Karol Ondrias

Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein

FK506-binding protein (FKBP12) was originally identified as the cytosolic receptor for the immunosuppressant drugs FK506 and rapamycin. The cellular function of FKBP12, a ubiquitously expressed 12,000-dalton proline isomerase, has been unknown. FKBP12 copurifies with the 565,000-dalton ryanodine receptor (RyR), four of which form intracellular Ca2+ release channels of the sarcoplasmic and endoplasmic reticula. By coexpressing the RyR and FKBP12 in insect cells, we have demonstrated that FKBP12 modulates channel gating by increasing channels with full conductance levels (by > 400%), decreasing open probability after caffeine activation (from 0.63 +/- 0.09 to 0.04 +/- 0.02), and increasing mean open time (from 4.4 +/- 0.6 ms to 75 +/- 41 ms). FK506 or rapamycin, inhibitors of FKBP12 isomerase activity, reverse these stabilizing effects. These results provide the first natural cellular function for FKBP12, and establish that the functional Ca2+ release channel complex includes FKBP12.

Regulation of the Inositol 1,4,5-Trisphosphate Receptor by Tyrosine Phosphorylation

Tyrosine kinases indirectly raise intracellular calcium concentration ([Ca2+]i) by activating phospholipases that generate inositol 1,4,5-trisphosphate (IP3). IP3 activates the IP3 receptor (IP3R), an intracellular calcium release channel on the endoplasmic reticulum. T cell receptor stimulation triggered a physical association between the nonreceptor protein tyrosine kinase Fyn and the IP3R, which induced tyrosine phosphorylation of the IP3R. Fyn activated an IP3-gated calcium channel in vitro, and tyrosine phosphorylation of the IP3R during T cell activation was reduced in thymocytes from fyn−/− mice. Thus, activation of the IP3R by tyrosine phosphorylation may play a role in regulating [Ca2+]i.

Protein Kinase A and Two Phosphatases Are Components of the Inositol 1,4,5-Trisphosphate Receptor Macromolecular Signaling Complex

The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed intracellular calcium (Ca2+) release channel on the endoplasmic reticulum. IP3Rs play key roles in controlling Ca2+ signals that activate numerous cellular functions including T cell activation, neurotransmitter release, oocyte fertilization and apoptosis. There are three forms of IP3R, all of which are ligand-gated channels activated by the second messenger inositol 1,4,5-trisphosphate. Channel function is modulated via cross-talk with other signaling pathways including those mediated by kinases and phosphatases. In particular IP3Rs are known to be regulated by cAMP-dependent protein kinase (PKA) phosphorylation. In the present study we show that PKA and the protein phosphatases PP1 and PP2A are components of the IP3R1 macromolecular signaling complex. PKA phosphorylation of IP3R1 increases channel activity in planar lipid bilayers. These studies indicate that regulation of IP3R1 function via PKA phosphorylation involves components of a macromolecular signaling complex.

Key bioactive reaction products of the NO/H2S interaction are S/N-hybrid species, polysulfides, and nitroxyl

Significance Reactions of sulfur-centered nucleophiles with nitrogenous species have been studied independently for more than a century for synthetic/industrial purposes; to understand geochemical, atmospheric, and biological processes; and to explain the origins of life. Various products and reaction mechanisms were proposed. We here identify a singular process comprising a network of cascading chemical reactions that form three main bioactive products at physiological pH: nitrosopersulfide, polysulfides, and dinitrososulfite. These anionic products scavenge, transport, and release NO/HNO or sulfide/sulfane sulfur, each displaying distinct chemistries and bioactivities. Our observations provide a chemical foundation for the cross-talk between the NO and H2S signaling pathways in biology and suggest that the biological actions of these entities can be neither considered nor studied in isolation. Experimental evidence suggests that nitric oxide (NO) and hydrogen sulfide (H2S) signaling pathways are intimately intertwined, with mutual attenuation or potentiation of biological responses in the cardiovascular system and elsewhere. The chemical basis of this interaction is elusive. Moreover, polysulfides recently emerged as potential mediators of H2S/sulfide signaling, but their biosynthesis and relationship to NO remain enigmatic. We sought to characterize the nature, chemical biology, and bioactivity of key reaction products formed in the NO/sulfide system. At physiological pH, we find that NO and sulfide form a network of cascading chemical reactions that generate radical intermediates as well as anionic and uncharged solutes, with accumulation of three major products: nitrosopersulfide (SSNO−), polysulfides, and dinitrososulfite [N-nitrosohydroxylamine-N-sulfonate (SULFI/NO)], each with a distinct chemical biology and in vitro and in vivo bioactivity. SSNO− is resistant to thiols and cyanolysis, efficiently donates both sulfane sulfur and NO, and potently lowers blood pressure. Polysulfides are both intermediates and products of SSNO− synthesis/decomposition, and they also decrease blood pressure and enhance arterial compliance. SULFI/NO is a weak combined NO/nitroxyl donor that releases mainly N2O on decomposition; although it affects blood pressure only mildly, it markedly increases cardiac contractility, and formation of its precursor sulfite likely contributes to NO scavenging. Our results unveil an unexpectedly rich network of coupled chemical reactions between NO and H2S/sulfide, suggesting that the bioactivity of either transmitter is governed by concomitant formation of polysulfides and anionic S/N-hybrid species. This conceptual framework would seem to offer ample opportunities for the modulation of fundamental biological processes governed by redox switching and sulfur trafficking.

Biphasic effects of doxorubicin on the calcium release channel from sarcoplasmic reticulum of cardiac muscle.

To define the mechanism of doxorubicin cardiotoxicity, the effects of doxorubicin and caffeine were examined on calcium release channels from cardiac sarcoplasmic reticulum. We found that calcium release from cardiac sarcoplasmic reticulum vesicles was induced by both compounds. When sarcoplasmic reticulum vesicles were incorporated into planar lipid bilayers, calcium-permeable channels were observed. Addition of caffeine (2.5-10 mM) increased channel open probability from less than 0.1% to 40%, and this effect persisted for a mean of 44 minutes. In contrast, doxorubicin (2.5-10 microM) had a biphasic effect; initially, doxorubicin activated the channel, whereas after a mean of 8 minutes, the channel became irreversibly inhibited. Although the degree of channel activation by doxorubicin was concentration dependent, the time needed to inactivate the channel was concentration independent. Pretreatment with dithiothreitol (0.2 mM) prevented doxorubicin-induced channel inactivation, and channel activity persisted for an average of 58 minutes. Dithiothreitol alone did not alter channel open probability. Our results support the hypotheses that 1) the integrity of sulfhydryl groups is important for some aspects of calcium release channel function and 2) activation and inactivation of the channel are separable processes. The biphasic effect of doxorubicin on channel function may also correspond to the clinically observed adverse effects of doxorubicin, a widely used chemotherapeutic agent that, after prolonged usage, causes a dilated cardiomyopathy.

INCREASED CYTOTOXICITY OF 3-MORPHOLINOSYDNONIMINE TO HEPG2 CELLS IN THE PRESENCE OF SUPEROXIDE DISMUTASE. ROLE OF HYDROGEN PEROXIDE AND IRON

3-Morpholinosydnonimine (SIN-1) is widely used to generate nitric oxide (NO) and superoxide radical (O). The effect of SOD on the toxicity of SIN-1 is complex, depending on what is the ultimate species responsible for toxicity. SIN-1 (<1 mM) was only slightly toxic to HepG2 cells. Copper, zinc superoxide dismutase (Cu,Zn-SOD) or manganese superoxide dismutase (Mn-SOD) increased the toxicity of SIN-1. Catalase abolished, while sodium azide potentiated, this toxicity, suggesting a key role for H2O2 in the overall mechanism. Depletion of GSH from the HepG2 cells also potentiated the toxicity of SIN-1 plus SOD. Although Me2SO, sodium formate, and mannitol had no protective effect, iron chelators, thiourea and urate protected the cells against the SIN-1 plus Cu,Zn-SOD-mediated cytotoxicity. The cytotoxic effect of Cu,Zn-SOD but not Mn-SOD, showed a biphasic dose response being most pronounced at lower concentrations (10-100 units/ml). In the presence of SIN-1, Mn-SOD increased accumulation of H2O2 in a concentration-dependent manner. In contrast, Cu,Zn-SOD increased H2O2 accumulation from SIN-1 at low but not high concentrations of the enzyme, suggesting that high concentrations of the Cu,Zn-SOD interacted with the H2O2. EPR spin trapping studies demonstrated the formation of hydroxyl radical from the decomposition of H2O2 by high concentrations of the Cu,Zn-SOD. The cytotoxic effect of the NO donors SNAP and DEA/NO was only slightly enhanced by SOD; catalase had no effect. Thus, the oxidants responsible for the toxicity of SIN-1 and SNAP or DEA/NO to HepG2 cells under these conditions are different, with H2O2 derived from O dismutation playing a major role with SIN-1. These results suggest that the potentiation of SIN-1 toxicity by SOD is due to enhanced production of H2O2, followed by site-specific damage of critical cellular sites by a transition metal-catalyzed reaction. These results also emphasize that the role of SOD as a protectant against oxidant damage is complex and dependent, in part, on the subsequent fate and reactivity of the generated H2O2.

The interaction of β-adrenoceptor blocking drugs with platelet aggregation, calcium displacement and fluidization of the membrane

beta-Adrenoceptor blocking drugs interfere with adenosine diphosphate-stimulated platelet aggregation. Alprenolol, exaprolol, Kö 1124 and propranolol inhibited the aggregation, metipranolol decreased the extent and rate of aggregation significantly. Atenolol potentiated the aggregation measured by amplitude significantly. The interaction of beta-adrenoceptor blocking drugs with aggregation correlated with the displacement of calcium ions from binding sites in isolated platelets and the fluidization of the whole platelets and isolated platelet membrane as measured with electron spin resonance of the spin probe. The most potent were highly liposoluble drugs alprenolol, exaprolol, metipranolol and propranolol which increased the calcium displacement and membrane fluidity, the least active was atenolol decreasing these phenomena. The inhibition by beta-adrenoceptor blocking drugs of stimulated platelet aggregation is rather a result of unspecific than specific receptor interaction.

The reaction products of sulfide and S-nitrosoglutathione are potent vasorelaxants.

The chemical interaction of sodium sulfide (Na2S) with the NO-donor S-nitrosoglutathione (GSNO) has been described to generate new reaction products, including polysulfides and nitrosopersulfide (SSNO(-)) via intermediacy of thionitrous acid (HSNO). The aim of the present work was to investigate the vascular effects of the longer-lived products of the Sulfide/GSNO interaction. Here we show that the products of this reaction relax precontracted isolated rings of rat thoracic aorta and mesenteric artery (but to a lesser degree rat uterus) with a >2-fold potency compared with the starting material, GSNO (50 nM), whereas Na2S and polysulfides have little effect at 1-5 µM. The onset of vasorelaxation of the reaction products was 7-10 times faster in aorta and mesenteric arteries compared with GSNO. Relaxation to GSNO (100-500 nM) was blocked by an inhibitor of soluble guanylyl cyclase, ODQ (0.1 and 10 µM), and by the NO scavenger cPTIO (100 µM), but less affected by prior acidification (pH 2-4), and unaffected by N-acetylcysteine (1 mM) or methemoglobin (20 µM heme). By contrast, relaxation to the Sulfide/GSNO reaction products (100-500 nM based on the starting material) was inhibited to a lesser extent by ODQ, only slightly decreased by cPTIO, more markedly inhibited by methemoglobin and N-acetylcysteine, and abolished by acidification before addition to the organ bath. The reaction mixture was found to generate NO as detected by EPR spectroscopy using N-(dithiocarboxy)-N-methyl-D-glucamine (MGD2)-Fe(2+) as spin trap. In conclusion, the Sufide/GSNO reaction products are faster and more pronounced vasorelaxants than GSNO itself. We conclude that in addition to NO formation from SSNO(-), reaction products other than polysulfides may give rise to nitroxyl (HNO) and be involved in the pronounced relaxation induced by the Sulfide/GSNO cross-talk.

Cdc2/Cyclin B1 Interacts with and Modulates Inositol 1,4,5-Trisphosphate Receptor (Type 1) Functions

The resistance of inositol 1,4,5-trisphosphate receptor (IP3R)-deficient cells to multiple forms of apoptosis demonstrates the importance of IP3-gated calcium (Ca2+) release to cellular apoptosis. However, the specific upstream biochemical events leading to IP3-gated Ca2+ release during apoptosis induction are not known. We have shown previously that the cyclin-dependent kinase 1/cyclin B (cdk1/CyB or cdc2/CyB) complex phosphorylates IP3R1 in vitro and in vivo at Ser421 and Thr799. In this study, we show that: 1) the cdc2/CyB complex directly interacts with IP3R1 through Arg391, Arg441, and Arg871; 2) IP3R1 phosphorylation at Thr799 by the cdc2/CyB complex increases IP3 binding; and 3) cdc2/CyB phosphorylation increases IP3-gated Ca2+ release. Taken together, these results demonstrate that cdc2/CyB phosphorylation positively regulates IP3-gated Ca2+ signaling. In addition, identification of a CyB docking site(s) on IP3R1 demonstrates, for the first time, a direct interaction between a cell cycle component and an intracellular calcium release channel. Blocking this phosphorylation event with a specific peptide inhibitor(s) may constitute a new therapy for the treatment of several human immune disorders.

Comparison of calcium release from sarcoplasmic reticulum of slow and fast twitch muscles

SummaryThe mechanism of Ca2+ release from the sarcoplasmic reticulum (SR) of slow and fast twitch muscle was compared by examining biochemical characteristics, ryanodine binding. Ca2+ efflux, and single Ca2+ channel properties of SR vesicles. Although many features of the Ca2+ release channel were comparable, two functional assays revealed remarkable differences. The comparable properties include: a high molecular weight protein from both types of muscle was immunologically equivalent, and Scatchard analysis of [3H]ryanodine binding to SR showed that theKd was similar for slow and fast SR. In the flux assay the sensitivity to the agonists caffeine, doxorubicin, and Ca2+ and the antagonists Mg2+, ruthenium red, and tetracaine differed only slightly. When SR vesicles were incorporated into lipid bilayers, the single-channel conductances of the Ca2+ release channels were indistinguishable. The distinguishing properties are: When Ca2+ release from passively45Ca2+-loaded SR were monitored by rapid filtration, the initial rates of Ca2+ release induced by Ca2+ and caffeine were three times lower in slow SR than in fast SR. Similarly, when Ca2+ release channels were incorporated into lipid bilayers, the open probability of the slow SR channel was markedly less, mainly due to a longer mean closed time. Our results indicate that slow and fast muscle have ryanodine receptors that are biochemically analogous, yet functional differences in the Ca2+ release channel may contribute to the different time to peak contraction observed in intact slow and fast muscles.