The role of Ca2+ and hemodynamics in the action of diltiazem on hepatic energy metabolism

Diltiazem causes vasoconstriction in the liver when present at high concentrations, an action that is strictly Ca²⁺-dependent. Diltiazem is also active on energy metabolism. This toxic action could be partly a consequence of hemodynamic effects. In the absence of Ca²⁺, the hemodynamic effects are no longer present and, consequently, Ca²⁺-free experiments are useful for distinguishing between hemodynamics-dependent and hemodynamics-independent effects. The experimental system used was the hemoglobin-free perfused rat liver from fed and fasted rats. Diltiazem was infused at various concentrations in the presence and absence of Ca²⁺. Several metabolic parameters were measured: lactate and pyruvate production (glycolysis), glycogenolysis, oxygen uptake, gluconeogenesis, and the cellular levels of lactate, pyruvate, glucose, AMP, ADP, and ATP. The effects of diltiazem can be divided into three groups: (1) Effects that are strictly dependent on the Ca²⁺-mediated hemodynamic action. This group comprises inhibition of oxygen uptake at all concentrations (50–500 mol/L) inhibition of lactate, pyruvate, and glucose release at high concentrations; the decrease in cellular ATP; the increase in cellular AMP; and the cellular accumulation of glucose and lactate. (2) Effects that are independent of the hemodynamic action. The most relevant effect of this type is inhibition of gluconeogenesis. (3) Effects that are influenced by Ca²⁺ but are independent of the hemodynamic effects. This is the typical case of lactate and glucose release from endogenous glycogen, whose stimulation by low diltiazem concentrations is more pronounced in the presence of Ca²⁺ than in its absence.     

Alterations of glial tumor cell Ca2+ metabolism and Ca2+-dependent cAMP accumulation by phorbol myristate acetate

C6 glial tumor cells exposed to phorbol myristate acetate (PMA) possessed lowered cAMP content, reduced ability to accumulate cAMP in response to norepinephrine or cholera toxin, and a 3-fold increase in the concentration of norepinephrine producing 50% of the maximal rate of cAMP accumulation. Detectable effects on cAMP accumulation occurred within 10 min of exposure to PMA, and prominent effects by 2 h. PMA similarly affected cells pretreated with cycloheximide. In contrast, Ca2+-depleted preparations of control and PMA-treated cells accumulated cAMP identically in response to norepinephrine or cholera toxin. Ca2+ restoration, which increased the rate of cAMP accumulation in control cells severalfold, did not enhance cAMP accumulation in PMA-treated cells. Neither high catecholamine nor high extracellular Ca2+ concentrations reversed the suppression of cAMP accumulation by PMA. Trifluoperazine, which inhibited the Ca2+-dependent component of norepinephrine-stimulated cAMP accumulation in control cells, did not significantly reduce norepinephrine-stimulated cAMP accumulation in PMA-treated cells. Cell free preparations of control and PMA-treated cultures did not differ significantly in calmodulin content or in Ca2+-stimulated adenylate cyclase, Ca2+-dependent cAMP phosphodiesterase, and (Ca2+-Mg2+)-ATPase activities. The Ca2+ content, however, of intact cells decreased with time of PMA treatment. Within minutes after exposure to PMA, the ability of Ca2+-depleted cells to take up 45Ca was significantly reduced. Both 45Ca uptake and Ca2+-dependent cAMP accumulation were reduced over the same PMA concentration range.     

Cytochrome c release occurs via Ca2+-dependent and Ca2+-independent mechanisms that are regulated by Bax

Release of cytochrome c from mitochondria is a key initiative step in the apoptotic process, although the mechanisms regulating this event remain elusive. In the present study, using isolated liver mitochondria, we demonstrate that cytochrome c release occurs via distinct mechanisms that are either Ca(2+)-dependent or Ca(2+)-independent. An increase in mitochondrial matrix Ca(2+) promotes the opening of the permeability transition (PT) pore and the release of cytochrome c, an effect that is significantly enhanced when these organelles are incubated in a reaction buffer that is based on a physiologically relevant concentration of K(+) (150 mm KCl) versus a buffer composed of mannitol/sucrose/Hepes. Moreover, low concentrations of Ca(2+) are sufficient to induce mitochondrial cytochrome c release without measurable manifestations of PT, though inhibitors of PT effectively prevent this release, indicating that the critical threshold for PT varies among mitochondria within a single population of these organelles. In contrast, Ca(2+)-independent cytochrome c release is induced by oligomeric Bax protein and occurs without mitochondrial swelling or the release of matrix proteins, although our data also indicate that Bax enhances permeability transition-induced cytochrome c release. Taken together, our results suggest that the intramitochondrial Ca(2+) concentration, as well as the reaction buffer composition, are key factors in determining the mode and amount of cytochrome c release. Finally, oligomeric Bax appears to be capable of stimulating cytochrome c release via both Ca(2+)-dependent and Ca(2+)-independent mechanisms.     

Endothelin contraction in pig coronary: Receptor types and Ca2+-mobilization

Endothelin is one of the most potent vasoconstrictors known. It plays an important role in the regulation of vascular tone and in the development of many cardiovascular diseases. This study focuses on the receptor types and the Ca²⁺ mobilization responsible for endothelin-1 (ET-1) contraction in de-endothelialized pig coronary artery rings. ET-1 contracted the artery rings with an EC₅₀ = 6.5 ± 1 nM and a maximum contraction which was 98.6 ± 9% of the contraction produced by 60 mM KCl. BQ123 (5 µM), an ET_A antagonist, reversed 78 ± 3% of the ET-1 contraction (50 nM). IRL1620, a selective ET_B agonist, produced 23 ± 3% of the total ET-1 contraction with an EC₅₀ = 12.7 ± 2 nM. More than 85% of the contraction due to 100 nM IRL 1620 was inhibited by 200 nMBQ788, an ET_B antagonist. Therefore, approximately 80% of the ET-1 contraction in this artery occurred via ET_A receptors, and the other 20% was mediated by ET_B receptors. To assess the Ca²⁺ pools utilized during the ET-1 response, ET-1 contraction was also examined in medium containing an L-type Ca²⁺ channel blocker nitrendipine, and in Ca²⁺ free medium containing 0.2 mM EGTA. In Ca²⁺ containing medium the contraction elicited by ET-1 was 98.6 ± 9% of the KCl contraction, however, in the presence 10 µM nitrendipine the ET-1 induced contraction was 54 ± 7% of the KCl contraction, and in Ca²⁺-free medium it was 13 ± 2%. Similarly, the IRL 1620 contractions in Ca²⁺ containing medium, in the presence of nitrendipine and in Ca²⁺-free medium were 22.4 ± 3%, 12 ± 3% and 11 ± 2% of the KCl response respectively. Thus, both ET_A and ET_B contractions utilize extracellular Ca²⁺ pools via L-type Ca²⁺ channels and other undefined route(s), as well as intracellular Ca²⁺ pools. In the pig coronary artery smooth muscle, ET-1 contractions occur predominantly via ET_A receptors, with ET_B receptors using similar Ca²⁺ mobilization pathways, but the ET_B receptors appear to use the intracellular Ca²⁺ stores to a greater extent.     

Termination of cAMP signals by Ca2+ and G(alpha)i via extracellular Ca2+ sensors: a link to intracellular Ca2+ oscillations

Termination of cyclic adenosine monophosphate (cAMP) signaling via the extracellular Ca(2+)-sensing receptor (CaR) was visualized in single CaR-expressing human embryonic kidney (HEK) 293 cells using ratiometric fluorescence resonance energy transfer-dependent cAMP sensors based on protein kinase A and Epac. Stimulation of CaR rapidly reversed or prevented agonist-stimulated elevation of cAMP through a dual mechanism involving pertussis toxin-sensitive Galpha(i) and the CaR-stimulated increase in intracellular [Ca2+]. In parallel measurements with fura-2, CaR activation elicited robust Ca2+ oscillations that increased in frequency in the presence of cAMP, eventually fusing into a sustained plateau. Considering the Ca2+ sensitivity of cAMP accumulation in these cells, lack of oscillations in [cAMP] during the initial phases of CaR stimulation was puzzling. Additional experiments showed that low-frequency, long-duration Ca2+ oscillations generated a dynamic staircase pattern in [cAMP], whereas higher frequency spiking had no effect. Our data suggest that the cAMP machinery in HEK cells acts as a low-pass filter disregarding the relatively rapid Ca2+ spiking stimulated by Ca(2+)-mobilizing agonists under physiological conditions.     

Ca2+-mediated activation of phosphoenolpyruvate carboxykinase occurs via release of Fe2+ from rat liver mitochondria

The addition of calcium chloride to rat liver homogenates resulted in activation of phosphoenolpyruvate carboxykinase by as much as 50%. The enhanced activity was inhibited by quinolinic acid; it was not additive with activation by FeCl2, and stimulation was prevented by 1,10-phenanthroline. Activation by calcium was lost when the particulate fractions of liver were removed, but an activating system could be reconstituted with isolated mitochondria, purified P-enolpyruvate carboxykinase, and purified ferroactivator. Iron-loaded mitochondria were more responsive to calcium than controls. A release of Fe2+ from washed mitochondria could be detected spectrophotometrically when 25-75 nmol of Ca/mg of protein were added to the mitochondrial suspension. If Ca2+ was buffered with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, the threshold of Ca2+ necessary for release of Fe2+ was approximately 10(-7) M, with peak response between 5 X 10(-7) and 10(-6) M. Total Fe2+ detected was normally 20-30 pmol of Fe2+/mg of protein. The synthetic activator of P-enolpyruvate carboxykinase, 3-aminopicolinic acid, as well as other picolinic acid derivatives, is capable of withdrawing Fe2+ associated with the mitochondrial fraction; after incubation with mitochondria, 3-aminopicolinate will activate phosphoenolpyruvate carboxykinase in the absence of exogenous metal.     

The cilia of Paramecium tetraurelia contain both Ca2+-dependent and Ca2+-inhibitable calmodulin-binding proteins.

To identify protein targets for calmodulin (CaM) in the cilia of Paramecium tetraurelia, we employed a 125I-CaM blot assay after resolution of ciliary proteins on SDS/polyacrylamide gels. Two distinct types of CaM-binding proteins were detected. One group bound 125I-CaM at free Ca2+ concentrations above 0.5-1 microM and included a major binding activity of 63 kDa (C63) and activities of 126 kDa (C126), 96 kDa (C96), and 36 kDa (C36). CaM bound these proteins with high (nanomolar) affinity and specificity relative to related Ca2+ receptors. The second type of protein bound 125I-CaM only when the free Ca2+ concentration was below 1-2 microM and included polypeptides of 95 kDa (E95) and 105 kDa (E105). E105 may also contain Ca2+-dependent binding sites for CaM. Both E95 and E105 exhibited strong specificity for Paramecium CaM over bovine CaM. Ciliary subfractionation experiments suggested that C63, C126, C96, E95, and E105 are bound to the axoneme, whereas C36 is a soluble and/or membrane-associated protein. Additional Ca2+-dependent CaM-binding proteins of 63, 70, and 120 kDa were found associated with ciliary membrane vesicles. In support of these results, filtration binding assays also indicated high-affinity binding sites for CaM on isolated intact axonemes and suggested the presence of both Ca2+-dependent and Ca2+-inhibitable targets. Like E95 and E105, the Ca2+-inhibitable CaM-binding sites showed strong preference for Paramecium CaM over vertebrate CaM and troponin C. Together, these results suggest that CaM has multiple targets in the cilium and hence may regulate ciliary motility in a complex and pleiotropic fashion.     

AMPA induced Ca2+ influx in motor neurons occurs through voltage gated Ca2+ channel and Ca2+ permeable AMPA receptor

The rise in intracellular Ca²⁺ mediated by AMPA subtype of glutamate receptors has been implicated in the pathogenesis of motor neuron disease, but the exact route of Ca²⁺ entry into motor neurons is not clearly known. In the present study, we examined the role of voltage gated calcium channels (VGCCs) in AMPA induced Ca²⁺ influx and subsequent intracellular signaling events responsible for motor neuron degeneration. AMPA stimulation caused sodium influx in spinal neurons that would depolarize the plasma membrane. The AMPA induced [Ca²⁺]_i rise in motor neurons as well as other spinal neurons was drastically reduced when extracellular sodium was replaced with NMDG, suggesting the involvement of voltage gated calcium channels. AMPA mediated rise in [Ca²⁺]_i was significantly inhibited by L-type VGCC blocker nifedipine, whereas ω-agatoxin-IVA and ω-conotoxin-GVIA, specific blockers of P/Q type and N-type VGCC were not effective. 1-Napthyl-acetyl spermine (NAS), an antagonist of Ca²⁺ permeable AMPA receptors partially inhibited the AMPA induced [Ca²⁺]_i rise but selectively in motor neurons. Measurement of AMPA induced currents in whole cell voltage clamp mode suggests that a moderate amount of Ca²⁺ influx occurs through Ca²⁺ permeable AMPA receptors in a subpopulation of motor neurons. The AMPA induced mitochondrial calcium loading [Ca²⁺]_m, mitochondrial depolarization and neurotoxicity were also significantly reduced in presence of nifedipine. Activation of VGCCs by depolarizing concentration of KCl (30 mM) in extracellular medium increased the [Ca²⁺]_i but no change was observed in mitochondrial Ca²⁺ and membrane potential. Our results demonstrate that a subpopulation of motor neurons express Ca²⁺ permeable AMPA receptors, however the larger part of Ca²⁺ influx occurs through L-type VGCCs subsequent to AMPA receptor activation and consequent mitochondrial dysfunction is the trigger for motor neuron degeneration. Nifedipine is an effective protective agent against AMPA induced mitochondrial stress and degeneration of motor neurons.     

Ca2+ stores regulate ryanodine receptor Ca2+ release channels via luminal and cytosolic Ca2+ sites

The free [Ca2+] in endoplasmic/sarcoplasmic reticulum Ca2+ stores regulates excitability of Ca2+ release by stimulating the Ca2+ release channels. Just how the stored Ca2+ regulates activation of these channels is still disputed. One proposal attributes luminal Ca2+-activation to luminal facing regulatory sites, whereas another envisages Ca2+ permeation to cytoplasmic sites. This study develops a unified model for luminal Ca2+ activation for single cardiac ryanodine receptors (RyR2) and RyRs in coupled clusters in artificial lipid bilayers. It is shown that luminal regulation of RyR2 involves three modes of action associated with Ca2+ sensors in different parts of the molecule; a luminal activation site (L-site, 60 microM affinity), a cytoplasmic activation site (A-site, 0.9 microM affinity), and a novel cytoplasmic inactivation site (I2-site, 1.2 microM affinity). RyR activation by luminal Ca2+ is demonstrated to occur by a multistep process dubbed luminal-triggered Ca2+ feedthrough. Ca2+ binding to the L-site initiates brief openings (1 ms duration at 1-10 s(-1)) allowing luminal Ca2+ to access the A-site, producing up to 30-fold prolongation of openings. The model explains a broad data set, reconciles previous conflicting observations and provides a foundation for understanding the action of pharmacological agents, RyR-associated proteins, and RyR2 mutations on a range of Ca2+-mediated physiological and pathological processes.     

Chick embryo muscarinic and purinergic receptors activate cytosolic Ca2+ via phosphatidylinositol metabolism

In dissociated cells from chick embryos or from chick limb buds, acetylcholine (ACh) induced an increase in cellular levels of inositol 1,4,5-trisphosphate (Ins-P3) and of inositol 1,3,4,5-tetrakisphosphate (Ins-P4). The concentration of Ins-P3 was enhanced transiently, whereas the level of Ins-P4 remained elevated for at least 20 min after addition of ACh. In most cases the increase in Ins-P4 levels was more pronounced than that of Ins-P3 levels. The inhibition of the ACh-induced inositol-phosphate response by atropine (half-maximal inhibition at 10 nM) indicates the involvement of muscarinic receptors, which in chick embryo cells induce a transient rise and a following persistent elevation of cytosolic Ca2+ activity (G. Oettling et al. (1989) J. Dev. Physiol. 12, 85-94). Adenosine 5'-triphosphate (ATP) elicited a similar transient rise in cytosolic Ca2+ activity, however, without a subsequent plateau. ATP also caused an increase in inositol-oligophosphate levels. Thus, both muscarinic and purinergic receptors in chick embryo cells are coupled to phospholipase C. The enzymatically formed Ins-P3 mediates the release of Ca2+ from internal stores. The Ca2+ signal could be involved in embryonic cell migration during morphogenesis.     

Effect of ethanol on intracellular Ca2+ metabolism

The aim of this review is to summarize current thinking on ethanol effects on the Ca2+ homeostasis in the excitable tissue cells. It has been shown that acute exposure to ethanol decreases cytoplasmic Ca2+ concentration due to Ca2+ channels inhibition and Ca2+ pumps activation. Whereas chronic exposure to ethanol increases the intracellular Ca2+ concentration in cells due to activation of passive Ca2+ transport systems and inhibition of energy-dependent Ca2+ transport systems. The emphasis is place on a possible role of pharmacologic agents that preserve Ca2+ homeostasis in protecting against ethanol-induced diseases.     

Ca2+ and phytochrome action in plants

Red light initiates many important morphogenetic responses in plants through the mediation of the pigment, phytochrome. How phytochrome promotes photomorphogenesis is unknown. The evidence that photoactivated phytochrome initiates calcium fluxes in cells is reviewed and how these fluxes could regulate several known red-light induced effects in plants is discussed.     

Crosstalk between cAMP and Ca2+ signaling in non-excitable cells

An impressive array of cytosolic calcium ([Ca2+](i)) signals exert control over a broad range of physiological processes. The specificity and fidelity of these [Ca2+](i) signals is encoded by the frequency, amplitude, and sub-cellular localization of the response. It is believed that the distinct characteristics of [Ca2+](i) signals underlies the differential activation of effectors and ultimately cellular events. This "shaping" of [Ca2+](i) signals can be achieved by the influence of additional signaling pathways modulating the molecular machinery responsible for generating [Ca2+](i) signals. There is a particularly rich source of potential sites of crosstalk between the cAMP and the [Ca2+](i) signaling pathways. This review will focus on the predominant molecular loci at which these classical signaling systems interact to impact the spatio-temporal pattern of [Ca2+](i) signaling in non-excitable cells.     

The effect of ruthenium red on Ca2+ transport and respiration in rat liver mitochondria

1. The effect of ruthenium red and K₂RuCl₆ on Ca²⁺ transport and mitochondrial respiration was studied.2. Low levels of ruthenium red (3–6 nmoles/mg mitochondrial protein) completely inhibited the stimulation of respiration by Ca²⁺ but had no effect on the response of respiration to ADP and 2,4-dinitrophenol.3. Ruthenium red (3–6 nmoles/mg mitochondrial protein) also inhibited respiration-dependent and ATP-supported Ca²⁺ uptake as well as the metabolism-independent, K⁺-driven translocation and the high- and low- affinity binding of Ca²⁺. However, it had no effect on the release of accumulated Ca²⁺. Respiration-dependent Sr²⁺ and Mn²⁺ uptake also were inhibited.4. At slightly higher concentrations (10 or more nmoles/mg mitochondrial protein) than those preventing Ca²⁺ binding and transport, ruthenium red markedly inhibited resting (State 4) respiration and altered, but did not prevent, the stimulation of respiration by ADP and P_i. 2,4-Dinitrophenol also overcame the ruthenium red inhibition of respiration.5. Latent ATPase was significantly inhibited by 4 nmoles of ruthenium red per mg mitochondrial protein. The Mg²⁺- and the 2,4-dinitrophenol-stimulated ATPase activities, on the other hand, were slightly, if at all, inhibited by as much as 40 nmoles of ruthenium red per mg protein. Phosphate-induced swelling of mitochondria also was not affected by high levels of ruthenium red.6. K₂RuCl₆ significantly inhibited State 4 respiration at a concentration of 2.5 μM (1 nmole/mg protein), essentially complete inhibition occurring at 10 to 50 μM (4–20 nmoles/mg protein). The inhibition of respiration by K₂RuCl₆ was overcome by Ca²⁺ just as effectively as by ADP and P_i. 2,4-Dinitrophenol also overcame the inhibition of respiration by K₂RuCl₆. Concentrations of K₂RuCl₆ 5–10 times higher than that which markedly inhibited respiration were required to inhibit Ca²⁺ transport.7. These results indicate that ruthenium red has a dual effect on mitochondrial metabolism; it interferes with the binding and transport of Ca²⁺ and at a slightly higher concentration it markedly inhibits respiration by interacting with the energy coupling pathway.     

Mechanism of action of GTP in the induction of Ca2+ release from hepatic microsomes

The mechanism by which GTP induces Ca2+ release from Ca2(+)-preloaded rat hepatic microsomes was studied. In the same concentration range as that for Ca2+ release, GTP inhibited the initial rate of ATP-driven Ca2+ uptake. It also inhibited the formation by ATP of the phosphorylated intermediate of Ca2(+)-ATPase, which had previously been identified by us as a 97-116 kDa protein (Fleschner, C.R., et al. (1985) Biochem. J. 226, 839). Vanadate, an inhibitor of Ca2(+)-ATPase, also caused Ca2+ release in a similar fashion, but its effect was not additive to that of GTP. Although the non-metabolizable GTP analogues, GMPPNP and GTP gamma S, did not cause Ca2+ release by themselves, GTP gamma S completely and GMPPNP partially blocked the effect of GTP. Pretreatment of vesicles with either cholera or pertussis toxin did not alter the responsiveness to GTP. These results indicate that GTP inhibits microsomal Ca2(+)-ATPase, independently of the Gs and Gi proteins. Because a decrease in Ca2+ uptake results in a net increase in Ca+ release, this effect of GTP seems to account, at least partially, for the GTP-induced Ca2+ release from microsomes.     

Influence of valproic acid on hepatic carbohydrate and lipid metabolism

Valproic acid (dipropylacetic acid), an antiepileptic agent known to be hepatotoxic in some patients, caused inhibition of lactate gluconeogenesis, fatty acid oxidation, and fatty acid synthesis by isolated hepatocytes. The latter process was the most sensitive to valproic acid, 50% inhibition occurring at ca. 125 microM with cells from meal-fed female rats. The medium-chain acyl-CoA ester fraction was increased whereas coenzyme A (CoA), acetyl-CoA, and the long chain acyl-CoA fractions were decreased by valproic acid. The increase in the medium chain acyl-CoA fraction was found by high-pressure liquid chromatography to be due to the accumulation of valproyl-CoA plus an apparent CoAester metabolite of valproyl-CoA. Salicylate inhibited valproyl-CoA formation and partially protected against valproic acid inhibition of hepatic metabolic processes. Octanoate had a similar protective effect, suggesting that activation of valproic acid in the mitosol is required for its inhibitory effects. It is proposed that either valproyl-CoA itself or the sequestration of CoA causes inhibition of metabolic processes. Valproyl-CoA formation also appears to explain valproic acid inhibition of gluconeogenesis by isolated kidney tubules. No evidence was found for the accumulation of valproyl-CoA in brain tissue, suggesting that the effects of valproic acid in the central nervous system are independent of the formation of this metabolite.