Tert-butyl ) -1,4-hydroquinone Research Articles

Acidic NAADP-releasable Ca2+ compartments in the megakaryoblastic cell line MEG01

BackgroundA novel family of intracellular Ca2+-release channels termed two-pore channels (TPCs) has been presented as the receptors of NAADP (nicotinic acid adenine dinucleotide phosphate), the most potent Ca2+ mobilizing intracellular messenger. TPCs have been shown to be exclusively localized to the endolysosomal system mediating NAADP-evoked Ca2+ release from the acidic compartments. ObjectivesThe present study is aimed to investigate NAADP-mediated Ca2+ release from intracellular stores in the megakaryoblastic cell line MEG01. MethodsChanges in cytosolic and intraluminal free Ca2+ concentrations were registered by fluorimetry using fura-2 and fura-ff, respectively; TPC expression was detected by PCR. ResultsTreatment of MEG01 cells with the H+/K+ ionophore nigericin or the V-type H+-ATPase selective inhibitor bafilomycin A1 revealed the presence of acidic Ca2+ stores in these cells, sensitive to the SERCA inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). NAADP releases Ca2+ from acidic lysosomal-like Ca2+ stores in MEG01 cells probably mediated by the activation of TPC1 and TPC2 as demonstrated by TPC1 and TPC2 expression silencing and overexpression. Ca2+ efflux from the acidic lysosomal-like Ca2+ stores or the endoplasmic reticulum (ER) results in ryanodine-sensitive activation of Ca2+-induced Ca2+ release (CICR) from the complementary Ca2+ compartment. ConclusionOur results show for the first time NAADP-evoked Ca2+ release from acidic compartments through the activation of TPC1 and TPC2, and CICR, in a megakaryoblastic cell line.

Olive tree wood phenolic compounds with human platelet antiaggregant properties

Oleuropein and (+)-cycloolivil are natural polyphenolic compounds with a significant radical scavenging activity present in olive tree. We have investigated the antiaggregant effects of oleuropein and (+)-cycloolivil isolated from an ethyl acetate extract of olive tree wood. Oleuropein and (+)-cycloolivil reduced the ability of thrombin to stimulate platelet aggregation. Both compounds reduced thrombin-evoked Ca 2+ release and entry to a similar extent to hydroxytyrosol. This effect was greater in platelets from patients with type 2 diabetes mellitus than in controls. Thrombin-, thapsigargin- and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ)-evoked protein tyrosine phosphorylation, which is involved in Ca 2+ signalling and platelet aggregation, is inhibited by oleuropein and (+)-cycloolivil. oleuropein and (+)-cycloolivil are natural oxygen radical scavengers that reduce thrombin-induced protein tyrosine phosphorylation, Ca 2+ signalling and platelet aggregation. These observations suggest that oleuropein and (+)-cycloolivil may prevent thrombotic complications associated to platelet hyperaggregability and be the base for the development of antiaggregant therapeutic strategies.

Intracellular Ca2+ store depletion induces the formation of macromolecular complexes involving hTRPC1, hTRPC6, the type II IP3 receptor and SERCA3 in human platelets

Endogenously expressed human canonical transient receptor potential 1 (hTRPC1) and human canonical transient receptor potential 6 (hTRPC6) have been shown to play a role in store-operated Ca2+ entry (SOCE) in human platelets, where two mechanisms for SOCE, regulated by the dense tubular system (DTS) or the acidic granules, have been identified. In cells preincubated for 1 min with 100 µM flufenamic acid we show that hTRPC6 is involved in SOCE activated by both mechanisms, as demonstrated by selective depletion of the DTS or the acidic stores, using thapsigargin (TG) (10 nM) or 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ) (20 µM), respectively, although it is more relevant after acidic store depletion. Co-immunoprecipitation experiments indicated that depletion of both stores separately results in time-dependent interaction between hTRPC1 and hTRPC6, and also between both hTRPCs and the type II IP3 receptor (IP3RII). The latter was greater after treatment with TG. TBHQ-induced coupling between hTRPC1 and 6 was transient and decreased after 30s of treatment, while that induced by TG increased for at least 3 min. TBHQ induced association between SERCA3, located in the acidic stores, hTRPC1, hTRPC6 and Orai1. TBHQ also evoked coupling between SERCA3 and IP3RII, presumably located in the DTS, thus suggesting interplay between both Ca2+ stores. Similarly, TG induces the interaction of SERCA2b with hTRPC1 and 6 and the IP3RII. The interactions between hTRPC1, hTRPC6, IP3RII and SERCA3 were impaired by disruption of the microtubules, supporting a role for microtubules in Ca2+ homeostasis. In conclusion, the present data demonstrate for the first time that hTRPC1, hTRPC6, IP3RII and SERCA3 are parts of a macromolecular protein complex activated by depletion of the intracellular Ca2+ stores in human platelets.

Functional relevance of the de novo coupling between hTRPC1 and type II IP3 receptor in store-operated Ca2+ entry in human platelets

Store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ entry in non-excitable cells, is regulated by the filling state of the intracellular Ca2+ stores. We have previously reported that a de novo conformational coupling between the type II IP3 receptor (IP3RII) and hTRPC1 channel occurs after depletion of the intracellular Ca2+ stores in human platelets, which might be involved in the activation of SOCE in these cells. Here we present for the first time direct evidence for the functional relevance of the coupling between hTRPC1 and IP3RII in SOCE in human platelets. Our data suggest that at least two pathways may contribute to SOCE in these cells. An early component, insensitive to cytochalasin D (Cyt D), is followed by a late component which is sensitive to Cyt D. Introduction of a peptide corresponding to IP3RII317–334 (IP3BD-peptide317–334) in the cells by electrotransjection impairs the coupling between hTRPC1 and IP3RII but not the interaction between hTRPC1 and STIM1 induced by store depletion. Coimmunoprecipitation experiments indicated that endogenously expressed hTRPC1 interacts with the IP3BD-peptide317–334. Electrotransjection of cells with IP3BD-peptide317–334, significantly attenuated the late stage of Ca2+ and Mn2+ entry induced by 10 nM thapsigargin (TG) or 20 μM 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ), providing evidence for a functional role of the de novo coupling between hTRPC1 and IP3RII in the activation of SOCE in human platelets.

SERCA2b and 3 play a regulatory role in store-operated calcium entry in human platelets

Two agonist-releasable Ca 2+stores have been identified in human platelets differentiated by the distinct sensitivity of their SERCA isoforms to thapsigargin (TG) and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Here we have examined whether the SERCA isotypes might be involved in store-operated Ca 2+entry (SOCE) activated by the physiological agonist thrombin in human platelets. Ca 2+-influx evoked by thrombin (0.01 U/mL) reached a maximum after 3 min, which was consistent with the decrease in the Ca 2+content in the stores; afterwards, the extent of SOCE decreased with no correlation with the accumulation of Ca 2+in the stores. Inhibition of SERCA2b, by 10 nM TG, and SERCA3, with 20 μM TBHQ, individually or simultaneously, accelerated Ca 2+ store discharge and subsequently enhanced the extent of SOCE stimulated by thrombin. In addition, TG and TBHQ modified the time course of thrombin-evoked SOCE from a transient to a sustained increase in Ca 2+ influx, which reveals a negative role for SERCAs in the regulation of SOCE. This effect was consistent under conditions that inhibit Ca 2+ extrusion by PMCA or the Na +/Ca 2+ exchanger. Coimmunoprecipitation experiments revealed that thrombin stimulates direct interaction between SERCA2b and 3 with the hTRPC1 channel, an effect that was found to be independent of SERCA activity. In summary, our results suggest that SERCA2b and 3 modulate thrombin-stimulated SOCE probably by direct interaction with the hTRPC1 channel in human platelets.

Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a versatile regulator of TRP channels. We report that inclusion of a PIP2 analogue, PIP2 1,2-dioctanoyl, does not induce non-capacitative Ca2+ entry per se but enhanced Ca2+ entry stimulated either by thrombin or by selective depletion of the Ca2+ stores in platelets, the dense tubular system, using 10 nM TG, and the acidic stores, using 20 μM 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Reduction of PIP2 levels by blocking PIP2 resynthesis with Li+ or introducing a monoclonal anti-PIP2 antibody, or sequestering PIP2 using poly-lysine, attenuated Ca2+ entry induced by thrombin, TG and TBHQ, and reduced thrombin-evoked, but not TG- or TBHQ-induced, Ca2+ release from the stores. Incubation with the anti-hTRPC1 antibody did not alter the stimulation of Ca2+ entry by PIP2, whilst introduction of anti-hTRPC6 antibody directed towards the C-terminus of hTRPC6 reduced Ca2+ and Mn2+ entry induced by thrombin, TG or TBHQ, and abolished the stimulation of Ca2+ entry by PIP2. The anti-hTRPC6 antibody, but not the anti-hTRPC1 antibody or PIP2, reduced non-capacitative Ca2+ entry by the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol. In summary, hTRPC6 plays a role both in store-operated and in non-capacitative Ca2+ entry. PIP2 enhances store-operated Ca2+ entry in human platelets, most probably by stimulation of hTRPC6 channels.

Dual role of tubulin-cytoskeleton in store-operated calcium entry in human platelets

Two mechanisms for store-operated Ca2+ entry (SOCE) regulated by two independent Ca2+ stores, the dense tubular system (DTS) and the acidic stores, have been described in platelets. We have previously suggested that coupling between the type II IP3 receptor (IP3RII) and hTRPC1, involving reorganization of the actin microfilaments, play an important role in SOCE. However, the involvement of the tubulin microtubules, located beneath the plasma membrane, remains unclear. Here we show that the microtubule disrupting agent colchicine reduced Ca2+ entry stimulated by low concentrations (0.1 U/mL) of thrombin, which activates SOCE mostly by depleting acidic Ca2+-store. Consistently, colchicine reduced SOCE activated by 2,5 di-(tertbutyl)-1,4-hydroquinone (TBHQ), which selectively depletes the acidic Ca2+ stores. In contrast, colchicine enhanced SOCE mediated by depletion of the DTS, induced by high concentrations of thapsigargin (TG), which depletes both the acidic Ca2+ stores and the DTS, the major releasable Ca2+ store in platelets. These findings were confirmed by using Sr2+ as a surrogate for Ca2+ entry. Colchicine attenuated the coupling between IP3RII and hTRPC1 stimulated by thrombin while it enhanced that evoked by TG. Paclitaxel, which induces microtubular stabilization and polymerization, exerted the opposite effects on thrombin- and TG-evoked SOCE and coupling between IP3RII and hTRPC1 compared with colchicine. Neither colchicine nor paclitaxel altered the ability of platelets to extrude Ca2+. These findings suggest that tubulin microtubules play a dual role in SOCE, acting as a barrier that prevents constitutive SOCE regulated by DTS, but also supporting SOCE mediated by the acidic Ca2+ stores.

Intracellular Ca 2+ homeostasis and aggregation in platelets are impaired by ethanol through the generation of H 2O 2 and oxidation of sulphydryl groups

The mechanisms involved in the effect of ethanol on Ca 2+ entry and aggregability have been investigated in human platelets in order to shed new light on the pathogenesis of alcohol consumption. Ethanol (50 mM) induced H 2O 2 production in platelets by Ca 2+-dependent and independent mechanisms. Ca 2+ entry induced by ethanol was impaired by catalase. Ethanol reduced SOCE mediated by depletion of the 2,5-di-( tert-butyl)-1,4-hydroquinone (TBHQ)-sensitive acidic stores but enhances SOCE regulated by the dense tubular system. This effect was abolished by treatment with catalase or the sulphydryl group reducing agent dithiotreitol (DTT). Similarly, the anti-aggregant effect of ethanol was prevented by platelet treatment with catalase or DTT. In conclusion we provide considerable evidence that ethanol alters Ca 2+ entry and reduces thrombin-induced aggregation as a result of the generation of H 2O 2 and the oxidation of sulphydryl groups in human platelets.

Effect of ADP on slow‐twitch muscle fibres of the rat: implications for muscle fatigue

Slow-twitch mechanically skinned fibres from rat soleus muscle were bathed in solutions mimicking the myoplasmic environment but containing different [ADP] (0.1 microm to 1.0 mm). The effect of ADP on sarcoplasmic reticulum (SR) Ca2+-content was determined from the magnitude of caffeine-induced force responses, while temporal changes in SR Ca2+-content allowed determination of the effective rates of the SR Ca2+-pump and of the SR Ca2+-leak. The SR Ca2+-pump rate, estimated at pCa (-log10[Ca2+]) 7.8, was reduced by 20% as the [ADP] was increased from 0.1 to 40 microm, with no further alteration when the [ADP] was increased to 1.0 mm. The SR Ca2+-leak rate constant was not altered by increasing [ADP] from 0.1 to 40 microm, but was increased by 26% when the [ADP] was elevated to 1.0 mm. This ADP-induced SR Ca2+-leak was insensitive to ruthenium red but was abolished by 2,5-di(tert-butyl)-1,4-hydroquinone (TBQ), indicating that the leak pathway is via the SR Ca2+-pump and not the SR Ca2+-release channel. The decrease in SR Ca2+-pump rate and SR Ca2+-leak rate when [ADP] was increased led to a 40% decrease in SR Ca2+-loading capacity. Elevation of [ADP] had only minor direct effects on the contractile apparatus of slow-twitch fibres. These results suggest that ADP has only limited depressing effects on the contractility of slow-twitch muscle fibres. This is in contrast to the marked effects of ADP on force responses in fast-twitch muscle fibres and may contribute to the fatigue-resistant nature of slow-twitch muscle fibres.

Alterations in intracellular calcium homeostasis and platelet aggregation induced by ethanol

The in vitro effects of ethanol on intracellular Ca 2+ homeostasis and tyrosine phosphorylation have been investigated in human platelets in order to clarify the cellular mechanisms underlying its described anti-aggregant effects. Ethanol (1–50 mM) reduced, in a dose-dependent manner, the rate and amplitude of aggregation and attenuated the phosphotyrosine content both induced by 0.1 U/ml of the physiological ligand, thrombin. Thrombin-induced Ca 2+ entry to the cytosol was significantly reduced, and capacitative Ca 2+ entry (CCE) significantly altered, by 50 mM ethanol, so that ethanol reduces CCE mediated by depletion of the 2,5-di-( tert-butyl)-1,4-hydroquinone (TBHQ)-sensitive store but enhances CCE induced by the TBHQ-insensitive pool. In conclusion, we provide considerable evidence that ethanol reduces thrombin-induced aggregation, which is likely a result of a significant inhibition of Ca 2+ entry, as well as a reduction in the activity of protein tyrosine kinases.

Two distinct Ca 2+ compartments show differential sensitivity to thrombin, ADP and vasopressin in human platelets

Recent studies propose the existence of two distinct Ca 2+ compartments in human platelets based on the expression of different SERCA isoforms with distinct sensitivity to thapsigargin and 2,5-di-( tert-butyl)-1,4-hydroquinone (TBHQ). Using fura-2-loaded human platelets we have found that depletion of the TBHQ sensitive store reduces thrombin—but not ADP—or vasopressin (AVP)-induced Ca 2+ release. Redistribution of cytosolic Ca 2+ after thrombin stimulation resulted in overloading of the TBHQ-sensitive store. This phenomenon was not observed with ADP or AVP. We found that NAADP decreases the Ca 2+ concentration into the stores in permeabilized platelets, which is prevented by depletion of the TBHQ-sensitive store. Nimodipine, an inhibitor of the NAADP receptor, reduced thrombin-induced Ca 2+ release from the TBHQ-sensitive stores, without having any effect on the responses elicited by ADP or AVP. Finally, the phospholipase C inhibitor, U-73122, abolished ADP- and AVP-induced Ca 2+ release, suggesting that their responses are entirely dependent on IP 3 generation. In contrast, treatment with both U-73122 and nimodipine was required to abolish thrombin-induced Ca 2+ release. We suggest that thrombin evokes Ca 2+ release from TBHQ-sensitive and insensitive stores, which requires both NAADP and IP 3, respectively, while ADP and AVP exert an IP 3-dependent release of Ca 2+ from the TBHQ-insensitive compartment in human platelets.

Two Pathways for Store-mediated Calcium Entry Differentially Dependent on the Actin Cytoskeleton in Human Platelets

A major pathway for stimulated Ca(2+) entry in non-excitable cells is activated following depletion of intracellular Ca(2+) stores. Secretion-like coupling between elements in the plasma membrane (PM) and Ca(2+) stores has been proposed as the most likely mechanism to activate this store-mediated Ca(2+) entry (SMCE) in several cell types. Here we identify two mechanisms for SMCE in human platelets activated by depletion of two independent Ca(2+) pools, which are differentially modulated by the actin cytoskeleton. Ca(2+) entry induced by depletion of a 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ)-sensitive pool is increased by disassembly of the actin cytoskeleton and that induced by a TBHQ-insensitive pool is reduced. Stabilization of the actin cytoskeleton prevented Ca(2+) entry by both mechanisms. We propose that the membrane-associated actin network prevents constitutive Ca(2+) entry via both pathways. Reorganization of the actin cytoskeleton permits the activation of Ca(2+) entry via both mechanisms, but only SMCE activated by the TBHQ-insensitive pool requires new actin polymerization, which may support membrane trafficking toward the PM.

Copper redox-dependent activation of 2- tert-butyl(1,4)hydroquinone: formation of reactive oxygen species and induction of oxidative DNA damage in isolated DNA and cultured rat hepatocytes

The biotransformation of butylated hydroxyanisole (BHA), a possible carcinogenic food antioxidant, includes o-demethylation to 2- tert-butyl(1,4)hydroquinone (TBHQ) which can subsequently be oxidized to 2- tert-butyl(1,4)paraquinone (TBQ). In this study, we have examined the capacity of Cu, a nuclei- and DNA-associated transition metal, to mediate the oxidation of TBHQ. In phosphate buffered saline (PBS), autooxidation of TBHQ to TBQ was not detectable, while Cu(II) at micromolar concentrations strongly catalyzed the oxidation of TBHQ to TBQ. Oxidation of TBHQ by Cu(II) was accompanied by the utilization of O 2 and the concomitant generation of H 2O 2. Using electron spin resonance spectroscopy, it was observed that Cu(II) mediated the one electron oxidation of TBHQ to a semiquinone anion radical. The formation of a semiquinone anion radical, the utilization of O 2 and the generation of H 2O 2 and TBQ could be completely blocked by bathocuproinedisulfonic acid (BCS) and reduced glutathione (GSH), two Cu(I)-chelators. 4-Pyridyl-1-oxide- N- tert-butylnitrone (POBN)-spin trapping experiments showed that the reaction of TBHQ with Cu(II) resulted in the generation of POBN-CH 3 and POBN-CH(OH)CH 3 adducts in the presence of dimethyl sulfoxide (DMSO) and ethanol, respectively, suggesting the formation of hydroxyl radical or a similar reactive intermediate. The formation of POBN-CH 3 adduct from the TBHQ/Cu(II)+DMSO could be completely inhibited by catalase, GSH or BCS, indicating that the hydroxyl radical or its equivalent is generated from the interaction of H 2O 2 with Cu(I). Incubation of supercoiled φX-174 plasmid DNA with the TBHQ/Cu(II) resulted in extensive DNA strand breaks, which could be prevented by catalase or BCS. Incubation of rat hepatocytes with TBHQ in PBS led to increased formation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in nuclear DNA. The TBHQ-induced formation of 8-OHdG was markedly reduced in the presence of cell permeable Cu(I)-specific chelator, bathocuproine or neocuproine, suggesting that a Cu(II)/Cu(I) redox mechanism may also be involved in the induction of oxidative DNA damage by TBHQ in hepatocytes. Taken together, the above results conclusively demonstrate that the activation of TBHQ by Cu(II) results in the formation of TBQ, semiquinone anion radical and reactive oxygen species (ROS), and that the ROS formed may participate in oxidative DNA damage in both isolated DNA and intact cells. These reactions may contribute to the carcinogenicity as well as other biochemical activities observed with BHA in animals. To our knowledge this study provides the first evidence that endogenous cellular Cu may be capable of bioactivating TBHQ, leading to oxidative DNA damage in cultured cells.

Effects of ADP on sarcoplasmic reticulum function in mechanically skinned skeletal muscle fibres of the rat.

1. The sarcoplasmic reticulum (SR) Ca(2+) content (expressed in terms of endogenous SR Ca(2+) content under physiologically resting conditions and measured from caffeine-induced force responses) and the effective rates of the SR Ca(2+) pump and SR Ca(2+) leak (measured from the temporal changes in SR Ca(2+) content) were determined in mechanically skinned skeletal muscle fibres of the rat at different [ADP] (< 0.10 microM to 1.04 mM). 2. The estimated SR Ca(2+) pump rate at 200 nM Ca(2+) did not change when [ADP] increased from below 0.10 microM to 10 microM but decreased by about 30 % when [ADP] increased from 10 microM to 1.04 mM. 3. The rate constant of SR Ca(2+) leak increased markedly with rising [ADP] when [Ca(2+)] in solution was 200 nM (apparent dissociation constant Kd(ADP) = 64 +/- 27 microM). Decreasing the [Ca(2+)] in solution from 200 nM to < 10 nM significantly increased the leak rate constant at all [ADP]. The SR Ca(2+) leak rate constant could be significantly reduced by blocking the SR Ca(2+) pump with 2,5-di(tert-butyl)-1,4-hydroquinone (TBQ). 4. The decrease in the SR Ca(2+) pump rate and the increase in the rate constant of SR Ca(2+) leak when the [ADP] increased from < 0.10 microM to 1.04 mM caused a 4.4-fold decrease in SR Ca(2+) loading ability at 200 nM Ca(2+). 5. The results can be fully explained by a mechanism whereby the presence of ADP causes a marked increase in the ADP-sensitive fraction of the phosphorylated pump protein, which can act as a Ca(2+)-Ca(2+) exchanger and demonstrates that ADP is an important modulator of SR function in skeletal muscle.

How does beta-adrenergic stimulation increase the heart rate? The role of intracellular Ca2+ release in amphibian pacemaker cells.

1. The mechanism by which sympathetic transmitters increase the firing rate of pacemaker cells was explored in isolated cells from the sinus venosus of the cane toad Bufo marinus. Intracellular calcium concentration ([Ca2+]i) was measured with indo-1 and membrane potential and currents were recorded with the nystatin perforated-patch technique. 2. Adrenaline or isoprenaline (2 microM) increased the transient rise in [Ca2+]i and increased the firing rate; these effects were blocked by propranolol (2 microM). 3. To determine whether the changes in [Ca2+]i might influence the firing rate we studied agents which affect either the loading or the release of Ca2+ from the sarcoplasmic reticulum (SR). Rapid application of caffeine (10 mM) to spontaneously firing cells caused a large Ca2+ release from the SR and the cells were then quiescent for 24 s. In the presence of beta-adrenergic stimulation the caffeine-induced [Ca2+]i was 14 % larger but the period of quiescence after application was reduced to 12 s. 4. Ryanodine, at either low (1 microM) or high (> 10 microM) concentration, stopped firing. However, when the SR store content of Ca2+ was tested with caffeine, at low ryanodine concentration the SR Ca2+ store was empty whereas at the high concentration the SR store was still loaded with Ca2+. beta-Adrenergic stimulation was not able to restore firing at the low concentration of ryanodine but did restore firing at the high ryanodine concentration. 5. An SR Ca2+ pump blocker, 2, 5-di(tert-butyl)-1,4-hydroquinone (TBQ) which depletes the SR store of Ca2+, also rapidly and reversibly stopped spontaneous firing. 6. The relation between the amplitude of the [Ca2+]i transient and firing rate established in the presence of ryanodine was similar when firing was restored by beta-stimulation. 7. In both spontaneously firing and voltage-clamped cells, depleting the SR store with either ryanodine or TBQ suggested that about half of the Ca2+ which contributes to the calcium transient is released from the SR. 8. These results show that the amplitude of the [Ca2+]i transient is an important factor in the firing rate of toad pacemaker cells and consequently agents which modify SR Ca2+ release influence firing rate. The effects of beta-stimulation on firing rate seem to be largely mediated by changes in amplitude of the [Ca2+]i transient.