Effect of different photoperiods on the diurnal rhythm of 5-methoxytryptamine in the pineal gland of golden hamsters (Mesocricetus auratus)

Summary This study tested the photo-dependency of the rhythmic synthesis of 5-methoxytryptamine (5-MT) in the pineal gland of golden hamsters. After pargyline administration, pineal 5-methoxytryptamine and melatonin were measured by HPLC in male golden hamsters kept under short and long photoperiod. In both photoperiodic regimes, a clear 5-MT rhythm was observed which fitted a sinusoidal function with high values occuring during the day-time and low values occuring during the night-time. The duration of the low nighttime levels was clearly proportional to the length of the dark phase. A marked rhythm of melatonin synthesis was also seen with low daytime levels and high night-time values. An inverse relationship between 5-MT and melatonin levels was observed. Thus, after pargyline administration, the rhythms of 5-MT and melatonin in the pineal gland of golden hamsters are photoperiod-dependent and show a reciprocal relationship.     

Selective effect of methoxyindoles on the lymphocyte proliferation and melatonin binding to activated human lymphoid cells

Three pineal methoxyindoles (melatonin (Mel), 5-methoxytryptamine (5-MTA) and 5-methoxytryptophol (5-MTO)) were studied for their ability to influence the proliferative response of human peripheral blood lymphocytes (PBL) and tonsillar lymphocytes (TL) following activation with concanavalin A (ConA) in vitro. The ConA-stimulated DNA synthesis was affected in a different dose-dependent mode by the methoxyindoles tested. Melatonin and 5-MTO inhibited and 5-MTA increased the ConA-induced [³H]thymidine incorporation in PBL and TL. The initial screening for 2-[¹²⁵I]iodomelatonin binding using a single point assay revealed significantly increased specific binding to PBL and TL after 72-h stimulation with ConA as compared to the non-activated cell cultures. Coincubation of separate lymphocyte cultures with ConA and Mel or 5-MTO resulted in inhibition of the specific 2-[¹²⁵I]iodomelatonin binding (85% and 74%, respectively). The specific binding determined in the presence of 5-MTA did not differ from control values. Series of saturation and competition experiments were performed to examine the binding characteristics of ConA-stimulated lymphocytes for 2-[¹²⁵I]iodomelatonin. The radioligand labelled binding sites of high affinity (K_d = 0.14 ± 0.03 nM) and low capacity (B_max = 6.8 ± 1.5 fM/mg protein). Competitive studies with a variety of indoles determined the following order of relative potency for inhibition of 2-[¹²⁵I]iodomelatonin binding in TL: 2-iodomelatonin > melatonin > > 5-methoxytryptophol. 5-Methoxytryptamine did not show displacement potency for the labelled ligand. Collectively, our data suggest that pineal hormones might be directly involved in the regulation of the T-lymphoproliferative response of human lymphoid cells. We show the availability of melatonin receptors, which seem to be an intrinsic characteristic of activated human lymphocyte populations. While the effects of Mel and 5-MTO can be linked to the binding sites described, it is unlikely that serotonin agonists like 5-MTA may act through the same sites to influence the mitogen-stimulated lymphocyte proliferation.     

Nerve terminal effects of indoleamine psychotomimetics on 5-hydroxytryptamine

The mode of action of indoleamine psychotomimetics has been closely linked to 5-HT. Early work showed increases in rat brain levels of 5-HT which were later localized to the nerve-ending fraction. With improved methodology, the 5-HT increment was further detected in the synaptic vesicle fraction. These effects were obtained with several indoleamine hallucinogens but not with mescaline. LSD has been most thoroughly studied and has served as the prototypical compound in ascertaining the mode of action of these drugs. Pretreatment with reserpine abolished the 5-HT effects of LSD in the vesicular fraction. However, a new compartment, termed “juxtavesicular,” displayed 5-HT increases following reserpine and LSD. A soluble binding site for 5-HT within the synaptoplasm has been postulated in confirmation of independent results by other groups of investigators. The origin of the 5-HT increment appears to be associated with newly synthesized amine. This was deduced from experiments involving various 5-HT synthesis blockers. To ascertain whether inhibition of raphé neuronal firing is responsible for the accumulation of 5-HT at the nerve terminal, two sets of experiments were performed. Destruction of the raphé cell bodies by radiofrequency lesions failed to abolish the LSD-induced 5-HT increase early after the lesion. Destruction of cortical 5-HT neurons with the neurotoxin 5,7-dihydroxytryptamine completely abolished the 5-HT effect of LSD. It was concluded that an intact nerve terminal is necessary for the expression of the LSD-mediated increases in 5-HT. A LSD “autoreceptor” is postulated, possibly identical to a 5-HT presynaptic receptor inhibiting the release of 5-HT.     

Nerve terminal effects of indoleamine psychotomimetics on 5-hydroxytryptamine

The mode of action of indoleamine psychotomimetics has been closely linked to 5-HT. Early work showed increases in rat brain levels of 5-HT which were later localized to the nerve-ending fraction. With improved methodology, the 5-HT increment was further detected in the synaptic vesicle fraction. These effects were obtained with several indoleamine hallucinogens but not with mescaline. LSD has been most thoroughly studied and has served as the prototypical compound in ascertaining the mode of action of these drugs. Pretreatment with reserpine abolished the 5-HT effects of LSD in the vesicular fraction. However, a new compartment, termed “juxtavesicular,” displayed 5-HT increases following reserpine and LSD. A soluble binding site for 5-HT within the synaptoplasm has been postulated in confirmation of independent results by other groups of investigators. The origin of the 5-HT increment appears to be associated with newly synthesized amine. This was deduced from experiments involving various 5-HT synthesis blockers. To ascertain whether inhibition of raphé neuronal firing is responsible for the accumulation of 5-HT at the nerve terminal, two sets of experiments were performed. Destruction of the raphé cell bodies by radiofrequency lesions failed to abolish the LSD-induced 5-HT increase early after the lesion. Destruction of cortical 5-HT neurons with the neurotoxin 5,7-dihydroxytryptamine completely abolished the 5-HT effect of LSD. It was concluded that an intact nerve terminal is necessary for the expression of the LSD-mediated increases in 5-HT. A LSD “autoreceptor” is postulated, possibly identical to a 5-HT presynaptic receptor inhibiting the release of 5-HT.     

The effect of thyroid hormones on rat pineal indoleamine metabolismin vitro

Summary The effect of TSH, triiodothyronine (T₃) and thyroxine (T₄) on basic and noradrenaline (NA)-induced indoleamine metabolism was investigated in rat pineal culture based on the observation that, under the experimental system used, melatonin is formed from its amino acid precursor tryptophan. When added to the culture medium, alone or in the presence of NA, TSH in dosages of 5 and 20 mU had no significant effect on pineal tryptophan metabolism. T₄ at 2.5g increased the melatonin concentration and, in the presence of NA 10^–4 M, also induced an increase in N-acetylserotonin (NAS). T₃, already at a dosage as low as 0.25g enhanced the melatonin concentration, while 2.5g produced significant increases in concentrations of all pineal indoleamines measured. The latter dosage of Ts also enhanced the NA-stimulated NAS. On adding larger quantities of T₄ or T₃ to the medium none of the changes encountered with the lower dosages could be seen. The results obtained may suggest a direct positive feedback between the thyroid and pineal glands.     

The influence of temperature upon depolarizing responses of rat isolated vagal nerve to 5-hydroxytryptamine

In isolated cervical segments of rat vagus nerve, over the temperature range 5-45 degrees C, reversible depolarizations to 5-hydroxytryptamine (5-HT) were maximal at 10 degrees C and negligible at 45 degrees C. Dose-response curves for this depolarization were compared at 20 degrees C and 37 degrees C, with a similar sensitivity but marked proportionate reduction at 37 degrees C vs 20 degrees C, whilst the 5-HT-induced depression of vagal compound action potentials seen at 20 degrees C was abolished at 37 degrees C; such changes at 20 degrees C or 37 degrees C were insensitive to inhibitors of 5-HT-uptake (zimelidine) or MAO (pargyline). 5-HT may participate in presynaptic inhibition of C-fibre afferents.     

Changes in the circadian rhythmicity of hydroxyindole-O-methyl transferase (HIOMT) activity in the synthesis of 5-methoxyindoles in the pineal gland of 28 day old male Wistar rats exposed to white,red and green light

Summary Hydroxyindole-O-methyl transferase activity for the synthesis of 5-methoxyindoles was investigated in the pineal gland of 28 day old male Wistar rats after exposure to white, red and green light for 12 hours daily. It could be demonstrated that, in comparison to white light, red light causes a shift of HIOMT activity for the synthesis of melatonin/5-methoxytrypto-phol towards an earlier period being highest between 12 and 16 hours. The synthesis of 5-methoxytryptamine and of 5-methoxytryptophan is shifted to 16 hours, while the synthesis of 5-methoxyindole-3-acetic acid, which normally peaks at 16 hours, is increased at this same hour.Green light shifts HIOMT activity for the synthesis of melatonin/ 5-methoxytryptophol to a later period, showing a peak at 24 hours. The synthesis of 5-methoxytryptamine is significantly increased at 24 hours. An increase is also observed in the synthesis of 5-methoxyindole-3-acetic acid at 12 and at 4 hours, times at which this synthesis is also maximal using white light, whereas the synthesis of 5-methoxytryptophan is generally decreased. A possible relationship between the present results and those obtained after exposure to different wavelengths of light on N-acetyltransferase activity, the effect of pterins on HIOMT activity and the effect of different wavelengths on gonadal growth are discussed.     

Inhibition of lateral vestibular nucleus neurons by 5-hydroxytryptamine derived from the dorsal raphe nucleus

Electrophysiological studies were performed to elucidate the effect of 5-hydroxytryptamine (5-HT) originating in the dorsal raphe nucleus (DR) on neuronal activity in the lateral vestibular nucleus (LVN) neurons, using cats anesthetized with alpha-chloralose. LVN neurons were classified into monosynaptic and polysynaptic neurons according to their responses to vestibular nerve stimulation. Conditioning stimuli applied to the DR inhibited orthodromic spikes elicited by vestibular nerve stimulation predominantly in polysynaptic neurons of the LVN. The iontophoretic application of 5-HT also inhibited orthodromic spikes of the LVN neurons. A close correlation was observed between the effects of DR conditioning stimulation and iontophoretically applied 5-HT in the same neurons. These inhibitions with both treatments were antagonized during the application of methysergide, a 5-HT antagonist. In the majority of LVN polysynaptic neurons that responded to antidromic stimulation of the ipsilateral or contralateral abducens nucleus, orthodromic spikes elicited by vestibular nerve stimulation were inhibited by DR conditioning stimulation and the iontophoretic application of 5-HT. In contrast, LVN neurons that responded to antidromic stimulation of the vestibulospinal tract were rarely affected by these treatments. These results indicate that 5-HT derived from the DR inhibits the synaptic transmission of LVN polysynaptic neurons ascending to the abducens nucleus, and suggest that 5-HT derived from the DR is involved in the regulation of the vestibulo-ocular reflex.     

Dynamics of 5-hydroxytryptamine released from dopamine neurons in the caudate putamen of the rat

Fast-scan cyclic voltammetry has been used for the detection of 5-hydroxytryptamine (5-HT) in vivo. The applied potential waveform was previously optimized to maximize response times to concentration changes. This technique has been used to reinvestigate 5-HT release from striatal dopaminergic terminals after pharmacological pretreatment as reported by Stamford et al. [13]. Our results concur with those set forth by Stamford and further show that the dopamine transporter is responsible for 5-HT uptake in this experiment.     

Rhythmic synthesis of various 5-methoxyindoles in the pineal gland of male adult golden hamsters,kept under the same artificial conditions throughout the year

Summary Until now the day/night and seasonal rhythmicity in the synthesis of 5-methoxyindoles (MI) is thought to be regulated by environmental factors, especially photoperiod and temperature. Endogenous factors are also implicated in the generation of N-acetyltransferase and hydroxyindole-O-methyltransferase activity rhythms. In the present experiments seasonal rhythmicity in the synthesis of MI in the pineal gland was investigated in hamsters kept under the same artificial conditions throughout the year. Though the environmental conditions were the same, day/night and seasonal rhythmicity in the production of MI in the pineal were observed indicating the existence of endogenous factors influencing the rhythmicities. In November, most of the MI showed the highest synthesis, MA and ML excepted, which were especially produced in July and September. The results obtained sustain the hypothesis that aMT is synthesized from MT rather than from aHT. Moreover, the rhythmicities in aMT synthesis are not identical to those found in aMT concentration as described in the literature. This indicates that synthesis and concentration of a compound are not comparable. At the end of the light period, when aMT injections have an antigonadotropic effect, a peak of aMT synthesis was always present. Although MI synthesis showed seasonal rhythmicity, no reproductive cycle occurred in the hamsters. At present, the concept that the pro- and/or antigonadal effects of the pineal are mediated by aMT seems to be the most acceptable. The present results, however, indicate that aMT and perhaps other MI, often regarded as factors influencing gonadal growth in golden hamsters, are not the only factors involved.In this paper the following abbreviations have been used (Smith, 1982): 5-hydroxyindoles, HI; 5-methoxyindoles, MI; tryptophan, W; 5-hydroxytryptophan, HW; 5-methoxytryptophan, MW; serotonin, HT; 5-methoxytryptamme, MT; N-acetylserotonin, aHT; melatonin, aMT; 5-hydroxyindoleacetaldehyde, HB; 5-methoxyindoleacetaldehyde, MB; 5-hydroxytryptophol, HL; 5-methoxytryptophol, ML; O-acetyl-5-hydroxytryptophol, aHL; O-acetyl-5-methoxytryptophol, aML; 5-hydroxyindole-3-acetic acid, HA; 5-methoxyindole-3-acetic acid, MA; hydroxyindole-O-methyltransferase, HIOMT; S-adenosyl-methionine, SAM; N-acetyltransferase, NAT.     

5-hydroxytryptamine and the mediation of pituitary-adrenocortical hormones in the extinction of active avoidance behaviour.

(1) The effect of corticosterone on the extinction of a one-way active avoidance task was correlated with hypothalamic and mesencephalic serotonin (5-HT) content. (2) Corticosterone treatment in a daily dose of 1·0 and 5·0 mg/kg i.p. facilitated the extinction of active avoidance behaviour while doses of 10·0 mg/kg corticosterone delayed it. (3) 1·0 and 2·0 mg/kg corticosterone increased the hypothalamic 5-HT content in normal and adrenalectomized animals, 5·0 mg/kg failed to influence it, and 10·0 mg/kg decreased it. (4) After 1·0, 5·0 and 10·0 mg/kg corticosterone treatment, changes in mesencephalic 5-HT followed a pattern similar to that observed in the hypothalamus. (5) Adrenocorticotrophic hormone (ACTH), in doses of 2·0 and 4·0 i.u./animal, resulted in an increased hypothalamic and mesencephalic 5-HT content in intact animals; however, there were no changes in cerebral 5-HT levels in adrenalectomized rats. (6) Decreased brain 5-HT content, brought about by parachlorophenylalanine (PCPA) (300 mg/kg), was able to prevent the behavioural effect of 1·0 and 5·0 mg/kg corticosterone. (7) PCPA treatment alone given every 72 hr caused a transitory delay in extinction 24 and 48 hr after administration; however, the tendency for extinction to occur did not change. (8) Decreased 5-HT content caused by mesencephalic raphé lesions delayed extinction. (9) Nialamide treatment (125 mg/kg), which increased brain 5-HT content, counteracted the effect of 10·0 mg/kg corticosterone on extinction. (10) These data suggest that the action of corticosterone on active avoidance behaviour is mediated, at least in part, via changed 5-HT metabolism in the brain.     

Plasma concentrations of 5-methoxytryptamine, 5-methoxytryptophol and melatonin after 5-methoxytryptamine administration of golden hamsters: physiological implications

Summary 5-Methoxytryptamine (5-MT), 5-methoxytryptophol (5-ML) and melatonin (Mel) were measured in the plasma after 2, 5, and 8 weeks administration of 25 g 5-MT to golden hamsters kept under long photoperiod. 5-MT showed a one compartment kinetic profile in the plasma with half lives of 14.8 min after 2 weeks, 15 min after 5 weeks and 19.1 min after 8 weeks. A rapid metabolism of 5-MT was shown, Mel and 5-ML being detected in the plasma following 5-MT administration. However it was also shown that the gonadal atrophy observed after 5-MT administration cannot be due to its metabolism into these 2 compounds. Indeed when exogenously administered at a dose generating the same plasma concentration as that observed after 5-MT, the gonadal regression observed after the association of 5-ML and Mel is much less than that observed after 5-MT. 5-MT is thus a compound of great physiological interest.     

The effect of different photoperiods on the methylating capacity of the pineal gland of adult,male golden hamsters,with special reference to 5-methoxyindoles

Summary Testes weight, plasma FSH and LH concentration and pineal methylating capacity were compared in hamsters housed under either long (LD 1410) or short (LD 816) photoperiods.Hamsters housed for 14 weeks under short photoperiod showed gonadal atrophy, which was complete after 6 weeks. Also plasma FSH and LH concentration showed a marked decline after transfer to short photoperiod. However, after 14 weeks the concentration of FSH and LH as well as testes weight increased again.Under both photoperiods day/night rhythms in plasma FSH and LH concentration were measured. Under both light regimes the concentrations did not show significant differences.Under long as well as short photoperiods in the pineal gland of animals no significant differences were found in the daily synthesis of various MI tested. Only the synthesis of ML was significantly higher in the pineal of hamsters housed under short photoperiod. The function of this higher synthesis of ML remains unknown.Although the maxima of the rhythm for the various MI found under different LD regimes did not differ in magnitude or duration, their location in respect to the onset of darkness was different. It is suggested that this specific location is of more physiological importance than the quantity or duration of synthesis, concentration or release of MI.At the moment the day/night rhythms were determined there were indications that recrudescence of the testes had already started. It is suggested that this recrudescence is responsible for the fact that no differences in the synthesis of MI were found comparing the influence of both photoperiods.After 14 weeks of exposure to short photoperiod, aML synthesis was, in contrast to the synthesis of the other MI, (not significantly) higher under LD 816. Moreover, opposite results for aMT and aML synthesis during darkness were found. It is suggested that the ratio of synthesis of these compounds is of physiological significance.Abbreviations have been used (Smith, 1982) MI 5-methoxyindoles - MW 5-methoxytryptophah - MT 5-methoxytryptamine - aMT melatonin - ML 5-methoxytryptophol - aML O-acetyl-5-methoxytryptophol - MA 5-methoxyindole-3-acetic acid - HIOMT hydroxyindole-O-methyltransferase - FSH follicle-stimulating hormone - LH luteinizing hormone - LD light/dark     

Comparison of some peptidic and proteic ovine pineal fractions with a bovine pineal E5 fraction

Summary Using rather simple and mild extraction and separation methods, three ovine pineal fractions (XM300 R-PP7.2, PP7.2 and PP7.2S) were obtained, which contain peptidic/proteic substances and which show fluorescence characteristics of indoles. The ovine fractions were compared with the bovine pineal E 5-fraction. The ovine fractions are chemically sensitive to normal laboratory light and stable in red light (>600nm). Immunologically, these fractions and the bovine E 5 fraction are stable.From the results of radioimmunological experiments it was concluded that the bovine pineal E 5 fraction as well as the ovine pineal fraction XM300R-PP7.2 and PP7.2S may contain (a) peptide(s) ending by the same carboxy terminal tripeptide Pro-Arg-Gly(NH₂).This work is supported by a FUNGO research grant No. 13-35-33.     

L-tryptophan treatment and the episodic secretion of pituitary hormones and cortisol.

(1) l-Tryptophan, the natural precursor of brain serotonin, was administered to healthy subjects. Plasma free and total tryptophan, somatotropin (growth hormone, GH), follitropin (FSH), lutropin (LH), prolactin and cortisol were analysed after the oral administration of 2 g and 100 mg/kg of l-tryptophan or after 1·28 g of l-leucine, at 08:30 and 11:30. (2) Plasma levels of free and total tryptophan were markedly increased after the oral administration of l-tryptophan. (3) Plasma somatotropin levels were significantly elevated after l-tryptophan treatment at different times of day, but this elevation was not dose-dependent. (4) l-Tryptophan or l-leucine treatment did not affect the pulsatile secretion of follitropin and lutropin. (5) Plasma prolactin was not significantly elevated after the oral administration of l-tryptophan. (6) The morning decline of plasma cortisol was significant with or without l-tryptophan. However, no decline was noticed after l-tryptophan in the middle of day.     

Effect of noradrenaline and 5-hydroxytryptamine depletion on locomotion in the cat

It has recently been hypothesized that stimulation of the mesencephalic locomotor region (MLR) can give rise to locomotion in mesencephalic cats due to activation of descending monoaminergic pathways to the spinal cord. This notion is based on the findings that monoamine agonists and precursors can induce hindlimb stepping in acute low spinal animals, and on the similarities between the effects of the noradrenaline (NA) precursor,l-DOPA, and stimulation of the MLR. The hypothesis that the descending monoamine systems comprose the only pathways which control the initiation of locomotion has been tested in the present study. NA was depleted from the CNS using intraspinal and intraventricular injections of 6-hydroxydopamine and i.v. injections of the NA synthesis inhibitor, α-methyltyrosine. Depletion of 5-hydroxytryptamine (5-HT) was achieved using intraventricular injections of 5,6-dihydroxytryptamine and i.p.p-chlorophenylalanine. These treatments did not abolish evoked locomotion in spite of substantial depletion of NA and 5-HT in the spinal cord and brain stem (maximal depletions of NA up to 14% of control in lumbar cord and 16% of control in pons; maximal depletions of 5-HT up to 19% of control in sacral cord and 25% of control in medulla). Combined depletion of NA and 5-HT did not abolish evoked locomotion in mesencephalic cats, although the treated animals displayed pronoounced ataxia prior to decerebration. Depletion of NA or 5-HT alone did not alter locomotion in otherwise intact animals. A previous report that phenoxybenzamine antagonizes the effects of MLR stimulation was not confirmed. The results therefore do not support the hypothesis that descending pathways containing monoamines are essential for locomotion evoked by brain stem stimulation.     

Paracrine relationship between incretin hormones and endogenous 5-hydroxytryptamine in the small and large intestine

Background

Enterochromaffin (EC) cell-derived 5-hydroxytryptamine (5-HT) is a mediator of toxin-induced reflexes, initiating emesis via vagal and central 5-HT₃ receptors. The amine is also involved in gastrointestinal (GI) reflexes that are prosecretory and promotile, and recently 5-HT's roles in chemosensation in the distal bowel have been described. We set out to establish the efficacy of 5-HT signaling, local 5-HT levels and pharmacology in discrete regions of the mouse small and large intestine. We also investigated the inter-relationships between incretin hormones, glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) and endogenous 5-HT in mucosal and motility assays.

Methods

Adult mouse GI mucosae were mounted in Ussing chambers and area-specific studies were performed to establish the 5-HT₃ and 5-HT₄ pharmacology, the sidedness of responses, and the inter-relationships between incretins and endogenous 5-HT. Natural fecal pellet transit in vitro and full-length GI transit in vivo were also measured.

Key Results

We observed the greatest level of tonic and exogenous 5-HT-induced ion transport and highest levels of 5-HT in ascending colon mucosa. Here both 5-HT₃ and 5-HT₄ receptors were involved but elsewhere in the GI tract epithelial basolateral 5-HT₄ receptors mediate 5-HT's prosecretory effect. Exendin-4 and GIP induced 5-HT release in the ascending colon, while L cell-derived PYY also contributed to GIP mucosal effects in the descending colon. Both peptides slowed colonic transit.

Conclusions & Inferences

We provide functional evidence for paracrine interplay between 5-HT, GLP-1 and GIP, particularly in the colonic mucosal region. Basolateral epithelial 5-HT₄ receptors mediated both 5-HT and incretin mucosal responses in healthy colon.     

Circadian rhythmicity in the methylation of 5-hydroxy-indoles and norepinephrine in the pineal gland of 10 day old rats

Summary In pineals of 10 day old rats 5-hydroxytryptophan, 5-hydroxyindole-3-acetic acid, N-acetylserotonin/5-hydroxytryptophol and norepinephrine are methylated following a circadian rhythm. During the night HIOMT and COMT activities were measured for the above mentioned substrates, while HIOMT activity for 5-hydroxytryptophan and N-acetylserotonin/5-hydroxytryptophol was also determined during daytime.     

Evidence for the involvement of microtubules in the ADP-induced secretion of platelet C-5-hydroxytryptamine

Platelet rich plasma was incubated with either saline or vincristine. Transmission electromicroscopy showed microtubules to be present in saline preincubated platelets, but absent from the platelets preincubated with vincristine. The release of ¹⁴C labelled 5-Hydroxytryptamine (5HT) by adenosine diphosphate (ADP) was inhibited in the vincristine treated platelets, although ADP induced aggregation was similar in the vincristine and saline treated platelets. It is concluded that microtubules have a role in ADP induced secertion of 5HT, but not in ADP induced aggregation.