Sensitivity of the mosquito Aedes aegypti (Culicidae) labral apical chemoreceptors to blood plasma components

The phagostimulants from the cellular fraction of blood induce gorging of Aedes aegypti (L.), and this process is enhanced by some plasma components. This project examines the responses of the labral apical chemoreceptors to plasma components enhancing phagostimulation. From the electrophysiological responses of the labral apical chemoreceptors four cells were identified by the waveform of their action potentials. Three of the cells (Cell 2, Cell 3 and Cell 4) responded in a dose dependent manner to NaCl. The responses of Cell 2 and Cell 3 to NaCl concentrations from 1 to 500 mmol/l can be described by a logarithmic equation. The response of Cell 2 to 150 mmol/l NaCl is modulated when a buffer is added. The magnitude of the modulation of the response is determined by the nature of the buffer: NaHCO(3) inhibits while Na(2)HPO(4) enhances the response. High osmotic pressure inhibits the response of Cell 4, regardless of how it is achieved. Cell 4 responds with a high frequency to the presence of L-alanine, the C-terminal amino acid of albumin, but shows a reduced response to the same concentration of albumin. From these results it can be concluded that labral apical chemoreceptors of A. aegypti are capable of detecting the plasma components involved in blood recognition.     

Factors modulating the blood feeding behavior and the electrophysiological responses of labral apical chemoreceptors to adenine nucleotides in the mosquito Aedes aegypti (Culicidae)

The feeding of Aedes aegypti (L.) on blood is induced by the presence of phagostimulants: adenine nucleotides. Three chemoreceptive cells in the labral apical sensilla can distinguish the presence of adenine nucleotides depending on the other stimulus components. This work aims at correlating the sensory information arising from the labral apical sensilla with the feeding behavior in response to the same stimuli. The saline stimulating solution, containing adenine nucleotides, is modulated by changing one of the following components: salt concentration, buffer or pH. Cell 3 that responds to NaCl in a dose dependent manner seems to have another unique modality. The response of this cell is unaffected by ATP when the stimulating solution is NaCl buffered by NaHCO(3). It responds at a higher spike frequency to the presence of ATP in a NaCl solution without NaHCO(3). Thus in the presence of ATP Cell 3 detects whether the NaCl solution is buffered by NaHCO(3). Both the blood feeding response and the sensory information from Cell 2 (which responds at high spike frequencies to the presence of ATP) are modulated by pH in a similar way. Both responses present a bi-modal response, with a major peak at pH 4.0 and a moderate peak at the most alkaline pH value tested.     

Feeding behaviour in response to blood fractions and chemical phagostimulants in the black-fly, Simulium venustum

ABSTRACT. Wild-caught black-flies ( Simulium venustum Say complex) were presented with diets at 37°C in an artificial feeding apparatus. Washed human red cells resuspended 1:1 in Ringer solution were potent phagostimulants, causing 89% of flies to gorge. Whole plasma was more potent (32% gorging) than platelet-poor plasma (2%). The ED₅₀ for red cells was 3.5%. Although ADP, contained in high-concentration in platelets, was confirmed as a more potent phagostimulant than ATP (ED₅₀ of 5πM V. 12μM), red cells were clearly more phagostimulatory than platelets, and with a potency more than adequate to trigger gorging in vivo. A high response to the ATP analogues, β, γ-methylene ATP and adenine phosphosulphate, supports the view that the phosphate chain is relatively unimportant in determining nucleotide potency to simuliids. The compounds phytic acid and 2,3-disphosphoglycerate, potent stimulants to Rhodnius prolixus , produced only moderate and no response, respectively at 1 mM; 5-hydroxytryptamine, another major constituent of platelets, also produced only a moderate response. Only flies caught while showing a characteristic probing behaviour would subsequently probe and feed in vitro; this 'blood-feeding mode' rapidly disappeared in the absence of stimuli eliciting actual probing, but for flies in this state a small temperature rise was sufficient stimulus for probing.     

ATP analogues and other phosphate compounds as gorging stimulants for Rhodnius prolixus

Five analogues of ATP and six other non-nucleotide compounds with phosphate groups were tested as gorging stimulants for second-instar larvae of Rhodnius prolixus to determine the importance of the phosphate chain. Only molecules with terminal phosphate groups were potent. Insertion of an imido group (5′-Adenylylimidodiphosphate, AMP-PNP) or a methylene group (β, γ-Methylene adenosine 5′-triphosphate, AMP-PCP) between the β and γ phosphates of ATP reduced the potency compared to ATP by ratios of 1.8 and 25.5, respectively. Substituting ribose (Adenosine 5′-diphosphoribose, AMP-PR) for the γ phosphate group or an amidate or a sulphate group (Adenosine 5′-phosphoramidate, AMP-N; Adenosine 5′-phosphosulphate, AMP-S) for the β and γ phosphate groups of ATP resulted in a complete loss of stimulatory activity.Some non-nucleotide phosphate compounds were potent phagostimulants. Pyrophosphate with an ED₅₀ of 64 μM had a potency ratio compared with ATP of 1:17. Methylene diphosphonic acid (ED₅₀ 680 μM) and even single phosphate ions (ED₅₀ 2.5 mM) had substantial potency. Two isomers of phosphoglyceric acid differ greatly in their ability to stimulate gorging; 2-PGA was active (ED₅₀ 160 μM) whereas 3-PGA had almost no activity.A summary of known phagostimulants to R. prolixus supports the hypothesis that ATP-like gorging stimulants act by forming a temporary binding to 3 sites on a receptor protein in the membrane of the chemosensory cell. The amino group on C₆ of adenine, the OH group on C₂ of ribose and the terminal phosphate group(s) determine potency, presumably by determining binding affinity. However, only the phosphate group appears essential to the chemosensory process.     

Stimulation of glycogenolysis by adenine nucleotides in the perfused rat liver.

Infusion of adenine nucleotides and adenosine into perfused rat livers resulted in stimulation of hepatic glycogenolysis, transient increases in the effluent perfusate [3-hydroxybutyrate]/[acetoacetate] ratio, and increased portal vein pressure. In livers perfused with buffer containing 50 microM-Ca2+, transient efflux of Ca2+ was seen on stimulation of the liver with adenine nucleotides or adenosine. ADP was the most potent of the nucleotides, stimulating glucose output at concentrations as low as 0.15 microM, with half-maximal stimulation at approx. 1 microM, and ATP was slightly less potent, half-maximal stimulation requiring 4 microM-ATP. AMP and adenosine were much less effective, doses giving half-maximal stimulation being 40 and 20 microM respectively. Non-hydrolysed ATP analogues were much less effective than ATP in promoting changes in hepatic metabolism. ITP, GTP and GDP caused similar changes in hepatic metabolism to ATP, but were 10-20 times less potent than ATP. In livers perfused at low (7 microM) Ca2+, infusion of phenylephrine before ATP desensitized hepatic responses to ATP. Repeated infusions of ATP in such low-Ca2+-perfused livers caused homologous desensitization of ATP responses, and also desensitized subsequent Ca2+-dependent responses to phenylephrine. A short infusion of Ca2+ (1.25 mM) after phenylephrine infusion restored subsequent responses to ATP, indicating that, during perfusion with buffer containing 7 microM-Ca2+, ATP and phenylephrine deplete the same pool of intracellular Ca2+, which can be rapidly replenished in the presence of extracellular Ca2+. Measurement of cyclic AMP in freeze-clamped liver tissue demonstrated that adenosine (150 microM) significantly increased hepatic cyclic AMP, whereas ATP (15 microM) was without effect. It is concluded that ATP and ADP stimulate hepatic glycogenolysis via P2-purinergic receptors, through a Ca2+-dependent mechanism similar to that in alpha-adrenergic stimulation of hepatic tissue. However, adenosine stimulates glycogenolysis via P1-purinoreceptors and/or uptake into the cell, at least partially through a mechanism involving increase in cyclic AMP. Further, the hepatic response to adenine nucleotides may be significant in regulating hepatic glucose output in physiological and pathophysiological states.     

The contribution of adenine nucleotide loss to ischemia-induced impairment of rat kidney cortex mitochondria.

Adenine nucleotides and respiration were assayed with rat kidney mitochondria depleted of adenine nucleotides by pyrophosphate treatment and by normothermic ischemia, respectively, with the aim of identifying net uptake of ATP as well as elucidating the contribution of adenine nucleotide loss to the ischemic impairment of oxidative phosphorylation. Treatment of rat kidney mitochondria with pyrophosphate caused a loss of adenine nucleotides as well as a decrease of state 3 respiration. After incubation of pyrophosphate-treated mitochondria with ATP, Mg2+ and phosphate, the content of adenine nucleotides increased. We propose that kidney mitochondria possess a mechanism for net uptake of ATP. Restoration of a normal content of matrix adenine nucleotides was related to full recovery of the rate of state 3 respiration. A hyperbolic relationship between the matrix content of adenine nucleotides and the rate of state 3 respiration was observed. Mitochondria isolated from kidneys exposed to normothermic ischemia were characterized by a decrease in the content of adenine nucleotides as well as in state 3 respiration. Incubation of ischemic mitochondria with ATP, Mg2+ and phosphate restored the content of adenine nucleotides to values measured in freshly-isolated mitochondria. State 3 respiration of ischemic mitochondria reloaded with ATP recovered only partially. The rate of state 3 respiration increased by ATP-reloading approached that of uncoupler-stimulated respiration measured with ischemic mitochondria. These findings suggest that the decrease of matrix adenine nucleotides contributes to the impairment of ischemic mitochondria as well as underlining the occurrence of additional molecular changes of respiratory chain limiting the oxidative phosphorylation.     

Effect of ATP analogues on the gorging response of Aedes aegypti

ABSTRACT. The ATP analogues adenylylimidodiphosphate and adenylylmethylenediphosphate are 3–5-fold more effective than ATP as gorging stimulants for Aedes aegypti. This increased potency is not due to the fact that the two analogues are not hydrolysed by the mosquito salivary apyrase, but most likely to their greater affinity to the mosquito gustatory receptor protein. The analogues 2'd ATP and 3'd ATP are about half as potent as ATP, while 2',3'-dideoxyadenosine triphosphate is 10-fold more potent than ATP in evoking the gorging response. It is proposed that removal of both hydroxyl groups eliminates binding of the stimulant at the ribose moiety, thus allowing the molecule greater freedom to rotate and bind more effectively to its two other binding sites at the amino group on the purine and at the terminal phosphate. Our data demonstrate that ATP activates the gorging response of Ae.aegypti merely by binding to its receptor protein and is not required as an exogenous source of energy. Gorging response to ATP is competitively inhibited by novobiocin.     

Regulation of human neutrophil functions by adenine nucleotides

Previous work has shown that platelet-derived adenine nucleotides modulate neutrophil superoxide anion (O2-) generation. Additional studies were undertaken to characterize the effects of authentic adenosine (ADO) and its nucleotide derivatives on the inflammatory functions of human neutrophils. Stimulus-specific inhibition of neutrophil O2- generation by ADO in response to FMLP was verified. In addition, the ability of ATP, ADP, and AMP to limit neutrophil O2- generation induced by FMLP (0.2 to 0.5 microM) was demonstrated. The concentration producing 50% inhibition for nucleotide inhibition of neutrophil O2- generation was in the rank order of ADO (0.1 microM) less than AMP (0.5 microM) less than ADP less than or equal to ATP (5 microM). Guanine and inosine nucleotides (0.01 to 100 microM) did not inhibit FMLP-stimulated neutrophil O2- generation. Neutrophil degranulation in response to FMLP was only modestly inhibited by adenine nucleotides and ADO. Adenosine and ADP failed to affect chemotaxis of neutrophils stimulated with FMLP. The inability of non-metabolizable analogs to mimic the inhibitory effects of authentic ATP or ADP on the neutrophil O2- response suggested that metabolism of added nucleotides is necessary for their effectiveness. Both TLC and HPLC confirmed that ATP and ADP were converted to AMP and ADO after their incubation with unstimulated or FMLP-activated neutrophils. The addition of adenosine deaminase to neutrophil reaction mixtures in which conversion of added nucleotides was apparent removed detectable ADO but failed to completely abrogate the inhibition of neutrophil O2- generation by accumulated AMP. The kinetics of inhibition of FMLP-induced neutrophil O2- generation by ATP and ADP also indicated that conversion of these nucleotides to ADO and/or AMP may be essential for their ability to reduce neutrophil responses.     

Opposite effects of uracil and adenine nucleotides on the survival of murine cardiomyocytes

We previously showed that the human heart expresses all known P2X and P2Y receptors activated by extra-cellular adenine or uracil nucleotides. Despite evidence that, both in humans and rodents, plasma levels of ATP and UTP markedly increase during myocardial infarction, the differential effects mediated by the various adenine- and uracil-preferring myocardial P2 receptors are still largely unknown. Here, we studied the effects of adenine and uracil nucleotides on murine HL-1 cardiomyocytes. RT-PCR analysis showed that HL-1 cardiomyocytes express all known P2X receptors (except for P2X(2)), as well as the P2Y(2,4,6,14) subtypes. Exposure of cardiomyocytes to adenine nucleotides (ATP, ADP or BzATP) induced apoptosis and necrosis, as determined by flow-cytometry. Cell death was exacerbated by tumour necrosis factor (TNF)-alpha, a cytokine implicated in chronic heart failure progression. Conversely, uracil nucleotides (UTP, UDP and UDPglucose) had no effect 'per se', but fully counteracted the deleterious effects induced by adenine nucleotides and TNF-alpha, even if added to cardiomyocytes after beginning exposure to these cell death-inducing agents. Thus, exposure of cardiomyocytes to elevated concentrations of ATP or ADP in the presence of TNF-alpha contributes to cell death, an effect which is counteracted by uracil-preferring P2 receptors. Cardiomyocytes do not need to be 'primed' by uracil nucleotides to become insensitive to adenine nucleotides-induced death, suggesting the existence of a possible 'therapeutic' window for uracil nucleotides-mediated protection. Thus, release of UTP during cardiac ischaemia and in chronic heart failure may protect against myocardial damage, setting the basis for developing novel cardioprotective agents that specifically target uracil-preferring P2Y receptors.     

Domain-specific purinergic signaling in polarized rat cholangiocytes

In cholangiocytes, adenine nucleotides function as autocrine/paracrine signals that modulate ductular ion transport by activation of purinergic receptors. The purpose of these studies was to identify cellular signals that modulate ATP release and nucleotide processing in polarized normal rat cholangiocytes. In Ussing chamber studies, selective exposure of the apical and basolateral membranes to ATP or adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) stimulated increases in short-circuit current. Apical purinergic receptor agonist preference was consistent with the P2Y(2) subtype. In contrast, basolateral ADP was more potent in stimulating transepithelial currents, consistent with the expression of different basolateral P2 receptor(s). Luminometric analysis revealed that both membranes exhibited constitutive ATP efflux. Hypotonic exposure enhanced ATP release in both compartments, whereas decreases in ATP efflux during hypertonicity were more prominent at the apical membrane. Increases in intracellular cAMP, cGMP, and Ca(2+) also increased ATP permeability, but selective effects on apical and basolateral ATP release differed. Finally, the kinetics of ATP degradation in apical and basolateral compartments were distinct. These findings suggest that there are domain-specific signaling pathways that contribute to purinergic responses in polarized cholangiocytes.     

Experiments on biting and gorging behaviour in the black fly, Simulium venustum

ABSTRACT. Factors which initiate 'biting' (i.e. probing, piercing and tasting collectively) and gorging responses, were studied in the black fly, S. venustum using an artificial feeding method. A positive relationship was found between biting activity and the magnitude of the temperature differential between the feeding surface and the air above it. Although many investigators consider temperature to be a probing stimulus, it is argued that an equally consistent interpretation could regard temperature as only a very short range host-location cue. The stimulus to probe could be contact with the feeding substrate. Of the compounds tested as gorging stimulants ATP and ADP proved most potent, followed by AMP and adenosine, followed by cAMP. The compounds GTP, CTP and UTP were all on the borderline of statistical significance as gorging stimulants. It is suggested that the host-location phase, including the biting responses, represents appetitive behaviour leading to the consummatory response of repetitive pumping (gorging) stimulated by ATP, etc.     

Degradation and resynthesis of adenine nucleotides in adult rat heart myocytes

The degradation and short-term resynthesis of adenine nucleotides have been examined in a preparation of isolated rat heart myocytes. These myocyte preparations are essentially free of vascular and endothelial cells, contain levels of adenine nucleotides quite comparable to those of intact heart tissue, and retain these components remarkably well for up to 2 h of aerobic incubation in the presence of 1 mM Ca2+. When the cells are rapidly and synchronously de-energized by addition of uncoupler, an inhibitor of respiration and iodoacetate, cellular ATP is degraded almost quantitatively to AMP. The AMP is then converted to either intracellular adenosine, which accumulates to high concentrations before release to the cell exterior, or to IMP. The relative contribution of these two pathways depends on the metabolic state of the cells just prior to de-energization, with IMP production favored when respiring cells are de-energized and adenosine formation predominant when glycolyzing myocytes are subjected to this treatment. Cells de-energized by anaerobiosis in the absence of glucose lose ATP and adenine nucleotides with the production of IMP and adenosine. Upon reoxygenation, these cells restore a high adenylate energy charge and about 60% of control levels of GTP. There is a net resynthesis of 5-7 nmol of adenine nucleotides.mg-1 protein with a corresponding decline in IMP. Added [14C]adenosine labels the adenine nucleotide pool, but little net resynthesis of adenine nucleotides via adenosine kinase can be detected. It therefore appears that a rapid regeneration of adenine nucleotides can occur via the enzymes of the purine nucleotide cycle in heart myocytes and is limited by the size of the IMP pool retained.     

Role of Blood Platelets in Haematophagy

RED blood corpuscles (RBC) suspended in saline induce gorging in many haematophagous insects because of their high intrinsic concentration of adenine nucleotides (ANS)^1–6. ANS are bound firmly inside the intact RBC, which raises the question of how they gain egress to contact the chemoreceptor surfaces and induce feeding. It has been suggested that saliva or secretions of the chemoreceptor surfaces act as ANS releasing agents¹. ANS release by haemolysis is discounted by the fact that all RBC found in the gut immediately after feeding are intact. Further, stereoscan electron microscopy of tsetse fly gustatory sensilla does not suggest that they operate by piercing the erythrocytes^7,8. Thus we decided to test the possibility that the chemoreceptors involved in blood identification receive an ANS stimulus from a source associated with, but not within the RBC.     

Potencies of combined doses of nucleotides as gorging stimulants for Rhodnius prolixus

The four nucleotides ATP, UTP, deoxyATP, and A(TETRA)P (adenosine tetraphosphate) were tested singly and in combinations for their potency in eliciting the gorging response of Rhodnius prolixus. All mixtures of ATP and dATP, and ATP and UTP, were more potent by a factor of 1.25 to 1.9 than predicted from their potencies when tested singly. No significant synergism or inhibition was seen with combinations of UTP and dATP, or ATP and A(TETRA)P. Lack of competitive inhibition suggests that physical fit between stimulating molecule and chemoreceptive protein rather than enzymatic modification of the stimulant is the mechanism of chemoreception of these compounds. The slight synergism seen is explained by competitive inhibition of a salivary ATPase.     

Decreased ecto-NTPDase1/CD39 activity leads to desensitization of P2 purinoceptors regulating tonus of corpora cavernosa in impotent men with endothelial dysfunction

Vascular responses to adenine nucleotides in human corpora cavernosa from men with vasculogenic erectile dysfunction were investigated. We also evaluated the catabolism of extracellular adenine nucleotides to probe its relevance to vascular hemodynamics in impotent men. Human corpora cavernosa have high NTPDase1/CD39 activity, converting ATP directly into AMP, without significant ADP formation. Extracellular ATP hydrolysis is slower in impotent patients. Adenine nucleotides have dual roles on phenylephrine-contracted strips of corpora cavernosa operated by P2X-contractant and P2Y-relaxant receptors. Prolonged exposure to endogenous ATP related to decreased NTPDase1/CD39 activity leads to P2-purinoceptor desensitization in impotent men. Shutting down ATP signaling in vasculogenic impotent men may represent a defense mechanism for preventing purinergic overstimulation.     

Decreased Ecto-Ntpdase1/Cd39 Activity Leads to Desensitization of P2 Purinoceptors Regulating Tonus of Corpora Cavernosa in Impotent Men with Endothelial Dysfunction

Vascular responses to adenine nucleotides in human corpora cavernosa from men with vasculogenic erectile dysfunction were investigated. We also evaluated the catabolism of extracellular adenine nucleotides to probe its relevance to vascular hemodynamics in impotent men. Human corpora cavernosa have high NTPDase1/CD39 activity, converting ATP directly into AMP, without significant ADP formation. Extracellular ATP hydrolysis is slower in impotent patients. Adenine nucleotides have dual roles on phenylephrine-contracted strips of corpora cavernosa operated by P2X-contractant and P2Y-relaxant receptors. Prolonged exposure to endogenous ATP related to decreased NTPDase1/CD39 activity leads to P2-purinoceptor desensitization in impotent men. Shutting down ATP signaling in vasculogenic impotent men may represent a defense mechanism for preventing purinergic overstimulation.     

Carboxyatractyloside-insensitive influx and efflux of adenine nucleotides in rat liver mitochondria

Unidirectional transport (influx and efflux) of adenine nucleotides in rat liver mitochondria was examined using carboxyatractyloside to inhibit rapid exchange of matrix and external adenine nucleotides via the adenine nucleotide translocase. Influx of adenine nucleotides was concentration-dependent. ATP was the preferred substrate with a Km of 2.67 mM and V of the preferred substrate with a Km of 2.67 mM and V of 8.33 nmol/min/mg of protein. For ADP, the Km was 14.7 mM and V was 10.8 nmol/min/mg of protein. Efflux of adenine nucleotides was also concentration-dependent, varying directly as a function of the matrix adenine nucleotide pool size. Any increase in the influx of adenine nucleotides was coupled to an increase in efflux. However, as the external ATP concentration was increased, influx was stimulated to a much greater extent than was efflux. This imbalance suggested that under certain conditions adenine nucleotide movement might be coupled to the movement of an alternate anion such as phosphate. Adenine nucleotide efflux increased as the external phosphate concentration was varied from 0.5 to 4 mM. Also, increasing the external phosphate concentration caused adenine nucleotide influx to decrease, suggesting competition. In the absence of external adenines and phosphate, no efflux occurred. Both adenine nucleotide influx and efflux were depressed if Mg2+ was omitted. Adenine nucleotide efflux in the presence of external phosphate was inhibited much less by lack of Mg2+ than was efflux in the presence of external ATP. This evidence supports a model in which either adenine nucleotides (probably with Mg2+) or phosphate can move across the mitochondrial membrane on a single carrier. Net adenine nucleotide movements can occur when adenine nucleotide movement is coupled to the movement of phosphate in the opposite direction.     

Some thoughts on the evolutionary basis for the prominent role of ATP and ADP in cellular energy metabolism

The predominance of the adenosine triphosphate/adenosine diphosphate (ATP/ADP) couple in cellular phosphorylation reactions, including those that form the basis for cellular energy metabolism, cannot be explained on thermodynamic grounds since a variety of "high energy phosphate" compounds (including ADP itself) found in the cell would, based on thermodynamic considerations, be at least as effective as ATP in serving as a phosphoryl donor. How then did present-day organisms come to rely on the ATP/ADP couple as the principal mediator of phosphorylation reactions? The early appearance of adenine compounds in the prebiotic environment is suggested by experiments indicating that, relative to other purine or pyridimine compounds, adenine derivatives are preferentially synthesized under simulated prebiotic conditions (Ponnamperuma et al., 1963). In addition to the roles of adenine nucleotides in phosphorylation reactions, other adenine derivatives (e.g. Coenzyme A, flavin adenine dinucleotide, puridine nucleotides) are employed in a variety of metabolic roles. The principal function of the adenine moiety in these latter cases is in the binding of these derivatives to the relevant enzyme. The capability for binding of the adenine moiety appears to have arisen early in evolution and been exploited in a multitude of contexts, a suggestion consistent with observed similarities between the binding sites of several enzymes employing adenine derivatives as substrate. The early availability of suitable adenine compounds in the biosphere and development of complementary binding sites on cellular proteins, coupled with the expected advantages in having a limited number of metabolites as central mediators of endergonic and exergonic metabolism could readily have led to the observed pre-eminence of adenine nucleotides in cellular energy metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy-dependent exchange of adenine nucleotides on chloroplast coupling factor (CF1)

1. [¹⁴C]ADP is incorporated into washed broken chloroplasts in the light. The bound labelled nucleotides which cannot be removed by washing are almost exclusively related to coupling factor CF₁. [¹⁴C]ADP binding exhibits a monophasic concentration curve with a K_m of 2 μM.2. By illumination of the chloroplasts, previously incorporated labelled nucleotides are released. A fast release is obtained in the presence of unlabelled ADP and ATP, indicating an energy-dependent exchange. A slow and incomplete release is induced by light in the absence of unlabelled adenine nucleotides. Obviously, under those conditions, an adenine nucleotide depleted CF₁ conformation is established.3. Re-binding of [¹⁴C]ADP by depleted membranes is an energy-independent process. Even after solubilization of adenylate-depleted CF₁, [¹⁴C]ADP is incorporated into the protein. By re-binding of ADP in the dark, CF₁ is converted to a non-exchangeable form.4. Energy-dependent adenine nucleotide exchange on CF₁ is suggested to include three different conformational states of the enzyme: (1) a stable, non-exchangeable form which contains firmly bound nucleotides, is converted to (2), an unstable form containing loosely bound adenine nucleotides. This conformation allows adenylate exchange; it is in equilibrium with (3) a metastable, adenylate-depleted form. The transition from state (1) to state (2) is the energy-requiring step.     

Heterogeneity of the calcium-induced permeability transition in isolated non-synaptic brain mitochondria

Calcium overload of neural cell mitochondria plays a key role in excitotoxic and ischemic brain injury. This study tested the hypothesis that brain mitochondria consist of subpopulations with differential sensitivity to calcium-induced inner membrane permeability transition, and that this sensitivity is greatly reduced by physiological levels of adenine nucleotides. Isolated non-synaptosomal rat brain mitochondria were incubated in a potassium-based medium in the absence or presence of ATP or ADP. Measurements were made of medium and intramitochondrial free calcium, light scattering, mitochondrial ultrastructure, and the elemental composition of electron-opaque deposits within mitochondria treated with calcium. In the absence of adenine nucleotides, calcium induced a partial decrease in light scattering, accompanied by three distinct ultrastructural morphologies, including large-amplitude swelling, matrix vacuolization and a normal appearance. In the presence of ATP or ADP the mitochondrial calcium uptake capacity was greatly enhanced and calcium induced an increase rather than a decrease in mitochondrial light scattering. Approximately 10% of the mitochondria appeared damaged and the rest contained electron-dense precipitates that contained calcium, as determined by electron-energy loss spectroscopy. These results indicate that brain mitochondria are heterogeneous in their response to calcium. In the absence of adenine nucleotides, approximately 20% of the mitochondrial population exhibit morphological alterations consistent with activation of the permeability transition, but less than 10% exhibit evidence of osmotic swelling and membrane disruption in the presence of ATP or ADP.