular Ca2+ oscillations with the same potency as they inhibited secretion from isolated cells. In addition to inhibiting basal secretion and spontaneous Ca2+ oscillations, NPY inhibited the basal production of cyclic AMP. On the basis of these results it is proposed that NPY inhibits secretion from Xenopus melanotropes by inhibiting cyclic AMP-dependent spontaneous Ca2+ oscillations through a Y1-like receptor.

Alpha 2-adrenoceptor-mediated inhibition of electrically evoked [3H]noradrenaline release from chick sympathetic neurons: role of cyclic AMP.

This study explores the role of cyclic AMP in electrically evoked [3H]noradrenaline release and in the alpha 2-adrenergic modulation of this release in chick sympathetic neurons. Along with an increase in stimulation-evoked tritium overflow, applications of forskolin enhanced the formation of intracellular cyclic AMP. Both effects of forskolin were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The forskolin-induced increase in overflow was abolished by the Rp-diastereomer of cyclic AMP-thioate, an antagonist at cyclic AMP-dependent protein kinases, and 1,9-dideoxy-forskolin, an inactive analogue at adenylyl cyclase, had no effect on the evoked overflow. A 24-h pretreatment with either cholera toxin or forskolin reduced the subsequent forskolin-induced accumulation of cyclic AMP and inhibited the stimulation-evoked release. Basal cyclic AMP production, however, remained unaltered after forskolin treatment and was enhanced after 24 h of cholera toxin exposure. The alpha 2-adrenergic agonist bromoxidine did not affect the formation of cyclic AMP stimulated by forskolin but reduced electrically evoked release. However, effects of bromoxidine on 3H overflow were attenuated by forskolin as well as by 8-bromo-cyclic AMP. Effects of bromoxidine on [3H]noradrenaline release were paralleled by an inhibition of voltage-activated Ca2+ currents, primarily through a delayed time course of current activation. This effect was abolished when either forskolin or 8-bromo-cyclic AMP was included in the pipette solution. Both substances, however, failed to affect Ca2+ currents in the absence of bromoxidine. These results suggest that the signaling cascade of the alpha 2-adrenergic inhibition of noradrenaline release involves voltage-activated Ca2+ channels but not cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of basal and FSH‐dependent cyclic AMP production in rat seminiferous tubules staged by an improved transillumination technique

The stage-dependent action of follicle-stimulating hormone (FSH) in the rat seminiferous epithelium was investigated in microdissected 1 mm tubule segments, where the precise stage of the cycle was identified by a rapid screening method of live cell squash preparations. For distinction of stages I and II and the substages of VII, new criteria were used. The step 16 spermatids with rapid assembly of outer dense fibers leading to marked increase of flagellar thickness were used for distinction of stages I and II. The form and density of the cytoplasmic lobes of step 19 spermatids was used for recognition of substages of VII. Highest basal production of cyclic AMP (cAMP, measured by radioimmunoassay) was found in stage II of the cycle and stages XIV-I-VI had higher values than did stages VII-XIII. A decline occurred during stage VII and an increase at stage XIV. When stimulated with FSH, highest cAMP secretion was found in stage IV of the cycle; again, stages XIV-I-VI had higher values than did other stages. A small but significant (P less than .01) stimulation was found at substage VIId. FSH-stimulated and basal cAMP productions of different stages were compared, highest values were found at stages IV and XIII, and lowest, at stages VIIa-c and IX of the cycle. Since the FSH-dependent cAMP production is confined to Sertoli cells, and the number of these cells is constant per unit length of seminiferous tubules, the Sertoli cells are obviously under a stage-specific paracrine control by the surrounding spermatogenic cells. Specific steps in cell differentiation, such as spermatogonial proliferation, final maturation of the spermatids (stages I-VII), onset of meiosis (substage VIId), and completion of meiotic divisions (stage XIV) may be involved in this interaction.

The effect of 1,25-dihydroxyvitamin D 3 on human osteoblast-like osteosarcoma cell: Modification of response to PTH

The influence of 1,25-dihydroxyvitamin D 3 [1,25 (OH) 2D 3] on adenylate cyclase responsiveness in cultured osteoblastic cells was studied using a human osteosarcoma cell line SaOS-2. 1,25(OH) 2D 3 treatment had no effect on cell growth, cell protein and alkaline phosphatase activity. 1,25(OH) 2D 3 did not alter the basal production of cyclic AMP (cAMP) in intact cells, but the cAMP formation in response to parathyroid hormone (PTH), isoproterenol (ISO) and cholera toxin was attenuated by 1,25(OH) 2D 3. The response to forskolin, however, was unaffected by 1,25(OH) 2D 3 treatment. Islet activating protein failed to modify these 1,25(OH) 2D 3 effect. In cell free experiments, 1,25(OH) 2D 3 showed similar effect — that is, PTH and ISO-stimulated adenylate cyclase activity were attenuated, but forskolin-stimulated adenylate cyclase was unaffected. 1,25(OH) 2D 3 treatment had no effect on the kinetics of PTH binding to PTH receptor and on the ADP ribosylation of GTP stimulatory binding protein (G s) in SaOS-2 cells. According to these results, 1,25(OH) 2D 3 appeared to change the coupling of G s with adenylate cyclase, but dose not affect receptor, G s and adenylate cyclase themselves, nor GTP inhibitory binding protein.

The effect of hydrogen peroxide on beta-adrenoceptor function in the heart.

A membrane preparation of calf heart left ventricle has been used to study the effect of radical stress on the beta-adrenoceptor complex. To this end the membranes were incubated for 30 minutes with several concentrations of hydrogen peroxide. This resulted in a dose dependent peroxidation of the membrane lipids. Preincubation with hydrogen peroxide in the concentration range 10(-7)-10(-3) M caused an increase in specific (-)-[125I]-Iodocyanopindolol binding, possibly due to a decrease in membrane fluidity as a result of lipid peroxidation, thus making the receptor protein more accessible. Higher concentrations H2O2 reduced the specific (-)-[125I]-Iodocyanopindolol binding, which is most likely the effect of deterioration of the receptor protein by the more pronounced radical stress induced by these higher concentrations. Also adenylate cyclase activity was affected by radical stress. Basal cyclic-AMP production and cyclic-AMP production induced by NaF (10(-2) M) or guanylylimidodiphosphate (10(-4) M), was suppressed after pretreatment with concentrations of H2O2 above 10(-4) M. This indicates a higher sensitivity of the adenylate cyclase toward radical stress when compared to the receptor protein. Our results show that radical stress can perturb beta-adrenoceptor function considerably in the heart.

The regulation of basal and dopamine-sensitive adenylate cyclase by salts of monovalent cations in brain of the American cockroach, Periplaneta americana L.

Production of cyclic AMP by cell-free preparations of cockroach brain is increased by dopamine and salts of monovalent cations. The effects of dopamine are GTP dependent with apparent plateaus in cyclic AMP production occurring at dopamine concentrations of between 1 and 10 μM and at 10 mM. Sodium chloride and potassium chloride elicit similar dose-dependent increases in cyclic AMP production ( K m = 15–20 mM) and choline chloride is slightly less effective ( K m = 29 mM). Sodium acetate has no effect of cyclic AMP production, sodium sulphate decreases cyclic AMP levels and sodium bromide enhances cyclic AMP production ( K m = 10 mM) in a manner similar to that of potassium chloride and sodium chloride. The cyclic AMP increase observed with sodium chloride is not dependent upon added GTP and doses of sodium chloride above 50 mM inhibit the stimulatory effects of 5′-guanylylimido-diphosphate. Forskolin enhances the effects of sodium chloride on cyclic AMP production by increasing maximal production and reducing the K m for sodium chloride. The dopamine-mediated (1.0 mM) increase in cyclic AMP production is reduced in the presence of sodium chloride, potassium chloride, choline chloride and sodium bromide with sodium chloride being most effective. Sodium acetate and sodium sulphate enhance the dopamine-mediated increase in cyclic AMP production. A fixed dose of sodium chloride (120 mM) shifts the dopamine dose-response curve to the right, thereby increasing the K m for the amine and reducing maximal stimulation. Inhibition of [ 3H]piflutixol ([ 3H]PIF) binding by dopamine is reduced in the presence of sodium chloride and lithium chloride but not by potassium chloride and choline chloride. The results indicate that the anionic moiety of the salt is most important in the stimulation of basal cyclic AMP production and in the inhibition of the dopamine-mediated increase in cyclic AMP. Halogens appear to be the most effective anions in mediating both effects. The ability of dopamine to inhibit [ 3H]PIF binding is reduced in the presence of the monovalent cations Na + and Li +.

Characterization of vasopressin receptors of rat urinary bladder and spleen.

By use of tritiated arginine-8-vasopressin (AVP), vasopressin specific binding sites were detected on Sprague-Dawley rat urinary bladder and spleen. In both tissues, one class of high-affinity binding sites was characterized with an equilibrium dissociation constant of 1.61 +/- 0.22 and 1.91 +/- 0.16 nM and a maximal binding capacity of 155 +/- 5 and 110 +/- 11 fmol/mg of protein, for bladder and spleen, respectively. In both tissues, several experimental arguments suggest that these receptors belong to the V1-vascular type: Highly significant correlations were found between the relative agonistic vasopressor activities of eight AVP agonists and their relative abilities to inhibit [3H]AVP binding to the receptors, whereas no such relationship existed when antidiuretic activities were considered. The same profile was also observed with the antagonistic activities of five AVP antagonists. Moreover, AVP (10(-12)-10(-5) M) did not modify the basal cyclic AMP production in either tissue. As cyclic AMP is known to respond to V2 stimulation, the data suggest that the receptors measured are the V1 type. In Dahl rats the receptor characteristics were modulated by salt diet. More interestingly, the number of spleen vasopressin binding sites was always lower in Dahl salt-resistant animals than in the Dahl salt-sensitive animals receiving either a sodium deficient or a 1% NaCl or an 8% NaCl-containing diet. The exploration of vasopressin receptors regulation should facilitate the comprehension of the role played by AVP in different models of experimental hypertension.

Serotonin 5-HT 1 receptors mediate inhibition of cyclic AMP production in neurons

In purified striatal and cortical neurons in primary culture, serotonin (5-HT) stimulated basal cyclic AMP production (EC 50, 0.5 μM) 2.5- and 1.5-fold, respectively. The 5-HT 1 selective agonists, RU 24969 and 8-hydroxy-2- (di-n-propylamino)tetralin (PAT), did not stimulate cyclic AMP production. However, 5-HT, RU 24969 and PAT inhibited VIP-stimulated cyclic AMP formation in a dose-dependent manner. The actions of selective agonists and antagonists at 5-HT receptors mediating attenuation of cyclic AMP production suggest that they may be of the 5-HT 1 subtype.

Functional GIP receptors in a hamster pancreatic beta cell line, In 111: Specific binding and biological effects

Specific binding sites for GIP have been characterized in a insulin-secreting pancreatic tumor cell line, In 111. The specific binding of 125I-GIP is time, temperature and cells concentration dependent. Under steady state conditions (2 hours at 13°C) specific binding of 125I-GIP (0.3 nM) is competitively inhibited by increasing concentrations of native GIP from 10 −10 to 10 −6M. Scatchard analysis reveals the presence of two types of sites: a high affinity (K D = 7 nM)/low capacity (3000 sites/cell) site and a low affinity (K D = 800 nM)/high capacity (150,000 sites/cell) site. No other peptide structurally related or not to GIP, interacts with GIP receptors. GIP (10 −10 to 10 −6 M) is able to potently stimulate insulin release in In 111 cells. At 37°C, the stimulation is rapid and reaches a maximum from 30 minutes of incubation. Half-maximal stimulation is elicited by 10 nM GIP and maximal effect reaches 3 times the basal level of insulin release. Concomitantly, GIP (10 −10 – 10 −6 M) increases the basal cyclic AMP level in the cells. Half-maximal stimulation is observed in the presence of 30 nM GIP, maximal stimulation induced by 10 −6 M peptide increases up to 4 times the basal cyclic AMP production. In conclusion, our data provide the first description of a functional GIP receptor in an insulin-secreting pancreatic beta cell.

Effect of furosemide on parathyroid hormone stimulated guinea pig renal adenylate cyclase and thyrotrophin and fluoride stimulated human thyroid adenylate cyclase.

The effect of furosemide 8 X 10(-4) mol/l an 8 X 10(-5) mol/l on parathyroid hormone stimulated adenylate cyclase was studied in renal tissue slices from guinea pigs. Furosemide caused a dose-dependent inhibition of the effect of parathyroid hormone on production of cyclic AMP, without having any significant effect on the basal cyclic AMP production. Furosemide in similar concentrations did not inhibit the stimulatory effect of thyrotrophin and fluoride in human thyroid homogenates suggesting that furosemide is not an universal inhibitor of adenylate cyclase and that the inhibition is not caused by a direct action of furosemide on the adenylate cyclase enzyme. Furosemide did not interfere with binding of cyclic AMP to cyclic AMP binding protein kinase from rabbit muscle. The results indicate that furosemide exerts an inhibitory influence either upon binding of parathyroid hormone to renal receptors or upon transmission of impulse from receptor to adenylate cyclase. The inhibitory influence of furosemide on parathyroid hormone action in kidney could explain the value of furosemide in the acute treatment of hypercalcaemia, but also suggest that chronic treatment with furosemide might interfere with normal calcium metabolism.

Effect of lutropin and cycloheximide on lutropin receptors and cyclic AMP production in Leydig tumour cells in vitro.

A system to study lutropin-induced desensitization of tumour Leydig cells in vitro has been investigated. Tumour Leydig cells were purified on a Percoll gradient and then incubated for 30 min with lutropin (0-1000ng/ml). The cells were then washed and incubated in suspension media at 32 degrees C. 125I-labelled human choriogonadotropin binding and basal and lutropin-stimulated cyclic AMP production were determined at various times. Initially the cells showed a dose-dependent decrease in human choriogonadotropin binding (1.18 and 0.13fmol/10(6) cells respectively) followed by an increase at 1 h (2.32 and 0.87fmol/10(6) cells respectively). Human choriogonadotropin binding remained elevated in the cells pre-incubated without lutropin, whereas the cells pre-incubated with lutropin showed a dose-dependent decrease over the next 10 h (2.20-0.18fmol/10(6) cells respectively). Basal production of cyclic AMP initially reflected the pre-incubation conditions (1.17-21.19ng/10(6) cells per h for 0-1000ng of lutropin/ml respectively). However, by 1 h there was a marked rise in basal cyclic AMP production which returned to the initial lower values by 4 h. At all time intervals studied, lutropin-induced cyclic AMP production showed a decrease that was proportional to lutropin concentration in the pre-incubated media. The decreases in human choriogonadotropin binding produced by pre-incubations with lutropin (100ng/ml) was partially inhibited by the presence of cycloheximide in the pre-incubation media and totally prevented by the continuous presence of cycloheximide. These results demonstrate that desensitization of tumour Leydig cells occurs after exposure to lutropin in vitro. This desensitization involves both a loss of plasma membrane receptors for lutropin and lutropin-stimulated adenylate cyclase. These events can be prevented by cycloheximide and are therefore probably dependent on protein synthesis.

Thyroid hormone regulation of the catecholamine effects in human adipose tissue.

The possibility of a relationship between the thyroid hormone level and the peripheral action of catecholamines was examined in 4 normal-weight and 19 obese euthyroid subjects as well as in 27 hyperthyroid subjects by comparing the serum thyroid hormone level and the in vitro effect of catecholamines on lipolysis and cyclic AMP accumulation in adipose tissue incubated with and without isopropyl noradrenaline (ISNA) or noradrenaline (NA). ISNA- and NA-induced rates of lipolysis and cyclic AMP production were significantly correlated with free thyroxine index (r = 0.63-0.74) and with the serum triiodothyronine level (r = 0.83-0.87). The thyroid hormone level was neither correlated with basal rate of lipolysis nor with basal cyclic AMP production (r < 0.2). These results suggest that the magnitude of catecholamine-induced cyclic AMP production by peripheral cells may be regulated by the level of circulating thyroid hormones. The effect of thyroid hormones on lipolysis appears to be specifically linked to the action of catecholamines.

Effect of somatomedin A and insulin on cyclic AMP generation in isolated rat hepatocytes.

Insulin and somatomedin A were shown to have inhibitory action on glucagon stimulated but not basal cyclic AMP production in isolated rat hepatocytes. The inhibition was dose-dependent and the potency per mol was about 100 fold higher for insulin than for somatomedin A.

Evidence concerning the anatomical location of the dopamine stimulated adenylate cyclase in the substantia nigra

The dopamine (DA)-sensitive adenylate cyclase in the substantia nigra was assayed in rats which had been subjected to 3 different kinds of brain lesion: (1) unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle; (2) unilateral lesions of the descending strio-nigral and pallido-nigral projections; (3) total lesions of the serotoninergic raphe-nigral pathway. Lesions of the medial forebrain bundle causing 97% depletion of striatal DA, 72% depletion of nigral tyrosine hydroxylase, and no change in nigral glutamate decar☐ylase (GAD), resulted in no change in basal or DA-stimulated cyclic AMP production ipsilateral to the injection. Lesions of the globus pallidus, causing 70% and 79% reductions in GAD and substance P respectively in the ipsilateral nigra, produced a reduction in basal cyclic AMP production and abolished the normal increase in cyclic AMP produced by DA on the side of the lesion. Lesions to the dorsal and median raphe nuclei did not affect the normal DA-sensitive adenylate cyclase response in the nigra. The results suggest that one of the neurotransmitter functions of DA in this brain region may be to modulate the release of γ-aminobutyric acid (GABA) or substance P from synaptic terminals afferent to the nigra.

Effects of an ATP analogue (α,β-methylene-adenosine-5′-triphosphate) on cyclic AMP and cyclic GMP levels, 45Ca efflux, and protein secretion from rat pancreas

The effects of the alpha,beta-methylene analogue of ATP (Ap(CH2)pp), described as a competitive inhibitor of adenylate cyclase (EC 4.6.1.1), were studied in the rat pancreas in vitro. The analogue did not alter basal cyclic AMP production and basal or carbachol-stimulated efflux of 45Ca from isotope-preloaded glands. On the other hand, Ap(CH2)pp reduced the secretory responses to carbachol, carbachol in the presence of dibutyryl cyclic AMP, pancreozymin (PZ), and the calcium ionophore, A-23187. Release of pancreatic protein in response to dibutyryl cyclic AMP itself was unaffected by the ATP analogue, suggesting that the other secretagogues tested share a common, Ap(CH2)pp-inhibitable pathway in their respective stimulatory actions. Though carbachol, PZ, and A-23187 all stimulated a rapid production (30 s) of pancreatic cyclic GMP, these responses were not affected by Ap(CH2)pp. Additional studies with the analogue indicated that it has a slow onset of action (30-45 min), i.e., its effect on secretion is preceded by secretagogue-induced changes in nucleotide levels and calcium efflux. Nonetheless, the analogue may affect cellular calcium homeostasis, if not during the initial events triggering secretion then during those events which maintain continued secretory output in the presence of a stimulus.

Morphine-like drugs inhibit the stimulation of E prostaglandins of cyclic AMP formation by rat brain homogenate.

In homogenate of rat brain, morphine, at concentrations obtainable in vivo, inhibited the stimulation by prostaglandin E1 or E2 of cyclic AMP formation, without inhibiting the basal production of cyclic AMP. Heroin was more atnd methadone less active than morphine, whereas dextrorphan was inactive and naloxone antagonised the effect of morphine. This inhibition may represent a mechanism whereby morphine-like drugs exert their analgesic or other effects.