Aspects of tic like behaviour and serotonergic control

Tic-like movements in rodents bear close similarities to those observed in humans both pharmacologically and morphologically. Pharmacologically, tics are modulated by serotonergic and dopaminergic systems and abnormalities of these systems have been reported in Tourette's Syndrome (TS). Therefore, serotonergic and dopaminergic modulation of tics induced by a thyrotrophin-releasing hormone (TRH) analogue were studied as possible models for TS. The TRH analogue MK771 induced a variety of tic like movements in mice; blinking fore-paw-licking and fore-paw-tremor were quantified and serotonergic and dopaminergic modulation was investigated. The selective dopamine D1 receptor antagonists SCH23390 and SCH39166 and dopamine D2 antagonists raclopride and sulpiride had no effect on MK771 induced blinking. The D1 antagonists attenuated fore-paw-tremor and -licking while the D2 antagonists were generally without effect on these behaviours. Ketanserin (5-HT2A/ alpha-1 antagonist) and ritanserin (5-HT2A/2C antagonist) were able to attenuate MK771-induced blinking and ketanserin, mianserin (5-HT2A/2C antagonist) and prazosin (alpha-1 adrenoceptor antagonist) were able to attenuate MK771-induced fore-paw-tremor and -licking. The 5-HT2C/2B antagonist SB200646A was without effect on blinking and fore-paw-licking but dose-dependently potentiated fore-paw-tremor. The 5-HT1A agonists 8-OH DPAT and buspirone attenuated blinking at the lower doses tested but were ineffective at the higher doses; the converse was found for fore-paw-licking and -tremor behaviours.The effects of these ligands appeared to be at a postsynaptic 5-HTlA site since para-chlorophenylalanine was without effect on the manipulation of these behaviours. (S)-W A Y100135 was without effect on MK771-induced behaviours, spontaneous and DOl-induced head shakes. Because kynurenine potentiates head shakes and plasma concentrations are raised in TS patients the effects of kynurenine on the 5-HT2A/2C agonist DOl mediated head shake were established. Kynurenine potentiated the DOl head shake. Attempts were made to correlate serotonergic unit activity with tic like behaviour in cats but this proved unsuccessful. However, the pharmacological understanding of 5-HTlA receptor function has been hampered because of the lack of selective antagonists for this site. For this reason the effects of the novel 5-HTlA antagonists (S)-WA Y- 100135 and WAY -100635 were tested on 5-HT single-unit activity recorded from the dorsal-raphe-nucleus in the behaving cat. Both drugs antagonised the suppression of unit activity caused by 8-0H DPAT. (S)-WA Y-100135 reduced unit activity whereas WAY-100635 increased it. This suggests that WAY-100635 is acting as an antagonist at the 5-HTlA somatodendritic autoreceptor and that (S)W A Y -100135 acts as a partial agonist at this site. Aspects of tic like behaviour and serotonergic control are discussed.

Drug action on anxiety models with special reference to serotonergic mechanisms

A study has been made of drugs acting at 5-HT receptors on animal models of anxiety. An elevated X-maze was used as a model of anxiety for rats and the actions of various ligands for the 5-HT receptor, and its subtypes, were examined in this model. 5-HT agonists, with varying affinities for the 5-HT receptor subtypes, were demonstrated to have anxiogenic-like activity. The 5-HT2 receptor antagonists ritanserin and ketanserin exhibited an anxiolytic-like profile. The new putatuve anxiolytics ipsapirone and buspirone, which are believed to be selective for 5-HT1 receptors, were also examined. The former had an anxiolytic profile whilst the latter was without effect. Antagonism studies showed the anxiogenic response to 8-hydroxy-2-(Di-n-propylamino)tetralin (8-OH-DPAT) to be antagonised by ipsapirone, pindolol, alprenolol and para-chlorophenylalanine, but not by diazepam, ritanserin, metoprolol, ICI118,551 or buspirone. To confirm some of the results obtained in the elevated X-maze the Social Interaction Test of anxiety was used. Results in this test mirrored the effects seen with the 5-HT agonists, ipsapirone and pindolol, whilst the 5-HT2 receptor antagonists were without effect. Studies using operant conflict models of anxiety produced marginal and varying results which appear to be in agreement with recent criticisms of such models. Finally, lesions of the dorsal raphe nucleus (DRN) were performed in order to investigate the mechanisms involved in the production of the anxiogenic response to 8-OH-DPAT. Overall the results lend support to the involvement of 5-HT, and more precisely 5-HT1, receptors in the manifestation of anxiety in such animal models.

Characterisation of calcitonin generelated peptide (CGRP) and amylin receptors

The aims of this study were to examine the binding characteristics of the rat CGRP receptor and to further the classification of CGRP and amylin receptors in guinea-pig tissue preparations. Binding characteristics of CGRP were investigated on rat splenic, cerebellar and liver membrane preparations. Human-α-CGRP, rat-α-CGRP and the CGRP receptor analogues Tyrº -CGRPC28-37) and [Cys (ACM)2,7 ]-human CGRP and the CGRP receptor antagonist CGRPC8-37) were utilised in competitive radioligand binding experiments to identify possible CGRP receptor subtypes in these tissues. There appeared to be no significant differences between the rat CGRP receptors examined. A panel of monoclonal antibodies (Mabs) raised against CGRP were employed to investigate the structure-activity relationships of CGRP and its receptor. No differences between the tissue receptors were observed using this panel of Mabs. The effects of human-α, human-β, rat-α-CGRP, human and rat amylin and adrenomedullin(13-52) were examined on the spontaneously beating right atria and on electrically evoked twitch contractions of isolated guinea-pig ileum, vas deferens and left atria. All of the peptides caused concentration-dependent inhibition of twitch amplitude in the ileum and vas deferens. CGRP produced positive inotropic effects in the right and left atria and positive chronotropic effects in the right atria. A variety of CGRP receptor antagonists and putative amylin receptor antagonists were used to antagonise these effects. CGRP(8-37) is currently used as a basis for CGRP receptor classification (Dennis, et al., 1989). Based upon results obtained using CGRP(8-37) it has been shown that the guinea-pig ileum contains mainly CGRP 1 receptors and the vas deferens contain CGRP2 receptors. Amylin was shown to act at receptors distinct from those for CGRP and it is postulated that amylin has its own receptors in these preparations. Experiments using CGRP (19-37) and Tyrº -CGRP(28-37) indicate that human and rat CGRP act at distinct receptors in guinea-pig ileum and vas deferens. The amylin receptor antagonist amylin(8-37) and the putative antagonist AC187 provide evidence to suggest human and rat amylin also act at receptors able to distinguish between the two types of amylin.

Adrenomedullin and calcitonin gene-related peptide receptors in endocrine-related cancers:opportunities and challenges

Adrenomedullin (AM), adrenomedullin 2 (AM2/intermedin) and calcitonin gene-related peptide (CGRP) are members of the calcitonin family of peptides. They can act as growth or survival factors for a number of tumours, including those that are endocrine-related. One mechanism through which this occurs is stimulating angiogenesis and lymphangiogenesis. AM is expressed by numerous tumour types and for some cancers, plasma AM levels can be correlated with the severity of the disease. In cancer models, lowering AM content or blocking AM receptors can reduce tumour mass. AM receptors are complexes formed between a seven transmembrane protein, calcitonin receptor-like receptor and one of the two accessory proteins, receptor activity-modifying proteins (RAMPs) 2 or 3 to give the AM1 and AM2 receptors respectively. AM also has affinity at the CGRP receptor, which uses RAMP1. Unfortunately, due to a lack of selective pharmacological tools or antibodies to distinguish AM and CGRP receptors, the precise receptors and signal transduction pathways used by the peptides are often uncertain. Two other membrane proteins, RDC1 and L1/G10D (the 'ADMR'), are not currently considered to be genuine CGRP or AM receptors. In order to properly evaluate whether AM or CGRP receptor inhibition has a role in cancer therapy, it is important to identify which receptors mediate the effects of these peptides. To effectively distinguish AM1 and AM2 receptors, selective receptor antagonists need to be developed. The development of specific CGRP receptor antagonists suggests that this is now feasible.

Receptor Activity Modifying Proteins (RAMPs) interact with the VPAC2 Receptor and CRF1 receptors and modulate their function

Background and Purpose Although it is established that the receptor activity modifying proteins (RAMPs) can interact with a number of GPCRs, little is known about the consequences of these interactions. Here the interaction of RAMPs with the glucagon-like peptide 1 receptor (GLP-1 receptor), the human vasoactive intestinal polypeptide/pituitary AC-Activating peptide 2 receptor (VPAC) and the type 1 corticotrophin releasing factor receptor (CRF) has been examined. Experimental Approach GPCRs were co-transfected with RAMPs in HEK 293S and CHO-K1 cells. Cell surface expression of RAMPs and GPCRs was examined by elisa. Where there was evidence for interactions, agonist-stimulated cAMP production, Ca mobilization and GTPγS binding to G, G, G and G were examined. The ability of CRF to stimulate adrenal corticotrophic hormone release in Ramp2 mice was assessed. Key Results The GLP-1 receptor failed to enhance the cell surface expression of any RAMP. VPAC enhanced the cell surface expression of all three RAMPs. CRF enhanced the cell surface expression of RAMP2; the cell surface expression of CRF was also increased. There was no effect on agonist-stimulated cAMP production. However, there was enhanced G-protein coupling in a receptor and agonist-dependent manner. The CRF: RAMP2 complex resulted in enhanced elevation of intracellular calcium to CRF and urocortin 1 but not sauvagine. In Ramp2 mice, there was a loss of responsiveness to CRF. Conclusions and Implications The VPAC and CRF receptors interact with RAMPs. This modulates G-protein coupling in an agonist-specific manner. For CRF, coupling to RAMP2 may be of physiological significance. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Expression of VEGF-C and activation of its receptors VEGFR-2 and VEGFR-3 in trophoblast

Placental villous development requires the co-ordinated action of angiogenic factors on both endothelial and trophoblast cells. Like vascular endothelial growth factor (VEGF), VEGF-C increases vascular permeability, stimulates endothelial cell proliferation and migration. In the present study, we investigated the expression of VEGF-C and its receptors VEGFR-3 and VEGFR-2 in normal and intrauterine growth-restricted (IUGR) placenta. Immunolocalisation studies showed that like VEGF and VEGFR-1, VEGF-C, VEGFR-3 and VEGFR-2 co-localised to the syncytiotrophoblast, to cells in the maternal decidua, as well as to the endothelium of the large placental blood vessels. Western blot analysis demonstrated a significant decrease in placental VEGF-C and VEGFR-3 protein expression in severe IUGR as compared to gestationally-matched third trimester pregnancies. Conditioned medium from VEGF-C producing pancreatic carcinoma (Suit-2) and endometrial epithelial (Hec-1B) cell lines caused an increased association of the phosphorylated extracellular signal regulated kinase (ERK) in VEGFR-3 immunoprecipitates from spontaneously transformed first trimester trophoblast cells. VEGF121 caused dose-dependant phosphorylation of VEGFR-2 in trophoblast cells as well as stimulating DNA synthesis. In addition, premixing VEGF165 with heparin sulphate proteoglycan potentiated trophoblast proliferation and the association of phospho-ERK with the VEGFR-2 receptor. VEGF165-mediated DNA synthesis was inhibited by anti-VEGFR-2 neutralising antibody. The results demonstrate functional VEGFR-2 and VEGFR-3 receptors on trophoblast and suggest that the decreased expression of VEGF-C and VEGFR-3 may contribute to the abnormal villous development observed in IUGR placenta.

Antagonistic effects of two novel GIP analogs, (Hyp3)GIP and (Hyp3)GIPLys16PAL, on the biological actions of GIP and longer-term effects in diabetic ob/ob mice

This study examines the actions of the novel enzyme-resistant, NH 2-terminally modified GIP analog (Hyp3)GIP and its fatty acid-derivatized analog (Hyp3)GIPLys16PAL. Acute effects are compared with the established GIP receptor antagonist (Pro3)GIP. All three peptides exhibited DPP IV resistance, and significantly inhibited GIP stimulated cAMP formation and insulin secretion in GIP receptor-transfected fibroblasts and in clonal pancreatic BRIN-BD11 cells, respectively. Likewise, in obese diabetic ob/ob mice, intraperitoneal administration of GIP analogs significantly inhibited the acute antihyperglycemic and insulin-releasing effects of native GIP. Administration of once daily injections of (Hyp 3)GIP or (Hyp3)GIPLys16PAL for 14 days resulted in significantly lower plasma glucose levels (P < 0.05) after (Hyp 3)GIP on days 12 and 14 and enhanced glucose tolerance (P < 0.05) and insulin sensitivity (P < 0.05 to P < 0.001) in both groups by day 14. Both (Hyp3)GIP and (Hyp3)GIPLys16PAL treatment also reduced pancreatic insulin (P < 0.05 to P < 0.01) without affecting islet number. These data indicate that (Hyp3)GIP and (Hyp 3)GIPLys16PAL function as GIP receptor antagonists with potential for ameliorating obesity-related diabetes. Acylation of (Hyp 3)GIP to extend bioactivity does not appear to be of any additional benefit. Copyright © 2007 the American Physiological Society.

Cancer cachexia

Purpose of review: Although cachexia has a major effect on both the morbidity and mortality of cancer patients, information on the mechanisms responsible for this condition is limited. This review summarizes recent data in this area. Recent findings: Cachexia is defined as loss of muscle, with or without fat, frequently associated with anorexia, inflammation and insulin resistance. Loss of adipose mass is due to an increased lipolysis through an increased expression of hormone-sensitive lipase. Adipose tissue does not contribute to the inflammatory response. There is an increased phosphorylation of both protein kinase R (PKR) and eukaryotic initiation factor 2 on the α-subunit in skeletal muscle of cachectic cancer patients, which would lead to muscle atrophy through a depression in protein synthesis and an increase in degradation. Mice lacking the ubiquitin ligase MuRF1 are less susceptible to muscle wasting under amino acid deprivation. Expression of MuRF1 and atrogin-1 is increased by oxidative stress, whereas nitric oxide may protect against muscle atrophy. Levels of interleukin (IL)-6 correlate with cachexia and death due to an increase in tumour burden. Ghrelin analogues and melanocortin receptor antagonists increase food intake and may have a role in the treatment of cachexia. Summary: These findings provide impetus for the development of new therapeutic agents. © 2010 Wolters Kluwer Health

Heterodimerisation between VEGFR-1 and VEGFR-2 and not the homodimers of VEGFR-1 inhibit VEGFR-2 activity

Vascular endothelial growth factor (VEGF) signaling is tightly regulated by specific VEGF receptors (VEGF-R). Recently, we identified heterodimerisation between VEGFR-1 and VEGFR-2 (VEGFR1–2) to regulate VEGFR-2 function. However, both the mechanism of action and the relationship with VEGFR-1 homodimers remain unknown. The current study shows that activation of VEGFR1–2, but not VEGFR-1 homodimers, inhibits VEGFR-2 receptor phosphorylation under VEGF stimulation in human endothelial cells. Furthermore, inhibition of phosphatidylinositol 3-kinase (PI3K) increases VEGFR-2 phosphorylation under VEGF stimulation. More importantly, inhibition of PI3K pathway abolishes the VEGFR1–2 mediated inhibition of VEGFR-2 phosphorylation. We further demonstrate that inhibition of PI3K pathway promotes capillary tube formation. Finally, the inhibition of PI3K abrogates the inhibition of in vitro angiogenesis mediated by VEGFR1–2 heterodimers. These findings demonstrate that VEGFR1–2 heterodimers and not VEGFR-1 homodimers inhibit VEGF-VEGFR-2 signaling by suppressing VEGFR-2 phosphorylation via PI3K pathway.

Characterization of receptors for calcitonin gene-related peptide and adrenomedullin on the guinea-pig vas deferens

1. The receptors which mediate the effects of calcitonin gene-related peptide (CGRP), amylin and adrenomedullin on the guinea-pig vas deferens have been investigated. 2. All three peptides cause concentration dependant inhibitions of the electrically stimulated twitch response (pD 2s for CGRP, amylin and adrenomedullin of 7.90 ± 0.11, 7.70 ± 0.19 and 7.25 ± 0.10 respectively). 3. CGRP 8-37 (1 μM) and AC187 (10 μM) showed little antagonist activity against adrenomedullin. 4. Adrenomedullin 22-52 by itself inhibited the electrically stimulated contractions of the vas deferens and also antagonized the responses to CGRP, amylin and adrenomedullin. 5. [ 125I]-adrenomedullin labelled a single population of binding sites in vas deferens membranes with a pIC 50 of 8.91 and a capacity of 643 fmol mg -1. Its selectivity profile was adrenomedullin > AC187 > CGRP = amylin. It was clearly distinct from a site labelled by [ 125I]-CGRP (pIC 50 = 8.73, capacity = 114 fmol mg -1, selectivity CGRP > amylin = AC187 > adrenomedullin). [ 125I]-amylin bound to two sites with a total capacity of 882 fmol mg -1. 6. Although CGRP has been shown to act at a CGRP 2 receptor on the vas deferens with low sensitivity to CGRP 8-37, this antagonist displaced [ 125I]-CGRP with high affinity from vas deferens membranes. This affinity was unaltered by increasing the temperature from 4°C to 25°C, suggesting the anomalous behaviour of CGRP 8-37 is not due to temperature differences between binding and functional assays.

Antidiabetic properties of zinc-alpha2-glycoprotein in ob/ob mice

Zinc-alpha(2)-glycoprotein (ZAG) is an adipokine associated with fat loss in cancer cachexia. The purpose of this study was to evaluate the ability of recombinant human ZAG to attenuate type 2 diabetes in the ob/ob mouse model. ZAG (50 microg daily, iv) induced a progressive loss of body weight (3.5 g in 5 d), without an effect on food or water intake but with a 0.4 C rise in body temperature, suggesting an increased energy expenditure. Despite an increased plasma glycerol, indicative of increased lipolysis, levels of glucose, triglycerides, and nonesterified fatty acids were decreased by 17, 25, and 62%, respectively, due to an increased use of both glucose and lipids by muscle and brown adipose tissue. The weight of the latter increased 2-fold, and there was increased expression of uncoupling proteins-1 and -3. Plasma insulin levels were reduced by 36%, whereas pancreatic insulin was increased 4-fold, and there was a 53% decrease in the total area under the glucose curve in the glucose tolerance test and reduced insulin requirement. There was an increase in skeletal muscle mass due to an increase in protein synthesis and a decrease in protein degradation. These results suggest that ZAG may potentially be effective in the treatment of type 2 diabetes.

Mediators of gram-positive septic shock

Septic shock can occur as a result of Gram-negative or Gram-positive infection and involves a complex interaction between bacterial factors and the host immune system producing a systemic inflammatory state that may progress to multiple organ failure and death. Gram-positive bacteria are increasingly becoming more prevalent especially Staphylococcus epidermidis in association with indwelling devices. Lipopolysaccaride (LPS) is the key Gram-negative component involved in this process, but it is not clear which components of Gram-positive bacteria are responsible for progression of this often fatal disease. The aim of this thesis was to investigate the effect of bacterial components on the immune systems. Lipid S, a short chain form of lipoteichoic acid (LTA) found to be excreted from bacteria during growth in culture medium was examined along with other Gram-positive cell wall components: LTA, peptidoglycan (PG) and wall teichoic acids (WTA) and LPS from Gram-negative bacteria. Lipid S, LTA, PG and LPS but not WTA all stimulated murine macrophages and cell lines to produce significant amounts of NO, TNF-a, IL-6 and IL-1 and would induce fever and tissue damage seen in inflammatory diseases. Lipid S proved to be the most potent out of the Gram-positive samples tested. IgG antibodies in patients serum were found to bind to and cross react with lipid S and LTA. Anti-inflammatory antibiotics, platelet activating factor (PAF), PAF receptor antagonists and monoclonal antibodies (mAbs) directed to LTA, CD14 and toll-like receptors were utilised to modulate cytokine and NO production. In cell culture the anti-LTA and the anti-CD14 mAbs failed to markedly attenuate the production of NO, TNF-a, IL-6 or IL-1, the anti-TLR4 antibody did greatly inhibit the ability of LPS to stimulate cytokine production but not lipid S. The tetracyclines proved to be the most effective compounds, many were active at low concentrations and showed efficacy to inhibit both lipid S and LPS stimulated macrophages to produce NO.

Adrenoceptors and intestinal fluid and electrolyte transport in the rat

Noradrenaline was found to significantly stimulate fluid and Na absorption across everted sacs of rat jejunum. Of a number of a1, and 2-adrenoceptor antagonists tested only prazosin significantly inhibited the stimulant effect of noradrenaline and further experiments revealed an antiabsorptive effect of prazosin alone. Theophylline reduced jejunal fluid and Na absorption and this effect was not reversed by 2-adrenoceptor stimulation in contrast to previous findings in vivo. Evidence suggests the everted sac preparation is not appropriate to the study of intestinal fluid and electrolyte transport. The investigation of Jejunal ion transport in vitro was continued using an Ussing chamber preparation. Selective 2-adrenoceptor stimulation was found to depress electrogenic anion secretion, as neurotoxin tetrodotoxin indicated that this was a direct epithelial effect. 2-adrenoceptor agonists have considerable therapeutic value as antisecretory agents and the model of rat jejunum in vitro represents a convenient experimental model for research in this area. The selective 2-adrenoceptor antagonist ICI 118551 decreased basal SCC and inhibited increases in SCC in response to isoprenaline or salbutamol indicating the presence of a 2-adrenoceptor mechanism mediating both secretory tone and increases in secretory processes. Many intestinal secretagogues elicit electrolyte secretion via the stimulation of intramural secretory nervous pathways. If these pathways involve the activation of 2-adrenoceptorsthe 2-adrenoceptor antagonists may be useful in the treatment of diarrhoeal diseases. A single pass lumen perfusion technique was used to investigate possible sympathetic tone over colonic fluid and electrolyte absorption in the rat colon in vivo. The technique employed appeared to lack the necessary resolution for this study and alternative approaches are discussed

Effects of some 5-hydroxytryptamine and related ligands in anxiety models

Drugs acting at 5-HT receptors were evaluated on three animal models of anxiety. On the elevated X-maze test the majority of 5-HT1 agonists were found to be anxiogenic. However, ipsapirone was anxiolytic and buspirone and gepirone were inactive. The 5-HT2 agonist DOI and the 5-HT2 antagonist ritanserin were anxiolytic while ICI 169,369, a 5-HT2 antagonist was inactive. All 5-HT3 antagonists tested were inactive in this test, while the indirect serotomimetics zimeldine and fenfluramine were anxiogenic. Neither beta-adrenoceptor agonists nor antagonists had reproducible effects on anxiety in this model. Combined beta-1/beta-2 adrenoceptor antagonists reversed the anxiogenic effects of 8-OH-DPAT while selective beta-1 or beta-2 antagonists did not. On the social interaction model the 5-HT1 agonists 8-OH-DPAT, RU 24969 and 5-MeODMT were anxiogenic and ipsapirone was anxiolytic. The 5-HT2 agonist DOI and the beta-adrenoceptor- and 5-HT- antagonist pindolol were anxiolytic, while the 5-HT2 and 5-HT3 antagonists were inactive. In the marble burying test, the 5-HT upake inhibitors zimeldine, fluvoxamine, indalpine and citalopram, the 5-HT1B/5-HT1C agonists mCPP and TFMPP and the 5-HT2/5-HT1C agonist DOI reduced marble burying without affecting locomotor activity. 5-HT1A agonists and the 5-HT2 and 5-HT3 antagonists were without effect. Lesions of the dorsal raphe nucleus reversed the anxiogenic effects of 8-OH-DPAT in the X-maze model. The implication of these results for the understanding of the pharmacology of 5-HT in anxiety is discussed.

Control of proliferation in the rat thymus

Quiescent rat thymocytes were stimulated to divide by a variety of agents. One such mitogen was the neurotransmitter acetylcholine which exhibited a biphasic action. Interaction with low affinity nicotinic receptors was linked with an obligatory requirement for magnesium ions whereas combination with high affinity muscarinic receptors induced mitosis only if calcium ions were present in the medium. Binding of acetylcholine to its muscarinic receptor enhanced calcium influx and increased intracellular calcium levels causing calmodulin activation, a necessary prelude to DNA synthesis and mitosis. Nicotinic receptor activation may be associated with a magnesium influx and stimulation of cells in a calmodulin-independent fashion. Parathyroid hormone and its analogues exhibited only a monophasic mitogenic action. This response was linked to calcium influx, a rise in cytosolic calcium and calmodulin activation. Parathyroid hormone did not stimulate adenylate cyclase in thymocytes and decreased cellular cyclic AMP concentrations. Picomolar amounts of interleukin-2 (IL-2) also stimulated division in thymocytes derived from 3-month old rats by binding to high affinity receptors. The response in thymocytes from newborn and foetal animals was greater reflecting the larger proportion of cells bearing receptors at this age. The mitogenic effect of IL-2 was abolished by a monoclonal antibody directed against the IL-2 receptor. Injections of IL-2 itself or the administration of IL-2 secreting activated syngeneic spleen cells also stimulated proliferation of both thymus and bone marrow cells in vivo. Likewise immunisation with pertussis toxin, which enhances endogenous IL2 production, also increased mitosis in these tissues. Calcium influx, increased cytosolic Ca2+ levels and calmodulin activation are associated features of the mitogenic action of IL-2. Interleukin-1 was also found to be mitogenic in thymic lymphocyte cultures. The responses to this mitogen and to parathyroid hormone and acetylcholine were not inhibited by the anti-IL2 receptor antibody suggesting that the thymic lymphocyte bears discrete receptors for these agents. Subtle interactions of hormones, neurotransmitters and interleukins may thus contribute to the turnover and control of lymphoid cells in the thymus and perhaps bone-marrow.