Proghrelin-derived peptides influence the secretion of insulin, glucagon, pancreatic polypeptide and somatostatin: a study on isolated islets from mouse and rat pancreas

Proghrelin, the precursor of the orexigenic and adipogenic peptide hormone ghrelin, is synthetized in endocrine (A-like) cells in the gastric mucosa. During its cellular processing, proghrelin gives rise to the 28-amino acid peptide desacyl ghrelin, which after octanoylation becomes active acyl ghrelin, and to the 23-amino acid peptide obestatin, claimed to be a physiological opponent of acyl ghrelin. This study examines the effects of the proghrelin products, alone and in combinations, on the secretion of insulin, glucagon, pancreatic polypeptide (PP) and somatostatin from isolated islets of mice and rats. Surprisingly, acyl ghrelin and obestatin had almost identical effects in that they stimulated the secretion of glucagon and inhibited that of PP and somatostatin from both mouse and rat islets. Obestatin inhibited insulin secretion more effectively than acyl ghrelin. In mouse islets, acyl ghrelin inhibited insulin secretion at low doses and stimulated at high. In rat islets, acyl ghrelin inhibited insulin secretion in a dose-dependent manner but the IC(50) for the acyl ghrelin-induced inhibition of insulin release was 7.5 x 10(-8) M, while the EC(50) and IC(50) values, with respect to stimulation of glucagon release and to inhibition of PP and somatostatin release, were in the 3 x 10(-12)-15 x 10(-12) M range. The corresponding EC(50) and IC(50) values for obestatin ranged from 5 x 10(-12) to 20 x 10(-12) M. Desacyl ghrelin per se did not affect islet hormone secretion. However, at a ten times higher concentration than acyl ghrelin (corresponding to the ratio of the two peptides in circulation), desacyl ghrelin abolished the effects of acyl ghrelin but not those of obestatin. Acyl ghrelin and obestatin affected the secretion of glucagon, PP and somatostatin at physiologically relevant concentrations; with obestatin this was the case also for insulin secretion. The combination of obestatin, acyl ghrelin and desacyl ghrelin in concentrations and proportions similar to those found in plasma resulted in effects that were indistinguishable from those induced by obestatin alone. From the data it seems that the effects of endogenous, circulating acyl ghrelin may be overshadowed by obestatin or blunted by desacyl ghrelin.     

Stimulation by prostaglandin E2 of glucagon and insulin release from isolated rat pancreas.

To ascertain whether prostaglandins (PG) may play a role in the secretion of glucagon and in an attempt to elucidate the conflicting observations on the effects of PG on insulin release, the isolated intact rat pancreas was perfused with solutions containing 1.1 x 10(-9) to 1.8 x 10(-5)m PGE2. In the presence of 5.6 mM glucose significant increments in portal venous effluent levels of glucagon and insulin were observed in response to minimal concentrations of 2.8 X 10(-8) and 1.4 X 10(-7) PGE2, respectively; a dose-response relationship was evident for both hormones at higher concentrations of PGE2. When administered over 60 seconds, 1.4 X 10(-6)M PGE2 resulted in a significant increase in glucagon levels within 24 seconds and in insulin within 48 seconds. Ten-minute perfusions of 1.4 X 10(-6)M PGE2 elicited biphasic release of both islet hormones; Phase I glucagon release preceded that of insulin. Both phases of the biphasic glucagon and insulin release which occurred in response to 15-minute perfusions of 10 mM arginine were augmented by PGE2. These observations indicate that PGE2 can evoke glucagon and insulin release at concentrations close to those observed by others in the extracts of rat pancreas. We conclude that PG may be involved in the regulation of secretion of glucagon and insulin and may mediate and/or modify the pancreatic islet hormone response to other secretagogues.     

Isolation of glucagon-secreting cell lines by cloning insulinoma cells

Summary Six glucagon-secreting cell lines designated as In-R1G1,-G3,-G7,-G9,-G10, and-G11 were isolated from insulinoma cells (In-111-R1) by single cell cloning. A small amount of insulin was also detectable in the incubation medium when hormone secretion was stimulated by the addition of arginine or theophylline. These cell lines grew as monolayers and the population doubling times varied from 16.8 to 28.8 h. Karyologically these clones were aneuploid and the modes of chromosome numbers were 61 to 70. Electron microscopic examination of one of these clones showed that these cells contained moderately developed Golgi apparatus and a few secretory granules, which more or less resembled α-cell granules. By gell filtration study of the incubation medium, glucagon and glucagonlike material were eluted. The molecular weight of the latter was approximately 9000, which suggested the concomitant secretion of rpoglucagon into the medium. The levels of secreted glucagon in basal state were 0.3 to 3.0 ng/10⁶ cells/2 h. Glucagon secretion was markedly enhanced in the presence of amino acids. Glucagon secretion increased slightly in the presence of high concentration of glucose in Hanks' balanced salt solution; however it was not affected by the varying concentrations of glucose when the cells were incubated in complete media with amino acids. Glucagon secretion was also stimulated by the addition of theophylline. These clonal cell lines seem to provide a useful tool for investigating the mechanism of glucagon secretion.     

Crystallization of mouse pancreatic ribonuclease

Mouse pancreatic ribonuclease has been crystallized in a form suitable for X-ray structure determination. The crystals grown from solutions of 2-methyl-2,4-pentanediol diffract to high resolution and belong to the hexagonal space group P6(1) (P6(5)) with unit cells dimensions a = b = 64.44 A, c = 53.91 A, y = 120 degrees and V = 1.94 x 10(5) A3 (1 A = 0.1 nm). There are six molecules per unit cell (1 molecule/asymmetric unit), and Vm = 2.3 A3/dalton.     

Distribution of neurotransmitters and their effects on glucagon secretion from the in vitro normal and diabetic pancreatic tissues

The distribution of adrenergic, cholinergic and amino acid neurotransmitters and/or their enzymes were examined in both the normal and diabetic pancreatic tissues in rat using immunohistochemistry to determine whether changes in the pattern of distribution of nerves containing these neurotransmitters will occur as a result of diabetes mellitus. In addition to this, the effect of noradrenaline (NA), adrenaline (ADR), acetylcholine (ACh) and gamma-amino butyric acid (GABA) on glucagon secretion from the isolated normal and diabetic pancreatic tissues was also investigated. Pancreatic fragments from the tail end of normal and diabetic rats were removed and incubated with different concentrations (10(-8)-10(-4) M) of these neurotransmitters. Glucagon secretion into the supernatant was later determined by radioimmunoassay. NA at 10(-6) M evoked a three-fold increase in glucagon secretion from normal pancreatic tissue fragments. In diabetic pancreatic tissue, NA at 10(-6) M was able to increase glucagon secretion 1.5 times the value obtained from diabetic basal. ADR (10(-8) M) increased glucagon secretion slightly but not significantly in normal pancreatic tissue. ADR inhibited glucagon secretion from diabetic pancreas at all concentrations. ACh (10(-8) M) induced a five-fold increase in glucagon secretion from normal pancreatic tissue. In a similar way, ACh evoked a two-fold increase in glucagon secretion from diabetic pancreas at 10(-4) M. In normal pancreatic tissue, GABA produced a slight but not significant increase in glucagon secretion at 10(-4) M. In contrast to this it inhibited glucagon secretion from diabetic pancreatic tissue fragments at all concentrations. In summary, tyrosine hydroxylase- and choline acetyltransferase-positive nerves are equally well distributed in both normal and diabetic rat pancreas. There was an increase in the number of glucagon positive cells and a decrease in the number of GABA-positive cells in diabetic pancreas. NA and ACh have a potent stimulatory effect on glucagon secretion from normal pancreatic tissue fragments, whereas ADR and GABA produced a small but not significant increase in glucagon secretion from normal pancreas. NA and GABA stimulated glucagon secretion from diabetic pancreas. In contrast, ADR and ACh inhibited glucagon secretion from diabetic pancreas. Neurotransmitters vary in their ability to provoke glucagon secretion from either normal or diabetic pancreas.     

Molecular organization of rat prolactin granules. I. In vitro stability of intact and "membraneless" granules.

Studies carried out on a number of secretory cell systems suggest that the specific cytoplasmic granules in which the secretion products are stored before their release are complex organelles which can possess a distinct molecular organization. For instance, it has been reported that in some granules the segregated secretion products are organized into crystalline structures (1-3) or large intermolecular aggregates (4-8). It is likely that all phenomena of this type are favorable to the economy of the cell, in the sense that they reduce the energy required for storage of the secretion products. The prolactin (LTH) granules of the rat pituitary possess a number of morphological features which strongly suggest that the molecules(s) of their content might be arranged in a relatively stable structure. Thus, these granules are remarkably polymorphic in shape, and their membrane is usually separated from their content by a clear space. Furthermore, identifiable LTH granules devoid of their membrane are often seen in the pericapillary space, suggesting that upon discharge by exocytosis they are dissolved only slowly (9). However, no studies specifically concerned with the mechanisms of LTH storage have been reported so far. In order to obtain some information on this question, we have studied the behavior of isolated granule fractions incubated in vitro under a variety of carefully controlled experimental conditions.     

Particle counting by fluorescence correlation spectroscopy. Simultaneous measurement of aggregation and diffusion of molecules in solutions and in membranes.

A method for simultaneous determination of molar weights (M) and lateral diffusion constants (D) of particles in three- and two-dimensional systems is described. Spontaneous concentration fluctuations in space and time are analyzed, by monitoring fluctuations in the fluorescence from fluorescein-labeled molecules (1 dye/molecule is sufficient), excited by a rotating laser spot. For particles in solution, M values are determined over the range of 3 x 10(2) to 3 x 10(11) daltons, and D values can be determined from approximately 10(-7) to 10(-10) cm2/s. The time for a determination is approximately 1 min. Aggregation can be followed by changes of either M or D. This method is used to study the calcium dependence of vesicle aggregation or fusion, and the time course of aggregate formation of porin (an Escherichia Coli outer membrane protein) in lipid monolayers. Essential parameters for the development of the method are described. Equations to estimate the signal-to-noise ratios and to find the optimal free parameters for a specific application are derived. The theoretical predictions for the correlation function of the signal and for the signal-to-noise ratio are compared with observed values.     

Specific localization of membrane dipeptidase and dipeptidyl peptidase IV in secretion granules of two different pancreatic islet cells.

Endocrine cells require several protein convertases to process the precursors of hormonal peptides that they secrete. In addition to the convertases, which have a crucial role in the maturation of prohormones, many other proteases are present in endocrine cells, the roles of which are less well established. Two of these proteases, dipeptidyl peptidase IV (EC 3.4.14.5) and membrane dipeptidase (EC 3.4.13.19), have been immunocytochemically localized in the endocrine pancreas of the pig. Membrane dipeptidase was present exclusively in cells of the islet of Langerhans that were positive for the pancreatic polypeptide, whereas dipeptidyl peptidase IV was restricted to cells positive for glucagon. Both enzymes were observed in the content of secretory granules and therefore would be released into the interstitial space as the granules undergo exocytosis. At this location they could act on secretions of other islet cells. The relative concentration of dipeptidyl peptidase IV was lower in dense glucagon granules, where the immunoreactivity to glucagon was higher, and vice versa for light granules. This suggests that, in A-cells, dipeptidyl peptidase IV could be sent for degradation in the endosomal/lysosomal compartment during the process of granule maturation or could be removed from granules for continuous release into the interstitial space. The intense proteolytic activity that takes place in the endocrine pancreas could produce many potential dipeptide substrates for membrane dipeptidase. (J Histochem Cytochem 47:489-497, 1999)     

Some taste molecules and their solution properties.

The solution properties of a variety of different sapid substances from all four basic taste modalities, namely, sweet (n = 24), salty (n = 7), sour (n = 11) and bitter (n = 2), have been investigated. Some multisapophoric molecules, i.e. molecules exhibiting more than one taste, have also been included in the study in an attempt to define their properties in relation to the tastes they exhibit; eight sweet-bitter and three salty-bitter molecules were used. The density and sound velocity of their solutions in water have been measured and their apparent volumes, apparent compressibilities and compressibility hydration numbers calculated and compared. Apparent molar volumes (phi(v)) and apparent specific volumes (ASV) reflect the state of hydration of the molecules, and thus their extent of interaction with water structure. The range of ASVs reported are 0.13-0.49 cm3/g for salty molecules, 0.55-0.68 cm3/g for sweet molecules, 0.53-0.88 cm3/g for sweet-bitter molecules and a much wider range (0.16-0.85 cm3/g) for sour molecules. Isentropic apparent specific compressibilities range from -2.33 x 10(-5) to -8.06 x 10(-5) cm3/g x bar for salty molecules, -3.38 x 10(-7) to -2.34 x 10(-5) cm3/g x bar for sweet molecules, +6.35 x 10(-6) to -2.22 x 10(-5) cm3/g x bar for sweet-bitter molecules and +6.131 x 10(-6) to -2.99 x 10(-5) cm3/g x bar for sour molecules. Compressibility hydration numbers are also determinable from the measurements of isentropic compressibilities and these reflect the number of water molecules that are disturbed by the presence of the solutes in solution. This study also shows that it is possible to group isentropic apparent molar compressibility values by the taste quality exhibited by the molecules in the same order as for ASV.     

Influence of gastric inhibitory polypeptide (GIP) and glucose on the regulation of glucagon secretion by pancreatic alpha cells

The effects of glucose and GIP on glucagon secretion were studied in perifused microdissected murine pancreatic islets. Glucagon levels were determined in effluent samples collected at 1-min intervals by radioimmunoassay using the glucagon-specific antibody, 30 K. There was no significant difference in the total amount (7740 +/- 212 pg vs 8630 +/- 36 pg, n = 10) of glucagon secreted over a 20 min period when the glucose concentration was alternately shifted between 5.5 mM and 11.1 mM, respectively. However, 22.2 mM glucose profoundly suppressed glucagon secretion. The suppressive effect of high glucose on glucagon release was partially, yet significantly, reversed by the presence of GIP, as glucagon secretion increased from a non-detectable level at 22.2 mM glucose alone to 10,175 +/- 145 pg, n = 10 (P less than 0.01). The glucagonotropic effect of GIP was dose-dependent in the range of 2 x 10(-9) - 2 x 10(-7) M, at 11.1 mM glucose. Our data show that GIP is able to substantially reverse the suppressive effect of a high glucose load on glucagon secretion.     

Distribution of NPY and SP and their effects on glucagon secretion from the in vitro normal and diabetic pancreatic tissues

Neuropeptides modulate the function of classic neurotransmitters in the regulation of body function. The role of neuropeptides in the regulation of endocrine secretion from the pancreas of diabetic rat is poorly understood. This study examined the pattern of distribution of neuropeptide-Y (NPY) and substance P (SP) in normal and diabetic rat pancreases. In addition to this, the effect of NPY and SP on glucagon secretion was also examined in the pancreases of normal and diabetic rats. Four weeks after the induction of diabetes, the pancreaseses of normal and diabetic rats were removed and processed for immunohistochemistry and glucagon secretion. The pattern of distribution of glucagon in the pancreas of diabetic rat was conspicuously deranged after the onset of diabetes. The pattern of distribution of NPY and SP was, however, similar in the pancreases of both normal and diabetic rats. Stimulation of normal rat pancreatic tissue with NPY (10(-12) and 10(-9) M) evoked large and significant (P < 0.001) increases in glucagon secretion compared to basal. In contrast to this, NPY inhibited glucagon secretion from the pancreas of diabetic rat. Treatment of pancreatic tissue fragments of normal rat with 10(-9) M SP resulted in significant (P < 0.03) increases in glucagon secretion. SP inhibited glucagon secretion from diabetic rat pancreas. In conclusion, NPY and SP stimulated glucagon secretion from the pancreas of normal rat. In contrast, NPY and SP inhibited glucagon secretion from diabetic rat pancreas.     

Crystallization of a tRNA . aminoacyl-tRNA synthetase complex. Characterization and first crystallographic data

A complex formed between the dimeric aspartyl-tRNA synthetase from yeast (Mr congruent to 125,000) and two molecules of its cognate yeast tRNAAsp (Mr = 24,160) was crystallized using ammonium sulfate as the precipitant. The crucial parameter which governs a successful crystallization is the enzyme tRNA stoichiometry. Crystals are only obtained when the starting solution precisely contains two tRNA molecules for one enzyme molecule. It was demonstrated by electrophoresis, biological activity assays, and crystallographic data that the crystals contain the two components in the same two to one stoichiometric ratio. The crystals, of cubic shape with edges up to 0.8 mm, belong to space group 1432. The cell parameter is 354 A and the asymmetric unit contains one particle of complex. The solvent content is about 78%, higher than the values commonly observed. Although particularly soft, the quality of the crystals is suitable for x-ray diffraction studies up to 7-A resolution.     

Model for glucagon secretion by pancreatic α-cells

Glucagon hormone is synthesized and released by pancreatic α-cells, one of the islet-cell types. This hormone, along with insulin, maintains blood glucose levels within the physiological range. Glucose stimulates glucagon release at low concentrations (hypoglycemia). However, the mechanisms involved in this secretion are still not completely clear. Here, using experimental calcium time series obtained in mouse pancreatic islets at low and high glucose conditions, we propose a glucagon secretion model for α-cells. Our model takes into account that the resupply of releasable granules is not only controlled by cytoplasmic Ca2+, as in other neuroendocrine and endocrine cells, but also by the level of extracellular glucose. We found that, although calcium oscillations are highly variable, the average secretion rates predicted by the model fall into the range of values reported in the literature, for both stimulated and non-stimulated conditions. For low glucose levels, the model predicts that there would be a well-controlled number of releasable granules refilled slowly from a large reserve pool, probably to ensure a secretion rate that could last for several minutes. Studying the α-cell response to the addition of insulin at low glucose, we observe that the presence of insulin reduces glucagon release by decreasing the islet Ca2+ level. This observation is in line with previous work reporting that Ca2+ dynamics, mainly frequency, is altered by insulin. Thus, the present results emphasize the main role played by Ca2+ and glucose in the control of glucagon secretion by α-cells. Our modeling approach also shows that calcium oscillations potentiate glucagon secretion as compared to constant levels of this cellular messenger. Altogether, the model sheds new light on the subcellular mechanisms involved in α-cell exocytosis, and provides a quantitative predictive tool for studying glucagon secretion modulators in physiological and pathological conditions.     

Effect of beta radiation on the crystallization of sodium chlorate from water: a new type of asymmetric synthesis.

Sodium chlorate is an achiral molecule that crystallizes from water in the chiral space group P2(1)3. In the absence of chiral perturbations, a random distribution of (+) and (-) crystals is obtained. Kondepudi(2) has shown that constantly stirring an evaporating NaClO(3) solution gives mostly either (+) or (-) crystals. Repeating this experiment many times gives equal numbers of (+) and (-) sets of crystals. Herein we report that when evaporating aqueous NaClO(3) is subjected to beta particles from an Sr-90 source, an asymmetric distribution of (+) and (-) crystals favoring the (+) crystals is obtained. The beta particles are energetic polarized electrons that are approximately 80% of left-handed helicity. By a poorly understood mechanism, the spin polarized electrons produce chiral nucleating sites that favor formation of the (+)-NaClO(3) crystals. Exposure of the evaporating solution instead to energetic positrons from an Na-22 source yields mainly (-)-NaClO(3) crystals. Polarized positrons are of predominantly right-handed helicity. One may conclude that the chirality of the radiation is correlated with the chirality of the crystals being generated.     

铃蟾肽(Bombesin)对离体灌流大鼠胃的胃泌素、生长抑素、胰升糖素及胃酸释放的影响

本研究用离体大鼠胃灌流技术来观察铃蟾肽对胃-肠激素及胃酸分泌的影响。2×10~(?)mol/L铃蟾肽以0.3ml/min速度作动脉内输注,可刺激胃酸的分泌,自2.50±0.05×10~(-1)增至5.50±1.50×10~(-1)mEq/min,但与外源性五肽胃泌素无协同作用。铃蟾肽引起两次性的门脉中胃泌索及生长抑素的释放,但抑制胰升糖素释放。这三种激素的基础释放率分别为:胃泌素62±8pg,生长抑素5.9±1.1ng,胰升糖素0.40±0.03ng/min;2×10~(-8)mol/L铃蟾肽以0.3ml/min作动脉内输注,胃泌素及生长抑素的峰值分别为1,000±20pg及12.2±2.0ng/min,胰升糖素的最低值为0.17±0.05ng/min,三种激素的反应均与铃蟾肽的浓度成正比。在胃腔流出液中也可测到上述三种激素,但量要少得多。     

Crystallographic studies of glutamate dehydrogenase. Preliminary crystal data.

Native and pyridoxal phosphate modified rat liver glutamate dehydrogenase crystals have been obtained and used for a preliminary x-ray crystallographic analysis. The space group is P6222 (P6422) having unit cell dimensions a = b = 101 A, c = 724 A and gamma = 120 A. The unit cell contains 36 subunits (six hexameric molecules) of molecular weight 56,000 and there is one half-molecule, i.e. three subunits, in the asymmetric unit. Packing considerations suggest that the glutamate dehydrogenase molecule has the point group symmetry 32 and that each subunit can be represented as a particle with approximate dimensions of 45 x 45 x 60 A.     

The endocrine cells in the gastroenteropancreatic system of the bowfin, Amia calva L.: an immunohistochemical, ultrastructural, and immunocytochemical analysis.

The gastroenteropancreatic (GEP) endocrine system of bowfin (Amia calva) was described using light and electron microscopy and immunological methods. The islet organ (endocrine pancreas) consists of diffusely scattered, mostly small islets and isolated patches of cells among and within the exocrine acini. The islets are composed of abundant, centrally located B cells immunoreactive to bovine and lamprey insulin antisera and D cells showing a widespread distribution and specificity to somatostatin antibodies. A and F cells are present at the very periphery of the islets and are immunoreactive with antisera against glucagon (and glucagon-like peptide) and several peptides of the pancreatic polypeptide (PP)-family, respectively. The peptides of the two families usually collocates within the same peripheral islet cells and are the most common immunoreactive peptides present in the extra-islet tissue. Immunocytochemistry and fine structural observations characterised the granule morphology for B and D cells and identified two cell types with granules immunoreactive to glucagon antisera. These two putative A cells had similar granules, which were distinct from either B or D cells, but one of the cells had rod-shaped cytoplasmic inclusions within cisternae of what appeared to be rough endoplasmic reticulum. The inclusions were not immunoreactive to either insulin or glucagon antisera. Only small numbers of cells in the stomach and intestine immunoreacted to antisera against somatostatin, glucagon, and PP-family peptides. The paucity of these cells was reflected in the low concentrations of these peptides in intestinal extracts. The GEP system of bowfin is not unlike that of other actinopterygian fishes, but there are some marked differences that may reflect the antiquity of this system and/or may be a consequence of the ontogeny of this system in this species.     

Toxicity of parasporal crystals of Bacillus thuringiensis subsp. israelensis to mosquitoes.

Toxicity of Bacillus thuringiensis subsp. israelensis (ONR-60A/WHO 1897) parasporal crystals to three medically important mosquito larvae is described. The numbers of larvae killed are in relation to crystal dry weight. The crystals are lethally toxic to Aedes aegypti Linnaeus (mean 50% lethal concentration [LC50] = 1.9 x 10(-4) micrograms/ml), Culex pipiens var. quinquefasciatus Say (LC50 = 3.7 x 10(-4) micrograms/ml), and Anopheles albimanus Wiedemann (LC50 = 8.0 x 10(-3) micrograms/ml). Purfied crystals of B. thuringiensis subsp. kurstaki, which are toxic to lepidopteran insects, are ineffective against the mosquito larvae. Likewise, B. thuringiensis subsp. israelensis parasporal crystals are not efficacious for larvae of the lepidopteran, Manduca sexta.     

Toxicity of parasporal crystals of Bacillus thuringiensis subsp. israelensis to mosquitoes.

Toxicity of Bacillus thuringiensis subsp. israelensis (ONR-60A/WHO 1897) parasporal crystals to three medically important mosquito larvae is described. The numbers of larvae killed are in relation to crystal dry weight. The crystals are lethally toxic to Aedes aegypti Linnaeus (mean 50% lethal concentration [LC50] = 1.9 x 10(-4) micrograms/ml), Culex pipiens var. quinquefasciatus Say (LC50 = 3.7 x 10(-4) micrograms/ml), and Anopheles albimanus Wiedemann (LC50 = 8.0 x 10(-3) micrograms/ml). Purfied crystals of B. thuringiensis subsp. kurstaki, which are toxic to lepidopteran insects, are ineffective against the mosquito larvae. Likewise, B. thuringiensis subsp. israelensis parasporal crystals are not efficacious for larvae of the lepidopteran, Manduca sexta.     

A novel technique for rapid determination of energy consumption in platelets. Demonstration of different energy consumption associated with three secretory responses

A novel method has been developed for rapid and quantitative determination of the rate of energy consumption in platelets. In platelets suspended in a cyanide-containing medium. ATP resynthesis is abruptly blocked by addition of 2-deoxyglucose and D-glucono-1,5-lactone. We demonstrate that the subsequent changes in the levels of cytoplasmic ATP and ADP reflect the velocity of energy consumption in the platelets immediately before addition of the inhibitors. Despite the arrest in ATP resynthesis the platelets remain responsive to stimulation by thrombin (5 units x ml-1) which triggers the secretion of the contents of dense, alpha- and acid hydrolase granules. Unstimulated platelets were found to consume about 3.5 and 0.5 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at 37 degrees C and 15 degrees C, respectively; the thrombin-treated platelets consumed respectively 16 and 2 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at these temperatures. When the velocity of energy consumption was varied by (a) changing the temperature and (b) preincubation with glyco(geno)lytic inhibitors, it was found to be linearly related to the initial rate of secretion from the three types of granules. The precise nature of this relationship differed between the three types of secretion responses and indicated an increasing requirement for metabolic energy for secretion from the three types of granules in the order: dense granule less than alpha-granule less than acid hydrolase granule. The results obtained with changes in temperature were superimposable on those obtained with the glyco(geno)lytic inhibitors for dense granule secretion and alpha-granule secretion, suggesting an apparent coupling between energy consumption and the rate of these secretion responses. The rate of secretion of acid hydrolase was always higher when energy consumption was varied by temperature changes than when glyco(geno)lytic inhibitors were used, probably as a result of metabolic changes prior to induction of secretion. On the basis of these experiments, we calculated an incremental energy consumption during complete secretion of dense, alpha- and acid hydrolase granule contents of 2.5, 4.2 and 6.7 mumol of ATP equivalents x (10(11) platelets)-1, respectively.