Type B monoamine oxidase and functions of Ca2+, Mg2+-dependent adenosinetriphosphatase in preparations from sarcoplasmic reticulum vesicles

Oxidative deamination of -phenylethylamine or benzylamine by type B monoamine oxidases (MAO) in preparations of sarcoplasmic reticulum vesicles from rabbit skeletal muscles is accompanied by inhibition both of active Ca²⁺ transport into the vesicles and of the activity of Ca²⁺, Mg²⁺-dependent ATPase, which is preventable by deprenil, a specific inhibitor of type B MAO. Aldehydes formed during enzymatic deamination of substrates of type B MAO may perhaps participate in the regulation of Ca²⁺, Mg²⁺-dependent ATPase, activity.Laboratory of Physicochemical Methods, Scientific-Research Institute for Biological Trials of Chemical Compounds, Ministry of the Medical Industry of the USSR. Laboratory or Biochemistry of Amines and Other Nitrogenous Compounds, Institute of Biological and Medical Chemistry, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Orekhovich.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 3, pp. 283–284, March, 1977.     

Investigation of the mechanism of action of ouabain and cyclic AMP of the transport of calcium ions by rat heart mitochondria

The action of ouabain and cyclic AMP on the Ca-accumulating capacity and outflow of Ca²⁺ ions from loaded rat heart mitochondria was studied by the tetracycline probe method. In the course of the investigations no effect of ouabain on these processes was found. Cyclic AMP did not act on Ca binding by the mitochondrial membrane but it induced rapid liberation of Ca²⁺ from organelles loaded with these ions.Department of Molecular Pharmacology, Medico-Biological Faculty, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR S. S. debov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 2, pp. 158–160, February, 1977.     

Role of membrane-bound calcium in changes in ATPase activity,permeability, and structural state of the human erythrocyte membrane

Experiments were carried out on reconstituted erythrocytes obtained by rapid reversible hemolysis. The free Ca²⁺ concentration in the reconstituted erythrocytes was maintained by means of Ca-EGTA and Ca-citrate buffers. The ouabain-inhibited component of ATPase activity with high affinity for Ca²⁺ (K_0.5=4 M) and a change in the passive and active permeability for K⁺ in the region of free Ca²⁺ concentrations up to 10 M could be found only by modifying the content of membrane-bound Ca²⁺. Reducing its content on the inner side of the membrane of the reconstituted erythrocytes was accompanied by a change in the hydrophobicity of the hydrocarbon regions of the membrane. it is suggested that Ca²⁺-induced changes in the structural state of the erythrocyte membrane may be the direct cause of the change in ATPase activity with high affinity for Ca²⁺ and in permeability for monovalent cations.Central Research Laboratory, Fourth Main Board, Ministry of Health of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 6, pp. 682–685, June, 1978.     

Synthesis of plasma membrane proteins in cells of the regenerating liver

Protein synthesis was studied by determining incorporation of [³H]glycine in cells of the regenerating rat liver. The rate of incorporation of [³H]glycine into total liver proteins, proteins of the microsomal fraction and of the hyaloplasm, and proteins of the plasma membranes, soluble and insoluble in 0.05 M K₂CO₃ was determined. The rate of incorporation of [³H]glycine into soluble proteins of the plasma membranes reached a maximum 1 h after partial hepatectomy. The maximal rate of synthesis of proteins of the other fractions occurred at the end of the g₁ period. The sharp increase in the rate of synthesis of plasma membrane proteins soluble in 0.05 M K₂CO₃ is evidently one of the earliest biochemical events in cells of the regenerating liver preparing to divide.Laboratory of Chemical Factors of Regulation of Growth and Cell Division, Institute of Biological and Medical Chemistry, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Orekhovich.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 5, pp. 589–591, May, 1978.     

Effect of drug resistance modulator,NO donor,on membrane structure and function of membrane-bound Ca<Superscript>2+</Superscript>-activated Mg<Superscript>2+</Superscript>-dependent ATPase

Exogenous NO donor 3,3-bis-(nitroxymethyl)oxetane (NMO) was synthesized at the Institute for Problems of Chemical Physics (Russian Academy of Sciences). This compound was shown to inhibit Ca²⁺-ATPase isolated from normal muscular cells and tumor cells. Both hydrolytic and transport functions of the enzyme were inhibited under these conditions. These changes were probably related to changes in membrane structure caused by NO donor. Our results suggest that changes in intracellular Ca²⁺ concentration can modulate the formation of tumor drug resistance. Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 146, No. 8, pp. 165–167, August, 2008     

Ischemic damage to the sarcoplasmic reticulum of skeletal muscles: The role of lipid peroxidation

The development of ischemia was shown to be accompanied by inhibition of the Ca²⁺ enzyme transport system (ETS) (a decrease in the Ca²⁺/ATP ratio and in activity of Ca²⁺-dependent ATPase), which correlates with accumulation of the primary and secondary molecular lipid peroxidation products (POL) in vivo and in the membranes of the sarcoplasmic reticulum (SR) of the skeletal muscles. Administration of antioxidants (2,6-di-tert-butyl-4-methylphenol, -tocopherol) prevents activation of POL in the ischemic muscle and partially protects the Ca²⁺ ETS against injury. Restoration of the blood flow after prolonged ischemia leads to further inhibition of the Ca²⁺ ETS while the concentration of POL products remains unchanged.A joint research project of the M. V. Lomonosov Moscow State University and the I. M. Sechenov First Moscow State Medical Institute.Laboratory of Transplantation of Organs and Tissues, Academy of Medical Sciences of the USSR. Department of Operative Surgery and Topographical Anatomy, I. M. Sechenov First Moscow Medical Institute. Laboratory of Physical Chemistry of Biomembranes, M. V. Lomonosov Moscow State University. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Kovanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 6, pp. 683–686, June, 1977.     

Comparative analysis of the action of papaverine,isoprenaline, La3+, and compound D-600 on depolarized guinea pig taenia coli smooth muscle

Experiments on strips of guinea pig taenia coli were carried out by the double sucrose gap method. Isometric contractions of the strip in the region of the artificial node were recorded during perfusion with a solution containing a high concentration of potassium (120 M KCl, 47mM NaCl), to which one of the myolytics and agents inducing contraction of depolarized muscle were successively added. Off-responses (OR) arising after the end of stimulation by square hyperpolarizing pulses (11 sec) also were studied. D-600 (10^–5 g/ml) and La³⁺ (2·10^–4 g/ml) were shown to reduce muscle tone (residual potassium contracture) and to abolish the contractile effects of histamine or of a tenfold increase in the Ca²⁺ concentration in the medium [Ca]₀. The amplitude of OR fell but its kinetics remained unchanged. Papaverine (10^–5 g/ml) caused a lasting decrease in tone, abolished the effects of an increase in [Ca]₀ or addition of histamine, but caused virtually no change in the amplitude of OR — the only effect was a sharp increse in the rate of muscle relaxation after the end of its contraction phase. Isoprenaline (10^–6 g/ml) lowered the tone of the depolarized muscle without changing the contractile effects of histamine, an increase in [Ca]₀ or OR. The results point to differences in the mechanisms of action of the various myolytics. D-600 and La³⁺ block chemically excitable and all types (fast, slow, and uninactivated) of electrically excitable Ca channels. The action of isoprenaline is evidently connected with activation of the system of cyclic AMP synthesis and, consequently, with increased sequestration of Ca²⁺ in the intracellular depots. To understand the nature of the action of papaverine, it has to be suggested that it can block chemically excitable Ca channels and also electrically excitable Ca channels that have been open for a long time.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Chernigovskii). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 6, pp. 553–556, June, 1979.     

Dimethylaminochalcone,an indicator of structural changes in theE. coli cell membrane produced by Ca++ cations and tris buffer

An attempt was made to find hypothetical structural changes in the cell membrane ofEscherichia coli under the influence of Ca⁺⁺ cations with the aid of the uncharged fluorescent proble 4-dimethylaminochalcone (DMC). Effects of Tris buffer (0.01 M) at 0°C and of other agents, namely Mg⁺⁺ cations and EDTA, also were tested for comparison. Treatment of theE. coli cells with Ca⁺⁺ cations was shown to cause structural changes in the surface of the cell membrane which differed from the changes produced by treatment with Tris buffer at 0°C, Mg⁺⁺ cations, and EDTA. DMC can be used with success as an indicator of structural changes in biomembranes.Laboratory of Molecular Microbiology, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. M. Zhdanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 7, pp 65–68, July, 1979.     

3-Isobutyl-1-methylxanthine (IBMX) affects potassium permeability in rat sensory neurones via pathways that are sensitive and insensitive to [Ca2+]in

The effects of externally applied 3-isobutyl-1-methylxanthine (IBMX), in millimolar concentrations, on the membrane currents in dorsal root ganglia (DRG) neurones isolated from newborn rats were investigated using the amphotericin-based perforated patch-clamp technique. In some experiments, simultaneous measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_in) were performed using fura-2 microfluorimetry. Applications of IBMX induced elevation of [Ca²⁺]_in resulting from Ca²⁺ release from caffeine-ryanodine-sensitive internal stores. In addition to Ca²⁺ release, IBMX produced a biphasic membrane current response comprised of an inward current transiently interrupted by outward current. The onset of the inward current slightly preceded the onset of the [Ca²⁺]_in transient, while the interrupting outward current developed synchronously with the [Ca²⁺]_in rise. The development of IBMX-induced outward current ultimately needed the [Ca²⁺]_in elevation. After the depletion of Ca²⁺ stores by IBMX or caffeine exposure, the subsequent IBMX challenge failed to produce both the [Ca²⁺]_in transient and outward membrane current, although the inward current remained unchanged. Both components of the IBMX-induced membrane current response had a reversal potential close to the K⁺ equilibrium potential and the IBMX-induced membrane current response disappeared while dialysing the cell interior with K⁺-free, Cs⁺-containing solutions suggesting their association with K⁺ channel activity. External administration of 10 mM tetraethylammonium chloride (TEA-Cl) evoked an inward current similar to that observed in response to IBMX; in the presence of TEA-Cl, IBMX application was almost unable to induce additional inward current. IBMX (5 mM) effectively (50%) inhibited K⁺ currents evoked by step depolarizations of membrane potential. We suggest that IBMX affects membrane permeability via activation of Ca²⁺-regulated K⁺ channels and direct inhibition of TEA-sensitive K⁺ channels.     

Ca2+ transport of the skeletal muscle sarcoplasmic reticulum during readaptation of rats after a 40-day load relief on the hind paws

The rate of Ca²⁺ accumulation in the sarcoplasmic reticulum of the skeletal muscle (m. gastrocnemius lateralis, m. vastus medialis, andm. soleus) is studied in rats under conditions of functional off-loading of the hind paws (suspending animals by the tail). The rate of Ca²⁺ transport in the sarcoplasmic reticulum is shown to be stepped up in all these muscles. In the sarcoplasmic reticulum ofm. gastrocnemius lateralis andm. vastus medialis the Ca²⁺ transport rate reliably drops, which does not occur inm. soleus. During a 2-week period of readaptation of animals suspended for 40 days, the Ca²⁺-transporting function of them. soleus sarcoplasmic reticulum gradually recovers to reach the control values, whereas the time course of recovery of Ca²⁺-pump activity in the sarcoplasmic reticulum ofm. gastrocnemius lateralis andm. vastus medialis has a phasic pattern. Translated fromByulleten' Eksperimental'noi Biologii i Meditsity, Vol. 118, N ^o 12, pp. 591–595, December, 1994 Presented by A. I. Grigor'ev, Member of the Russian Academy of Medical Sciences     

Incorporation of glycine-14C into plasma membrane proteins of the normal and regenerating liver

Incorporation of glycine-¹⁴C into plasma membrane proteins of the intact and regenerating liver was studied at the beginning of the C₁-period of the mitotic cycle. Electrophoretic analysis of plasma membrane proteins of the regenerating liver, labeled with glycine-¹⁴C and soluble in 0.05M Na₂CO₃, showed that uptake of label was greatest into proteins with a molecular weight of about 60,000. The difference between incorporation of glycine-¹⁴C into plasma membrane proteins of the intact and regenerating liver insoluble in 0.05M Na₂CO₃ during electrophoretic separation was not significant.Laboratory of Chemical Factors of Regulation of Growth and Cell Division, Institute of Biological and Medical Chemistry, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Orekhovich.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 12, pp. 674–676, December, 1979.     

Contracture of myocardial fibers of the frog ventricle during high-frequency stimulation after blocking of the calcium channels by manganese ions

The strength of contraction of a stimulated (0.5 Hz) strip of frog ventricular myocardium was reduced to 3–5% of its original value by perfusion with Ringer's solution containing 2.5 mM Mn²⁺; the duration of the action potential under these circumstances was sharply reduced. An increase in the frequency of stimulation to 5 Hz led to the development of a contracture, the magnitude of which reached 30% of the original strength of contraction recorded in normal perfusion solution. The amplitude of the contracture was more than doubled by the addition of ouabain (2·10^–6 g/ml). In analogous experiments with La³⁺ (2.5 mM) contracture did not develop in response to an increase in the frequency of stimulation; ouabain was ineffective in these experiments. It is postulated that the contracture induced in the presence of Mn²⁺ is due to nonelectrogenic Ca²⁺ transport in the muscle fibers.Laboratory of Electrophysiology of the Heart, All-Union Cardiological Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR E. I. Chazov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 4, pp. 407–410, April, 1978.     

Effect of binding of palmitoly-CoA with adenine-nucleotide translocase on mitochondrial energization

During inhibition of oxidative phosphorylation by oligomycin, palmitoyl-CoA (p-CoA) reduces the velocity of energy-dependent reduction of acetoacetate and the Ca⁺⁺-capacity of the mitochondria in medium with phosphate. Energy-independent osmotic swelling of the mitochondria in medium with NH₄NO₃, which depends on the proton conductance of the inner membrane, is inhibited by ADP and accelerated by p-CoA. All effects of p-CoA are abolished by carnitine and competitively by ADP. It is concluded that the lowering of the energization level by p-CoA is connected with increased permeability of the inner membrane for H⁺ as a result of binding of the inhibitor with adenine-nucleotide translocase.Institute of Clinical and Experimental Medicine, Siberian Branch, Academy of Medical Sciences of the USSR, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR V. P. Kaznacheev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 9, pp. 297–299, September, 1979.     

Effect of the central cholinolytic cyclosil on rat liver mitochondrial function

During incubation of rat liver mitochondria in the presence of the central cholinolytic cyclosil (0.04 mg/ml) the effectiveness of formation of high-energy compounds is increased. The rate of O₂ utilization remained unchanged. Some tendency was observed for the inward K⁺ transport into the mitochondria to be increased in the presence of cyclosil. The results point to an increased degree of energization of the mitochondria in the presence of cytosil.Leningrad. (Presented by Academician of the Academy of Medical Sciences of the USSR S. N. Golikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 4, pp. 431–434, April, 1978.     

Effect of presynaptic neurotoxins from bee and cobra venom on spontaneous mediator secretion from mouse motor nerve ending

Phosphorylases A₂ (PLA) from bee and cobra venoms cause triphasic changes in the frequency of miniature endplate potentials (MEPPs) recorded in the mouse diaphragm: an initial decrease of liberation of mediator followed by an increase, followed in turn by complete blocking of liberation. Removal of Ca⁺⁺ from the perfusing solution (to concentrations below 10^–9M) prevented the presynaptic effect of the two PLAs. If the PLA was washed off with calcium-free solution, subsequent treatment with standard solution (2 mM Ca⁺⁺) caused an increase in the frequency of MEPPs. Agents increasing the Ca⁺⁺ concentration in the axoplasm (K⁺ ions, hypertonic sucrose, and the uncoupling agent 4, 5, 6, 7-tetra-2-trifluoromethylbenzimidazole) caused an increase in the MEPP frequency in muscles poisoned with PLA. Blocking the liberation of mediator evidently was not caused by exhaustion of its reserves.Department of Human and Animal Physiology, M. V. Lomonosov Moscow State University. Group for Biophysics of Synaptic Processes, I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Leningrad. Laboratory of Protein Chemistry, M. N. Shemyakin Institute of Bioorganic Chemistry, Academy of Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 5, pp. 396–399, May, 1979.     

Effect of Ca++ and GABA on oxidation of glutamate and glutamine by rat brain synaptosomes

Experiments in vitro showed that the addition of Ca⁺⁺ inhibits respiration of rat brain synaptosomes in the presence of glutamate and glutamine. The addition of GABA potentiates the inhibitory effect of Ca⁺⁺ on the oxidation of glutamine but not of glutamate. GABA itself has no effect on the oxidation of either glutamate or glutamine.Laboratory of Functional Neurochemistry, I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Chernigovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 9, pp. 293–295, September, 1978.     

45Ca exchange in rat cortex slices in conditions of long-term potentiation

Existing data on the role of Ca²⁺ ions in the development of long-term potentiation were used as a basis for studying changes in different Ca²⁺ compartments in cells in living rat olfactory cortex slices during potentiation. The kinetics of⁴⁵Ca²⁺ exchange were studied at 5, 15, and 30 min of potentiation. During the induction phase (1–5 min) of long-term potentiation, the fraction of tightly-bound intracellular Ca²⁺ decreased. There were no changes in the content of Ca²⁺ ions in other fractions at this stage. During maintenance of potentiation, which lasted 15–25 min, Ca²⁺ levels in the extracellular and intracellular compartments did not differ from controls. At 30 min, during extinction of long-term potentiation, there was a significant redistribution of Ca²⁺ in cells: the levels of free and loosely-bound Ca increased, as did extracellular Ca²⁺. Laboratory of Functional Neurochemistry, I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, 6 Makarov Bank, St. Petersburg 199034, Russia. Translated from Rossiiki Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 83, No. 7, pp. 45–49, July, 1997.     

Bradykinin and inositol 1,4,5-trisphosphate-stimulated calcium release from intracellular stores in cultured bovine endothelial cells

The relative importance of intracellular and extracellular Ca²⁺ in the release of endothelium-derived relaxing factor (EDRF) and the mechanisms involved in the release of intracellular Ca²⁺ were investigated in cultured bovine endothelial cells. The release of EDRF by bradykinin, determined by bioassay, was dose-dependent showing an EC₅₀ of 4×10^–10 M. The bradykinin-induced EDRF release from endothelial cells was maintained in the presence of extracellular Ca²⁺. However, in the absence of external Ca²⁺, bradykinin-induced EDRF release was both attenuated and transient. In cells loaded to isotopic equilibrium with⁴⁵Ca, bradykinin increased the⁴⁵Ca efflux into both calcium-containing and calcium-free solutions, with an EC₅₀ for the increase in⁴⁵Ca efflux induced by bradykinin of 1.3×10^–9 M. The involvement of an intracellular Ca²⁺ store and the participation of a second messenger in its release were investigated in saponin-permeabilized endothelial cells. In saponin-permeabilized cells, ATP-sensitive calcium uptake was Ca²⁺,Mg²⁺-ATPase-dependent. The ATP-sensitive uptake of calcium at different free Ca²⁺ concentrations showed at least two compartments involved in the uptake of Ca²⁺. The⁴⁵Ca uptake into the compartment with the lowest affinity and highest capacity could be inhibited by sodium azide, suggesting that this uptake was into mitochondria. The majority of the⁴⁵Ca uptake into the azide-insensitive store could be released by inositol-1,4,5-trisphosphate (IP₃). The IP₃-induced release was not affected by apyrase or exogenous GTP. The EC₅₀ for the release of Ca²⁺ by IP₃ was 1.0 M and was unaffected by an inhibitor of IP₃ breakdown (2,3-diphosphoglyceric acid). The results suggest that the release of EDRF is dependent on extracellular Ca²⁺ influx and the release of intracellular Ca²⁺. The release of calcium from one of the high affinity intracellular Ca²⁺ stores is mediated by the intracellular second messenger, IP₃.     

Second messengers contribute to cholinoceptor modulation by amiridine inHelix lucorum neurons

The effect of amiridine on the local inward acetylcholine current and its volt-ampere characteristic are studied by the two-electrode method of membrane voltage clamp in identified RPa3 and LPa3helix lucorum neurons pretreated with forskolin, sodium nitroprusside, A23187, and EGTA. The results suggest that second messengers (Ca²⁺, NO, cGMP, and cAMP) are implicated in the amiridine-mediated regulation of cholinoceptors inHelix lucorum neurons. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 11, pp. 470–473, November, 1994 Presented by Yu. A. Romanov, Member of the Russian Academy of Medical Sciences     

Effect of thapsigargin and caffeine on Ca2+ homeostasis in HeLa cells: implications for histamine-induced Ca2+ oscillations

Several studies have already established that the stimulation of H1 receptors by exogenous histamine induces intracellular Ca²⁺ oscillations in HeLa cells. The molecular mechanism underlying this oscillatory process remains, however, unclear. A series of fura-2 experiments was undertaken in which the nature of the Ca²⁺ pools involved in the histamine-induced Ca²⁺ oscillations was investigated using the tumour promoter agent thapsigargin (TG) and the Ca²⁺-induced Ca²⁺-release promoter, caffeine. The results obtained indicate first that TG causes a gradual increase in cytosolic Ca²⁺ without inducing internal Ca²⁺ oscillations, and second that TG and histamine share common internal Ca²⁺ storage sites. The latter conclusion was derived from experiments performed in the absence of external Ca²⁺, where the addition of TG before histamine resulted in a total inhibition of the Ca²⁺ response linked to H1 receptor stimulation, whereas the addition of histamine before TG decreased by more than 90% the TG-induced Ca²⁺ release. Finally, TG was found to inhibit irreversibly histamine-induced Ca²⁺ oscillations when added to the bathing medium during the oscillatory process. The effect of caffeine at concentrations ranging from 1 mM to 10 mM on intracellular Ca²⁺ homeostasis was also investigated. The results obtained show that caffeine does not affect systematically the internal Ca²⁺ concentration in resting and TG-stimulated HeLa cells, but increases the Ca²⁺ sequestration ability of inositol-trisphosphate (InsP ₃)-related Ca²⁺ stores. These results suggest either that TG acts on InsP ₃-sensitive as well as InsP ₃-insensitive Ca²⁺ pools, so that no final conclusion on the nature of the pools involved in Ca²⁺ spike generation can be currently drawn, or that the contribution of an InsP ₃-insensitive Ca²⁺-induced Ca²⁺-release process is not essential to the Ca²⁺ oscillation machinery in these cells. It is also concluded that a release of Ca²⁺ by caffeine may not be directly accessible to fura-2 measurements in this cellular preparation, but that the inhibitory effect of caffeine on the Ca²⁺ mobilization process triggered by InsP ₃ can be clearly documented using this experimental approach.