Pharmacology of scorpion (Lychas laevifrons Pock) venom with special reference to its neuromuscular activity

L. laevifrons venom caused irreversible blockade of electrically induced twitch responses on phrenic nerve diaphragm and chick biventer cervicis preparation. The venom lowered cat blood pressure, caused a brief cardiac arrest and increased cutaneous capillary permeability. It contracted several smooth muscle preparations. The quick contraction produced on guinea pig ileum was partly antagonized by mepyramine and completely by methysergide. The residual slow contraction was antagonized by SC 19220, a prostaglandin blocker. Haemolysis was not produced by the venom on human RBC. LD50 of crude venom in mice was 13.8 mg/kg (iv).     

Evidence for two adenine derivative receptors in rat ileum which are not involved in the nonadrenergic, noncholinergic response.

The receptors mediating inhibition of the rat ileum by adenosine and adenine nucleotides were studied. ATP and ADP were more potent than AMP or adeonsine. Theophylline antagonized the effects of adenosine and AMP but not those of ATP or ADP. Preparations desensitized to ATP or ADP were still inhibited by adenosine and vice versa. The nonadrenergic, noncholinergic inhibition produced by field stimulation or nicotine was not attenuated by the presence of theophylline or desensitization to ATP. These data indicate that more than one adenine derivative receptor is present in rat ileum and that ATP and adenosine are unlikely candidates for the unknown transmitter.     

Neurotransmitters in the intestine of the Atlantic cod, Gadus morhua

The effects of the putative neurotransmitters acetylcholine, adrenaline, adenosine, ATP, bombesin, 5-hydroxytryptamine, met-enkephalin, neurotensin, somatostatin, substance P and VIP have been investigated in the perfused intestine of the cod, Gadus morhua. The presence and distribution of the different types of nerves was investigated with immunohistochemistry and Falck-Hillarp fluorescence histochemistry. A spontaneous rhythmic activity of the perfused preparations usually occurred within a few minutes from the start of the experiment. This activity was diminished or abolished by addition of atropine, methysergide or tetrodotoxin to the perfusion fluid. Acetylcholine, 5-hydroxytryptamine or substance P caused a contraction of the intestinal wall. The response to acetylcholine was blocked by atropine but not by tetrodotoxin, while the response to 5-hydroxytryptamine was blocked by methysergide and usually also by tetrodotoxin. This indicates that the effect of acetylcholine is direct on the muscle cells, while the effect of 5-hydroxytryptamine may be at least partly via a second neuron. All adrenergic agonists (adrenaline, isoprenaline and phenylephrine) had a dominating inhibitory effect on the intestine. Experiments with antagonists showed that the inhibition is due to stimulation of both alpha-adrenoceptors and beta-adrenoceptors. ATP, adenosine and somatostatin also caused a relaxation of the intestinal wall, often followed by a contraction. Met-enkephalin produced variable responses, either a relaxation, a contraction or both. Bombesin caused a weak inhibition, if anything. Neurotensin and VIP did not visibly affect the intestinal motility. 5-HT-, substance P- and VIP-like immunoreactivity and catecholamine fluorescence were observed in the myenteric plexus, submucosa and muscle layers in all parts of the intestine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological actions of tentacle extract of the jellyfish, Acromitus rabanchatu, occurring in the Bay of Bengal

Tentacle extract of A.rabanchatu, produced a fall of blood pressure in cat, rat and guinea pig. Hypotension produced in cat remained unantagonized by blockers of acetylcholine, histamine and 5-HT. On isolated guinea pig heart, the extract significantly reduced the rate and amplitude of contraction leading to irreversible cardiac arrest. In cats and rats, the respiratory rate and amplitude was decreased significantly and resulted in temporary apnoea. The extract also produced vasoconstriction in perfused rat hindquarter preparation and increased cutaneous capillary permeability. The extract produced contraction in several isolated smooth muscle preparations. Contraction on guinea pig ileum was partly antagonized by atropine and cyproheptadine. On isolated rat phrenic nerve diaphragm and chick biventer cervicis, the extract produced irreversible blockade of the electrical stimulation-induced twitch responses. Haemolytic and myonecrotic activity was exhibited by the extract. LD50 was found to be 7.7 mg/kg (iv, mice).     

Nicotinic action of anatoxin-a on guinea pig ileum antagonized by thymopentin

(+)-Anatoxin-a (ANTX) stimulated guinea pig ileum contraction with a potency similar to that of acetylcholine (ACh); the stimulation was blocked by tubocurarine, hexamethonium, or atropine. Although the contraction stimulated by ANTX was blocked by atropine, no specific inhibition of the binding of [3H]N-methylscopolamine to ileum membranes was observed in the presence of ANTX. Furthermore, ANTX failed to stimulate the secretion of alpha-amylase from pancreatic acinar cells, a process that is activated by cholinergic agonists at the muscarinic receptors. When the ileum itself was stimulated by ACh, the contraction was not blocked by either hexamethonium or tubocurarine. Preincubation of the ileum with hemicholinium caused a 50% reduction in the ability of ANTX to stimulate contraction. Based upon these data, it was inferred that ANTX binds to postganglionic synaptic nicotinic receptors in the ileum, thus releasing endogenous ACh, which in turn causes ileum contraction by interacting with the postsynaptic muscarinic receptors. It was also observed that thymopentin (TP-5), a pentapeptide corresponding to positions 32-36 of thymopoietin, blocked the stimulation of ileum contraction by ANTX.     

Different modes of action of substance P in the motor control of the feline stomach and pylorus

In an experimental in vivo model to study gastropyloric motility in the cat a contraction of the stomach and the pyloric sphincter was regularly obtained in animals subjected to electrical vagal nerve stimulation or local intraarterial (i.a.) injection of substance P (SP). Much more infrequently contractile motor responses were recorded at splanchnic nerve stimulation. The contractile effects of SP were sensitive to atropine or local infusion of a SP analogue, (d-Pro²,d-Trp^7,9)-SP, indicating that SP activated a final common cholinergic neuron in both stomach and pylorus. However, there seemed to be separate transmission mechanisms in these two regions based on the results of the physiological studies. The vagally induced pyloric contraction was noncholinergic, nonadrenergic, but sensitive to ganglionic blockade (hexamethonium) or the SP analogue, indicating involvement of SP in a peptidergic pathway to the sphincter. The infrequent splanchnically induced pyloric contraction was sensitive to atropine, the SP analogue or ganglionic blockade (hexamethonium) in favour of SP acting on a final cholinergic neuron in this system. On the other hand the gastric contraction, obtained at either extrinsic nerve stimulations or local i.a. injection of SP, was sensitive to atropine or the SP analogue but hexamethonium resistant. These findings suggest antidromic activation of SP-containing axon collaterals of the extrinsic nerves terminating on cholinergic neurons of the gastric wall. When afferent C-fibres of the vagal nerve were selectively activated by local heating, pyloric contraction and gastric relaxation were obtained via vago-vagal reflexes. After cervical vagotomy heating of the distal end of the vagal nerve elicited a gastric contraction, previously demonstrated to be atropine sensitive and hexamethonium resistant, but no pyloric motor response. This suggests that the antidromic activation mechanism was present only in the stomach, not in the pylorus.     

Responses of the rectum and oesophagus of the snail Helix aspersa to purine nucleotides and nucleosides

1. Purine compounds were examined for pharmacological activity in the rectum and oesophagus of the garden snail Helix aspersa.2. In the rectum, adenosine, AMP, ADP and ATP (above 10μM) and acetylcholine (above 1 nM) consistently caused concentration-dependent contractions. The slope of the dose-response curve for ADP in the rectum was significantly steeper than for the other purine compounds. The contractile responses to the nucleotides and acetylcholine, but not adenosine, were selectively potentiated by physostigmine (1μM). Atropine (1 μM) and tubocurarine (30 μM) failed to block the responses to the purines or acetylcholine.3. In the oesophagus, adenosine, AMP, ADP and ATP (above 10 μM) and acetylcholine (above 1 nM) caused concentration-dependent contractions that were antagonised by atropine (l μM). Tubocurarine (30 μM) failed to block the responses to the purine compounds or acetylcholine. Physostigmine (1 μM) potentiated the responses to ADP and acetylcholine but not ATP, AMP or adenosine.4. In both the rectum and the oesophagus, the synthetic analogues of purine compounds inclucling 2-chloroadenosine, α, β -methylene ATP and 2-methylthio ATP were inactive up to a concentration of 100 μM.5. Electrical field stimulation of the rectum and oesophagus produced consistent contractions which were unaffected by atropine (1 μM), tubocurarine (30 μM) or physostigmine (1 μM). These responses were not modulated by any of the purine compounds or their stable analogues.6. The responses obtained appear novel even within known invertebrate purinergic systems, suggesting a differentiation of purinoceptor subtypes in this species. There is evidence in the rectum for AMP, ADP and ATP causing the release of acetylcholine; physostigmine potentiated responses to AMP, ADP and ATP, but not to adenosine. This indicates that activity may be mediated via different types of purinoceptors, perhaps equivalent to the P₁- and P₂-purinoceptors identified in vertebrates.     

Multiple responses to electrical field stimulation in circular muscle of canine gastric corpus

Innervation of circular muscle of the canine stomach studied in vitro was investigated by subjecting muscle strips to electrical field stimulation. Strips were cut from the lesser curvature of the gastric corpus and stimulated with 10-s trains of 0.5-ms pulses at 0.5-20 Hz, 40 V. Most responses were classified into one of three types. In general, field stimulation tended to elicit sequences of varying magnitudes of transient on-contraction, on-relaxation, off-relaxation, off-contraction. Responses were abolished by tetrodotoxin. On-contraction was almost abolished by atropine plus desensitization by 5-hydroxytryptamine (5-HT) or substance P. On-relaxation and off-relaxation were not affected by adrenergic blockade, methysergide, apamin, or 4-aminopyridine. ATP usually caused contraction and slightly diminished relaxation to field stimulation. Vasoactive intestinal polypeptide (VIP) had little effect on tone and response to field stimulation. Relaxation disappeared after scorpion venom treatment. This probably resulted from depletion of the transmitter which mediates relaxation. Off-contraction was reduced by atropine, desensitization by 5-HT or substance P, cromoglycate, indomethacin or ATP, but was not affected by adrenergic blockade, hexamethonium, methysergide, mepyramine, or VIP. The findings suggest that innervation of gastric corpus circular muscle included excitatory cholinergic and both excitatory and inhibitory noncholinergic, nonadrenergic innervation. However, the responses of circular muscle to field stimulation in vitro were drastically different from those obtained previously in vivo, suggesting damage or altered inputs to circular muscle when strips of circular muscle are studied.     

Acute Caffeine Treatment Increases Extracellular Nucleotide Hydrolysis from Rat Striatal and Hippocampal Synaptosomes

The psychostimulant caffeine promotes behavioral effects such as hyperlocomotion, anxiety, and disruption of sleep by blockade of adenosine receptors. The availability of extracellular adenosine depends on its release by transporters or by the extracellular ATP catabolism performed by the ecto-nucleotidase pathway. This study verified the effect of caffeine on NTP-Dase 1 (ATP diphosphohydrolase) and 5-nucleotidase of synaptosomes from hippocampus and striatum of rats. Caffeine and theophylline tested in vitro were unable to modify nucleotide hydrolysis. Caffeine chronically administered in the drinking water at 0.3 g/L or 1 g/L for 14 days failed to affect nucleotide hydrolysis. However, acute administration of caffeine (30 mg/kg, ip) produced an enhancement of ATP (50%) and ADP (32%) hydrolysis in synaptosomes of hippocampus and striatum, respectively. This activation of ATP and ADP hydrolysis after acute treatment suggests a compensatory effect to increase adenosine levels and counteract the antagonist action of caffeine.     

Pharmacological and haematological study of shol fish (Channa striatus) skin extract on experimental animal

Snake head fish Channa striatus (locally called 'shol') skin extract (SFSE) was examined for certain pharmacological and haematological effects on experimental animals. LD50 of SFSE was found to be 6 mg/20gm (iv) in male albino mice. SFSE potentiated pentobarbitone induced sleeping time in male albino mice and produced hypothermia. Low dose of SFSE decreased respiratory rate in rat and guineapig and high dose produced apnoea leading to death. On isolated toad and guineapig heart, SFSE significantly decreased rate and amplitude of contraction leading to temporary blockade, which returned after repeated wash. On isolated nerve muscle preparations, SFSE produced irreversible blockade of twitch response. SFSE induced quick contraction on isolated guineapig ileum, which was antagonised by atropine and cyproheptadine. SFSE did not possess haemolytic and haemorrhagic activity but produced anaemia in male albino mice. A neurotoxic compound (fluoroscent and ninhydrin positive) was isolated from SFSE by thin layer chromatography. This compound (CS-NT) was lethal in male albino mice, produced death by apnoea in rat and produced irreversible blockade of isolated nerve-muscle preparation. This study confirms that the skin of Channa striatus possesses toxic, and lethal components, which needs further detailed study.     

Interdependence of mitochondrial ATP production and extramitochondrial ATP utilization in intact spermatozoa

The dependence of both respiration and total activity of ATP-consuming reactions on the cellular adenine nucleotide pattern was investigated in intact bovine spermatozoa. ATP consumption was manipulated by inhibition with vanadate and activation with caffeine, leading to a decrease or increase in the rate of respiration up to 70% or 20%, respectively. Oligomycin blocked the respiration to the same extent as did vanadate, suggesting that the total extramitochondrial ATP-consuming activity is vanadate-sensitive. The major part of ATP utilization must be linked to dynein ATPase, since inhibition of (Na⁺, K⁺) ATPase by ouabain showed only a small effect on respiration (−17%). Being a potent inhibitor of dynein ATPase, vanadate drastically reduced the amount of motile cells, whereas caffeine tended to increase the intensity of motion. The effects of vanadate or caffeine on respiration were paralleled by changes in cellular ATP, reflecting the response of mitochondrial respiration on the cellular ATP/ADP ratio. Respiration was found to depend on changes in the ATP/ADP ratio in the range from about 3 (+ caffeine) to 9 (+ vanadate). The range of response of ATP consumption to the ATP/ADP ratio was determined by varying the mitochondrial ATP production via the concentration of lactate which was used as substrate. The measured effects on both respiratory rate and ATP/ADP ratio suggested that ATP consumption was markedly dependent on ATP/ADP ratios below 5. It is concluded that lactate concentrations above 1 mM sufficiently supply bovine spermatozoa with substrate and the energy turnover is mainly limited by the activity of dynein ATPase rather than by the capacity of mitochondrial oxidative phosphorylation.     

Changes of gastric mucosal biochemistry in ethanol-treated rats with and without acute surgical vagotomy

The biochemical background of ethanol-(ETOH) induced gastric mucosal damage was studied in rats with intact vagus and after acute surgical vagotomy. Observations were carried out on Sprague-Dawley (CFY) strain rats of both sexes. Gastric mucosal lesions were produced by intragastric administration of 1 ml 96% ethanol. Bilateral truncal surgical vagotomy was carried out 30 min before ETOH administration. The number and severity of gastric mucosal lesions was noted 1 h after ETOH administration. Biochemical measurements (gastric mucosal level of ATP, ADP, AMP, cAMP and lactate) were carried out from the total homogenized gastric mucosa. The adenylate pool (ATP + ADP + AMP), energy charge ((ATP + 0.5 ADP)/(ATP + ADP + AMP)) and ratio of ATP/ADP were calculated. It was found that: 1) ATP transformation into ADP increased, while ATP transformation in cAMP decreased in ethanol-treated animals with intact vagus nerve, while these transformations were quite the opposite in vagotomized animals; 2) no significant changes were found in the tissue level of lactate: and 3) the extent of biochemical changes was significantly less after surgical vagotomy. It is concluded that an intact vagus is basically necessary for the metabolic adaptation of gastric mucosa.     

Assays of the metabolic viability of single giant mitochondria. Experiments with intact and impaled mitochondria

Single giant mitochondria isolated from mice fed cuprizone were assayed for their metabolic viability. Two tests were devised. One test optically detected the accumulation of calcium phosphate within the mitochondria under massive loading conditions (including the presence of succinate and ATP). The accumulation corresponds to a test of energy coupling from either electron transport or the hydrolysis of ATP since it is blocked by either antimycin A or oligomycin. The other assay tested for the production of ATP from ADP and Pi, using myofibrils. Myofibrils prepared from glycerinated rabbit psoas muscle contract only in the presence of ATP and not in the presence of ADP. Myofibrillar contraction is unaffected by the presence of antimycin A or oligomycin. However, myofibrils in the presence of mitochondria that are phosphorylating ADP to ATP do contract. This contraction is blocked by antimycin A and/or oligomycin. Hence, the ATP which causes myofibrillar contraction is produced by oxidative phosphorylation. At low mitochondrial concentration, only the myofibrils in close proximity with mitochondria contract in the presence of ADP. Therefore the assay can be used to test the viability of individual mitochondria. Individual giant mitochondria were found to be viable, using both of these assays. Comparable results were obtained in mitochondria impaled with microelectrodes. The potentials and resistances were unaffected by concomitant calcium phosphate accumulation or oxidative phosphorylation.     

Nucleotide coronary vasodilation in guinea pig hearts

The role of P1 receptors and P2Y1 receptors in coronary vasodilator responses to adenine nucleotides was examined in the isolated guinea pig heart. Bolus arterial injections of nucleotides were made in hearts perfused at constant pressure. Peak increase in flow was measured before and after addition of purinoceptor antagonists. Both the P1 receptor antagonist 8-(p-sulfophenyl)theophylline and adenosine deaminase inhibited adenosine vasodilation. AMP-induced vasodilation was inhibited by P1 receptor blockade but not by adenosine deaminase or by the selective P2Y1 antagonist N6-methyl-2'-deoxyadenosine 3',5'-bisphosphate (MRS 2179). ADP-induced vasodilation was moderately inhibited by P1 receptor blockade and greatly inhibited by combined P1 and P2Y1 blockade. ATP-induced vasodilation was antagonized by P1 blockade but not by adenosine deaminase. Addition of P2Y1 blockade to P1 blockade shifted the ATP dose-response curve further rightward. It is concluded that in this preparation ATP-induced vasodilation results primarily from AMP stimulation of P1 receptors, with a smaller component from ATP or ADP acting on P2Y1 receptors. ADP-induced vasodilation is largely due to P2Y1 receptors, with a smaller contribution by AMP or adenosine acting via P1 receptors. AMP responses are mediated solely by P1 receptors. Adenosine contributes very little to vasodilation resulting from bolus intracoronary injections of ATP, ADP, or AMP.     

ATP hydrolysis pathways and their contributions to pial arteriolar dilation in rats

ATP is thought to be released to the extracellular compartment by neurons and astrocytes during neural activation. We examined whether ATP exerts its effect of promoting pial arteriolar dilation (PAD) directly or upon conversion (via ecto-nucleotidase action) to AMP and adenosine. Blockade of extracellular direct ATP to AMP conversion, with ARL-67156, significantly reduced sciatic nerve stimulation-evoked PADs by 68%. We then monitored PADs during suffusions of ATP, ADP, AMP, and adenosine in the presence and absence of the following: 1) the ecto-5'-nucleotidase inhibitor α,β-methylene adenosine 5'-diphosphate (AOPCP), 2) the A(2) receptor blocker ZM 241385, 3) the ADP P2Y(1) receptor antagonist MRS 2179, and 4) ARL-67156. Vasodilations induced by 1 and 10 μM, but not 100 μM, ATP were markedly attenuated by ZM 241385, AOPCP, and ARL-67156. Substantial loss of reactivity to 100 μM ATP required coapplications of ZM 241385 and MRS 2179. Dilations induced by ADP were blocked by MRS 2179 but were not affected by either ZM 241385 or AOPCP. AMP-elicited dilation was partially inhibited by AOPCP and completely abolished by ZM 241385. Collectively, these and previous results indicate that extracellular ATP-derived adenosine and AMP, via A(2) receptors, play key roles in neural activation-evoked PAD. However, at high extracellular ATP levels, some conversion to ADP may occur and contribute to PAD through P2Y(1) activation.     

The adenine nucleotide carrier of plant mitochondria: contraction induced by adenosine diphosphate

Energised mitochondria show an ADP-induced contraction which is partially resistant to oligomycin, uncouplers or respiratory inhibitors but sensitive to atractyloside.The addition of ADP or ATP, but not AMP, to non-energised corn mitochondria induces a contraction with a K_d of approx. 1 μM. Titration of the ADP-induced contraction with atractyloside produces an inhibition curve closely resembling the atractyloside inhibition curve of phosphorylating respiration. Partial recovery of atractyloside-inhibited contraction occurs in the presence of bongkrekic acid.It is suggested that these changes reflect changes in orientation of the adenine nucleotide (AdN) carrier in the inner mitochondrial membrane.     

The effects of adenosine and adenine nucleotides on the guinea-pig vas deferens: evidence for existence of purinergic receptors.

The effect of adenosine 5′-triphosphate (ATP) and its congeners on the alpha-adrenergic neuroeffector transmission in the isolated vas deferens of the guinea pig was evaluated. Both intracellular activity and contractile response of the smooth muscle of the vas deferens were recorded by using the sucrose-gap method. Adenosine, adenosine diphosphate (ADP) and adenosine monophosphate (AMP) influenced alpha-adrenergic receptor-mediated excitatory responses by depolarizing the cell membrane potential. ATP, on the other hand, produced action potentials rather than sustained depolarization, and its activity was blocked by theophylline and 2, 2′-pyridylisatogen, an ATP antagonist, but not blocked by either phentolamine or phenoxybenzamine, which inhibit alpha-adrenoreceptor responsiveness caused by norepinephrine or phenylephrine. Furthermore, dipyridamole, an adenosine uptake blocker, potentiated both ATP and adenosine activities. These findings indicate that adenosine and adenine nucleotides may exert their action at an extracellular site. From these results, it may be speculated that alpha adrenoreceptors and purinergic receptors do indeed exist on the smooth muscle of the vas deferens.     

IMP production and energy metabolism during exercise in rats in relation to age.

IMP production in and force exerted by rat quadriceps muscle in situ during various types of exercise were examined in relation to age. During continuous isometric exercise with constant stimulation time, the amount of IMP was linearly and inversely related to the age of the animals; a higher IMP concentration was found in intermittent isometric and dynamic exercise. No relationship was found between the total AMP deaminase activity and age. Exercise influenced neither the total activity nor the activity in the soluble fraction. From the results it is concluded that: the IMP concentration is linearly related to the free intracellular ATP4-/ADP3- ratio and the free AMP2- concentration; older animals are better able to maintain a high intramuscular ATP4-/ADP3- ratio and a low AMP2- concentration; IMP is produced in particular under conditions when the muscle has to work under extreme stress. IMP possibly exerts a feed-back control on the contraction system.     

Extracellular ATP has a potent effect to enhance cytosolic calcium and contractility in single ventricular myocytes

The effect of extracellular ATP on the contraction of single rat cardiac myocytes was investigated, together with the effect on the transient change in cytosolic Ca2+ (Cai) elicited by excitation and on the relationship between these two parameters. In unstimulated single myocytes, ATP caused a small increase in Cai (measured as the ratio of fluorescence of Indo-1 at 410 to that at 490 nm. In myocytes bathed in a medium containing 1.0 mM [Ca2+] at 23 degrees C and stimulated at 1 Hz, ATP (1 microM) resulted in a two-threefold increase in amplitude of contraction, as measured by video cinemicrographic techniques. The duration of the Cai-transient was not altered but its amplitude was markedly enhanced, as was the amplitude of contraction. The relation between Cai and contraction-amplitude was not altered by ATP, when measured over a range of extracellular [Ca2+], suggesting that ATP does not affect the myofilament-Ca2+ interaction. The primary site of action of ATP in increasing Cai is at the sarcolemma since the addition to suspensions of myocytes of caffeine (10 mM), which depletes the sarcoplasmic reticulum Ca2+ load, does not prevent the subsequent increase of Cai due to ATP. Further, lowering of the extracellular [Ca2+] to less than 1 microM with EGTA abolishes the response of Cai to ATP, though not the response to caffeine. Thus in rat cardiac myocytes ATP stimulates trans-sarcolemmal influx of Ca2+: ADP, AMP and adenosine are ineffective. ATP markedly augments the amplitude of the Cai transient elicited by electrical stimulation thus rendering it a potent inotropic agent.     

Inhibition by ADP of prostaglandin induced accumulation of cyclic AMP in intact human platelets

The influence of extracellular ADP on cyclic AMP accumulation within intact human platelets was studied. ADP inhibited the increase in cyclic AMP which occurs when platelets are exposed to prostaglandin E1 or I2. The degree of inhibition varied in the range 70-95% , and was half maximal at ADP concentrations of between 0.3 and 2 microM. Other naturally occurring diphosphates, i.e. GDP, IDP and UDP, were at least 100 fold less effective than ADP, and UDP at 1mM partially reversed the effect of ADP. The effect by ADP was completely reversed by ATP, but only attenuated to a minor degree of 10 mM EDTA. Increasing concentrations of ADP caused a progressive degree of inhibition of cyclic AMP accumulation, and the kinetics of this inhibition were compatible with a simple saturable process with no cooperativity. ADP added 10 seconds after prostaglandin E1 blocked cyclic AMP accumulation within 1-2 seconds, and addition of ATP after ADP and prostaglandin I2 relieved the inhibition due to ADP within 2-3 seconds. The action of ADP was blocked by sulphydryl reagents including N-substituted maleimides, cytochalasin A, NBD chloride and p-mercuribenzene sulphonate. The data were considered to be consistent with mediation of the ADP effect through a sulphydryl-bearing specific extracellular receptor coupled to the adenylate cyclase.