Purine metabolism by the avian malarial parasite Plasmodium lophurae

Extracts of normal duckling erythrocytes catabolized AMP to IMP, inosine and hypoxanthine; adenosine and adenine were not formed from AMP. When erythrocyte-free Plasmodium lophurae, prepared by antibody lysis, were incubated in the presence of [¹⁴C]hypoxanthine approximately 60% of the label was recovered as purine nucleotides and there was no evidence for extracellular alteration of added hypoxanthine. However, when adenosine was added to suspensions of antibody- or saponin-prepared parasites extensive conversion to inosine and hypoxanthine occurred. This conversion was found to be the result of parasite lysis with release of cytosolic purine salvage pathway enzymes; plasmodial surface membrane ecto-enzymes were not responsible for adenosine catabolism. It appears that in vivo the intracellular plasmodium utilizes the normal erythrocytic process of purine turnover to avail itself of hypoxanthine, the red cell's end-product, and at the same time the parasite avoids direct competition for adenosine essential to erythrocyte survival. Since the blood plasma of infected ducklings contained increased amounts of hypoxanthine it is possible that P. lophurae also utilizes this as a purine source.     

Release of Adenosine-like Material from Isolated Perfused Dog Adipose Tissue Following Sympathetic Nerve Stimulation and its Inhibition by Adrenergic α-Receptor Blockade

Following the intraarterial infusion of ^sH-adenine to isolated perfused canine subcutaneous adipose tissue, its adenine nucleotides are labelled. A continuous release of radioactivity, comprised of non-nucleotide material, was observed. The rate of this release was markedly enhanced by sympathetic nerve stimulation. The major components of the enhanced release appeared to be inosine and adenosine. Adrenergic α-receptor blockade (phentolamine or Hydergin®) abolished the enhanced nucleoside release, while glycerol release was enhanced. The release of radioactivity was decreased during mechanical blood flow reduction and enhanced afterwards. However, the magnitude of this enhancement of release after clamp was much less than following nerve stimulation. The results suggest that adenosine or a closely related compound is released from canine subcutaneous adipose tissue by sympathetic nerve stimulation and that the release is related to adrenergic α-receptor stimulation. Since adenosine is a potent inhibitor of catecholamine induced lipolysis in this tissue the possibility of a regulatory role must be considered.     

Mechanism of Ca++ Release from the Sarcoplasmic Reticulum: A Computer Model

The proposed model describes myocyte calcium (Ca⁺⁺) cycling, emphasizing the kinetics of sarcoplasmic reticulum (SR) Ca⁺⁺ release channels. The suggested SR channel regulating mechanism includes two types of Ca⁺⁺ binding sites: (1) low affinity sites with high binding rates, regulating the opening of Ca⁺⁺ channels and (2) high affinity sites with low binding rates, which regulate their closing. The amount of Ca⁺⁺ released from the SR and the peak value of Ca⁺⁺ ion concentration [Ca⁺⁺] in the cytoplasm were found to depend on the rate of the increase of [Ca⁺⁺], similar to Ca⁺⁺ induced Ca⁺⁺ release experiments. The model describes spontaneous release of Ca⁺⁺ from overloaded SR. The dependence of the control mechanism on the activating and inactivating sites is substantiated by simulations of ryanodine intervention, providing results similar to experimental results. Simulations under conditions of isolated SR vesicles produced Ca⁺⁺ release results similar to measured data. Consequently, it is suggested that the recovery of Ca⁺⁺ release channels represents the rate limiting factor in the process of mechanical restitution. © 1998 Biomedical Engineering Society. PAC98: 8722Fy, 8710+e     

Inositol trisphosphate and cyclic adenosine diphosphate-ribose increase quantal transmitter release at frog motor nerve terminals: possible involvement of smooth endoplasmic reticulum

The release of chemical transmitter from nerve terminals is critically dependent on a transient increase in intracellular Ca²⁺.6., 25. The increase in Ca²⁺ may be due to influx of Ca²⁺ from the extracellular fluid¹⁵ or release of Ca²⁺ from intracellular stores such as mitochondria.1., 8., 18. Whether Ca²⁺ utilized in transmitter release is liberated from organelles other than mitochondria is uncertain. Smooth endoplasmic reticulum is known to release Ca²⁺, e.g., on activation by inositol trisphosphate or cyclic adenosine diphosphate-ribose,² so the possibility exists that Ca²⁺ from this source may be involved in the events leading to exocytosis. We examined this hypothesis by testing whether inositol trisphosphate and cyclic adenosine diphosphate-ribose modified transmitter release. We used liposomes to deliver these agents into the cytoplasmic compartment and binomial analysis to determine their effects on the quantal components of transmitter release. Administration of inositol trisphosphate (10⁻⁴ M) caused a rapid, 25% increase in the number of quanta released. This was due to an increase in the number of functional release sites, as the other quantal parameters were unaffected. The effect was reversed with 40 min of wash. Virtually identical results were obtained with cyclic adenosine diphosphate-ribose (10⁻⁴ M). Inositol trisphosphate caused a 10% increase in quantal size, whereas cyclic adenosine diphosphate-ribose had no effect. The results suggest that quantal transmitter release can be increased by Ca²⁺ released from smooth endoplasmic reticulum upon stimulation by inositol trisphosphate or cyclic adenosine diphosphate-ribose. This may involve priming of synaptic vesicles at the release sites or mobilization of vesicles to the active zone. Inositol trisphosphate may have an additional action to increase the content of transmitter within the vesicles. These findings raise the possibility of a role of endogenous inositol phosphate and smooth endoplasmic reticulum in the regulation of cytoplasmic Ca²⁺ and transmitter release.     

Purine salvage at the cholinergic nerve endings of the Torpedo electric organ: the central role of adenosine

The uptake and metabolism of adenosine, adenine, inosine and hypoxanthine were studied at the cholinergic nerve endings of the Torpedo electric organ. In isolated synaptosomes there is a linear uptake (measured up to 60 min) for adenosine and adenine at concentrations of 0.3 μM Uptake of adenosine exceeds that of adenine by a factor of 10. Adenosine is transported into synaptosomes via a saturable uptake system (K_m, 2 μM;V_max, ～- 30 pmols/min/mg protein). 2′-Deoxyadenosine is a competitive inhibitor of synaptosomal adenosine uptake. The nerve terminal possesses anabolic pathways for the formation of adenosine 5′-triphosphate from both adenosine and adenine. Adenosine becomes phosphorylated rapidly after entry into synaptosomes to form adenosine 5′-monophosphate; adenosine 5′-diphosphate and adenosine 5′-triphosphate were also major metabolites (70%). Adenine, inosine and hypoxanthine first accumulate in the synaptosomes. However, adenine leads to major formation of nucleotides (41% adenosine 5′-triphosphate after 60 min). Only traces of adenosine-3′:5′ cyclic monophosphate are formed from both adenosine and adenine. If adenosine 5′-triphosphate is added to a suspension of intact synaptosomes it becomes degraded to adenosine.We conclude that cholinergic nerve endings in the Torpedo electric organ possess an effective purine salvage system. Adenosine 5′-triphosphate released from either a pre- or a postsynaptic source would become degraded to adenosine in the extra-cellular medium and be re-used via an uptake system for renewed synthesis of adenosine 5′-triphosphate in nerve terminals.     

Extracellular levels of adenosine and its metabolites in the striatum of awake rats: inhibition of uptake and metabolism

The level of purines in the striatum of awake, freely moving rats was studied using microdialysis. The calculated extracellular concentration of adenosine and its metabolites inosine and hypoxanthine was very high immediately after implantation of the dialysis probe but decreased within 24 h to a level which remained stable for two days. Using in vitro calibration to determine the relative recovery of the dialysis probes we estimated resting levels in the striatal extracellular space to be 40, 110 and 580 nM, respectively. Inhibition of adenosine deaminase by deoxycoformycin produced a significant 1.4-fold increase in extracellular adenosine levels and a fall in inosine and hypoxanthine. A combination of three uptake blockers (dipyridamole, lidoflazine and nitrobenzylthioinosine), caused a 4.5-fold increase in extracellular adenosine levels without any change in inosine or hypoxanthine levels. After uptake inhibition deoxycoformycin did not have any significant effect. The present results show that the microdialysis technique can be used to determine levels of purines in the extracellular fluid of defined brain regions in awake animals. The high levels recorded during the first several hours after implantation may be artefactually high and reflect trauma. The results also show that adenosine levels can be altered in vivo by inhibitors of adenosine transport and adenosine deaminase. The present results indicate that the physiological adenosine level in striatal extracellular space is in the range 40-460 nM.     

The effect of high-intensity training on purine metabolism in man

The effect of intermittent high-intensity training on the activity of enzymes involved in purine metabolism and on the concentration of plasma purines following acute short-term intense exercise was investigated. Eleven subjects performed sprint training three times per week for 6 weeks. Muscle biopsies for determination of enzyme activities were obtained prior to and 24 h after the training period. After training, the activity of adenosine 5′-phosphate (AMP) deaminase was lower (P < 0.001) whereas the activities of hypoxanthine phosphoribosyl transferase (HPRT) and phosphofructokinase were significantly higher compared with pre-training levels. The higher activity of HPRT with training suggests an improved potential for rephosphorylation of intracellular hypoxanthine to inosine monophosphate (IMP) in the trained muscle. Before and after the training period the subjects performed four independent 2-min tests at intensities from a mean of 106 to 135 % of Vo_max. Venous blood was drawn prior to and after each test. The accumulation of plasma hypoxanthine following the four tests was lower following training compared with prior to training (P < 0.05). The accumulation of uric acid was significantly lower (46% of pre-training value) after the test performed at 135% of Fo_2mM (P < 0.05). Based on the observed alterations in muscle enzyme activities and plasma purine accumulation, it is suggested that high intensity intermittent training leads to a lower release of purines from muscle to plasma following intense exercise and, thus, a reduced loss of muscle nucleotides.     

The role of augmented breaths (sighs) in bronchial asthma attacks

Summary In an attempt to test the hypothesis whether adenosine is involved in the regulation of coronary flow, adenosine, inosine and hypoxanthine were measured in the effluent perfusate and in the tissue of isolated guinea pig hearts under various experimental conditions. In addition, the release of¹⁴C-adenosine,¹⁴C-inosine and¹⁴C-hypoxanthine was determined after prelabeling cardiac adenine nucleotides with¹⁴C-adenine.The decrease in coronary resistance induced by hypoxic perfusion (30% and 20% in the gas phase) and during autoregulation was associated with a considerable increase in the release of adenosine, inosine and hypoxanthine. Under both conditions the concentrations of adenosine in the effluent perfusate were clearly within the coronary vasodilating range of exogenously administered adenosine. The tissue content of adenosine also increased significantly when the perfusion pressure was reduced. The release of¹⁴C-adenosine closely paralleled the changes in coronary resistance during hypoxic perfusion, autoregulation and during reactive hyperemia. The specific activity of adenosine in the effluent perfusate, however, decreased substantially upon reduction of the oxygen supply to the heart, indicating that the release of¹⁴C-adenosine does not provide an absolute measure of total adenosine release by the heart.Our data indicate that the greater part of the adaptive changes of vascular resistance during hypoxia and autoregulation can be attributed to adenosine which is formed at an enhanced rate under these conditions. However, other factors might be involved as well.A preliminary report of these studies was given at the VI. Annual Meeting of the International Study Group for Research in Cardiac Metabolism, Freiburg i. Br., September 1973 and appeared in Recent advances in studies on cardiac structure and metabolism, Vol. 7, Editors: P. Harris, R. J. Bing, and A. Fleckenstein, pp. 171–175. Urban & Schwarzenberg 1976     

Compartmentation of cardiac adenine nucleotides and formation of adenosine

Summary After prelabeling the adenine nucleotides (ATP, ADP, AMP) of isolated perfused guinea pig hearts with either¹⁴C-adenine or¹⁴C-adenosine for 35 min, labeled adenosine, inosine, hypoxanthine and cyclic 35-AMP (cAMP) were continuously released into the cardiac perfusate. Determination of the specific activities (SA) of the adenine nucleotides, cAMP, and their breakdown products (adenosine, inosine, hypoxanthine) in tissue and perfusate revealed: Under steady state conditions the SA of adenosine and cAMP in the perfusate were of the same order of magnitude and proved to be many times higher than the SA of the respective precursor adenine nucleotides. This difference was observed regardless whether adenine or adenosine was used as prelabeling substance. The SA of inosine and hypoxanthine in the perfusate were constantly lower than the SA of adenosine. Cardiac ischemia of 6 min, which resulted in a markedly increased formation of adenosine, led to a pronounced decrease in the SA of adenosine released from the heart.Our findings provide evidence that at least two different adenine nucleotide compartments of the heart serve as precursors for the formation of adenosine and cAMP, one characterized by a high, the other by a lower SA. Under normoxic conditions adenosine and cAMP released into the cardiac perfusate are derived mainly from a nucleotide fraction of high SA, which appears to be rather small. During ischemia a second compartment of much lower SA in addition contributes to the formation of adenosine.A preliminary report of part of this work appeared in Biochemistry and Pharmacology of Myocardial Hypertrophy, Hypoxia and Infarction Vol. 7 of Recent advances in studies on cardiac structure and metabolism. (P. Harris, R. J. Bing, A. Fleckenstein, eds.), pp. 171–175. München: Urban & Schwarzenberg 1976A preliminary report of part of this work appeared in Biochemistry and Pharmacology of Myocardial Hypertrophy, Hypoxia and Infarction Vol. 7 of Recent advances in studies on cardiac structure and metabolism. (P. Harris, R. J. Bing, A. Fleckenstein, eds.), pp. 171–175. München: Urban & Schwarzenberg 1976     

Trophic effects of purines in neurons and glial cells

In addition to their well known roles within cells, purine nucleotides such as adenosine 5' triphosphate (ATP) and guanosine 5' triphosphate (GTP), nucleosides such as adenosine and guanosine and bases, such as adenine and guanine and their metabolic products xanthine and hypoxanthine are released into the extracellular space where they act as intercellular signaling molecules. In the nervous system they mediate both immediate effects, such as neurotransmission, and trophic effects which induce changes in cell metabolism, structure and function and therefore have a longer time course. Some trophic effects of purines are mediated via purinergic cell surface receptors, whereas others require uptake of purines by the target cells. Purine nucleosides and nucleotides, especially guanosine, ATP and GTP stimulate incorporation of [3H]thymidine into DNA of astrocytes and microglia and concomitant mitosis in vitro. High concentrations of adenosine also induce apoptosis, through both activation of cell-surface A3 receptors and through a mechanism requiring uptake into the cells. Extracellular purines also stimulate the synthesis and release of protein trophic factors by astrocytes, including bFGF (basic fibroblast growth factor), nerve growth factor (NGF), neurotrophin-3, ciliary neurotrophic factor and S-100beta protein. In vivo infusion into brain of adenosine analogs stimulates reactive gliosis. Purine nucleosides and nucleotides also stimulate the differentiation and process outgrowth from various neurons including primary cultures of hippocampal neurons and pheochromocytoma cells. A tonic release of ATP from neurons, its hydrolysis by ecto-nucleotidases and subsequent re-uptake by axons appears crucial for normal axonal growth. Guanosine and GTP, through apparently different mechanisms, are also potent stimulators of axonal growth in vitro. In vivo the extracellular concentration of purines depends on a balance between the release of purines from cells and their re-uptake and extracellular metabolism. Purine nucleosides and nucleotides are released from neurons by exocytosis and from both neurons and glia by non-exocytotic mechanisms. Nucleosides are principally released through the equilibratory nucleoside transmembrane transporters whereas nucleotides may be transported through the ATP binding cassette family of proteins, including the multidrug resistance protein. The extracellular purine nucleotides are rapidly metabolized by ectonucleotidases. Adenosine is deaminated by adenosine deaminase (ADA) and guanosine is converted to guanine and deaminated by guanase. Nucleosides are also removed from the extracellular space into neurons and glia by transporter systems. Large quantities of purines, particularly guanosine and, to a lesser extent adenosine, are released extracellularly following ischemia or trauma. Thus purines are likely to exert trophic effects in vivo following trauma. The extracellular purine nucleotide GTP enhances the tonic release of adenine nucleotides, whereas the nucleoside guanosine stimulates tonic release of adenosine and its metabolic products. The trophic effects of guanosine and GTP may depend on this process. Guanosine is likely to be an important trophic effector in vivo because high concentrations remain extracellularly for up to a week after focal brain injury. Purine derivatives are now in clinical trials in humans as memory-enhancing agents in Alzheimer's disease. Two of these, propentofylline and AIT-082, are trophic effectors in animals, increasing production of neurotrophic factors in brain and spinal cord. Likely more clinical uses for purine derivatives will be found; purines interact at the level of signal-transduction pathways with other transmitters, for example, glutamate. They can beneficially modify the actions of these other transmitters.     

Purine metabolism in the intact sporozoites and merozoites of Eimeria tenella

Intact Eimeria tenella sporozoites and merozoites did not incorporate radiolabeled formate or glycine into their purine nucleotides suggesting a lack of de novo purine synthesis. However, [U-14C]glucose was incorporated into the cellular purine and pyrimidine nucleotide pools of both forms probably via conversion to radiolabeled ribose-1-phosphate and/or 5'-phosphoribosyl-1-alpha-pyrophosphate and the resulting action of various purine and pyrimidine salvage enzymes. Both forms of the parasite salvaged radiolabeled purine bases and nucleosides in a similar fashion. These purines were incorporated into ribonucleotides and into RNA and DNA. Adenine and inosine were transformed to hypoxanthine. Adenosine was converted to both inosine and hypoxanthine. Hypoxanthine and xanthine were not oxidized to uric acid but were metabolized to nucleotides. Guanosine was cleaved to guanine; guanine was deaminated to xanthine. The results demonstrate the presence of several purine salvage pathways. Purine phosphoribosylating and nucleoside phosphorylating activities as well as purine nucleoside cleaving and adenosine, adenine and guanine deaminating activities were evident. The metabolic evidence suggests the enzymes required to convert the newly formed nucleoside monophosphates to ATP and GTP were present also.     

Tissue content of adenosine,inosine and hypoxanthine in the rat kidney after ischemia and postischemic recirculation

Summary The effect of renal ischemia of 15 s to 60 min duration on the tissue levels of adenosine, inosine and hypoxanthine was investigated in Sprague Dawley rats. A sharp increase in the tissue levels of adenosine from 5.13±0.56 to 31.3±2.96 nmol/g wet weight after 1 min of ischemia was found. The tissue levels of inosine and hypoxanthine in the controls were 3.62±0.51 and 3.19±0.76 nmol/g wet weight, respectively. Maximal levels of adenosine (38.1±6.3 nmol/g wet weight) were reached after 10 min of ischemia. The hypoxanthine levels rose steadily up to 922±183 nmol/g wet weight after 60 min of ischemia. Recirculation of 15 min after 60 min ischemia resulted in a fall of adenosine and inosine levels to values comparable to controls, whereas hypoxanthine was elevated above control values. In a second experimental series with tracing of renal blood flow (RBF) by a means of an electromagnetic flow meter a transient marked reduction of RBF after occlusion of the renal artery for 30 s was observed. The 3-fold increase of adenosine tissue levels within 30 s of renal artery occlusion and the inhibition of the postocclusive RBF reduction by theophylline (3.3 mol/100 g body weight) make it likely that this phenomenon may be caused by intrarenal adenosine.Parts of this investigation were presented at the 46th Meeting of the German Physiological Society in Regensburg, March 15–20, 1976.Supported by the Deutsche Forschungsgemeinschaft Os 42/2     

Uptake and supply of purine compounds by the liver.

We have analyzed for purine compounds entering and leaving the liver in lightly anesthetized rabbits and rats and for the export of utilizable purine from liver perfused with oxypurine. The in vivo results indicate that roughly 80% of hypoxanthine, xanthine, and urate is removed in a single passage of blood through liver. Conversely, the adenosine concentration of hepatic venous blood is increased 10-fold over portal or arterial levels. When the liver is isolated and perfused with hypoxanthine there is significant release of adenosine, whether measured quantitatively by microbiological assay or qualitatively by analysis of the radioactive purines released from liver that has been prelabeled with [14C]hypoxanthine. These results provide direct evidence for the clearance of hydroxylated purines and the release of utilizable adenine derivatives by liver.     

Oxytocin release from isolated posterior pituitary lobes of adult male rats as determined by radioimmunoassay

Summary In vitro oxytocin (OXT) release from isolated posterior pituitary lobes (PPL) of adult male Wistar rats was measured under basal and K⁺-stimulated conditions using a specific, sensitive radioimmunoassay. A basal release of 0.95±0.20 ng OXT/lobe/10 min was estimated in standard Locke's bathing solution. An excess of K⁺ (56 mmol/1) augmented the OXT secretion to 18.1±2.24 ng OXT/lobe/10 min in the presence of 2.2 mmol/1 Ca⁺⁺. A stimulatory effect of K⁺ excess was also determined in Ca⁺⁺-free medium and in Ca⁺⁺free medium enriched with 0.7 mmol/1 EGTA. An inhibitory effect on K⁺-stimulated OXT release was achieved by raising the Mg⁺⁺ concentration from 1.0–8.0 mmol/1 of bathing fluid. During prolonged K⁺ stimulation the rate of OXT release declined exponentially. Estimation of the OXT content of PPLs after prolonged stimulation with K⁺ excess revealed that the lobes still contained 80% of their original OXT content.Abbreviation EGTA ethylene glycol-bis(-aminoethyl ether) - N,N tetra-acetic acid     

Inhibition of histamine release and ionophore-induced calcium flux in rat mast cells by lidocaine and chlorpromazine

We studied the effects of lidocaine (L) and chlorpromazine (C), two compounds known to affect the binding of calcium to cell membranes, on histamine release and⁴⁵calcium uptake by purified mast cells upon challenge with the ionophore A23,187 or with compound 48/80. At low concentrations L and C inhibited the Ca⁺⁺ flux as well as histamine release while higher concentrations caused enhancement in this function. Evidence was obtained that L 10^–4 M may displace Ca⁺⁺ from the cell membranes.Presented in part at the Annual Meeting of The American Academy of Allergy, San Juan, P.R., March 5, 1976.     

Ionic changes in cerebrospinal fluid and feeding, drinking and temperature of sheep

The effects of third ventricular injections of Ca⁺⁺, Mg⁺⁺, Na⁺ and K⁺ on feeding, drinking and intraperitoneal (IP) temperature were studied in sheep. The chloride salts were dissolved in synthetic CSF (control) and injected (0.5ml) at a rate of 0.2 ml/min into satiated sheep. Ca⁺⁺ and Mg⁺⁺ injections elicited dose dependent feeding responses but Ca⁺⁺ resulted in greater feeding. Doses as little as 1.5 μmoles/animal of either Ca⁺⁺ or Mg⁺⁺ elicited feeding with a latency of about 2 min and lasted for 15 min postinjection. With larger doses, the duration of the feeding response increased but the latency stayed the same. A 20 or 40 μmole dose of K⁺ elicited a decrease in feed intake. Na⁺ given at similar doses produced an increase in water consumption without affecting feeding. Injections of either Na⁺ or K⁺ 10 min prior to either Ca⁺⁺ or Mg⁺⁺ resulted in only normal feeding. K⁺ inhibited the feeding response to both Ca⁺⁺ and Mg⁺⁺ at dose levels lower than the doses of Na⁺ required (40 vs 550 μmoles). The inhibitory effect of Na⁺ on Ca⁺⁺ feeding was overcome by either increasing the dose of Ca⁺⁺ or decreasing the dose of Na⁺. Water intake was generally increased after injections of Ca⁺⁺, Mg⁺⁺ and Na⁺. Intraperitoneal temperatures increased after injections of Na⁺ and Mg⁺⁺ but remained unchanged after injections of Ca⁺⁺, K⁺ or synthetic CSF. We conclude that the CNS mechanisms for the control of feeding in sheep are sensitive to changes in the ionic environment of brain tissue, probably the hypothalamus. Ca⁺⁺ and Mg⁺⁺, known neurodepressants, probably exert their action by decreasing metabolism and neuroconductivity of inhibitory nerve fibers acting on the lateral hypothalamus. Preinjections with the neurostimulants Na⁺ or K⁺ would tend to neutralize such changes in neuroexcitability, so that the combination of ions would result in a net no effect, as far as feeding in concerned. Feeding and drinking behaviors were more sensitive than IP temperature to the ionic changes in the cerebrospinal fluid of sheep.     

Heterogeneity of calcium channels in mast cells and basophils and the possible relevance to pathophysiology of lung diseases: A review

Calcium plays a critical role in the formation and secretion of a wide variety of chemical mediators. Calcium slow-channel blockers,e.g. nifedipine and verapamil, have been shown to inhibit the synthesis of SRS (SRS-A, leukotrienes) in human and guinea pig lung tissue, thromboxane A₂ formation in rat lung and platelet activating factor in human neutrophils. Verapamil and nifedipine also prevent the release of lysosomal enzymes from rabbit and human polymorphonuclear neutrophils. Calcium-channel blockers produce variable inhibitory effects on allergic and nonallergic histamine secretion. Ca⁺⁺-entry blockers also inhibit the Ca⁺⁺ uptake (influx) into mast cells. Many of these inhibitory effects of Ca⁺⁺ antagonists are antagonized by an increased extracellular Ca⁺⁺ ion concentration. The magnitude of the inhibitory influences of Ca⁺⁺-channel blockers on allergic and nonallergic release of chemical mediators appears to depend on the cell source, species, nature and the concentration of the secretory stimuli as well as on the composition and pH of buffers and the concentration of Ca⁺⁺-entry blockers used. The data summarized in this review suggest the existence of a functional heterogeneity of Ca⁺⁺ channels in leukocytes, mast cells and basophils. Interference with the Ca⁺⁺-dependent steps involved in the formation and/or release of chemical mediators appears to be the primary mode of action for Ca⁺⁺-channel blockers in these cells.The differential effects of Ca⁺⁺ antagonists on Ca⁺⁺-dependent activation of phospholipase A₂, 5-lipoxygenase, and calmodulin (or other intracellular Ca⁺⁺-binding proteins) in different cell types (mast cells, basophils, leukocytes, lung tissue, etc.) may explain the variation of their effectiveness in inhibiting the synthesis/release of chemical mediators and antagonizing bronchoconstriction in response to diverse stimuli.During the process of hypersensitization and in immediate hypersensitivity diseases, Ca⁺⁺ homeostasis (uptake, mobilization, distribution, relocation, etc.) may be altered in leukocytes (mast cells, basophils) and lung tissues. The altered Ca⁺⁺ homeostasis could be responsible for the induction of airway hyperreactivity in asthmatics and for hyperreleasability of chemical mediators from leukocytes, mast cells and other cell types.The development of drugs (Ca⁺⁺-channel blockers, antiallergic agents) that are capable of selectively altering Ca⁺⁺-dependent functions in leukocytes (mast cells, basophils, macrophages) and lung tissue in disease staes would offer an attractive alternative and an effective therapeutic approach for obstructive respiratory diseases,e.g. allergic asthma, exercise-induced asthma and a variety of other mediator-dependent allergic disorders.     

Synaptic and non-synaptic mechanisms underlying low calcium bursts in the in vitro hippocampal slice

Summary 1. The epileptiform activity generated by lowering extracellular [Ca⁺⁺] was studied in the CA1 subfield of rat hippocampal slices maintained in vitro at 32° C. Extracellular and intracellular recordings were performed with NaCl and KCl filled microelectrodes. 2. Synaptic potentials evoked by stimulation of the stratum radiatum and alveus were blocked upon perfusion with artificial cerebrospinal fluid (ACSF) containing 0.2 mM Ca⁺⁺, 4 mM Mg⁺⁺. Blockade of synaptic potentials was accompanied by the appearance of synchronous field bursts which either occurred spontaneously or could be induced by stimulation of the alveus. 3. Both spontaneous and stimulus-induced low Ca⁺⁺ bursts recorded extracellularly in stratum pyramidale consisted of a negative potential shift with superimposed population spikes. This extracellular event was closely associated with intracellularly recorded action potentials rising from a prolonged depolarization shift. Steady hyperpolarization of the cell membrane potential decreased the amplitude of the depolarizing shift suggesting that synaptic conductance were not involved in the genesis of the low Ca⁺⁺ burst. 4. Spontaneous depolarizing inhibitory potentials recorded in normal ACSF with KCl filled microelectrodes were reduced in size in low Ca⁺⁺ ACSF. However, small amplitude potentials could still be observed at a time when low Ca⁺⁺ bursts were generated by hippocampal CA1 pyramidal neurons. 5. Bicuculline methiodide, an antagonist of -aminobutyric acid (GABA), was capable of modifying the frequency of occurrence and the shape of synchronous field bursts. The effects evoked by bicuculline methiodide were, however, not observed when 81–100% of NaCl was replaced with Na-Methylsulphate. Hence, it was concluded that in low Ca⁺⁺ ACSF even though large release of transmitter such as those following electrical activation of stratum radiatum or alveus cannot be observed, small spontaneous release of the inhibitory transmitter GABA seems to persist. 6. Substitution of NaCl with Na-Methylsulphate also caused changes in the synchronous field bursts which were different from those observed following application of bicuculline methiodide. These findings suggest that in low Ca⁺⁺ ACSF, in addition to residual GABAergic Cl^- mechanisms, non-synaptic Cl^- conductances might play a role in controlling the excitability of hippocampal neurons.Supported by grants from the MRC of Canada (MA-8109) and Sick Children Foundation to MA     

Adenosine triphosphatase in non-secreting and secreting mast cells

A Ca⁺⁺–Mg⁺⁺ ATPase has been demonstrated in the plasma membrane of rat peritoneal mast cells. The enzyme is localized by electron microscopy on the outer surface of the membrane. This agrees with the biochemical findings. A Ca⁺⁺–Mg⁺⁺ activated ATPase has also been shown to be present in the granule membrane. The optimal pH of the plasma membrane enzyme is close to the optimal pH for histamine release. All the 14 inhibitors of plasma membrane ATPase tested-which caused varying degrees of inhibition of the enzyme-also inhibited histamine release induced by antigen, compound 48/80 and the divalent ionophore A23187. The conclusion from the study with the inhibitors is that a mild inhibition of the enzyme is compatible with histamine release, but a pronounced inhibition of the enzyme is always associated with inhibition of histamine release. ATP in low concentrations potentiates the release.     

Release of adenosine-like material from isolated perfused dog adipose tissue following sympathetic nerve stimulation and its inhibition by adrenergic alpha-receptor blockade.

Following the intraarterial infusion of 3H-adenine to isolated perfused canine subcutaneous adipose tissue, its adenine nucleotides are labelled. A continuous release of radioactivity, comprised of non-nucleotide material, was observed. The rate of this release was markedly enhanced by sympathetic nerve stimulation. The major components of the enhanced release appeared to be inosine and adenosine. Adrenergic alpha-receptor blockade (phentolamine or Hydergin) abolished the enhanced nucleoside release, while glycerol release was enhanced. The release of radioactivity was decreased during mechanical blood flow reduction and enhanced afterwards. However, the magnitude of this enhancement of release after clamp was much less than following nerve stimulation. The results suggest that adenosine or a closely related compound is released from canine subcutaneous adipose tissue by sympathetic nerve stimulation and that the release is related to adrenergic alpha-receptor stimulation. Since adenosine is a potent inhibitor of catecholamine induced lipolysis in this tissue the possibility of a regulatory role must be considered.