Intracellular ADP modulates the Ca2+ release-activated Ca2+ current in a temperature- and Ca2+-dependent Way.

Abstract The rat basophilic cell line RBL-1 is known to express high levels of the Ca current activated by store depletion, known as Ca release-activated Ca current (I), the main Ca influx pathway so far identified in nonexcitable cells. We show here that, as reported in other cell types, metabolic drugs strongly inhibit the Ca influx operated by store depletion in RBL-1 cells also. We have tested the hypothesis that intracellular adenine and/or guanine nucleotide levels act as coupling factors between I and cell metabolism. Using the whole cell configuration of the patch-clamp technique, we demonstrate that addition of ADP to the intracellular solution significantly reduces I induced by inositol 1,4,5-trisphosphate. This phenomenon differs from other regulatory pathways of I, since it is highly temperature-dependent, is observable only in the presence of low intracellular Ca buffering capacity, and requires a cytosolic factor(s) which is rapidly lost during cell dialysis. Moreover, the inhibition is specific for ADP and is partially mimicked by ADPβS and AMP, but not by GDP or GTP

Purinergic Receptors Mediate Two Distinct Glutamate Release Pathways in Hippocampal Astrocytes

The purinergic P2X(7) receptor (P2X(7)R) can mediate glutamate release from cultured astrocytes. Using patch clamp recordings, we investigated whether P2X(7)Rs have the same action in hippocampal astrocytes in situ. We found that 2- and 3-O-(4-benzoylbenzoyl)ATP (BzATP), a potent, although unselective P2X(7)R agonist, triggers two different glutamate-mediated responses in CA1 pyramidal neurons; they are transient inward currents, which have the kinetic and pharmacological properties of previously described slow inward currents (SICs) due to Ca(2+)-dependent glutamate release from astrocytes, and a sustained tonic current. Although SICs were unaffected by P2X(7)Rs antagonists, the tonic current was inhibited, was amplified in low extracellular Ca(2+), and was insensitive to glutamate transporter and hemichannel inhibitors. BzATP triggered in astrocytes a large depolarization that was inhibited by P2X(7)R antagonists and amplified in low Ca(2+). In low Ca(2+) BzATP also induced lucifer yellow uptake into a subpopulation of astrocytes and CA3 neurons. Our results demonstrate that purinergic receptors other than the P2X(7)R mediate glutamate release that evokes SICs, whereas activation of a receptor that has features similar to the P2X(7)R, mediates a sustained glutamate efflux that generates a tonic current in CA1 neurons. This sustained glutamate efflux, which is potentiated under non-physiological conditions, may have important pathological actions in the brain

Generation of inositol phosphates, cytosolic Ca2+, and ionic fluxes in PC12 cells treated with bradykinin

Abstract Accumulation of inositol phosphates (Ins-Ps, revealed by high performance liquid chromatography), changes of the cytosolic free Ca2+ [( Ca2+]i, revealed by fura-2), membrane potential and ionic currents (revealed by bis-oxonol and patch clamping) were investigated in PC12 cells treated with bradykinin (BK). The phenomena observed were (a) due to the activation of a B2 receptor (inhibitor studies) and (b) unaffected by pertussis toxin, cAMP analogs, and inhibitors of either cyclooxygenase or voltage-gated Ca2+ channels. During the initial tens of s, three interconnected events predominated: accumulation of Ins-1,4,5-P3, Ca2+ release from intracellular stores and hyperpolarization due to the opening of Ca2+-activated K+ channels. Phorbol myristate acetate partially inhibited Ins-1,4,5-P3 accumulation at all [BK] investigated, and the [Ca2+]i increase at [BK] less than 50 nM. In PC12 cells treated with maximal [BK] in the Ca2+-containing incubation medium, Ins-1,4,5-P3 peaked at 10 s, dropped to 20% of the peak at 30 s, and returned to basal within 5 min; the peak increase of Ins-1,3,4-P3 was slower and was variable from experiment to experiment, while Ins-P4 rose for 2 min, and remained elevated for many min thereafter. Meanwhile, influx of Ca2+ from the extracellular medium, plasma membrane depolarization (visible without delay when hyperpolarization was blocked), and increased plasma membrane conductance were noticed. Evidence is presented that these last three events (which were partially inhibited by phorbol myristate acetate at all [BK]) were due to the activation of a cation influx, which was much more persistent than the elevation of the two Ins-P3 isomers. Our results appear inconsistent with the possibility that in intact PC12 cells the BK-induced activation of cation influx is accounted for entirely by the increases of either Ins-1,3,4-P3 or Ins-1,4,5-P3 (alone or in combination with Ins-1,3,4,5-P4), as previously suggested by microinjection studies in different cell types

A Role for Calcium Influx in the Regulation of Mitochondrial Calcium in Endothelial Cells

By using an endothelial cell line (ECV304), derived from human umbilical vein and transfected with recombinant aequorin targeted to the mitochondrial matrix, we find that stimulation with ATP evokes long lasting increases in mitochondrial Ca2+ ([Ca2+]m) that largely depend on Ca2+ influx. In these cells, the release of stored Ca2+ is inefficient at elevating [Ca2+]m. Consequently it appears that in ECV304 cells, bulk cytosolic Ca2+ ([Ca2+]c) is the main determinant of [Ca2+]m changes. In ECV304 cells4% of mitochondria are within 700 nm of the endoplasmic reticulum as opposed to 65% in HeLa cells, whereas 14% are within 700 nm of the inner surface of the plasma membrane, compared with6% in HeLa cells. Following Ca2+ depletion, readdition of extracellular Ca2+ evokes an increase in [Ca2+]m but not in [Ca2+]c. Under these conditions, microdomains of high [Ca2+]c may occur beneath the plasma membrane of ECV304 cells resulting in the preferential elevation of Ca2+ in mitochondria located in this region. A model is discussed in which the localization of mitochondria with respect to Ca2+ sources is the main determinant of their in situ Ca2+ uptake kinetics. Thus, in any given cell type mitochondria may be localized to suit the energy and metabolic demands of their physiological actions

Ca2+ Homeostasis in the Agonist-sensitive Internal Store: Functional Interactions Between Mitochondria and the ER Measured In Situ in Intact Cells

Mitochondria have a well-established capacity to detect cytoplasmic Ca2+ signals resulting from the discharge of ER Ca2+ stores. Conversely, both the buffering of released Ca2+ and ATP production by mitochondria are predicted to influence ER Ca2+ handling, but this complex exchange has been difficult to assess in situ using conventional measurement techniques. Here we have examined this interaction in single intact BHK-21 cells by monitoring intraluminal ER [Ca2+] directly using trapped fluorescent low-affinity Ca2+ indicators. Treatment with mitochondrial inhibitors (FCCP, antimycin A, oligomycin, and rotenone) dramatically prolonged the refilling of stores after release with bradykinin. This effect was largely due to inhibition of Ca2+ entry pathways at the plasma membrane, but a significant component appears to arise from reduction of SERCA-mediated Ca2+ uptake, possibly as a consequence of ATP depletions in a localized subcellular domain. The rate of bradykinin-induced Ca2+ release was reduced to 51% of control by FCCP. This effect was largely overcome by loading cells with BAPTA-AM, highlighting the importance of mitochondrial Ca2+ buffering in shaping the release kinetics. However, mitochondria-specific ATP production was also a significant determinant of the release dynamic. Our data emphasize the localized nature of the interaction between these organelles, and show that competent mitochondria are essential for generating explosive Ca2+ signals

Voltage-dependent activation and inactivation of calcium channels in PC12 cells. Correlation with neurotransmitter release.

The existence and mechanisms of inactivation of voltage-gated Ca2+ channels are important, but still debatable, physiological problems. By using the Ca2+ indicators quin2 and fura-2, we demonstrate that in PC12 cells voltage-gated Ca2+ channels undergo inactivation dependent on both voltage and [Ca2+]i. Inactivation, however, is never complete and a small number of channels remains open during prolonged depolarization, explaining the steady state elevation of [Ca2+]i observed in cells depolarized with high KCl. A close parallel exists between Ca2+ channel inactivation and the transient nature of neurotransmitter release: secretion is rapidly stimulated during the first 30 s of depolarization, when a transient overshoot in [Ca2+]i can be demonstrated, while it is negligible during the following period, despite the persistence of an elevated [Ca2+]i; predepolarization in Ca2+-free medium and subsequent addition of Ca2+ (a condition which allows the development of the voltage inactivation) abolishes the fast phase of secretion, while not modifying the steady state [Ca2+]i eventually attained; and increases in the intracellular Ca2+ buffering decreases the amplitude of the fast secretion phase induced by KCl without altering the steady state [Ca2+]i. We suggest that localized [Ca2+]i gradients form close to the plasma membrane shortly after depolarization and that the [Ca2+]i reached in these regions is the relevant parameter in the regulation of secretion

Direct Monitoring of the Calcium Concentration in the Sarcoplasmic and Endoplasmic Reticulum of Skeletal Muscle Myotubes

Direct monitoring of the free Ca2+ concentration in the sarcoplasmic reticulum (SR) was carried out in rat skeletal myotubes transfected with a specifically targeted aequorin chimera (srAEQ). Myotubes were also transfected with a chimeric aequorin (erAEQ) that we have demonstrated previously is retained in the endoplasmic reticulum (ER). Immunolocalization analysis showed that although both recombinant proteins are distributed in an endomembrane network identifiable with immature SR, the erAEQ protein was retained also in the perinuclear membrane. The difficulty of measuring [Ca2+] in 100-1000 microM range was overcome with the use of the synthetic coelenterazine analogue, coelenterazine n. We demonstrate that the steady state levels of [Ca2+] measured with srAEQ is around 300 microM, whereas that measured with erAEQ is significantly lower, i.e. around 200 microM. The effects of caffeine, high KCl, and nicotinic receptor stimulation, in the presence or absence of external calcium or after blockade of the Ca-ATPase, were investigated with both chimeras. The kinetics of [Ca2+] changes revealed by the erAEQ were similar, but not identical, neither quantitatively nor qualitatively, to those monitored with the srAEQ, indicating that at this stage of muscle development, differences exist between SR and ER in their mechanisms of Ca2+ handling. The functional implications of these findings are discussed