Neuromodulation and Eating Disorders.

PURPOSE OF REVIEW: We review recent evidence on the use of neuromodulation for treating eating disorders (EDs), including anorexia nervosa, bulimia nervosa and binge eating disorder. We evaluate studies on (a) modern non-invasive methods of brain stimulation, such as transcranial magnetic stimulation (rTMS) and transcranial direct current stimulation (tDCS), (b) electroconvulsive therapy (ECT) and (c) more invasive techniques, including deep brain stimulation (DBS). RECENT FINDINGS: Most reports on the clinical applications of neuromodulation in EDs are limited to case studies, case series and small clinical trials. The majority have focused on severe, enduring and hard-to-treat cases of AN. In this population, data suggest that both rTMS and DBS have therapeutic potential and are safe and acceptable. High-quality clinical trials in different ED populations are needed which investigate different stimulation methods, sites and parameters, the use of neuromodulation as stand-alone and/or adjunctive treatment, as well as the mechanisms of action.

Repetitive transcranial magnetic stimulation reduces cortisol concentrations in bulimic disorders.

BACKGROUND: In people with bulimic eating disorders, exposure to high-calorie foods can result in increases in food craving, raised subjective stress and salivary cortisol concentrations. This cue-induced food craving can be reduced by repetitive transcranial magnetic stimulation (rTMS). We investigated whether rTMS has a similar effect on salivary cortisol concentrations, a measure of hypothalamic-pituitary-adrenal axis (HPAA) activity. METHOD: We enrolled twenty-two female participants who took part in a double-blind randomized sham-controlled trial on the effects of rTMS on food craving. Per group, eleven participants were randomized to the real or sham rTMS condition. The intervention consisted of one session of high-frequency rTMS delivered to the left dorsolateral prefrontal cortex (DLPFC). Salivary cortisol concentrations were assessed at four time points throughout the 90-min trial. To investigate differences in post-rTMS concentrations between the real and sham rTMS groups, a random-effects model including the pre-rTMS cortisol concentrations as covariates was used. RESULTS: Salivary cortisol concentrations following real rTMS were significantly lower compared with those following sham rTMS. In this sample, there was also a trend for real rTMS to reduce food craving more than sham rTMS. CONCLUSIONS: These results suggest that rTMS applied to the left DLPFC alters HPAA activity in people with a bulimic disorder.

Neurocognition in bulimic eating disorders: a systematic review.

UNLABELLED: Van den Eynde F, Guillaume S, Broadbent H, Stahl D, Campbell IC, Schmidt U, Tchanturia K. Neurocognition in bulimic eating disorders: a systematic review. OBJECTIVE: The aim of this study was to review the literature on neurocognition comparing people with a bulimic eating disorder in the acute phase of the illness with healthy controls (HC). METHOD: The review follows the PRISMA (preferred reporting items for systematic reviews and meta-analysis) statement guidelines. Three databases (Medline, Web of Science, and Scopus) were searched combining the search terms 'bulimic disorder', 'bulimia nervosa (BN)', 'binge-eating disorder (BED)' with terms referring to cognitive domains (e.g. 'executive functions'). RESULTS: Thirty-seven studies on people with BN and four on people with BED were selected for review. Overall, sample sizes were relatively small [bulimic disorders: median and range 22 (12-83); HC: 27 (13-172)]. The diversity in methodology precluded a meta-analytical approach. People with a bulimic disorder did not present with a clear neurocognitive profile. Inclusion of salient, disorder-related stimuli (e.g. body weight/shape words) in the neurocognitive paradigms tended to generate differences between people with a bulimic disorder and HC. CONCLUSION: Neurocognition in bulimic eating disorders is under researched, and the available evidence is inconclusive. This review outlines strategies for further research in this area.

Immediate cognitive effects of repetitive Transcranial Magnetic Stimulation in eating disorders: a pilot study.

OBJECTIVE: The aim of this study was to evaluate the effects of high frequency repetitive Transcranial Magnetic Stimulation (rTMS), delivered to the dorsolateral prefrontal cortex, on selective attention in people with a bulimic disorder. METHOD: Participants (N=33) were randomised to a single session of real or sham rTMS. They performed a Stroop colour word task before and after the rTMS intervention. Interference scores were calculated as the time difference between completing cards with congruent and incongruent stimuli. RESULTS: Analysis of covariance comparing the interference scores post-rTMS with the pre-rTMS scores as covariates showed no differences between the real and sham groups [F(1,32)=1.110; p=0.301]. DISCUSSION: While methodological issues warrant a cautious interpretation, these pilot data suggest that selective attention is unaffected by a single session of rTMS.

Social appearance anxiety and bulimia nervosa.

Social appearance anxiety is an unexplored concept in eating disorders (ED). It refers to social anxiety surrounding overall appearance, including body shape, and fear of negative evaluation by others. It is potentially relevant to those with bulimia nervosa (BN) as both social anxiety and body image disturbance are commonly experienced by patients. Thirty women with BN and forty healthy controls (HC) completed the Social Appearance Anxiety Scale (SAAS), a 16-item self-report questionnaire. ED cognitions and behaviours were assessed with the Eating Disorders Examination-Questionnaire. Women with BN have significantly higher SAAS scores than HC (z=-6.79, p<0.001). In BN, SAAS scores show significant positive correlation with global ED subscales and dietary restraint. In HC, SAAS scores are correlated with shape, weight, eating concern, and global eating disturbance subscales. Preliminary findings suggest the SAAS is potentially useful in future research concerning overall risk factors for eating disturbance and treatment outcome in BN.

Brain-derived neurotrophic factor (BDNF) and set-shifting in currently ill and recovered anorexia nervosa (AN) patients.

BACKGROUND: Studies of patients with anorexia nervosa (AN) have shown that they do not perform well in set-shifting tasks but little is known about the neurobiological correlates of this aspect of executive function. The aim of this study was to measure serum brain-derived neurotrophic factor (BDNF) and to establish whether set-shifting difficulties are present in people with current AN and in those recovered from AN, and whether serum BDNF concentrations are correlated with set-shifting ability. METHOD: Serum BDNF concentrations were measured in 29 women with current AN (AN group), 18 women who had recovered from AN (ANRec group) and 28 age-matched healthy controls (HC group). Set-shifting was measured using the Wisconsin Card Sorting Test (WCST). Eating-related psychopathology and depressive, anxiety and obsessive-compulsive symptomatology were evaluated using the Eating Disorder Examination Questionnaire (EDEQ), the Hospital Anxiety and Depression Scale (HADS), and the Maudsley Obsessive-Compulsive Inventory (MOCI) respectively. RESULTS: Serum BDNF concentrations (mean+/-s.d.) were significantly lower in the AN group (11.7+/-4.9 ng/ml) compared to the HC group (15.1+/-5.5 ng/ml, p=0.04) and also compared to the ANRec group (17.6+/-4.8 ng/ml, p=0.001). The AN group made significantly more errors (total and perseverative) in the WCST relative to the HC group. There was no significant correlation between serum BDNF concentrations and performance on the WCST. CONCLUSIONS: Serum BDNF may be a biological marker for eating-related psychopathology and of recovery in AN. Longitudinal studies are needed to explore possible associations between serum BDNF concentrations, illness and recovery and neuropsychological traits.

Multidimensional self reports as a measure of characteristics in people with eating disorders.

This study used multidimensional self report assessments to measure perfectionism, impulsivity and obsessive compulsive characteristics in females with anorexia nervosa (AN), bulimia nervosa (BN) and in matched healthy controls (HC). The Frost Multidimensional Perfectionism Scale (FMPS), Barrett Impulsivity Scale (BIS) and Obsessive Compulsive Inventory-Revised (OCI-R) scale were completed by 107 participants (AN=30, BN=26, HC=51), in parallel with clinical measures. Results show that people with AN have the highest scores on the dimensions of the FMPS as well as on the overall score; the AN and BN groups have the highest scores on the dimensions and on the overall score of the OCI-R; on the BIS, the AN and BN groups have the highest scores on the attention subscale, but there are no group differences on the overall BIS scores. In relation to the FMPS, the global score, and the subscales 'concern over mistakes' and 'doubts about actions' are all highly correlated with both eating pathology (Eating Disorder Examination Questionnaire, EDE-Q) and low global functioning (Structured Clinical Interview for DSM IV, SCID). The subscale 'obsessing' on the OCI-R shows a strong correlation with eating pathology. The overall score and also the subscales of the BIS do not show strong correlations with eating pathology or poor global functioning. In conclusion, therapies should seek to address these specific areas which are highly correlated with eating disorder pathology.

The impact of an emotionally expressive writing intervention on eating pathology in female students.

Introduction: Previous research demonstrating emotional influences on eating and weight suggest that emotionally expressive writing may have a significant impact on reducing risk of eating pathology. This study examined the effects of writing about Intensely Positive Experiences on weight and disordered eating during a naturalistic stressor. Method: Seventy-one female students completed an expressive or a control writing task before a period of exams. Both groups were compared on BMI (kg/m2) and the Eating Disorder Examination - Questionnaire (EDE-Q) before the writing task and at 8-week follow-up. A number of secondary analyses were also examined (to identify potential mediators) including measures of attachment, social rank, self-criticism and self-reassurance, stress and mood. Results: There was a significant effect of intervention on changes in the subscales of the EDE-Q (p = .03). Specifically, expressive writers significantly reduced their dietary restraint while those in the control group did not. There was no significant effect of the intervention on changes in BMI or the other subscales of the EDE-Q (Eating, Weight and Shape Concern). There was also no effect of writing on any of the potential mediators in the secondary analyses. Discussion: Emotionally expressive writing may reduce the risk of dietary restraint in women but these findings should be accepted with caution. It is a simple and light touch intervention that has the potential to be widely applied. However, it remains for future research to replicate these results and to identify the mechanisms of action.

Affect systems, changes in body mass index, disordered eating and stress: an 18-month longitudinal study in women.

Background: Evidence suggests that stress plays a role in changes in body weight and disordered eating. The present study examined the effect of mood, affect systems (attachment and social rank) and affect regulatory processes (self-criticism, self-reassurance) on the stress process and how this impacts on changes in weight and disordered eating. Methods: A large sample of women participated in a community-based prospective, longitudinal online study in which measures of body mass index (BMI), disordered eating, perceived stress, attachment, social rank, mood and self-criticism/reassurance were measured at 6-monthly intervals over an 18-month period. Results: Latent Growth Curve Modelling showed that BMI increased over 18 months while stress and disordered eating decreased and that these changes were predicted by high baseline levels of these constructs. Independently of this, however, increases in stress predicted a reduction in BMI which was, itself, predicted by baseline levels of self-hatred and unfavourable social comparison. Conclusions: This study adds support to the evidence that stress is important in weight change. In addition, this is the first study to show in a longitudinal design, that social rank and self-criticism (as opposed to self-reassurance) at times of difficulty predict increases in stress and, thus, suggests a role for these constructs in weight regulation.

Interaction between cytosolic monoamine oxidase and spin-labeled amphetamine and its modification by clorgyline and pargyline.

Interactions between a monoamine oxidase (monoamine: oxygen oxidoreductase deaminating, EC 1.4.3.4) obtained from rat liver cytosol by high speed centrifugation and a biologically active, spin labeled analog of amphetamine have been analyzed. The acetylenic monoamine oxidase inhibitors, pargyline and clorgyline, have been used to modulate the binding of spin labeled amphetamine. Broadening of electron spin resonance lines induced by immobilization of the probe on binding has been used to determine the concentration of bound probe. Pargyline was found to inhibit binding of spin labeled amphetamine by cytosolic monoamine oxidase. Bound spin labeled amphetamine was also displaceable by pargyline. In contrast, clorgyline enhanced the binding of spin labeled amphetamine to the cytosolic monoamine oxidase preparation. Inhibition or enhancement of amphetamine binding was very rapid and occurred during the reversible stage of interaction between the enzyme and the acetylenic compounds.

Selective and nonselective monoamine oxidase inhibitors: behavioral disturbances during their administration to depressed patients.

The occurrence of behavioral disturbances during four-week treatment of depressed patients with the nonselective monoamine oxidase (MAO) inhibitor, phenelzine sulfate (N = 14), the selective MAO-type A inhibitor, clorgyline (N = 12), and the partially selective MAO-type B inhibitor, pargyline hydrochloride (N = 13), was studied. Behavioral disturbances were encountered during treatment with each of the MAO-inhibiting drugs, with an overall incidence of 15% (six of 39 patients). All but one episode met criteria for mania or hypomania. Patients with bipolar illness experienced significantly greater incidences of behavioral disturbances in comparison with patients with unipolar illness (35.3% v 4.5%, respectively). The earliest latency to onset of a behavioral disturbances was 18 days, whereas the mean latencies were 22 to 26 days. Episodes of hypomania were observed after discontinuation of drug treatment in individual patients with unipolar and bipolar illness. Repeated MAO-inhibitor treatment, as part of a crossover study of clorgyline and pargyline, produced an increased severity of behavioral disturbances and a significantly shortened latency to onset.

Phenoxybenzamine partially inhibits alpha 2-adrenoceptors without affecting their presynaptic function.

The turnover of alpha-adrenoceptors was assessed by administering phenoxybenzamine (PBZ) intraperitoneally to rats in order to block the receptors irreversibly. The reappearance of the binding of [3H]prazosin, [3H]clonidine and [3H]rauwolscine in membranes from cerebral cortices was then measured. Maximum inhibition of binding occurred 3 hr after administration of phenoxybenzamine. The binding of [3H]prazosin was inhibited by 95% after administration of phenoxybenzamine (2 X 4 mg/kg, i.p.), and the half life (t1/2) for the alpha 1-adrenoceptor was 1.87 days. The "turnover" of binding for the alpha 2-adrenoceptor ligands ([3H]clonidine and [3H]rauwolscine) was similar: with doses of phenoxybenzamine up to 15 mg/kg (i.p.), the binding of both ligands was inhibited to a maximum of 30%. Maximum recovery occurred 3 days after treatment with phenoxybenzamine and the alpha 2-adrenoceptor has an apparent half life for recovery of 12 hr. Since only partial blockade of alpha 2-adrenoceptors was possible with phenoxybenzamine the possibility that these blocked sites included functional presynaptic autoreceptors was investigated. Clonidine (1 microM) attenuated K+-induced release of preloaded [3H]noradrenaline from cortical synaptosomes prepared from control rats by some 35%. Clonidine inhibited this release of [3H]noradrenaline to the same extent in synaptosomes prepared from rats treated with phenoxybenzamine 3 hr prior to sacrifice. This indicates that the alpha 2-adrenoceptors which are blocked by phenoxybenzamine are not part of the functional receptor population.

The kappa-opiate agonist U50488H decreases the entry of 45Ca into rat cortical synaptosomes by inhibiting N- but not L-type calcium channels.

The selective kappa-opiate agonist U50488H (1-100 microM) significantly reduced the uptake of 45Ca into cortical synaptosomes from the brain of the rat, in a time- and dose-dependent manner. In physiological medium, the maximum inhibition occurred after 2 min; this was approximately 55% (at 100 microM) and the IC50 was 80 nM. Nifedipine (1 microM) had no significant effect on the influx of Ca2+ in physiological medium (containing 5 mM K+), though, in fact, there was an approximately 20% decrease in the presence of 100 microM of drug. Nifedipine, however, did cause a significant blockade of the entry of 45Ca in medium containing 10 or 15 mM K+, demonstrating that L-type channels on synaptosomes were operational under depolarising conditions. Under these depolarising conditions, there was an additive inhibitory effect on entry of 45Ca into synaptosomes when U50488H (1 microM) and nifedipine (1 microM) were incubated together. Treatment of synaptosomes with omega-conotoxin (omega-CgTx, 0.5 microM) resulted in a 35% reduction in the uptake of 45Ca. omega-Conotoxin (0.5 microM) or naloxone (20 microM) abolished the inhibitory effect of U50488H on the uptake of 45Ca, but naloxone did not alter the blockade of L-type Ca2+ channels, caused by nifedipine. In conclusion, the data demonstrate that under depolarising conditions, there are functional L-type calcium channels on nerve endings in the CNS.(ABSTRACT TRUNCATED AT 250 WORDS)

Subcellular distribution of beta-adrenoceptors in brain following administration of antidepressant drugs.

The distribution of specific binding of [3H]dihydroalprenolol([3H]DHA) in sucrose gradients (0.2-1.75 M), containing homogenates of the cortex of rat brain, centrifuged to equilibrium (110,000 g/16 hr), was examined in controls and after treatment with antidepressant drugs. There were no significant changes in the specific binding of [3H]DHA after acute administration of desipramine (DMI, 50 mg/kg) or clorgyline (20 mg/kg), either in terms of the number of receptors or distribution in the sucrose gradient. There was a significant decrease (29%) in the number of beta-adrenoceptors after the chronic regimen with DMI, but again no apparent alteration in the density of the receptor-containing membranes, both samples having a maximum distribution at approximately 1.1 M sucrose. Non-specific binding was maximal at 0.65 M sucrose. Electron microscopy showed that the non-specific binding was largely to myelin and the fraction containing most specific binding was composed of membrane fragments. The activity of Na+ K+ ATPase had a single broad peak (maximum at 1.1 M sucrose). Thus, at the times studied, in vivo desensitisation/internalisation of cortical beta-adrenoceptors did not apparently occur following large acute doses of antidepressant drugs and furthermore the down-regulation which followed the chronic regimen with DMI did not involve migration of receptors into "light density fractions" which are reported to be present after acute exposure to agonists in vitro.

Changes in alpha- and beta-receptor densities in rat brain as a result of treatment with monoamine oxidase inhibiting antidepressants.

The effects of chronic administration of the nonselective monoamine oxidase (MAO) inhibitor, phenelzine, and two selective inhibitors, clorgyline, and pargyline on adrenergic receptor binding and MAO activity were studied in the rat brain. Chronic but not acute administration of both phenelzine and the MAO-A inhibitor, clorgyline, resulted in significant decreases in cortical beta-adrenoreceptor binding ([3H]dihydroalprenolol) of 47 and 24% respectively. In contrast pargyline, which only partially inhibited MAO-A, caused only a nonsignificant (7%) change in rat cortical beta-receptor binding. In a more detailed study, chronic administration of clorgyline also caused changes in alpha-adrenergic receptor binding: by day 3 of treatment, significant changes were already observable in alpha-2-adrenergic receptor binding ([3H]clonidine), although changes in alpha-1 ([3H]WB4101)- and beta ([3H]dihydroalprenolol)-cortical receptor binding were not present until day 10. After 21 days of clorgyline treatment, the Bmax's of alpha-2-, alpha-1- and beta-adrenergic cortical receptors were reduced by 62, 36 and 34% respectively. In brainstem, alpha-2- and beta-receptors were reduced by 60 and 74%. The magnitude of these changes and particularly the rapidity of the alpha-2 changes suggest that clorgyline-induced alpha-2 autoreceptor changes may precede and contribute to the changes in postsynpatic alpha-1- and beta-receptors. These sequential alterations in adrenergic receptors may be pertinent to the efficacy and delay in onset of the clinical changes which follow treatment with these antidepressants.

Effect of desipramine, phenoxybenzamine and yohimbine on beta-adrenoceptors and cyclic AMP production in the rat brain.

In rat brain, the number of beta-adrenoceptors and activity of noradrenaline-dependent adenylate cyclase were examined after treatment with desipramine (7.5 mg kg-1 day-1) for three days alone or in combination with the alpha 2-adrenoceptor antagonist, yohimbine (2 mg kg-1 12 hr-1), or with phenoxybenzamine (7.5 mg kg-1 day-1), which is a more potent inhibitor of alpha 1 than alpha 2-adrenoceptors. The only treatment which significantly decreased the specific binding of the beta-adrenoceptor antagonist, [3H]dihydroalprenolol was the combination of desipramine with yohimbine. Desipramine alone and desipramine with yohimbine also significantly reduced the formation of cyclic AMP in response to incubation with noradrenaline, the response to the drug combination being accounted for by addition of the individual effects of the drugs. The results showed that decreases in the activity of noradrenaline-dependent adenylate cyclase could become apparent before decreases in beta-adrenoceptor numbers. Whether these rapid changes in noradrenaline-dependent adenylate cyclase or in numbers of beta-adrenoceptors which are produced by combination of desipramine with an alpha 2-adrenoceptor antagonist, are of therapeutic value remains to be elucidated.

Circadian regulation of prion protein messenger RNA in the rat forebrain: a widespread and synchronous rhythm.

Although the expression of the normal prion protein in the host is critical to the development of transmissible spongiform encephalopathies, the physiological role of this protein and the processes regulating its expression remain obscure. We now report that the messenger RNA for the prion protein is regulated in the rat brain in a marked circadian manner not only in the suprachiasmatic nuclei, the principal site for the generation of mammalian circadian rhythms, but also in other forebrain regions. The data show a remarkable consistency in the concurrence of a single peak of prion protein messenger RNA at each of the sites early in the animal's phase of increased locomotor activity; behavioural arousal does not, however, appear to affect this expression. We believe this to be the first study demonstrating that the expression of prion protein messenger RNA can change over a relatively short period in vivo. The results are discussed with reference to the range of recently discovered "clock-related" transcripts which also have widespread tissue expression; these include the messenger RNAs for D-box binding protein and thyroid embryonic factor, transcription factors which bind to the prion protein promoter.

On the pharmacology and biochemistry of the amine-uptake mechanism in human blood platelets.

1. The uptake of 5-hydroxytryptamine (5-HT) by human blood platelets in vitro has been studied with the object of identifying the biochemical mechanisms involved.2. Drugs active in adrenergic systems are only moderate inhibitors of uptake, although prenylamine is as active as the less potent tricyclic anti-depressive drugs; phenoxybenzamine is almost inactive as a competitive inhibitor but is effective if pre-incubated with the platelets beforehand. This parallels its pharmacological pattern of action.3. Inhibitors of oxidative phosphorylation do not inhibit 5-HT uptake, but iodoacetate inhibits, if pre-incubated with the platelets; p-chloromercuribenzoate does also, when the platelets are suspended in synthetic medium, but not in plasma.4. Ouabain causes significant inhibition at 10(-7)M; by 10(-6)M it achieves its maximal effect, namely 40% inhibition; in K(+)-deficient medium, uptake falls to 30% of normal; the K(+)-dependent fraction of the uptake includes the ouabain-sensitive component. Mg(++) has no effect.5. A drug not possessing the imipramine structure, which has been tried in the treatment of depressive illness, 4-phenyl bicyclo (2,2,2) octan-1-amine, is a highly potent inhibitor of 5-HT uptake.

An examination of experimental design in relation to receptor binding assays.

1. Real and computer-simulated data were used to examine the efficiency of designs for receptor binding assays. 2. Initially, several different concentrations of [3H]-ketanserin were used in receptor binding studies, using membrane preparations from rat cerebral cortex to establish the form of the binding curves and to investigate the relationship between the variance of the binding measurements and their means. 3. The data showed that the specific binding could be modelled by a simple rectangular hyperbola, and were consistent with the assumption that the binding measurements have a constant coefficient of variation (0.1). 4. Computer-simulated data, with a coefficient of variation of 0.1, were then used to look at the precision of estimates of KD and Bmax obtained through the use of assay designs based on replicate incubations at two radioligand concentrations, or through the use of a geometric sequence of 5-7 radioligand concentrations. In each case, the influence of varying amounts (3.8-50%) of non-specific binding at KD on the precision of these estimates was monitored. 5. The results (a) illustrate the problems which arise in the analysis of receptor binding data when there are relatively high amounts of non-specific binding in combination with a constant coefficient of variation, (b) calculate the errors involved and (c) quantitate the relative merits of 2, 5, 6 and 7 point saturation curves in the estimation of Bmax and KD.

Studies of receptor-mediated inhibition of 45Ca accumulation into synaptosomes.

1. The effects of alpha 2-adrenoceptor and kappa-opiate receptor activation on 45Ca accumulation into rat cortical synaptosomes were examined. 2. Clonidine (1 microM) and U50488H (1 microM) significantly reduced 45Ca accumulation under both resting (5 mM K+) and depolarizing (15-30 mM K+) conditions. 3. The inhibitory effects of the agonists on 45Ca accumulation into synaptosomes were enhanced in the presence of the Na(+)-Ca2+ exchange inhibitor sodium orthovanadate (vanadate, 2 mM), and were not present in mitochondrial preparations. 4. When the agonists were used together, their inhibitory effects were not additive but were, in fact, attenuated. 5. In the presence of the alpha 2-adrenoceptor antagonist idazoxan (1 microM), the inhibitory effect of U50488H on 45Ca accumulation was enhanced. A similar increase in the inhibitory effectiveness of clonidine was observed in the presence of naloxone (20 microM). 6. When synaptosomes were pretreated with 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM), dibutyrylcyclic AMP (db-cyclic AMP, 10 microM) or 8-bromo-cyclic AMP (8Br-cyclic AMP, 10 microM), the inhibitory effects of clonidine and U50488H were abolished, suggesting that a decrease in cyclic AMP production is part of the receptor-effector coupling mechanism of both receptor systems. 7. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.05 microM) increased 45Ca accumulation but did not alter the inhibitory effects of clonidine or U50488H, thus showing that the effects of the agonists are not mediated by protein kinase C. 8. We conclude that alpha 2-adrenoceptor and Kappa-opiate receptor activation dramatically reduce 45Ca influx through Ca21 channels (e.g., by 50%), that there is a functional antagonism between the two receptor systems and that in both cases, the receptor effector mechanism involves a decrease in cyclic AMP production.